O N O The Art and Science of Analysis NH 2 · 2020. 4. 19. · N H CO 2 H N H OH OH O HN O O H N H...

N H CO 2 H N H OH OH HO HN H N H N O Cl O O O O O O N H N NH 2 O O HO HO 2014 Eastern Analytical Symposium & Exposition EAS.org The Art and Science of Analysis Abstract Book November 17–19, 2014 Garden State Exhibit Center Somerset, New Jersey

Transcript of O N O The Art and Science of Analysis NH 2 · 2020. 4. 19. · N H CO 2 H N H OH OH O HN O O H N H...

Page 1: O N O The Art and Science of Analysis NH 2 · 2020. 4. 19. · N H CO 2 H N H OH OH O HN O O H N H N O l O O O O O O N H OH e NH 2 O O O O O 2014 Eastern Analytical Symposium & Exposition



























2014 Eastern Analytical Symposium & Exposition


The Art and Science of Analysis

Abstract BookNovember 17–19, 2014 Garden State Exhibit CenterSomerset, New Jersey

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November 20142014 EAS Abstracts

1000961-EAS_17x11_Spread_PREP.indd 1 10/20/14 8:43 AM

2014 EAS AbstractsThis volume contains the final abstracts for the oral and poster presentations which take place Monday, November 17, through Wednesday, November 19, 2014. Additional abstracts received after this volume was finalized are provided in the Addendum to the Final Program. If an abstract is not provided in this volume or the Addendum, then the presenting author did not supply an abstract. For each abstract provided, a complete mailing address for the presenting author is shown. Additional authors are indicated, however, their mailing addresses are not provided.

Schedule and meeting room information for the technical sessions, as well as information concerning short courses, exhibitor workshops, and the exposition, are contained in the Final Program Book.

More InformationTo obtain answers to EAS-related questions after the meeting:

EAS Hotline 732-449-2280 EAS E-mail [email protected] EAS Web Site www.EAS.org

Eastern Analytical Symposium & Exposition, Inc.P.O. Box 185

Spring Lake, NJ 07762

Save the DateThe 2015 EAS

November 16 - 18, 2015Somerset, NJ

We want you to be a part of the54rd Eastern Analytical Symposium!

2014 Call for PapersMarch 1 – April 15, 2015

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1 Finger Prick Blood Analysis: The Omega-3 IndexKen D. Stark, University of Waterloo, 200 University Ave. West, Waterloo, ON N2L3G1 Canada

The fatty acid composition of blood can be used to determine dietary intake pat-terns, but also to indicate the potential risk of chronic disease. While over thirty individual fatty acids can be determined, blood biomarkers of omega-3 polyunsat-urated fatty status such as “the Omega-3 Index” have been developed to simplify interpretation and to enable usage in clinical settings. However, these omega-3 blood biomarkers can still be influenced by sample collection and storage, chemical preparation of specimens, gas chromatography methods, and data processing. The interpretation of omega-3 biomarkers therefore requires consideration of the type of blood sample analyzed (i.e., plasma, erythrocytes or whole blood), whether individu-als are consuming their habitual intake or undergoing dietary modification, and other biological factors known to effect fatty acid metabolism such as sex, age and genet-ics. Metabolically, measured fatty acid levels have been dictated by the metabolic balance between synthesis and oxidation, but also by the selective incorporation into complex lipid structures such as triacylglycerols and phospholipids. Differences in the incorporation of eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) into blood with fish oil feeding and the use of omega-3 blood biomarkers to confirm dietary adherence in clinical studies are examined, specifically. Insights based on rodent models that examine the relationship between blood and tissue levels including the expression of genes involved in fatty acid metabolism are also presented. Future directions in lipid biomarkers, including the potential application of lipidomic analytical techniques, are also discussed.

2 Forensic DNA Analysis: The Gold Standard for Criminal Investigation of Biological EvidenceLawrence Kobilinsky, John Jay College of Criminal Justice, 524 West 59th St., New York, NY 10019

Criminalists are charged with the analysis of biological and other physical evidence found at a crime scene in order to identify the chemical nature of the item (identifi-cation) and its source (individualization). Biological evidence can usually be iden-tified using chemical and serological methods. However, determining the origin of such evidence poses a greater challenge. There has been an evolution of tech-niques over the past several decades all of which attempt to determine genetic information for the source. This paper presents an overview of methods which have been employed to identify and individualize biological evidence. Methods have be-come more sensitive and more specific with the newer methods taking advantage of polymerase chain reaction amplification of DNA extracted from evidence and more sophisticated instrumentation and software to determine which alleles (genes) are present. Currently, forensic scientists can perform PCR-STR DNA analysis to routinely obtain genetic profiles of individuals at 24 loci using GlobalFiler (Applied Biosystems) or PowerPlex Fusion (Promega) commercial kits. The introduction of Next Generation Sequencing is revolutionizing the process of individualization even with exceedingly small evidentiary specimens. Although the NGS systems are not as yet validated for forensic casework, there is more interest in adopting this new technology. It has the capability of performing autosomal s Short tandem repeat hort tandem repeat (STR) analysis, X STR and Y STR analysis, and mitochondrial DNA analysis simultaneously from the same evidentiary item. Testing will soon become even more cost effective, more rapid, and more informative with respect to individ-ualizing the specimen at hand.

3 Doing a Lot with a Little: Ambient Ionization and MicrochemistryRobert B. Cody, Jeol USA Inc., 11 Dearborn Rd., Peabody, MA 10960

When Gene Hall invited me to speak at the symposium honoring him with the Amer-ican Microchemical Society’s 2014 Benedetti-Pichler Award, my first thought was that ambient ionization has little to do with microchemistry. Reading about the “Fa-ther of American Microchemistry,” Anton Alexander Benedetti-Pichler, I realized that the two have much in common! “Spur-of-the-moment improvisations” are a familiar situation and “uncomplicated solutions” are the goal in our lab. Although mass spec-trometry has excellent detection limits, a practical factor in handling small samples is the preparation steps that precede mass spectrometric analysis. Open-air “am-bient” ion sources like direct analysis in real-time (DART) present an opportunity for chemistry on a small scale. Each day in our laboratory, we encounter new and completely different analytical challenges. We treat the gap between the ion source and the mass spectrometer as a small chemistry laboratory. Samples are often an-alyzed on the sealed end of a melting point tube, or positioned in the gas stream with vacuum tweezers. Microextractions, in-situ derivatization of small samples, and micro-scale reactions such as chelation and hydrogen–deuterium exchange are among the many “tricks” that enhance our ability to analyze a wide range of sam-

ples with DART. For samples that cannot be analyzed by DART, we can apply other ambient ionization methods such as paper spray or several forms of inlet ionization with minimal effort. Therefore, in the spirit of Father of American Microchemistry, I look forward to celebrating Professor Gene Hall as he is awarded the 2014 Bene-detti-Pichler Award!

4 Chemical Microscopy for the 21st Century: Surface-Enhanced Raman Microspectroscopy for the Ultrasensitive Detection of Organic ColorantsMarco Leona, The Metropolitan Museum of Art, 1000 Fifth Ave., New York, NY 10028

Surface-enhanced Raman scattering (SERS) is an ideal microchemical technique for the analysis of dyes and organic pigments in cultural heritage material. Its sen-sitivity makes it possible to identify organic pigments in samples often smaller than those required for polarized light microscopy. SERS is now moving beyond the clas-sical microsampling and microanalytical approach, with the use of gels as sampling aids for quasi-non-invasive analysis, and with inkjet system to precisely and repro-ducibly deliver silver colloid nanodroplets directly on the objects to be analyzed. Fi-nally, the coupling of laser ablation with SERS microscopy is promising micrometer scale spatial resolution in the analysis of complex samples.

5 From Archimedes Palimpsest to Water Analysis: My 35 Years of Instrumental Analytical Chemistry at RutgersGene S. Hall, Rutgers University, Chemistry Department, 610 Taylor Rd., Piscataway, NJ 08854

This presentation focuses on my 35 years of analytical chemistry in the chemistry department at Rutgers. I share with you some of the exciting research projects that I have been involved with based on the use of several analytical instruments (Raman, gas chromatography flame ionization detection mass spectrometry (GC-FID-MS), MS-MS, energy dispersive X-ray fluorescence (EDXRF), Fourier transform infrared (FTIR), high-performance liquid chromatography (HPLC), and thin-layer chroma-tography -FID) in my toolbox. Teaching has been an exciting method to pass on my knowledge to up-and-coming students wishing to pursue a career in analytical chemistry. My research focus is on environmental, biological, and forensic prob-lems. Inductively coupled plasma (ICP) -MS is used for tap water analysis to deter-mine sources of Pb by measuring its stable isotopes. ICP-MS was used to monitor blood-lead concentrations from a gunshot victim. Lead isotope ratios from sequen-tial blood samples were compared to ratios from Pb bullet fragments revealed inter-esting Pb exchange. Recently, we have used shotgun lipidomics to characterize de-signer omega-3 dietary supplements used to provide docosahexaenoic acid (DHA) to Alzheimer’s patients. Forensic science is an exciting venture in our laboratory especially for student participation. In this regard, I share with you our micro EDXRF method to recover text for the first time on pages from the Archimedes Palimp-sest. Keeping with the theme of micro analytical chemistry, results using Raman microscopy, micro attenuated total reflectance (ATR)-FTIR, and micro EDXRF for non-destructive analyses of analyzing postage stamps, jewelry (counterfeit Rolex watches), counterfeit US and British banknotes, Coach handbags, mouse embryos, and krill are also presented. Use of chemometrics with digital spectral libraries and database mining software assisted in data interpretation.

6 Vibrational Optical Activity: An Established Tool for Research and Routine Analytical ApplicationsLaurence A. Nafie, Syracuse University, Department of Chemistry, Syracuse, NY 13244

Both infrared (IR) and Raman forms of vibrational optical activity (VOA) were dis-covered experimentally nearly 40 years ago. [1] The IR form is vibrational circular di-chroism (VCD) and the Raman form is Raman optical activity (ROA). For the first 20 years of VOA, VCD and ROA passed through a period of gradual growth spearhead-ed by a few academic laboratories. Then in mid-1990s two developments occurred for VCD, followed a few years later for ROA. These were the commercial availability of instrumentation for measurements and software for calculations. The comparison of measured and calculated VOA allows the determination of the absolute structure and conformation of chiral molecules. In the past two decades VOA has undergone a period of rapid growth and maturation. Academic labs now use VOA primarily to determine the conformation of biological molecules, including the use of VCD to probe the supramolecular chiral structure of amyloid fibrils. Pharmaceutical compa-nies use VOA to determine the absolute configuration of new chiral drug substances and to probe the higher order structure of biopharmaceutical molecules. This pre-sentation shows how hundreds of scientists across a wide range of disciplines now use VOA on a regular basis to determine absolute molecular structure and solve

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stereochemical problems of practical importance.


[1] Laurence A. Nafie, Vibrational Optical Activity: Principles and Applications, John Wiley & Sons, Ltd., Chichester, 2011.

7 Two-Dimensional Correlation Spectroscopy in Materials DevelopmentIsao Noda, University of Delaware, 201 DuPont Hall, Newark, DE 19716

Two-dimensional (2-D) correlation spectroscopy has gained considerable popularity over the last 25 years in not only academic but also industrial laboratories as gen-erally applicable and versatile technique to analyze spectral data. The technique is readily adaptable to many different fields, including materials design and develop-ment. In 2-D correlation spectroscopy, a set of spectra defined by two independent variables, e.g., IR wavenumbers, is generated by applying a correlation analysis to the changes in spectral signal intensities induced by an external perturbation, such as mechanical deformation and change in temperature. Notable features of 2-D correlation spectra are: simplification of complex spectra consisting of many overlapped peaks, enhancement of spectral resolution by spreading peaks over the second dimension, and establishment of unambiguous assignment through correlation of bands selectively coupled by various interaction mechanisms. 2-D correlation spectroscopy has played a key role in the materials development effort in my laboratory. An illustrative example of the successful industrial applications of 2-D correlation spectroscopy will be discussed, focused especially on the design and development of bio-based biodegradable plastic material with excellent physi-cal properties, which is now in commercial production.

8 Electrospun PHA (polyhydroxyalkanoate) Fibers: Influence of Processing on StructureBruce Chase, University of Delaware, Department of Materials Science, 210 Dupont Hall, Newark, DE 19716, Liang Gong, Isao Noda, John Rabolt

Over the past decade, the production of polymeric fibers by electrospinning has become a major focus for a large number of research groups. The ability to pro-duce sub-micron diameter fibers, often in structures and morphologies different from those found in conventional fiber spinning is potentially a powerful tool which one can use to modify bulk properties for use in a variety of applications. Poly-hydroxyalkanoates (PHAs), a class of biodegradable and biocompatible aliphatic polyesters synthesized by a variety of bacteria as intracellular carbon and energy storage materials, have properties which make them attractive for environmental, electrical, pharmaceutical, and biomedical applications. Poly [(R)-3-hydroxybutyr-ate-co-(R)-3-hydroxyhexanoate] (PHB-HHx) nanofibers have been electrospun and collected on aluminum foil with an air-gap or on a rotary disk with a sharp edge. The collection method has a significant effect on the morphology, crystal structure and chain conformation found in the resultant fibers as demonstrated using scan-ning electron microscope (SEM), wide-angle X-ray diffraction (WAXD) and Fourier transform infrared (FTIR). In addition, for the first time in the copolymer PHB-HHx, we have succeeded in generating the strain-induced metastable β-form crystalline structure, with the chains extended and adopting a planar zig-zag conformation. This crystalline structure was observed in macroscopically aligned electrospun PHB-HHx fibers collected across the air-gap or on the sharp edge of the rotary disk.

9 UV Resonance Raman Spectroscopic Studies of Protein Structure and DynamicsSanford (Sandy) A. Asher, University of Pittsburgh, Chevron Science Center, Rm. 701, Pittsburgh, PA 15260

UV Raman excitation into the ~200 nm peptide bond electronic transitions enhance peptide bond amide vibrations of the backbone. A particular band (the amide III3) reports on the Ramachandran psi angle and peptide bond hydrogen bonding. This band is Raman scattered independently by each peptide bond with insignificant coupling between adjacent peptide bonds. Isotope editing of a peptide bond (by replacing the Calpha

- H with Calpha- D) allows us to determine the frequency of individ-

ual peptide bonds within a peptide or protein to yield their psi angles. Consideration of the Boltzmann equilibria allows us to determine the psi angle Gibbs free energy landscape along the psi (un)folding coordinate that connects secondary structure conformations. The psi angle coordinate is the most important reaction coordinate necessary to understand mechanism(s) of protein folding. We examine the details of peptide folding conformation dynamics with laser T-jumps where the water tem-perature is elevated by an 1.9 µM IR nsec laser pulse and we monitor the ~200 nm UV Raman spectrum as a function of time. These spectra show the time evolution of conformation. We discuss the role of salts on stabilizing conformations in solution.

10 Application of UPLC-High Resolution MS in Drug Product Comparability StudiesPilsoo Kang, Genzyme, a Sanofi Company, 45 New York Ave., Framingham, MA 01701, Jianmei Kochling, Yimin Hua, Robert W. Donaldson

A comparability study is conducted in order to evaluate the impact of the changes to drug product for every biological manufacturing change, whether caused by site, scale, or process. The comparability study will involve not only demonstration of analytical equivalence for protein structures, but also verify that the products have similar quality attributes and equivalent functionality. The analytical demonstration in similarity commonly involves the use of forced degradation methods, by which structure quality attributes as well as product degradation pathways are compared using analytical methods. In this talk, comparability studies utilizing the state-or-art technology with ultra-performance liquid chromatography (UPLC)-high resolution mass spectrometry (MS) technique are presented.

11 Quality-by-Design Method Development of the 2nd Generation Peptide Map Assays for Therapeutic ProteinsYimin Hua, Genzyme, a Sanofi Company, 68 New York Ave., Framingham, MA 01701, Francisca Gbormittah, Alekhya Pochiraju, Qian Guan, Robert W. Donaldson, Jianmei Kochling

Analytical method development process has evolved along with industry’s signif-icant understanding of the “quality-by-design (QbD)” concept. Quality-by-design approach analytical methods development relies upfront understanding of targeted method attributes and acceptance criteria, process and product knowledge, regula-tory requirements and the incorporation of the modern technology. In this presenta-tion, the QbD method development process of peptide map assays utilizing UPLC and mass spectrometry is presented. A platform approach applying for multiple ther-apeutic proteins is discussed and case studies are provided.

12 Withdrawn by the author.

13 The Influence of Biocompatible Coating Thickness of Non-porous Polymer Beads on Protein Recoveries and ResolutionMichael Lu, BioChrom Labs, 1719 S. 13th Street, PO Box 2404, Terre Haute, IN 47802

The thickness of biocompatible coating was determined by molecular weight, viscosity, and chemical composition of coating reagents. It can be changed. The thickness of biocompatible coating of non-porous polymer beads affected protein recoveries. The low coating thickness of hydrophilic beads had poor recoveries in comparison with optimized and high coated thickness of hydrophilic beads. The derivation of hydrophilic beads with hydrophobic groups such as C3 showed the effects of coating thickness on the recoveries and resolution of proteins on hydro-phobic interaction media. The low coating thickness of media, C3 NP10 showed poor recoveries and long retention time. The optimized coating thickness of media showed good recoveries, retention and resolution. The high coating thickness of media showed good recoveries, but short retention. The further derivation of C3 NP10 with oxidizing agents showed the effects of coating thickness on the recov-eries and resolution of proteins on weak cation exchange media. The low coating thickness of media CM NP10 showed good resolution, longer retention time and poor recoveries. The optimized coating thickness of media showed good resolution and recoveries. The high coating thickness of media showed poor resolution, but good recoveries. In Summary, the low coating thickness of polymer beads showed poor recoveries. High coating thickness of polymer beads showed short retention and poor resolution. The optimized coating thickness of polymer beads shows good recoveries and resolution. The article shows the comparison of chromatograms of the different biocompatible coating thickness of polymer beads on protein recover-ies and resolution.

14 Development, Validation and Comparison of a Competitive Ligand Binding and a Functional Cell-Based Assay for Neutralizing Antibody (NAb) DetectionWeifeng Xu, Bristol-Myers Squibb, Route 206 & Province Line Rd., Lawrenceville, NJ 08550, Michael Sank, Jennifer Cummings, Xuefeng Li, Robert Dodge, Renuka Pillutla, Binodh DeSilva

Two assays were developed and validated to detect NAb against Bristol-My-ers Squibb post-marketing mono-clonal therapeutics in clinical samples: an ELI-SA-based competitive ligand-binding assay (CLB) and a cell-based functional assay. Both assays had minimal drug interference at the high concentration of drug expected in clinical samples. In the competitive ligand binding assay, drug competed with Ruthenium-labeled ligand for binding to plate-coated drug target. Presence of NAb blocked binding of drug to its target thus increased signal. For the functional bioassay, Jurkat cells overexpressing cell-surface restricted drug target were incubated with ligand-expressing Raji cells. Without drug, drug target engaged

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with ligand and inhibited luciferase-reporter expression while presence of the drug blocked this interaction and led to increased reporter expression. Presence of NAb blocked drug and facilitated the interaction between drug and its ligand and de-creased Luciferase-reporter expression. Since high drug concentration are present in most of the clinical samples, a bead extraction method followed by acid dissoci-ation was developed to remove both drug and serum factors. However, unlike CLB assay, cells used in the functional bioassay were very sensitive to a broad range of acid buffer commonly used for this purpose. Much more efforts were focused on improving the extraction efficiency and buffer compatibility for the cell assay.

15 Withdrawn by the author.

16 USP <233> Analysis of DMSO Soluble Drug Substances and Excipients Using NexION ICP-MSJonathan L. Sims, PerkinElmer, Chalfont Rd., Seer Green, HP9 2FX United Kingdom, Fadi Abou-Shakra

Compliance with the requirements of United States Pharmacopeia (USP) <232> that “Elemental impurity levels present in drug substances and excipients must be known, documented and made available on request” is demonstrated through the application of NexION inductively coupled plasma mass spectrometry (ICP-MS) fol-lowing the procedure presented in USP <233> for samples prepared directly as organic solutions. A range of common excipients and drug substances that are used in the formulation of readily available high dose oral medicines have been studied and the levels of the 15 target elements discussed.

17 Determination of Elemental Impurities: A Practical Approach from a Contract LabSamina Hussain, Exova, 9240 Santa Fe Springs Rd., Santa Fe Springs, CA 90670

There are several challenges a contract lab faces with the determination of elemen-tal impurities in finished drug products, active pharmaceutical ingredients (APIs), and excipients. Due to the expense and complexity of inductively coupled plasma instrumentation, a contract lab is often contacted by manufacturers to implement compliance with the new elemental impurities chapters, such as United States Phar-macopeia (USP) <232>, USP <233>, EP 5.20, and International Conference on Harmonization (ICH) Q3D. A common issue arising during the method development and validation process concerns setting appropriate specifications for a particular material. An evaluation of the specifications and how they relate to each and every drug product, raw material, and the manufacturing process as a whole is required. Regulatory compliance in different regions of the world may require a risk assess-ment involving different elements. Mastering sample preparation and analysis meth-ods for a wide range of materials and specifications requires experience in these sensitive techniques. Contamination controls at ultra trace levels is also a challenge. An understanding of potential interferences associated with the instrumentation and specific analytical approach is essential. Finally, there is a concern about analytical capacity of existing contract labs for scheduling the numerous method validations required once the chapters have been finalized. This presentation addresses these challenges.

18 Use of EDXRF for the Determination of Elemental Impurities in Pharmaceutical MaterialsNancy Lewen, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903

United State Pharmacopeia (USP) and International Conference on Harmonization(ICH) requirements for elemental impurity monitoring in pharmaceutical productsare changing and moving towards performance based requirements, not partial tospecific techniques. Although USP <233>, which describes the methods for elemen-tal impurity testing, still focuses on inductively coupled plasma (ICP) and ICP-mass spectrometry for historical reasons, other techniques are now allowed. Due to typi-cal ICP challenges like sample digestion, waste streams and operation costs, com-panies seek for more simple, fast and cost effective means to perform these anal-yses. This study focuses on X-ray fluorescence (XRF), which is now a compendial technique, using a PANalytical Epsilon 5 Energy Dispersive (ED) XRF spectrometer as an alternative to ICP for determination of elemental impurities in pharmaceutical products in a non-destructive manner.

19 An Update on General Chapters <232> and <233> and Implementation TimelinesKahkashan Zaidi, United States Pharmacopeia, 12601 Twinbrook Parkway, Rockville, MD 20879

Heavy metals tests have been available in the United States Pharmacopeia and The National Formulary (USP-NF) for many years. The screening tests in <231> are non-element-specific limit tests. Sample preparation and instrumental technology available today (e.g., closed-vessel microwave digestion followed by atomic absorp-

tion spectroscopy and inductively-coupled plasma spectroscopy with various modes of detection) permit the quantification of a broad range of elements at low levels with excellent precision and accuracy. USP has updated its heavy metals testing to pro-cedures that are element specific and performance based. General chapters <232> Elemental Impurities – Limits and <233> Elemental Impurities – Procedures have been official since February 1, 2013 but will have a delayed implementation date of December 1, 2015. This presentation discusses these general chapters, their implementation and future directions based on the ICH Q3D Expert Working Group.

20 Simple Magnification, Optical Phase Contrast Microscopy, and Desktop SEM-EDS: Characterizing a Cleaning Agent John Scott, New York Conservation Foundation, 261 Fifth Ave., Rm. 2000, New York, NY 10016

No abstract submitted by the author.

21 SEM-EDS and Multi-Instrumental Analysis: Studying the Corrosion of Ag 999 and Ag 925 Inside and Outside VitrinesPaula Homem, Faculdade de Letras da Universidade do Porto, Via Panorâmica s/n, Porto, 4150-564 Portugal

A study about fine silver and sterling silver tarnishing has been conducted in un-controlled NW Portuguese exhibition contexts, not confined to vitrines. Outdoor/indoor/inside vitrines atmospheric deposition, temperature and relative humidity (RH) were studied. Ag 999 and Ag 925 samplers were exposed at 45° angle and used to model artifacts’ reactions, in response to seasonal conditions and annual exposure. A multi-instrumental analysis approach was adopted to study interactions effects, considering scanning electron microscopy coupled with X-ray microanalysis (SEM-EDS), X-ray photoelectron spectroscopy (XPS), linear sweep voltammetry (LSV) and colorimetry. The corrosion process proved to be complex and sensitive to seasonal conditions, tending to slow down with time. Ag 925 is always more reactive than Ag 999. Globally, the corrosion products detected were: Ag2O, Ag2O2, AgCl2¯, AgCl, AgHS, Ag2S, Cu2O, Cu(OH)2, CuO, CuCl, CuCl2¯, CuCl2 and 3Cu(OH)2.CuCl2. Formation of oxides and hydroxides correlated with relative absence of chlo-ride ions inside vitrines and on down-facing surfaces, and was favored by relatively high O3 in spring and summer and by relatively high NO2 and RH in autumn and winter. Relatively high presence of chloride ions outside vitrines and on upward-fac-ing surfaces, correlated with formation of chlorides and hydroxichlorides. Formation of silver sulfides, detected only after a year-long exposure and mainly on Ag 999, correlated with relative absence of chloride ions inside vitrines and on down-facing surfaces. Surprisingly, their presence was only detected in association with particles of sodium and ammonium sulfate. SEM-EDS contributions to the results are pre-sented and discussed.

22 Accessible Microanalysis in Authentication and Attribution: A Case Study Soraya Alcalá, The Hispanic Society of America, 613 W 155th St., New York, NY 10032, Hèléne Fontoira, Thiago Assan Piwowazrczyk, Marcelo Myada Redígolo

Small to medium collections often lack ready access to advanced analytical exper-tise and services to augment curatorial and connoisseurship inputs in authentication of works of art. However, with careful organization a lot may be accomplished using basic photon microscopy, sometimes augmented by readily accessible methods of elemental and molecular analysis. In this presentation we show how this is possi-ble, by using basic materials analytical microscopical techniques to study a painting from the collection of Fordham University, NY, NY. We wished to help determine the period of the painting, which seems to be High Baroque or neo-Baroque Italian. Our painting evokes the European Counter-Reformation, especially certain Italian masters, but it has also been credibly suggested that this work is from Mexico. If the provenance is Mexican, the painting could still be Italian, since Mexicans imported much Italian art starting in the colonial period before 1821, and throughout the 19th century. Moreover, the Academy of Fine Arts of San Carlos, established at Mexi-co City in the late 18th century, sent Mexican artists to Rome throughout the 19th century, and their works reflect Italian influence. Our aim was to test attributed pro-venience and period, possibly involving painting techniques much associated with Mexican artists, and to note alterations attributable to aging or to prior treatment.

23 Scientific Analysis of Pietro da Cortona’s The Triumph of DavidKristen E. Watts, Villanova University, 800 E. Lancaster Ave., Villanova, PA 19085, Amanda Norbutus, Anthony Lagalante, Kristin deGhetaldi

A two-year restoration campaign at Villanova University has begun on a 17th centu-ry masterpiece attributed to baroque master painter Pietro da Cortona. The massive painting, colloquially known as The Triumph of David (144”×228”), has been located in a library reading room since its donation to Villanova University from Italy after sustaining damages during the Battle of Nemi in World War II. The painting requires a varnish replacement, canvas mends, and corrections to prior, less-scrupulous res-toration campaigns. In order to provide the best conservation and treatment plan for

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the painting, it is important to employ a multitude of analytical techniques to obtain the most complete understanding of the painting’s provenance, history, and place in Pietro’s oeuvre. Through scientific research, we can obtain a better understanding of the materials and methods utilized in the painting’s original construction and sub-sequent restoration campaigns. This presentation summarizes our findings using a host of traditional analytical methods to image and characterize the pigments and binders present in the original and restoration layers of the painting, including: fluo-rescence microscopy, infrared microscopy, portable X-ray fluorescence, and scan-ning electron microscopy paired with energy dispersive X-ray spectroscopy. New research in our laboratory is aimed at employing desorption electrospray ionization mass spectrometry (DESI-MS) to image binding materials present in paint layer cross-sections. DESI-MS is capable of 100 micron image resolution and can readily distinguish between areas of oil paint versus areas of more modern materials based upon the stratigraphy of characteristic molecular ions.

24 Synchrotron X-Ray Microtomography, Confocal Laser Microscopy and SEM Imaging Applied to Characterization of Fungal Biodeterioration of Paper in the Context of Cultural Heritage Collections Hanna M. Szczepanowska, Smithsonian Institution, 4210 Silver Hill Rd., Room E2101, Suitland, MD 20746, Diwaker Jha, Thomas G. Mathia

Biodeterioration of paper induced by fungi is one of the most complex phenomena due to involvement of living organisms thriving on heterogeneous substrate such as paper. Both materials, paper and fungi, are complex structurally and chemically, making their characterization very challenging. Black stains produced by Dematia-ceous (black fungi) and Chaetomiaceae family fungi were analyzed. Their spatial and temporal distribution on the paper surface and in the matrix were investigat-ed with Confocal Laser Scanning Microscopy (CLSM) and X-ray Microtomography (XµT) complementing scanning electron microscopy (SEM-VP) and transmitted light microscopy (TLM) imaging. The XµT data was obtained on ID 19 beamline at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. The 3D modeling of fungi in the paper matrix and statistical analysis of cells-size revealed presence of yeast , information which could not be obtained solely from the surface or microscopic analysis. This new analytical multi-scale approach combining sever-al techniques elucidates dynamic interactions of fungi with paper, deposition of bio-logical cells in the paper matrix and patterns of fungal growth on the surface of paper providing base for designing preservation strategies for cultural heritage collections.

25 Investigation of Body Products on Worn Clothing Substrate Found at a Fire Scene Conflicting with Ignitable Liquid Residue Identification by GC-MSGina M. Guerrera, University of New Haven, 300 Boston Post Rd., West Haven, CT 06516, Brooke W. Kammrath, Erika Chen, Michael Valetutti

The question of whether cosmetic and medicinal body product deposits on clothing as well as the composition of clothing are being mistaken for ignitable liquids is of considerable importance for criminalists in forensic science laboratories. Body products and oil secretions can have similar chemical profiles to ignitable liquid residues (ILR) as a result of comparable chemical compounds that are found in both sources. This research investigates the substrate contribution of worn and unworn clothing due to possible body oil, lotions, perfume/cologne, medicinal creams as well as other body products. Clothing samples of varying fiber content were col-lected from three individuals having applied a body product provided for them by the researcher before wearing the clothing item. Passive headspace by activated charcoal strips (ACS) and desorption with carbon disulfide (CS2) was the method of choice for sample preparation. The samples were then analyzed by gas chroma-tography-mass spectrometry with a quadrupole mass analyzer, and evaluated as to whether the residues could be mis-identified as belonging to a class of ignitable liquid residues. This research has important implications for criminal justice because the identification of an ILR on clothing could result in a person being charged with an arson crime, thus it is critical to be able to differentiate substrates from ILRs.

26 Differentiation of Human, Animal, and Synthetic Hair by ATR FTIR SpectroscopyJeremy M. Manheim, University at Albany, 1400 Washington Ave., Albany, NY 12222, Kyle Doty, Greg Mclaughlin, Igor K. Lednev

Hair and synthetic fibers are a common form of trace evidence found at crime scenes. Currently, the methodology of microscopic examination of potential hair ev-idence is lacking statistical probability and is inherently subjective. Here attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy along with chemometric analysis was used to identify and differentiate human hair from a syn-thetic fiber and animal hair with a specific confidence and solely from its spectrum. Two partial least squares-discriminant analysis (PLS-DA) models were constructed: one to differentiate natural hair fibers from synthetic fibers (binary) and the second discriminating human hair from animal hair (species specific). Both models were successful in internally differentiating the classes examined; synthetic hair was completely separated from actual hair in the binary approach and all human sam-

ples were correctly predicted as human in the species specific model. An external validation was performed using hair and fiber samples, which were not used in the training set. Overall, this method is able to quantitatively identify a sample of hair as being of human origin with a high degree of confidence, using a non-destructive and rapid approach, without the need for sample preparation or specialization, proving its significance to the field of forensic science. The differentiation of animal species based on the hair analysis is discussed.

27 Quantitation of Dyes in Forensic Fibers Using UHPLC-TOF MS and Building a Dye Database with AxION EC ID SoftwareJoanne Mather, PerkinElmer, 940 Winter St., Waltham, MA 02451, Sharanya Reddy, Bonnie Marmor

Fibers originating from textiles in carpet, furniture, and clothing are often used as evidence at crime scenes. Fiber analysis is traditionally performed using vari-ous non-destructive techniques including optical microscopy, visible and infrared micro-spectrophotometry. Chromatography techniques such as poor resolving thin-layer chromatography or higher resolution high-performance liquid chromatog-raphy (HPLC) with UV or diode array detection (DAD) can also be used for fiber identification. In this study, we show analysis of dyes extracted from fibers using ul-tra HPLC coupled to time-of-flight mass spectrometry (TOF- MS), providing a more specific and sensitive technique. TOF-MS provides a higher degree of discrimina-tion between closely resembling dyes with similar functional groups of detection compared to UV or DAD detection using accurate mass measurements and isotope ratio confirmations. We also provide a method to build a database for forensic dyes using accurate mass and isotope profile information acquired from the time-of-flight mass spectrometer along with the proprietary AxION® EC ID™ software.

28 Characterization of the Binding Medium Used in Roman Encaustic Paintings on Wall and WoodRuben M. Savizky, The Cooper Union, 41 Cooper Square, New York, NY 10003, John L. Bove, Pedro Cuni, Jorge Cuni, Brielle Eisen

The characterization by means of attenuated total reflectance Fourier transform infrared spectroscopy and gas chromatography-mass spectrometry of the binding medium present in eight samples of Roman wall paintings coming from three ar-chaeological sites in Spain and a sample of a Roman-Egyptian mummy portrait on wood showed strong evidence that the medium in all the studied samples was composed of beeswax and soap. These results suggest for the first time that Roman artists used in wall and easel paintings a water soluble encaustic paint of beeswax and soap. Experimental studies with a wax-and-soap technique showed that this painting technique allows reproduction of the physical characteristics of many Ro-man-Egyptian encaustic mummy portraits with greater accuracy than the hot wax encaustic paint and the alkali-treated encaustic paint often considered to be the painting techniques used in these portraits. Wax-and-soap encaustic also showed greater accuracy in reproducing the physical characteristics of Roman wall paintings than the fresco painting technique, generally thought to be the technique used to execute such paintings. This study suggests that wax-and-soap encaustic could be a common painting technique among Roman artists, and its composition could cor-respond to a lost ancient encaustic formulation searched for the last five centuries by many artists and researchers dissatisfied with the former reconstructions of the ancient encaustic painting technique.

29 Withdrawn by the author.

30 Differentiating Authentic from Counterfeit Drugs by Raman MicroscopyFran Adar, Horiba Scientific, 3880 Park Ave., Edison, NJ 08820, Pauline Leary, Thomas A. Kubic

The introduction of counterfeit drugs into the United States marketplace is important for two reasons. These products appear without the oversight of regulatory agen-cies such as the Food and Drug Administration who can confirm the safety and efficacy of the products. In addition, their sale deprives the legitimate corporations (both innovator and generic companies) from benefiting from the results of the re-search and development that have been spent to produce these products. Raman microscopy provides at least two means of screening these products. On the one hand, the tablet coating can be removed enabling the acquisition of a Raman map from which the components’ identities and distributions can be measured. On the other hand, spectra of the packaging and the printing colorants can be measured for comparison of suspected counterfeits to the authentic products. Examples of both are shown in this talk.

31 Withdrawn by the author.

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32 Fit-for-Purpose Miniature NIR Spectroscopy for Solid Dosage Continuous ManufacturingKrizia Karry, Rutgers University, 98 Brett Rd., Piscataway, NJ 08854

Near-infrared (NIR) spectrometers have long been the process analytical technolo-gy (PAT) tools of choice for pharmaceutical and industrial scientists seeking to gain both physical and chemical information from a sample. More recently, the develop-ment of fast and inexpensive miniature NIR instruments have made possible the real-time monitoring of individual unit operations within a process. For continuous manufacturing operations this means that, NIR-based multivariate models can be used as supervisory tools for closed loop control and eventually real-time release of the drug product. In this presentation we discuss different case studies for which the implementation of ultraportable spectrometers of low wavelength ranges (950 – 1650 nm) aided in formulation and process trouble-shooting and ultimately, how these were used as PAT tools for the continuous manufacturing of tablets and oro-dispersible polymer films.

33 The Next Generation of Long Wavelength Handheld Raman Spectrometers for Raw Material Inspection and BeyondClaire Dentinger, Rigaku Raman Technologies, 14 New England Executive Park, Suite 102, Burlington, MA 01803, Mark Mabry, Claude Robotham

Handheld Raman analyzers are very well suited for material identification outside the analytical laboratory and they are becoming ever more accepted and used for portable measurements in such diverse areas as the pharmaceutical industry, safe-ty and security professionals and academic institutions. We have recently designed a smaller and higher performance handheld spectrometer with a 1064 nm excitation wavelength. The use of 1064 nm excitation reduces fluorescence for most mate-rials. This allows the 1064 nm excitation Raman to measure many materials that cannot be measured at lower excitation wavelength as well those materials which can. Using a handheld Raman, incoming raw material inspection can be done in a warehouse or loading dock not just in analytical laboratory. This can reduce material hold times and analysis costs. Using 1064 nm excitation allows a wider range of materials to be investigated. For example polysorbates are often high purity and used in sterile applications, this combined with the fact that they will degrade upon exposure to oxygen makes analyzing these materials in the original amber bot-tles highly advantageous. We investigate how these Raman measurements in the sealed containers compare at different excitation wavelength and for different poly-sorbates. Other incoming raw materials and pharmaceutical products are discussed in terms of analysis with different Raman wavelengths and specificity of Raman to identify closely related materials.

34 Mixture Analysis Using Handheld Raman SpectrometerDawn Yang, B&W Tek, 19 Shea Way, Newark, DE 19713

Over the past few years, Raman technology has been increasingly applied in fields that require equipment that is portable, gives quick results, and is easy to use. While the data (spectra) collected using Raman on pure chemical compounds are known for their distinct peaks and current software with advanced algorithms allow for great selectivity, attempting to define materials of impure compounds and mixtures re-quires a mixture analysis function with new and effective algorithms. As most mate-rials found in the field are mixtures, a handheld Raman spectrometer that is easy to carry around and is simple to use is ideal for onsite applications when equipped with mixture analysis capabilities. In this study, mixture analysis on a handheld Raman spectrometer is reviewed. Results for different types of mixtures are discussed.

35 Miniaturized Multi-Spec (UV-NIR, Raman and Hyperspectral) Instruments in Practical In-situ AnalysisFredrick Haibach, BaySpec, 1101 McKay Dr., San Jose, MA 95131

Using “orthogonal” techniques that provide additional, and complementary data, provides additional confidence in analytical models. The advantage can be powerful if the data arises from the same source and the combination of instrumentation is sufficiently reliable. Chromatography has become an extremely useful and powerful tool due to the use of hyphenated techniques that use this “orthogonal information” principle. BaySpec is creating advances in device miniaturization and integration, efficiently combining ultraviolet near-infrared (UV-NIR), Raman and hyperspectral imaging into reliable, and affordable, instrumentation that we call “Multi-Spec.” The hyphenated methods have been described before; these instruments provide a straightforward path to measurements or integration into a predefined sampling setup. Improved reliability and stability arises from the use of volume phase gratings (VPG), solid-state light sources and detectors. We report on a variety of BaySpec’s newly developed miniaturized systems including the world’s first multi-excitation wavelength miniature Raman spectral engines (Raman-Raman), Amphi-spec™ (NIR-Raman), and recently, handheld hyperspectral imagers (spatial-NIR). Advan-tages and specific, real-world examples of the different techniques are presented. Raman-Raman instruments allow the efficient investigation of samples that exhibit fluorescence, resonance Raman signatures, absorbance, or low Raman scatter-ing cross-sections. NIR-Raman systems combine the specificity of Raman and its

easily-interpretable spectrum with the increased volume sampling in NIR and its sensitivity to physiochemical industrial processing parameters. Hyperspectral imag-ing has clear advantages, and has been brought to a number of types of instrumen-tation. We anticipate providing example data from chemical and biological material analysis, such as precision agriculture, threat detection, contamination control, fo-rensics, anti-counterfeiting, point-of-care medical diagnostics, and numerous other in-field applications.

36 Off-Line and In-Line Monitoring of Mulling ProcessesSavitha S. Panikar, Rutgers University, 19 Paulus Blvd., New Brunswick, NJ 08901, Suyang Wu, Benjamin J. Glasser, Rohit Ramachandran

The characterization of mulling/extrusion of alumina, for the purposes of making catalyst supports, is investigated. Specifically: 1) the optimal instrument set-up of a near-infrared (NIR) spectrometer and, 2) monitoring and quantifying critical qual-ity attributes (CQAs) of a continuous mulling process is reported. For this, off-line and in-line calibration standards of alumina/nitric acid granules with known water concentrations were prepared. An optimized unit operation was set up to produce extrudable granules. Subsequently, the relationship between sample-detector dis-tance and prediction accuracy was investigated for a JDSU microNIR spectrometer. It was found that sample-detector distance was optimal around 15mm at 15mm of sample thickness and 25 of scan number. Additionally, set-ups that could be used for in-line monitoring have been investigated including monitoring from the side and/or bottom surface through glass or plastic. Next, after ensuring adequate method accuracy, NIR prediction models were built for the in-line measurement of critical quality attributes: granule water content and agglomerate size.

37 Achieving Greater Process Understanding through On-Line Reaction MonitoringMichael L. Hall, SABIC, 1 Noryl Ave., Selkirk, NY 12158, Nancy L. Jestel, Eylem Tarkin-Tas, Mark Denniston, Carolyn Degonzague

It has been well demonstrated across different industrial environments that on-line process analytical technology for reaction monitoring can provide chemists and en-gineers greater understanding of chemical processes. By simultaneously collecting data from multiple sensors, quick transitions can be detected that likely would be missed by off-line techniques due to delays associated with sampling and analysis. During this talk, examples are presented where lab scale oxidative coupling polym-erizations were monitored real-time by Fourier transform infrared (FTIR), near-in-frared (NIR) and ultraviolet-visible (UV-VIS) spectroscopies. In the NIR spectra, the signal reduction of hydroxyl overtones associated with phenolic monomers can be observed while bands associated with oligomers and polymers were more easily detected by FTIR. UV-VIS spectroscopy was employed to gain greater sensitivity for the detection of low level reaction intermediates. These data sets were comple-mented by real-time temperature and agitation data streams. Chemometric decom-position and correlation techniques were employed to extract information from the individual and combined data sets. The process understanding gained from these models was leveraged to make more informed decisions during lab scale process development.

38 In-Line PAT Method Development for Flowing Pharmaceutical Blend Homogeneity MonitoringFan Zhang-Plasket, Merck, Sumneytown Pike and Broad St., West Point, PA 19486, John P. Higgins, Cat MacConnell, Mano Ramasamy, Robert F. Meyer, Charles E. Miller, Jennifer Pai

In-line, real-time measurement of flowing pharmaceutical blend homogeneity is a key element for demonstrating the feasibility of continuous blending technology. The same measurement can be used for the quantitative blend homogeneity in a feed frame during tablet compression to detect powder segregation. Process analytical technology (PAT) analysis could potentially be used for process control and/or re-al-time release. The development of quantitative multivariate models using in-line near-infrared and Raman data is presented. The performance of the models on a continuous blending process and during a tablet compression process along with long term model robustness is discussed.

39 NIR Analyses in the Field; How to get better AnswersFranklin E. Barton, Light Light Solutions Instruments, 165 Sunnybrooke Dr., Athens, GA 30605, James A. de Haseth

For over a half century near-infrared (NIR) has been used to make analyses of ag-ricultural commodities and crops easier, quicker and provide improved products to all. In this half century the instrumentation has become easier to use, less expensive and more reliable. The software has evolved from “homegrown” programs to very powerful, user friendly chemometric packages. We have become very comfortable in our heated, air conditioned, well-lighted laboratories and that is a good result of years of research effort. However, there are issues which remain to be resolved. First, there is the issue of sample preparation. This issue has been with us from the beginning and there have been great improvements in instrument sampling geome-tries and ways to make the analysis with minimal sample preparation. Even so there

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is always the risk of some change in the sample that is correlated to the sample’s modification more than to its composition. Second, the delay in analysis caused by transport from the source, storage at the laboratory and subsequent preparation can affect the results and therefore the accuracy of NIR methods. In recent years we have spent our efforts to move NIR analyses from the laboratory to the manu-facturing plant and the field. This presents a new and different set of challenges and is illustrated by the applications we have developed. These challenges and their solutions have resulted in new instrumentation that is flexible, lightweight, and robust. It has required new approaches to software and the hardware, particularly to scan the sample.

40 High-Throughput Virtual Slit Technology: Benefits for Polymer ExtrusionYusuf Bismilla, Tornado Spectral Systems, 555 Richmond St. West, PO Box 218, Toronto, ON, M5V 3B1 Canada, Jeffrey T. Meade, Bradford B. Behr, Andrew T. Cenko, Brandon DesRoches, Jared Slaa, Arsen R. Hajian

High-throughput virtual slit (HTVS) technology is a revolutionary new design para-digm for dispersive spectrometers that enables slit-like spectral resolution without the associated throughput loss, significantly enhancing performance for any given system size. This makes the HTVS particularly useful for real-time monitoring appli-cations where speed and sensitivity are both required. Raman chemical identifica-tion is a highly sensitive method of determining the chemical makeup of samples as well as inferring information on structural properties of the chemicals themselves. On a manufacturing line, fast product verification leads to increased quality and low-er costs—HTVS technology allows for significantly decreased measurement time, providing substantial productivity increases. Raman measurements are tradition-ally signal limited due to the low Raman signal intensity. Slit-based spectrometer designs must balance throughput and resolution requirements, severely impacting system sensitivity when high spectral resolution is required. HTVS technology im-proves measurements by mitigating the standard tradeoffs between spectrometer throughput and resolution. HTVS technology allows for size and weight reductions while maintaining performance. This enables significant enhancements where small and/or lightweight systems are required. HTVS spectrometers have the capability to open entirely new regimes to spectroscopic work. We will present an in-depth application case study showing the benefits of HTVS to polymer extrusion. Real time measurements of the polymer melt in an extruder provide valuable information on melt properties, both chemical and mechanical, and allow for quantitative and qualitative analysis.

41 Analytical Testing for the Cannabis Industry: A New Era Presents New OpportunitiesChristopher J. Hudalla, ProVerde Laboratories, 420 Fortune Blvd., Milford, MA 01757, Nathan M. LaCroix

The cannabis industry has been thriving for many years, with the use of cannabis tracing back thousands of years. However, the illicit status of the herb throughout much of the world has stifled commercialization and research, forcing most activities underground, often times with high risk and minimal accountability. As a result of this, technological advances in science and analytical instrumentation have found little to no application to this diverse field. Recent advances throughout the world in legislation, regulation and public acceptance have opened the door for legitimacy of this industry. This provides the new opportunity for the use of the latest advances in scientific instrumentation and methodologies to be applied to different aspects of this industry, including ensuring consumer safety, basic research, optimization of cultivation practices, and the design and development of marijuana infused prod-ucts (MIPs). Like most natural products, cannabis is comprised of an intrinsically complex chemical profile. To further complicate our understanding of the complexity, the high potential from contaminants due to human cultivation practices and from microbiological contamination makes safety profiling critical for ensuring consumer safety. Here we demonstrate the application of modern technology to the study of cannabis, employing state-of-the-art chromatographic, mass spectrometric and mi-crobiological methodologies to provide a better understanding of the plant constitu-ents and the possible impurities or contaminants present. The high quality analytical data generated leads to improved cultivation and manufacturing practices, and most importantly, increased safety for patients and consumers.

42 Impact of Method Robustness to the Success of Product Commercialization - Lesson LearnedXin Bu, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903, John Castoro, Joel Young

The robustness of an analytical method becomes increasingly important when a new pharmaceutical entity transitions from the development stage to the commer-cialization stage. Whether a manufacturing process transfer from development to commercial is deemed successful is heavily dependent on the analytical results, thus the ability of the method to consistently deliver accurate and precise results is of significant importance. In this presentation, an example from a pharmaceuti-

cal project is used to demonstrate how significant the method robustness impacts the success of a product commercialization. The analytical method for the deter-mination of assay and impurities of an oral solid dosage form was developed and proven to be robust via successful validation and technology transfers. However, when used to analyze samples from the manufacturing process transfer batches, low potency results were observed. It is critical to understand whether it is due to method robustness or product manufacturing issues which warrant further pro-cess optimization to ensure the success of commercialization. A comprehensive investigation on the analytical method looking into many relevant parameters, such as diluent composition and sample preparation procedure was conducted. It was concluded that the method is sufficiently robust to consistently generate accurate and precise results. With the demonstrated proof of confidence in the method, the consistent analytical results allowed for the discovery of the low potency root cause. The manufacturing process was subsequently optimized and the success of product commercialization was ensured.

43 Incorporation of Benchtop NMR Spectroscopy into Undergraduate Laboratories: An Active-Learning ApproachSusanne Riegel, Nanalysis, Bay 4, 4500 5 St., NE, Calgary, AB, T2E 7C3 Canada

Nuclear magnetic resonance (NMR) Spectroscopy is one of the most widely used characterization techniques in chemistry. Despite pedagogical shifts towards ac-tive-learning and guided-inquiry approaches, incorporation of NMR spectrometers directly into undergraduate curriculum has remained largely limited due to mitigating factors of size, cost and availability of high-field spectrometers. As a result, students do not gain hands-on access to this instrumentation, particularly in the beginning states of their programs. An emergence of a new class of benchtop NMR spec-trometers (42 – 60 MHz) that are affordable, portable and do not require weekly upkeep or maintenance can facilitate the introduction of this technique at all stages of chemical education. They also offer sufficient resolution and sensitivity for struc-ture elucidation, reaction monitoring, and basic quantitation. Herein, we describe methods for the incorporation of the NMReady benchtop spectrometer into com-mon undergraduate laboratory experiments and illustrate how students can learn the proper technique to use an NMR spectrometer, prepare samples, monitor and characterize reaction mixtures. In combination with modern, easy-to-use interfaces and network accessibility, these spectrometers are perfect for teaching and training students use in the elementary chemistry laboratory.

44 Analytical Pipetting of Serum and Serum-Like LiquidsJohn T. Bradshaw, Artel Inc., 25 Bradley Dr., Westbrook, ME 04092, Richard H. Curtis, Rachel Parshley

Many core types of chemical analyses are based upon analytical techniques in-volving accurate delivery of liquid components. A common tool developed to deliver these liquid components is the handheld micropipette, as well as automated ver-sions of the same. Handheld and automated pipettors have become commonplace tools, especially used in many biological and pharmaceutical laboratories. While these tools are familiar to many, their performance differences when pipetting dif-ferent types of solutions are often over-looked and neglected aspects that relate directly to the accuracy and reproducibility of pipetting performance. For example, it is commonly known that water pipettes differently than serum. This presentation addresses the difference in performance of a handheld micropipette when dispens-ing water versus various types of animal and human serum. These differences are quantifiable and can be accounted for through careful experimentation and attention to physical pipetting details. Details on achieving ideal performance when pipetting serum are also discussed.

45 S.M.A.R.T. Laboratory PracticesJulius Neil Buenconsejo, Keppel Seghers Engineering, 108 Pasir Panjang Rd, Singapore 118535

In recent years, laboratories are facing immense pressure to continuously improve quality while keeping up with increasing demand in efficiency, productivity and re-liability of test results. From large testing facilities to small independent analytical centers, the complex and intricate nature of the challenges to run optimum opera-tions requires tailor-made solutions for various diversified and distinctive conflicting needs to achieve maximum throughput. S.M.A.R.T. (S – Specific, M – Measurable, A – Attainable, R – Relevant, T – Time-based) laboratory practices are simple but practical approach in improving laboratory operations utilizing invaluable tools and work-related enhancement programs. Proper application and execution not only reduce waste and save energy, but also shorten turnaround times (TAT) through efficient allocation of staff man-hours and resources - ensuring quick delivery of analytical results and timely reporting of relevant information in accordance with quality standards and regulatory compliance. For new and existing laboratories, implementation of such programs drives higher cost-efficiency, improves cost-effec-tiveness and develops sustainable environment for the next generation of laboratory professionals.

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46 Taking the Pain Out of Chromatographic Peak IntegrationPeter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA 94085, Shaun Quinn

Peak detection and integration are fundamental tasks in chromatography, most often done using chromatography software. Enabling software to detect and inte-grate the peaks as desired (or as required by laboratory rules) is challenging and time-consuming. Common challenges in peak detection include distinguishing peaks from noise, correctly identifying the underlying baseline, maintaining correct peak and baseline detection throughout a sequence of chromatograms, and cor-rectly handling rider peaks and other unresolved peaks. Ideally, these challenges should be addressed using detection parameters so that the same treatment can be applied across multiple chromatograms automatically, thereby minimizing vari-ations introduced by different operators. However, finding parameter combinations that produce the desired results has often been a tedious process, causing many chromatographers to give up and resort to manual integration, which is subjective and labor-intensive. In this presentation we look at a new peak detection algorithm that addresses these issues by separating peak detection from baseline determi-nation. It uses second-derivative signal analysis, providing reliable and consistent peak detection even if the underlying baseline shape changes. An easy to use inter-face that guides the user through the correct set-up of peak detection parameters is also shown. Examples of how the tools quickly solve problems with real-world chromatograms are given.

47 Innovative Approach to Helium Carrier Gas Conservation in Analytical Gas ChromatographyLori Dolata, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA 94085, Edward B. McCauley, Massimo Santoro, Paolo Magni, Alexander N. Semyonov

The global helium shortage and price increase cause more and more analytical lab-oratories employing gas chromatography (GC) to re-assess their consumption pat-terns of this non-renewable noble carrier gas. The existing solutions to this problem include changing analytical column, migrating to another carrier gas (e.g., hydro-gen) or passively reducing the helium consumption by reducing its usage during an-alytical runs or switching the GC or GC-mass spectrometry over to nitrogen during the longer idling periods. In all these cases a considerable amount of time is spent developing new methods or wasting daily the time needed for the instruments to come back to normal operations. A new, innovative approach to the conservation of helium carrier gas will be presented, comprised of separation of the flows inside the standard split/splitless injector. While preserving the analytical GC column flow with helium, the septum purge and the split flows are maintained with another inert gas like nitrogen - even during the analytical run. On average, the helium consumption is reduced such that a standard cylinder of compressed helium gas can last 3-6 years vs. 3-6 months without and changes in the analytical methods or conditions. This new patented technology for saving Helium is presented, along with the data validating its use; the additional practical benefits introduced to the quality of the chromatographic data are also discussed.

48 Automated, Rapid, Reliable Determination of Dissolved Gases in Water by Static Headspace Gas ChromatographyLori Dolata, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA 94085, Andrea Caruso, Richard Jack, Massimo Santoro

Hydraulic fracturing, the fracturing of a rock by a pressurized liquid is a well stim-ulation technique in which typically water is mixed with sand and chemicals, and the mixture is injected at high pressure into a wellbore to create small (< 1mm) fractures in order to maximize fluid removal and well productivity. While this once-very diffused technique makes accessible big amounts of formerly non-accessible hydrocarbons, the dissolved gases have become a controversial environmental and health matter with some countries completely banning the practice. Public outcry over preservation of water quality has led the U.S. EPA and other state agencies to investigate the impact of hydraulic fracturing on the quality of environmental waters. Some procedures for testing waters for dissolved gases through static headspace sampling exist already, like RSK 175 standard operating procedure, but since the target compounds are light hydrocarbons from methane through propane, are ex-tremely volatile, a closed sampling system is required. A high- throughput test meth-od using robust, automated and relatively inexpensive instrumentation like static headspace and gas chromatography with flame ionization detection is used in this paper, and data is shown for the quantitative determination of dissolved gases in ground, waste and drinking waters.

49 Assessing Qualitative and Quantitative NMR Limits in a Drug Metabolism EnvironmentGregory Walker, Pfizer, Eastern Point Rd., Groton, CT 06340, Raman Sharma, Shuai Wang

With the introduction of cryo-probes the minimum sample requirements for the acquisition of interpretable and quantifiable one- and two-dimensional (1-D/2-D) nuclear magnetic resonance NMR data has decreased significantly. Over the last

decade sample requirements have decreased even further with the advent of low volume (1.7 mm) cryo-probes. The sensitivity limits of these systems have been well parameterized with samples made from serial dilutions of known compounds. While these are important experiments, the practical aspects of creating usable samples at these levels from biological isolates are daunting. Concomitant with these de-velopments in the field of NMR, drug metabolism scientists have honed several in vitro methods of incubation that can produce metabolites that reflect those found in animal models. These methods include isolated cell lines, sub-cellular organelles or recombinant enzyme systems. Together, these disparate developments have creat-ed a powerful tool for the structural elucidation and quantitation of metabolites much earlier in the drug discovery/development paradigm. Using the in-vitro methods dis-cussed above in conjunction with liquid chromatographic isolation and cryogenic NMR probes, samples in the 10 nmole range can be routinely produced of sufficient quality to allow full 1H and 13C characterization. In situations where full 13C character-ization is not necessary the amount of material required may be decreased to less than 5 nmole. Once characterized, these samples are of sufficient quantity that they may be subsequently quantitated and used as reference standards in a variety of discovery efforts. In this presentation several examples are discussed that highlight the details and limitations of this process.

50 Pulsed Field Gradient Nuclear Magnetic Resonance (PFGNMR) Studies of Nanoparticle Surfaces and their InteractionsKebede Beshah, The Dow Chemical Company, 400 Arcola Rd., Collegeville, PA 19426, Aslin Izmitli, Antony Van Dyk

Hydrophobically modified ethoxylated urethane resins (HEUR), telechelic polymers, with molecular weights greater than 30 KDa, have been used to control viscosity of nanoparticle dispersions. Extensive work has been done and published over the past three decades on the associative nature of these amphiphilic HEUR polymers in water. A molecular level study that shows the interaction between the HEUR poly-mer and the nanoparticles was conspicuously missing. The major reason for this is the inability to detect the critical low level hydrophobic end group and urethane linker signals in an overwhelming matrix of aqueous phase materials of the dis-persions. We applied diffusion filtered one-dimensional (1-D) and two-dimensional (2-D) nuclear magnetic resonance (NMR) experiments that enabled the elimination of unwanted signals based on their higher diffusion coefficients and/or dynamics of the various species to detect the desired HEUR signals without any perturbation of the system. With these pulsed field gradient (PFG) NMR techniques, we are able to probe HEUR polymer-nanoparticle interactions as a function of HEUR end group and nanoparticle surface hydrophobicity. The method is used to obtain adsorption isotherms of the composite in-situ providing unperturbed dynamic equilibrium sys-tems compared to the widely used centrifugation method. Our observation has led to a new model of HEUR-nanoparticle interaction that sheds light on the elusive mechanism of high shear viscosity (10,000 s-1) of HEUR-latex composites. The de-tails of the diffusion and dipolar filter 1-D and 2-D NMR techniques and implications of these molecular level interactions to shear thinning rheology of HEUR-nanoparti-cle composites are discussed.

51 High-Throughput Screening of Solid Pharmaceuticals with 35Cl Solid-State NMRRobert W. Schurko, University of Windsor, 401 Sunset Ave., Windsor, ON, N9B3P4 Canada, Michael J. Jaroszewicz, Anthony R. Sandre, Andrew M. Namespetra, Kristopher J. Harris, Adonis Lupulescu, Lucio Frydman

Structural screening of solid active pharmaceutical ingredients (APIs) is a priority in the pharmaceutical industry, and a major problem currently confronting solid-state chemists. More than 80% of APIs are manufactured, stored and/or ingested as sol-ids; therefore, they must undergo a rigorous quality assurance process to moni-tor their identities, structures and purities. Since hydrochloride (HCl) salts of APIs constitute more than 50% of known pharmaceutical solids, 35Cl solid-state nuclear magnetic resonance (SSNMR) is an appealing choice for their characterization. 35Cl is a quadrupolar nucleus (spin = 3/2), with NMR powder patterns influenced by both chemical shift (CS) and quadrupolar interactions (QI). Though the patterns are quite broad, and in some cases challenging to acquire, they are rich with structural information, and provide a unique fingerprint for each API, as well as associated polymorphs, solvates, hydrates, disproportionation forms and impurities. In this lec-ture the latest advances from my research group in the development of 35Cl SSN-MR as a technique for high-throughput screening of APIs are presented. Discussion includes: 1) the utilization of 35Cl SSNMR and plane-wave DFT calculations for fingerprinting, identification and discovery of solid phases of APIs; 2) the application of two powerful pulse sequences, WURST-CPMG and BRAIN-CP, which are used for the rapid acquisition of broad 35Cl powder patterns associated with APIs; and 3) the use of powerful spectral editing techniques, including two-dimensional relax-ation-assisted separation (2-D RAS) methods which can be used to differentiate broad overlapping patterns and aid in making spectral assignments.

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November 20142014 EAS Abstracts

52 Strategies to Enhance the Sensitivity and Reduce the Ambiguity of NMR Spectra of Complex Biological AssembliesGalia Debelouchina, Princeton University, Department of Chemistry, Princeton, NJ 08540

Multidimensional nuclear magnetic resonance (NMR) spectroscopy is capable of providing atomic-level structural information of complex and heterogeneous biolog-ical systems. As the size and the complexity of these systems increases, however, the interpretation of NMR spectra becomes more challenging due to poor sensitivity and ambiguity in the assignment of the observed interactions. Dynamic nuclear po-larization, a method that significantly improves the sensitivity of magic angle spin-ning NMR experiments, can be used to generate spectra with greatly improved sig-nal-to-noise ratios and unprecedented savings in data acquisition times. Advanced isotopic labeling strategies such as site-specific, sparse and segmental labeling, on the other hand, can be used to simplify the informational content of the spectra and facilitate the interpretation of structural constraints. This talk focuses on the combined use of dynamic nuclear polarization and specific labeling strategies used to elucidate the higher order structural organization of complex protein assemblies such as amyloid fibrils.

53 Understanding the Behavior of Different Tablet Disintegrants Using High-Resolution FLASH Magnetic Resonance ImagingAnuji Abraham, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903, Dolapo Olusanmi, David Good, Denette Murphy, Daniel McNamara, Venkatramana Rao, Andrew J. Ilott, Alexej Jerschow

Understanding the behavior of tablet disintegrants is valuable in the development of pharmaceutical solid dosage formulations. High-resolution real-time magnetic resonance imaging (MRI) allows viewing the full cross section of the tablet disin-tegration process. The fast low angle shot (FLASH) sequence has been used to understand the behavior and mechanism of disintegration. Furthermore, FLASH se-quence minimizes experimental time allowing high resolution images to be collected in < 10s. In the current work, FLASH MRI methods have been used to understand tablet disintegration mechanisms resolving swelling, gelling and erosion processes. The study was done on a variety of disintegrants, such as sodium starch glycolate, crospovidone and croscarmellose sodium. By studying different grades of these disintegrants a correlation could be established between their disintegrating mech-anism and rates with their chemical compositions, cross linking, hydrophilic nature and ionic strengths. Quantification of the disintegration rates was possible using a bespoke voxel analysis. Videos based on the MRI results were also produced help-ing to distinguish between different disintegration mechanisms. Overall, FLASH MRI methods have been implemented and shown to be a useful tool for understanding fast tablet disintegration giving valuable insights into the development of pharma-ceutical formulations.

54 Cleaning Verification: TOC or HPLC?Elizabeth C. Moroney, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903, Mariann Neverovitch

Validated cleaning verification methods are required by federal and international regulatory agencies to comply with good manufacturing practice standards. Clean-ing verification ensures the safety and purity of the product, satisfies regulatory requirements and assures internal control over processes. Cleaning verification methods are typically developed for each pharmaceutical compound individually, as well as for detergents. Traditionally, high-performance liquid chromatography (HPLC) has been the method of choice for analyzing cleaning verification samples due to the widespread availability of instrumentation and the ability to quantify low levels of product. Recently, total organic carbon (TOC) analysis has become more prevalent owing to the simplicity of use and acceptance of the method by regu-latory authorities. In this presentation, I summarize and compare the techniques and explore a case study that highlights the challenges encountered during method transfer/covalidation.

55 Justification of Applying Acceptable Residual Limit (ARL) Based on Toxicological Data and Visual InspectionMariann Neverovitch, Bristol Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903

Acceptable residual limit (ARL) value is based on subject exposure limit (SEL), minimum batch size, maximum daily dose and total surface area. For total organic carbon (TOC) analysis, the ARL value is corrected for percentage of carbon in the molecule, and measured in parts per million units (ppm). Based on this calculation and the molecular formula of a given compound, the value of ARL may vary from very low (<0.1ppm C) to very high (>200 ppm C). If the ARL is lower than 1 ppm C, the TOC technique will not be applicable due to background contribution from the blank. If the ARL is too high, then the method limit should be evaluated and an alert limit should be introduced. Historical cleaning validation data indicates that the vast majority of drug product residue is visible at a level of 1 ppm C. The system suitabil-ity solution (KHP) for TOC analysis is prepared at 5 ppm C. Therefore it is scientif-ically justifiable that the value of the alert limit should be between 1 and 5 ppm C.

56 Automated Kinetic Forced Degradation Development and ValidationLina Liu, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065, Timothy Rhodes, Roy Helmy, Keri Joy McNamara, Donna Carroll

Forced degradation studies are an important element in assessing a compound’s drug-like properties and understanding the chemical liabilities that will need to be addressed in preclinical development. Forced degradation studies help in early de-velopment to devise and to validate stability-indicating analytical methods. Kinetic stability data allow for evaluation of degradation rates and can provide an under-standing of the sensitivity to different degradation pathways and can even help to distinguish between primary and secondary degradation products. Finally, this data can help in devising a strategy to minimize excipient induced degradation and can help to differentiate between excipient derived adducts and drug substance degra-dation products. Forced degradation studies are conducted multiple times over the life of a drug development to serve a various purposes and, most often, the work spans across multiple groups. In order to harmonize the procedures of forced deg-radation, an automated method for forced degradation was developed, utilizing the CTC LEAP PAL workstation automation system. The automated forced degradation approach significantly reduces the amount of manual labor used to perform the tests and harmonizes the operational procedures of forced degradation. The automated forced degradation system is user-friendly and is intended to be used as a “walk-up system” that is able to prepare forced degradation and linearity samples, perform on-line high-performance liquid chromatography analysis including sampling at mul-tiple time points, and generate reports automatically using Empower customized report template. The details of the system are discussed along with a number of case studies demonstrating its use.

57 Resolving the Isolation Challenges of Color Impurities in NOXAFILWeidong Tong, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065

NOXAFIL, a marketed posaconazole IV injection formulation, is proven to help pre-vent invasive fungal infections from Aspergillus (mold) and Candida (yeast). Both fungi are relatively harmless in healthy people but can cause serious illness – and even death – for individuals who have suppressed or severely weakened immune systems. During stabillity study of the drug formulation solutions, a few low level (0.02 area %) degradation impurities were observed. Initial attempts to purify and identfy the impurities using liquid chromatography tandem mass spectrometry (LC-MS-MS), or extractions for impurities enrichment, however, proved to be unsuc-cessful. Instead, by using Amberchrom CG161C resin treatment and eluting with water and organic solvents with adjusted pH, we were able to successfully remove sulfobutyl-β-cyclodextrin, and enriched impurities to about 0.1 to 3% in a few frac-tions. Following that, prep high-performance liquid chromatography was employed to successfully separate and purify the impurities.

58 Analytical Challenges Encountered in Compatibility Testing of Parenteral Dosage Formulations Used in Preclinical StudiesLara D. Penn, Merck, 770 Sumneytown Pike, West Point, PA 19486, Samantha Devine, Oksana Leidy, Michelle Mathe, Julie Novak, Elizabeth Sander

Medical devices used for the storage and/or delivery of parenteral dosage formulations to preclinical species involve plastic, rubber, glass, and stainless steel. It is important to evaluate the compatibility of parenteral dosage formulations with materials used for dose administration and storage. Ideally, an important performance characteristic of the materials used in medical applications is chemical inertness, and material compatibility is critical prior to conducting any preclinical toxicology assessment. There exists a high level of concern for parenteral dosage formulations that are solutions and suspensions since there is a higher risk for interaction between storage and/or delivery component(s) and parenteral dosage formulations. The three parameters typically evaluated in compatibility studies are drug adsorption, device induced active degradation, and the appearance of leachables and/or extractables. Analytical challenges encountered during the evaluation of the compatibility of administration sets and storage containers with preclinical formulations are discussed.

59 Migration of Ink Components into Transdermal PatchKenneth Wong, SGS Life Science Services, 75 Passaic Ave., Fairfield, NJ 07004, Richard Bunnell, Sushmeet Singh, Gayatri Trivedi, Terry Panetta, Xinjie Song

Food and drug packaging is printed with colorful labels; however, there is limited knowledge about the potential migration of printed ink components into the product. With no global legislation available and ill-defined terminology in the scientific com-munity, formulating ink used in packaging becomes a challenge for suppliers and users who are concerned about potential migration of harmful components. Possi-ble sources of migratable compounds in inks and coatings include UV photo-initia-tors, mineral oils, and resins. No single analytical method can detect all components in the ink due to the complexity of the composition and solubility of individual com-ponents. Hence, the aim of this study is to apply the most effective methods using

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November 20142014 EAS Abstracts

minimal extraction combined with instrumental analysis to determine the amount of ink components migrating into the contact adhesive layer of a transdermal patch with printing directly on it. The instrumental analytical tests include gas chroma-tography flame ionization mass spectrometry (GC-FID-MS) for volatile compounds (benzyl alcohol as ink reducer), ultra-performance liquid chromatography diode array detection mass spectrometry (UPLC-DAD-MS) for non-volatile compounds (polyamide, photo-initiators and their decomposition products) and inductively cou-pled plasma optical emission spectrometry (ICP-OES) for metal analysis (D&C Red as color dye). The calibration curves showed linearity in the range of 0.1 - 5.0 µg/mL for all three instrumental analyses, GC-FID-MS, UPLC-DAD-MS and ICP-OES with r2 no less than 0.998. The limits of detection and the recoveries of individual ink components from extraction samples were 89-102% and 0.2 µg/mL, respectively. The proposed methods will be useful for the quantification of ink migration from the transdermal patch.

60 Direct Determination of Native N-linked Glycans by HPLC with Charged Aerosol DetectionDavid H. Thomas, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA 01824, Ian N. Acworth, Marc Plante, Matthew W. Neely

Glycoproteins of biological, diagnostic or therapeutic interest owe key aspects of their normal function to the oligosaccharides attached to the protein backbone. Changes in the number, type, composition or linkage pattern of these glycans may serve as a biomarker of disease or influence the efficacy of a biotherapeutic prod-uct. For this reason, the ability to correctly identify and measure these glycans is of scientific interest, and to do so reliably, quickly and inexpensively is of practi-cal benefit. This work explores direct detection of native glycans as an alternative to the common techniques for glycan analysis that rely on derivatization reactions to render glycans detectable. The lack of a detectable chromophore in native gly-cans is overcome by using high-performance liquid chromatography (HPLC) with charged aerosol detection, a detector that can quantitatively measure any non-vol-atile compound. N-linked glycans were released from proteins by PNGase-F. The native glycans were separated by ultra HPLC on a column that employs both weak anion exchange and hydrophilic interaction liquid chromatography (HILIC) separa-tion mechanisms. The native glycans were detected directly without derivatization by using charged aerosol detection. Sialylated glycans released from bovine fetuin were analyzed by a fast separation according to charge as well as by a higher resolution separation based on size, charge and polarity. Precision, detection limits and dynamic range of quantitative measurements are presented. Typical figures of merit include low-nanogram on-column sensitivity, over two orders of magnitude of dynamic range, and peak area precision of approximately three percent relative standard deviation.

61 Identification of a Unique, Metformin Related, Impurity in a Drug Product Stability SampleJonathan Marshall, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903, Yande Huang, David Ayers, Michael Peddicord, Scott Miller

Metformin is an oral antidiabetic drug which is prescribed primarily for the treatment of type 2 diabetes. Metformin is also marketed in several fixed dose combination products. A new impurity was recently observed during stability testing for a fixed dose combination (FDC) drug product which contained Metformin as one of the two active ingredients. Characterization of the impurity by liquid chromatography (LC-MS) resulted in a structure proposal indicative of a reaction of Metformin with formaldehyde. Formaldehyde is a known degradation product of polyethylene glycol (PEG) which is used as a component in the coating of the FDC drug product. This presentation will describe the LC-MS characterization performed on the impurity as well as the details of its synthesis, purification, and nuclear magnetic resonance characterization for absolute structural assignment.

62 Ultra-High Speed USP Analysis within the New USP General Chapter 621 Allowed LimitsKenichiro Tanaka, Shimadzu Scientific Instruments, 7102 Riverwood Dr., Columbia, MD 21046, William Hedgepeth

The United States Pharmacopeia (USP) defines allowed adjustments of high-per-formance liquid chromatography (HPLC) and gas chromatography (GC) parameters in the general chapter <621> chromatography. If system suitability is met, method parameters can be changed within the allowed limits without revalidation. The gen-eral chapter 621 was revised in the first supplement to USP37-NF32 published on February 1, 2014 and will be official after August 1, 2014. The feature of the new general chapter 621 is that a column packed with small particles can be used as long as column length and particle ratio (L/dp) is kept constant between the desig-nated and modified column. This enables the high speed analysis of USP methods more than ever. In this poster, an adjustment of a USP method for speed is shown. As a result, analysis time and solvent consumption were reduced to 1/10 and 1/15, respectively, with the system suitability requirements met. Additionally, running a USP method on the Nexera X2 UHPLC system is shown. The system suitability

requirements were easily met on the ultra high-performance liquid chromatography (UHPLC) system similar to a standard HPLC system. This result means that this system is also suitable for those who are running traditional USP methods on a standard HPLC system and considering the adoption of a UHPLC system for high speed analysis of USP methods in the future.

63 Quantitation and Characterization of Polysorbate in Biotherapeutic Products Using Two-Dimensional HPLCKenichiro Tanaka, Shimadzu Scientific Instruments, 7102 Riverwood Dr., Columbia, MD 21046, William Hedgepeth, Keiko Yamabe

Polysorbates are commonly used for biotherapeutic products to prevent aggrega-tion and surface adsorption, as well as to increase the solubility of biotherapeu-tic compounds. A reliable method to quantitate and characterize polysorbates is required to evaluate the quality and stability of biotherapeutic products. Several methods for polysorbates analysis have been reported, but most of them require time-consuming sample pretreatment such as derivatization and alkaline hydrolysis because polysorbates do not have sufficient chromophores. Those methods also require an additional step to remove biotherapeutic compounds. Here we report a simple and reliable method for quantitation and characterization of polysorbates in biotherapeutic products using two-dimensional high-performance liquid chromatog-raphy (HPLC).

64 High Sensitivity Profiling of Glycans Using High-Performance Liquid Chromatography with Fluorescence DetectionPeter Ratsep, Shimadzu Scientific Instruments, 19 Schoolhouse Rd., Somerset, NJ 08873, Kenichiro Tanaka, William Hedgepeth

Glycans in antibody drugs may play roles in the antigenicity, pharmacokinetics and high-order structural stability of drugs, which could adversely affect drug safety and effectiveness. It is therefore necessary to investigate which glycans are present in antibody pharmaceuticals. Non-uniformity of glycans due to the instability of culture conditions used for antibody drugs is a concern which requires rigorous manage-ment of the production process. Here, using an amide column TSKgel Amide-80 and a Prominence high-performance liquid chromatograph, we introduce an example of the analysis of glycans. For detection, the high-sensitivity fluorescence detector RF-20AXS was used. A mixture of three glycans was injected to the system and limit of detection was found to be around 1 fmol.

65 A Two Level Limit Test for the Detection of Trace Hydrazine in a Drug Substance by In-situ Derivatization Headspace GC-MSSarah G. Westerbuhr, Array BioPharma, 3200 Walnut St., Boulder, CO 80301, Peter A. Lobue, Philip N. Anderson

Hydrazine and its derivatives are common building blocks for the synthesis of drug substances. However, hydrazines are known genotoxic compounds and must be controlled in drug substances at very low levels. A method to detect trace hydrazine in drug substance samples was developed and validated. The method is a two level limit test for 1 and 8 ppm limits when the drug substance samples are prepared at 100 mg/mL. The method employs in-situ derivatization of hydrazine with acetone to produce the acetone azine, which is then introduced via headspace sampling, chro-matographed using gas chromatography, and detected using mass spectrometry in single-ion monitoring mode. The limit of detection is 129 ppb. The two level limit test approach allows for a distinction between samples with a relatively low concentra-tion of hydrazine from higher concentration samples. However, because of the less stringent validation requirements of a limit test, the method validation required only minimal time and resource.

66 Coupling of PXRD, ssNMR and HPLC for Impurity Identification in a Batch of an Anti-Diabetic AgentRoxana F. Schlam, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903, Anuji Abraham, Ian Young, George Crull

Upon completion of the Bristol-Myers Squibb - A toxicology batch, a new peak was observed in the powder X-ray diffraction (PXRD) pattern that did not correspond to any allowed reflections of the selected crystalline form of the active pharma-ceutical ingredient (API). The issue had to be resolved prior to the upcoming First in Human study in order to facilitate API release for clinical use. A combination of PXRD, solid-state nuclear magnetic resonance (SSNMR) and high-performance liquid chromatography (HPLC) was used to identify the nature of the extra peak observed in the tox batch. PXRD and ssNMR ruled out the possibility of amorphous or other crystalline phases. Analysis of HPLC data identified very low levels of phos-phine ligand utilized in the synthetic process which was not completely removed. PXRD together with 13C and 31P ssNMR confirmed that the extra peak corresponds to the phosphine ligand. This knowledge allowed Chemical Development to design a strategy to control the level of the ligand, bellow the allowed safety limits, in future clinical deliveries.

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November 20142014 EAS Abstracts

67 Effect of Organic Additives in Running Buffer on Capillary-Electrophoretic Analysis of Amino AcidsJinmo Huang, The College of New Jersey, PO Box 7718, Ewing, NJ 08628, Margaret Chen, Kimberly Benitez

Organic additives have been used in running buffer to enhance the separation of analytes in capillary electrophoresis. In this research, organic solvents including acetonitrile, methanol, and ethanol are respectively added to phosphate running buffer used in the capillary-electrophoretic analysis of some light-absorbing amino acids. The effect of these organic additives on the separation and determination of the amino acids are compared and discussed.

68 A New Ion Chromatography Method for the Trace Level Analysis of Hydrazine in Povidone and CrospovidoneLawrence J. Feeley Jr., Ashland Specialty Ingredients, 1005 US Highway 202/206, Bridgewater, NJ 08807, James E. Brady

An accurate and precise ion chromatography (IC) method for the quantitative mea-surement of trace level residual hydrazine (N2H2) in Povidone and Copovidone has been developed. The IC method has numerous advantages over the thin-layer chromatography (TLC) monograph method, United States Pharmacopeia (USP) 37 including: (a) less cost per sample, (b) analysis speed and (c) increased sensitivity and dynamic range. The IC method can monitor hydrazine levels over three orders of magnitude from an MQL of 8 ppb to 8 ppm for a 6% sample solution and can be extended with sample dilution. Sample preparation is fast and the method can be automated. Finally, since there is no derivatization step, the analysis is direct. This method is based on previous work by Jagota, et al, (Jour. Pharm Biomed Analysis (1998) 16, 1083-7), in which N2H4 is oxidized to N2 and that reaction is directly monitored.

69 Examining the Thermal Stability of Compound A and Methacrylic Copolymer Solid Dispersion formulationSanjaykumar Patel, Merck, 556 Morris Ave., Summit, NJ 07901, Pranav Gupta

Compound A is a weak base with steep pH dependent solubility. To enhance solubil-ity at the intestinal pH a solid dispersion based formulation was made using hot-melt extrusion (HME). In solid dispersion matrix active pharmaceutical ingredient (API) was stabilized in methacrylic copolymer enteric polymer. To define the appropriate HME processing temperature window, it is important to understand the thermal sta-bility of methacrylic copolymer and compound A. For stability of methacrylic copo-lymer, a reverse-phase liquid chromatography charged aerosol detection ultraviolet (RPLC-CAD/UV) analysis method was developed. The polymer was monitored us-ing Corona detector and monomers are volatile hence monitored using UV detector. Polymer cross linking can form a large molecular weight polymer while the back-bone breakage could result in the formation of low molecular weight species. Either of these impacts the molecular weight distribution of the polymer and its function-ality and to confirm this, a size exclusion method with refractive index detector was developed. API thermal stability was monitored using a stability indicating assay method. Compound A and methacrylic copolymer extrudates were prepared with temperature ranging between 145 °C to 180 °C on an 18 mm twin screw extruder. RPLC-CAD/UV analysis method demonstrated that there was no change in polymer assay over the range of processing temperature and this was further confirmed with size exclusion chromatography. Therefore, methacrylic copolymer is chemically sta-ble during hot-melt extrusion processing. API specific method exhibited increase in level of Deg A as a function of processing temperature hence processing tempera-ture need to be below 180 °C. Overall processing temperature between 145 °C to 170 °C was recommended for compound A HME solid dispersion.

70 Finding the Needle in the Haystack – Characterization of Trace Crystallinity in a Commercial Formulation of Paclitaxel Protein-Bound Particles by Raman Spectroscopy and Synchrotron X-Ray Diffraction Enabled by Second Harmonic Generation MicroscopyPaul D. Schmitt, Purdue University, 560 Oval Dr., West Lafayette, IN 47907, Niraj S. Trasi, Shijie Zhang, Fengyuan Deng, Lynne S. Taylor, Garth J. Simpson

Second harmonic generation (SHG) microscopy was used to rapidly identify re-gions of interest for localized confocal Raman spectroscopy and synchrotron X-ray diffraction (XRD) measurements in order to quantify crystallinity within lyophilized Abraxane® powder (protein bound paclitaxel for injectable suspension). Water insoluble noncentrosymmetric crystalline particles ranging from ~1 micrometer to 120 micrometers were identified by SHG, with wide variability in crystal size and frequency observed between several lots of Abraxane®. By targeting the Raman and XRD analysis to these localized regions identified by SHG, the corresponding reduction in background lowered the limits of detection for both Raman and XRD by 2-3 orders of magnitude relative to ensemble averaged measurements. Experimen-tal Raman spectra of SHG active domains in Abraxane® were in good agreement with experimental spectra of pure crystalline paclitaxel. Peaks identified by syn-chrotron experiments exhibited significant preferred orientation effects complicating

quantitative comparisons with powder patterns. However, the XRD peak positions were generally consistent with calculated peaks for paclitaxel crystal forms found in the Cambridge Structural Database. These collective results are consistent with up to 30% of the active ingredient present as poorly soluble crystalline particulates in some batches of Abraxane®.

71 Comparison of the Performance and Spray Behavior of a New Nasal Microemulsion to the Commercial Suspension Tina Masiuk, Merck, 556 Morris Ave., Summit, NJ 07901, Elaine Pu, Zhenyu Wang, Oscar Liu, Julianne Berry, Samir Shah

The mometasone furoate (MF) microemulsion nasal spray and mometasone furo-ate/azelastine (MF/AZ) microemulsion nasal spray were developed to treat allergic rhinitis as lifecycle extensions to the Nasonex suspension. The aim of the micro-emulsions is to increase the solubility and reduce the dose strength of mometasone. The new formulations solubilize drug(s) using surfactant and oil, and have a viscos-ity resembling water. These differences in viscosity, and surface active excipients could change the spray behavior, which could affect nasal cavity deposition. Since Nasonex has been shown to deposit a majority of its dose into the posterior nasal cavity (Shah, 2011), this study was undertaken to compare its performance to the microemulsions. Actuations from Nasonex (500 mcg/g MF), MF microemulsion (31 mcg/g) and MF/AZ microemulsion (31 mcg/g and 2.055 mcg/g) were character-ized by plume geometry, spray pattern, droplet size distribution, and spray content uniformity. Statistical analysis assessed the effect of formulation. Small differences between microemulsions and suspension were observed and are likely due to the differences in viscosity and excipient surface activity. While statistically significant, it is uncertain if these in-vitro differences are practically significant, given studies finding no changes in deposition due to small changes in in-vitro spray tests (Suman 2006). Therefore, deposition from these formulations will likely be similar. Since the performance of the microemulsions and suspension are comparable, it is expected that any efficacy differences may be attributed to the active ingredients and formu-lation, not to spray behavior. Therefore, the microemulsions have the performance capability of Nasonex.

72 Using Skin-Pampatm for Transdermal Patch TestingDavid A. Kwajewski, Pion Inc., 10 Cook St., Billerica, MA 01821, Balint Sinko

Using skin as absorption site presents interesting features that have facilitated the progression of transdermal drug delivery in the past decades. Efforts in drug re-search have been devoted to find a quick and reproducible model for predicting the skin penetration of molecules. The parallel artificial membrane permeability assay (PAMPA) has been recently extended by this group for the prediction of transdermal penetration (Skin PAMPATM)1. This commercially available system has been mod-ified to make it suitable for transdermal patch testing. Four active pharmaceutical ingredients (nicotine, fentanyl, ketoprofen and rivastigmin) have been investigated, each applied in 1-3 marketed transdermal patches. The permeation vs. time profile demonstrated linear release profile in every case, though the cumulative permeated amount was about 30% higher than expected that can be caused by the edge ef-fect reported by Hadgraft and co-workers2. In-vitro/in-vivo correlation of permeation profiles were performed based on manufacturers’ data and resulted in acceptable correlation. Skin PAMPA system appears to be a useful tool for transdermal patch comparisons, though standard protocol needed to be modified. Results can be used for patch comparison and for ranking; therefore Skin PAMPA can provide valuable information for transdermal patch development.

73 Pharmaceutical Residual Solvent Analysis Using (FT-MRR) Spectroscopy: Fourier Transform Molecular Rotational ResonanceBrent J. Harris, BrightSpec, 770 Harris St. #104B, Charlottesville, VA 22903, Robin L. Pulliam, Roger Reynolds, Justin L. Neill, Matthew T. Muckle, Dave McDaniel, Brooks H. Pate

Residual solvent content is a critical quality attribute (CQA) in pharmaceutical manu-facturing that has motivated the search for new process analytical technology (PAT). The standard gas chromatography (GC) methods require hours of calibration and suitability testing that are incompatible with process monitoring applications. Fourier transform molecular rotational resonance (FT-MRR) offers a powerful alternative for fast, direct analysis of volatiles (including water) in pharmaceutical drug development and process monitoring applications. A single analyte can be detected at 100 ppm (m/m) levels in as few as 10 seconds. The technology platform is inherently selective and adaptable to enable monitoring of an extensive list of volatiles without chromatography or chemomet-rics. Detection targets can be re-assigned arbitrarily by selecting pre-defined measure-ment protocols that include a double resonance analysis for unambiguous identification of analytes in the mixture. FT-MRR performance for residual solvent analysis is present-ed using the headspace of a custom mixture with a GC verified composition of common solvents dissolved in water and dimethylacetamide (DMAC). The results summary in-cludes cycle time, detection limits, linearity, and reproducibility. The broader implications include applications of FT-MRR to in-line batch drying analysis, high throughput residual solvent analysis, genotoxic impurity analysis, and chiral analysis.

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November 20142014 EAS Abstracts

74 HPLC Separation Polar Compounds with a Focus on the Role Stationary PhaseMatthew Przybyciel, ES Industries, 701 South Route 73, West Berlin, NJ 08091

The chromatographic separation and the analysis of polar compounds using high-performance liquid chromatography (HPLC) has always been a challenging. It would seem that HPLC analysis would be well suited for the determination of the polar compounds; however routine reverse-phase HPLC analysis has yielded poor quality results for these types of compounds. The analysis of polar compounds via routine HPLC analysis has been deficient in several main aspects including poor retention, unacceptable low k’ values and poor peak shapes. One of few HPLC tech-niques capable of analyzing polar compounds has been traditional normal-phase chromatography (polar stationary phase with a non-polar mobile phase). Unfortu-nately, normal phase chromatography suffers from several major deficiencies in-cluding poor retention time reproducibility, poor column to column reproducibility and very low or no solubility of the target analytes. A number of HPLC stationary phases have introduced to improve the analysis polar compounds. These HPLC stationary phases include high-aqueous stable phases, hydrophilic interaction chro-matography (HILIC) phases and polar embedded phases all of which utilize various levels of water in the mobile phase. It is the focus of this presentation to compare and contrast the HPLC stationary phases developed for the analysis of polar com-pounds. In addition, we introduce new stationary phases optimized for the separa-tion of polar compounds. The ultimate goal of this work is to present a strategy for the HPLC analysis of polar compounds and to provide the chromatographer with the information necessary to match their polar compounds/samples with the appropriate HPLC technique.

75 Characterization of Copaxone® by Viscotek TDAmaxRevital Krispin, Teva Pharmaceutical, Hatruffa 12, Netanya, 4250483 Israel, Arthur Komlosh

Copaxone® is an aqueous solution containing 20 mg/mL of the active ingredient Glatiramer Acetate (GA) and 40 mg Mannitol. Glatiramer Acetate is a complex mix-ture of synthetic amino acid copolymer composed of a huge number of indiscrim-inate sequences of L-alanine, L-lysine, L-glutamate and L-tyrosine. This complex mixture of linear polypeptides results in a varying chain sizes, therefore the molec-ular weight distribution of the GA components span over wide range of about 2,500 – 20,000 Daltons. Viscotek TDAmax is a dedicated multi-detector size exclusion chromatography (SEC) system for polymers and macromolecules analysis. The Tri-ple Detector Array (TDA) is comprised of three detectors that act simultaneously; refractive index (RI), viscometer and light scattering detector (LSD) providing com-plimentary characteristic data on the sample being analyzed including molecular weight and molecular size. In this study Viscotek TDAmax was used for charac-terization of Copaxone® by certain molecular weight characteristics (such as Mn, Mw, and Mz), Polydispersity, hydrodynamic radius and intrinsic viscosity properties. These values are physical characteristics of the polypeptide chains and are derived from their chemical structure. Using this technique Copaxone® displayed a good batch to batch consistency, whereas purported generic copies showed differences within their characteristic parameters. The Viscotek TDAmax method demonstrated to be specific and discriminatory, and thus suitable for the analysis of Copaxone®.

76 Charged Aerosol Detection and Evaporative Light Scattering Detection – Fundamental Differences Affecting Analytical PerformanceDavid H. Thomas, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA 01824, Ian N. Acworth, Marc Plante, Qi Zhang, Matthew W. Neely

At a fundamental level, both charged aerosol and evaporative light scattering detec-tion share some similarities in that mobile phase exiting the column is first nebulized and then dried to form analyte particles. However, the mechanism by which these techniques measure analyte mass differ markedly and this has major impact on an-alytical performance. In charged aerosol detection, charged particles are measured by an electrometer generating a signal that is proportional to particle size (ie., the mass of analyte). For evaporative light scattering detection, signal is also propor-tional to particle size, but this relationship is much more complex, as the magnitude of scattered light varies depending on particle size, resulting in sigmoidal response curves. Unlike charged aerosol detection, evaporative light scattering detection uses non-contiguous signal attenuation. As each attenuation setting has its own unique sensitivity, response, calibration curve and dynamic range, samples may have to be reanalyzed multiple times in order to quantify analytes occurring at dif-ferent levels. In this poster the analytical performance of charged aerosol and light scattering detection are evaluated and include: sensitivity, dynamic range, inter-an-alyte response, linearity, reproducibility and the effects of mobile phase flow rate.

77 Automated Pre-Column Derivatization and High Speed LC Separation of Primary and Secondary Amino AcidsWilhad M. Reuter, PerkinElmer, 710 Bridgeport Ave., Shelton, CT 06484

For any liquid chromatography (LC) application incorporating automated pre-column

derivatization to be effective, it is important that both the derivatization procedure as well as the chromatography be robust and reproducible. This is highlighted herein via the automated pre-column derivatization and chromatographic separation of 17 primary/secondary amino acid hydrolysates. This approach also takes advantage of in-line photodiode array (PDA) and fluorescence (FL) detection, as well as stressing the importance of low peak dispersion. In addition, this application demonstrates that, at times, running superficially porous particle columns under ultra high-perfor-mance LC conditions can be quite beneficial.

78 Determination of Zonyl FSA Surfactant in Anti-Bleed Additive Using Ion-Pairing, Light Absorption Spectrophotometry, and HPLC MethodsPeter Yeh, MacDermid, 245 Freight St., Waterbury, CT 06702, Shelby Hale

Zonyl FSA surfactant is used in the anti-bleed additive to prevent or minimize bleed-ing or hazing in the deposition of fine-line metal traces. Analytical methods have been developed for determining the Zonyl surfactant concentration in the anti-bleed additive. In one of the analytical methods, methylene blue is added to react with the Zonyl surfactant to form an ion-pair which, when extracted into chloroform, can be determined spectrophotometrically. This analytical method is simple, fast, ac-curate, and sensitive, capable of detecting and determining low concentrations of the surfactant in the process solution. Due to its unique light absorption behavior in the UV region, Zonyl FSA surfactant in the additive can be determined spectropho-tometrically with high accuracy and precision. Taking advantage of their different chromatographic elution behavior, high-performance liquid chromatograph (HPLC) analysis method has also been developed for determining the Zonyl FSA surfactant and other active ingredient concentrations in the additive and process solutions. The HPLC method utilizes a reversed-phase C18 column for separation in con-junction with an acidic mobile phase solution containing methanol for elution. The separated and eluting components are detected using a spectrophotometric detec-tor set at a selected wavelength. The same HPLC method can be used to detect impurities present in the additive and process solutions for product quality control and anti-bleed process control. In this paper, analytical methods developed in our laboratory for determining Zonyl FSA surfactant in the anti-bleed additive and pro-cess solutions are described and the accuracy and precision of the analytical results obtained will be discussed.

79 Characterization of HILIC Columns with Zwitterionic Functional Groups: Correlation between Retention, Selectivity, Stationary Phase and Water Layer ThicknessDavid Lentz, EMD Millipore, 290 Concord Rd., Billerica, MA 01821, Tobias Jonsson, Phuoc Dinh, Patrik Appelblad

Liquid chromatograph mass spectrometry (LC-MS) with hydrophilic interaction liq-uid chromatography (HILIC) separations facilitates identification and quantification of hydrophilic species due to the orthogonality and enhanced sensitivity compared to reversed phase. Zwitterionic columns, purposely having a 1:1 balanced ratio of positive and negative charges, provide separation by HILIC partitioning plus weak attraction and repulsion interactions. However, the limited information on station-ary phases provided by several manufacturers might hamper the selection of best column for a particular analysis situation. In this work we conducted several sepa-rations of hydrophilic model compounds with relevance to cell metabolism and sig-naling, including nucleotides, nucleosides, nucleobases and catecholamines. Re-sults were correlated to measured characteristics of the stationary phases and their ability to absorb water. Characterization of five different commercial silica-based zwitterionic columns were carried out at typical HILIC conditions, more specifically in eluents containing acetonitrile at 70-95% mixed with aqueous buffers of ammonium acetate or formate at different pH. Columns were also assessed for background in electrospray mass spectromentry in the range of 20-2000 m/z under these elu-ent conditions. Column materials were examined for stationary phase content and surface area by elemental analysis and the BET-method, respectively. A subset of materials was selected for further testing by determining the amount of bound water using Karl Fischer titration.

80 Analysis of Phytosterols in Whole Blood by HPLC-ECDMarc Plante, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA 01824, Bruce Bailey, Ian Acworth, Michael Hvizd

Phytosterols are a group of naturally occurring steroid alcohols found in plants. They are key structural components of plant cell membranes, assuming the role that cholesterol plays in mammalian cells. There is considerable interest in phytos-terols as dietary supplements as they are reported to lower cholesterol levels and also have a positive impact on cardiovascular diseases. However, recent research suggests that phytosterol supplementation may aggravate atherosclerosis and lead to aortic valve stenosis. Phytosterols are typically measured by gas chromatogra-phy. However, this approach is time-consuming since it requires saponification of the sample, several extractions steps, and then derivatization. Presented here is a simplified method using reversed-phase, high-performance liquid chromatogra-

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phy (HPLC) and electrochemical detection (ECD) using a boron doped diamond electrode. Five standards, campesterol, cholesterol, stigmasterol, ß-sitosterol, and stigmastanol were resolved in < 6 minutes using a solid core C8 phase. The limit of detection (LOD) was ≤ 1 ng on column for all analytes. The method presented here was evaluated by characterizing the phytosterol content of whole blood The HPLC method with electrochemical detection is simple to implement, has good linearity and sensitivity, and is capable of measuring numerous phytosterols in animal and plant extracts. This approach can be used to examine product purity, supplement content, and adulteration. The use of the BDD electrode extends the range of ana-lyte to those whose structured are normally considered to be electrochemically inert when using conventional carbon- or metal-based working electrodes.

81 The Raptor™ ARC-18 Stationary Phase: Selectivity AcceleratedTy W. Kahler, Restek Corporation, 110 Benner Cir., Bellefonte, PA 16823, Art Marigliano, Hanna Rutter

With Raptor™ LC columns, Restek chemists became the first to combine the speed of superficially porous particles (SPP) with the resolution of highly selective USLC® technology. This new breed of chromatographic column allows you to more easily achieve peak separation and faster analysis times without expensive ultra high-per-formance liquid chromatography (UHPLC) instrumentation. The new Raptor™ ARC-18 column was designed to stand up to even the harshest acidic mass spectrom-etry (MS) conditions. It utilizes a proprietary bonding procedure that arranges our sterically protected ligand to resist acid hydrolysis and, therefore, also resist phase degradation and bleed. Designed and intended specifically for use on liquid chroma-tography (LC) MS-MS systems, the Raptor™ ARC-18 column features a well-bal-anced retention profile without the drawbacks of using an ordinary C18 in the harsh, acidic mobile phases needed for mass spectrometry. Even after extended use in these low-pH (≤ 2.0) conditions, the sterically protected ARC-18 offers consistent retention, peak shape, and response for charged bases, neutral acids, small polar compounds, and more. The Raptor™ ARC-18 column has the performance needed for critical multiclass workflows in any industry or lab.

82 The Raptor™ Biphenyl Stationary Phase: Selectivity AcceleratedHanna Rutter, Restek Corporation, 110 Benner Cir., Bellefonte, PA 16823, Ty W. Kahler, Art Marigliano

With Raptor™ LC columns, Restek chemists became the first to combine the speed of superficially porous particles (SPP) with the resolution of highly selective USLC® technology. This new breed of chromatographic column allows you to more easily achieve peak separation and faster analysis times without expensive ultra high-per-formance liquid chromatography (UHPLC) instrumentation. Our top priority when de-veloping our new SPP line was to create a version of our innovative Biphenyl. The Biphenyl ligand provides greater aromatic selectivity than commercially available phe-nyl-hexyl columns and a greater degree of dispersion than conventional phenyls. This unique selectivity mechanism allows greater retention of dipolar compounds, conju-gated compounds, and compounds containing strong electron withdrawing groups. The industry-leading Biphenyl is Restek’s most popular LC stationary phase because it is particularly adept at separating compounds that are hard to resolve or that elute early on C18 and other phenyl chemistries. Increasing retention of early-eluting can limit ionization suppression, and the heightened selectivity helps eliminate the need for complex mobile phases that are not well-suited for mass spectrometry detection.

83 Withdrawn by the author.

84 A Novel Core-Shell HPLC Column with Unique Shape SelectivityKen Tseng, Nacalai USA, 10225 Barnes Canyon Rd., A103, San Diego, CA 92121, Toshi Ono, Tsunehisa Hirose, Kazuhiro Kimata

A cholesterol-bonded high-performance liquid chromatography (HPLC) column allows for separation of structurally similar compounds under the reversed-phase condition. The column has similar hydrophobicity as a C18 column. However, the relatively rigid structure of the cholesterol functional group gives it better shape se-lectivity than a typical C18 column. We have applied this phase to the core-shell particle technology to further improve the column performance. Core-shell columns have better performance and throughput than the fully-porous ones. We routinely obtain the same separation performance on a 2.6- mm-particle core-shell column at half the back pressure as a 1.7-mm fully porous column. Examples of method development using this cholesterol-bonded core-shell column are shown with the catechin mixture and other compounds. We have conducted experiments to show the performance differences between fully porous 5-mm and 2.5-mm particle col-umns to the 2.6-mm core-shell.

85 The Use of Sequential Elution Liquid Chromatography of Increasingly Complex Combinations of Compounds for Increased Peak Capacity and Decreased Separation DisorderErin J. Ennis, Drexel University, 3141 Chestnut St., Philadelphia, PA 19104, Michael R. Fletcher, Donna M. Blackney, Catherine A. Kita, Joe P. Foley

Sequential-elution liquid chromatography (SE-LC), through the use of multiple se-quential elution modes, can separate analytes by class. By employing a pH gradient followed by a solvent gradient, analytes composed of weak acids, weak bases, and neutrals can be separated by class and also within each class (i.e. each analyte from another). Previous work [1] shows a greater probability of successful separa-tion utilizing SE-LC with increased peak capacity and decreased separation dis-order over traditional liquid chromatography for the separation of a 12-component sample. In this work, SE-LC experiments are extended to include additional elution schemes, including serially coupled columns, as well as new and increased combi-nations of compounds. Experimental emphasis is on the optimization of chromato-graphic figures of merit and applications for future use are explored.


[1] A. Socia and J. P. Foley, J. Chromatogr. A, 2014, 1324, 36-48.

86 Increasing Efficiency while Decreasing Solvent Usage: The Development of a High-Throughput uHPLC Method for Determination of the Aerodynamic Particle Size Distribution (APSD) by Andersen Cascade Impaction (ACI) and Emitted Dose Uniformity (EDU) for Dry Powder Inhalers (DPI)Jagruti Patel, Merck, 556 Morris Ave., Summit, NJ 07901, Justin Pennington

As part of Merck’s commitment to the green chemistry, the Merck Research Labs (MRL) has been exploring ways to reduce the formation of hazardous waste. Chromatographic analysis generates a significant amount of solvent waste in the analytical laboratories, providing great potential to decrease solvent usage while increasing efficiency. The current example involves the implementation of a high-throughput ultra high-performance liquid chromatography (UHPLC) method for determination of aerodynamic particle size by Andersen cascade impaction (ACI) of dry powder inhalers. The ACI test segregates particles based on their aerodynamic size on a series of plates and provides insight into drug product aerodynamic prop-erties. This information comes at the expense of a significant number of samples for extraction and chromatographic analysis. A typical 5 inhaler ACI evaluation results in 50 samples (10 / inhaler) providing a significant opportunity to increase efficiency of analysis while reducing solvent usage. The resulting method was developed by optimization of UHPLC parameters and sample preparations steps with the overall goal of a one minute chromatographic run time. The newly developed one minute UHPLC method has proven to be a significant improvement when compared to historical methods which range from ~5-14 minutes. For a typical analysis set of 5 inhalers the UHPLC method uses only ~250 mL of solvent resulting in a 75-90% reduction from typical HPLC methods for a total reduction of waste up to 1000-2750 mL. The efficiency gain results in a time savings of 8-24 hours per ACI set allowing for a significant increase in analytical throughput.

87 Use of Additives for Improving LC-MS AnalysisShyam Verma, Supelco/Sigma-Aldrich, 595 North Harrison Rd., Bellefonte, PA 16823, Rudolf Kohling

Ultra high-performance liquid chromatography (UHPLC) using ultraviolet (UV) or mass spectrometry (MS) detection is now a routine technique in most analytical lab-oratories. The development of instruments and LC columns in the recent years im-proved the performance of fast and highly resolved separations significantly. Fused Core or the new Titan particles represent key developments that enable the full performance of a UHPLC system with a maximum of sensitivity and chromatograph-ic efficiency. Unfortunately these enhancements can be undermined by the use of unsuitable solvents. The ion suppression effect is still a major issue of the most common electrospray ionization (ESI) source and has not yet been addressed. The results presented in this poster show the positive effect of a sample clean-up step prior to LC-MS analysis. Complex sample matrices, such as food samples, often cause ion suppression by co-eluting components, such as sugars or phospholipids. These issues can be resolved by using high purity additives.

88 Retention Characteristics of Fluorophenyl HPLC Stationary PhasesBrittany George, Ursinus College, Department Chemistry; 601 E. Main St., Collegeville, PA 19426, Eric Williamsen

Fluorinated stationary phases are commercially available and are useful in separa-tions of compounds of interest to the pharmaceutical as well as in other industries. The retention characteristics of these phases however, are not well characterized, and few comparisons between fluorinated and similar non-fluorinated phases have been reported. Retention of approximately 40 analytes with phenyl, alcohol, nitro, cyano, halogenated, and other functionality was measured on a fluorophenyl sta-

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tionary phase at temperatures ranging from 5.0 to 65.0 ˚C and with various organic/aqueous mobile phase compositions, ranging from 10 – 100% organic. Retention measurements on a traditional, similar phenyl stationary phase have also been made under the same conditions. By analyzing the retention data through van’t Hoff and selectivity plots, and quantitative structure retention relationships, similarities and differences in the retention characteristics of phenyl and fluorophenyl stationary phases under various mobile phase and temperature conditions are described and discussed.

89 Analysis of the Retention Characteristics of Fluorinated and Non-Fluorinated Octyl HPLC Stationary PhasesPeter Willard, Ursinus College, Department of Chemistry; 601 E. Main St., Collegeville, PA 19426, Eric Williamsen

Although high-performance liquid chromatography (HPLC) is the one of the most commonly used and successful separation techniques, work continues on com-pletely understanding the processes that leads to retention. Fluorinated stationary phases are commercially available and have been shown to be more useful than traditional stationary phases in the separation of a number of compounds of phar-maceutical interest. To better understand the similarities and differences of retention on fluorinated and similar hydrocarbon phases, retention of over 40 analytes with phenyl, alcohol, nitro, cyano, halogenated, and other functionality have been mea-sured on fluoro octyl and octyl stationary phases. Measurements have been made at temperatures ranging from 5.0 to 65.0 ˚C and with various organic/aqueous, mo-bile phase compositions. Because of the large number of variables, multivariate analysis techniques, such as cluster analysis and principal component analysis, have been used to emphasize distinctive patterns in the data. In order to better un-derstand the underlying physical interactions that lead to retention, linear solvation energy relationships are also used. The results of these analyses are presented and discussed.

90 The Concept of Standard Adsorption Isotherms: Comparison of Excess Adsorption of Binary Aqueous Organic Mixtures on Classical Packing Material Modified with Alkylated and Fluorinated LigandsMargaret Figus, Seton Hall University, 400 South Orange Ave., South Orange, NJ 07079, Yuri V. Kazakevich, Alexander Y. Fadeev

The absorbent surface area, bonded phase chemistry, and bonding density are the major factors affecting the reverse-phase separation. The effect of bonded chain chemistry, bonding density, chain length on adsorbent pore volume, and void volume of high-performance liquid chromatography (HPLC) columns are described in this work. A series of covalently attached monolayers of alkanes and perfluorinated-al-kanes were prepared via solution phase reaction of CnH2n+1Si(CH3)2N(CH3)2 where n = 1, 4, 8, 18, and CnF2n+1C2H2Si(CH3)2N(CH3)2 where n = 1, 4, 6, 8 with well characterized silica substrate adsorbent. The energies of adsorption, surface area, and pore volumes of modified and unmodified silica were calculated using nitrogen adsorption isotherm. Grafting density of bonded ligands was determined from the weight percent of carbon and fluorine. The excess adsorption isotherms of acetonitrile and methanol from water were measured on in-house packed columns. Comparison of the excess adsorption isotherms measured on these columns and expressed in surface specific form demonstrated significant similarity of the adsorp-tion properties, which allows us to introduce the “standard adsorption isotherm” for reversed-phase alkane type columns and suggests that adsorption depends on the type of ligands and bonding density of the ligands.

91 Outperforming Sub-2-µm Totally Porous Particles using Fused-Core TechnologyRobert E. Moran, Advanced Materials Technology, 3521 Silverside Rd., Quillen Bldg., Ste. 1-K, Wilmington, DE 19810, Stephanie A. Schuster, Barry E. Boyes, William L. Miles

Fused-core particles have shown distinct advantages over comparable totally porous particles for separating molecules of all sizes and characteristics. The in-troduction of a new 2.0-µm particle size to the Fused-Core product line continues to advance high speed, high efficiency separations into the future. These 2.0-µm Fused-Core particles permit separation speeds and resolution improvements great-er than sub-2-µm totally porous particles, but with about 20% lower column back pressure. Based on chromatographic theory, the trend of using smaller particle sizes has been accepted as a way to increase efficiency. Coupled with this trend, the detrimental effects of extra column band broadening have become more significant. This report describes the effect of these new particles on multiple factors of sep-aration performance, including reduced plate height, column efficiency, and extra column band broadening.

92 Selection of Mobile Phase Modifiers for High Efficiency HILIC SeparationsWilliam L. Miles, Advanced Materials Technology, 3521 Silverside Rd., Quillen Bldg., Ste. 1-K, Wilmington, DE 19810, Robert E. Moran, Stephanie A. Schuster, Barry E. Boyes

In high-performance liquid chromatography (HPLC) separations, the use of mobile phase modifiers (buffers, and other additives) has become common practice to achieve better separations in both reverse-phase (RPLC) and hydrophilic interac-tion chromatography (HILIC). Trifluoroacetic acid (TFA) is widely used, but other acidic additives such as formic acid (FA) and buffers like ammonium formate (AF) have become more popular. The use of these and other additives were examined for HILIC separations of small bases, acids, zwitterions, and peptides. Highly effi-cient superficially porous particles (Halo Fused-Core) bonded with the Penta-HIL-IC stationary phase was employed. Separations of small molecules and peptides exhibited excellent peak symmetry, column efficiency, and load tolerance when appropriate mobile phase additives were used. We show examples of how these improvements can be enjoyed in liquid chromatography-mass spectroscopy (LC-MS) applications with appropriate selection of volatile mobile phase modifiers.

93 Integrating Real-World Chemical Separation Research Projects into Analytical Chemistry CurriculumYuegang Zuo, University of Massachusetts-Dartmouth, 285 Old Westport Rd., North Dartmouth, MA 02747

Analytical chemistry is increasingly becoming an integral part of all science and engineering disciplines, and it plays a vital role in broader societal concerns. Thus, today’s analytical chemistry education must be more relevant to modern analytical laboratory practices. Although traditional undergraduate chemistry curricula provide a solid foundation in the fundamental principles of analytical chemistry, they do not formally value practical skills that enable students to adapt and be successful in today’s rapidly changing and competitive analytical workplace. To bridge the current gap between “real work” experiences and university training in analytical chemistry, the author has integrated real world chemical separation research projects[1] into an-alytical chemistry curriculum to prepare our students academically for what the real world wants from them. The integrated research projects challenged students to think creatively and improved their skills in communication, teamwork, and problem-solv-ing. The details of this innovative curriculum project are given at the presentation. Reference:[1] Y. Zuo, 2014, High-Performance Liquid Chromatography (HPLC): Principles,

Procedures and Practices. Nova Science Publishers, Inc., New York.

94 Withdrawn by the author.

95 Quantitation of Creatinine and Albumin Biomarkers in Mouse Urine by HPLC and Affinity Chromatography Joe Shao, Bristol-Myers Squibb, 311 Pennington-Rocky Hill Rd., Pennington, NJ 08534, Yingru Zhang, Preeti Sejwal

Creatinine and albumin are two key renal biomarkers present in urine. A urinary albumin/creatinine ratio (UACR) can be used to diagnose and monitor kidney dis-ease. Clinically creatinine/albumin is tested using Jaffe’s colorimetric method, en-zymatic or immunoassay method with low specificity. In this study, the optimized reverse-phase high-performance liquid chromatography (HPLC) method for creati-nine utilized a Zorbax Bonus-RP column and sodium phosphate buffer/methanol as mobile phases. The method demonstrated no interference in urine samples from six types of mouse models. The creatinine data obtained from the HPLC method and enzymatic method were within 86-117% consistency. For albumin analysis, affinity chromatography with commercial resin-bound single-domain mono-specific antibody fragments coupled with MW cutoff membrane filtration was used for sep-aration of albumin from the urine matrix. The sample was further quantified using size exclusion chromatography with UV detection at 215 nm. Non-specific binding, molecular cutoff membrane filter selection, pH/salt effect and in-house packing of an affinity ligand column are discussed. The HPLC methods developed were able to quantify creatinine and albumin with UV detection, and can be applied as the reference or calibration methods for quantitation of UACR in urine.

96 Simultaneous Analysis of APIs and their Counterions Using a Mixed-Mode ColumnPeter Ratsep, Shimadzu Scientific Instruments, 19 Schoolhouse Rd., Somerset, NJ 08873, Kenichiro Tanaka, William Hedgepeth

Approximately 50% of all drug molecules used in pharmaceutical products are reported to be ionic compounds. Ion chromatography is generally appropriate to analyze inorganic or organic ions, but not suitable for active pharmaceutical ingredi-ents (APIs) analysis due to their hydrophobicity. On the other hand, reversed-phase liquid chromatography (RPLC) is mainly applied for analysis of APIs, but cannot retain commonly-used ions for drugs. Consequently, it is difficult to analyze APIs and their counter-ions simultaneously. In this study, we evaluated the ReDual col-

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umn, our newly-developed mixed-mode column, for simultaneous analysis of APIs and their counter-ions. Mixed-mode column usage has increased because of the ability to analyze a wide range of compounds in a single run by multimode retention mechanisms. The ReDual column was able to analyze APIs and their counter-ions simultaneously. In addition, we investigated how retention behavior changes with parameters such as pH, ion strength, and concentration of organic solvents in the mobile phase.

97 Fast GPC Analysis of Residual Lauryl Methacrylate in Polymer ProductsTian Lu, Ashland, 1005 US 202/206, Bridgewater, NJ 08807, Fan Wu

Lauryl methacrylate (LMA) is an important monomer in polymer chemistry. The long alkyl chain makes it a key component for manipulating the hydrophobicity of poly-mers or copolymers. The determination of residual LMA in polymer products is re-quired in product and application development. Both gas chromatography (GC) and high-performance liquid chromatography (HPLC) methods have limitations for LMA residual determination in a polymeric matrix. If the polymer is thermally labile, the polymer will undergo degradation in the GC injector and column which can interfere with quantitative LMA detection. Poor recovery is observed when HPLC is used as the analytical approach. Therefore, a fast gel permeation chromatography (GPC) method was developed for residual LMA analysis. LMA can be well separated from other components and impurities and quantified directly from the polymeric matrix on a fast GPC column platform.

98 Use of New YMC-Meteoric Core Core-Shell and YMC-Triart Hybrid Material for an Improved USP Chlorhexidine Gluconate Assay MethodJeffrey A. Kakaley, YMC America, 941 Marcon Blvd., Allentown, PA 18109, Ernest J. Sobkow

United States Pharmacopeia (USP) methods are used widely in the pharmaceuti-cal industry and are often not optimized for use with modern instrumentation and packing materials. As it becomes increasingly important to boost throughput in the modern laboratory, new instrumentation and smaller particle size packing materials are allowing scientists to achieve these goals while also realizing cost savings in terms of man-hours and solvent usage. In this poster we investigate the use of 3-µm YMC-Triart C18 and 2.7-µm YMC-Meteoric Core C18 stationary phases for improv-ing a USP assay method for chlorhexidine gluconate.

99 A Rapid Hydrophilic Interaction Liquid Chromatography (HILIC) Method for Determination of Trace Nitrate and Nitrite in Snow and Rain SamplesXiaofei Lu, University of Massachusetts-Dartmouth, 285 Old Westport Rd., Dartmouth, MA 02747, Yuegang Zuo

Nitrite and nitrate are two common ions in natural waters. It is of great significance to determine the concentration of these ions because they can put potential risk to human and environment health. A rapid, specific, and sensitive method for deter-mining nitrite and nitrate in snow and drinking water has been developed based on high-performance liquid chromatography. A hydrophilic amino column was used to separate the nitrite and nitrate which were then detected by an ultraviolet-vis-ible spectroscopy detector at wavelength of 215 nm. The mobile phase consists of acetonitrile and acetate buffer solution. It was found that the retention times of nitrite and nitrate increased with decrease of pH of mobile phase or decrease of proportion of buffer solution in mobile phase. For the effect of acetate concentration, the retention times of nitrite and nitrate increased with the increase of concentration under low acetate concentrations range (below 2.5 mM), whereas in the high con-centration range (2.5 mM to 10 mM), they decreased with increase of concentration. The results may indicate an ion exchange mechanism to the separation of nitrite and nitrate on amino column. When determining the nitrite and nitrate in snow and rain water samples, the average recovery of the two compounds are 97.86% and 98.39%, respectively. Nitrate concentration varied from 0.072 to 1.39 mg/L in the in snow or rain sample. The nitrite was quantified only in one snow sample with concentration of 0.012 mg/L.

100 Two-Dimensional GPC Analysis for Blends of PolymersYejia Li, Arkema, 900 First Ave., King of Prussia, PA 19406, Martin Nosowitz, Mark Lavach, Joseph Mitchell, Robert Barsotti, Florence Mehlmann

Blending of different types of polymers offers a cost-effective way to develop high performance materials. However when the polymers are miscible, determination of each polymer’s molecular weight can be challenging. The two-dimensional gel permeation chromatography (2D-GPC) method is a versatile technique for the sep-aration and analysis of polymer blend’s molecular weight. Using liquid adsorption chromatography (LAC) the blend is first separated by polarity. A second separation using GPC provides molecular weight information for fractions eluted from the LAC separation. Examples of analysis using the 2D-GPC system are discussed.

101 High-Speed Amino Acid Analysis (AAA) on a New 2.7-µm Superficially Porous High pH Stable Reversed-Phase (RP) Column William J. Long, Agilent Technologies, 2850 Centerville Rd., Wilmington, DE 19808, Anne E. Mack, Jason Link

Amino acid analysis (AAA) is commonly used food and fermentation testing as a tool to determine the precise amino acid (AA) makeup of samples. In order to do this in a timely fashion a short turnaround time is needed; to do it with limited sample amounts, a highly sensitive method is needed. And to maximize laboratory pro-ductivity, an automated method is desirable. In many cases these samples contain particulates which can clog many high resolution columns or necessitate addition-al sample clean-up, and the analysis is further complicated by the often rigorous conditions used, resulting in poor column lifetimes. This work describes methodol-ogy using a high pH stable superficially porous column with a 2.7-μm engineered particle size. These particles have 90% of the efficiency of sub two micron totally porous columns at 50% of the pressure and offer longer lifetimes under the higher pH conditions typically used in this analysis. Additionally since they incorporate a 2-μm frit, they are as resistant to clogging as 3.5 and 5-μm columns. In this work, an automated pre-column OPA/FMOC amino acid method previously developed on 3.5 and 1.8-μm columns is expanded to include 2.7-μm superficially porous columns. This column shows good lifetime and transferability to different column dimensions which are shown in this work. Applications of this column to fermentation products are also shown.

102 Lead and Cadmium in Counterfeit Cigarettes: Implications for Public Health and Research on the Illegal Cigarette TradeYi He, John Jay College, 524 W 59th St., New York, NY 10019, Klaus von Lampe, Laura Wood, Marin Kurti

Lead (Pb) and cadmium (Cd) are two toxic heavy metals commonly found in regu-lated amounts in genuine cigarettes. However, information regarding their concen-tration in counterfeit cigarettes is limited. This study compares the concentration of Pb and Cd in twenty-three samples of counterfeit cigarettes seized by federal, state and local law enforcement agencies in the United States with that in genu-ine cigarettes using inductively coupled plasma-mass spectrometry (ICP-MS) after microwave digestion. Significantly higher Pb and Cd concentrations were found in the majority of counterfeit samples. The average Pb and Cd concentrations in coun-terfeits were 5.13 ± 0.17 mg/kg (n=23) and 5.13 ± 1.95 mg/kg (n=23), compared to 0.59 ± 0.08 mg/kg (n=9) and 1.08 ± 0.02 mg/kg (n=9) respectively in the genuine samples. The counterfeit cigarette samples with the highest concentrations of Pb and Cd originated from the People’s Republic of China. A sample originating from Paraguay and classified as counterfeit had similar Pb and Cd content as genuine brands. Analytical results suggest that if consumed prevalently, typically in an eco-nomically disadvantaged area, counterfeit cigarettes potentially pose a higher risk to public health. In addition, the elemental distribution was related to the geographical origin of the counterfeit products. This work provides important information regard-ing the illicit cigarette trade including the level of organization among counterfeiters, who broker between availability of supplies (i.e., premium tobacco leaves, filter tips, cigarette paper, etc.) and consumer demand for cheaper product that is assumed to be genuine.

103 Blood Alcohol Determination Using Static Headspace Analysis with Optimized Sample ThroughputAnne Jurek, EST Analytical, 503 Commercial Dr., Fairfield, OH 45014, Mike Moses, Lindsey Pyron, Kelly Cravenor

There are several ways to determine the amount of alcohol that is in a person’s system. The most common methods are breath analysis in the field and blood anal-ysis in the lab. Blood alcohol determination in the laboratory is used predominantly when a person refuses a breath test. In order to determine blood alcohol content, a person’s blood has to be withdrawn as soon as possible after the occurrence. Furthermore, the blood needs to be collected in duplicate in order to confirm the test results. This poster will examine static headspace sampling of alcohol standards using gas chromatography for separation and flame ionization detection for analy-sis. The linearity of the compounds of interest will be examined and compared using a secondary column for confirmation. Additionally, as many forensic labs have an excess of samples to examine, the use of software innovations will aid in optimizing sample throughput.

104 Application of Infrared Microscopy for the Chemical Analysis of Security DocumentsRonald Rubinovitz, Thermo Fisher Scientific, 4410 Lottsford Vista Rd., Lanham, MD 20706, Monica Joshi

Government issued identity documents include credentials such as passports, driv-er’s licenses and travel visas. These documents generally incorporate various se-curity features that are used to authenticate them. Many of these security features can be verified visually or through the use of an alternate light source. Forensic document examination includes the assessment of these security features and oth-er characteristics of the identity documents. When a document is suspected of being

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fraudulent the document is subjected to more scrutiny of its physical characteristics and the chemical analysis of inks or paper. Several recent high profile cases have involved the use of counterfeit passports, visas and state issued ID’s. Document examination and improving methods for document examination is essential to main-taining the integrity of homeland security and immigration services. In this poster we explore the utility of Fourier transform infrared (FTIR) microscopy for the chemical analysis of the inks and the substrates used in identity documents. Establishing ref-erence collections and databases of inks and substrates used in security documents is a crucial aspect of document examination. We present the chemical analysis of representative documents in the categories of passports, visas and Pennsylva-nia driver’s licenses. We highlight the advantages of FTIR microspectroscopy in conducting rapid, highly discriminatory and non-destructive chemical analysis of inks and plastics used in security documents. The evaluation and optimization of different spectral search methods to facilitate the use of a document component database is also presented.

105 The Use of Automated Direct Sample Analysis (DSA-TOF) for the Rapid Screening of Illicit Street Drugs,Bonnie Marmor, PerkinElmer, 940 Winter St., Waltham, MA 02451, Joanne Mather, Sean Daugherty

Screening methods for drugs of abuse currently require long chromatographic methods and sample preparation. These methods can be time-consuming and la-borious. Due to the variety of the illicit and abused drugs available, it is important to monitor the entire spectral range, which is easily performed using time-of-flight mass spectrometry (TOF-MS). The ability to analyze samples rapidly is desirable, as drug seizures may result in thousands of samples. A method was developed for screening various classes of street seized illicit designer drugs, drugs of abuse and abused human growth hormones in solid, liquids as well as in urine of possible illicit drug offenders using the DSA-TOF. We present a fast screening and confirmation method by AxION® direct sample analysis with time-of-flight mass spectrometry (DSA™ TOF-MS). Fast screening of seized samples in seconds, without sample preparation, is easily performed using DSA-TOF-MS. We present a real life case study for the rapid analysis of 369 unknown drugs from seized pills, vials, powders and urine samples. The presence of the target analytes was confirmed using AxION Solo™ software by exact monoisotopic masses of precursor ion and product ions.

106 Lawsone’s Derivatives for Fingerprint DetectionJoo Meang, John Jay College, Science Department, 524 W 59th St., New York, NY 10019, Natalia Fernandez, Gloria Proni

Lawsone, 2-hydroxy-1,4-naphthoquinone, is a naturally occurring coloring agent. It is a relatively new reagent for developing latent fingerprints compared to ninhydrin and 1,8-diazofluoren (DFO) that are more routinely used for the same purpose. Lawsone has the property of coloring and fluorescing in a single step, which is a beneficiary property when used to develop fingerprints. In this research, deriva-tives of the lawsone compound with selected fluorophores are being analyzed for the enhanced properties in development of fingerprints using mass spectrometry and nuclear magnetic resonance spectrometry. This enhancement is expected to be seen in both coloring and fluorescing properties. The synthesized compounds were studied after individually adding several amino acids and treating fingerprints using ultraviolet-visible spectroscopy and fluorescence spectroscopy to view and compare their fluorescence properties. The furthermost objective of the research is to find the best compound with upgraded properties of both fluorescence and solubility for fingerprint detection.

107 Analytical and Synthetic Studies on Substituted 1-n-Pentyl-3-Benzoylindoles: Isomeric Synthetic CannabinoidsKarim M. Abdel-Hay, Auburn University, 3306 Walker Building HSOP, Auburn, AL 36849, Jack DeRuiter, Forrest Smith, C. Randall Clark

Gas chromatography mass spectrometry (GC-MS), GC-time-of-flight (TOF)-MS and GC-IRD techniques provide complimentary data in the differentiation of regioiso-meric and isobaric piperazine drugs of abuse and controlled substances belonging to synthetic cannabinoids. The mass spectra for regioisomeric and isobaric pipera-zines are almost identical with no marked differences that would allow for differentia-tion between these compounds. Perfluoroacylation of the secondary amine nitrogen of these isomeric piperazines gave mass spectra with differences in the relative abundance of some fragment ions but did not alter the fragmentation pathway to provide unique ions for discrimination among these isomers. Gas chromatography coupled with time-of-flight mass spectrometric detection provided a means of dis-crimination among the isobaric piperazines by confirming the elemental composition of the major fragment ions in the mass spectra of these compounds. On the other hand, GC-TOF-MS was not successful in differentiating between regioisomeric pip-erazines. The vapor phase infrared spectrum obtained by GC-IRD was successful in differentiating among the regioisomeric and isobaric piperazines by the charac-teristic bands in the region 700 – 1700 cm-1. These techniques have been applied to the analysis of the ring substituted methoxy-, methylenedioxy-, dimethoxy- and bromodimethoxy-, in both the benzyl and benzoylpiperazine series. In addition to

that, isotope labeling experiments such as deuterium and carbon 13 labeling were used to confirm mass spectrometric fragmentation mechanisms that result in the formation of some key fragment ions or to confirm the elemental composition of these fragment ions. The gas chromatographic resolution is described for these piperazines on a variety of stationary phases.

108 High-Throughput Methodology for Mechanical Testing of Polymer FilmsSara Reynaud, Arkema, 900 First Ave., King of Prussia, PA 19406, Jun Fang, Liang Fang, Evan Crocker

The measurement of mechanical properties of thin polymer films under high speed impact is often difficult. Standard weight dropping testing is performed at high strain rate but it is not suitable for thin samples and does not provide information on stress-strain. Other techniques such as tensile/flex or nanoidentation can only be performed at low strain rate. A high-throughput methodology for mechanical testing (HTMECH) of freestanding thin films under high speed impact is introduced in this work. The main advantages of this technique are: high instrumental sensitivity; test rapidity and simplicity of the data analysis. Toughness properties of polymers sup-plied by Arkema Inc. were measured and compared with dynamic mechanical analy-sis (DMA). Interesting correlations between HTMECH and DMA results were found. In particular, highest impact performance was observed for lower Tg polymers.

109 Withdrawn by the author.

110 Method Implementation for THC, THC-OH, and THC-COOH Testing and Application for Blood in Experimental SubjectsAlex J. Krotulski, The Forensic Sciences Mentoring Institute, 2300 Stratford Ave., Willow Grove, PA 19090, Kathryn Peavy, Ian Wietecha-Reiman, Samiyah Cornwell

Cannabis sativa, or marijuana, is a plant containing over 70 psychoactive com-pounds. Delta-9-tetrahydrocannabinol (THC) is the primary psychoactive compound and is metabolized to 11-hydroxy-delta-9-tetraydrocannbinol (THC-OH) and 9-car-boxy-11-nor-delta-9-tetrahydrocannbinol (THC-COOH) during first pass metabolism in the liver. This project aimed to develop and optimize a method for detecting and quantifying low levels of THC, THC-OH, and THC-COOH in blood. After smoking, THC levels quickly spike in blood and then fall rapidly due to metabolism. Blood taken at varying times after inhalation will be analyzed so it is important to be able to quantify high and low levels of THC and its metabolites. Solid-phase extraction was used to separate out the analytes from blood. Once the analytes were extracted, the samples were dried down and derivatized with BSTFA + 1% TMCS for analy-sis by gas chromatography-mass spectrometry (GC-MS). Calibration models were created using analyte standards spiked into sheep blood. Correlation coefficients for the calibrations curves produced were 0.995 for THC, 0.996 for THC-OH, and 0.999 for THC-COOH. Recovery of our analytes after solid-phase extraction was lower than expected at 49% for THC, 41% for THC-OH, and 51% for THC-COOH. Precision studies were also carried out and determined to be acceptable (<20%) for all analytes at 5 ng/mL and 200 ng/mL. Once the method is validated, real-world blood samples will be analyzed from known marijuana users. The results gathered from the blood samples will be used to correlate blood concentrations with driving records, as part of a larger driving under the influence of drugs study.

111 Development of an Analytical Method for Nootropic “Smart” Drugs in Biological FluidsMollie M. Mares, The Forensic Sciences Mentoring Institute, 2300 Stratford Ave., Willow Grove, PA 19090, Iryna Kurochka, Rini Gupta, Dymere Taylor, Mandi Mohr, Barry Logan

Smart drugs, or nootropics, are stimulants that allegedly boost brain function and cognition. The media attention on these drugs has increased within the last few years. The drugs have developed an underground following and are commonly sold on-line and in illicit supply chains. Most have not been approved or scheduled in the United States, and are therefore of concern to regulators such as the Food and Drug Administration (FDA) and Drug Enforcement Administration (DEA). There are ongo-ing investigations in the applications of smart drugs in the treatment of Alzheimer’s disease, Huntington’s disease, and attention deficit hyperactivity disorder (ADHD). The stimulant properties of the drugs have led to their use in academic doping and as drugs of abuse. Some drugs are also prohibited by the World Anti-Doping Agen-cy (WADA). The goal of this project was to develop a single analytical method for screening, confirmation, and quantification of a series of the widely known smart drugs in blood and urine using supported liquid extraction (SLE). Gas chromatog-raphy-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) were investigated to determine the optimum approach for sensitivity and ability to detect a broad range of compounds, specifically piracetam, pramiracetam, aniracetam, modafinil, adrafinil, ciproxifan, and noopept. Due to the thermal instabil-ity of the compounds and the inability to produce single, stable chromatographable analytes, LC-MS provided a superior means of analysis. All seven analytes were

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successfully extracted from blood, providing an effective method for the analysis of nootropics in biological fluids.

112 Automating Liquid-Liquid Extractions Using a Bench-Top WorkstationFredrick D. Foster, Gerstel, 701 Digital Dr., Suite J, Linthicum, MD 21090, Oscar G. Cabrices, Jacqueline A. Whitecavage, John R. Stuff, Edward A. Pfannkoch

Liquid-liquid extractions have long been performed manually and are used to extract and concentrate analytes from aqueous matrices. Inclusion of liquid-liquid extraction in many official methods attests to the wide acceptance of the technique. Following solvent extraction it is also common to include an evaporation and reconstitution step to improve detection limits or exchange solvents for compatibility with subse-quent chromatographic separations. Modern analytical labs are looking to automa-tion to help reduce solvent usage and increase sample throughput while ensuring the high quality of the resulting data. A single robotic X-Y-Z coordinate autosampler commonly used for sample introduction in gas chromatography (GC) or high-per-formance liquid chromatography (HPLC) can be used to perform a wide variety of sample preparation techniques using a single instrument and controlling software. The sampler can be configured as part of a GC or LC system or can be configured as a bench-top workstation and can also include a six position evaporation station. In this report, the automation of liquid-liquid extractions by the robotic autosampler is discussed. Examination of a new, automated vortexing option that allows samples to be rapidly and effectively mixed using speeds of up to 3000rpm is described. Automated liquid-liquid extractions methods for a variety of analytes from different matrices are examined and resulting precision and accuracy data are provided.

113 New Stationary Phases for Large Volume SPEPranathi R. Perati, Thermo Fisher Scientific, 1214 Oakmead Pkwy., Sunnyvale, CA 94085, Jing Hong, Rosanne Slingsby

Five new polymeric stationary phases were developed for large sample volume solid-phase extraction (SPE). The phase chemistries include a hydrophilic re-versed-phase material as well as mixed mode, strong and weak, anion and cation exchange. Base particles are grafted with polymeric and monomeric moieties to achieve a useful balance between reversed-phase and ion exchange capacities. The hydrophilic reversed-phase material bears specific functionality to retain poly-phenolic and azo-containing species through a charge transfer mechanism and H-bonding. This allows the retention of such species as humic acids along with the ability to elute other organic species using conventional reversed-phase techniques. These new resins are formatted to be compatible with sample volumes larger than 20 mL in automated SPE instrumentation. Several applications are shown. The targets include triclosan, a popular but very hydrophobic bactericide, anionic and cationic surfactants, the weak base atenolol and others with specific advantage for the phases. Recoveries greater than 95% for sample volumes of 20-200 mL at load-ing flow rates of 5-30 mL/min in different matrices ranging from tap water, surface waters to the American Society for Testing and Materials formulation of wastewater were observed. Comparisons illustrating the right choice of bed weight to minimize cost are also presented.

114 Adsorption of Steroid Hormones on Filter Membranes during Analytical Sample PreparationJingjing Xie, University of Massachusetts-Dartmouth, 285 Old Westport Rd., North Dartmouth, MA 02747, Si Zhou, Yuegang Zuo

Determination of steroid hormones (SH) in aquatic environments has been a chal-lenge for environmental scientists because of the extremely low analyte concen-tration levels and complex matrix composition. Consequently, it is necessary to preconcentrate the selected analytes and clean-up the samples from interfering components, especially in the presence of humic subtances or suspended particles in waste and surface waters. In this study, the effects of filter materials on the con-centration of SH were systematically examined with commonly used commercially available filter membranes, including glass fiber, acetate cellulose, polycarbonate and nylon membranes. The pH and electrolyte concentration of the sample solu-tions can also affect the SH adsorption on the filter membranes. Under all conditions tested, nylon membrane has the highest adsorption efficiency for SH. Approximately 64% of 17α-ethinylestradiol and 55% estrone was adsorbed on the nylon membrane after 10 mL of 5.0 mg L-1 of these analytes were filtered through the membrane.Abstracts 115 - 116

115 New Advances in Environmental Air MonitoringCharles Haws, Markes International, 11126-D Kenwood Rd., Cincinnati, OH 45242, Pete Grosshans, Nicola Watson

As the harmful effects of low-level exposure to hazardous organic air pollutants become more evident, there is constant pressure to improve the detection limits of indoor and ambient air monitoring; for example, by collecting larger air volumes and by optimizing the sensitivity of the analytical detector. However, at the other end of the scale, rapid industrialization in the developing world and growing pressure to

reclaim derelict industrial land for house building is driving the need for air mon-itoring methods that can reliably accommodate very high concentration samples in potentially aggressive matrices. Many techniques are available for the sampling and measurement of volatile organic compounds in air/gas, but which are the best to use? Several international standard methods have been introduced that dictate the sampling and analytical methodology for the study of air toxics, with sampling generally carried out using evacuated canisters or sorbent tubes. In response to the global interest in this application area, a number of instrument developments have been made to address this analysis. This work describes a cryogen-free, analytical system designed to offer flexibility of sampling methodology, fully compliant with United States Environmental Protection Agency Methods TO-15 (canisters) and TO-17 (sorbent tubes).

116 Increasing Extraction Efficiency of Pesticides and Dioxins from Wet Samples Using a Novel New Polymer during Accelerated Solvent ExtractionAaron Kettle, Thermo Fisher Scientific, 1214 Oakmead Parkway, Sunnyvale, CA 94085, Pranathi Perati, Kannan Srinivasan, Rahamt Ullah

Accelerated solvent extraction (ASE) is a high-temperature and high-pressure ex-traction technique that is widely used for sample extractions in the environmental, chemical and food analysis industries. Extraction efficiencies when extracting vol-atile or semi volatile analytes from wet solids are often low, as the analyte of inter-est may partition between the extracting solvent and the water phase. Traditional pre or post extraction methods of heat evaporation cannot be used for volatile and semi-volatile compounds. Drying techniques that involve mixing the wet samples with an inorganic salt that has a high affinity for the aqueous phase are unsuitable for in-cell extractions. Drying methods with inorganic salts suffer from the limitations of clumping or precipitation making post extraction clean-up difficult. Off-line drying methods like freeze drying are extremely tedious and time consuming. This study presents the use of a novel new polymer designed to remove moisture from wet samples like soil, tissue and food products and increase the extraction efficiency of volatile and semi volatile compounds. The polymer has a high capacity for water removal and does not suffer from some of the limitations of clumping or precipita-tion observed in some of the traditional drying methods. Data showing recoveries of organochlorine pesticides, polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and polychlorinated biphenyls in different matrices are presented.

117 Automated Solid-Phase Extraction of Organochlorine Pesticides from Drinking WaterAaron Kettle, Thermo Fisher Scientific,1214 Oakmead Parkway, Sunnyvale, CA 94085, Rosanne Slingsby, Jing Hong, Pranathi Perati

Organochlorine pesticides are chlorinated hydrocarbon compounds that have a history of wide spread use both in the United States and globally. Organochlorine compounds degrade slowly and can bio-accumulate over time, with increasing con-centrations in animals high in the food chain. Their ability to volatilize in warm re-gions allows them to spread over long distances, with measurable concentrations detected near the Arctic Circle and in alpine areas where they have not been used. Organochlorine pesticides have found their way into sediments and drinking water supplies posing serious health risks. Organochlorines have a wide range of both acute and chronic health effects, including cancer, neurological damage, and birth defects. In response to growing health concerns, the United States and Europe has banned several of these compounds such as DDT, dieldrin and chlordane. Others still in use include lindane, endosulfan and methoxychlor. This study evaluated ex-traction recoveries of twenty five chlorinated pesticides from drinking water using automated solid phase extraction (SPE). This study also compared recoveries of the twenty five pesticides when using automated SPE to the traditional liquid-liq-uid phase extraction. The quantitative determinations of the twenty five chlorinated pesticides were performed by gas chromatography followed by electron capture detection.

118 Development of Jatropha Oil Extraction from Biodiesel Feedstocks Using Accelerated Solvent ExtractionPeter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA 94085, Aaron Kettle, Linda Lopez

The determination of oil content in biodiesel feed stocks can be performed using several methods, including mechanical press, solvent extraction, and nuclear mag-netic resonance (NMR). For the feedstock quality control in terms of oil content, it is important that the applied method is universally accepted so as to obtain results that can be compared with those reported from alternate sources. The European Norm (EN) has specified two methods for the determination of oil content in oil seed crops: conventional Soxhlet extraction and NMR imaging. However, these methods have several disadvantages. Both methods are time, labor, and cost intensive, and both require highly skilled labor and a significant amount of sample. These methods are also unfriendly to the environment. The Thermo Scientific™ Dionex™ Acceler-ated Solvent Extractor (ASE™) has great potential to overcome these constraints.

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Furthermore, ASE also has a high potential for application to oil-content testing of third generation biodiesel feed stocks (e.g., micro algae). In this poster we present the extraction of oil from Jatropha seeds in compliance with accuracy and reproduc-ibility requirements described in the EN method. Oil extraction using an ASE system requires only one to one-and-a-half hours as compared to nine hours consumed by the Soxhlet extraction. In this research, the effects of ASE system conditions on the percentage of oil extracted, the optimum conditions for oilseed extraction, and the minimum amount of oilseeds required per test are also investigated.

119 Accelerated Solvent Extraction for Alternative Fuel ResearchPeter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA 94085, Aaron Kettle

As global interest in alternate fuel sources increases, many laboratories are re-searching effective ways to test, develop, and produce fuel from renewable energy sources. One of these is the production of alcohol from biomass, called bioalcohol. Alcohol produced from biomass has several benefits of fossil fuels because it is pro-duced from crops, a renewable energy source, and causes little environmental pol-lution when burned. As bioalcohol is produced from biomass via sugar fermentation, it is necessary to research which types of plants produce the best yields of usable sugars. Thermo Scientific Dionex ASE Accelerated Solvent Extractors provide a fast and efficient way to extract various biomass samples for sugar analysis to determine sample viability for alcohol production. The Dionex ASE 150 and ASE 350 Acceler-ated Solvent Extractors with the pH-hardened pathways provide additional benefits to the method by allowing pre-hydrolyzed biomass samples or those with acidified solvents to be extracted. This allows samples to be hydrolyzed in the extraction cell. This poster describes methods for the extraction of sugars from biomass using acid-ified solvents (in-cell hydrolysis) and extracting pre-hydrolyzed samples.

120 Applying a New and Unique Microwave Sample Digestion Technique towards the Determination of Metals Present in Wine, Berry and Soil SamplesReynhardt Klopper, Anton Paar, 10215 Timber Ridge Dr., Ashland, VA 23005

The presence and concentration of several metals can have a large impact on the quality and preservation of wines. Various metals can have a negative impact on several properties of wines, which include color, clarity, flavor and oxidation stability. Generally the source of these metals is environmental, i.e., soils, berries. Metals present in wine can be determined by a number of analytical techniques, the most common include AAS, inductively coupled plasma optical emission spectrometry (ICP-OES) and ICP-mass spectrometry. Although liquid samples can be introduced directly into the ICP instruments, certain wines which contain high amounts of solids or sugars need to be digested or mineralized before introduction. Moreover, solid sample matrices such as soils and berries also need to be completely digested to prevent spectral interferences during the analysis step. In this study we illustrate how new and unique microwave digestion technology allows for the efficient and fast processing of several wine and beverage industry related sample types which contain high amounts of sugar. The compact and lightweight Multiwave GO micro-wave system features the patent pending directed multimode cavity (DMC) technol-ogy, which enables efficient and focussed application of microwave energy to a very compact cavity system, while allowing for the simultaneous processing of up to 12 samples in 18 minutes, yielding clear digestions of multi-gram samples.

121 SmartSample™ Titration Weighing TechnologyMatt Eby, Mettler Toledo, 1900 Polaris Pkwy, Columbus, OH 43240, David Zavich

SmartSample technology eliminates transcription and ordering errors, while optimiz-ing efficiency by wirelessly transferring sample information and weights to a titration beaker to be automatically read during a titration on an InMotion™ Autosampler.

122 Trace Determination of Some Heavy Metals by Solid-Phase Extraction and Atomic Absorption Spectrophotometry Sevgi Kocaoba, Yildiz Technical University, Faculty of Art and Science, Department of Chemistry, Davutpasa İstanbul, 34210 Turkey, Zeynep Aslan, Gonul Celen

The presence and movement of potentially toxic metals such as Cd, Pb, Cu, Zn and Ni in soils and aqueous systems have created concerns for environmental and hu-man health. Heavy metals can be introduced into soils and aqueous environments either naturally or as a result of human activities such as agricultural, industrial and mining operations. The adsorption of Cd(II), Pb(II), Cr(III) and Mn(II) onto dolomite and sepiolite was examined in aqueous solutions with respect to the pH, concentra-tion, adsorbent dosage, contact time, eluent type and volume of the sample solution, flow rate of solution on the retention of the metal ions have been studied. Also, for investigation of exchange equilibria different amounts of dolomite and sepiolite contacted with a fixed volume and concentration of a heavy metal bearing solutions. Sorption data have been interpreted in terms of Langmuir and Freundlich equations.

The linear Langmuir and Freundlich models were applied to describe equilibrium isotherms and both models fitted well.[1-3]


[1] Yang G, Fena W, Lei C, Xiao W, Sun H. Study on solid phase extraction and graphite furnace atomic absorption spectrometry for the determination of nickel, silver, cobalt, copper, cadmium and lead with MCI GEL CHP 20Y as sorbent. J. Hazard. Mater. 2009; 162:44-49.

[2] Malekpour A, Hajialigol S, Taher MA. Study on solid-phase extraction and flame atomic absorption spectrometry for the selective determination of cadmium in water and plant samples with modified clinoptilolite. J. Hazard. Mater. 2009; 172: 229–233.

[3] Eren E, Tabak A, Eren B. Performance of magnesium oxide-coated bentonite in removal process of copper ions from aqueous solution. Desalination 2010; 257: 163–169.

123 New Sources for Ambient Desorption/Ionization Mass SpectrometryGary M. Hieftje, Indiana University, 800 East Kirkwood Ave., Bloomington, IN 47405, Jacob T. Shelley, Steven J. Ray, Kevin P. Pfeuffer, Gregory T. Schilling

Ambient desorption/ionization mass spectrometry burst upon the analytical scene roughly ten years ago in the form of two novel sources: desorption electrospray ionization (DESI) and direct analysis in real time (DART). Since then, more than 40 additional sources for the same field have been introduced. Of these sources, more than half have been based on atmospheric-pressure electrical discharges. In this presentation, the features of some of these sources are examined, with particular emphasis on the flowing atmospheric-pressure afterglow (FAPA) developed at Indi-ana University. The FAPA is an unusually simple source, operated in flowing helium and powered by a dc supply. There are two basic FAPA designs, one that uses a hole or capillary in the discharge chamber and the other that generates a toroidal (halo-shaped) discharge in the open air. Both have advantages, limitations, and optimal areas of application. These aspects are explored in detail and tales spun to show how they relate to the many achievements of Professor Joseph Caruso.

124 The Liquid Sampling-Atmospheric Pressure Glow Discharge (LS-APGD) Microplasma: An Example of What I Learned from Joe Caruso – It’s More Fun Living (a little) Outside of the BoxR. Kenneth Marcus, Clemson University, Department of Chemistry, Clemson, SC 29634

While the field of analytical atomic spectrometry has a long rich history, no one can say that it is a history of high evolution in terms of the base tools. Beyond the ven-erable inductively-coupled plasma (ICP), flame, and graphite furnace sources, the vast majority of advances have occurred on the sample introduction and analyzer components of the instruments. Sample introduction strategies in atomic spectrom-etry have centered on application-driven developments. For this, Joe Caruso looked outside of the box and put the power of the ICP to work in the solving of real-world problems dealing with enviro/bio/medicinal chemistries. It is this sort of pragma-tism-driven research, driving hardware developments, where Joe has left his mark and showed me a path through my career. He demonstrated that this approach is a valuable way to attract high quality students of highly diverse interests and talents (oh ya, and funding from diverse sources). In this presentation, I pay homage to Joe Caruso in describing the developments of the liquid sampling-atmospheric pressure glow discharge (LS-APGD) microplasma as a source for elemental, isotopic, and molecular species analysis by optical emission and mass spectrometry. While the Caruso group led the way in sample introduction for ICP- optical emission spec-trometry mass spectrometry, the LS-APGD had as its genesis the desire to have a chromatography-friendly source for those cases where the ICP was not needed. Our latest results in the development and characterization of this microplasma will point to new directions in its use, a little outside of the box.

125 Plasma Sources Pushing the Limits in Analytical ChemistryDavid W. Koppenaal, Pacific Northwest National Laboratory, EMSL, PO Box 999, Richland, WA 99352

Plasma sources continue to be the source of choice for most elemental analyses, approximately 50 years following their introduction to the community during the 1960’s. The use of plasma sources as ionization sources, although more recent (1980’s introduction), has revolutionized inorganic mass spectrometry. In this paper, the use of both standard and non-standard plasma sources for elemental/isotopic analyses is discussed, with emphasis on new approaches and techniques designed to make such sources even more powerful and adaptable to a wide range of scien-tific problems. In our work we are using both inductively coupled plasma (ICP) and glow-discharge (GD) plasma sources for environmental and biological applications. In the latter category, and in recognition of this year’s Field’s award winner, we are especially interested in the development of metallomic applications of these sources.

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126 Spectroelectrochemistry as a Strategy for Improving Sensor SelectivityWilliam R. Heineman, University of Cincinnati, Department of Chemistry, PO Box 210172, Cincinnati, OH 45221, Samuel A. Bryan

Spectroelectrochemistry improves selectivity for sensors by electrochemically changing the optical signal associated with the analyte and thereby distinguishing it from interfering species whose signal is constant. The sensor consists of an optical-ly transparent electrode (OTE) coated with a thin polymer film that preconcentrates the target analyte adjacent to the electrode for spectroelectrochemical detection. The OTE serves as an optical waveguide for attenuated total reflectance (ATR) spectroscopy, which detects the analyte that has partitioned into the polymer film by UV-vis absorption spectroscopy. Alternatively, ATR can provide the excitation light for fluorescence detection, which is generally more sensitive than absorption. The analyte partitions from the sample into the film where it is electrochemically reduced or oxidized at the OTE surface. The change in the spectral response associated with the change in oxidation state of the analyte is used for quantification. Absorp-tion sensors for metal ion complexes such as [Fe(CN)6]4- and [Ru(bpy)3]2+ and fluorescence sensors for [Ru(bpy)3]2+ and the polycyclic aromatic hydrocarbon 1-hydroxypyrene have been developed. The sensor concept has been extended to binding assays for a protein as demonstrated using avidin–biotin and 17β-estradiol–anti-estradiol antibodies. The sensor has been demonstrated to measure analytes in complex samples such as nuclear waste and natural water.

127 From Solution Chemistry to Mass Spectrometry – My Fields of AnalyticalJoseph A. Caruso, University of Cincinnati, PO 210172, Cincinnati, OH 45221

Following an MSU Ph.D. with Alex Popov and a postdoc at Texas Austin with Joe La-gowski, my first years at Cincinnati continued the solution physical/analytical chem-istry in non-aqueous solvents, but inevitably moved to atomic spectrometry. Our first AA studies were on lead in evaporated milk – much to the consternation of the “contented cow folks,” since the cans at that time were Pb soldered, which the milk clearly showed. With inspiration from a Velmer Fassel lecture, we moved modestly into a low-power microwave plasma, which required vapor generation for sample introduction. The Varian mini-atomizer fit the bill and was heavily utilized, leading us to a brief study with a PAR Vidicon system for multielement work; perhaps one of the earliest studies of analytical plasma emission with an array detector. As a means of sample vaporization at about the same time, we began hydride generation for sample introduction and found good results for As and Se, especially. With better power coupling methods, the TM010 cavity allowed us to use helium plasmas and the ultimate departure into ICPMS and many speciation studies, such as, metal species translocation in plants. Further, while the glow-discharge was primarily used for solids analysis, we showed that these plasmas could be used for vapor sample analysis. Ultimately, speciation studies were to move us to biological/clinical studies of metals, metal species and metalloproteins. With the help of Agilent Technologies, our work with proteomics became possible. This presentation tiptoes through my fields of analytical.

128 Regulatory Perspectives on Good Analytical PracticesLinda Ng, United States Food and Drug Administration, WO 51-4324, 10903 New Hampshire Blvd, Silver Springs, MD 20993

The selection of analytical procedures, method validation or verification, and in-strument qualification are critical backbones for reliable analytical data to support product quality in pharmaceuticals approved by the United States Food and Drug Administration (US FDA). The current regulatory issues on analytical procedures especially for the generic industry with the related requirements for method valida-tion and instrument qualification will be shared. Discussion may include Internation-al Conference on Harmonization (ICH) Q2, Q3, Q6 and Q7; USP <621>, <1225>, <1226>, <1010>, <1058>, <1086>; and related FDA guidances. The definition of terms, tools, responsibilities of the respective parties, and current practices for selecting scientifically appropriate analytical methods will be covered. In addition, recommendations on information that should be included in the chemistry, man-ufacturing, and controls portions of drug applications to optimize the efficiency of the review process, and data stored in the laboratory for the investigative team are shared. Discussions also include Field Alert Reports, 483 observations and warning letters attributed to the laboratories generating these data. These observations can be avoided by an understanding of CGMP and the Agency’s expectations.

129 Impact of New Stability Guidance on Generic Drug DevelopmentKim Huynh-Ba, Pharmalytik Consulting and Training, 32 Forest Ridge, Newark, DE 19711.

Stability is defined as a critical quality attribute to determine an expiry and storage condition for pharmaceutical products. The new abbreviated new drug application (ANDA) stability guideline was issued in September 2012 established the regulatory requirements for submitting an Abbreviated NDA based on International Conference on Harmonization (ICH) requirements; however, over the past couple of years, The

Office of Generic Drugs has received numerous questions related to this document. Therefore, an addendum was published to clarify many of these requirements. This session introduces the stability requirements and how it impacts the analytical de-velopment sector of generic industry.

130 Advances on the Modernization of USP MonographsLeonel Santos, United States Pharmacopeia, 12601 Twinbrook Parkway, Rockville, MD 20878, Clydewyn M. Anthony

The modernization of monographs continues to be a key initiative at United States Pharmacopeia (USP) after a year of formal implementation. The key benefit of the modernization scheme is the evaluation of the identity, strength, and purity of other genetic drugs. A summary of the past year’s accomplishments and specific exam-ples of the influence of monograph modernization of generic drugs is presented. Important issues and challenges affecting modernization of monographs as well as future plans to modernize drug product monographs based on the over-the-counter (OTC) family of monographs are presented and discussed.

131 Comparison of Dissolution Profiles Including Bioequivalence, Method Transfer/Validation, and f2Vivian Gray, V. A. Gray Consulting, 9 Yorkridge Trail, Hockessin, DE 19707

In dissolution, one of the most important tasks is the comparison of profiles. Minimiz-ing variability is paramount so that when comparing profiles significant differences can be observed. In method validation, the comparison of profiles is expected with intermediate precision, manual versus automated testing, and robustness determi-nations. The appropriate criteria for method validation will be discussed. Sources of variability and failures during method transfer will be explored. The comparison of dissolution profiles is also critical to determine bioequivalence, as in the case of f2. The improper applications of the f2 criteria, some ideas to explain failures, and possible remedies for these failures are presented.

132 3-D Digital Microscopy and Cultural Heritage: A Successful MarriageIlaria Cacciari, IFAC-CNR, Via Madonna del Piano 10, Sesto Fiorentino, 50019 Italy, Salvatore Siano

The need for observing the microscopic details on artwork surface often occurs during the course of the same restoration work. Standard optical systems such as eyepieces, videomicroscopes or stereomicroscopes represent powerful aids for the restorer during the examination of the preservation status, the decision of the treat-ment to be applied and interpretation of the peculiarity of the decoration process. In recent years there has been an increasing use of digital videomicroscopes due to their low cost and high convenience, but where the observation of the depth is an important element in the assessment, they cannot fully replace the stereomi-croscope. The “feeling” of three-dimensionality provided by the latter can allow to decode textural aspects of interest, but in turn is limited if quantitative data on the depth of the surface is needed. In this case, a versatile solution is represented by the three-dimensional (3-D) digital microscopes. Since 2007 we are directly involved in the development of devices for 3-D reconstruction of surfaces, and now we im-prove, promote and accelerate the technology transfer to increase the access to this technology. Here, after an introduction on the main features of the 3-D digital micro-scopes we have developed, a review of several case studies is presented. These are representative of microrelief problems which can arise in practice in cultural her-itage domain. In particular we report the micromorphological studies of the punch-works on medieval painting, the granulation motives decorating jewels, the minting toolmarks on Roman coins, and the test of laser cleaning on marble artifacts.

133 Pairing 3-D Laser Scanning Confocal Microscopy with Reflectance Transformation Imaging to Gain Insight into the Techniques Used to Create Relief and Contour Lines on Greek Red-Figure VasesPhilip A. Klausmeyer, Worcester Art Museum, 55 Salisbury St., Worcester, MA 01609, Paula Artal-Isbrand

This presentation offers an overview of research published in Studies in Conser-vation (Oct 2013, vol. 58:4 pp 338-359) in which the relief and contour lines on a group of ancient Greek red-figure vases and vase fragments in the collection of the Harvard Art Museums and the Worcester Art Museum were examined using two surface examination methods – reflectance transformation imaging and three-di-mensional (3-D) laser scanning confocal microscopy. These methods helped re-solve longstanding debate among art historians, archaeologists, and scholars in the field, regarding the tools, techniques, and production sequence used by Greek vase painters to decorate vessels. This research also incorporated fabricated mock-ups to gain a better understanding of the ancient technology and ultimately established that two distinct types of relief lines exist on these ancient vases: the ‘laid line’ and the ‘pulled line’. This study also determined the sequence of application between re-lief and contour lines. Based on observations made during this research, the authors propose a likely evolution for the techniques used to produce decorative features throughout the period of red-figure vase production. The role of 3D laser scanning

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confocal microscopy in this research is presented along with a brief introduction to how the technique differs from better-known types of confocal microscopy.

134 Optical Coherence Tomography for the Non-Invasive 3-D Volume Imaging of Cultural HeritageHaida Liang, Nottingham Trent University, School of Science & Technology, Nottingham, NG11 8NS United Kingdom

Optical coherence tomography (OCT) is a non-invasive, non-contact imaging technique based on a fast scanning Michelson interferometer capable of imaging surface and subsurface structure in three-dimensional (3-D). In the last 10 years, optical coherence tomography has been successfully applied to art conservation, art history and archaeology. OCT has the potential to become a routine non-invasive tool in museums allowing cross-section imaging anywhere on an intact object where there are no other methods of obtaining subsurface information. While current OCTs have shown potential in this field, they are optimized for biomedical applications. Some major limitations are: 1) lower depth resolution compared to conventional microscopic examination of sample cross-sections; 2) limited probing depth through highly scattering material such as paint. Increasing the depth resolution involves broad band laser sources and increasing the probing depth needs sources at longer wavelength than conventionally used in biomedical imaging. To achieve this, we have recently built two new OCT systems so that when used in conjunction, they would aim to match the information from conventional invasive microscopic exam-ination of sample cross-sections: 1) a long wavelength (2000nm) OCT optimized for deeper penetration suitable for imaging cultural heritage objects consisting of highly scattering materials; 2) an ultra-high depth resolution OCT at a central wavelength of 800nm (axial resolution ~1.2 microns) targeted towards applications where the highest resolution is required but the material is relatively transparent, such as im-aging of multiple thin varnish layers on painting surfaces and gel layers on degraded glass.

135 Pump-Probe Microscopy Enables Noninvasive, Three-Dimensional Imaging of ArtworkWarren S. Warren, Duke University, Department of Chemistry, Durham, NC 27708, Tana Villafana, Martin Fischer

The layering structure of a painting contains a wealth of information about the artist’s choice of materials and working methods, but currently, no 3-D noninvasive method exists to replace the taking of small paint samples in the study of the stratigraphy. Here, we adapt femtosecond pump-probe imaging, previously shown in tissue, to the case of the color palette in paintings, where chromophores have much greater variety. We show that combining the contrasts of multispectral and multidelay pump-probe spectroscopy permits nondestructive 3-D imaging of paintings with molecular and structural contrast, even for pigments with linear absorption spectra that are broad and relatively featureless. We show virtual crosssectioning capabilities in mockup paintings, with pigment separation and nondestructive imaging on an intact 14th century painting (The Crucifixion by Puccio Capanna)[1]. Our approach makes it possible to extract microscopic information for a broad range of applications to cultural heritage. Additional applications to paper, pottery, and photodegradation analysis are presented.


[1] T. E. Villafana, W. P. Brown, J. K Delaney, M. Palmer, W. S. Warren, and M. C. Fischer, Femtosecond Pump-Probe Microscopy Generates Virtual Cross-Sec-tions in Historic Artwork, Proc Natl Acad Sci U S A. 2014 Feb 4;111(5):1708-13. doi: 10.1073/pnas.1317230111

136 Diffuse Reflections on Chemical ImagingNeil Lewis, Malvern Instruments, 7221 Lee Deforest Dr., Columbia, MD 21046

The maturation of chemical (spectroscopic) imaging as a valuable problem solving tool in the life sciences, pharmaceutical and polymer industries is well document-ed. In particular near-infrared (NIR) chemical imaging has proven to be one of the most rugged and versatile implementation when industrial, routine or even process analytical measurements are desired. This is due in part to the fundamental robust measurement characteristics that it inherits from conventional or single-point NIR spectroscopy. Commercial near-infrared imaging platforms are particularly flexible with respect to the range of sample sizes that can be evaluated, addressing micro-scopic and remote sensing (satellite) applications and are in some cases capable of generating chemical images in real-time. This flexibility enables NIR chemical imaging to interrogate the characteristics of many different types of spatially and chemically complex manufactured or naturally occurring materials delivering in-formation not readily available using more traditional analytical methods such as high-performance liquid chromatography (HPLC) or ‘point average’ spectroscopic measurements. That is, information about not only what or how much of a partic-ular component or chemical species is in a sample, but where it is, and how it is distributed. This insight into the structure/function relationship of complex materials ultimately enables an understanding of the factors that can influence, or be used to

modify, the functional properties that drive the quality or performance of numerous industrial products. In this presentation I reflect on a variety of developments, appli-cations, perspectives and insights that describes not just how we measure chemical images but just as importantly why.

137 Historical Development of Mid-Infrared SpectroscopyJohn Reffner, John Jay College, Sciences Department, 524 West 59th St., New York, NY 10019

When William Weber Coblentz began his graduate studies at Cornell University in 1900, infrared spectroscopy was in a primitive state. He built and calibrated his own infrared spectrometer. Collecting infrared spectra with his spectrometer was te-dious. Manual alignment of the spectrometer required skill. Tabulating wavelengths and recording the radiometer’s response was laborious. Coblentz plotted all his infrared spectra point by point. By 1905, he had collected several hundred spectra. His pioneering research was published by the Carnegie Institution of Washington D.C. in a book titled: Investigations of Infrared Spectra. His 40-year career at the National Bureau of Standards (now NIST) is the cornerstone for the science of infra-red spectroscopy and radiometry. Until the late 1930’s, infrared spectroscopy was an academic curiosity. Analytical chemists took little note of its utility. As World War II approached, the US government recognized its need to develop synthetic rubber. On June 28, 1940, President Franklin D. Roosevelt designated rubber as a strategic and critical material. Scientists from academia and industry joined forces to produce synthetic rubber. Mid-infrared analysis of polymers and the raw materials needed to make synthetic rubber was a pivotal part of this program. After the war, the sale of commercial infrared spectrometers soared. By the mid-1960’s, the market matured and other instrumental methods became serious competition. Beginning in the late 70’s, the development of Fourier transform IR spectrometers resurrected infrared spectroscopy. The higher through-put, noise reduction, wavelength stability and computer integration revived infrared analysis.

138 Historical Development of Raman Imaging - the Impact of the Instrumentational Developments on the Quality of the ResultsFran Adar, Horiba Scientific, 3880 Park Ave., Edison, NJ 08820

After calculations showed that the loss of signal produced by small sample volumes would be more than compensated by highly effective sampling optics, the Raman microscope was conceptualized as a molecular analogue of the electron micro-scope, and then prototyped and commercialized, in northern France in the early 1980s. Originally the microscope was mounted on a scanning double monochro-mator which detected one wavelength at a time. As the instrumentation evolved, it became faster, more sensitive and less subject to fluorescence interference. Con-sequently many new materials could be studied, and the number of successful ap-plications exploded. Each new success promoted new instrumentation innovations. In addition, the software became an integral part of the instrumentation, simplify-ing data acquisition, as well as data treatment and interpretation. This talk reviews these developments, indicating the synergism between the evolution of the instru-mentation and software, with the expansion of the applications. In particular, with the maturation of the technology, the implementation of Raman imaging which was key in the original concept of the early 1970’s, is now a practical reality.

139 Terahertz Spectroscopy: Past, Present and a Very Bright FutureRichard A. McKay, Advantest America, 508 Carnegie Center, Suite 102, Princeton, NJ 08540

The terahertz (THz) spectrometer has, only recently, entered the realm of robust and user-friendly laboratory tools. This mode of vibrational spectroscopy extends the reach of the spectroscopist further down the electromagnetic frequency spec-trum and is becoming an important complement to laboratory mainstays such as near-infrared and Raman spectroscopy. There has been a lag in implementation of this spectroscopic method due to the so-called ‘terahertz gap’, a technological limitation in our ability to reliably and efficiently generate and detect THz waves. Recent improvements in this field have made this technology more accessible. This talk explores the history of terahertz spectroscopy, its current status as a growing field, and it’s very promising future.

140 Paper on Glass: A Practical Guide to the Implementation of an Electronic Lab NotebookJames Exarchakis, Colgate-Palmolive Company, 909 River Rd., Piscataway, NJ 08855

Electronic laboratory notebooks (eLN) have proliferated throughout modern labo-ratories. This presentation describes the implementation of eLN within a multi-dis-ciplinary analytical department. The transition from traditional notebooks to an electronic system was facilitated by a paper on glass philosophy. Well trained power-users helped define the eLN processes in terms that the end-users already practiced eliminating the need to change behavior and practices of record keeping. An emphasis on end-user return on investment of their time and on consistency in formatting contributed to a speedy adoption. Additional experiences with instrument integration, internal audits, and the relationships the IT department are discussed.

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141 Implementing a Process Intelligence System for Pharmaceutical DevelopmentEdward Keefe, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903, Megerle Scherholz

For drug formulation understanding the process is paramount. For most formulators retrieving all the data necessary to do any type of analysis is often a laborious en-deavor. The data, which includes process parameters, analytical results and batch genealogy, is often dispersed across different electronic systems, exists in paper records, and resides with multiple individuals. Once the data collection is complete the formulator faces another challenge. Since data capture is not standardized the data must be normalized before any analysis can be conducted. In this type of environment where process intelligence can have a beneficial impact on the drug development process the knowledge must be extracted in a reasonable time frame. This presentation discusses the implementation of a process intelligence system along with the benefits, challenges, and lessons learned in two distinct areas of pharmaceutical development: oral solid dosage formulation and parenteral formu-lation. It focuses primarily on three tools: the electronic lab notebook (Symyx ELN), lab information management system (LabWare LIMS) and process intelligence soft-ware (Discoverant). It also presents actual examples showing some of the benefits that can be derived from a process intelligence system.

142 The Role of the BIOVIA (Accelrys) Laboratory Execution System (LES) and Inventory Management System (IM) in a CRO cGMP EnvironmentKathy Shanks, PPD, 8551 Research Way, Suite 90, Middleton, WI 53562

To improve data quality and security, gain efficiencies, decrease review and turn-around time, and to eliminate manual data entry into analytical reports, the PPD LLC cGMP Laboratory implemented a laboratory execution system. In addition, the laboratory deployed a system for the control and maintenance of consumables. A synopsis of the implementation of the BIOVIA (Accelrys) LES in conjunction with Inventory Manager is discussed including lessons learned, best practices, benefits derived, and the plan going forward.

143 Electronic Orchestration of Knowledge Creation and ManagementJoel Young, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903, Ronald Behling, Larry Fulton

Due to the disconnected and un-integrated nature of the various operational soft-ware solutions across the pharmaceutical development workspace (e.g., chemical development, formulation development, analytical), it is extremely challenging to manage, aggregate and connect the diverse, but related, knowledge inputs lead-ing to tremendous inefficiency and rework. The ability to transform these collective data inputs to knowledge is limited, sporadic, and inconsistent across platforms and applications. A solution to orchestrate the creation of pharmaceutical development knowledge (with a focus on analytical development) is discussed.

144 Incorporating Research Method Development into the Analytical LabKimberley Frederick, Skidmore College, 815 N Broadway, Department of Chemistry, Saratoga Springs, NY 12866, Maryuri Roca, Lisa Quimby

Many types of chemical research involve the process of analytical method develop-ment making it the most transferrable part of an analytical laboratory course. Unfor-tunately, traditional labs in analytical chemistry focus on giving students a broad sur-vey of wet and instrumental techniques. We have chosen to replace the second half of the lab experience with a research-based method development project. Students work in teams with a faculty member or community member to develop, optimize and validate an analytical method that supports a research project. Teams have de-veloped preparative separation methods for an organic research lab, optimized and validated detection methods for a geosciences lab group and devised and optimized a detection reaction for a microfluidic assay. Because the results are of interest to the research group or community member that the students are partnering with, stu-dents are more engaged, willing to work many additional hours and more invested in the quality of their results. Students have been shown to be able to transfer the skills learned in this course to new situations as well. As a bonus, their results have also made important contributions to research projects and they have been listed as co-authors on papers resulting from their project.

145 Integrating Teaching and Research: Strategies and Practices for Building a Research-Supportive CurriculumRoger S. Rowlett, Colgate University, Department of Chemistry, 13 Oak Dr., Hamilton, NY 13346

Undergraduate research is a high-impact educational practice that should be a high priority for incorporation into science curricula. Developing a research-rich, research supportive curriculum is an excellent way to accomplish this goal. This presentation addresses the challenges of transitioning from a traditional content-based curricu-

lum to a research-rich, research supportive curriculum, and provide some practical advice and examples of how to do this.

146 Integration of Research into the Curriculum Using XRD and NMR Structural AnalysisBenny Chan, The College of New Jersey, PO Box 7718, Ewing, NJ 08628, Abby R. O’Connor

The College of New Jersey has recently received two National Science Foundation - Major Research Instrumentation grants to perform structural studies using single crystal X-Ray diffraction (XRD) and nuclear magnetic resonance (NMR) spectros-copy. The inorganic faculty has integrated instruments into our curriculum through research projects in our courses required of chemistry majors. Students have de-veloped organometallic projects and studied the metallation of a variety of potential ligands. Students either purchase the ligands or perform ligand design synthesis strategies. Ligands chosen are studied through NMR and XRD. The metallation studies are first performed via NMR and then crystals of the complex are studied. The two faculties are particularly interested in these projects as they are novel re-search area and are soon entering the publication stage after only two cycles of our courses.

147 Involving Undergraduate Students in Collaborative Research: The Union College Aerogel TeamMary K. Carroll, Union College, Department of Chemistry, 807 Union St., Schenectady, NY 12308

The Aerogel Team is a cross-disciplinary research group established at Union Col-lege by the presenter, an analytical chemist, and Prof. Ann Anderson, a mechanical engineer. Founded in 1795, Union College was the first college to be chartered in New York State, to create a bachelor’s degree in science and mathematics (1822), and to establish an engineering degree program within the context of the liberal arts (1845). An analytical chemistry laboratory course was first offered at Union in 1857. Since 2001-02, more than 90 undergraduate students have participated in aerogel research at Union, working alongside faculty mentors, using state-of-the-art instrumentation, presenting and, in some cases, publishing their results. The Union College Aerogel Team has received considerable internal and external (ACS PRF, NSF MRI, NSF RUI, NSF I-Corps) support. Undergraduate students are involved in all aspects of the research: they learn about the statistical design of experiments, employ cutting edge instrumentation and data acquisition systems, perform exper-iments and reduce and interpret the results. In addition to fundamental studies of aerogel materials, the team investigates applications of aerogels in areas as diverse as chemical sensing, catalysis, drag reduction and drug delivery. Being part of a cross-disciplinary team provides students with a valuable opportunity to learn how to work beyond the confines of their own core disciplines. This approach engages students, excites them to construct their own investigations, gives them practical experience, and prepares them well for postgraduate study and careers in science, technology, engineering, and mathematics fields.

148 Structure/Function Studies of Obscurin Ig58/59Nathan T. Wright, James Madison University, 901 Carrier Dr., Harrisonburg, VA 22807

The giant protein obscurin (~800-900 kDa) plays a role in cytoskeletal organiza-tion and myofibrillogenesis. A mutation in the 58th Ig domain of this highly modular protein causes hypertrophic cardiomyopathy in humans, possibly through ablation of obscurin binding to its molecular target titin. Here, we analyze this obscurin-titin interaction in more detail. We present the high resolution structure of Ig58, Ig59, and a model of Ig58/59 together. While both obscurin domains are necessary to bind to titin, the domains do not interact extensively with each other, and in fact exist in a semi-extended conformation. Further analysis shows that the disease-causing mu-tation is on the surface of the Ig58 domain, and disrupts an extensive electrostatic surface of the molecule.

149 Liquid State, High-Resolution 14N-NMR of Symmetrically Substituted Nitrogen-Containing CompoundsBenjamin Liu, Princeton University, 4589 Frist Center, Princeton, NJ 08540, Istvan Pelczer

Nuclear magnetic resonance spectroscopy (NMR) is one of the most powerful tools available to chemists today. Conventional high-resolution liquid state NMR exper-iments, involving 1H, 13C, 19F, 31P and 15N routinely are used for structure determi-nation, kinetic studies and quantitative analysis. However, one nucleus that has not been studied in much detail is 14N, the primary isotope of nitrogen (99.7% natural abundance). Nitrogen-14 is a nucleus has a nuclear spin of 1, meaning it has a quadrupole moment. This quadrupole moment makes studying the nucleus more difficult as severe line broadening often is observed. However, if the nitrogen atom has symmetric substitutions, this can effectively collapse the quadrupole moment and the peak narrows. Conventionally, the substances that have been looked at have mostly been tetrahedral ammonium salts that can completely collapse this

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quadrupole moment. However, through an assay of common functional groups con-taining nitrogen we have identified a surprisingly large family of compounds suit-able for high-resolution 14N–NMR. We have found that nitrogen atoms of both linear and planar symmetry can also be visible in the NMR spectra with little broadening, greatly expanding the substances available for this type of spectroscopy. Since this nucleus is highly selective for molecules with appropriate symmetry, 14N-NMR po-tentially has a great number of applications. Complex or biological mixtures can easily be analyzed with no background, since only the symmetrically substituted nitrogen-containing compounds will appear in these spectra. We are in the process screening a large variety of nitrogen-containing substances to exploit this opportu-nity.

150 Noncanonical Polyubiquitin Chains: Insights from Structural and Dynamical Studies, Carlos Castañeda, Syracuse University, Chemistry Department, Syracuse, NY 13244, Susan Krueger, Ashton Cropp, David Fushman

Polyubiquitination, an important post-translational modification of proteins, signals for a wide variety of cellular events including proteasomal degradation, DNA repair, cell cycle regulation, etc. The molecular basis of the diversity of polyubiquitin signal-ing lies in the different structural and dynamical properties of polyubiquitin chains, imparted by the different possible covalent linkages between the C-terminus of one ubiquitin (Ub) and the ε-NH2 of a lysine side chain (K6, K11, K27, K29, K33, K48 or K63) or α-NH2 of M1 on a second Ub. While the canonical K48-linked and K63-linked chains have been well characterized, little is known about the structural and functional properties of so-called noncanonical polyUb chains (K6, K11, K27, K29, K33) largely due to a lack of linkage-specific Ub-conjugating enzymes for these polyUb chains. Recent studies suggest these chains are implicated in many non-proteolytic functions, including innate immune system activation and mRNA stability. To study these chains biochemically and biophysically, we devised a chemical as-sembly strategy to make polyubiquitin chains composed of every lysine linkage, of specific length, and with selective isotopic labeling. Solution nuclear magnetic res-onance measurements, including chemical shift perturbations, 15N relaxation mea-surements and residual dipolar couplings, in conjunction with small angle neutron scattering (SANS) measurements, have enabled us to describe the structural and dynamical properties of free polyubiquitin chains of every lysine linkage for the first time. These studies provide a foundation for future work in elucidating the molecular recognition properties of these chains.

151 Using Complexity to Make Separations SimplerThomas L. Chester, University of Cincinnati, Department of Chemistry, PO Box 210172, Cincinnati, OH 45221

Chromatography is becoming more complex as the ranges of parameters are pushed beyond conventional boundaries. This is not to be feared because increas-ing complexity provides new opportunities for creating rapid separations that more fully satisfy the underlying analysis needs of resolution and speed. However, knowl-edge and expertise are required to get the most benefit. Short courses and on-line training resources have rarely met the needs of developing frontier-pushing knowl-edge in the workplace. The next level of complexity in supercritical fluid chromatog-raphy (SFC) is to link reversed-phase liquid chromatography, hydrophilic interaction liquid chromatography, and SFC into a seamless, unified behavior. This seemingly impossible task is theoretically possible and has been demonstrated piecewise. The key to accomplishing continuity is to develop understanding of phase behavior and the formation and role of excesses of mobile phase components on stationary phase surfaces. The ability to control surface excesses and to predict how solute re-tention in SFC is affected by parameter changes is a largely unexplored opportunity that will lead to easily adjustable selectivity among dissimilar solutes. This flexibility, combined with accurate models to quickly sort through complexity, will provide the simplest and fastest separations within the allowed ranges of parameter values.

152 Liquid Chromatographic Estimates of Octanol - Water Partitioning: Is there a Rational Stationary Phase Choice?John G. Dorsey, Florida State University, Department of Chemistry, Tallahassee, FL 32306

There is a 40 year history of modern liquid chromatography being used for estimat-ing octanol – water partition coefficients and hydrophobicity. Even with the many hundreds of papers that have appeared, there is not a consensus of how best to make these measurements. Mobile phase composition and additives have been extensively studied, approaches to measure or estimate k’w, the retention factor in 100% water are many, but there are very few studies of the effect of the choice of stationary phase on these estimates. Snyder developed the hydrophobic subtrac-tion model for characterizing reversed phase stationary phases, and this has proven a useful tool in picking alternative columns for methods development. We are now asking if this model might provide a rational approach for choosing an appropriate stationary phase for octanol – water measurements. We are investigating a diverse set of compounds of varying structure, size and functional group on a series of

columns representing the range of possibilities from the column database. We com-pare results from different columns to assess the feasibility of this approach.

153 Ultrahigh Performance Supercritical Fluid Chromatography of Lipophilic Compounds with Emphasis on Synthetic and Commercial BiodieselLarry T. Taylor, Virginia Tech, Department of Chemistry, 117 Surge Building, Blacksburg, VA 24061, Mehdi Ashraf-Khorassani, Kenneth J. Fountain, Giorgis Isaac

Ultra high-performance supercritical fluid chromatography (UHPSFC) in combina-tion with sub-2 micron particles and either diode array UV, evaporative light scatter-ing or mass spectrometric detection has been shown to be a valuable technique for the determination of acylglycerol composition in soybean, corn, sesame, and tobac-co seed oils. Excellent resolution on an unendcapped single C18 column (3.0 x 150 mm) with a mobile phase gradient of acetonitrile and carbon dioxide in as little as 10 minutes served greatly as an improvement on first generation SFC instrumentation. Separation of residual tri-, di-, mono-acylglycerols, glycerol itself, fatty acid ethyl es-ters, and free fatty acids at 0.2% (w/w) was easily obtained. The methodology was applied to an in house synthetic biodiesel (i.e., fatty acid ethyl esters) derived from tobacco seed oil both before and after purification by column chromatography on bare silica. The established UHPSFC mass spectrometry (MS) approach has poten-tial application in lipidomics and food testing as a complementary method alongside high-performance liquid chromatography-MS and gas chromatography-MS, as the former can separate both polar and nonpolar lipids to improve both detection limits and peak shape.

154 Thinking Outside the Valve: Pressure Control and Other Important Questions in Supercritical Fluid Chromatography-Mass Spectrometry (SFC-MS)J. David Pinkston, Kellogg Company, WK Kellogg Institute, 2 East Hamblin Ave., Battle Creek, MI 49017

SFC bears many similarities to high-performance liquid chromatography (HPLC), but there are important differences. One of these is the importance of controlling mobile phase pressure at the column outlet to maintain a single chromatographic mobile phase and to control the strength of the mobile phase. This presentation describes novel approaches for pressure control and interfacing in SFC-MS which eliminate some of the disadvantages of a traditional mechanical valve and of flow splitting. I also highlight differences with a comparison of the applicability of SFC-MS to that of LC-MS for a wide range of pharmaceutical compounds. Finally, nature and function of mobile phase additives in HPLC and SFC can be quite different. For example, HPLC has traditionally been the only technique routinely used for peptide and protein separations, but I show that large, hydrophilic peptides can be eluted in SFC-MS when the proper mobile phase additives are used. These additives are not usually needed in HPLC.

155 Spherical Nanoparticle Supported Lipid Bilayers for the Study of Membrane ProteinsFang Tian, Pennsylvania State University, Biochemistry and Molecular Biology, Penn State Medical School, Hershey, PA 17033, Richard L. Gill Jr.

We are developing spherical nanoparticle supported lipid bilayers (SSLBs) to mod-el various degrees of membrane curvature and study membrane proteins. SSLBs were first introduced in 1990, and preserve many characteristics of cell membranes such as lateral fluidity, impermeability to ionic species, and flexibility along the fatty acyl chain. SSLBs are easy to prepare with high reproducibility and stability. More-over, unlike the commonly used liposomes SSLBs can be homogeneously prepared with well-defined sizes. In our preliminary study, we found that spectra from SSLBs and liposomes are nearly identical, validating the use of SSLBs for the structural study by solid-state nuclear magnetic resonance. Furthermore, spectra of SSLB samples display higher resolution than those of liposome preparations.

156 Probing Dynamic Protein-Glycosaminoglycan Interactions Using Paramagnetic LigandsXu Wang, Arizona State University, Physical Sciences, Bldg. D 102, Tempe, AZ 85287

Glycosaminoglycans (GAG) are large, sulfated, linear polysaccharides ubiquitous to all mammalian cells. Their polyanionic nature sets them apart from other glycans, and allows GAGs to act as receptors for many classes of signaling molecules. How-ever, studying GAGs’ interactions with proteins has been challenging for nuclear magnetic resonance (NMR) because non-labile protons needed by intermolecular Nuclear Overhauser Enhancement Spectroscopy (NOESY) experiments are rarely found at the intermolecular interfaces of these complexes. To improve the sensitivity of detection, we have developed methods to functionalize GAG ligands specifically with paramagnetic tags. Investigations of several GAG-binding proteins showed that not only can the ligands indicate the location of the GAG-binding epitopes, but they can also reveal orientations of the bound ligands on the protein, and, because of the

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sensitivity of NMR signals to paramagnetic relaxation enhancement effects, uncover existence of binding conformation heterogeneity as well as secondary binding sites. The application of this technology to the investigation of GAGs’ interactions with the pro-inflammatory chemokine CCL5/RANTES is discussed in detail. In particular, we were able to confirm that CCL5 possessed more than one major GAG binding epitope, and its interactions with GAGs are exquisitely sensitive to the sulfation patterns of the GAG ligands. Use of these ligands in the investigations of other GAG-binding systems is also discussed.

157 NMR Studies of Bacterial Masonry that Provide Protective BarriersCharles V. Rice, University of Oklahoma, Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, Norman, OK 73019

Although a fundamental aspect of bacterial physiology; little is known about the or-ganization of peptidoglycan and teichoic acid in the cell wall of Gram positive bacte-ria. Both components influence lysozyme function, repulsion of antibiotics, and met-al binding. In bacterial spores, the coat proteins and peptidoglycan cortex provide a means of survival against conditions that normally kill vegetative bacteria. Peptido-glycan is a network of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) groups modified with a peptide. The peptides form crosslinks between glycan chains, creating the cell wall framework. Interlaced within the peptidoglycan are the teichoic acid molecules, phosphodiester polymers decorated with D-Alanine and NAG. With nuclear magnetic resonance (NMR) spectroscopy, we are able to identify how teichoic acid interacts with the peptidoglycan. Studies of metal absorp-tion reveal that strong binding helps metals stabilize the peptidoglycan structure. However, teichoic acid has a weaker interaction with metals, allowing diffusion into the bacterial cytoplasm. Peptidoglycan also forms a protective shield around bacte-rial spores and additional NMR studies reveal that this shield affects the penetration of sterile water into the bacterial spore and may explain resistance to oxidative at-tack by hydrogen peroxide.

158 Challenges and Triumphs in Glycoprotein NMRMegan Macnaughtan, Louisiana State University, 437 Choppin Hall, Baton Rouge, LA 70803

Glycosylation is one of the most common post-translational modifications of human proteins, but its effect on the proteins’ biophysical properties is poorly understood. Almost all human proteins are modified with a form of glycosylation. Small O-linked glycans, like elongated O-fucose and intracellular O-GlcNAc, have been shown to directly regulate enzyme function, but the molecular mechanism is unclear. Nuclear magnetic resonance (NMR) spectroscopy is recognized tool to study proteins, but it is also an effective method to study glycans. One limitation of studying glycopro-teins with NMR is the production of isotopically labeled sample. Some proteins can be modified in-vitro with glycosyltransferases, after metabolic labeling in E. coli, to produce isotopically labeled glycoprotein. Notch1 epidermal growth factor-like (EGF) repeats can be modified with O-fucose, and even elongated to the tetra-saccharide form, with glycosyltransferases in-vitro. Wh ile some EGF repeats do not show structural changes upon O-fucosylation, EGF27 of Notch1, a key play-er in the activation of Notch signaling, shows significant changes in structure. A second strategy to produce glycoproteins for NMR analysis is to co-express the glycosyltransferase with the substrate protein in E. coli. This method provides the advantage of isotopically labeling the protein and the glycans. We have strived to produce a co-expression system to produce O-GlcNAc-modified proteins using hu-man O-GlcNAc transferase. With the discovery that E. coli NagZ sabotages the in-vivo production of O-GlcNAc-modified proteins, we have engineered a functional, co-expression system and produced O-GlcNAc-modified human cAMP responsive element-binding protein (CREB1) and human Abelson tyrosine-kinase 2 (ABL2).

159 Solid-State Characterization Techniques for Inhalation Product DevelopmentMatthew Lamm, Merck, 556 Morris Ave., Summit, NJ 07901

In this presentation, solid-state characterization methods specific for inhaled drug product active pharmaceutical ingredient phase screening and selection as well as formulation development are presented. Case studies and retrospectives are used to highlight the importance of specific phase attributes with respect to millability, par-ticle size stability in the presence of moisture, and phase stability during drug prod-uct processing and storage. The utility of solid-state characterization techniques such as X-ray diffraction, solid-state nuclear magnetic resonance, Fourier trans-form-Raman spectroscopy, electron microscopy, thermal analysis, laser diffraction and dynamic vapor sorption is discussed.

160 Real-Time Inhalation Analytics Using Single Particle Aerosol Mass Spectrometry (SPAMS)David P. Fergenson, Livermore Instruments, 2038 Livingston St., Suite B, Oakland, CA 94606, Maria Balaxi, David Kohler, Anna Susz, Bradley D. Morrical

Single particle aerosol mass spectrometry (SPAMS) is a general method of ana-

lyzing individual aerosols in real-time that we have applied to the analysis of in-haled pharmaceuticals. A SPAMS draws individual particles directly from the aero-sol phase at atmospheric pressure and directs them across the paths of two laser beams. The first is a continuous wave laser with a square profile and duration of a particle’s light scattering as it crosses it is related to the aerodynamic diameter of the particle. As the particle passes out of the square laser and into the path of the second laser, that second laser is pulsed, ionizing material from the particle. The ionization event takes place at the center of the source region of a dual polarity time-of-flight mass spectrometer and positive and negative ion mass spectra are acquired from the particle. In this manner, the aerodynamic diameter and chemical composition are determined for each individual particle that the SPAMS analyzes. Up to 250 particles per second ranging from 0.1 to more than 8-µm in aerodynamic diameter may be analyzed. Results from three series of experiments on inhalational pharmaceuticals are reported. In the first, different formulations of individual and combination commercial products were analyzed and the internal versus external mixing states determined at the single particle level. In another, analytical consis-tency of the SPAMS was demonstrated over weeks in measuring a commercial for-mulation of indacaterol. A third series of experiments demonstrated the quantitation of nebulized ciprofloxacin.

161 Using Particle and Polymer Properties to Tailor Aerosol FormulationsChristopher Kuehl, University of Kansas, 2030 Becker Dr., Lawrence, KS 66047, Nashwa El-Gendy, Laird Forrest, Cory Berkland

The prevalence of asthma has increased steadily in the United States from 20 mil-lion to 30 million people over the past decade and is associated with over $56 billion dollars in healthcare costs annually. Asthma is expected to increase worldwide from 300 million to 400 million patients by 2025. To help treat asthma and its growing prevalence, there has been a push for longer duration treatments by utilizing chem-ical approaches: by chemically modifying therapies such as beta-2 agonists or by using combined therapeutics such as beta-2 agonists and corticosteroids to ex-tend duration from 6 to 24 hours recently. This work looks to increase duration and efficacy by employing a physical approach compared to a chemical method. The first approach uses agglomerated nanoparticles called NanoClusters to improve deposition into the peripheral lung and enhance dissolution of corticosteroids. The other technique is to employ a polymer, hyaluronic acid (HA), as a delivery vehi-cle specifically for biomolecules. By choosing the appropriate molecular weight of HA, different body compartments can potentially be selectively accessed from the delivery vehicle into the lungs. The drainage kinetics and distribution from delivery to the lungs (based on HA molecular weight) can enhance the persistence in the lungs, thereby increasing the therapeutic efficacy of the therapeutic. By utilizing physical as opposed to purely chemical methods, better aerosol formulations can be achieved with enhanced duration for inflammatory respiratory diseases.

162 Using Spray Pattern Area as a Surrogate for SAC in Optimizing pMDI Test MethodsZachary Pitluk, Proveris Scientific, 290 Donald Lynch Blvd., Ste. 100, Marlborough, MA 01752, Dino Farina

No abstract submitted by the author.

163 Near-Real Time Detection of Counterfeit PharmaceuticalsWilliam E. Mayo, Veracity Networks Inc., 745 North Dr., Melbourne, FL 32934

X-ray diffraction (XRD) is an accepted characterization method (e.g., United States Pharmacopeia <941>) that can be used to detect counterfeit pharmaceuticals. However, the method is limited to laboratory environments, is relatively slow (15 – 60 min) and is a destructive ex-situ technique. Hence, it can only be used as a screening tool that is unsuitable for field use or 100% floor inspection. To overcome these obstacles, a novel technique has been developed based on energy disper-sive X-ray diffraction (ED-XRD), which can detect sub potency, contamination and counterfeit formulations with high speed (<1 min), high detection rate (>99%) and low false alarm rate (<1%). The instrument is a table top unit with low power and no external cooling requirements. Moreover, nondestructive testing is performed in-situ and can inspect pharmaceuticals inside bottles and blister packs, with a maximum throughput of 250,000 bottles per year per machine. The ED-XRD method relies on scattering of incident X-ray beams by the crystalline and amorphous components of the tablet/capsule, which produces a molecular fingerprint that is unique to that ma-terial. A test sample is then determined to be legitimate or counterfeit by comparing its diffraction pattern against the reference standard in the material database. High speed is achieved by use of an energy dispersive detector that collects the entire diffraction pattern simultaneously, which eliminates the sample movements required in conventional XRD analyses. The system can be used by virtually anyone and minimally adds to the wholesale average cost (WAC), thus providing an incentive to screen non-salable returns and overstock acquisitions.

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164 Analytical Testing of Counterfeit Drugs in the Field Using Vibrational Spectroscopy and GC-MSPauline Leary, Graduate Center, City University of New York, 1934 Bulls Head Rd., Stanfordville, NY 12581, John A. Reffner

The counterfeit-drug problem is complex and integrates matters of science, law, criminal justice, public health, and public policy. Proposed solutions require an in-tegrative approach. Forensic science, local laws and law enforcement, drug-devel-opment regulations, international law, public treaties and policies, funding of drug development, and intellectual property (IP) rights and enforcement all need to be considered if an appropriate solution is to be presented. The impact of these goods is devastating; ranging from localized fatalities resulting from small-scale product adulteration, to large-scale public-health crises due to the development of drug re-sistance and fatal diseases caused by substandard medications. These goods pres-ent a significant regional and global problem and are a serious threat to public health and safety. The nature of these goods as well as the internet and other factors that have streamlined global trade are rendering current practices ineffective. The field of forensic science struggles to establish analytical methods to identity composition and provenance of counterfeit goods. No method has been shown to be universally applied to achieve this goal. The authors present results of research performed on counterfeits to identify and source these drugs in the field. Methods based upon infrared spectroscopy, Raman spectroscopy and gas chromatography-mass spec-trometry (GC-MS) are presented. Method limitations as well as discrimination po-tential are reviewed. In addition, challenges to admissibility in court are discussed.

165 Paper Test Cards for Presumptive Identification of Falsified and Very Low Quality PharmaceuticalsMarya Leiberman, University of Notre Dame, Department of Chemistry and Biochemistry, 271 Stepan, South Bend, IN 46556

Finding falsified or very low quality medications in low resource settings is a very different task for the analytical chemist than finding them in a developed-world set-ting. Among the constraints: very limited access to instrumental analysis; lack of funding, difficulty in obtaining reagents, solvents, supplies, and lab space; unreliable electricity, water, and mail service, shortage of trained technicians and chemists. We developed a paper test card that can be used to quickly screen pharmaceutical dosage forms. The test card combines a dozen chemical color reactions and can be evaluated by comparison to stock images or via an image analysis program. In ten minutes, a user can detect the presence of unauthorized fillers or substitute active pharmaceutical ingredients (APIs), and with a little more effort can spot formulations in which the concentration of API is below 50%. We are working with pharmacists and regulatory agencies in Kenya to improve supply chain testing and follow up on adverse medical outcomes. I show how the cards work, then focus on recent results from genuine samples of falsified medications and from anti-infective and antimalar-ial drugs collected in Kenya and South East Asia.

166 Analysis of Counterfeit Pharmaceutical Products Using Visible and Vibrational Spectroscopies Inside and Outside of the LaboratoryAdam Lanzarotta, United States Food and Drug Administration, Forensic Chemistry Center, 6751 Steger Dr., Cincinnati, OH 45237, Nicola Ranieri

Counterfeit pharmaceuticals pose a serious health problem worldwide, and the United States Food and Drug Administration’s Forensic Chemistry Center (FCC) has been involved in their detection and characterization for several years. FCC analysts employ handheld devices for field examinations and utilize state-of-the-art bench-top instrumentation in the laboratory for confirmatory analyses. This presen-tation discusses some recent advances by the FCC for the screening and analysis of counterfeit pharmaceuticals including the use of handheld alternate light sources, infrared spectrometers and Raman spectrometers as well as the use of bench-top alternate light sources, macroscopic infrared spectrometers, infrared spectroscopic imaging microscopes and Raman microspectrometers.

167 Jackson Pollock’s Number 1A (1948): Using Spatially Resolved XRF and Multivariate Analysis to Characterize the Artist Palette and ProcessAna Martins, Museum of Modern Art, 11 W53rd St., New York, NY 10019, Jim Coddington, Donald Dahlberg, Geert van der Snickt, Birgit van Driel, Joris Dik

Analysis of historical paintings using non-destructive spatially resolved X-Ray fluo-rescence (XRF) microscopy has brought a tremendous insight into the materials and methods used by artists. The volume of information to interpret, however, can be as overwhelming as the amount of data that is gathered for a single object. Multivariate image analysis (MIA) can assist in the interpretation by extracting the meaningful information contained in the XRF hyper-spectra and by presenting this information in two dimensional maps. This presentation shows how multivariate image analysis methods were used to simultaneously characterize and map the different paints used by Jackson Pollock in Number 1A (1948). Principal component analysis (PCA) and multivariate curve resolution (MCR) were used to elucidate the characteristic

XRF profile of the different paints and help identify the pigments and fillers in them. The different paints where then mapped to help visualize the artist strategy for laying in the composition through several application techniques, including brushwork, oil paint dragged directly from the tube or applied to his hands and pressed on the canvas and industrial house paints dripped or poured across the surface.

168 Multiphoton Microscopy: An Efficient and Promising Tool for In-Situ Characterization of Historical ArtifactsGaël Latour, Imagery and Modelisation in Neurobiology et Cancerology (IMNC), Batiment 440, Orsay, 91405 France, Laurianne Robinet, Jean-Philippe Echard, Marie Didier, Marie-Claire Schanne-Klein

Characterization of coatings (stratigraphy, composition) is of the utmost importance for the understanding and conservation of historical artifacts. In the past few years, optical coherence tomography (OCT) has become a well-established technique for in-situ three-dimensional (3-D) imaging with a micrometer-scale resolution. How-ever, discrimination of the various components is strongly limited with OCT. Multi-photon microscopy (MPM), also called non-linear optical microscopy, appears as a promising alternative imaging technique for investigating cultural heritage artifacts. This technique performs non-invasive 3-D imaging with micrometer-scale resolution based on an intrinsic optical sectioning. A key advantage of MPM is its multimodal capability with different modes of contrasts that are directly linked to the structural and chemical nature of the materials. Two-photon excited fluorescence (2PEF) is emitted by a wide range of materials (fluorophores) in historical artifacts with specific absorption and emission spectra. As an example, we used spectral discrimination of 2PEF signals to separate gelatin-based film from sandarac film and to perform 3-D imaging of cochineal lake pigments. Second harmonic generation (SHG) signals are specific for non-centrosymmetric structures, with no counterpart in usual (linear) optical techniques. In historical artifacts, we observed this signal in plaster particles composed of bassanite crystals, crystalline cellulose and fibrillar collagen. We stud-ied the potential of this imaging technique for the investigation of varnishes, musical instruments and parchments. Our results demonstrate that multimodal MPM is an efficient and promising technique for 3-D in-situ investigation of historical artifacts and woods.

169 Advances in the Imaging and Microscopy of Semiconductor Pigments: From Laser-Based Time-Resolved Luminescence Imaging to Micro X-Ray Fluorescence Analysis Using Synchrotron RadiationAustin Nevin, Institute of Nanotechnologies and Photonics, Department of Physics, Piazza Leonardo da Vinci 32, Politecnico di Milano, Milano, 20133 Italy, Sara Bellei, Anna Cesaratto, Valentina Capogrosso, Gianluca Valentini, Daniela Comelli, Caroline Tokarski, Hervé Vezin

Semi-conductor pigments are found in paintings and include oxides and sulphides of zinc and cadmium-based sulphoselenides. While today these pigments are sta-ble and very pure, due to advances in synthetic processes and purification during production, in the past impurities in pigments may have a significant impact on the long-term stability of historical paint. The analysis of historical and modern Zn- and Cd- based semiconductor pigments is presented using a variety of non-destructive techniques to probe their chemical and physical properties with an aim to define useful markers for their detection in paintings and other indicators for their deg-radation. Micro-photoluminescence imaging and spectroscopy using laser-based analysis are presented for the detection of emissions and lifetimes from the band-gap and trap states; differences in signals are ascribed to defects and impurities in the pigments. Complementary analysis of pigments using alternative approaches provides data regarding the chemical nature of the impurities and their distribution in micro samples. High-resolution X-ray spectroscopy (using both micro-X-ray fluo-rescence mapping and X-ray absorption spectroscopy), and electron paramagnetic resonance (EPR) demonstrate the presence of various transition metal impurities, some of which were intentionally added to pigments during production to improve . Finally, examples of the application of luminescence imaging to paintings by Van Gogh and other 19th C. artists are shown.

170 Laser Ablation Surface-Enhanced Raman MicrospectroscopyPablo Londero, The Metropolitan The Metropolitan Museum of Art, 1000 Fifth Ave., New York, NY 10028, Marco Leona, Anna Cesaratto, John R. Lombardi

Surface-enhanced Raman spectroscopy (SERS) has become a powerful tool for the identification of organic colorants in cultural heritage studies, particularly when only trace amounts of sample are available. However, it has been limited by the protocols and instrumentation with which it is applied. These generally require water solubility and, for the best spatial resolution, mechanical removal of a micro-sample typically >20 µm. We have developed a new approach based on laser ablation in the desorption regime, in which a microscopic region is visualized, selected, and vaporized directly onto a SERS-active active substrate which one can immediately excite with a Raman laser to detect a spectroscopic signature. High quality spec-troscopic signatures are obtained with up to 4-5 µm resolution and ~100 attomole

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1:1 SNR sensitivity, though 10-100 picomoles is more likely for most samples. This provides comparable performance to some mass spectrometry measurements and opens the door to a number of new SERS-based studies. Measurements of insolu-ble pigments and the stratigraphy of thin paint layers have been performed, on both reference samples and genuine works of art.

171 Method Development with Zirconia Based Stationary PhasesClayton McNeff, ZirChrom Separations, 617 Pierce St., Anoka, MN 55303, Daniel Nowlan, Kelly S. Johnson, Richard A. Henry

The surface chemistry of zirconia-based reversed-phase high-performance liquid chromatography (RP-HPLC) stationary phases differs considerably from the bulk of silica and polymeric HPLC phases. These differences impart zirconia-based phases with both extraordinary chemical stability (pH 1-14 and up to 200 °C) and unique chromatographic selectivity. However, these differences must be taken into account in method development in order to achieve optimal results. This presentation will cover the appropriate selection of pH, buffer type and organic modifier type when using a zirconia-based phases as well as the use of elevated temperature. Carefully selected applications highlighting zirconia-based stationary phase method develop-ment techniques are presented.

172 High-Pressure Ion Chromatography: Following the Lead of Ultra High-Pressure Liquid ChromatographyPeter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA 94085, Frank Hoefler, Maria Rey

Typical ion chromatography systems and analytical columns in standard 4 mm and microbore 2 mm formats operate below a maximum pressure of 3000 psi using well established reagent-free ion chromatography (RFIC) system techniques. Howev-er, when using smaller particle-size analytical column, a higher-pressure system may be required. Capillary high-pressure ion chromatography (HPIC) systems can operate continuously at up to 5000 psi without eluent generation. The modified system used here extends the high system backpressure capabilities to the entire flow rate range of the system. Running any columns at increased flow rates may cause a decrease in peak efficiencies. However, the superior chromatographic fi-delity achieved using the new 4-μm columns, minimizes such losses, to provide better separation of ions in complex matrices and can provide faster analysis times and higher throughput. Higher peak efficiencies can be achieved, even when run-ning at faster flow rates, providing shorter analysis times and increased productivity. This poster demonstrates the use of smaller-particle-size (4-μm) analytical columns combined with high-pressure ion chromatography to achieve efficient separations, fast analyses, and high-throughput.

173 Achieving Maximum Kinetic Performance from UHPLC ColumnsRichard A. Henry, Sigma-Aldrich/Supelco, 595 North Harrison Rd., Bellefonte, PA 16823, William H. Campbell, David S. Bell

During the past decade, developments in high-performance liquid chromatography (HPLC) columns confirmed predictions of Knox and Saleem in 1969 that smaller particles and much flatter van Deemter (h- ν) curves would have a dramatic impact on HPLC speed and resolution, if suitable instruments could be developed. Instru-ments have made necessary advances in pressure and extra-column dispersion, and porous and core-type silica ultra-HPLC columns in the 2-μm range perform well with small, nonpolar test solutes at high velocities, but it is not that simple. Modern UHPLC columns still show larger C terms that reduce separation performance for high molecular weight, complex molecules at high mobile phase velocities. In 1988, Horvath and Neue described fundamental equations behind this mass transfer lim-itation. The magnitude of this undesired resistance to mass transfer will be docu-mented for different polar solutes, columns and operating conditions. Techniques are demonstrated for plotting plate height, efficiency and resolution as a function of flow velocity to define practical operating limits for high speed separation of real sample mixtures on modern columns. Certain limitations could be reduced or even eliminated by learning more about the nature of separation processes (diffusion, partition and adsorption) that occur inside pores of silica particles. The full benefits of modern, small-particle columns and instruments cannot be realized until flat van Deemter curves are achieved with samples containing larger, polar and ionic solutes on silica-based columns in reversed-phase, hydrophilic interaction liquid chroma-tography and other operating modes. Several areas for possible improvement in columns and instruments are described.

174 Halophase: A Novel Stationary Phase with Tunable Retention Properties through Functionalized Carboxylate Deposition of Halogenated Small MoleculesBrian A. Logue, South Dakota State University, Box 2202, Brookings, SD 57002, Michael W. Stutelberg, Venkataiah Mallam, Robert P. Oda, Raj K. Bhandari

The current state-of-the-art liquid chromatography stationary phase materials are produced by bonding chloro- or methoxysilanes to surface hydroxyls of highly porous silica solid supports. A promising gas-phase deposition technique, coined

functionalized carboxylate deposition (FCD), was used to rapidly deposit a self-as-sembled monolayer of targeted alpha-carbon modified carboxylic acid containing small molecules to porous silica solid supports. The FCD process rapidly (15-30 min) produces novel stationary phases, including a stationary phase coined Halo-phase. Halophase is created by depositing haloacetic acids via FCD and has shown tunable retention properties based on their electronegativity and substitution (i.e., mono, di, or tri).

175 The Use of Elevated Temperature in HPLC Biomolecule Separation to Shorten Analysis Time and Improve Peak Shape with Wide-Pore, Core-Type Silica ColumnsStacy Squillario, Sigma-Aldrich/Supelco, 595 North Harrison Rd., Bellefonte, PA 16823, Roy Eksteen, Hillel Brandes

Temperature elevation has proven to be beneficial for decreasing the analysis time and improving the peak shape of small molecule separations by high-performance liquid chromatography (HPLC). Antia and Horvath demonstrated in 1988 that signif-icant advantages are realized when columns containing superficially porous silica particles rather than fully porous particles are utilized in these elevated temperature separations. In this talk, we extend this concept to the analysis of biomolecules by HPLC, demonstrating a similar trend. Briefly, several globular proteins and various monoclonal antibodies were studied using commercial HPLC columns with large pore diameters suitable for reversed-phase chromatography of biomolecules. The operating capability of columns packed with either superficially or fully porous silica particles was examined for stability at temperatures up to 90 °C for a prolonged period of time. In general, wide pore columns packed with superficially porous silica particles were found to provide higher peak heights, narrower peak widths, and in some cases larger peak areas under conditions of elevated temperature than columns packed with fully porous particles. Larger peak areas were due to greater biomolecule recovery on columns containing the superficially porous silica particles. These results show the importance of temperature as well as particle morphology in biomolecule separation. This data benefits analysts who are tasked to find optimum conditions for the separation of proteins or peptides during drug development or biomarker research.

176 Easy Method Transfer and Scalability with Superficially Porous ParticlesAnne E. Mack, Agilent Technologies, 2850 Centerville Rd., Wilmington, DE 19808, William J. Long, Wu Chen, Xiaoli Wang, Jason Link, Maureen Joseph

Superficially porous particles make transferring and scaling existing liquid chroma-tography (LC) analyses flexible and easy. Compared to similarly sized totally porous particles, superficially porous particles have a shorter mass transfer distance and substantially narrower particle size distribution, resulting in more efficient analyses and possible time savings. In many cases methods using totally porous particle columns can be directly transferred to superficially porous particle columns without adjustment. This is particularly true when similar or identical bonding chemistries are available on both types of particles. Method scalability is also facilitated by the increase in choices of particle sizes. Together, these qualities allow for quick and reliable method scaling or transfer, ranging from method development for prepara-tive laboratories to methods for high throughput analyses, while also catering to the use of a wide range of instruments and pressure limitations. In this presentation, we share various practical examples of successful method transfer and scaling using superficially porous particles with both isocratic and gradient elution.

177 Monolithic Silica’s for “Dirty Sample” HPLC Analysis with Less Sample PreparationEgidijus Machtejevas, EMD Millipore, Frankfurter 250, Darmstadt, 64293 Germany

In contrast to conventional particle-packed columns, monolithic silica columns are made of a continuous piece of high purity porous silica. High permeability and porosity of the silica skeleton and the resulting low backpressure allow for more flexible flow rates compared to particulate columns. Monolith columns are just as versatile and, in addition, provide several advantages over packed columns espe-cially for “dirty” sample analysis. Especially the much higher tolerance for sample matrix makes monolith columns the preferred choice for “dirty” sample analysis. This presentation contains several methods, with workflows; including calibration curves, recovery calculations, and method robustness overview for “heavy” samples such us beer, milk, sun lotions, lipsticks, etc., with minimal sample preparation. For example 2700 analysis of Bisphenol A in baby milk formulation (total volume of injected sample was 27 ml + 65 l of mobile phase) were performed, and the results demonstrated the excellent long-term stability and method robustness obtained with monolithic silica columns.

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178 Discriminating Power of Fiber Associations in Forensic Examination PracticeEdward G. Bartick, George Washington University, Department of Forensic Sciences, 2100 Foxhall Rd. NW, Washington DC 20007, Stephen L. Morgan, Kevin Roberts

The objective of this work was to evaluate the statistical significance of measure-ment variance of like and unlike fibers using multivariate statistical methods, and to determine the discriminating power for the comparison of sets of red cotton and acrylic fibers was determined. Using visual light microscopy, visible microspectro-photometry (MSP) and Fourier transform infrared spectroscopy, data was collected on fibers. Studies were done on 21 red cotton and 21 red acrylic fibers using mul-tivariate analysis. The absorption spectra of visible microspectrophotometry scans on each fiber were compared by using principal component (PCA) and linear dis-criminant analysis (LDA) to determine how well fiber color matched on similarly dyed fibers. The discriminating power (DP) for the classification performance achieved was calculated as the ratio of the number of discriminated pairs of fibers divided by the total number of pairs of fibers under examination. The discriminating power was calculated for the red cotton fibers based on the discrimination achieved by PCA and LDA of the MSP data. On the 21 red acrylic fibers, the discrimination of the dyes was also done by MVA. The IR spectra were discriminated by spectrometer search software and the fiber diameters and cross-sectional shapes were discriminated by visual light microscopy. The final discriminating power based on all the parameters measured was then calculated.

179 Raman Microspectroscopy and Advanced Statistics for the Analysis of Biological Stains and Gunshot ResidueIgor K. Lednev, University at Albany-SUNY, 1400 Washington Ave., Albany, NY 12222, Greg McLaughlin, Kyle C. Doty, Justin Bueno

The identification of traces of body fluids discovered at a crime scene is a ma-jor part of forensic investigation today. The main problem with current tests is the destruction of the sample. The forensic community is in great need of a reliable, non-destructive, on-field method for identification of all common body fluids. We report here on the latest development of a new method for identification of body fluid traces using Raman spectroscopy combined with advanced statistics. Multidimen-sional Raman spectroscopic signatures of dry traces of sweat, vaginal fluid, semen, saliva, and blood were developed. The ability of identification of contaminated and/or mixed samples, differentiating menstrual and peripheral blood as well as deter-mine species (human vs. animal) based on dry traces of blood is discussed. The development of a novel and alternative method for detection, identification and dis-crimination of gunshot residue (GSR) is also discussed. We implemented advanced statistics for differentiating experimental spectra collected from non-equivalent GSR samples. Specifically, the differentiation of GSR particles originating from two dif-ferent caliber ammunitions was achieved with high confidence. Automated Raman microspectroscopic mapping was utilized for GSR detection and identification on an adhesive tape used for GSR collection. Our preliminary results indicate that this new technique is more comprehensive than all currently accepted methods of GSR identification.

180 Chemometrics in Action: Deployment of Handheld Spectroscopic Devices for First RespondersSuzanne Schreyer, Thermo Fisher Scientific, 2 Radcliff Rd., Tewksbury, MA 01876, Lin Zhang, Michael Hargreaves

In recent years, there is an increasing trend of moving analytical chemistry test-ing from laboratory environments to field based material identification. The use of analyzers in the field has characteristics which clearly distinguishes itself from the use in a traditional laboratory setting. Portable instruments in the field need to be able to give rapid, consistent results over a range of conditions – from testing in a warehouse to high stress situations involving emergency personnel and unknown conditions. This puts additional requirement on the software and underlying data treatment. For field based applications, merely presenting a spectrum is not enough. Rather the instrument is required to present a clear and unambiguous result that can be quickly interpreted by the user. This puts greater emphasis on analytical method development and is a big hurdle for wide adoption of new analyzers. In-telligent chemometrics algorithms must be used in these cases to give users clear answers for their intended use in the field. In this presentation, an overview is given on recent developments of chemometrics algorithms for handheld analyzers based on Raman, infrared and near-infrared spectroscopy. Specific examples are given of field based applications for safety and security applications, narcotic detection, and forensic analysis.

181 Hyperspectral Imaging and its Applications in Forensics ScienceJeffrey Beckstead, ChemImage Corporation, 7301 Penn Ave., Pittsburgh, PA 15208, Carley Chwal

Hyperspectral imaging has been shown to improve the detection of challenging samples within many forensic applications such as question document, fingerprint and biological fluids. Hyperspectral imaging combines high fidelity spatial imaging

with high fidelity spectroscopy. By combining spatial and spectral (color) informa-tion, and the use of statistical processing methods, the isolation of objects with very similar color or objects within complex backgrounds can be achieved. ChemImage and its customers have used hyperspectral imaging in many forensic applications. This presentation gives a background of hyperspectral imaging and its application to forensic investigations. Applications shall include question documents, finger prints, biological fluids, gunshot residue and bruise detection.

182 Fate and Effects of Engineered Nanomaterials in Agricultural SystemsJason White, Connecticut Agricultural Experiment Station, 123 Huntington St., New Haven, CT 06504, Joseph Hawthorne, Roberto De la Torre-Roche

Although engineered nanomaterial (NM) use has increased, the fate and effects of these substances in the environment is poorly understood. This lack of understand-ing is of particular concern in agriculture, where food chain contamination may oc-cur. The accumulation and transfer of NM from soil through terrestrial food chains is being measured. Bulk or nanoparticle (NP) cerium oxide (CeO2) was added to soil at 0 or 1000 mg/kg and zucchini were planted. After 28 d, the plant Ce content was determined by inductively coupled plasma mass spectrometry (ICP- MS). Leaves from each treatment were fed to crickets for 14-d; crickets were harvested and fed to wolf spiders for 7 days. The cricket and spider Ce content was measured. CeO2 exposure had no effect on total biomass but NP CeO2 significantly suppressed reproductive tissue production. The Ce content of zucchini was significantly greater when exposure was in the NP form. The flowers, leaves, stems, and roots of bulk CeO2-exposed zucchini was 93.3, 707, 331, and 119,000 ng/g, respectively; levels in NP-exposed plants were 153, 1510, 479 and 567,000 ng/g, respectively. Crickets fed bulk and NP CeO2-exposed leaves contained 13.5 and 19.7 ng/g, respectively. Feces from bulk and NP-fed crickets contained Ce at 393 and 1010 ng/g, respec-tively. Spiders that consumed crickets from bulk treatments contained non-quantifi-able Ce amounts but NP-fed spiders contained Ce at 4.9 ng/g. These findings show that NP CeO2 accumulates in zucchini at levels significantly greater than equivalent bulk materials and that this greater NP intake may result in trophic transfer and food chain contamination.

183 Locating Metal Oxide Nanoparticle Transformation in Plants Using Synchrotron X-RaysJorge Gardea-Torresdey, University of Texas-El Paso, Department of Chemistry, El Paso, TX 79968, Jose Hernadez-Viezcas, Hiram Castillo-Michel

With the increased use of engineered nanomaterials such as ZnO and CeO2 nanoparticles (NPs), these materials will inevitably be released into the environ-ment, with unknown consequences. In addition, the potential storage of these NPs or their biotransformed products in edible/reproductive organs of crop plants can cause them to enter into the food chain, and the next plant generation. Few reports thus far have addressed the entire life cycle of plants grown in NP-contaminated soil. Soybean (Glycine max) seeds were germinated and grown to full maturity in organic farm soil amended with either ZnO NPs at 500 mg/kg or CeO2 NPs at 1000 mg/kg. At harvest, synchrotron micro X-ray fluorescence (μ-XRF) and micro X-ray absorption near edge spectroscopy (μ-XANES) analyses were performed on soybean tissues, including pods, to determine the forms of Ce and Zn in NP-treated plants. The X-ray absorption spectroscopy studies showed no presence of ZnO NPs within tissues. However, µ-XANES data showed O-bound Zn, in a form resembling Zn-citrate, which could be an important Zn complex in the soybean grains. On the other hand, the synchrotron μ-XANES results showed that Ce remained mostly as CeO2 NPs within the plant. The data also showed that a small percentage of Ce(IV), the oxidation state of Ce in CeO2 NPs, was biotransformed to Ce(III). To the authors’ knowledge, this is the first report on the presence of CeO2 and Zn compounds in the reproductive/edible portion of the soybean plant grown in farm soil with CeO2 and ZnO NPs.

184 Determination of Carbon Nanotube Fate and Exposure Using a Microwave-Induced Heating MethodJacyln Cañas-Carrell, Texas Tech University, PO Box 41163, Lubbock, TX 79409, Amanda Parra, Sabrina Deleon, Fahmida Irin, Mohammad Saed, Micah Green

Carbon nanotubes (CNTs) are currently one of the most widely used nanomate-rials. However, there are currently limited analytical methods to quantify carbon nanotubes. A novel technique utilizing microwave-induced heating was developed to determine carbon nanotube concentrations in biological samples. The new method has been used to determine multi-walled carbon nanotube (MWNT) and single walled carbon nanotube (SWNT) uptake and bioaccumulation in terrestri-al and aquatic organisms. Various forms of carbon nanotubes (functionalized and non-functionalized MWNTs and SWNTs) were taken up into corn exposed to various concentrations in soil for 40 d. Translocation of nanotubes to the above ground portion of the plant (leaves and stem) was very low. The new method has also been

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used to evaluate the potential for bioaccumulation of CNTs in earthworms. Carbon nanotubes were also taken up into earthworms, but did not bioaccumulate with a low bioaccumulation factor of 0.015 ± 0.004. Finally, the new method has been used to investigate uptake of nanotubes in aquatic organisms such as Daphnia and fish. Considering the lack of detection and quantification methods for carbon nanotubes in biological samples, this new method is not only novel but will be useful in the risk assessment of carbon nanotubes.

185 Identification and Avoidance of Potential Artifacts and Misinterpretations in Nanomaterial Ecotoxicity MeasurementsElijah Petersen, National Institute of Standards and Technology, 100 Bureau Dr., Gaithersburg, MD 20899,Vincent Hackley, Teresa Kirschling, Theodore Henry, Jian Zhao, Baoshan Xing, Robert MacCuspie, Marina Dobrovolskaia, Jason White

Novel physicochemistries of engineered nanomaterials (ENMs) offer considerable commercial potential for new products and processes, but also the possibility of unforeseen and negative consequences upon ENM release into the environment. Investigations of ENM ecotoxicity have revealed that the unique properties of ENMs and a lack of appropriate test methods can lead to results that are inaccurate or not reproducible. The occurrence of spurious results or misinterpretations of results from ENM toxicity tests that are unique to investigations of ENMs (as opposed to traditional toxicants) have been reported, but have not yet been systemically re-viewed. Our objective in this talk is to highlight artifacts and misinterpretations that can occur at each step of ecotoxicity testing: procurement or synthesis of the ENMs and assessment of potential toxic impurities such as metals or endotoxins, ENM storage, dispersion of the ENMs in the test medium, direct interference with assay reagents and unacknowledged indirect effects such as nutrient depletion during the assay, and assessment of the ENM biodistribution in organisms. We recommend thorough characterization of initial ENMs including measurement of impurities, im-plementation of steps to minimize changes to the ENMs during storage, inclusion of a set of experimental controls (e.g., to assess impacts of nutrient depletion, ENM specific effects, impurities in ENM formulation, desorbed surface coatings, the dis-persion process, and direct interference of ENM with toxicity assays), and use of orthogonal measurement methods when available to assess ENMs fate and distri-bution in organisms.

186 Silicon Surface Functionalization to Control the Oxidation State of Copper during Metalorganic Chemical Vapor Deposition of Cu(I) and Cu(II) PrecursorsYichen Duan, University of Delaware, 114 Lammot DuPont Laboratory, Newark, DE 19716, Andrew Teplyakov

This work focuses on the surface-limited deposition reactions leading to the for-mation of copper-containing nanoparticles on functionalized Si(111) surface. The study compares the previous work on the deposition of Cu(I) precursors and a new-ly developed deposition of solid-state Cu(acac)2 compound. H-terminated Si(111) surface is prepared by modified RCA method. Then the sample is loaded into a high vacuum chamber with the base pressure 1.0 x 10-6 torr. The precursor, Cu(acac)2 powder, is placed in a metal-organic solid state doser which is directly attached to the high vacuum chamber. The precursor is sublimed by heating and dosed into the chamber where it reacts with the H-terminated Si(111) surface. A reduction reac-tion from Cu(II) to Cu(I) is confirmed by X-ray photoelectron spectroscopy analysis. Atomic force microscopy images reveal the morphology of the nanoparticles formed on the surface following deposition. A reaction pathway is proposed based on the densityfunctional-theory calculations to explain the mechanism of this surface-limit-ed reduction reaction and to compare with the deposition of Cu(I) precursor, Cu(h-fac)VTMS.

187 Mechanism of Electrical Passivation of Silicon Surfaces with QuinhydroneMeixi Chen, University of Delaware, 201 Dupont Hall, Newark, DE 19716, Nicole A. Kotulak, Nikolas J. Schreiber, Robert L. Opila

Quinhydrone in methanol is known to interact with hydrogen terminated Si surfaces in such a way that this interaction greatly diminishes excited electron recombination at the surface. In this work quinhydrone has been separated into its constituent parts, p-benzoquinone and hydroquinone, and each was dissolved in methanol in order to test the electrical passivation of the silicon surfaces. P-benzoquinone is the active component; however, hydroquinone solutions improve in performance when in contact with the substrates for a longer duration. The effect of light was also examined. Substrates passivated with benzoquinone in full ambient light conditions displayed the highest effective carrier lifetimes. The passivation effect of solutions exposed to light during preparation, but with measurements taken in the dark, were compared with that of substrates passivated using a process completely performed in the dark. The passivation effect from these procedures is much less effective than full light passivation. It is confirmed that the presence of light facilitates the passiv-ation. The presence of a free proton in the solution (i.e., the hydroxyl proton) is also found to play an important role in passivation. Surface analyses (X-ray photoelec-

tron spectroscopy and Fourier transform infrared) demonstrate that benzoquinone has reacted with the surface. Density functional calculations are consistent with a photoexcited benzoquinone species reacting with the surface.

188 Electron Beam Induced Reactions of Adsorbed π-Allyl Ruthenium Tricarbonyl Bromide: Towards Design of Electron Beam Induced Deposition (EBID)-Specific PrecursorsJulie A. Spencer, Johns Hopkins University, Department of Chemistry, Baltimore, MD 21218, Rachel Thorman, Michael Barclay, Howard Fairbrother, Joseph A. Brannaka, Lisa McElwee-White, Oddur Ingólfsson

Electron beam induced deposition (EBID) is a resistless process with a great deal of promise in depositing well-defined, metal-containing nanostructures. To date, EBID of nanostructures has used precursors designed for thermal processes, such as chemical vapor deposition (CVD); this underscores the need to design precur-sors specifically for EBID. This surface science study focuses on elucidating the reaction mechanism involved in EBID of π-allyl ruthenium tricarbonyl bromide (π-C3H5Ru(CO)3Br), an organometallic precursor synthesized specifically to test its suitability as an EBID precursor. Experiments studied the effects of 500 eV in-cident electrons on nanometer scale films of π-C3H5Ru(CO)3Br under ultrahigh vacuum conditions using X-ray photoelectron spectrometry complemented by mass spectrometry. Preliminary results indicated that the initial reaction step involved electron stimulated desorption of one to three carbonyl ligands from the parent π-C3H5Ru(CO)3Br molecules, while continued electron processing or annealing both resulted in almost full loss of Br from the thin film. A parallel gas phase single electron impact study on π-C3H5Ru(CO)3Br evaluated the dissociative ionization and dissociative electron attachment mechanisms potentially involved in EBID. This parallel study indicated that loss of a single CO ligand is most prevalent, with mini-mal loss of Br, in agreement with surface science experiments; conversely, the gas phase study showed significant loss of the π-allyl group, which was not observed in surface experiments. Insights from π-C3H5Ru(CO)3Br studies are compared with results from other organometallic precursors (e.g. cis-dicarbonyldichloro plati-num(II), cis-PtCl2(CO)2) and discussed with regards to increased understanding of EBID processes and potential precursor design implications.

189 Formation of High-Coverage Nanoparticle Monolayers through Click ChemistryMackenzie G. Williams, University of Delaware, 111 Lammot DuPont Laboratory, Newark, DE 19716, Yue Liu, Timothy Miller, Andrew Teplyakov

Gold and silicon surface modification was accomplished using click chemistry. Mod-ified nanoparticles were attached to this functionalized surface through a 1,3-dipo-lar cycloaddition click reaction between terminal alkyne and azide groups. Silica nanoparticles were functionalized with azide and alkyne groups using 2-azidoetha-nol and 4-pentyn-1-ol respectively. In a separate set of experiments, magnetic iron (III) oxide nanoparticles were functionalized with alkyne groups using 10-undecyn-ylphosphonic acid and 5-hexynoic acid. The chemistry of nanoparticle deposition was verified through scanning electron microscopy (SEM), single-point attenuated total reflectance infrared spectroscopy (ATR-IR), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM) studies. The quality of the monolayers formed and the coverage obtained were interrogated by SEM and AFM. The suc-cessful application of the click reaction was verified by the presence of the triazole ring and disappearance of azide and alkyne groups in ATR-IR and XPS results and compared to density functional theory (DFT) calculations. Continuing work will focus on the formation of a complete monolayer and the development of pre-defined mul-tilayers controlled by click chemistry.

190 Correlation of Carbon Growth on Ru-Capped Multilayer Mirrors Irradiated by Extreme-Ultraviolet Light and ElectronsMichael S. Barclay, Johns Hopkins University, 3400 North Charles St., Baltimore, MD 21218, Nadir S. Faradzhev, Howard Fairbrother, Shannon B. Hill, Thomas B. Lucatorto

Extreme-ultraviolet lithography (EUVL) is the next likely step in improving chip pro-duction for the semiconductor industry. Using a shorter (13.5 nm) wavelength of light, manufacturers can mass-produce chips with even smaller nanoscale features than possible with current lithographic techniques. Due to the nature of 13.5 nm light, EUVL must be performed in a vacuum chamber. This exposes the highly sen-sitive optics in the EUVL tool to contamination by outgas products from the photo-resists used during the lithographic process. To protect the delicate optics, industry has established a resist-outgas testing protocol to determine a resist’s efficacy be-fore introducing it to the EUVL tool. Unfortunately, a key component of this protocol is the use of a dedicated, bright, EUV source. To mitigate the large capital invest-ment necessary for such a source, an electron beam is often used as an analog for the EUV source. To this end, we have measured rates of carbon growth on samples of Ru-capped multilayer optics exposed to both electron and EUV irradiation in the presence of admitted hydrocarbon vapors of two model species: benzene and tet-radecane. We find that the carbon growth rate for both exposure methods have

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sub-linear pressure dependence at low irradiance which transitions to linear scaling at higher irradiance. The growth rates at which this transition occurs, however, are different for EUV and e-beam irradiation.

191 Controlled Synthesis and Optical Studies of ZnO NanostructuresZhengxin Li, University of Delaware, Department of Chemistry and Biochemistry, 105 Lammot DuPont Laboratory, Newark, DE 19711, Jolie Blake, Lars Gundlach

In this work, various ZnO nanostructures were synthesized via chemical vapor depo-sition, and hydrothermal method. The morphology change of ZnO nanostructures was investigated by changing growth parameters. Scanning electron microscope, X-ray diffraction, and fluorescence measurements are compared for differently pre-pared ZnO nanostructures. Ultrafast time-resolved luminescence measurements can reveal preparation dependence of electronic parameters. Controllable synthesis of ZnO nanoparticles has great potential in design of desirable ZnO nanostructures for nanoscale applications such as solars cells, photochemical analysis, and laser devices.

192 Detection of Olive Oil Adulteration Using Benchtop NMR SpectroscopySusanne Riegel, Nanalysis, Bay 4, 4500 5 St. NE, Calgary, AB, T2E 7C3 Canada

Olive oil adulteration remains of considerable concern on a global scale. Methods and techniques for rapid, easy identification of adulterants in olive oil have gen-erated considerable interest. Typical techniques rely on hyphenated chromatogra-phy-mass spectrometry (MS) systems (typically liquid chromatography-MS or gas chromatography-MS). These methods can be time consuming, requiring extensive sample preparation and data interpretation; and, depending upon the scope of the method being used, it can be possible to miss certain contaminants if, for example, they have different retention times or potential dangerous volatiles. We address the efficacy of a robust, easy-to-use 60 MHz nuclear magnetic resonance spectroscopy for rapid sample preparation and data acquisition.

193 Conditions Using Accelerated Solvent Extraction (ASE) and LC-MS-MSChangling Qiu, South Dakota State University, Chemistry and Biochemistry Department, Avera Bldg.131, Brookings, SD 57007, Douglas Raynie

Perfluorooctanoic acid (PFOA) is used as a polymerization aid in the production of fluoropolymers. These polymers provide oil and water repellency as well as stain re-sistance, which make them as ideal coating materials for non-stick cookware. PFOA is bio-accumulative and potentially harmful to humans. PFOA is not supposed to be found in the final products of non-stick cookware after processing. This study pres-ents a method to determine the potential leaching of PFOA from the cookware under simulated cooking conditions. PFOA cookware was extracted with ethanol/water mixture using accelerated solvent extraction (ASE), and the extraction parameters such as pressure, temperature, and time were optimized. The resulting extracts were analyzed by liquid chromatography tandem mass spectrometry (LC-MS-MS).

194 Determination of Perfluorooctanoic Acid (PFOA) from the Surface of Cookware under Simulated Cooking HPLC Analysis of a Commercial Whirlpool Hopping Process in the Beer Brewing IndustryJessica S. Henry, University of New Hampshire, 23 Academic Way, Durham, NH 03824, Kelsey A. Packard, Elizabeth A. Brady, Sterling A. Tomellini, Austin D. Gregoire

Hops are a key ingredient in the brewing industry due to the bittering and aromatic qualities they contribute to the final product. In their raw form, hops contain little bitterness; however, adding hops to the wort kettle and boiling the hops in the wort will cause an isomerization of insoluble α-acids to soluble iso-α-acids, which are the main component of the bitter flavor. An addition of hops can also occur during the whirlpool, when no boiling occurs. This late addition of hops is believed to increase the aroma of the final product, yet little is known about the efficiency of the isomer-ization during this process. This study aimed to investigate the effects of whirlpool hopping on the iso-α-acids in wort. A series of brews were conducted with different kettle hopping methods to substantiate previous work. These test brews assisted in removing assumptions that brewers use when talking about hop utilization that were used in past reports. The test brews where also hopped and fermented as the standard operating procedure brews at the brewery and then put in front of professional sensory panels for taste evaluation. The analysis utilized a modified high-performance liquid chromatography (HPLC) method to monitor the concentra-tion of iso-α-acids over time in wort samples collected from an industrial brewhouse. Results show the increase of the iso-α-acids concentration over time.

195 Development of SERS Methods for Rapid Detection of Multi-Pesticides in FoodLili He, University of Massachusetts-Amherst, 102 Holdsworth Way, Amherst, MA 01003, Hua Zhang

Pesticide residues in food are under strict regulations due to their potential toxicity to environmental and human health. Due to the large variety of insecticides, herbi-cides, and fungicides, it is challenging and time consuming for factory, governmental agency or test labs to test every possible pesticide residue in food. There is an increasing need for rapid detection of pesticides in food, as traditional high-per-formance liquid chromatography (HPLC) and gas chromatography (GC) methods are usually time consuming. Here I demonstrate the superior capacity of surface enhanced Raman spectroscopy (SERS) for rapid detection of pesticides on apples, lettuce and in apple juices; this research is funded by the United States Department of Agriculture (USDA). SERS is a combination technique of Raman spectrosco-py and nano-techniques. The use of nanostructures like silver dendrites increas-es the Raman signals by more than a million times. Contaminants can be either swabbed from apple surfaces or captured in food slurries with or without selective agent (i.e., aptamer), then rapidly measured using a Raman instrument. Our results show SERS can detect multiple pesticides in different food products and at parts per million (ppm) level. All detections can be done within 30 minutes including the sample preparation. A portable Raman instrument can be used to detect pesticides on site. In summary, the use of SERS shows great success in rapid detection of pesticides in food.

196 Very Large Range Pesticide Screening in Food Using GC Triple Quadrupole MSLori Dolata, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA 94085, Massimo Santoro

Greater than before ease of access to high selectivity gas chromatography mass spectrometry (GC-MS) is allowing more rapid and generic sample preparation in pesticide testing, is permitting consolidation of multiple analyte lists and matrices into one analytical qorkflow. While GC-MS-MS is well suited to multi-residue anal-ysis in a diverse range of matrices, an increased number of targeted compounds also amplify the complexity of method optimization and analytical performance may become problematic. Furthermore, there is sometimes a desire to look beyond tar-geted lists for other potentially harmful food contaminants. Presented is the use of smart instrument control and data processing software applied to GC-MS-MS analysis of >600 pesticides in matrix to mitigate analytical performance degradation through MS duty cycle optimization. Also discussed is combining this optimized tar-geted quantitation with general unknown analysis through fullscan-MRM.

197 Analysis of Beer, Wine and their Agricultural Constituents (Hops, Grapes, Grains) for Pesticides Using QuEChERS Extraction and High-Throughput Sample PreparationPatricia L. Atkins, SPEX CertiPrep, 203 Norcross Rd., Metuchen, NJ 08840

There are hundreds of commercial pesticides in use in industrial and private agricul-ture. The concern over human pesticide exposure over the past few decades has led to the monitoring of these pesticides. Increasing concern over the health effects of residual pesticides on fruits and vegetables has led to increased testing of these products to determine the levels of pesticides on produce when it goes to market. In this study, commercial red wine and beer samples were examined for their pesticide concentrations. In addition to the examination of the finished alcoholic beverage, the constituent agriculture products of wine and beer production: grains, malts hops and wine grapes; were also examined to determine the levels of pesticides found in those products. The sample preparation and extraction process efficiency and recovery were examined by processing samples using manual versus high-through-put techniques. The QuEChERS (quick, easy, cheap, effective, rugged, and safe) method was used to process a greater number of samples in a shorter period of time than other extraction methods.

198 Vacuum Ultraviolet Detection for the Identification and Quantification of Pesticides by Gas ChromatographyHui Fan, University of Texas-Arlington, Department of Chemistry and Biochemistry, 700 Planetarium Pl., Arlington, TX 76019, Doug D. Carlton Jr., Ian Sawicki, Kevin A. Schug, Phillip Walsh, Dale Harrison

Pesticide analysis by gas chromatography (GC) is a tedious process due the activ-ity, thermal instability, and the prevalence of isomeric analyte species. Identification of these analytes, typically by mass spectrometry, also becomes taxing, since the ionization routes for many of the compounds result in low analyte response and similar spectra used for database identification. The vacuum ultraviolet detector which we have recently introduced is capable of measuring gas phase absorbance spectra within the wavelength range of 115-185 nm (VUV region) and up to 240 nm. Within this region, all species absorb energy and possess a unique spectral response, even for ionizably labile pesticides which limit mass spectrometry. Specif-ically, chlorinated pesticides have sections of fine structure absorbance throughout

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this wavelength range, further aiding to identification. The mass spectral responses for captafol, folpet, and methidathion have reported low spectral database matching capabilities for qualitative identification. The VUV absorbances of these pesticides, along with various others that are isomers, structurally similar, or tend to coelute, are very unique, allowing for confident identification and spectral deconvolution if analyte or matrix coelution occurs. The ability to deconvolute is demonstrated by quantifying captan under fast GC conditions used to reduce thermal decomposition on column and the coeluting pair of fenthion and chlorpyrifos.

199 Development of a Sensitive LC-UV Method to Determine Two Trace Aldehydes in MaltitolGeorge Wang, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903, Zhen Wu, Holly M. Shackman, Mark S. Bolgar, David K. Lloyd

A high-performance liquid chromatography (HPLC) method for the determination of glyceraldehyde (GA) and pyruvic aldehyde (PA) in maltitol, based on derivatization with the 2, 4-di-nitrophenylhydrazine (DNPH), is presented. Maltitol is a commonly used excipient in drug formulations. It may contain trace levels of GA and PA, which can react with primary or secondary amines in drug actives to generate impuri-ties. In Baraclude oral solution, maltitol is present at 380 mg/mL while the primary amine-containing active ingredient is only 0.05 mg/mL. Both high maltitol concen-tration and low drug loading pose challenges for the method sensitivity. The deri-vatization of GA and PA by DNPH facilitated the detection with UV absorbance. As a bi-molecular reaction, the derivatization rate is proportional to the concentrations of both aldehydes and DNPH. Since the aldehydes are at trace level, to allow the reaction to proceed quickly, the concentration of DNPH needs to be relatively high. It was found that some GA-DNPH converted to PA-DNPH when stored for an extend-ed period. A mechanism based on enolate tautomerization is proposed for this con-version. To prevent maltitol from forming a gel, the content of acetonitrile should be kept low (<25%) in the sample diluent. Finally, the method sensitivity was enhanced by optimizing HPLC conditions including detection wavelength, injection volume and maltitol concentration. The finalized method has easy sample preparation and is suitable for use in a quality control environment. The method was validated with a quantitation limit of 0.01 ug/mL (~25 ppb relative to maltitol).[1]


[1] C. Pathirana et al, Tetrahedron Letters, 54 (2013) 132–133.

200 Particulate Contamination Control - Current Technology versus State-of-the-PastOliver Valet, rap.ID Inc., Princeton Corporate Plaza, 11 Deer Park Dr., Ste. 201, Monmouth Junction, NJ 08852

The thorough root cause investigation of particle contamination in pharmaceutical production requires reliable chemical identification techniques. Particle identification techniques including: scanning electron microscopy / energy dispersive spectrome-ter, Fourier transform infrared (FTIR) or Raman microscopy are necessary to unveil a particles chemical nature. Traditional laboratory methods use different equipment for each technique, meaning micro-objects must be transferred between methods. It is difficult and time consuming to continuously locate small particles on microme-ter scales, and errors can occur. Besides manual obstacles, every instrument has its own software and chemometric system, producing independent data-sets. The pharmaceutical industry works in a regulated environment, and 21 CFR Part 11 compliance is stringent regarding the collection of raw data. State of the art equip-ment combines multiple techniques in one system and produces traceable, reliable, and compliant data. This data enables chemical characterization, and matches mi-cro-particle materials with materials used in manufacturing. The rap.ID SPE unit eliminates need for sample transfer between machines. Furthermore, analysis is completely automated and user friendly, thus there is no need for a spectroscopic expert; results are compared to a built-in matrix database, and corresponding re-ports generated. Once the database establishes a match for the samples spectral fingerprints, differentiation between samples can be made and their origins deter-mined. Raman spectroscopy and laser-induced breakdown spectroscopy (LIBS) techniques are vital for the accurate identification of microscopic materials. Raman provides information regarding molecular structure, while LIBS yields elemental composition. Thus, their combination provides a unique tool for identifying unknown particles such as foreign particulate matter.

201 Determination of Crystallinity in Amorphous Solid DispersionsCynthia S. Randall, Particle Sciences, 3894 Courtney St., Bethlehem, PA 18017, Ajoy Koomer, Rabi Bello

Solid dispersions are an attractive option for formulating poor water soluble drugs. But the inherent physical instability of amorphous materials is a major concern, and methods are needed to detect the onset of crystallization. X-ray diffraction (XRD) and Fourier transform (FT)-Raman were evaluated for this purpose in analysis of Compound X solid dispersions. The basic approach involved spiking pure amor-phous material with known amounts of the crystalline drug form. Spiking was done with the amorphous drug substance alone, as well as with amorphous solid disper-

sions containing Compound X and a stabilizing excipient; the same spiked mixtures were analyzed by XRD and FT-Raman. Results indicate that both techniques can be exploited for identification and quantification of crystalline content in initially amor-phous samples. An XRD method alone may be sufficient for routine characteriza-tion of these dispersions, especially when measurement parameters are optimized. However, XRD in conjunction with FT-Raman can provide more definitive evidence of crystallinity. FT-Raman also offers information about the state of the drug in the formulation matrix.

202 Nanoparticle Synthesis Using Aerosol ReactionsHo Yeon Yoo, Stanley Bruckenstein Chemical Consulting, 115 Foxpoint W, Williamsville, NY 14221, Stanley Bruckenstein

Nanoparticles were synthesized by merging two aerosols containing the different constituents of the nanoparticles. The feasibility of this concept for three classes of reactions in aerosols was demonstrated using the chemiluminescent reaction between Luminol and Potassium Ferricyanide, the pH sensitive fluorescence of Umbelliferone and the precipitation of silver chromate by reaction of silver nitrate with potassium chromate. We postulate that using oppositely charged aerosols are more efficient synthetically and will produce a narrower distribution of nanoparticle sizes than using uncharged aerosols. We also describe how to construct a desk top apparatus to carry out the synthesis of nanoparticles.

203 Withdrawn by the author.

204 Moisture Determination in Rubber Stoppers Using Relative Humidity Sensing Technology; A Karl Fischer Titration AlternativeGarrett Rowe, Arizona Instrument, 3375 N. Delaware St., Chandler, AZ 85225, Christopher Altamirano

The use of rubber stoppers in the pharmaceutical industry has an important purpose of keeping the active ingredients within drugs protected from the external environ-ment. Keeping the contents within a vial dry (or at least within a controlled humidity range) is essential for long term storage and drug efficacy. Although rubber stoppers provide an airtight seal to a vial, the rubber material itself is inherently hygroscopic and prone to moisture absorption. Therefore, knowing the baseline moisture level of certain rubber stoppers during drug processing and packaging has become part of the quality assurance monitoring of many pharmaceutical companies. Moisture analysis of rubber stoppers is traditionally performed using a Karl Fischer (KF) ti-tration method, which tend to be lengthy and consume expensive wet chemical reagents. Using a moisture specific relative humidity (RH) sensor to detect inher-ent moisture within rubber stopper provides fast and accurate results that can be used without the use of wet chemical reagents. The % moisture of various rubbers stoppers analyzed by both the RH and KF methods statistically correlate between various data sets. Moreover, using the RH sensor method proved to yield quicker test times with a smaller standard deviation when compared to the traditional KF method.

205 Detection and Quantification of As, Cd, Hg, and Pb in API and FDP Using Procedure 2 of Updated USP <233>Michael Murphy, Intertek Pharmaceutical Services, PO Box 470, Whitehouse, NJ 08807, Philip Costello, Diana Buro

The United States Pharmacopeia (USP) has replaced the non-specific colorimet-ric test with two new chapters <232> (Limits) and <233> (Procedures). The limits used to assess active pharmaceutical ingredients (API) and formulated drug prod-ucts (FDP) will be updated this year pending the adoption of guidance International Conference on Harmonization (ICH) Q3D. The limits between the USP and ICH documents will not be perfectly aligned and hence the limits chosen for testing will depend on the intended market of the API or FDP. A simplified testing scheme will allow rapid transfer of methodology between different APIs and FDPs reducing/elim-inating the time required to develop adequate sample preparation/analytical condi-tions. The Milestone Ultrawave microwave digestion system and an Agilent 7700X ICP-MS can be used to develop a general methodology for sample preparation and analysis. Data presented for multiple sample types demonstrates how this type of procedure can streamline method development/validation for the required USP elements at the new monograph limits.

206 Impact of Croscarmellose Sodium on Recovery of Active from Formulated CapsulesSongling Yu, Celgene Corporation, 86 Morris Ave., Summit, NJ 07901, Xiaolu Liao, Mauro Solorzano, Naijun Wu

A new formulation with 0.3 mg and 1 mg capsule strengths was developed for a weakly basic developmental compound. During the development of assay and re-lated impurity methods by high-performance liquid chromatography (HPLC), low drug recoveries were observed. However, no significant impurities or degradation products were found in the sample. In this study, the effects of formulation excipient, sample diluent pH, diluent composition, and sonication time on assay recovery were

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investigated. The results showed that the low recovery was related to the use of 4% w/w Croscarmellose sodium disintegrant. The drug recovery was improved signifi-cantly by using low pH diluents and less impacted by acetonitrile percentage of the diluents or sample sonication time. The data suggests that the low recovery could be attributed to interactions between the amino group of drug and the carboxylic group of Croscarmellose sodium. The interaction between drug and Croscarmellose sodium is pH dependent and can be minimized using acidic diluents. Accordingly, assay and related impurity methods were optimized and a recovery of 100.3% was achieved using an ACN:0.1% HCl, 20:80 (v/v) diluent at pH ~2.

207 Characterizing Ligand Binding to Serum Albumin Using Taylor Dispersion AnalysisDavid Goodall, Paraytec, York House, Outgang Lane, Osbaldwick, York, YO19 5UP United Kingdom, Thomas Allen, Alexander Chapman

Characterizing the interaction of small molecule ligands with selectors is important for drug discovery in the pharmaceutical industry. For serum albumin, the binding affinities for ligands are routinely screened in order to characterize plasma protein binding, with the gold standard method equilibrium dialysis. The objective of this work was to determine whether unbound fractions and binding affinities in the milli- and micro-molar ranges can be measured by Taylor Dispersion Analysis (TDA). A Viscosizer 200 (Malvern Instruments) was used for TDA, with wavelengths for study 214, 254 and 280 nm. Human serum albumin (HSA) was the selector, with naproxen and warfarin as ligands. The method uses less than 10 µL of material, and is shown to be applicable in biorelevant media (40 mg/mL HSA and plasma). New TDA meth-odology is demonstrated showing how binding information can be extracted over a range of 2 orders of magnitude variation of sample concentration in a single run for injection of ligand as sample into a carrier containing a fixed concentration of HSA. At low absorbance/low concentration the TDA profile is that of the bound ligand, whereas at high absorbance / high concentration the profile is that of the free ligand. Advantages relative to alternative techniques (e.g., surface plasmon resonance, affinity chromatography) include having the protein in free solution, with no immobi-lization required which might change affinity and binding properties.

208 Dual Wavelength Imaging of Whole Tablet DissolutionAlexander Chapman, Paraytec, York House, Outgang Lane, Osbaldwick, York, YO19 5UP United Kingdom, David Goodall

The aim of this study was to provide proof of concept for whole tablet imaging with dual wavelength detection. The instrumentation used an ActiPix UV area imaging module (Paraytec Ltd). A standard 1 cm fused silica spectrophotometer cuvette was used as the dissolution vessel. The tablet was mounted on a fused silica tube, held in the teflon cap. A miniature stir bar motor was positioned underneath the cuvette. The light sources were 255 (UV) and 505 nm (Vis) light emitting diodes, with se-quential on/off switching at frame rate 2 Hz. The sample tested was Sulazine EC, an enteric-coated sulfasalazine delayed release tablet. Measurements were carried out in 0.1 N HCl and in PBS. A narrow boundary layer develops in both the UV and Vis, consistent with initial swelling of the polymer film coating the tablet. In PBS there is a sharp increase in the gradient of absorbance with time at 42 minutes, with UV-Vis absorbance ratio 10:1 mirroring the ratio of extinction coefficients of sul-fasalazine at 255 and 505 nm and indicating the start of the main phase of release of sulfasalazine. This timing agrees well with literature data (Bali et al, Int. J. Biol. Pharm. Res. 2013, 4, 491-494). Spatially- and time-resolved images provide an im-mense amount of detail and prove the benefits of both visualization and quantitative measurements of absorbance at the two wavelengths, one in the UV and one in the Vis to characterize physical and chemical changes occurring during the tablet dissolution process.

209 Development of a 2-Butanol Background Electrolyte for Non-Aqueous Chiral Capillary ElectrophoresisErin J. Ennis, Drexel University, 3141 Chestnut St., Philadelphia, PA 19104, Joe P. Foley

Non-aqueous capillary electrophoresis (NACE), an alternative to aqueous elec-trokinetic chromatography, is a technique that utilizes organic modifiers to enhance the separation of charged hydrophobic compounds. NACE can be applied to the separation of enantiomers by utilizing chiral additives or an enantiopure solvent, although the latter has received little attention. The separation of charged enantio-mers will be examined for various preparations of non-aqueous enantiopure sol-vent to optimize experimental methods. While previous work examined 2-butanol as a potential NACE background electrolyte at both low (mainly aqueous) and high (mainly non-aqueous) concentrations, this work examines a novel and completely non-aqueous background electrolyte comprised of various concentrations of 2-bu-tanol and acetonitrile for the separation of charged pharmaceutical enantiomers. Experimental emphasis is on the optimization of resolution, migration time, and en-antioselectivity. Chromatographic figures of merit are examined and applications are explored for future work.

210 Determination of the Enantiomeric Purity of Dextromethorphan via PolarimetryMark Canestrano, Anton-Paar USA, 10215 Timber Ridge Dr., Ashland, VA 23005

Methorphan is a chiral drug that differs in its effects and pharmacology with respect to its two enantiomers. The dextrorotatory dextromethorphan is applied as an anti-tussive drug in cough medicines, whereas the levorotatory enantiomer levometho-rphan is a strong opioid analgesic that is listed as a schedule II drug in the United States. As dextromethorphan is approved for use in over-the-counter drugs, accu-rate control of enantiomeric purity is highly essential. Enantiomeric purity is con-trolled by measurement of optical rotation, which is dependent on the wavelength. The specific rotation of dextromethorphan is increased approximately 10 fold when measuring in the UV range, compared to values obtained from measurements per-formed at the sodium D line that is typically used in pharmaceutical applications. As the UV range wavelength results in an increased optical rotation that also correlates with a higher sensitivity of the measurement, international pharmacopeias demand the measurement of optical rotation in dextromethorphan solutions at 325 nm. To determine optical rotation in dextromethorphan according to international pharma-copeias at 325 nm, Anton Paar developed the MCP 500/MW325 polarimeter.

211 Determination of Carbohydrates in Various Matrices by Capillary HPAE-PADTerri T. Christison, Thermo Fisher Scientific, 1214 Oakmead Parkway, Sunnyvale, CA 94088, Peter Bodsky, Carl Fisher, Hua Yang, Monika Verma, Linda Lopez

Monosaccharide and disaccharide determinations are important to various indus-tries from ensuring quality product to researching biological pathways and disease states. Because carbohydrates are poor chromophores, chemical derivatization is needed for absorption. However, derivatization is costly, labor-intensive and may cause changes in molecular configuration. High-performance anion-exchange chro-matography with pulsed amperometric detection (HPAE-PAD) is a proven sensitive method to directly and selectively determine carbohydrates. In HPAE-PAD, carbo-hydrates are ionized in strong base and separated by anion-exchange chromatog-raphy. The carbohydrates are detected by PAD with a gold working electrode using a four-potential waveform selective and sensitive for carbohydrates. This sensitivity allows carbohydrate analysis down to pmole concentrations or when the samples are limited. This sensitivity is moderated in beverage samples which contain g/L concentrations by minimizing the flow path combined with moderate dilution. Here we combine the advantages of a reagent-free ion chromatography (RFIC) sys-tem and a capillary format IC to determine monosaccharides and disaccharides in various applications, from low concentrations in synthetic urine samples to high concentrations in beverage samples. In an RFIC system, the hydroxide eluent is electrolytically generated inline to deliver accurate and precise concentrations for isocratic or gradient separations by only adding deionized water. Eluent generation eliminates carbonate contamination and errors from manual preparation. A capillary scale system with µL/min flow rates can run 24/7, always on and always ready for samples. Eluent consumption and waste generation are reduced to 5.2 L/yr and eluent generator cartridges can last up to 18 months.

212 High-Throughput and Highly Reproducible Sub-4 min Separation of Proteins and Antibodies Using Size Exclusion ChromatographyJustin Steve, Tosoh Bioscience, 3604 Horizon Dr., King of Prussia, PA 19406, Atis Chakrabarti

Aqueous size exclusion chromatography (SEC), popularly known as gel filtration chromatography (GFC) is a powerful analytical tool in the separation of protein species of differing size and their molar mass variants and impurities. Traditionally, GFC columns with a dimension of 7.8 mm ID x 30 cm L are used for analytical purpose. The longer column dimension lead to longer run times, sample dilution as well as substantial solvent waste. Instead a 4.6 mm ID x 15 cm L column may give high-throughput separation with shorter run time, high resolution and minimal solvent waste using conventional high-peformance liquid chromatography (HPLC). Here we show the use of a 4.6 mm ID x 15 cm L TSKgel SuperSW mAb HTP SEC column for the highly reproducible separation of proteins and antibodies in less than 3.5 minutes by using a moderate flow rate of 0.75 mL/min. Even at the slower flow rate of 0.35 mL/min, the overall run time was less than 7 minutes without consider-able decrease in resolution. Here we report the lifetime study and performance of the TSKgel SuperSW mAb HTP column in the separation of mAb using fast assay conditions. The retention time and other peak parameters were reproducible with a low percentage relative standard deviation (%RSD). This study illustrates adequate stability of the TSKgel SuperSW mAb HTP column for high speed, sub-4 minute separations of monoclonal antibodies.

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213 Environmentally Friendly HPLC Methods for the Determination of Renal Function Biomarkers, Creatinine and Uric Acid, in Human FluidsSi Zhou, University of Massachusetts Dartmouth, 285 Old Westport Rd., North Dartmouth, MA 02747, Zhuo Zhu, Ruiting Zuo, Xiaofei Lu, Yuegang Zuo

Two environmentally-friendly high-performance liquid chromatography (HPLC) methods for simultaneous determination of creatinine and uric acid in human urine samples have been developed. Human urine samples were pretreated by dilution, protein precipitation, centrifugation and filtration, followed by reversed-phase (RP) or hydrophilic HPLC separations. The developed methods provide simple, rapid separation and sensitive detection for the interest species using a C18 or an Agilen Zorbax SB-CN column (4.6 x 150 mm) in 6 minutes with UV detection at 205 nm by using an isocratic elution containing highly aqueous mobile phase at pH 4.75.[1] Quantification was carried out by relating the peak areas of identified compounds to that of the cimetidine as an internal standard. The detection limits for creatinine and uric acid were 0.04 µg/mL and 0.06 µg/mL, respectively. The recoveries of the standards added to urine samples were 91.6% - 97.7% for creatinine and 89.7% - 100.9% for uric acid and the relative standard deviation for both analytes was less than 6.0%. For the RP-HPLC, a simple retention recovery procedure is required after each run. These methods are suitable for the estimation of creatinine and uric acid in human urine and other bio-fluid samples.


[1] Y. Zuo, 2014, High-Performance Liquid Chromatography (HPLC): Principles, Procedures and Practices. Nova Science Publishers, Inc., New York.

214 Simultaneous Determination of 4-Cumylphenol, 2,4-Bis-(dimethylbenzyl)phenol and Bisphenol A in Blue Crab and Prawn SamplesZhuo Zhu, University of Massachusetts-Dartmouth, 285 Old Westport Rd., North Dartmouth, MA 02747, Mohammed Alshanqiti, Joseph Michael, Sarah Pereira, Yuegang Zuo

Bisphenol A (BPA), 4-cumylphenol (4-CP) and 2,4-bis-(dimethylbenzyl)phenol (2,4-DCP) are all high production volume chemicals and widely used in plastic and other consumer products. During the past two decades, BPA has attracted a great deal of scientific and public attention due to its presence in the environment and estro-genic property. Although 4-CP and 2,4-DCP are much more estrogenic and toxic than BPA, little information is available about their occurrence and fate in the en-vironment. In this study, a rapid, selective, accurate and reliable analytical method was developed for the simultaneous determination of 4-CP, 2,4-DCP and BPA in blue crab and prawn Macrobrachium rosenbergii. The method comprises an ultra-sound-accelerated extraction followed by capillary gas chromatographic (GC) sep-aration. The detection limits range from 1.50 to 36.4 ng kg−1 for the three alkylphe-nols. The calibration curves are linear over the concentration range tested with the coefficients of determination, R2, greater than 0.994. The developed method was successfully applied to the simultaneous determination of 4-CP, 2,4-DCP and BPA in prawn samples. The peak identification was confirmed using GC mass spectrom-etry. Bisphenol A, 2,4-bis-(dimethylbenzyl)phenol and 4-cumylphenol were found in blue crab and prawn samples in the concentration ranges of 0.67–5.51, 0.36–1.61, and 0.00–1.96 ng g−1 (wet weight), respectively. All relative standard deviations are less than 4.8%. At these environmentally relevant concentration levels, 4-CP, 2,4-DCP and BPA may affect the reproduction and development of aquatic organisms, including negative influence on crustaceans’ larval survival, molting, metamorphosis and shell hardening. This is the first study reported on the occurrence of 4-CP, 2,4-DCP and BPA in blue crab and prawn M. rosenbergii.

215 The Building Blocks of Life: Comparing Methods for Amino Acid AnalysisWilliam Hedgepeth, Shimadzu Scientific Instruments, 7100 Riverwood Dr., Columbia, MD 21046, Kenichiro Tanaka, Steve Wishnies

Amino acids are the building blocks of proteins and contain an amine and carbox-ylic acid functional group. High-performance liquid chromatography (HPLC) is the most popular technique for analyzing amino acids, and their usage has increased as supplements in the health food industry. Traditionally, amino acid analysis has been run with cation exchange chromatography with post-column derivatization. Pre-column derivatization with higher speed reversed-phase ultra-high pressure liquid chromatograhy has become more popular to shorten analysis times. Addition-ally, analysis time can be further shortened with automation and the use of a mass spectrometry detector. This poster discusses the various methods used to analyze amino acids by LC and the benefits and disadvantages of each method.

216 Glycerophospholipids Analysis by Comprehensive Two-Dimensional Liquid ChromatographyWilliam Hedgepeth, Shimadzu Scientific Instruments, 7100 Riverwood Dr., Columbia, MD 21046, Kenichiro Tanaka, Tetsuo Iida, Yoshiyuki Watabe, Junichi Masuda, Yoshihiro Hayakawa

Comprehensive two-dimensional liquid chromatography (LCxLC) is a powerful tool for the analysis of complex samples including pharmaceutical, biological, and nat-ural products. It can provide higher resolution that traditional LC (1-D/LC) can ever achieve because it combines two separation modes in a single analysis. Recently, the use of ultra high-performance liquid chromatography (UHPLC) has been suc-cessfully employed to remarkably decrease analysis time in the second dimension of comprehensive LC, and it is becoming a popular way to speed up 2-D/LC sepa-rations. In this poster, we present glycerophospholipids (GPLs) analysis using our newly-developed comprehensive two-dimensional liquid chromatograph system. GPLs are the major component of biological membranes. They can not only act as a barrier from the external environment, but can also play a key role in a variety of biological processes including membrane trafficking and signal transduction. Thus, analysis of GPLs is one of the most important studies in the metabolomics field. Although reversed-phase (RP) HPLC coupled with electrospray ionization tandem mass spectrometry (ESI-MS-MS) is an effective strategy for lipidomics, there is still room for further improvement of the analytical methods. One drawback to perform-ing determination of GPLs is ion suppression by co-eluting compounds. To obtain reliable results, complete separation of target GPLs by comprehensive two-dimen-sional liquid chromatography with ESI-MS-MS is an effective strategy.

217 Development of Stability Indicating HPLC Assay and Degradation Product Methods for a Peptide Drug ProductKaimeng Zhou, Merck, 556 Morris Ave., Summit, NJ 07901, Jing Zhang, Zheng Zhao, Wendy Zhong, Claudia Neri, Yun Mao

Peptide separation is very challenging especially for peptide drug product contain-ing impurities and degradants whose structures are highly similar to their parent peptide. To guide peptide formulation optimization, good stability-indicating meth-ods which provide baseline separation of peptide and its impurities and degradants is essential. This presentation focuses on the development of reversed-phase high-performance liquid chromatography (HPLC) methods for peptide A impurities and degradants. Our analytical strategy to develop good stability-indicating methods involves multiple HPLC modes to achieve the separations of complex peptide impu-rities and degradation products for the peptide stability characterization. A number of conventional and new columns including C18 and phenyl-hexyl stationary phases were screened. To optimize resolution, several mobile phases containing different organic modifiers and salts were examined. A broad range of separation parameters were evaluated. It was found that column temperature and gradient have strong impact to the critical pair resolution and need to be carefully tuned and controlled. The optimum conditions for assaying Peptide A and its impurities were obtained with a mobile phase containing acetonitrile with perchlorate (NaClO4) at acidic pH as an ion-pairing reagent. A mobile phase containing trifluoroacetic acid (TFA) was found to provide better resolution for the peptide degradation products. Liquid chromatog-raphy tandem mass spectrometry was utilized to identify the degradants and verify the separation. This degradant method has been successfully validated and applied in stability monitoring of clinical formulations.

218 Carbohydrate Analysis in Beverages and Food Using Pulsed Amperometric and Charged Aerosol DetectionQi Zhang, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA 01824, Marc Plante, Bruce Bailey, David Thomas, Ian Acworth, Michael Hvizd

Carbohydrates are important food components affecting taste and nutrition. The determination of the types and levels of carbohydrates in foods is important for energy evaluation, nutritional labeling, quality control and for identifying possible product adulteration. Separation and detection of carbohydrates can be challenging. Simple carbohydrates are highly polar, uncharged, and as they lack a chromophore cannot be measured directly by UV absorbance detectors. High-performance liq-uid chromatography (HPLC) with various detection techniques has been used for carbohydrate analysis, such as infrared, mass spectrometry, UV or fluorescence following derivatization. Although methods utilize derivatization improve the chro-matographic resolution and detector sensitivity, they can lead to increased assay variability. This poster presents two approaches for carbohydrate analysis that solve the challenges for separation and detection of carbohydrates without requirement for laborious derivatization: high performance anion exchange chromatography with pulsed amperometric detection, hydrophilic interaction liquid chromatography and charged aerosol detection. The electrochemical detector now with pulsed ampero-metric detection capabilities when coupled with a gold working electrode provides high sensitivity and selectivity for the measurement of carbohydrates in complex food sample matrices. The charged aerosol detector is a mass-sensitive detector that can measure all non-volatile, and many semi-volatile compounds in a sample,

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typically with low nanogram sensitivity. Application examples for various juice sam-ples, milk and other dairy products, syrup and honey are discussed.

219 Targeted Research of Peptides by LCxLC-MS-SQDJoanne Mather, PerkinElmer, 940 Winter St., Waltham, MA 02451, Noelle Elliott, Sean Daugherty, Sabine Heinisch

One-dimensional liquid chromatography coupled to mass spectrometry detection (LC-MS) is a widely used analytical method which can be applied to a broad variety of analysis fields such as pharmaceuticals, biological compounds as well as envi-ronmental samples. However, the complexity of such samples causes two relevant issues in LC-MS which can be overcome with comprehensive two-dimensional liq-uid chromatography coupled to MS (LCxLC-MS): 1) The maximum peak capacity that can be achieved in 1D-LC for peptide analysis, taking into account the current equipment constraints, is about 1000-1500 at the expense of very long gradient times and very long columns. Conversely, the theoretical peak capacity in LCxLC is the product of peak capacities in each dimension and hence could lead, for in-stance, to 2500 considering a peak capacity as low as 50 in each dimension. 2) In LC-MS, poorly separated compounds reach together the mass spectrometer source and as a result are subjected to an ion suppression phenomenon which may com-promise their detection. The increase in total peak capacity in LCxLC, and hence the better separation of compounds before entering the ionization source is expected to reduce ion suppression effects. This study shows the great benefit of LCxLC-MS for peptide analysis via reverse-phase (RPLCxRPLC) separations of protein digests. These separations led to much higher peak capacities than those expected in LC-MS. A significant reduction of ion suppression effects is brought out. Moreover, these results are associated to a huge decrease in analysis time. Our results point out the right adequacy between a simple quadruple mass spectrometer coupled to LCxLC, and the targeted research of peptides in complex protein tryptic digests.

220 Benefits of Using Wide Pore Superficially Porous Particles for Biomolecule SeparationsWu Chen, Agilent Technologies, 2850 Centerville Rd., Wilmington, DE 19808, Anne Mack, Jim Martosella, Kunqiang Jiang

Superficially porous particles with pore size ranging from 90Å to 120Å have been a great success for fast separation of small molecules over totally porous particles in the recent years. However, superficially porous particles with wide pore size of ≥300Å provide even better benefits over totally porous particles for separation of large biomolecules such as proteins and monoclonal antibodies (mAbs) because the analyte diffusion length inside a particle plays a more important role for fast separation in large molecule separation than in small molecule separation. Wide pore superficially porous particles in 5 µm size have been used for protein separa-tions for a decade.[1] And more recently, wide pore superficially porous particles in smaller size have been developed to meet the need of continuing interest in larger therapeutic molecules by biopharmaceutical companies.[2] This report includes fast separations of intact protein mixtures, as well as examples of very high-resolution separations of larger monoclonal antibodies and associated variants by using wide pore superficially porous particles, and their advantages over wide pore totally po-rous particles.


[1] J. Chromatogr. Sci.; 2008; 46(3); 261-8; Ricker R. D., Woodward C. B. 3rd, Forrer K., Permar B. J., Chen W.; Options for rapid analysis of peptides and proteins, using wide-pore, superficially porous, high-performance liquid chromatography particles with unique bonded-phase ligands.

[2] J. Chromatogr. A; 2013; 1315; 118-26; Schuster S. A., Wagner B. M., Boyes B. E., Kirkland J. J.; Optimized superficially porous particles for protein separations.

221 Analysis of Phenolic Compounds in New Hampshire Sugar Maple Sap by LC-MSElizabeth Brady, University of New Hampshire, 23 Academic Way, Durham, NH 03824, Walter C. Shortle, Sterling Tomellini, Martha Carlson, Barrett N. Rock

A liquid chromatography-mass spectrometry (LC-MS) method has been developed for the analysis of selected phenolic compounds in sugar maple sap. The method employs a direct injection analysis in order to avoid expensive and time consum-ing sample preparation steps. Interest in phenolic compounds has grown in recent years as research has demonstrated their potential antioxidant, antiradical and an-timutagenic capabilities. While many studies have focused on these compounds in relation to human health, relatively less is focused on the relationship of phenolic compounds to the health of the plant in which they are produced. The LC-MS meth-od developed in this study provides the capability to detect selected phenolic com-pounds in sugar maple sap samples provided by volunteers throughout the state of New Hampshire using only a syringe filter for sample preparation. Quantitative data from the 2011, 2012 and 2013 tapping seasons were analyzed for trends and vari-ability. Preliminary observations show that a wide range of variability are possible for these compounds over individual tapping seasons and significant differences

exist in their mean concentrations over the three years studied. Signals for as of yet unidentified compounds also displayed seasonal and yearly trends. These results are presented along with a discussion of the challenges of identifying the selected phenols in the maple sap matrix.

222 Rapid Identification and Determination of Polyphenols in Herbal Plants by UPLC/MS/MSSaliha Esin Çelik, Istanbul University, Faculty of Engineering, Department of Chemistry, 34320 Avcılar, Istanbul, Turkey 34320, Burcu Bekdeşer, Ayşe Nur Tufan, Mustafa Özyürek, Kubilay Güçlü, Reşat Apak

This study reports the characterization of polyphenolic compounds in pennyroyal (Mentha pulegium), marjoram (Origanum marjoram) and lavender (Lavandula vera) extracts by using ultra performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS-MS) in negative mode of electron spray ioniza-tion (ESI). 9 phenolic acids (gallic acid, protocatechuic acid, vanillic acid, caffeic acid, ferulic acid, sinapic acid, syringic acid, p-coumaric acid, rosmarinic acid) and 9 flavonoids (catechin, rutin, hesperidin, morin, fisetin, quercetin, naringenin, kae-mpferol and apigenin) were monitored within 12 minute gradient elution program. The developed method was validated with good precision (RSD % 0.54- 2.72 for intra-day, 1.71-4.64 for inter-day), reproducibility (REC% 95.1-104.6) and linearity (r=0.9989-0.9999). Polyphenols were quantitatively analyzed in the plant extracts. Total antioxidant capacity and total phenolic content of samples were measured by using CUPRAC (cupric reducing antioxidant capacity) and Folin assays. Microwave assisted extraction (MAE) technique was used to extract phenolic antioxidants in herbal plants. The extracts exhibited high antioxidant capacity (ranged from 0.33 mmol T/g to 0.70 mmol T/g). The proposed method was found to be easy, fast, reproducible and convenient for quantitative analysis of polyphenols.

223 Determination of Corilagin by Hybrid Solid-Phase Extraction (SPE) Ultra-Performance Liquid Chromatography Coupled with Triple Quadrupole Mass Spectrometry in Rat Plasma and its Application to Pharmacokinetic and Bioavailability StudiesAtul S. Rathore, Poona College of Pharmacy, Bharati Vidyapeeth University, Erandawane, Paud Rd., Pune 411038, India, Sathiyanarayanan Lohidasan, Kakasaheb R. Mahadik

A sensitive, specific and rapid hybrid SPE ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS-MS) method has been established to study pharmacokinetic properties of corilagin. Corilagin is an ellagitannin, was determined in rat plasma with ellagic acid as internal standard. Chromatographic separation was performed on a Waters ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm, 1.7-µm) with gradient elution system at a flow rate of 0.3 mL/min. The mobile phase was composed of 0.1% formic acid water and 0.1% formic acid acetonitrile solution. Analysis was performed under a triple-quadruple tandem mass spectrometer with an electrospray ionization (ESI) source via the multiple reaction monitoring (MRM) mode to determine corilagin at [M-H]- m/z 633.0 → m/z 301.0 and that of IS at [M-H]- m/z 300.9 → m/z 145.0 within 3 min. The assay method exhibited good separation of corilagin from the interference of endogenous substances. The lower limit of quantification (LLOQ) was 20 ng/mL, with a good linearity within the concen-tration range of 20–1000 ng/mL (r = 0.9993). Intra-day and inter-day precision RSD was less than 7.83%; intra-day and inter-day accuracy was 90.26% and 102.12% respectively. The extraction recoveries of corilagin was over 90% in rat plasma, and the extraction RSD was less than 2.06%. The established UPLC–ESI-MS-MS meth-od is rapid and sensitive, which has been successfully applied to a pharmacokinetic study of corilagin in rats for the first time following oral (10 mg/kg) and intravenous (1 mg/kg) administration.

224 Rapid Quantitative Determination for Comparative Analysis of Biologically Active Piperamides, Phenolics, Flavonoids and Terpenoid in Fruits and Leaves of Ten Piper Species using UPLC-ESI-MS-MSPreeti Chandra, Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Rd., Lucknow 226031 India, Renu Pandey, Brijesh Kumar

Piper species are of high commercial and economic importance have been used extensively in traditional medicines. Piperamides are the main bioactive compounds in the species other than, phenolics, flavonoids and terpenoid. To determine the distribution and content of these compounds in different plant parts (fruits & leaves) of ten Piper species, a reliable method has been established using ultra high-perfor-mance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometry (UPLC-ESI-MS/MS). Thirteen constituents, namely piperamides (piperine, piperlongumine and piperlonguminine), phenolics (caffeic acid, ferulic acid, protocatechuic acid, rosamarinic acid and vanillic acid), flavonoids (quercetin, kaempferol, apigenin and luteolin) and terpenoid (ursolic acid) were simultaneously determined in 8 min. The analysis was accomplished on an Acquity UPLC BEH C18 (2.1 mm × 50 mm, 1.7-µm) column using gradient elution with 0.1% (v/v) formic acid water and acetonitrile. Piperamides were detected in positive mode while phe-

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nolics, flavonoids and terpenoid were detected in negative electrospray ionization tandem mass spectrometry operating in multiple-reaction monitoring (MRM) mode with continuous polarity switching. A good linear regression relationship for each analyte was obtained over the range from 1-250 ng/ml. The intra- and inter-day assay precision ranged from 0.63 to 3.37% and 0.91 to 4.09% respectively. The recovery relative standard deviation (RSD) measured at three concentration levels varied from 0.78-2.78%. The method sensitivity expressed as limit of quantitation was typically 0.06-3.88 ng/ml. The present study can provide necessary information for the rational utilization and quality control of Piper species.

225 Analysis of Emulsifiers in Foods by High Pressure Liquid Chromatography and Charged Aerosol DetectionMarc Plante, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA 01824, Bruce Bailey, Ian Acworth, Qi Zhang

Emulsifiers are used to maintain a uniform suspension of immiscible materials. These compounds are typically surfactants, and can be designed for use in spe-cific applications and products. Acylglycerols are used in food products containing oil and water (e.g. margarine, mayonnaise); lecithin is commonly found in choco-late and spray oils; acid esters of monoglycerides are used for dough conditioners; and hydroxypropylmethyl cellulose (HPMC) is use to thicken dairy products and help improve flavor characteristics. HPMC is also an important emulsifier used in the pharmaceutical industry. The analysis of emulsifiers is becoming increasingly important, for product quality, consistency and stability properties. High-pressure liquid chromatography is one of the more prevalent methods for analyzing these compounds. However, the majority of analytes do not contain suitable chromophore characteristics for UV detection, which then requires the use of a universal detector, such as evaporative light scattering, refractive index, or charged aerosol detection. The charged aerosol detector was used in the analyses of two emulsifiers (HPMC and lecithin) that were extracted from food products. The charged aerosol detector, a sensitive mass-based detector, is ideally suited for the direct measurement of emulsifiers, as they are non-volatile and non-chromophoric compounds. It offers excellent sensitivity (down to low nanogram amounts on column), a dynamic range of over four orders of magnitude, and similar inter-analyte response independent of chemical structure.

226 Analysis of Phospholipids in Natural Samples by Normal Phase HPLC and Corona Charged Aerosol DetectionMarc Plante, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA 01824, Bruce Bailey, Ian Acworth, Qi Zhang

Phospholipids are a broad class of lipids that can be divided into glycerophospholip-ids (GPLs) and sphingolipids. Both groups show great structural diversity. Phospho-lipids are amphiphilic molecules, having a hydrophilic head group, and a lipophilic fatty acid tail. Several families of GPLs exist biologically, differing in the type of polar head group present, and include: phosphatidylcholine (PC), phosphatidylethanol-amine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylcholine (DPPC), and lysophophatidylcholine (LPC). Each compound contains many spe-cies resulting from differences in their fatty acid composition. For example, PC may contain several different fatty ligands, which will result in multiple peaks by reversed phase chromatography. Differences in polar moieties were used to separate these analytes by normal phase liquid chromatography and provided superior quantitative data with less effort. Analysis of phospholipids in red palm oil and krill oil are provid-ed. The Corona Veo charged aerosol detector, a sensitive mass-based detector, is ideally suited for the direct measurement of phospholipids, as they are non-volatile and non-chromophoric compounds. It offers excellent sensitivity (down to low nano-gram amounts on column), a dynamic range of over 4 orders of magnitude, and similar inter-analyte response independent of chemical structure. The developed method is based on an original publication by Rombaut, R., et al., (J. Dairy Sci., 2005, 88, 482), that enables the direct measurement of a number of GPL and sphin-golipid species, each as near-single peaks.

227 Proteomic Investigation of Saliva from People with Smith-Lemli-Opitz Syndrome (SLOS) and ControlsEmmalyn J. Dupree, Clarkson University, 49 Market St., Apt. 3, Potsdam, NY 13699, Megan M. Borland, Costel C. Darie, Alisa G. Woods, Kelly L. Wormwood

Saliva samples were analyzed from people with Smith-Lemli-Opitz Syndrome (SLOS) and controls using sodium dodecyl sulfate polyacrylamide gel electropho-resis (SDS-PAGE), in-gel trypsin digestion, and nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) to investigate the differences between the proteomes of people with SLOS and controls. The pending results will hopefully provide more information about the protein changes that occur in SLOS, potentially leading to identification of treatment targets. These results may also shed some light on the possible link between SLOS and Autism Spectrum Disorder (ASD), as ASD is almost always co-morbid with SLOS. This could possibly lead to better diagnosis, treatment and understanding of ASD as well.

228 Investigation of Tumor Differentiation Factor (TDF)-Induced Cell Differentiation Using Mass Spectrometry Based ProteomicsDevika Channaveerappa, Clarkson University, 8 Clarkson Ave., Potsdam, NY 13699

Tumor differentiation factor (TDF) is a protein, produced by the pituitary gland and is secreted into the bloodstream. TDF and TDF derivatives induce differentiation of breast and prostate cells, but not of other cells. However, the mechanism of action of TDF to induce differentiation is unknown. Therefore, one option to identify the mem-bers of the TDF pathway is to investigate the proteomes of the TDF-treated and –untreated human breast and prostate cells. In our preliminary studies, we observed substantial differences in the sodium dodecyl sulfate polyacrylamide gel electro-phoresis (SDS-PAGE) protein patterns of untreated cells and cancer cells treated with TDF analogs. Hence, the current study is focused on the proteomic analysis of TDF-untreated and TDF treated MCF7 and DU145 human breast and prostate cancer cells, on the functional investigation of TDF as well as on the localization of TDF. These experiments will help elucidate the mechanism through which TDF induces cell differentiation. To carry out these investigations, MCF-7 human breast and DU-145 human prostate cancer cells in culture were grown to confluence under standard cell culture conditions and transferred to six-well plates in a serum-free medium. The cells were treated with various concentrations of TDF analogs (0 to 10 µg), lysed and the proteins were separated by SDS-PAGE for qualitative inspec-tion and direct comparison of the samples. The gel pieces were digested and the different peptide mixtures were analyzed using a nanoAcquity ultra-performance liquid chromatography coupled with a quadrupole time-of-flight micro mass spec-trometer followed by data analysis. Further validation and functional studies will also be performed.

229 Reverse-Phase UPLC Assay for Purity and Concentration Determination during Admixtrue Compatibility Study of a Therapeutic Protein ProgramShenjiang Yu, Merck, 2000 Galloping Hill Rd., Kenilworth, NJ 07033, Shuai Shi, Joseph Rizzo, Venus Hashemi, Chakravarthy Narasimhan, Valenthyn Antochshuk, Daisy Richardson, Mohammed Shameem

A pharmaceutical admixture consists of a drug product mixed with a suitable dilu-ent in an intended dosing/delivery device for the purpose of parenteral infusion to the patient. Regulatory agencies have specific requirements for the demonstration of the compatibility of the drug product with reconstitution diluents, because many of the therapeutic proteins are dosed intravenously in the form of admixtures. A reverse-phase liquid chromatography (RPLC) method has been developed to mon-itor the concentration and purity during the admixture study. A ultra-performance liquid chromatography (UPLC) system and sub-2-µm column have been utilized to obtain improved separation. An appropriate column pore size has chosen for the therapeutic protein. Protein loss through the dosing device and infusion lines at low concentration level has been discovered. A preventive action has been taken by conditioning the infusion lines by protein sample. In addition, some new impurities during the admixture have been detected by the RPLC method. Some preliminary investigation about the impurities has been reported.

230 The Use of Diamond-Based Core-Shell HPLC Particle in the Analysis of ProteinsDavid S. Jensen, Diamond Analytics, 1260 S 1600 W., Orem, UT 84660, Janusz Zukowski, Andrew E. Dadson

Diamond core-shell particles are composed of a solid, spherical carbon core and a functionalized porous nano-diamond shell. The composite material utilizes nano-di-amond to create a permeable, but extremely stable shell that can tolerate high tem-peratures and extreme pH separation conditions. Nano-diamonds also have a much higher thermal conductivity when compared to silica. This innovative material offers a new standard in liquid chromatography performance, providing a powerful tool for difficult drug and protein separations when compared to more traditional columns. Herein, a variety of protein applications are demonstrated under conditions that will normally stress and degrade a traditional high-performance liquid chromatography (HPLC) column. Resolution of oxidized, native and reduced forms of Interferon vari-ants is shown which were separated under highly acidic conditions. Additionally, a systematic approach is discussed on how to use this diamond-based HPLC material for protein separations.

231 Accelerated Solvent Extraction and In-Cell Sample Clean-Up of Marine Sediment for the Analysis of Persistent Organic Pollutants by GC-MS-MSEmma L. Gatley, University of Connecticut, 3107 Horsebarn Hill Rd. U-4210, Storrs, CT 06269, Emmanuel O. Omari, Aliaksandr V. Yeudakimau, James D. Stuart, Christopher R. Perkins, Molly B. Williams, Anthony A. Provatas

Contamination of sediment is an important factor when evaluating the overall qual-ity of water since it holds a crucial role in ecosystem and human health. Continu-ous pollution from non-point source run-off, petroleum production and transport,

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000961-EAS_17x11_Spread_PREP.indd 1 10/20/14 8:43 AM

EAS is pleased to announce that we have partnered with Pharma Webinars to offer new low-cost

webinars to all analytical scientists launching in 2015

FebruaryOverview of Protein Analysis: Analytical Testing and

SpecificationsRobert Johnson, Consultant

MarchICP and Elemental Impurities Testing in

Pharmaceutical Analysis

Stay tuned to EAS.org; more details to follow

Follow us for more info:

NEW IN 2015

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Garden State Exhibit Center | Somerset, NJNovember 16–18, 2015EAS.org


2015 Eastern Analytical Symposium & Exposition

Three-day technical program

State-of-the-art exposition featuring analytical equipment and services

Extensive selection of short courses and professional development workshops

Employment bureau, and more

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Call for PapersMarch 1–June 15, 2015


Capillary Electrophoresis


Conservation Science

Environmental Analysis

ESR Spectroscopy

Food Analysis

Forensic Analysis

Gas Chromatography




Industrial Hygiene

Ion Chromatography

IR Spectroscopy

Laboratory Automation

Laboratory Management

Laboratory Miniaturization

Ligand Binding Assays





Near-Infrared (NIR) Spectroscopy

NMR Spectroscopy

Pharmaceutical Analysis

Process Analytical Science

Protein Analysis



Raman Spectroscopy

Sample Preparation

Science Education


Size Exclusion Chromatography

Solid State Analysis

Space Analytics

Supercritical Fluid Chromatography

Surface Science

Very High-Pressure LC/ Ultra High-Pressure LC

Vibrational Spectroscopy

EAS seeks contributed abstracts in these and other analytical fields:

Please help EAS serve you better. If you have suggestions for a short course offering, or would like to teach a short course, please contact us at [email protected]. If you have suggestions for program, or would like to organize a session, please contact us at [email protected].

Abstracts received from June 16–Sept 30, 2015 will be reviewed for quality to be included in the poster session. You will be notified via email when/if the abstract is placed.

Call for Proposals

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November 20142014 EAS Abstracts

incomplete combustion of fossil fuels, industrial operations, agricultural spraying, and improper disposal of industrial materials all contribute to the concentration of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and pesticides in sediment. Elevated levels of PAHs within the environment are consid-ered hazardous to human health, with exposure proven to have carcinogenic and mutagenic effects. Similarly, PCBs and pesticides also are carcinogenic while con-tributing to reproductive complications and nervous system damage respectively. Subsequently, various methods have been developed to detect PAHs, PCBs, and pesticides in sediment with the majority involving extensive clean-up steps such as gel permeation chromatography (GPC) and solid-phase extraction. The objec-tive of this project was to analyze for Environmental Protection Agency’s 16 priority PAHs, select PCBs and pesticides in marine sediment utilizing accelerated solvent extraction and in-cell alumina clean-up for rapid sample preparation. This was fol-lowed by gas chromatography tandem mass spectrometry (GC-MS-MS) analysis in a “soft” Electron ionization setting of 40 eV instead of the conventional 70 eV to increase analyte sensitivity. To expedite the evaporation process, a Genevac EZ-2 evaporator was utilized. This validated methodology is presented for 16 PAHs, 22 PCBs and 19 pesticides of interest.

232 Characterization of the Points and Extent of Glycosylation in Steviol GlycosidesLi Chen, International Flavors & Fragrances, 1515 State Highway 36, Union Beach, NJ 07735, Richard Hiserodt

Steviol glycosides are a sweet principal of Stevia rebaudiana Berotoni.They are up to 450 times sweeter than sucrose. Steviol can be glycosylated at the C19-carboxyl group or the C13-hydroxyl group. The position and extent of glycosylation will affect the sweetness and mouth feel attributes of the steviol glycosides. The aim of this study is to evaluate the extent to which mass spectrometry can be used to predict the position and extent of glycosylation. To accomplish this, the product ion spec-tra of fourteen steviol glycosides were acquired using a Linear Trap Quadropole Orbitrap Discovery high resolution, accurate mass-mass spectrometer. The data from this study showed that using both full scan and product ion spectra (MS2), the position and extent of glycosylation can be predicted for these compounds.

233 Evaluation of Low- and High-Resolution Mass Spectrometry Techniques for Metabolite Profiling and IdentificationKhushbu G. Gajjar, Kean University, 1000 Morris Ave., Union, NJ 07083, Dil Ramanathan

Preclinical species drug metabolism studies are integral part of drug discovery process to understand and predict the metabolism of drugs in humans. Loratadine (LOR) is a second-generation histamine H1 receptor antagonist drug which is used in the treatment of allergic symptoms. In the present study, LOR and its primary me-tabolites such as desloratadine (DL), hydroxy desloratadine (OH-DL) and glucuron-ide metabolites of LOR and DL in plasma and urine of male and female rats treated with a single oral 8 mg/kg dose of LOR were analyzed by a low resolution linear trap quadropole (LTQ) and a high resolution Orbitrap mass spectrometers with a run time of 3 min. The main purpose of this analysis is to compare the two types of mass spectrometers to understand which type of mass spectrometer should be used to accurately and precisely identify and characterize drugs and their metabolites. Plas-ma and urine from rats were collected at different time intervals over 48 hours to identify and profile circulating and excretory metabolites. The pharmacokinetics of LOR and DL were also studied.

234 Gas-Phase Fragmentation of Metal-Cationized Acid SaltsRobert D. Hale, Stevens Institute of Technology, Department of Chemistry, Hoboken, NJ 07030, Athula B. Attygalle, Carl S. Weisbecker

Very few detailed investigations have been undertaken on the mass spectromet-ric fragmentation of cationic adducts of salts of small acidic molecules. Generally, collision-induced mass spectra of small cationized molecules are considered to be rather uninformative, because most spectra show only a single product-ion peak for the ejected metal cation. Nonetheless, in our exploration of oxalate and car-bonate salts, we found that these ions fragment in an unusual manner, generating product ions that retain all three metal atoms of the precursor ion. Upon collision in-duced dissociation (CID), the [Na3C2O4]+ (m/z 157); [39K3C2O4]+ (m/z 205); [7Li-3C2O4]+ (m/z 109); and [Na3CO3]+ (m/z 129) ions ejected a 44-Da fragment even at a collision energy as low as 2 eV. DFT calculations predicted that the product ions, [Na3C2O4]+ and [Na3CO3]+ are planar in geometry , and that the ejection of CO2 requires less energy than an ejection of a Na+. When the sodium adducts from the labeled compounds [Na2C218O4] (m/z 165) and [Na2C18O3] (m/z 135) were subjected to CID, the spectra showed a peak at m/z 117 and m/z 87, respective-ly, which confirmed that the ejected fragment was CO2. Furthermore, product-ion spectra for [Na2C2O4•Na+], [39K2C2O4•39K+] and [Na2CO3•Na+] recorded at higher collision energies showed an additional peak for an even-electron ion formed by a loss of an M• from the m/z 113 ion to form the M3O+ ion, which violates the even-electron rule.

235 Recognition of N-Oxides and Quaternary Ammonium Compounds by Chloride Ion Attachment LC-MSShirley A. Rodriguez, Boehringer Ingelheim, 900 Ridgebury Rd., PO Box 368, Ridgefield, CT 06877, Keith B. McKellop, Scott Pennino, Daniel L. Norwood

Gas phase chloride ion attachment negative ion atmospheric-pressure chemical ionization (APCI) liquid chromatography mass spectrometry (LC-MS) has been ap-plied to the analysis of drug molecules, such as nitroglycerin, which are difficult to ionize by more conventional LC-MS methods. [M+Cl]- ions are formed by non-cova-lent gas phase interactions of analyte molecules with Cl- derived from the addition of chlorinated reagents (e.g., chloroacetonitrile) to the high-performance (HPLC) mobile phase. At the EAS 2013 we presented a comparison of two approaches (APCI and electrospray ionization) to evaluate the selectivity of chloride attachment ionization to a diverse group of compounds including hydroxylated (sugars), phe-nols, nitro, N-oxides and sulfur containing, and aliphatic vs. non-aliphatic structures using standard LC-MS platforms and ionization conditions. From these studies we observed that N-oxides showed an interesting pattern of attachment ions, in cases where the attachment worked, of [M+Cl]- and [M+Cl+HCl]-. N-oxides can be geno-toxic impurities in pharmaceuticals and identification by MS can be inconclusive and many times requires confirmation by other techniques, such as, LC-nuclear mag-netic resonance. Therefore, the work has continued on the recognition of N-oxides and quarternary ammonium compounds by chloride ion attachment LC-MS and is presented here.

236 Analysis of Drugs of Abuse by LC-TOF MS: A Unique Quantitation Viewer for Streamlining WorkflowsLeslie Sullivan, PerkinElmer, 740 Bridgeport Ave., Shelton, CT 06484, George Perkins, John Moncur, Scott Campbell, Stephen Roberts, Bill Hahn

Workplace drug screening has been in existence in the United States since the 1980’s. Employers commonly use drug screening to test prospective employees for the existence of alcohol and illegal drugs. It is also common during an accident at the job site. Most illegal substances remain in the body for at least 24 hours follow-ing use. The Substance Abuse and Mental Health Services Administration (SAMH-SA) panel (formally referred to as the NIDA panel) classes are chosen to represent the most commonly abused drugs in the general public, this list includes cocaine, marijuana, amphetamines, opiates and phencyclidines. Liquid chromatography tan-dem mass spectrometry (LC-MS-MS) is often used to quantitate drugs of abuse in biological fluids due to its sensitivity and selectivity. However, triple quadrupole tech-niques can have an undesirable high cost and lack the ability to easily identify new or unknown compounds. This becomes significant with the constant development of drugs, making it harder to identify and quantify them as they appear in the samples. We present an alternative technique to quantitate drugs of abuse in urine utilizing a rapid dilute and shoot with LC separation method in combination with time-of-flight mass spectrometry. We use a unique quantitation viewer to rapidly review the data for integrity and final results, thus streamlining workflows.

237 High-Throughput UPLC-MS-MS Method Development for Phenolic Acids (Chlorogenic Acids) Determination in American CranberryYifei Wang, Rutgers University, RM 379, Foran Hall, 59 Dudley Rd., New Brunswick, NJ 08901, Nicholi Vorsa, Ajay P. Singh

Phenolic acids are the class of compounds abundant in Rubiaceae family and oth-er plant species including Vaccinium species. Based on biosynthetic pathway the accumulation of phenolic acids may vary in the different organs of the plant e.g. seeds, stem, leaf, fruit epidermis and fruit flesh. Chlorogenic acids (CGA) are a family of esters formed between quinic acid and certain trans cinnamic acids such as caffeic acid, ferulic acid, p-coumaric acid and their dimers. As per literature the chlorogenic acids absorbed in humans through dietary digestion, and have been re-ported to possess anti-oxidant, anti-hypertensive activities as important implications to human health. A numerous diversity of structures exist in the CGA family, varying by the identity, number and position of their acyl residues, identification of the var-ious species has been difficult and problematic due to their structural similarities. A high-throughput ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS-MS) method has been developed to identify 10 indi-vidual CGA compounds simultaneously, including 3 caffeoylquinic acids (CQA), 3 di-caffeoylquinic acids (diCQA), 1 feruloylquinic acid (FQA) and 3 caffeoylferuloylquin-ic acids (CFQA). The method was successfully applied to compare different varietal cranberry organ extractions for their CGA composition. Six CGAs were successfully identified, including 3/4/5-CQA, 5-FQA, 3,5-diCQA and 4,5-diCQA. Quantitation of characterized CGAs in cranberry organs was determined by multiple reaction moni-toring (MRM) mode in precursor/product ion pair of m/z 353.2/191.5 for 3/4/5-CQA, m/z 366.7/191.0 for 5-FQA and m/z 515.3/353.1 for 3,5-diCQA and 4,5-diCQA.

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November 20142014 EAS Abstracts

238 Screening and Sequencing of Gangliosides from Human Glioblastoma Multiforme by High-Performance Mass Spectrometry Loredana M. Lupu, Clarkson University, Department of Chemistry and Biomolecular Science 5810, Potsdam, NY 13699, Megan Borland

Glioblastoma multiforme represents one of the most common types of brain can-cer known to man. It is a grade IV primary neoplasm of the central nervous sys-tem, usually located in the cerebral cortex. Following drastic surgical resection and chemotherapy the prognosis is very poor. At the present time one strategy for the investigation of tumor treatments is to target invading tumor cells by using spe-cific ligands. Gangliosides, syalilated glycosphingolipids represent a class of such ligands; therefore characterization of their expression and structure in gliblastoma tumor is of a high clinical importance. In the present work, a systematic profiling of ganglioside composition followed by structural analysis of individual species in a glioblastoma specimen vs. normal brain tissue was carried out using the advanced mass spectrometric (MS) method based on chip-nano electrospray ionization quad-rupole time-of-flight (QTOF) MS. It was discovered that the total number of distinct individual gangliosides is actually much larger than reported before. Tandem MS provided for the first time the complete and accurate set of structural elements for glioma-associated GD2. The results represent a comprehensive evaluation of the ganglioside heterogeneity and structural elucidation of several species considered as human glioma-associated structures.

239 Identification of the O-Glycoforms Expressed in the Urine of a Patient Suffering from Schindler Disease Using Chip-Based Nanoelectrospray Tandem Mass SpectrometryAdrian C. Robu, Clarkson University, Department of Chemistry and Biomolecular Science, 8 Clarkson Ave., Box 5810, Potsdam, NY 13699, Megan Borland, Costel C. Darie, Mirela Sarbu, Aurel Vlaicu, Jasna Peter-Katalinic, Alina D. Zamfir

Schindler disease is a rare, however severe condition, recently recognized as an autosomal recessive disorder, defined as a α-N-acetylgalactosaminidase (α-NAGA) deficiency. Deficient activity of the lysosomal enzyme α-NAGA leads to an abnor-mal accumulation of sialylated and asialo-glycopeptides and oligosaccharides with α-N-acetylgalactosaminyl (GalNAc) residues, causing a 100 times higher concen-tration of O-glycans in patient urine than in healthy controls. Screening, structural characterization and complete identification of O-glycosylated amino acids and pep-tides extracted from patient urine using tools of high sensitivity, reproducibility and accuracy is nowadays of major clinical importance. In this study an integrative mass spectrometry (MS) approach based on fully automated chip-nanoelectrospray quad-rupole time-of-flight mass spectrometer was optimized and applied for the discovery and structural characterization of O-glycopeptides in a fraction from the urine of a patient diagnosed with Schindler disease type I. A mixture of O-glycopeptides ex-tracted and purified from an age matched healthy subject served as control. Struc-tures with relevant biological significance, previously not described, such as O-fu-cosylated tetrasaccharides and chains up to pentadecamers O-linked to threonine, serine or serine-proline were identified in the pathological urine and characterized by tandem MS (MS/MS). 49 glycoforms were discovered in the investigated urine fraction from Schindler disease vs. only 14 in control urine. A number of species with long chain glycans were not previously reported as associated to this condition. All glycopeptides were detected in only 1 min analys is time, with a sensitivity situated in the low picomole range.

240 Air Sampling of Fragrance Compounds Using the GERSTEL GSS-FP Automated SamplerJohn Stuff, Gerstel, 701 Digital Dr., Linthicum, MD 21090, Jacqueline Whitecavage

Air fresheners are commonly used in households to mask unpleasant odors. Many types of fragrances are commercially available. The fragrance can be a complex mix of many components. The effectiveness of the freshener to mask the off odors over time can be directly related to the air concentration of the fragrance components. Therefore, an important aspect in product development is airborne concentration of the fragrance components. This study describes the use of the GERSTEL GSS-FP sampler for the collection of fragrance compounds in air. The GSS-FP is a 28 position active sorbent tube gas/air sampling system designed to collect sample onto sorbent tubes. The sampler can collect multiple samples on a pre-programmed schedule or on demand from the user interface. Air fresheners were dispersed in a room and samples collected using the GSS-FP. The sorbent tubes were analyzed by thermal desorption gas chromatography mass spectrometry. Several fragrance components contained in the dispersed products were monitored and quantified over time. The study also shows validation of an air sampling method for the com-pounds. This type of experiment can be used to show the rate at which compounds decay in the room air as a function of time as well as identifying which compounds are important contributors to the perceived odor in the room.

241 An Investigation of Biodiesel Stability: A Study of the Behavior of Antioxidants in Soybean and Canola Derived BiodieselCasey Camire, University of Connecticut, 100 Oak St., Meriden, CT 06450, James D. Stuart, Anthony A. Provatas

Biodiesel is a renewable fuel that is synthesized via the transesterification of the triglycerides found in vegetable oil, used cooking oil, or animal fats. Unlike ethanol fuels, which often can take the place of important food supplies, biodiesel precur-sors are usually made from waste products. Biodiesel can be used on its own as a replacement for regular, petroleum-based diesel, but is more commonly used as a blend with the usual petroleum-based diesel in order to reduce fossil fuel consump-tion and greenhouse gas emissions. However, biodiesel can be prone to undergo degradation thru a series of radical reactions of oxygen and other species involving the conjugated double bonds of the long chain fatty acids and resulting in the for-mation of shorter fatty acids, carboxylic acids and networked polymers. In order to minimize these effects, antioxidants have been added to both prevent oxygen from interacting with the biodiesel and to inhibit the free-radical degradation of the ester tails. A standardized test has been developed which provides a measure of oxida-tive stability of biodiesel. A modification of this method can be used to determine the kinetics of antioxidants added to biodiesel. This project seeks to use that method to assess the kinetics of commonly used antioxidants in canola and soy derived biodiesels.

242 Developing a Sensitive and Robust Method for Container Closure Integrity (CCI) TestingCasey A. Tyrrel-Pawlowic, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903, Nikunj Vasoya, Chris Knutsen, Jayshree Patel, Steve Klohr, Antonio Fernandez

Container closure integrity testing (CCIT) is performed to measure the ability and quality of a container closure system to provide protection and maintain efficacy and sterility of a sterile drug product. Methylene blue dye has typically been used for dye ingress testing of syringes and vials, but we found it unsuitable when used for sen-sitive detection of breaches in drug product filled containers. Changing regulatory expectations necessitated the development of CCIT methods for stability studies, so sensitive detection of breaches in the presence of drug product was required. Here we describe the use of fluorescein dye to enable development of robust, sensitive, and quality control friendly CCIT methods suitable for long-term stability (LTS) and market-life stability (MLS) studies.

243 Characterizing Nanoparticles in Cosmetic Formulations to Quantitate and Compare the Contribution of Absorbance from Large Particle and Nanoparticle ScatteringChris Lynch, PerkinElmer, 940 Winter St., Waltham, MA 02451, Jeffrey Taylor

Nanoparticles have been growing in use in a large number of application areas. Cosmetic products such as sun screens are one major application area. Nanopar-ticles are commonly used to enhance the protection from harmful ultraviolet (UV) radiation by scattering it away from the skin. Characterization and quantitation of nanoparticles used in creams and lotions is a key element in understanding how homogenously materials are distributed and the overall effectiveness of these nanoparticles. The unique use of UV-visible spectroscopy coupled with a 150 mm integrating sphere and center mount sample holder provides important information to quantitate and compare the contributions of absorbance, large particle scattering, and nanoparticle scattering.

244 Authentication of Extra Virgin Olive Oil Using Fluorescence and Fourier Transform Infrared SpectroscopyFred Behringer, Surveillant LLC, 81 Halls Rd., Ste. B, Old Lyme, CT 06371, Emily Paine

Extra virgin olive oil (EVOO) is considered susceptible to economic adulteration. Rapid and sensitive screening methods would be useful to aid in the identification of impure products. Multivariate models based on fluorescence and Fourier transform infrared (FTIR) spectra of EVOOs were developed and evaluated for the ability to detect blends with various non-EVOOs. Spectra obtained from 43 brands of EVOO purchased from retail outlets over a 14 month period were used to build models that factored in the normal variation of EVOO. Canola, corn, grape, hazelnut, peanut, rice, soybean, sunflower, and safflower oils were blended with EVOO to assess limits of detection. At least three brands of each type of non-EVOO were evaluated and the brand representing the mean was chosen for blending. The non-EVOOs were detected from a low of 5% for rice bran oil to 20% for sunflower oil. The other oils were detected at either 10% or 15%. The model based on fluorescence resulted in lower detection limits than the FTIR model for a majority of the adulterants. How-ever, the FTIR model provided lower detection limits for soybean and sunflower oils. The results suggest that fluorescence and FTIR used together can provide a rapid method for detecting adulteration of EVOO with other types of oils.

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November 20142014 EAS Abstracts

245 Analysis of Cocoa Content in Chocolate Using Tribo-Rheometry and its Correlation to MouthfeelAlina Higham-Latshaw, TA Instruments, 159 Lukens Dr., New Castle, DE 19720

A large amount of resources in the food science industry are directed to under-standing properties of food products such as texture and mouthfeel before, during, and after the consumption of food and beverages. These properties play a large role in consumer selection and acceptability, directly impacting the success of new formulations. Unfortunately, many sensory properties of food are difficult to charac-terize. Numerous studies have been shown to correlate some properties, such as firmness, fattiness, and creaminess with bulk rheological properties and recently, studies have begun to focus on thin-film tribological properties. Tribology is defined as the study of friction, wear, and lubrication between two interacting substrates in relative motion. Within the mouth alone, the number of interacting substrates is quite plentiful: teeth-teeth, tongue-teeth, tongue-palette, tongue-food, just to name a few. It is believed that tribology can be used to analyze friction properties of sub-strates within the mouth and food surfaces to correlate with consumer perception and mouthfeel. In this poster, the tribo-rheometry accessory in conjunction with a rotational rheometer are described and the tribological properties of commercial-ly-available chocolates. We demonstrate how differences in chocolate composition, such as fat and cocoa content, affect the properties and behavior of chocolate thin films between two sliding surfaces. The coefficient of friction as a function of sliding velocity and load force is given for the chocolate samples.

246 Analysis of Lead in Commercial Wine Samples by Graphite Furnace Atomic Absorption SpectroscopySamuel M. Abegaz, Columbus State University, 4225 University Ave., Columbus, GA 31907, Matthew Mireles, Kydric Smith

The purpose of this study was to investigate the level of lead in red and wine sam-ples produced in different parts of the world. Forty five wine samples (twenty five red and twenty white) were obtained from local wine stores and treated before analysis. The thermal behavior of lead during pyrolysis and atomization stages was studied with and without matrix modifier. The analytical procedure was validated using stan-dard reference material of water origin. The concentrations of lead ranged from 12.4 – 35.0 µg/L. These concentrations were well below the maximum permissible levels set by the International Organization of Vine and Wine. The precision was better than 5% for 95% of the samples.

247 The Determination of Preservatives in Cosmetic Products by LC-MS-MSEmily A. Myers, Cedar Crest College, 100 College Dr., Allentown, PA 18104, Thomas A. Brettell, Thomas H. Pritchett

Preservatives are natural or synthetic ingredients that are commonly added to prod-ucts in order to prevent spoilage, including but not limited to microbial growth or undesirable chemical changes, ultimately extending the products shelf life. Without the addition of preservatives cosmetic products can easily become contaminated leading to product degradation increasing the risk of irritation or infection. A liquid chromatography tandem mass spectrometry (LC-MS-MS) method has been devel-oped which identifies and quantifies multiple preservatives in cosmetic products. LC-MS-MS data was acquired using an ABI Sciex 3200 QTRAP triple quadrupole mass spectrometer interfaced with a Shimadzu LC system. The instrument utilized electrospray ionization (ESI) and all samples were run in positive-ion mode moni-toring. Chromatography was performed on a 5.0 cm x 3.0 mm x 2.7-µm Raptor® biphenyl capillary column (Restek®). The strong mobile phase used was 0.1% for-mic acid in 2-propanol and the weak mobile phase used was 0.1% formic acid in HPLC grade methanol. Various cosmetic product samples were prepared by adding approximately 100 mg to 5 mL of methanol:acetonitrile (1:1) and sonicated for 10 minutes. After sonication the solution was placed into centrifuge tubes and centri-fuged for 5 minutes at 3000 rpm. After centrifugation the supernatant was carefully removed using disposable pipettes and filtered using a 0.2-µm Millipore filter. Ap-proximately 1 mL of the supernatant was added to a vial along with 60 µL of the internal standard (BHA). Lastly, 2.0 µL of sample was injected onto the LC column.

248 Rapid and Inexpensive Method for Olive Oil Adulteration and IdentificationHarri Harma, University of Turku, Tykistokatu 6A 5th Floor, Turku, 20300 Finland

Olive oil is the most adulterated food product. Recent reports show that fraudulent activities are obvious as olive oil is sold at a higher price than other vegetable oils. To preserve its quality and authenticity new analytic methods are required. We have developed a chemical detection fingerprinting technique to detect subtle changes in the content of vegetable oils. The core of the technique bases on time resolved luminescence (TRL), label chemistries and a fingerprint that provides a unique sample-specific signature of virtually any liquid or liquidized sample. TRL chelating structures are highly unstable, and the sample interacts non-specifically with the chelating structures modulating the TRL luminescence signal. The array is created

through multiple different chelating agents as samples interact differently with each chelating structures. In the current protocol the sample is simply mixed with the chelates and signal is measured within five minutes. Until now the technology plat-form has been shown to be powerful in a number of food applications. Here olive oil adulteration (spiked) with canola oil down to 1% is measured with a large dynamic range as the signal-to-background ratio of assay was more than 500. Identification of vegetable oils is also presented.

249 Forensic Genetic Analyses of Mitochondrial DNA Haplogroups and Control Region Variation in the Korean PopulationWook Kim, Dankook University, Department of Biology Science, 1st Science Hall #406-1, Cheonan, South Korea 330-714, Ki Cheol Kim, Seung Beom Hong

We analyzed the distribution of mitochondrial DNA (mtDNA) haplogroups and control region sequence variation using a 20-plex SNaPshot assay/sequencing to evaluate the possible genetic structure and differentiation as well as forensic pur-pose from 704 unrelated males residing in six major provinces in Korea. The most common mtDNA haplogroups were found to be D4 and B4, followed by A, D4a, and M7, which are prevalent in South/Northeast Asian populations. Based on the result of control region variation, a total of 558 different haplotypes characterized by 271 polymorphic sites were identified, of these 471 haplotypes were unique. The gene diversity and random match probability were 0.9989 and 0.0025, respectively. According to the pairwise comparison of the 704 control region sequences, a mean number of pairwise differences between individuals found to be 13.47 ± 6.06. Pair-wise FST genetic distances revealed population homogeneity of six Korean prov-inces on a peninsula level, except samples from Jeju Island. In contrast, statistically significant distances were observed between Asian populations (p < 0.001). The present data may help not only in personal identification but also in determining maternal lineages for forensic purpose. These data will be available on the EMPOP database via accession number EMP00661.

250 Using Pure Shift HSQC to Characterize Microgram Quantities of MetabolitesYong Liu, Merck, 126 E. Scott Ave., Rahway, NJ 07065, Mitchell D. Green, Rosemary Marques, Tony Pereira, Roy Helmy, Robert T. Williamson, Wolfgang Bermel, Gary E. Martin

Difficulties in isolating samples from complex biological matrices and sensitivity lim-itations have long stymied the utilization of heteronuclear two-dimensional (2-D) nu-clear magnetic resonance (NMR) for the characterization of drug metabolites. Small diameter cryogenic NMR probes have largely ameliorated sensitivity limitations and the recently reported pure shift heteronuclear single quantum coherence (HSQC) 2-D NMR pulse sequence offers a further and marked improvement in both resolu-tion and sensitivity. Using a 7.4 µg sample of the commercially available metabolite 3-hydroxy carbamazepine dissolved in 30 µL of deuterated solvent and a 600 MHz NMR equipped with a 1.7 mm cryogenic NMR probe, it was possible to acquire high signal-to-noise pure shift HSQC data in just over 30 minutes. A conventional HSQC spectrum acquired with identical parameters had approximately half the sig-nal-to-noise of the pure shift HSQC spectrum. Collapsing the vicinal homonuclear couplings in the pure shift HSQC spectrum also significantly improves resolution. A practical, real world application of the technique is illustrated with the chromato-graphically isolated metabolite 3-hydroxy amiodarone from incubation with CYP2J2 recombinant enzyme. High quality pure shift HSQC data were recorded in slightly over 14 h for a 3 µg sample of the metabolite.

251 The Power of the Combination of HILIC-MS-MS and 31P-NMR Techniques for the Characterization of Phospholipid Compositions in Different Lecithin ProductsYing Yang, International Flavors and Fragrances, 1515 Highway 36, Union Beach, NJ 07735, Richard Hiserodt, Jing Li

The chemical composition of phospholipids strongly influences the physicochemical properties and performance of different types of lecithin. Consequently, a rapid and reliable method to characterize phospholipid composition is necessary for many applications. However, due to the complex nature and poor solubility of these phos-pholipids in water, it is not practical to use the most common analytical approach of reverse-phase high-performance liquid chromatography tandem mass spectrometry (HPLC-MS-MS). In the present study, an hydrophilic interaction liquid chromatog-raphy (HILIC)-electrospray ionization (ESI)-MS-MS method was employed for the separation and identification of the chemical composition of different phospholipid classes and molecular species by ion trap MS-MS technique. SIEVE TM was used for post-acquisition data analysis in order to rapidly achieve a comprehensive struc-tural characterization for key components in a variety of lecithin products. Quantita-tive results for each different phospholipid class were obtained using 31P-nuclear magnetic resonance (NMR). The results from the present study revealed the differ-ent strengths of these two techniques and demonstrated the power of the combi-nation of HILIC-MS-MS and NMR for a rapid and comprehensive characterization of phospholipids.

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252 Ultrasensitive Carbohydrate-Peptide Microarray for Diagnosing IgE Mediated Peanut Allergy Using Surface Plasmon Resonance ImagingAmit A. Joshi, University of Connecticut, 55 North Eagleville Rd., Storrs, CT 06269, Mark W. Peczuh, Challa V. Kumar, James F. Rusling

Quantification of allergen specific IgEs specific to individual peptide and carbohy-drate epitopes is a promising strategy to reliably predict severity of allergic response. Here the first prototype microarray for IgE detection using peptide and carbohydrate epitopes is shown. A surface plasmon resonance imaging (SPRi) microarray is spot-ted with a 28-mer peptide and a β-xylosyl glycoside (BXG). Both are known to be antigenic epitopes from the Arachis hypogaea h2 (Ara h2) peanut glycoprotein. IgEs from human serum are precaptured onto to magnetic beads, loaded with polyclonal anti-IgE antibodies, and injected into the SPRi microarray to identify IgEs in dilute serum. As low as 0.1 attomoles of IgEs are detected in 45 min assay. Amounts of IgEs binding to the Ara-h2 peptide and BXG are identified by SPRi in patient sam-ples and compared to standard immunoCAP assay values.

253 Versatile Low-Cost Electrochemical Microfluidic Immunosensors for Cancer DiagnosticsBrunah A. Otieno, University of Connecticut, 55 North Eagleville Rd., Storrs, CT 06269, Colleen E. Krause, Gregory W. Bishop, James F. Rusling

Rapid, accurate and sensitive detection of multiple biomarker proteins holds sig-nificant promise for early diagnosis of cancer and personalized therapy guidance. Here we describe a simple, low-cost, modular microfluidic system for on-line capture and detection of cancer protein biomarkers. The system features a small chamber for on-line protein capture from serum by magnetic beads labeled with many cop-ies of analyte-specific antibodies and signal-transducing enzyme labels, positioned upstream of a detection chamber housing a nanostructured 8-electrode sensor ar-ray. Microfluidic chambers are made by templating polydimethylsiloxane (PDMS) channels on machined aluminum molds and mounting on hard flat poly(methyl methacrylate) (PMMA) plates equipped with inlet and outlet panels. The chambers are interfaced with a sample injector, syringe pump and switching valves to deliver sample and reagents. Gold immunosensors fabricated by ink-jet printing of 4 nm alkylthio l gold nanoparticles ($0.2) or commercial screen printed carbon sensors coated with 5 nm glutathione-gold nanoparticles($6) are fitted into the microfluidic detection chamber to achieve high sensitivity. Ultralow detection limit in the low fM range was achieved for multiplexed detection of four oral cancer biomarker proteins from as little as 5 µL sample within 30 minutes. The incorporation of electrochemical immunoassays for protein biomarkers in a microfluidic platform with nanostructured sensors thus provides a rapid, sensitive and effective tool for cancer diagnostics.

254 A Novel Device for Plasma Micro-Sampling Technique Developed for BioanalysisJi Zhang, Takeda Pharmaceutical International, 40 Landsdowne St., Cambridge, MA 02139, David Lok, Jesse Gray, Kelly Connolly, Steve Grossman

Micro-sampling technique was widely used in the past five years and its benefits have been recognized throughout the pharmaceutical industry. Traditionally, plasma is gold standard matrix of choice. But it is a technical challenge to accurately and precisely sample plasma directly from derived plasma layer at micro volume level. Here, we report a novel device for isolating plasma from red blood cells at micro volume level (as low as 20 mL) which allows direct pipetting plasma from generated plasma layer. All experiments were performed API 5500 Qtrap mass spectrometer interfaced with a Shimazu high-performance liquid chromatography (HPLC). Twenty or 40 µL mouse blood was pipetted into the novel device developed in-house for plasma micro-sampling followed by 6 min centrifugation at 14000 rpm. Pipette 5 or 10 µL plasma from top layer derived plasma for Bioanalysis. Roughly 10 or 20 µL plasma can be isolated from 20 or 40 µL blood in the devices after centrifuga-tion. Five or 10 µL plasma can be easily pipetted out. Side-by-side comparison of this new plasma micro-sampling and conventional plasma collection method using in-house discovery compounds has demonstrated that the mean percentage dif-ference between two methods is less than 15%. The precision (%CV) falls within ±15%. Animal studies have also been performed to show good agreement between the data obtained from conventional plasma collection and that from micro-sampling method using this new device. The device is novel, simple to use and cost-effective.

255 Use of New 1.9-µm YMC-Triart C18 and 2.7-µm YMC-Meteoric Core C18 Bio Stationary Phases for Fast Peptide Mapping of Monoclonal AntibodiesJeffrey A. Kakaley, YMC America, 941 Marcon Blvd., Allentown, PA 18109, Ernest J. Sobkow

Peptide mapping methods have historically been lengthy, time-consuming meth-ods requiring large amounts of solvent. Modern instrumentation and smaller particle size stationary phases now allow for these methods to be scaled down; improving throughput, saving solvent, and increasing scientists’ overall productivity. In this

poster we investigate the use of YMC-Triart C18 1.9µm hybrid and YMC Meteoric Core C18 2.7µm core-shell materials for improving peptide mapping methods.

256 Development of a Quantitative Method for Synthetic Cannabinoid Metabolites in Urine Using Liquid Chromatography Tandem Mass Spectrometry (LC-MS-MS)Craig T. Leopold, The Forensic Sciences Mentoring Institute, 2300 Stratford Ave., Willow Grove, PA 19090, Alonzo Elias, Christina Bangura, Monica Marcuse

Over the past few years, synthetic cannabinoids have become increasingly pop-ular in an effort to achieve a “legal” high. At the federal level in the United States, most are quickly regulated as Schedule I substances, so “manufacturers” produce compounds that are structurally different, but still give similar effects to achieve that high. JWH-018, UR-144, ADB-PINACA, AKB-48, PB-22, and 5F-PB-22 have been scheduled by the Drug Enforcement Agency (DEA); while AB-PINACA, ADBICA, and BB-22 are currently not scheduled but have recently been identified as com-ponents in synthetic cannabis blends in Japan and the United States. An updated method was developed to extract, identify, and quantify the N-pentanoic acid me-tabolites and the 3-carboxyindole metabolites of the compounds listed above from urine, using liquid-liquid extraction (LLE) followed by liquid chromatography tandem mass spectrometry. Samples were made strongly acidic using HCl and extracted into chloroform, isopropyl alcohol, and n-heptane (50:17:33) and the LC-MS-MS was operated in positive ionization mode. The method achieved baseline separation for all analytes and identification was based on their multiple reaction monitoring (MRM) transitions. Calibration models were produced in the range of 1-100 ng/mL for JWH-018, UR-144, AKB-48, PB-22, and 5F-PB-22 metabolites; 1-50 ng/mL for ADBICA, AB-PINACA, and ADB-PINACA metabolites; and 5-100 ng/mL for BB-22 metabolite (R2 > 0.98). The limits of detection were ≤ 2 ng/mL and limits of quantita-tion were ≤ 5 ng/mL. Currently, the method is being validated for use as quantitative assay in the field of forensic toxicology as it incorporates several scheduled com-pounds and recently emerging synthetic cannabinoids.

257 Metabolic Profile Determination of Novel Psychoactive Substances by Use of Human Liver MicrosomesSarah E. Wolf, The Forensics Mentoring Institute, 2300 Stratford Ave., Willow Grove, PA 19090, Noah Gubernick, Timothy Martin, Grace Pak

Novel psychoactive substances have been increasingly abused, particularly within the electronic dance music (EDM) scene. With the continued popularity of “designer drugs,” the market is constantly changing, requiring continued research, including the metabolic pathways of these newly emerging drugs. The aim of this research was to develop and optimize a method for in-vitro metabolism using human liv-er microsomes (HLM). In-vitro metabolism using pooled HLMs was first optimized by incubating the microsomes with diazepam, and monitoring for the presence of nordiazepam and temazepam. Analysis occurred using liquid chromatography tri-ple quadrupole tandem mass spectrometry. The incubation mixtures contained a phosphate buffer (pH= 7.4), magnesium chloride, nicotinamide adenine dinucleo-tide phosphate, reduced (NADPH), diazepam, and HLM, and were incubated at 37 °C. After optimizing the time of incubation and the amounts of NADPH, HLM, and substrate, the following values of each were used: incubations were incubated for two hours with 25 µL of microsomes (20 mg/ml), 50 µL NADPH (10 mM), and 5000 ng substrate with a final volume of 600 µL. Once it was established that the mic-rosomal incubation mixture was effectively producing metabolites as seen in-vivo, this optimized method was used to produce the metabolites of the drugs of interest, including α-PVP. For this, analysis occurred by liquid chromatography quadrupole time-of-flight mass spectrometry to produce exact mass data.

258 LC-MS-MS Method Development and Analysis of Sofosbuvir Metabolites Using PGC (Porous Graphitic Carbon) Column: Determination of the Metabolites in Human Liver and Lung Cell Lines.Prabhakar G. Reddy, Emory Institute for Drug Development, 954 Gatewood Rd. NE, Atlanta, GA 30329, Taylor J. Evers, Adhitya R. Katkam, Gregory Bluemling, Michael G. Natchus, Damien Kuiper, Manohar Saindane, Shuli Mao, Mark Lockwood, Michael Hager, Richard F. Arrendale

A sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS-MS) method was developed for Sofosbuvir metabolites (monophosphate, tri-phosphate, intermediate metabolite, and nucleoside), and the method was used to determine these metabolites in human liver (Huh7) and lung (A549) cell lines. The method utilized a porous graphitic carbon (PGC) stationary phase, Hypercarb (Ther-mo), under conditions suitable for liquid chromatography mass spectrometry (LC-MS). The separation of the Sofosbuvir metabolites was optimized using gradient elution with acetonitrile and ammonium bicarbonate buffer. The pH of the ammoni-um bicarbonate buffer solution proved to be critical for selectivity and retention of the mono and triphosphate metabolites of Sofosbuvir. Baseline separation of the mono-phosphate, triphosphate, intermediate metabolite, and the nucleoside was achieved

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in less than 10 minutes. An internal standard spiking technique was used to produce a quantifiable linear range for these metabolites from 1 ng/ml to 1000 ng/ml when using the appropriate blank cell extract for preparation of calibration curves. Sofos-buvir was incubated with cells from human liver (Huh7) and human lung (A549) cell lines for up to 24-hours, and the formation of the Sofosbuvir metabolites were mon-itored and quantitated. ATP levels were also monitored to assess the reproducibility of the cell incubation procedure. This presentation outlines the method development and application of this methodology to these cellular metabolism studies.

259 Supported Liquid Extraction as a Bioanalytical Sample Preparation TechniqueMatthew Cleeve, Kinesis Ltd., 9 Orion Court, Ambuscade Rd., St Neots, PE19 8YX United Kingdom

An increasingly diverse range of drug candidates present challenges in bioanalysis. The selected sample preparation method should be simple, efficient and optimized for high sample throughput. One of the key challenges is to remove interferences present in biological fluid samples, commonly plasma. Protein precipitation, liq-uid–liquid extraction (LLE) and solid-phase extraction (SPE) are commonly used techniques to extract drug molecules. It is clear traditional techniques are not al-ways suited to removing protein and phospholipid interferences, with phospholipids significantly affecting the reliability of results. Removing phospholipids and proteins is key to the effectiveness of the sample prep procedure. Recently, diatomaceous earth has gained popularity in bioanalytical sample preparation. Its porous channel structure provides excellent dispersive properties, trapping large molecules includ-ing phospholipids. When the plasma sample passes through the material, it pene-trates the pores, forming an aqueous film on the surface. When a water-immiscible solvent is passed through LLE occurs. This is referred to as supported liquid ex-traction (SLE). Using this approach, analytes will be eluted while lipids are retained. It is believed the acidic property of the hydroxyl groups and metal centers on the substrate contribute to the retention of phospholipids via the interaction with its chlo-rine and phosphate groups. In this study, we have compared LLE, PPT, SPE and SLE methods to extract dexamethasone in plasma prior to liquid chromatography tandem mass spectrometry (LC-MS-MS). Spiked plasma samples containing 10ng/ml, 100ng/ml, and 1000ng/ml dexamethasone were used in each case. The results presented will show the responses from SLE are between four and six times higher than the other techniques.

260 Extraction of Indomethacin and Ibuprofen from Small Volume Biological Fluid SamplesMatthew Cleeve, Kinesis Ltd., 9 Orion Ct., Ambuscade Rd., St Neots, PE19 8YX United Kingdom

Developments in liquid chromatography tandem mass spectrometry (LC-MS-MS) detection levels have seen biological fluid sample volumes reduced to such an ex-tent that conventional solid-phase extraction (SPE) formats are not always suitable, or provide considerable analyte dilution and extended evaporation times. When ex-tracting from sample volumes of less than 100 µl, it is important elution volumes and the sample prep format are fully compatible with the original sample volumes being processed. The TELOS MicroPlate is a modular design, allowing for flexibility in sample numbers. Full or partially populated plates can be processed using vacuum or positive pressure. In addition, the well outlet design ensures good collection plate penetration, removing any possibility of well to well cross contamination. This poster summarizes a solid-phase extraction method for two drugs from human plasma, In-domethacin and Ibuprofen using TELOS neo PRP, a water-wettable polymer-based SPE sorbent. Analyte concentrations range from 5 to 50ng/ml. The data highlights the minimum elution volumes that can be achieved from the 5mg sorbent mass, providing high reproducible recoveries and RSDs. Excellent linearity was achieved, along with LODs of 0.5 and 1ng/ml for Indomethacin and Ibuprofen respectively.

261 Development of an Ultra-Sensitive Affinity Capture Method for the Quantification of Microdosed Dog Toxicology SamplesLorell N. Discenza, Bristol-Myers Squibb, Route 206 & Province Line Rd., Princeton, NJ 08543, John Mehl, Georgia Cornelius, Charu Chaudhry, Randy Talbott, Jinping Gan, Celia D’Arienzo, Timothy Olah

Because a millamolecule of interest was not well tolerated in dogs at 0.01 mg/kg, a new study was designed and dosed at 0.0001, 0.0003, and 0.001 mg/kg to evaluate toxicity, tolerability and to provide guidance for future toxicology studies. In order to measure the low concentration levels expected from this study, an affinity capture sample preparation method was developed. With this improved methodology, we were able to lower the lower limit of quantitation (LLOQ) ~ 100 fold down to 0.076 nM. Further reduction of the LLOQ was achieved by analysis on a recently acquired Waters Xevo TQ-S with ionKey technology. How this strategy was implemented and future applications is discussed.

262 Finding Order Amid Chaos: In-Vitro Challenges Working with Boronic AcidsTeresa Mulder, Genentech, 1 DNA Way, S. San Francisco, CA 94618, Xiaorong Liang, Chenghong Zhang, Hoa Le, Matt Baumgardner, Quynh Ho, Savita Ubhayakar, Yuan Chen, S. Cyrus Khojasteh, Cornelis E.C.A. Hop, Peter Fan

As bacteria are becoming increasingly resistant to antibiotics, novel therapies are becoming necessary. Compound A is a boron-containing compound we are as-sessing for this purpose. Its boronic acid component is similar to that of Bortezo-mib, the dipeptidyl boronic acid proteasome inhibitor used to treat myeloma. Both compounds undergo rapid deboronation in the conditions typical of in-vitro DMPK screening assays such as protein binding and liver microsome stability. The mecha-nism of deboronation may be the same in both compounds, although the abundant metabolite/degradant of Compound A is different from that of Bortezomib. This may be due to an amine offering intra-molecular stabilization of the abundant metabolite in compound A. With similar mechanisms causing instability, we were able to use published Bortezomib conditions to accelerate our discovery of in-vitro methods. Comparing metabolite percentages from microsomes incubated with and without nicotinamide adenine dinucleotide phosphate (NADPH) allowed us to determine the NADPH-dependent component we were interested in. For our plasma protein binding assay, Compound A was more stable in freshly prepared plasma than the frozen plasma that was provided to us. These in vitro methods can be used to test the properties of similar antibiotic candidates in the future, and to guide the develop-ment of further in vitro tests as they become necessary.[1]


[1] S Wu, et al. J. Pharm. Sci. (2000) 758-765.

263 Aggregation Behavior of Ionic Liquids in Low Polarity SolventsElise Cade, The College at Brockport, 41 Stuart Rd., Churchville, NY 14428, Markus Hoffmann

Ionic liquids are salts that are liquid below 100 oC. Normally, salts do not dissolve in solvents of low polarity. However, many ionic liquids are very soluble or completely miscible in low polarity solvents and their physical chemistry is not well understood. The goal of our ongoing research is to elucidate the speciation (freely dissolved ions, ion pairs and aggregates) present for ionic liquids dissolved in solvents of low polarity. We present experimental results for concentration and temperature dependent self-diffusion coefficients measured by nuclear magnetic resonance spectroscopy in conjunction with viscosity measurements to determine the average hydrodynamic radii of the present species. Five systems were investigated: [C6mim][NTf2] dichloromethane , tetrahydrofuran , and chlorobenzene , as well as [C4mim][NTf2] and [C2mim][NTf2] in dichloromethane.

264 Linked Scanning of He and Compensation Voltage to Improve the Resolving Power of Differential Ion Mobility SeparationsRachel Harris, University North Carolina-Chapel Hill, Caudill Laboratories Rm. 326, 131 South Rd., Chapel Hill, NC 2514, Brandon Santiago, Samantha Isenberg, Gary Glish

One of the benefits of gas phase separations such as differential ion mobility spec-trometry (DIMS) is the ability to improve signal-to-background in mass spectrome-try-based analyses. Helium can be added to the DIMS carrier gas to improve the resolving power of the separations. However, the addition of He also leads to a loss of ion transmission, especially for ions of higher differential ion mobility. In an effort to improve both the resolving power and ion transmission of DIMS separations, the “linked scan” was developed, which varies the DIMS compensation voltage and carrier gas He content simultaneously in a fixed relationship. Throughout the linked scan, as the compensation voltage increases, the He content of the carrier gas is decreased. Experiments were performed using dispersion fields of 24, 26, 28, 30, 32, and 34 kV/cm. The compounds analyzed include Agilent ESI tuning mix and ubiquitin. Linked scans were performed by varying the carrier gas He content during the CV scan. CV scans with a constant helium composition were run with He compositions ranging from 0% to 60% He in 5% increments. Resolving powers of peaks from linked scans are compared to those from static scans at the nearest He composition. For both analytes, peak widths are reduced and resolving power increased when using linked scans. Generally, higher dispersion fields (ED = 32 kV/cm) and larger He ranges give larger increases in resolving power. The average percent increase in resolving power across all experiments is 37±18% for tuning mix and 51%±17% for ubiquitin.

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265 Photochemical and Thermal Control over Electrolessly Gold-Plated Film Structure on Thin Silicon Nitride to Target Sensing ApplicationsCaitlin M. Masterson, University of Rhode Island, Department of Chemistry, 51 Lower College Rd., Kingston, RI 02881, Brian D. Velleco, Buddini Iroshika Karawdeniya, Julie C. Whelan, Y.M. Nuwan D.Y. Bandara, Jason R. Dwyer

Thin silicon nitride films are a common building block of nanofabricated devices. They offer favorable physical properties, such as the ability to form free-stand-ing <100nm-thick films, but their complex surface chemistry presents challenges. We developed a process to electrolessly plate thin gold films onto silicon nitride membranes, and into through-membrane pores, so that we could use thiol-based chemistry to more readily modify the surface chemistry. We discovered that the gold film grain structure allowed us to perform surface-enhanced Raman spectroscopy (SERS). Thermal annealing of the films dramatically changed the gold film structure, as revealed by both the SERS signal and field-emission scanning electron micros-copy (FESEM). Mean grain radii, for example, could be readily increased by tens of nanometers by post-deposition annealing under suitable conditions. We exerted control over the film spatial distribution across longer length scales by preceding the plating with a photochemical surface protection route that allowed us to generate patterned gold films. We will describe the various gold film plating approaches and present applications.

266 Quantification of Local Bonding Environments in Cesium Silicate Glasses Using Si-29 Magic–Angle Flipping NMRPyae Phyo, Berea College, Chemistry Program, CPO 1119, Berea, KY 40404, Jay H. Baltisberger, Kevin Sanders, Eric Keeler, Philip J. Grandinetti

Cesium silicate glasses were studied using Si-29 magic-angle flipping Nuclear Mag-netic Resonance (NMR) spectroscopy. The chemical shift anisotropy parameters were extracted for each glass with a variety of Q(4) and Q(3) sites. The Q(4) site increases in width as the amount of cesium is increased. This is consistent with an increasingly disordered environment around these sites as the neighboring sites have more non-bridging oxygen atoms. Two distinct Q(3) sites are seen with very different anisotropies at the low cesium content and become more similar as cesium is added. Previous work has shown that larger anisotropies correspond to short non-bridging Si–O bond distances for Q(3) sites. This leads to a general interpreta-tion that the large anisotropy site arises from an increasing number of coordinating cesium cations near that site. The other Q(3) site with increasing anisotropy with increasing cesium content must arise from either a smaller number of coordinating cations or a reduced local microscopic density to increase the Si-O bond lengths as cesium is added. This model indicates a microscopically phase separated mixture of isolated cesium cations scattered throughout the silica matrix, as well as some channels filled with clustered cesium cations. No macroscopic evidence for phase separation is seen in these glasses, however the degree of homogeneity of the melt is difficult to quantify given that samples were quenched from 1200 ˚C. Smaller cat-ions such as sodium, rubidium, and potassium have been studied in silicate glasses with no evidence of multiple Q(3) sites.

267 Mass Spectrometry Analysis of NXS/T Glycosylation Sites in Recombinant GlycoproteinsArmand G. Ngounou Wetie, Clarkson University, 8 Clarkson Ave., Potsdam, NY, 13699, Izabela Sokolowska, Urmi Roy, Alisa G. Woods, Costel C. Darie

To fulfill the United States Food and Drug Administration’s requirements, therapeutic proteins have to be fully characterized with regard to their size, sequence and their post-translational modifications (PTMs). One of the most important PTMs that play a role in the solubility, stability and function of therapeutic proteins is glycosylation. In this study, we investigated N-glycosylation at NXS/T sequons of a fusion pro-tein using a mass spectrometry-based proteomic strategy to determine if the intro-duction of new N-glycosylation sites into a chimeric protein would influence known N-glycosylation sites. Therefore, a chimeric protein IgG-Fc-ZP3E7, composed of the Fc part of IgG1 heavy chain and of exon 7 of the zona pellucida (ZP) protein, was over-expressed and the protein product purified. As a control, IgG-HC (Fc part of IgG1 heavy chain) was used. Samples were treated with and without PNGase F and analyzed by SDS-PAGE, Western blotting, trypsin-AspN double digest followed by nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) using data dependent analysis (DDA) and information dependent analysis (IDA or DDA with inclusion list). Our results show that addition of a new potential NXS/T glyco-sylation site within a fusion or chimeric protein could prevent the glycosylation of original NXS/T glycosylation sites.

268 Using Nuclear Magnetic Resonance (NMR) Techniques to Study Small (d = 2-3 nm) Gold-Transition Metal Nanoparticle AlloysLauren Marbella, University of Pittsburgh, Chevron Science Center, 219 Parkman Ave., Pittsburgh, PA 15227, Christopher M. Andolina, Jill E. Millstone

Small gold-transition metal nanoparticle alloys (diameter = 2 – 3 nm) display physi-cal behaviors distinct from either their bulk or molecular counterparts, and are desir-able for applications ranging from heterogenous catalysis to therapeutics. However, nanoscale alloyed architectures are difficult to synthesize because metal mixing behavior exhibits length scale dependence. We hypothesize that alloy formation at the nanometer scale can be manipulated by controlling surface chemistry. Using classical colloidal methods, we have been able to synthesize a range of gold-tran-sition metal nanoparticle alloys with fully tunable compositions, regardless of bulk miscibilities. With a unique combination of NMR-based molecular characterization techniques and traditional materials characterization strategies, we are able to pro-vide crucial insight into particle formation, as well as directly correlate particle com-position with particle physical properties.

269 Toxicity of Silver Nanoparticles in the EnvironmentMaral P.S. Mousavi, University of Minnesota, Department of Chemistry, 207 Pleasant St. South East, Minneapolis, MN 55455, Ian L. Gunsolus, Carlos E. Pérez De Jesús, Christy L. Haynes, Philippe Bühlmann

Silver nanoparticles, Ag NPs, are widely used in many consumer products. As a result, they can leach into the environment during product usage, raising concern about their toxicity in the ecosystem. Natural organic matter, (NOM), which is abun-dant in water supplies, soil, and sediments, can interact with Ag NPs and influence their environmental persistence. Moreover NOM can bind with Ag+ that is released from Ag NPs resulting in formation of silver-species with altered bioavailability and toxicity. Using fluorous-phase Ag+ ion-selective electrodes (ISEs), we studied Ag+ binding to NOM and showed that Ag+ binding to NOM is favored by a higher pH. Moreover, effect of NOM chemical composition on kinetics and extent of binding was investigated. It was demonstrated that NOM samples with a low sulfur and nitrogen content have weak Ag+ binding, while stronger binding was observed for Pony Lake NOM, which has higher amounts of these elements. Fast kinetics of Ag+ and NOM binding was confirmed. Using fluorous-phase Ag+ ISEs it was shown that presence of NOM does not alter the concentration of Ag+ released from Ag NPs. NOM samples with high sulfur and nitrogen content were shown to reduce silver toxicity to the bacterium Shewanella oneidensis. More generally, this work has shown that fluorous-phase Ag+ ISEs are effective tools for studying Ag NP toxicity in the environment.

270 Enzyme Modulator Screening Using Droplet Samples and Sub-Second Microchip ElectrophoresisErik D. Guetschow, University of Michigan, Department of Chemistry, Ann Arbor, MI 48109, Robert T. Kennedy

Traditional high-throughput screening (HTS) utilizes multiwell plate based assays and fluorescence detection to perform tens of thousands of assays per day. While this method has been successful, fluorogenic reagents or coupled reactions are required and multi-analyte detection is difficult. We have developed a novel method to couple multiwell plate-based assays to microchip electrophoresis (MCE) using droplet microfluidics. Samples contained in multiwell plates are reformatted in to nanoliter volume plugs segmented by an oil carrier phase. Droplet streams are cou-pled to a hybrid polydimethylsiloxane-glass microfluidic device capable of selective-ly extracting the aqueous samples from the droplet stream and rapidly analyzing droplet contents by MCE. To validate this new method, a test library containing 140 small molecules was screened for inhibitors or protein kinase A (PKA). Each sample was reformatted as two droplets allowing at least 6 MCE injections per sample. Sub-second separation of three analytes – rhodamine, substrate peptide, and prod-uct peptide – was achieved in 5 mm using an applied electric field of 2000 V/cm with an efficiency of 16,000 plates/second. Using 700 injections total, 96 samples were analyzed in 12 minutes (0.13 Hz) and several small molecule inhibitors of PKA were identified and verified through dose-response experiments. While this method has been applied to screening applications, the device layout and operation is amenable to many applications including coupling two-dimensional separations and chemical sensing. This work was supported by funding from the National Institutes of Health.

271 Hard-Modelling - From Titrations to Solving Climate TribulationsGraeme Puxty, Commonwealth Scientific and Industrial Research Organization, PO Box 330, Newcastle, NSW 2300 Australia

In the chemometrics/chemistry world hard-modelling is the fitting of models that represent some sort of chemical phenomena to measurements of that phenomena. A simple example is the fitting of a straight line to spectrophotometric measure-ments of absorbance at a characteristic wavelength as a function of concentration of the absorbing chemical species. In its most complex it is the fitting of a chemical model incorporating reaction kinetics and equilbria (between species and phases), thermodynamics, and physical processes to multidimensional measurements of dif-

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ferent type. This presentation covers a range of examples where a combination of intelligent measurement combined with appropriate model selection, development and fitting has led to understanding, insights and developments of chemistry and chemical processes. From the fitting of a pH curve to determine the pKa of an acid, to multidimensional potentiometric and spectrophotometric titrations and stopped-flow kinetics to elucidate the equilibrium constants, rate constants and thermody-namics of complex multi-step reactions. Finally, how this has developed into new and improved absorbents and processes for the capture of carbon dioxide from gas streams. Developments that are now leaving the lab and moving to real-world large scale testing.

272 Hybrid Hard and Soft-Modeling and Local Rank Analysis: Two Relevant Ideas to Describe and Explore Chemical SystemsAnna de Juan, University of Barcelona, Chemometrics Group, Department of Analytical Chemistry, Diagonal, 645, Barcelona, 08028 Spain

No abstract submitted by the author.

273 Calculation of Feasible Bands for Visualizing the Constraint Effects in Model-Free MethodsHamid Abdollahi, Institute for Advanced Studies in Basic Sciences, Department of Chemistry, PO Box 4, Zanjan, 5195-1159 Iran

The curve resolution (CR) methods magnetize substantial research efforts aimed to discover knowledge of multicomponent systems. The soft methods establish a specified part in CR methods, as they allow going into insight the data without prior knowledge of the studied system. Using soft methods, the outcome of the analysis of one system is not usually the true one due to rotational ambiguity. The rotational ambiguity leads to a range of possible solutions that fulfill constraints and represent the measured data correctly. Consequently, taking into consideration of all feasible solutions can provide useful information about the system and the process under study when there is no unique solution for the system. Additionally, the calculation of feasible bands is dramatically advantageous not only for quantitative and qualitative application of the resolved profiles but also for visualizing and deep understand-ing the effects of constraints on results of using soft-modeling methods. Different methods have been proposed in the literature to calculate the feasible solutions. Analytical methods for calculating the feasible solutions in two and three-component systems developed as Lwton-Sylvester and Borgen-Rajko plots. Then the exact and explicit calculations of range of feasible solutions were approximated by numeri-cal methods as grid search procedures up to four component systems. The micro structure of chemical data can be visualized by showing the feasible solution re-gions in abstract spaces under considered constraints. The effects of non-negativity, equality, selectivity-zero regions and hard modeling constraints on micro structure of chemical data have been investigated basically and some new conclusions have been obtained from interpretation of observations.

274 Chemometrics: The Balance Between ‘Chemistry’ and ‘Metrics’Marcel Maeder, University of New Castle, Department of Chemistry, University Dr., Newcastle, 02308 Australia

Chemometrics is a term that does not have a clear definition; it is rather a substan-tial collection of computational methods that are used by chemists for the analysis of usually large data sets. Selecting the ‘optimal’ method, the one that delivers the maximum amount of useful information is clearly an important but also challenging task. One way of bringing together and differentiating between the diverse methods of chemometrics is to investigate the nature and amount of prior information used for the analysis. Specifically, we will consider the amount of chemical insight that is used as the backbone for the analysis of the process under investigation. There is little doubt that the more such prior information is available and efficiently used, the more robust the analysis and also, at least potentially, the more useful the extracted information. Conversely, it is not immediately clear how such prior information can be incorporated into the different algorithms. It is interesting to investigate the histo-ry of chemometrics, e.g., by compiling the chapter headings of the bi-annual reviews on Chemometrics in Analytical Chemistry: the trend is clearly in the direction of less rather than more chemical content in the analyses. The presentation argues that this is a ‘sub-optimal’ development.

275 History of DBS and its Development from Clinical to Pharmaceutical ApplicationDaniel L. Norwood, Boehringer Ingelheim, 900 Ridgebury Rd., PO Box 368, Ridgefield, CT 06877

Dried blood spots (DBS) on filter paper have been employed since the early 1960s for the collection, storage and transport of small (< 100 µL) samples for the purpose of newborn screening. Originally, newborns were screened for phenylketonuria by measurement of phenylalanine using a bacterial inhibition assay. Advances in tech-nology over the last two decades of the 20th century led to a significant increase in the number of disease conditions assessed in newborns by DBS analysis, as well as virtually universal application of DBS analysis for newborn screening in the United

States. The advent of tandem mass spectrometry (MS-MS) coupled with “soft” ion-ization techniques significantly increased the number of disease states amenable to detection by DBS analysis. This presentation describes the advent of DBE analysis with tandem mass spectrometry, and traces its history from the earliest applications to modern mass newborn screening, toxicokinetics, pharmacokinetics, and thera-peutic drug monitoring.

276 Dried Blood Spots - From Newborn Screening to Forensic ScienceDonald Chace, Pediatrix Medical Group, 1301 Concord Terrace, Sunrise, FL 33323

The most frequent application of dried blood spot analysis is its clinical use as a blood collection matrix for metabolic screening of infants for genetic disease screen-ing (newborn screening) and forensic pathology (postmortem screening). The dried blood spot is collected between 24-48 hours after birth and tested for more than 40 conditions using a variety of technologies such as immunoassays, fluorescence as-says, electrophoresis and tandem mass spectrometry. Metabolic diseases screened by tandem mass spectrometry analysis of primarily amino acids and acylcarnitines which account for more than half of all disorders screened. This comprehensive analysis of more than 50 metabolites is achieved by a single sampling (a punch) of the dried blood spot and in an analysis that takes approximately two minutes per sample. The forensic pathology analysis uses primarily tandem mass spectrometry for primarily acylcarnitines in dried blood samples which are collected at autopsy (the metabolic autopsy) from unexplained infant or child deaths or in medico-legal cases. The dried blood spot is unique because it requires small volumes of blood and can be easily shipped to a laboratory for analysis. These advantages coupled with mass spectrometry analysis are continuing to lead to new applications in areas such as premature infant metabolism and nutrition.

277 Recent Progress in Implementing Dried Blood Spot Technology in Drug DevelopmentQin Ji, Bristol-Myers Squibb, Rte. 206 & Province Line Rd., Princeton, NJ 08540

No abstract submitted by the author.

278 Automated Bioanalysis of Dried Blood Spots and Dried Plasma Spots Coupled with High Resolution Mass Spectrometry and Selected Reaction Monitoring LC-MS TechnologiesJack Henion, Quintiles Bioanalytical and ADME Labs, 19 Brown Rd., Ithaca, NY 14850, Robert Sturm, Regina Oliveira

The pharmaceutical industry has recently shown interest in dried blood spot (DBS) techniques which has expanded to other important biological matrices including plasma (DPS), cerebrospinal fluid, saliva, bile, etc. DBS and DPS techniques rep-resent micro sampling techniques which are also of increasing interest when limited quantities of biological samples are available. If large numbers of DBS cards require liquid chromatography mass spectrometry (LC-MS) analysis which is beginning to occur in certain drug discovery programs, it is very helpful for automation which can preclude tedious off-line manual punching and extraction of these micro samples. Challenges from micro samples include the need for high sensitivity, but modern LC-MS techniques can meet these needs. In contrast, the use of “DBS techniques” has raised new issues including the effects of hematocrit on the distribution of whole blood on the cellulose substrate. New strategies are being explored to deal with these issues. For our DBS bioanalytical studies commercially available cards were employed coupled with commercially available automated DBS extraction robotic equipment coupled with high resolution accurate mass using a quadrupole time-of-flight MS. For our DPS studies we have produced a home-made laminated DPS card device that is well-suited for micro sampling low volumes of whole blood (10to 25 µL) to produce dried plasma without the need for centrifugation. These devices have undergone accuracy and precision LC-MS experiments employing the pro-drug, guanfacine and its stable isotope internal standard. A comparison of automat-ed DBS and DPS analysis with the title techniques is presented.

279 Enhanced-Fluidity Liquid Chromatography (EFLC) for Highly Polar Biologically Relevant CompoundsSusan Olesik, Ohio State University, Department of Chemistry and Biochemistry, 100 West 18th Ave., Columbus, OH 43016, Martin Beres, Rafael Bennett

Enhanced-fluidity liquids (EFLs) are organic solvents or organic/aqueous solvents mixed with high proportions of liquefied gases. These solvents share the positive attributes of supercritical fluids (fast diffusion rates and low viscosities) and the posi-tive attributes of common liquids (high solvent strength). These liquids when used in liquid chromatography provide high efficiency and decreased separation time. Our group previously illustrated that EFLC when combined with hydrophilic interaction liquid chromatography (HILIC) stationary phases is quite effective for the separation of highly polar nucleotides and nucleosides without the need for gradient conditions. This talk presents data illustrating the separation of even more polar biologically

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relevant compounds using HILIC and ion-exchange stationary phases. The range of operating conditions that are important in EFLC-HILIC or EFLC-IE is discussed and a deep characterization of the retention mechanism for various operating conditions is included.

280 Recent Advances in Supercritical Fluid Chromatography ApplicationsJennifer Van Anda, Agilent Technologies, 330 Locust Grove Rd., West Chester, PA 19382, Rick Wikfors

The key market for the application of supercritical fluid chromatography (SFC) has been in the pharmaceutical industry, primarily in chiral separations. Many recent improvements have enabled the use of SFC in a variety of other application areas previously deemed unavailable. Previously, major vendors had limited the support-ed mass spectrometers to single quadrupole systems. Recently, the coupling and support of triple quadrupole and time of flight spectrometers have become avail-able. These additional detectors will allow increased sensitivity in application areas such as drug residue analyses and with additional discrimination power, applica-tions such as lipids and metabolomics. A recently introduced flow splitter contains componentry for enhancing robustness and allowing either split or direct interfaces to various mass spectrometers. When using the flow splitter, the split flow across a gradient is more constant, assuring greater accuracy in peak quantitation after the splitter. The use of a solventless injection mode has been developed with minimal additional hardware. Solventless injection enables very large volumes, comparable to a high-performance liquid chromatography (HPLC) injection, to be injected. Addi-tionally, the solventless injection allows for previously incompatible sample solvent injections including an aqueous injection. Examples of applications utilizing the sol-ventless injection mode, high discrimination mass spectrometry outside of the chiral arena are shown.

281 Effect of Particle Size on the Speed and Resolution of Chiral Separations Using Supercritical Fluid ChromatographyMirlinda Biba, Merck, PO Box 2000, MS: RY818-B218, Rahway, NJ 07065, Erik L. Regalado, Christopher J. Welch, Naijun Wu

Fast chiral supercritical fluid chromatography (SFC) separations have become important due to the increasing use of high-throughput experimentation (HTE) in organic synthesis. These HTE experiments can generate hundreds of samples for chiral analysis that need to be assayed in a short time. In general, chiral SFC can provide much faster analysis times compared to liquid chromatography (LC). Addi-tionally, columns packed with smaller particles can provide faster and more efficient separations. In this study, the effect of the particle size on the speed and resolution of chiral separations by SFC was evaluated. The performance of Chiralcel OD col-umns packed with either 5-µm or 3-µm particles were compared using van Deemter or other kinetic plots. The benefits of using smaller particle columns for chiral SFC analysis are illustrated.

282 The Power of Ultra Performance Convergence Chromatography: Theoretical Considerations and some Examples of UseFrancois J. Huby, The Dow Chemical Company, 400 Arcola Rd., Collegeville, PA 19426, Robert M. Campbell

Supercritical carbon dioxide (CO2) enhanced chromatography (supercritical fluid chromatography and ultra performance convergence chromatography) is comple-mentary to gas chromatography (GC) since it can separate higher boiling com-pounds and to high-performance liquid chromatography (HPLC) since it can sepa-rate and detect closely related compounds which may co-elute or be hard to detect by liquid chromatography. New instrumentation has allowed implementing various CO2 mediated separation techniques with greater efficiency and ease of use than earlier versions. Theoretical evidence and examples of use supporting these state-ments are presented.

283 Comparison of GCMS, EIA, and LC-QTOF Screening Methods for Novel Psychoactive Substances in Urine SamplesJill Yeakel, Lehigh Valley Toxicology, 3864 Courtney St., Ste. 150, Bethlehem, PA 18017, Mandi Mohr, Barry Logan

Designer drugs, such as synthetic cathinones and substituted phenethylamines have been introduced into the drug user community over the last 5 years. US au-thorities have moved to control dangerous compounds as they are discovered, and in response, illicit manufacturers make structural modifications to the drugs in an ef-fort to create legal yet potent stimulant and hallucinogens. The ever-changing drug market has presented a unique problem for medical professionals and toxicologists in identifying the compounds responsible for intoxication or adverse medical effects. Attendees at electronic dance music (EDM) festivals have been documented as having high rates of use of novel psychoactive substances (NPS). We obtained biological samples including urine from anonymous volunteer attendees at a large EDM festival in the US in early 2014 to determine the patterns of use of known NPS as well as to identify new and emerging compounds. The urine samples were screened via a battery of tests including immunoassay (EIA), full scan GC-MS,

LC-QTOF, and RapidFire-MS-MS. The effectiveness of these different screening methods was compared; 75 of the 104 urine samples collected (72%) were positive for drugs of interest by one or more screening techniques, with good agreement between the various methods. This presentation describes the results from four complementary approaches in screening these samples and evaluates ease of use, breadth of analyte detection, sensitivity and ability to detect unknown compounds.

284 Screening Novel Psychoactive Substances (NPS) by High Resolution Accurate Mass Spectrometry (HRAMS)Jared Castellani, South Carolina Law Enforcement Division, 4400 Broad River Rd., Columbia, SC 29212, Alex L. Magitti III, Barry K. Logan, Francis X. Diamond, Matthew M. McMullin

In the realm of forensic toxicology, recent interest has developed over the use of HRAMS for drug screening casework due to its superior specificity and sensitivity to historical screening methods. Time-of-flight (TOF) identifies compounds based on a peak’s retention time and accurate mass relative to the expected retention time (when available) and each compound’s exact mass based on its molecular formula and constant calibration. The issue with this type of testing on complex forensic specimens is that artifacts, minor metabolites and isomers can create false positives that lead to unnecessary confirmatory testing and additional manual evaluation/ma-nipulation to characterize potential candidate compounds. This study demonstrates techniques that have the ability to fragment the molecular ion, allowing for identifi-cation of fragment ions to enhance specificity. Using an Agilent 1290/6530 high-per-formance liquid chromatography (HPLC)-quadropoleTOF mass spectrometer, par-ent and respective fragment masses can be monitored to identify positive findings. Three fragmentation modes were evaluated using LC-QTOF including: quadru-pole-TOF liquid chromatography tandem mass spectrometry (QTOF LC-MS-MS), LC-all product ions-MS with collision induced dissociation in the source (CIDS) and LC-all product ions-MS with collision induced dissociation in collision cell (CIDCC). Three fragmentation modes were evaluated by screening patient whole blood and spiked synthetic blood for NPS. Using an Agilent developed database containing MS-MS spectra for 140 target compounds, the three modes were evaluated. Assay performance was comparable across all three methods at elevated drug concen-trations for the spiked blood samples, but at lower concentrations both CIDS and CIDCC performed superior to QTOF with respect to spiked drug identification.

285 Determination of 22 Antiepileptic Drugs in Post-Mortem Blood, Serum and Plasma Using LC-MS-MS with Focus on their Rule in Forensic CasesKaren Scott, Arcadia University, 450 S. Easton Rd., Glenside, PA 19038, Shaza Deeb

In the last decade, there has been a growth in reports of antiepileptic drugs (AEDs) being misused both on their own and in combination with other drugs of abuse in a variety of toxicological case types such as drug abuse, suicide, overdose and drug facilitated crime. To date, there are no methods for the simultaneous analysis of the most commonly encountered AEDs in post mortem whole blood and clinical plasma/serum samples. A simple, accurate and cost effective liquid chromatography tandem mass spectrometric (LC-MS-MS) method was developed and validated for the quantification of carbamazepine and its metabolite carbamazepine-10,11-epox-ide, eslicarbazepine acetate, oxcarbazepine and S-licarbazepine as a metabolite, gabapentin, lacosamide, lamotrigine, levetiracetam, pregabalin, phenobarbital, phe-nytoin and its metabolite 5-(p-hydroxyphenyl)-5-phenylhydantoin, retigabine (ezo-gabine) and its metabolite N-acetyl retigabine, rufinamide, stiripentol, topiramate, tiagabine, valproic acid, vigabatrin and zonisamide in post-mortem whole blood, serum and plasma which is suitable for routine forensic toxicological analysis and therapeutic drug monitoring. Drugs were extracted from biological matrix using a simple effective liquid liquid extraction. All AEDs were detected and quantified within 17 minutes without endogenous interferences. The correlation coefficient (R2) was greater than 0.994 for all drugs with accuracy ranging from 90 to 113% and precision <13% for all analytes. The recovery ranged from 70% to 98%. No carryover was observed in a blank control injected after the highest standard and the matrix effect was acceptable and ranged from 90% to 120%. The method has been successfully verified using authentic samples from clinical, postmortem and prison case samples.

286 Development of a Pyrrolidinophenone Panel in Blood and Detection in Toxicology Case WorkDonna Papsun, NMS Labs, 3701 Welsh Rd., Willow Grove, PA 19090, Barry K. Logan, Robert Middleberg, Carol Beck

Pyrrolidinophenones are an emerging group of compounds characterized as novel psychoactive substances (NPS) as part of the designer drug movement. Routine forensic toxicology assays may not be detecting these compounds, consequent-ly underestimating their use. For this reason, it was necessary to develop screen and confirmation tests for blood to identify compounds in this group, which includ-ed alpha PVP, naphyrone, pyrovalerone, alpha PBP, alpha PPP, MePPP, MDPPP, MOPPP, MPBP, and MPHP. Traditional screening tools use gas chromatogra-phy-mass spectrometry (GC-MS) library matching; however, validated reporting lim-

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its for this group of pyrrolidinophenones varied from 5 to 80 ng/mL, which poses a significant problem when trying to identify small amounts of drugs in biological spec-imens. A second screening technique, Liquid Chromatography Time of Flight Mass Spectrometry (LC-TOF-MS), demonstrated 100% sensitivity and specificity for all compounds at a reporting limit of 10 ng/mL. A quantitative method was developed using Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (HPLC-ESI-MS-MS) operated in electrospray positive ionization multiple reaction monitoring mode. Calibration was satisfactory for all 10 analytes from 5-500 ng/mL. Accuracy data were within the acceptance interval of +/- 15% of the nominal values for all drugs. Within-day (repeatability) and intermediate precision data were within 20% relative standard deviation (RSD). After analysis of de-identified patient cases, positives were seen for alpha PVP and alpha PBP. These findings suggest that a combination of LC-TOF-MS and LC-MS-MS provides two valuable tools for the sen-sitive and specific analytical identification and measurement of pyrrolidinophenones in forensic casework.

287 How Photoacoustic and Nanomechanics Combine to Perform IR Spectroscopy at the NanoscaleAlexandre Dazzi, University of Paris-Sud, Laboratory of Physical Chemistry, Batiment 349, Orsay 91405 France

Atomic force microscope-based infrared spectroscopy (AFM-IR) has been devel-oped in recent years providing extremely high spatial resolution chemical charac-terization and imaging. The technique is based on the combination of a tunable infrared laser with an atomic force microscope that can locally map and measure thermal expansion of nanoscale regions of a sample resulting from the absorption of infrared radiation. The results obtained these last years in microbiology and poly-mer sciences show how this approach is a powerful tool. The principle is based on detecting the local thermo expansion of the sample, irradiated at the wavelength of its absorption bands. This expansion is detected by the AFM tip in contact mode. As the duration of expansion and relaxation of the sample is always shorter than the response time of the cantilever in contact, the excitation transmitted to the cantile-ver acts as an acoustic shock, exciting oscillations at resonant frequencies of the cantilever. The technique can create nanoscale IR absorption spectra by recording the amplitude of these oscillations as a function of wavelength and chemical maps by measuring the oscillation amplitude as a function of position. [1] Because the AFM probe tip can map the thermal expansion on very fine length scales, the AFM-IR technique provides a robust way to obtain interpretable IR absorption spectra at spatial resolution scales well below the diffraction limit.


[1] Dazzi et al. J Appl. Phys. 107, 124519 2010

288 Extending AFM-Based Infrared Spectroscopy to a Wide Range of ApplicationsCraig Prater, Anasys Instruments, 325 Chapala St., Santa Barbara, CA 93101

Atomic force microscope-based infrared spectroscopy (AFM-IR) is a growing tech-nique for nanoscale chemical characterization of materials. Initial AFM-IR instru-ments had two key limitations: 1) Samples had to be prepared as thin sections with a very limited thickness range; and 2) the thin sample sections needed to be mounted on an infrared transparent prism. Recent research has enabled instrumentation that overcomes both of these limitations. Now samples can be measured on almost ar-bitrary substrates, opening up nanoscale chemical analysis to a much wider range of samples, including many in-situ measurements. In addition, recent innovations have enabled dramatic improvements in AFM-IR measurement sensitivity. Using resonant enhanced AFM-IR, it is possible to perform spectroscopic analysis and chemical imaging on films as thin as a single molecular monolayer. This presenta-tion describes recent innovations in AFM-IR technology and applications in various fields including energy, materials and life sciences.

289 AFM-IR: Nanoscale IR Spectroscopy for the Materials and Life SciencesCurtis Marcott, Light Light Solutions, 2558 High Hammock Rd., Johns Island, SC 29455, Craig Prater, Qichi Hu, Michael Lo, Kevin Kjoller

Atomic force microscope-based infrared spectroscopy (AFM-IR) has been devel-oped in recent years providing extremely high spatial resolution chemical characteri-zation and imaging. The technique is based on the combination of a tunable infrared laser with an atomic force microscope that can locally map and measure thermal expansion of nanoscale regions of a sample resulting from the absorption of infrared radiation. Because the AFM probe tip can map the thermal expansion on very fine length scales, the AFM-IR technique provides a robust way to obtain interpretable IR absorption spectra at spatial resolution scales well below the diffraction limit. Sev-eral applications of AFM-IR spectroscopy and imaging to problems in the materials and life sciences are presented.

290 Tip-Enhanced Infrared Nanospectroscopy via Molecular Expansion Force DetectionMikhail A. Belkin, University of Texas-Austin, Department of Electrical and Computer Engineering, Austin, TX 78759, Feng Lu, Mingzhou Jin

Mid-infrared photoexpansion nanospectroscopy, or atomic force microscopy infra-red (AFM-IR), is a powerful technique for nanoscale chemical analysis in which light absorption is detected by measuring associated local sample thermal expan-sion with an atomic force microscope (AFM). Originally developed by Alexandre Dazzi and co-workers, this approach required high-power laser pulses to induce significant sample heating and samples of ~100 nm or more in thickness. Recently, we demonstrated that the sensitivity of this technique may be improved to take nanoscale spectra of samples as thin as molecular monolayers with better than 30 nm spatial resolution. This was achieved by sending low-power laser pulses at a repetition frequency that is tuned in resonance with the mechanical vibrational frequency of the AFM cantilever and by employing tip-enhancement of the optical field below a sharp gold-coated AFM tip. I describe the details of our approach and present the latest results, including nano scale spectra of samples in aqueous en-vironment. This project was supported by the Welch Foundation grant F-1705 and STTR program from the DOE.

291 NanoIR to Investigate Parchment and its DegradationLaurianne Robinet, Center of Research for Conservation, 36, rue Geoffroy Saint Hilaire, Paris, 75005 France, Gaël Latour, Ariane Deniset-Besseau, Alexandre Dazzi, Marie-Claire Schanne-Klein

The characterization of cultural heritage materials is essential to understand the artefact technology and its alteration, so to develop suitable restoration treatment or conservation conditions. These analyses are often complex due to the heterogene-ity of the material at different length scales and its composite nature originating from the manufacturing or the degradation. Additionally, artefacts are generally fragile or rare thus sampling is often restricted or impossible. For these reasons non- or mi-cro-invasive analytical approaches using sensitive techniques are being developed. Parchment was the main writing material in the Middle Ages in Western Europe up to the growth of paper production in the 14-15th centuries. Made from an untanned animal skin it was preserved by liming, scraping and drying the skin under tension. Parchment is very sensitive to water, which can cause denaturation of collagen, its main constituent. This degradation, known as gelatinization, induces modifications at all structural levels in the molecule, thus complementary analytical techniques are required to better understand the involved mechanisms. Infrared spectroscopy is used to examine parchment modifications at the molecular level; however the struc-tural heterogeneity within a collagen fiber and the interference of the carbonate sig-nal (from the liming process) in the spectra often renders the interpretation difficult when working at the micro scale. To overcome this limitation, infrared analysis at the nanoscale is crucial. This presentation presents the first results in parchment fibers analysis at the nanoscale using atomic force microscopy infrared (AFM-IR) and dis-cusses the potential of coupling nanoIR with other microscopy imaging techniques.

292 Quantitating Protein Panels and Normalizing Hematocrit from Dried Blood Spots (DBS) Using an Automated SISCAPA® Mass Spectrometry WorkflowMorteza Razavi, SISCAPA Assay Technologies, Box 53309, Washington DC 20009, Selena S. Larkin, Terry W. Pearson, Leigh Anderson

The use of dried blood spots (DBS) for bioanalysis has gained considerable mo-mentum with reports demonstrating that both small molecules and proteins can be reliably extracted and analyzed by mass spectrometry. However, lack of a standard-ized method to normalize for sample hematocrit and volume variations has slowed the adoption of DBS in clinical settings. Moreover, standard separative means used in mass spectrometry are either overly complicated and low-throughput or do not provide enough sensitivity to measure low-abundance proteins. To address these challenges, we devised an automated approach using a Bravo liquid handling ro-bot (Agilent Technologies, CA) whereby proteotypic peptide targets are selective-ly enriched from digested human blood using high-affinity anti-peptide antibodies and quantitated based on their relative ratio to internal stable isotope standards; an approach known as SISCAPA®. The approach was complemented with a propri-etary algorithm that normalizes sample-to-sample hematocrit and volume variations based on measurement of high-abundance proteins of plasma and red blood cells. Using this novel strategy, we successfully measured normalized levels of two 11-plex protein panels, with abundance spanning 8 orders of magnitude, from a single ¼” diameter DBS punch. By monitoring longitudinal DBS specimens, we were able to track the kinetics of changes in concentration of these proteins through condi-tions such as a common cold, pneumonia and kidney infection. We also observed an interesting ‘protein baseline-concentration fingerprinting pattern’ unique to each individual under study. These results bode well for personalizing medicine through longitudinal collection of dried-blood-spots.

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November 20142014 EAS Abstracts

293 Validated Determination of Dapagliflozin and Qualitative Determination of Its Glucuronide Metabolite in Human Dried Blood Spot: Multiple-Site Pediatric Clinical ExperienceXiaohui Xu, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ 08540, Jane Liu, Jim Shen, Melanie Pe Benito, Marsha Epstein, Guowen Liu, David Boulton, Pathanjali Kadiyala, Mark E. Arnold, Qin Ji, Michael Waldron

Dry blood spot (DBS) technology has garnered acceptability as a microsampling technology to support drug discovery and development process due to the ease of sample storage and shipment. Bristol-Myers Squibb has extensively applied this technique in toxicokinetic studies and started to extend the application into pedi-atric studies. DBS sample collection, including collections from both venous and finger-stick draws, were incorporated in a pediatric study protocol for dapagliflozin (DAPA, Farxiga™). Plasma sample collection was also made in parallel and com-pared against DBS data to ascertain the fitness of the DBS as a sampling scheme. We have developed a DBS liquid chromatography tandem mass spectrometry (LC-MS-MS) assay to simultaneously quantify DAPA while qualify the major glucuronide metabolite (GLU) in dry blood spots with a volume 15-µL of blood collected on What-man FTA DMPK-C cards. Each analysis utilizes a circular punch size of 6mm in di-ameters. Analytes were eluted from the cards with 75:20:5 water / 1.0 M ammonium acetate / formic acid, v/v/v which was cleaned up via solid-phase extraction and analyzed under negative ion electrospray LC-MS-MS detection in multiple reaction monitoring. A lower limit of quantitation of 1 ng/mL was achieved for DAPA and 2.0 ng/mL for GLU, which were suitable for the pediatric study. The between-run %CV from assay qualification batches was ≤ 8.60 and the within-run %CV was ≤ 8.03. The mean % deviation from nominal concentration was within ±8.61. The concentra-tion data from DBS venous and finger stick draws, and plasma draw are comparable and we have demonstrated the utility of DBS in this pediatric clinical study.

294 Advantages and Challenges of Applying LC-MS-MS to Regulated Protein BioanalysisHao Jiang, Bristol-Myers Squibb, Route 206 & Province Line Rd., Princeton, NJ 08543, Jianing Zeng, Craig A. Titsch, Linlin Luo, Dharmesh D. Desai, Anne F. Aubry, Binodh S. DeSilva, Mark E. Arnold

Liquid chromatography-mass spectrometry (LC-MS) has been recently applied to protein bioanalysis due to its ease of measuring multiple analytes, unique selectivity, and significant flexibility. In this presentation, the authors discuss the advantages and challenges of applying LC-MS in regulated protein bioanalysis, followed by elaborating the strategy of supporting protein bioanalysis using either LC-MS or li-gand-binding assays (LBA). A recent case study comparing the two assay formats in the bioanalysis of monoclonal antibody (mAb) drugs is presented. In this regulated toxicokinetic study, two co-administered mAb drugs in monkey serum were simulta-neously quantitated with LC-MS by monitoring a pair of tryptic peptides (quantitation peptide and confirmatory peptide) derived from different regions of each mAb, after a simple and efficient protein pellet digestion. Stable isotopically labeled peptides with flanking amino acids on C- and N-terminals were used as internal standards to minimize the variability during sample processing and detection. In the assay, the confirmatory peptides played a critical role in confirming quantitation accuracy and the integrity of the drugs in the study samples. Meanwhile, two separate LBAs were applied for quantitation of each mAb for the same set of samples. The equivalen-cy of the data between LC-MS and LBA was demonstrated from cross-validations using quality control samples and incurred study samples. The comparable bioana-lytical concentrations, toxicokinetic parameters, and statistical correlations from the both assay formats confirm that LC-MS is a reliable and complementary approach for mAb bioanalysis.

295 Improved LC-MS Method Development for Pain Management PanelsThomas A. Russell, Shimadzu Scientific Instruments, 7102 Riverwood Dr., Columbia, MD 21046

In order to maximize turnaround time in a pain management lab setting there has been a focus on combining multiple drug panels in a short analysis time. This may present challenges when considering isobaric pairs and fast polarity switching. This study was undertaken to demonstrate the ability to quickly develop a rugged method using ultra-performance liquid chromatography tandem mass spectrometry (UHPLC-MS-MS). Method development software is used to efficiently determine the best mobile phase and column combination for one method. This assay meets re-quirements for sensitivity and quantitation for all classes of drugs of abuse: barbitu-rates, THC metabolites, amphetamines, benzodiazepines, cocaine and metabolites, opiates and opioids. The focus is on selectivity of all compounds, especially isobaric pairs, while optimizing the shortest runtime possible.

296 Identification of Volatile Organic Compounds in Beer by TurboMatrix Headspace Trap System-Gas Chromatography-Mass SpectrometryHetal Rana, Kean University, 1000 Morris Ave., Union, NJ 07083, Dil Ramanathan

Beer is the most popular drink around the world, after water and tea. It has be-come a standard and most consumed alcoholic beverage. Beer is made by the saccharification of starch and fermentation of sugar, a process called brewing. The saccharification enzymes, required for fermentation process, are extracted from malted cereal grains like wheat and barley. Female flowers called hops are used as flavoring agents, which also give bitterness to the beer and act as a natural preser-vative. Beer is a complex mixture of various compounds such as alcohols, sugars, esters, proteins, ketones and acids. In this study, a rapid and sensitive method is developed to identify and quantify many of these compounds using a TurboMatrix headspace trap (TMHST) system connected to gas chromatography-mass spec-trometry (GC-MS). The selectivity and inertness of the stationery phase, the chro-matography column (60 m x 0.25 mm x 1.0 µm Elite-5MS), and the gaseous mobile phase give precise retention times to separate the peaks. In the MS, each peak will be further separated according to their mass-to-charge ratio and then identified and characterized. The method developed is used to create a library of volatile organic compounds present in beer. In the results all beers contain 3-methylbutanol and all hops contain Limonene which is responsible for citrus aroma. The 3-methylbutanol is one of the fusel oils or fusel alcohols associated with fermentation of beer.

297 Assuring Water Purity by Monitoring Water Contaminants at Ultratrace LevelsSatinder (Sut) Ahuja, Ahuja Consulting, 1061 Rutledge Court, Calabash, NC 28467

After air, water is the most essential material for human survival. Without water, life as we know it in this world would not be possible. Even though our earth is composed largely of water, fresh water comprises only 3% of the total water avail-able to us. Of that, only 0.06% is easily accessible. An estimated 1.2 billion people drink unclean water today. Drinking water comes mainly from the following sources: rivers, lakes, wells, and natural springs. These sources are exposed to a variety of conditions that can contaminate the water. The failure of safety measures relat-ing to production, utilization, and disposal of a large number of inorganic/organic compounds (from arsenic to zinc) can cause contamination of our water supplies. Whereas zinc amounts is in small desirable, arsenic is harmful over 10 parts per billion (ppb). The most notable case is the massive arsenic contamination of ground-water in Bangladesh. Arsenic contamination of water has also been observed in the US, Australia, Asia, Europe, and South America. Inorganic arsenic above 10 ppb level can increase the risk of lung, skin, bladder, liver, kidney, and prostate cancer. This lecture discusses the impact of such effects on the basis of studies in Ban-gladesh where 60 million people are at risk. Analytical chemists can play a leading role in the prevention of a number of illnesses by monitoring our water supplies by ultra-trace analysis at or below ppb level, using various sophisticated techniques.

298 Recent Developments in Stationary Phases for Ion ChromatographyChristopher A. Pohl, Thermo Fisher Scientific, 445 Lakeside Dr., Sunnyvale, CA 94085

Although ion chromatography is a relatively mature science, research into the devel-opment of new stationary phases for ion chromatography has continued at an active pace for the more than 35 years since the original introduction of ion chromatogra-phy as an analytical technique. In this work we review the latest developments in new ion exchange phases developed specifically for ion chromatography. We cover a variety of different stationary phase architectures and include examples illustrating their application to water quality analytical challenges.

299 Novel Electrochemical and Reflectance Photometric Techniques for the Measurement of Trace Arsenic in WaterAbul Hussam, George Mason University, 4400 University Dr., Fairfax, VA 22030, Douglas Mays, Jinsoo Hong, Joan Rozario

The measurement of trace arsenic in the environment at a low cost is a continuing analytical challenge. Here, we explore some promising analytical techniques cost-ing less than $10,000 and field deployable for large scale measurement efficiency. Recent advances in electrochemical techniques using B-doped diamond electrode, Au-NP modified electrode, Au-micro electrode in flow cell, and electrochemical de-tection of gas phase arsine appears promising. In a novel reflectance photometric technique the formation of Agx-AsHy (x=1,2,3 y= 2,1,0) (s) complex by the reaction of Ag+ and AsH3(g) was monitored as a function of time. The concentration of trace arsenic was determined by a kinetic method for faster analysis.

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300 Pharmaceuticals in Environment and Water Quality: The Role of Analytical ChemistryHua Yao, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903

Thanks to advances in analytical chemistry, trace levels of active pharmaceutical ingredients (APIs) have been discovered in the environment, especially in water systems, around the globe. Pharmaceuticals in the Environment (PIE) raises public interest and concerns among regulatory, scientific, environmental and media stake-holders. Although environmental risk assessment on APIs is now required by regu-latory agencies such as the Food and Drug Administration and European Medicines Agency prior to new drug approval, a framework is still lacking to evaluate and monitor the potential impact on the environment once a new drug is launched. Only a small percentage of the total of over 3000 APIs have been studied for their occur-rence and fate in environment. Models have been proposed to estimate the concen-trations of APIs in aquatic environment. At Bristol-Myers Squibb collective efforts have been initiated to advance the understanding of its drug products’ impact on the environment. Analytical chemistry plays an important role by providing analytical tools to monitor the occurrence of APIs post drug approval. Quantitative analytical methods for twelve APIs have been developed using solid-phase extraction and liq-uid chromatography with electrospray ionization source tandem mass spectrometry. Multiple APIs are analyzed simultaneously. Stable isotopically labeled analogs or structure analogs have been used as internal standards to improve method perfor-mance. Lower limits of quantification as low as parts per trillion are achieved.

301 Withdrawn by the author.

302 Analysis of High and Low Dielectric Constant Oxides for Microelectronics ApplicationsRobert Opila, University of Delaware, 201 DuPont Hall, Newark, DE 19716, Jonathon Church, James Krajewski, Dan Yang, Venkateswara Pallem, Conan Weiland, David Gidley

Electronic materials are becoming compositionally and structurally more compli-cated. Thus, characterization of these materials is becoming more and more of a challenge. We discuss two examples from a collaboration between Air Liquide and the University of Delaware. In the first study, atomic layer deposition (ALD) of ZrO2 and Nb2O5 thin films has been studied using high-energy synchrotron-based X-ray photoemission spectroscopy. Given the relatively high thickness of the layers (tens of nanometers), the X-rays are able to effectively probe the interface of ZrO2 and Nb2O5 layers with their respective substrates. Binding energy shifts associated with the chemical environment at the interface of up to 1.4 eV and 1.1 eV for the ZrO2 and Nb2O5 films respectively are shown. These energy shifts suggest significant charge transfer at the interface between the high-k oxide and Si. In addition, the composition and chemistry of the films as a function of thickness has been deter-mined non-destructively by converting a series of spectra taken with different inci-dent X-ray energies (and simultaneously, different photoelectron sample depths) to depth profiles using Bayesian analyses. In the second example, the characterization of porosity of porous low-k dielectric films is a challenge in part due to the limited sampling volume. The application of three non-destructive porosimetry techniques: positron annihilation lifetime spectroscopy, ellipsometric porosimetry, and X-ray re-flectivity to characterize porous low-k thin films, are examined. The advantages and limitations of each technique, especially the sensitivity to pore interconnectivity and its relationship to the mechanical properties, are discussed.

303 Interface Analysis of Nano-scale MaterialsLeonard Feldman, Rutgers Institute for Advanced Materials, Devices and Nanotechnology, 607 Taylor Rd., Piscataway, NJ 08854

Modern material research has provided the means of creating structures controlled at the atomic scale. Examples include the formation of hetero-structures grown with atomic precision, monolayer (graphene and graphene-like) films, nanostructures with designed electronic properties, shaped nano-plasmonic materials and new or-ganic structures employing the richness of organic chemistry. The current forefront of such nano-materials research includes the creation and control of new materials for energy, bio/medical and electronics applications. The performance of these di-verse materials systems is invariably determined by their fabrication and their inter-facial structure. Interfaces are the critical component and least understood aspect of such materials-based structures. Interfacial analysis is described in the context of a number of projects underway at the Rutgers Institute for Advanced Materials, Devices and Nanotechnology. These include: 1) self-assembled monolayers on or-ganic single crystals resulting in enhanced surface mobility; 2) interfacial analysis of complex oxide hetero-structures to elucidate the enhanced two-dimensional elec-tron mobility; 3) characterization of the semiconductor-dielectric interface in SiC/SiO2 for energy efficient power transmission. We describe two new analytical tools: the Zeiss-Orion 0.25nm He ion beam and the Rutgers-NION Scanning Transmis-sion Electron Microscope with ~ 12 meV electron energy loss resolution. Collabo-

rators: R. A. Bartynski, E. Garfunkel, T. Gustafsson, H.D. Lee, D. Mastrogiovanni, V. Podzorov, L. S. Wielunski, G. Liu, J. Williams, S. Dhar, P. Cohen, E. Conrad, Yi Xu, P. Batson

304 Structural Motifs in Carbon Nanoparticle Films: Nanoparticle Functionalization and NanorystallizationJanice Reutt-Robey, University of Maryland, Department of Chemistry and Biochemistry, College Park, MD 20742

We explore structural motifs that can be achieved in monolayer films of C60 and C70, and their phenyl-butyric-acid methyl ester derivatives, at neat and chemical-ly-modified Au (111) and Ag (111) substrates. On metallic substrates, we generate glassy films of C60-PCBM and C70-PCBM (nanoparticle) monolayers through a microaerosol deposition method. We then reveal how the ordering of the films is frustrated by nanoparticle-nanoparticle interactions through UHV-STM measure-ments. Transition to crystalline (hcp) fullerene arrangements proves to be highly activated and dependent on local PCBM density. Anisotropic PCBM-PCBM interac-tions account for the observed packing arrangements in PCBM films, which include disordered, dimer-row phases, and hcp phases, and barriers for nanocrystallization. Additionally, we present comparative studies of nanostructure evolution in binary C60-ZnPc and C60-PCBM-ZnPC films. In the first case, we identify and explore the formation mechanisms of a serpentine C60 chain phase structure that arises at the ZnPc/Ag(111) interface. In the case of functionalized carbon nanoparticles, we explore the vertical phase separation that results in the formation of PCBM/ZnPc/Au(111) stacked film structures. We show how molecular Electrostatics – including contributions from the interface dipole and from the intrinsic molecular dipoles – provide key driving forces that lead to remarkable - and predictable - pattern forma-tions. Implications for functionalized carbon nanoparticles in organic electronic ap-plications are discussed. This research is supported by the NSF-Chemistry (MSN) through Award # CHE1310380.

305 Understanding the Electron Stimulated Surface Reactions of Organometallic Complexes to Enable Design of Precursors for Electron Beam Induced DepositionHoward Fairbrother, Johns Hopkins University, Department of Chemistry, 3400 North Charles St., Baltimore, MD 21218, Julie Spencer, Mike Barclay, Samantha Rosenberg, Lisa McElwee-White

Standard practice in electron beam induced deposition (EBID) is to use precursors designed for thermal processes, such as chemical vapor deposition (CVD). How-ever, organometallic precursors that yield pure metal deposits in CVD often create EBID deposits with high levels of organic contamination. This contamination nega-tively impacts the deposit’s properties (e.g., increasing resistivity, decreasing cata-lytic activity) and severely limits the range of potential applications for metal-contain-ing EBID nanostructures. To provide the information needed for the rational design of precursors specifically for EBID, we have employed an ultra-high vacuum (UHV) surface science approach to elucidate the elementary reactions of organometallic precursors during EBID. Our results have shown that the initial electron induced depositions of surface bound organometallic precursors proceed through desorption of one or more of the ligands present in the parent compound. In specific cases this deposition step has been shown to proceed via dissociative electron attachment (DEA), involving low energy secondary electrons generated by the interaction of the primary beam with the substrate. However, electron beam processing of the surface bound species produced in the initial deposition event usually causes decomposi-tion of the residual ligands, creating non-volatile fragments. This process is believed to be responsible for the bulk of the organic contaminants typically observed in EBID nanostructures. Our results also suggest that simple ligands architectures that involve a combination of CO and halogen atoms as the exclusive ligands may yield deposits with significantly higher metal contents compared to the values that can currently be attained with CVD precursors.

306 Reducing Solvent Consumption in High-Performance Liquid Chromatography during Column Re-Equilibration after Gradient ElutionMichael R. Fletcher, Drexel University, 3141 Chestnut St., Philadelphia, PA 19104, Erin J. Ennis, Donna M. Blackney, Catherine A. Kita, Joe P. Foley

In traditional high-performance liquid chromatography (HPLC), column re-equilibra-tion between runs is necessary to prepare a column for subsequent experiments when using gradient elution. This process replaces the final mobile phase in the col-umn, interstitially (between the particles), intra-partically (within the pores), and in-terfacially with the initial mobile phase. Column re-equilibration, which is presumably limited by the rate of diffusion within the pores, traditionally consumes a large quan-tity of mobile phase due to the high flow rates commonly employed. In the interest of green chemistry, an alternative method of column re-equilibration after gradient elution is presented. This work aims to minimize solvent consumption by substan-tially reducing the flow rate during column re-equilibration, thus allowing more time

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for new mobile phase to diffuse into all regions of the column while consuming less solvent. A variety of column sizes and composition are studied to optimize equilibra-tion time, flow rate, and solvent consumption.

307 The Importance of Tolerance Limits in UHPLC Method Modelling for Robustness EvaluationImre L. Molnár, Molnár-Institute, Schneeglöckchenstr.47, Berlin, 10407 Germany, Hans-Jürgen Rieger

From over 100,000 diseases only 20,000 can be treated with drugs, as the develop-ment costs of the drugs are too high to cover the investments because the numbers of patients are too small. This can only be changed, if ultr high-performance liquid chromatography (UHPLC) method development becomes faster and more efficient and allows to simplify also the complexity of regulatory interactions. By modelling the separation, the development process, which is based on solid science, can be dramatically shortened. Multifactorial influences can be modeled in seconds and the best separation with the best column can be selected in a revolutionary way, much faster than in the past. Tolerance limits will be varied and the separation quality will be unically visualized. The presentation shows how to develop UHPLC meth-ods with high efficiency and excellent robustness, based on modern computational technologies and reliable chromatographic rules. Case studies support the presen-tation in an effective way demonstrating a breakthrough in modern UHPLC method development.

308 The Use of Eluent Generation in Ion ChromatographyPeter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA 94085, John Madden

In ion analysis, mobile phases and regenerant solutions are typically prepared using acidic (ie: HCl) or basic (ie: NaOH) stock solutions. Manual preparation of these reagents requires the need to handle acids or bases and thus a skilled chemist. Manual preparation is also labor intensive and can often lead to errors if the re-agents used to make the solutions and the inevitable dilutions are not made pre-cisely. Reagent-free ion chromatography (RFIC) use electrolytic technologies to generate eluents from deionized water. Using RFIC, laboratories spend less time on equilibration, calibration, method verification, troubleshooting, and consistency checks because the systems minimize unintentional variations in the preparation of eluents and regenerants. In this presentation, we review how RFIC or eluent gener-ation works and provide examples demonstrating that it can be superior to manually prepared mobile phases and regenerats.

309 What Performance can we Expect from Microfluidic LC-MS System?Martin Gilar, Waters, 34 Maple St., Milford, MA 01757, Thomas S. McDonald, Gregory Roman, Jay S. Johnson, James P. Murphy

When dealing with sample limited applications or when striving for lower limits of detection, microfluidic liquid chromatography (LC) is a promising alternative to con-ventional scale chromatography. In this presentation, we discuss the implications of dispersion in microfluidic chromatography, both pre-column (sample injection, sam-ple trapping) and post-column (connection to the detector), and illustrate how these affect the results of an analysis. We illustrate how the holistic design of a microfluidic LC-mass spectrometry (MS) system can minimize many of these difficulties, provid-ing the analytical scientist with robust methods and a high level confidence in data quality. Theoretically predicted and experimental results are shown to demonstrate the performance of a well optimized microfluidic LC system. We discuss the perfor-mance of the recently introduced microfluidic systems (0.15 mm ID) and compare its performance to industry standard 2.1-mm I.D. Ultra-performance LC columns. We show that microfluidic LC does not have to be difficult to use, and ease of use does not have to be traded for an increased sensitivity.

310 Investigation of Multidimensional Chromatography for Chromatographic Separation of Complex Mixtures of Closely Related SpeciesErik L. Regalado, Merck, 126 E. Lincoln Ave., PO Box 2000, Rahway, NJ 07065, Christopher J. Welch

In this presentation we describe our recent investigations into the separation of complex mixtures of constitutional isomers, stereoisomers and other closely-related species using a variety of single- and multi-dimensional chromatography approach-es. Strategies and approaches for application of chiral or achiral stationary phases, mass spectrometry deconvolution and the use of tandem supercritical fluid chroma-tography, offline two-dimensional (2-D) liquid chromatography and comprehensive 2-D liquid chromatography are discussed, and several examples of the practical use of multi-dimensional chromatography methods for resolution of multicomponent mixtures are presented.

311 Determination of Common Counter and Impurity Anions in Pharmaceutical Using a High-Pressure Modular Capillary Ion Chromatography System (HPIC) with Suppressed Conductivity (CD) and Charge (QD) DetectionHua Yang, Thermo Fisher Scientific, 1214 Oakmead Pkwy., Sunnyvale, CA 94085, Linda Lopez

Ion analysis is important for pharmaceutical industry because many active phar-maceutical ingredients (APIs) exist in their salt form. Pharmaceutical products are strictly regulated by the United State Food and Drug Administration (FDA) and oth-er regulatory agencies, and must be tested for composition to verify their identity, strength, quality and purity. Recently, identification and quantification of ions in early stage drug development has gained increasing attention, because the APIs maybe contaminated with different counter ions from synthesis steps, and because se-lecting the counter ion to enhance APIs’ solubility and stability is becoming a key step in formulation development. Since many of the ions are non-chromophoric, Ion chromatography (IC) with suppressed conductivity detector (CD) is an established method used for ion determination. The recently introduced high-pressure capable capillary IC systems (HPIC) combined with 4-µm particle ion-exchange columns have improved separation efficiency and significantly reduced the eluent consump-tion to 5.2 L/year which saves money and time. Use of a newly invented charge detector (QD) combined with CD permits peak purity assessment to further simplify identification and confirmation of ions. This study demonstrates the identification and quantification of 22 commonly found anions in pharmaceuticals in a single run using a high-pressure capillary IC system (HPIC) with 4-µm particle ion –exchange column, and CD-QD dual detectors.

312 Recent Compendial Activity for Dissolution Testing when Gelatin Crosslinking is an IssueGregory P. Martin, Complectors Consulting, 804 Bauman Cir., Pottstown, PA 19465, Vivian A. Gray

Crosslinking of gelatin in pharmaceutical dosage forms is known to cause dissolu-tion slowing, and the USP allows use of enzymes to mitigate this. There are many issues with the two tier testing allowed in United States Pharmacopeia (USP) Gen-eral Chapter Dissolution <711> where enzyme is added to media when dissolution failures are observed on stability with capsules shell/gelatin products. New pro-posed revisions to Dissolution General Chapter <711> and new General Chapter on Capsules <1094> are discussed, including when enzyme use is appropriate, strategies when enzyme activity is adversely affected by the dissolution medium and consideration of use of additional enzymes in the pH region where pepsin and pancreatin have little enzyme activity.

313 Characterizing the Dynamic Formation of Disease Protein Aggregates Using Dark-State Exchange Saturation Transfer (DEST) NMRNicolas L. Fawzi, Brown University, Department of Molecular Pharmacology, Physiology, and Biotechnology, 70 Ship St., Rm. 119, Providence, RI 02903

A common feature of numerous neurodegenerative diseases is the formation of neurotoxic inclusions of proteins. However, the structures along the protein as-sembly pathway, made up of large, disordered, and transient aggregates, are often invisible to traditional atomic resolution techniques. Here we describe techniques in solution nuclear magnetic resonance (NMR) spectroscopy to characterize the aggregation intermediates of proteins associated with neurodegenerative diseases. Alzheimer’s disease is characterized by the accumulation of amyloid β peptides. Recently, the 43-amino-acid peptide Aβ43 has garnered attention as a highly ag-gregation-prone, neurotoxic species shown to deposit sooner and with higher likeli-hood than its shorter variants. We present the biophysical characterization of Aβ43 structure and dynamics in the monomeric and protofibrillar states to determine if and why this peptide has enhanced aggregation propensity. Slower dynamics and chemical shift differences are present across residues 30-43 in the monomeric state of Aβ43 as compared to Aβ42. Dark-state exchange saturation transfer NMR demonstrates that, compared to Aβ42, Aβ43 is less often directly-bound to the sur-face of protofibrils, but the C-terminus directly binds more frequently. Dynamics in protofibril-bound states are slowed at the C-terminus. Our results suggest that faster aggregation of Aβ43 is due to slowed dynamics and the formation of aggregate-fa-voring structure in the C-terminal region in both the monomeric and protofibril-bound states of Aβ43. Recent extensions of the DEST method to methyl sidechains in Aβ and application to aggregation of prion-like domains of RNA-binding proteins in amytrophic lateral sclerosis are also reviewed.

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314 Structure, Dynamics, and Function of Opa60: A Neisserial ß-Barrel Membrane Protein that Mediates Host PhagocytosisLinda Columbus, University of Virginia, Department of Chemistry, McCormick Rd., PO Box 400319, Charlottesville, VA 22904, Alison K. Criss, Peter M. Kasson

The family of Opa proteins from Neisseria gonorrhoeae and N. meningitidis are eight-stranded β-barrel proteins that induce phagocytosis of the bacterium by en-gaging three different host receptors: carcinoembryonic antigen cellular adhesion molecules (CEACAM), heparansulfate proteoglycans (HSPG), or integrins via HSPG and fibronectin or vitronectin. The receptor engaged depends on the se-quence of two hypervariable (HV) regions in two extracellular loops, which differ be-tween isolates. There are hundreds of HV sequences identified; however, multiple sequence alignment of the HV loops does not reveal specificity motifs among the family of Opa proteins due to the extreme variability in the amino acid sequences. Towards understanding the molecular recognition required to gain entry into human cells, the structure of Opa60, which binds CEACAM1, 3, 5 and 6 was determined. Structure determination of membrane proteins is challenging and even more so for proteins that have large portions of both soluble and membrane-embedded re-gions. Thus, Opa proteins presented some methodological obstacles in both solu-tion nuclear magnetic resonance (NMR) assignments and structure calculation and refinement. Strategies for the assignment of the protein included trypsin cleavage, peptide synthesis, and assignment at various temperatures. To overcome struc-ture refinement obstacles, a hybrid method that used the restraints determined with NMR spectroscopy in detergent micelles in conjunction with molecular dynamics (MD) simulations in a lipid bilayer. The resulting Opa60 structure, coupled with in-vivo and in-vitro Opa reconstituted liposome assays, has facilitated investigations of the molecular determinants of Opa-receptor interactions.

315 Characterization of the Active Site of Vanadium Chloro-peroxidase by 51V Solid-State NMR SpectroscopyRupal Gupta, University of Delaware, Department of Chemistry, Newark, DE 19716, Guangjin Hou, Tatyana Polenova

Vanadium-dependent haloperoxidases (VHPOs) are enzymes found in seaweeds, fungi, and lichens that catalyze a two-electron oxidation of halides. Understanding the electronic structure and coordination geometry of the vanadium site remains a challenge because vanadium is in a “spectroscopically silent” diamagnetic V(V) state throughout the catalytic cycle, precluding its characterization by EPR and UV-vis. We have previously demonstrated that 51V Magic Angle Spinning (MAS) NMR spectroscopy provides a detailed glimpse of the geometry and electronic structure at the vanadium site in VHPOs.[1-2] 51V MAS NMR spectra are dominated by the quadrupolar and chemical shift anisotropy interactions, which are highly sensitive probes of the active site environment and report on protonation state and coordina-tion geometry. In this work, we present a 51V MAS NMR investigation of the resting state of vanadium chloroperoxidase (VCPO), a 67-kDa member of this family, and of its mutant (P395D/L241V/T343A) showing 100-fold increased brominating activity, as a function of pH. We demonstrate that, with fast MAS (40 kHz), the required sam-ple amounts and the experiment time are drastically reduced, permitting acquisition of high-quality spectra. This work is supported by the US-Israel Binational Science Foundation (Grant 2011077).


[1] N. Pooransingh-Margolis, R. Renirie, Z. Hasan, R. Wever, A. J. Vega, and T. Polenova, J. Am. Chem. Soc., 2006, 128, 5190-5208.

[2] T. Polenova, N. Pooransingh-Margolis, D. Rehder, R. Renirie, and R. Wever, In “Vanadium, The Versatile Metal”, Eds. Ken Kustin, Debbie Crans, and Jose Costa-Pessoa, Oxford University Press, ACS Publications, 2007, pp. 178-203.

316 13C-Detect NMR and Mass Spectrometry Applied to Study the Effects of Post-Translational Modifications on Disordered ProteinsEric B. Gibbs, The Pennsylvania State University, 104 Chemistry Building, University Park, PA 16802, Debashish Sahu, Scott A. Showalter

Recent advances in spectrometer and cryogenic probe technology have resulted in the re-emergence of 13C-direct detection spectroscopy as a tool for the study of pro-teins in solution. For intrinsically disordered proteins (IDPs), a highly dynamic class of biomolecules that lack temporally stable secondary/tertiary structure, 13C-direct detect NMR has become indispensable for the characterization of structural and dynamic properties at atomic resolution. Our biomolecular “tool-kit” built from 13C′-15N and 13C′-13Cα heteronuclear correlation spectroscopy, has provided enhanced peak dispersion and line width narrowing. This along with the use of multi-dimen-sional spectra and non-uniform sampling has greatly improved the spectral res-olution achievable for these systems while also reducing acquisition times. With these techniques we can now address fundamental questions about IDP structure function relationships. For example, we are interested in whether post-translational modifications like serine phosphorylation, enact long range structural changes or merely influence structure locally. The carboxy-terminal domain of RNA polymerase II (CTD), an essential regulator of Eukaryotic gene expression, becomes phosphor-

ylated at multiple serine residues in-vivo. A growing body of evidence supports the hypothesis that phosphorylation modulates the binding of transcription factors that are required for mRNA biogenesis, yet little is known about how phosphorylation affects CTD structure or dynamics. Here we present the combined application of 13C-direct detect NMR and mass spectrometry to address the hypothesis that serine phosphorylation fundamentally alters the structure and dynamics of the CTD.

317 Studying Networks of Weak Protein Interactions by NMRAssen Marintchev, Boston University School of Medicine, Department of Physiology and Biophysics, Boston, MA 02118, Nabanita Nag, Jielin Yu, Devika Nadkarni, Katherine A. Edmonds, Boriana Marintcheva

Macromolecular complexes are built from networks of interactions, many of which are weak, allowing dynamic remodeling. Such weak, and often transient, interac-tions are notoriously difficult to study because they usually cannot be observed in isolation, when the respective proteins are not part of the large complex. Nucle-ar magnetic resonance (NMR) is uniquely suited to provide structural information about such weak interactions because binding is observed at equilibrium, at high protein concentrations. Furthermore, transient interactions are readily observed, including those involving intrinsically disordered peptides. The translation initiation complexes represent an example of macromolecular complexes, whose assembly, remodeling and regulation involve a number of weak interactions. Two eukaryotic translation initiation factors (eIFs), eIF1A and eIF5B are at the center of an intricate network of protein-protein and protein-RNA interactions. We used a combination of NMR, site-directed mutagenesis, and deletion analysis to identify both intra- and intermolecular interactions involving eIF1A and eIF5B and to characterize the re-spective binding interfaces. Further, we analyze these interfaces in the context of the recently determined positions of these proteins in ribosomal complexes. Some of these interactions appear to be enabled by ribosome binding, while others are hindered. For example, ribosome binding is expected to disrupt an intramolecular interaction interface within eIF1A, exposing a binding site for eIF5B. Taken together, the observed overlaps in intra- and intermolecular contact surfaces offer a mech-anism for coordination of the interactions of eIF1A and eIF5B with each other, the ribosome and with other eIFs.

318 My Contributions to NIR SpectroscopyMark Westerhaus, FOSS, 2379 Corinna Ct., State College, PA 16803

Since 1978, I have worked with near-infrared (NIR) spectra developing new ways to make NIR predictions more reliable and accurate. Instrument standardization re-moved some of the differences between instruments. Repeatability files made cal-ibrations less sensitive to variation not included in the calibration dataset. Modified partial least squares (PLS) improved the calibration accuracy for some constituents. Local calibrations provided accurate predictions when the calibration set was too variable for linear models to handle. Global H and Neighborhood H values provided confidence that a new spectrum looked like the calibration spectra. Good product definition detected non-conforming spectra in a process environment. Many recom-mendations were also developed over the years. The history and current status of these developments are described.

319 Classification and Interpretation of NIR Data: Recursive Weighted Partial Least Squares Discriminant Analysis (rPLS-DA)Søren B. Engelsen, University of Copenhagen, Rolighedsvej 30, Frederiksberg, 1958 Denmark

Near-infrared (NIR) spectra contain holographic information about the samples be-ing investigated. The same information is repeated and the spectral variables are thus highly correlated (collinear data structure). For this type of data, multivariate projection methods are very suitable and indeed the combination of multivariate data analysis and NIR spectroscopy is the working principle in high throughput qual-ity control (QC) and process analytical technology (PAT). Despite the high redun-dancy in NIR spectra, the multivariate methods can often be improved by variable selection such as for example interval partial least squares (iPLS). The primary reason for the improvements is the reduced number of interferences in the reduced set of variables. Perhaps even more important than improved and parsimonious models is the improved interpretation. This paper introduces the recently developed recursive weighted PLS algorithm (rPLS) which is related to jack-knifing, but instead of iteratively eliminating variables, rPLS iteratively uses the regression coefficients to magnify important variables and thus down-weight less important variables. The rPLS model has the advantage that it will converge to a limited number of variables (good for interpretation) but it will exhibit optimal performance before normally in-cluding co-linear neighbor variables. This talk demonstrates the performance for regression (rPLS) and classification (rPLS-DA) on selected NIR data sets.

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320 Two Major Advanced Algorithms in NIRS: Cloning Instruments and Local CalibrationPierre Dardenne, Walloon Agricultural Research Centre, Chaussee de Namur, Gembloux, 5030 Belgium

This lecture presents an overview of two major algorithms implemented into the package called Winisi: clone and local. Already in the middle of the 80’s, Prof. John Shenk and Mark Westerhaus proposed in their package these methods to increase the performances of the NIR instruments networks. Cloning (matching or standardization) consists of correcting the spectra from “slave” instruments to make them as close as possible to the spectra of a “master” instrument. The method has been applied successfully for 30 years inside numerous networks. Nowadays the standardization between instruments of the same brands or series is adjusted by the manufacturers and the need of correction becomes less important. Anyway, the method remains a very practical tool to transfer data bases between instruments of different brands (i.e., dispersive to Fourier transform or reverse). The second algo-rithm concerns the concept of “local” calibration. It is well known that including large variations in a common data set improves the robustness but can affect negatively the prediction performances. To analyze one sample, the “Local” method selects N close samples from a big data set based on the highest correlations between the spectrum to be processed and the library. Trials with real large data sets show that “Local” outperforms the others methods as classical partial least squares, artificial neural network and even support vector machines. Mark Westerhaus is the author of the computer codes to run these algorithms included in the Foss WinISI package and his contributions are recognized as major impacts to the development of the NIR technology.

321 Application of Non-Linear Chemometric Models to NIR DataLars Nørgaard, FOSS, Foss Allé 1, Hillerød, DK-3400, Denmark, Morten With Pedersen, Pedro Højen-Sørensen

Linear partial least squares regression modeling is extensively used for near-in-frared (NIR) spectroscopic calibration modeling despite the fact that many NIR spectroscopic data sets often exhibit different levels of non-linearities between the spectra and the property of interest. In this paper the background for the applica-tion of artificial neural networks (ANN) from an industrial research and development perspective is provided including examples of the benefit by this approach. The talk also touches upon alternative machine learning methods like the Gaussian process regression that has recently been introduced to the chemometric society as an al-ternative to both linear and non-linear modeling methods like partial least squares and ANN.

322 Solid-State NMR of Viral Fusion ProteinsDavid Weliky, Michigan State University, Department of Chemistry, 578 S. Shaw Lane, East Lansing, MI 48824

Solid-state nuclear magnetic resonance (SSNMR) has been applied to probe the structures and membrane locations of domains of the HIV gp41 and the influenza vi-rus HA2 membrane fusion proteins. Advantages of SSNMR include: 1) use of mem-brane bilayers without detergent and with lipid and cholesterol composition similar to that of host cells; and 2) preparation of samples under conditions very similar to those used for functional vesicle fusion assays. The N-terminal ~25-residue HA2 fusion peptide (IFP) domain has helix-turn-helix structure and in detergent there are reports of: 1) an open interhelical topology with a hydrophobic interhelical pocket and inverted V membrane insertion; and 2) a closed topology with tightly-packed antiparallel helices and membrane contact with a hydrophobic protein surface. SSN-MR measurements in membranes lacking cholesterol show that the closed structure is predominant. Both the IFP and the corresponding N-terminal gp41 fusion peptide (HFP) have predominant oligomeric beta structure in membranes with 30 mole% cholesterol which is typical for viral and host cell membranes. SSNMR measure-ments for HFP show that there is antiparallel arrangement of adjacent stands with a broad distribution of antiparallel registries. The central regions of the antiparallel sheets are deeply inserted in the membrane and contact the methyl termini of the lipid acyl chains and cholesterol. This antiparallel distribution is also observed for a much larger segment of gp41 that includes the fusion peptide. A very different regis-try distribution is detected for the non-functional V2E mutant and suggests that the hydrophobicity of the antiparallel sheet is correlated to membrane fusion.

323 Structural Examination of Amyloid Proteins and Graphene-Based Nanomaterials by Solid-State NMRYoshitaka Ishii, University of Illinois-Chicago, 845 W Taylor St., Department of Chemistry, SES Rm. 4500, Chicago, IL 60607

In this study, we report two separate topics on solid-state nuclear magnetic res-onance (SSNMR) methodologies and its applications to amyloid proteins and graphene-based carbon nano-materials. In the first topic, we discuss structural fea-tures of amyloid fibrils and spherical intermediates of 42-residue Alzheimer’s amy-loid-β (Aβ(1-42)). In particular, we present the first high-resolution structure model of Aβ(1-42) amyloid fibril. The model based on SSNMR data displays structural fea-tures, which are critically different from reported structures of less pathogenic Aβ(1-

40) fibrils. Remarkably, Aβ(1-40) is not compatible with the structure of Aβ(1-42) fibril as seeding the Aβ(1-42) fibrils did not promote conversion of monomeric Aβ(1-40) into fibrils via cross-replication. The results provide crucial structural insight into how Aβ(1-42)-selective amyloid propagation is introduced in early-stage Alzhei-mer’s without involving Aβ(1-40). In the second topic, we present characterization of graphene-based materials derived from graphene/graphite oxide by various SSN-MR methods. We examine structural changes including graphene-edge modifica-tions through reduction of graphite/graphene oxide by hydrazine (H2-N-N-H2).[1] We present the structural features of other reduced graphene/graphite oxide systems obtained via milder modifications by azide (N3-)[2] or time-controlled thermal reduc-tion.[3] Other topics on recent development of solid-state NMR methods are covered.


[1] Park, S. et al. Nature Communications 2012, 3, 638.

[2] Eigler, S. et al. Nanoscale 2013, 5, 12136.

[3] Wen, Y. Nature Communications 2014, 5, 5033.

324 Structural and Dynamic Studies of Protein Assemblies by Solid-State NMR SpectroscopyChristopher P. Jaroniec, Ohio State University, 100 West 18th Ave., Columbus, OH 43221

In this talk I discuss our recent structural and dynamic studies of large protein as-semblies by magic-angle spinning solid-state nuclear magnetic resonance (NMR) techniques. The main focus of the talk is on our progress toward providing an atomic level understanding of the phenomena of amyloid strains and cross-seeding barriers for Y145Stop prion protein variants. Our recent results related to the characteriza-tion of flexible histone N-terminal tail domains in the context of large nucleosome arrays under experimental conditions corresponding to extended, folded and highly condensed chromatin are also presented.

325 Solid-State NMR of Protein Assemblies: New Methods and New ResultsRobert Tycko, National Institutes of Health, Bldg. 5, Rm. 112, Bethesda, MD 20892, Jun-Xia Lu, Marvin Bayro, Alexey Potapov, Eric Moore, Dylan Murray, Kent Thurber, Wai-Ming Yau

Our lab works on solid-state nuclear magnetic resonance (NMR) methods and on the application of these methods to structural problems in biological systems. We are particularly interested in non-crystalline protein assemblies, including amyloid fibrils and viral capsids, where solid-state NMR can provide truly unique information. In the area of Alzheimer’s disease (AD), we have recently reported the first detailed structural studies of amyloid-beta fibrils that develop by spontaneous self-assembly in brain tissue of AD patients, including the first detailed molecular structural model (Lu et al., Cell 2013; PDB 2M4J). I discuss this work and describe ongoing efforts to determine whether patient-to-patient variations in fibril structures, as revealed by solid-state NMR, correlate with variations in clinical history and neuropathology. In the area of AIDS research, we are investigating the molecular structures of tubular and non-tubular assemblies formed by the HIV-1 capsid (CA) protein. I describe how solid-state NMR data can be used to identify structurally ordered, disordered, and dynamic segments of CA within CA tubes and to characterize conformational chang-es that accompany CA self-assembly. I also describe methodological developments that enable progress in these areas, including the use of ultra-low-temperature dy-namic nuclear polarization in studies of transient intermediates in the amyloid-beta self-assembly process.

326 Finding Homes for all of the Orphan Assays Brian Buckley, Environmental and Occupational Health Sciences Institute (EOHSI), Rutgers University, 170 Frelinghuysen Rd., Piscataway, NJ 08854, Andreia Valente, Ill Yang, Min Liu, Kyle Buckley, Frances Acevedo Mariani, Stephanie Marco, Elizabeth McCandlish

Just as there are orphan drugs looking for homes to be either tested or manufac-tured, there are orphan assays looking for separation techniques that have not been made available because there is not enough interest to make them commercially viable. Most of the separation applications; solid phases, clean-ups, etc., are driven by the large commercial needs of the various industries. There are a handful of solid phases for high-performance liquid chromatography and essentially two phases for ion chromatography offered commercially with the belief that all your needs can be met with these products and the right gradient. When an orphan application comes around, e.g., potentially toxic small molecule adducts to chemotherapeutic agents or pesticides separated from small quantities of brain, there is no application guide to visit and no column recommended by a manufacturer. The chromatographer is left to their own ingenuity, trying to make the column fit the application. This presen-tation focuses on the many challenges faced by methods development analysts, challenged by assays that others have not explored. This is because the application is new or there is not enough interest in supporting the columns to make the sepa-ration commercially viable. In addition to the aforementioned examples other sepa-

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ration challenges are discussed including bile acids without buffer and microbiome profiling of isoprostanes.

327 Agroecosystem Analysis Using Chromatographic MethodsJeffrey S. Buyer, United States Department of Agriculture, Bldg. 001, Rm. 124, BARC-West, Beltsville, MD 20705

The agroecosystem is a complex community of organisms in a heterogeneous en-vironment which is altered periodically by agricultural management. Soil microbial communities, which carry out biochemical functions essential to ecosystem sus-tainability, are the most diverse and complex biological assemblages known. These communities are poorly understood, but chemical, biochemical, and genomic meth-ods are revealing their structure and function. In this talk we discuss three chro-matographic methods used to analyze different components of the agroecosystem that are relevant to microbial community structure and function. First, phospholipid fatty acid analysis by gas chromatography (GC) and GC-mass spectrometry (MS) is used to measure soil microbial biomass and broad-scale microbial community structure. Second, the chemical composition of soil organic matter is analyzed by pyrolysis-GC-MS. Third, microbial metabolites extracted from agricultural samples are analyzed by derivatization and GC-MS. These analyses illustrate just a few of the many chromatographic methods useful in agricultural and ecosystem analysis.

328 Deciphering 4-Billion Year Old Meteorite Chemistry through Liquid Chromatography-Mass SpectrometryMichael P. Callahan, National Aeronautics and Space Administration Goddard Space Flight Center, 8800 Greenbelt Rd., Greenbelt, MD 20771

Laboratory analyses of meteorites can provide a window into the extraterrestrial organic chemistry that took place during the formation of the Solar System. The detection of numerous biologically-relevant organic compounds in carbonaceous chondrites, a class of chondritic meteorites, has led to the assertion that meteorites delivered important organic compounds for the origin of life on early Earth. I present recent investigations of amino acids and nucleobases in meteorites and discuss potential formation pathways for these compounds, their significance for the origin of life, the challenges associated with analyzing meteorites, and the application of emerging analytical technologies (such as liquid chromatography coupled to Or-bitrap mass spectrometry) in the field of meteoritics.

329 Utilizing a Semi-Automated Sample Preparation Workflow to Achieve Accurate, Precise, Rugged and Robust Analytical Assay Methods for Pharmaceutical Solid Oral Dosage FormsKhanh Ha, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903

Sample extraction of pharmaceutical solid oral dosage forms is a critical step of any assay method. An accurate, precise, and robust data set typically include a well prepare standard solution and a sample preparation that has a robust extraction process. The overall sample preparation process can be challenging and very time consuming. Traditionally, manual weighing, agitation, such as shaking and/or son-ication are used to deliver a known accurate amount of material, and facilitate dis-persion / dissolution of the solid dosage form over a given period of time. In addition, a well-developed sample preparation method needs to be rugged and robust with short sample preparation times to ensure accurate and precise method execution by analysts with varying analytical experiences at different facilities over the life of the pharmaceutical product. This presentation discusses the evaluation of an automated weighting system, and novel sample extraction apparatus that employs homogenizing multiple samples of solid oral dosage forms in parallel that signifi-cantly reduces the sample extraction time and increases sample throughput. This semi-automated sample workflow achieves faster sample turnaround time, resourc-es saving while ensuring extraction ruggedness and robustness for the analysis of pharmaceutical solid dosage forms.

330 Matrix-Assisted Ionization: A Simple Low Energy Ionization Approach for Volatile and Nonvolatile CompoundsCharles N. McEwen, University of the Sciences, 600 South 43rd St., Philadelphia, PA 19104, Khoa Hoang, Milan Pophristic

The initial finding that laser ablation of a small molecule matrix at atmospheric pres-sure (AP) produces ESI-like charge states of incorporated analyte[1] was later shown not to require the laser.[2] Ionization occurs simply by passing the matrix:analyte sample through a heated inlet tube linking AP and the vacuum of a mass analyzer. Trimpin, et al.[3] later demonstrated that by selecting the proper matrix, ionization occurs spontaneously when the matrix:analyte sample is simply exposed to the vacuum inherent with any mass spectrometer, even without added heat. Thus, ma-trix:analyte preparations, identical to those used in matrix-assisted laser desporp-tion-ionization, can be introduced to a mass spectrometer through any AP inlet to produce ions regardless of analyte volatility. An ion source is not necessary. In this presentation, the utility of this simple and highly sensitive matrix assisted ioniza-tion method for high throughput analysis of peptides, proteins, small molecules and

surfaces is demonstrated, and its potential use with field portable and clinical mass spectrometers is discussed along with mechanistic insights.


[1] Trimpin, S.; Inutan, E. D.; Herath, T. N.; McEwen, C. N. Mol. Cell. Proteomics 2010, 9, 362.

[2] McEwen, C. N.; Pagnotti, V. S.; Inutan, E. D.; Trimpin, S. Anal. Chem. 2010, 82, 9164.

[3] Trimpin, S.; Inutan, E. D. J. Am. Soc. Mass Spectrom. 2013, 24, 722.

331 From Laser Ablation and Electrospray Fundamentals to Ambient Ionization by LAESIAkos Vertes, George Washington University, Department of Chemistry, Washington DC 20052, Tarek R. Mansour, Sylwia A. Stopka, Hang Li, Bindesh Shrestha

Mass spectrometry inherently relies on converting neutral molecules in the sample into ions. In recent years, there has been a proliferation of ionization techniques to expand the variety of sample types and sampling conditions to include, for example: single cells and ambient ionization, respectively. Many of the new ionization tech-niques rely on laser-matter interactions and electrosprays produced from liquids. Both of these phenomena have been extensively studied from the perspective of fundamental processes under idealized conditions. This presentation describes the path from the fundamental understanding of mid-infrared laser ablation of samples with high water content to the microsampling of biological cells and tissues, from de-scribing the nanophotonic interactions between a laser pulse and silicon nanopost arrays (NAPA) to ultratrace analysis of adsorbates, and from the disintegration of an electrified liquid meniscus at the end of an electrified capillary to the electrospray ionization of a laser ablation plume. Recent applications of the high-throughput am-bient ionization method, laser ablation electrospray ionization (LAESI) are demon-strated to biological tissue imaging and to metabolic profiling of lipid production in microalgae. The combination of LAESI-mass spectrometry with stable isotope la-beling pulse-chase experiments sheds a new light on the dynamics of the metabolic network in these microorganisms. The ultratrace analysis capability of NAPA opens the door for the metabolic analysis of single human cells.

332 Mass Spectrometry: A Tool for Studying MetabolomicsAnn M. O’Brien, DuPont, 200 Powder Mill Rd., Wilmington, DE 19803

Metabolomics is the systematic study of the unique chemical fingerprints that re-sult from specific cellular processes. Metabolomics studies can be targeted and non-targeted. In a targeted approach the metabolites are known and experiments are conducted to measure the flux of the metabolites as a function of changing cellu-lar processes. In an untargeted approach the metabolites are unknown and must be identified. Mass spectrometry is a valuable tool for characterizing small molecules with high sensitivity. However, because metabolites vary greatly in chemical nature a full metabolomics profile cannot be accomplished by applying only one analytical technique alone. This presentation aims to discuss development, validation and im-plementation of a suite of mass spectrometric and separation methods in order to have a comprehensive strategy to characterize the cellular metabolites. Additionally, this presentation addresses the importance of robust, validated sample preparation protocols in support of proper interpretation of data in the context of the biological experiment.

333 Biological Applications of FTICR Imaging Mass Spectrometry: Overcoming the Analytical Challenges of Direct Tissue AnalysisJeffrey Spraggins, Vanderbilt University, Rm. 9160, 465 21st Ave. South, Nashville, TN 37232, Raf Van de Plas, Richard Caprioli

While imaging mass spectrometry (MS) development has traditionally focused on spatial resolution and throughput, mass resolution and accuracy are becoming im-portant new focal points. Driving factors include the need to identify spatially re-solved ions directly from tissue and the difficulties introduced by nominally isobaric species. Low-resolution instrumentation can produce ion images that are in fact merged images of several species that fall under a given peak. Not only can this make ion identification extremely difficult, in many cases biologically relevant in-formation is lost. Matrix-assisted laser desporption/ionization Fourier transform ion cyclotron resonance (MALDI FTICR) provides the unique performance capabilities to overcome these challenges while maintaining high-throughput, sensitivity and dynamic range. In this work, a series of case studies that demonstrate the analytical power of MALDI FTICR imaging mass spectrometry (IMS) for lipid, peptide and pro-tein analysis are highlighted. All imaging experiments were performed using a 15T solariX MALDI FTICR MS (Bruker Daltonics, Billerica, MA) equipped with an Apollo II dual ion source and 355 nm solid-state laser. Images were acquired with spatial resolutions ranging from 10-150 µm and resolving powers up to 1,000,000. Initial results have demonstrated the necessity for high performance molecular imaging to overcome the challenges of biological complexity and structural identification.

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334 Quantitative Analysis of Moisture in Intact Lyophilized Protein Samples by NIRSSuzanne Schreyer, Thermo Fisher Scientific, 2 Radcliff Rd., Tewksbury, MA 01876, Michelle Pressler

Lyophilization is a common process for the storage of biopharmaceuticals yet the challenge remains on how to analyze the moisture content once the vials of protein are sealed. Near-infrared spectroscopy (NIRS) has been used effectively and reli-ably to measure moisture in a wide variety of pharmaceutical samples due to the large overtone band for water in NIR, and relatively high levels of accuracy for re-sulting predictive models. In this study NIRS, using a portable instrument, is used to generate a predictive model for the quantitative determination of moisture in protein samples. The use of a portable instrument is also evaluated and compared with a lab based NIR instrument.

335 Noninvasive In-Vivo Glucose Sensing on Human Subjects Using Mid-Infrared LightSabbir Liakat, Princeton University, F310 G4 EQUAD, Olden St., Princeton, NJ 08544, Kevin A. Bors, Callie M. Woods, Claire F. Gmachl

The realization of a clinically accurate, noninvasive, in vivo glucose sensor would improve the quality of life of the more than 343 million diagnosed diabetics in the world because it would eliminate the necessity of drawing blood to monitor blood glucose levels. We have made significant progress towards such a sensor by uti-lizing fundamental vibrational resonances of the glucose molecule between 1000-1200 cm-1, which yield stronger absorption cross sections than overtone absorption or Raman scattering. Previously, we showed promising initial results of clinically accurate glucose concentration prediction using human in-vivo mid-infrared spectra. Here we report recent improvements to our prediction results for human subjects utilizing an integrating sphere to enhance collection efficiency, as well as derivative spectroscopy for analysis. In-vivo spectra of backscattered light from human fore-arms was recorded between 1020 and 1220 cm-1 (a prominent glucose absorption feature centers around 1080 cm-1) after propagating through an integrating sphere, which improved collection efficiency by allowing the collection of all backscattered angles. This reduced noise present in consecutive human spectra by 5%. Spectral datasets were created for multiple subjects, with expected concentration quantified using a commercially available glucose monitor. A small subset of spectra was set aside for calibration, and PLSR predicted concentrations of the rest. By performing regression on first derivative spectra, clinically accurate predictions were achieved approximately 80% of the time, a net 6% improvement from our previously present-ed results. We continue to work on improving our optical setup and data processing to achieve greater accuracy.

336 Surface Enhanced Raman Scattering Characterization of one Hydroxyl-polymethoxylflavonesChangchu Ma, University of Massachusetts-Amherst, 100 Holdsworth Way, Chenoweth Lab 247, Amherst, MA 01003, Hang Xiao, Lili He

Polymethoxylflavones (PMFs), a unique class of flavonoids mainly found in citrus fruits. Our previous study using surface enhanced Raman spectroscopy (SERS) found that the presence and numbers of hydroxyl played an important role on the interaction with sliver (Ag) dendrites, the SERS substrate. The objective of this study is to further characterize PMFs, and investigate the role of hydroxyl position using SERS. Serial concentrations of 3’-demethylnobiletin (3DMN), 4’-demethylnobiletin (4DMN), and 5-demethylnobiletin (5DMN) in methanol were incubated with Ag den-drites. Below saturation, 5DMN had the highest peak intensity, while 3DMN had the lowest peak intensity. The high-pressuer liquid chromatography (HPLC) analysis revealed that 36.13% ±1.06% 5DMN, following 18.40% ± 3.31% 4DMN and 9.66% ± 0.94% 3DMN were bound to Ag. This result indicates that higher peak intensi-ty may due to higher binding affinity of the molecule onto Ag dendrites. Different binding affinity also influenced the saturated concentration. Meanwhile, the overall spatial conformation of PMFs bound on Ag surface was determined by the position of hydroxyl. 3DMN and 4DMN tended to form relative vertical conformation on Ag. The lower binding of 3DMN on Ag surface was further interfered by the nearby methyl. 5DMN tended to lay on the Ag surface and occupied larger space resulting in low saturated concentration. The carboxyl nearby could enhance binding affin-ity by chelating Ag. This study reports how the position of hydroxyl in these one hydroxyl-PMFs is crucial for the interaction with Ag dendrites, and provides value information for further application of the SERS technique for molecular characteri-zation of flavonoids.

337 Bacterial Identification Using Raman Spectroscopy: How does the Technique Perform with many Species of Bacteria?Bradford G. Clay, bioMérieux, Inc., 595 Anglum Rd., Hazelwood, MO 63042, Pierre Mahe, Maud Arsac, Brenna Moss, Olivia Jobes, Chris Huff, Virginia D. Shortridge, Ron Robinson, Sonia Chatellier, Alex van Belkum

Raman spectroscopy has been discussed by many researchers as a potential, next-generation technology for bacterial identification. To date, an in-depth study

with a large number of bacterial and yeast species (e.g., >20) has not been pub-lished. Current clinical identification systems; e.g., matrix-assisted laser desporp-tion-ionization time-of-flight (MALDI-TOF) mass spectrometry, have the capability to correctly identify over 180 species. As such it has been difficult to increase inter-est in commercialization of Raman bacterial identification systems. To this end, we have recently completed a study using multiple strains from 40 clinically relevant microorganisms to evaluate the ability of Raman to correctly identify the each of the species. Raman results are compared to correct identification of the same strains that have been evaluated using a MALDI-TOF mass spectrometer with a commer-cially available database. This talk reviews clinical microbiology work flow, sample preparation for either Raman or MALDI-TOF analysis and the comparative results of the two methods as well as the clinical significance of the findings.

338 Terahertz Spectral Profiling and Imaging of Human Skin for early Detection of CancerAisha Masud, Applied Research & Photonics, 345 East 37th St., Ste. 317, New York, NY 10016, Babar Rao, Aunik Rahman, Anis Rahman

Terahertz scanning reflectometry was used to investigate both the surface and the sub-surface of human skin biopsy. The non-ionizing nature of T-ray eliminates radi-ation damage or perturbation of sensitive tissues while able to probe disease condi-tions in the deeper layers leading to an effective early diagnostic tool. In this study, a terahertz technique has been developed that is comprised of terahertz scanning reflectometry, terahertz time-domain spectroscopy and terahertz imaging (all instru-ments from Applied Research & Photonics, Harrisburg, PA 17111) for detection of cancerous skin with basal cell carcinoma (BCC) in comparison to benign skin sam-ples. Two groups of samples were studied; the first group samples are benign skin and the second group samples are biopsy from cancerous area. Thickness profile exhibits significant differences in profiles of the respective skin samples both in their layer structure and also in their total reflected intensities; thus indicating presence and lack of cellular order for respective specimens. Similarly, terahertz spectra ac-quired in transmission exhibit quantifiable differences for both groups. More inter-estingly, terahertz image of the benign skin shows regular cell patterns while the image of a sample with BCC exhibit no clear cell pattern. The lack of clear cell order in the skin, thus, may be used as an indication of cancerous area and this finding may be used as an early diagnosis tool. It is notable that this is the first of such a concerted observation of benign versus BCC skins from three different experiments. The results are consistent from individual experiments and collectively provide a conclusive means of early diagnosis of skin cancer detection.

339 Microscopy’s Importance in Cases I Have WorkedPeter DeForest, Forensic Consultant, 16 Bryant St., Hartsdale, NY 10530

A revolution has taken place in the field of analytical chemistry over the last 70 years, made up of sweeping, profound changes. Physical methods have replaced chem-ical ones for the vast majority of chemical analyses conducted. This is especially true for analyses conducted in high-volume testing facilities. In these high-volume facilities, scientists have been replaced by technicians, as large quantities of funda-mental knowledge of chemistry and chemical reactions is set aside and forgotten. This presentation will not argue for a return to the “good old days” in such modern laboratories; automated physical methods are ideal for routine high-volume applica-tions. The difficulty arises when laboratories encounter non-routine problems. Prob-lems in the forensic science laboratory provide an example. Even forensic science laboratories deal with sets of routine problems, but non-routine problems, intellec-tually challenging and difficult to solve, frequently arise in complex cases. For this reason, microscopy and chemical microscopy remain exceedingly powerful tools in the forensic science laboratory. Illustrative case examples are discussed.

340 Microscopy of Tattoo InksMichelle Miranda, SUNY- Farmingdale, 2350 Broad Hollow Rd., Farmingdale, NY 11735

No abstract submitted by the author.

341 Microscopy of Shotgun Buffers (Not your Common pH)Peter Diaczuk, John Jay College, Department of Sciences, 524 W 59th St., New York, NY 10019, Jack Hietpas

A multi-projectile shotgun shell is more complex and consists of more components compared to a standard piece of handgun or rifle ammunition. Besides the shell, primer, and propellant, an unfired shotgun shell is available in a multitude of payload options, consisting of a single projectile or a variety of pellet or “shot” sizes. Some shotgun ammunition manufacturers use an additive, called “buffer,” in their prod-ucts, which are small polymer granules added to shotgun shells to cushion larger sized pellets, named buckshot, and some smaller pellets, such as turkey shot. The comingling of softer polymer buffer with harder metal pellets provides a cushioning effect that reduces deformation of the pellet’s spherical shape. When a buffered shotgun shell is fired, numerous polymer granules are discharged with the pellets and wad, which imbeds in close range wounds or clothing and becomes scattered

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about downrange of the muzzle for several yards. Being able to associate a sample of buffer to a specific ammunition manufacturer offers useful information to shooting scene reconstructions when spent shells are not present. Several brands of ammu-nition were obtained, representative shotgun shells were dissected and buffers were examined microscopically in pre- and post-discharged instances. Comparison of post-discharged to pre-discharged buffer samples revealed no significant changes in buffer size or shape. However, stereomicroscopy showed morphological differ-ences in unfired buffer used by different manufacturers. Using particle micrometry and infrared spectroscopy, it was possible to determine and differentiate among different buffers used in different brands of shotgun shells.

342 Microscopy of Dust SamplesSkip Palenik, Micro Trace, 790 Fletcher Dr., Ste. 106, Elgin, IL 60123

No abstract submitted by the author.

343 Enhancement of Curcumin Fluorescence by Ascorbic Acid in Bicontinuous MicroemulsionMaurice O. Iwunze, Morgan State University, 1700 E. Coldspring Lane, Baltimore, MD 21251

Bicontinuous microemulsion is a thermodynamically stable and isotropic solvent system that has been found to solubilize both hydrophobic and hydrophilic solutes in a given reaction. In this work bicontinuous microemulsion made up of a four com-ponent system (water:oil:surfactant and co-surfactant (amyl pentane)) in the ratio of 0.35:0.12:0.18:0.35 w/w is used to solubilize curcumin that is known to be of very low solubility in water. However, it was found to be very soluble in the prepared bicontinuous microemulsion. The oil used in the preparation is tetradecane and the surfactant is cetyltrimethylammonium bromide (CTAB). The observed fluorescence of this compound was enhanced by introduction of ascorbic acid to the solution. The association constant, Ka, that resulted between curcumin and the ascorbic acid was determined using the Benesi-Hildebrand type of equation. The value obtained is 6.19 x 106 with the free energy of association, ∆Ga, of -23.92 kJ/mole. The impli-cation of this observation is discussed with regards the synergism of the two com-pounds for combating the reactive oxygen species, ROS that is very prevalent in any given metabolic system.

344 An Investigation of Solution Phase Copper ESR Spectra as a Means to Study Speciation by Ligands in Aqueous Solution, Including those of Geochemical SignificanceChristopher J. Bender, Fordham University, 441 E Fordham Rd., Bronx, NY 10458

The electron spin resonance (ESR) spectrum of the copper (II) ion in aqueous solu-tion appears as one of three forms that are indicative of the degree of sequestration by anionic ligands. A survey of Cu(II) ESR spectra in aqueous solutions containing inorganic acids, mono- and di-basic organic acids, amines, amino acids and am-ides was conducted on samples prepared according to complexometric titrimetric procedures described by Schwarzenbach. A consistency of behavior is observed regarding the transition of ESR lineshape among three forms that differ with re-spect to effective g-value and resolution of the copper hyperfine lines. The degree of copper sequestration is accompanied by an improved resolution of the copper hyperfine structure and a shift of the effective g-value towards the pure spin g-val-ue. Thus one may investigate copper speciation in mixed ligand solutions by using ESR spectroscopy to identify and make quantitative determinations of magnetically distinct species. In cases of two different ionizable functional groups on the ligand, one may detect and resolve the spectra of complexes formed by these independent ionic species. For example, among the aliphatic alpha-amino acids, first indication of copper coordination appears at approximately pH 2.5; a second complex makes its appearance in the form of a second ESR line with g-value closer to 2 as the pH is raised past 5. Subtractive methods of data analysis permit one to recover the spectrum of each copper species along the ligand concentration or pH coordinate.

345 Raman, Infrared and Microwave Spectra, r0 Structural Parameters, and Conformational Stability of IsopropylisocyanateBhushan S. Deodhar, University of Missouri-Kansas City, 5100 Rockhill Rd., Kansas City, MO 64110, James R. Durig

The microwave spectrum of isopropylisocyanate, (CH3)2CHNCO, has been inves-tigated from 11,000 to 21,000 MHz and 18 transitions for the more stable trans conformer were assigned and A=6693.23(15), B=2263.10(3), C=1960.05(2) MHz were obtained. By utilizing these rotational constants along with ab initio MP2(-full)/6-311+G(d,p) predicted structural values, adjusted r0 parameters have been ob-tained for the trans conformer along with estimated values for the gauche conform-er. Variable temperature Raman and infrared spectra of xenon solutions have been recorded and a ΔH value of 115±11 cm-1 (1.38±0.13 kJ/mol) has been determined. The conformational stabilities have been predicted from ab initio calculations utiliz-ing several different basis sets up to cc-PVQZ for MP2(full) and 6-311+G(3df,3pd) for density functional theory calculations by the B3LYP method. From the MP2(full)/cc-PVQZ calculations an energy difference of 87 cm-1 (1.04 kJ/mol) is predicted

between the trans and the gauche forms and 168 cm-1 (2.01 kJ/mol) for the cis form which is a transition state.

346 Comparison of Target Detection and Maximum Signal FactorsNeal B. Gallagher, Eigenvector Research, 160 Gobblers Knob Ln., Manson, WA 98801

The mathematics behind target detection and maximum signal factors (MSF) can be shown to be identical. The difference in the approaches is that target detection is a supervised pattern recognition technique while MSF is an exploratory analysis method. Target detection typically employs known, well-characterized, library spec-tra for detection and classification. Unfortunately, due to measurement related dif-ferences between the library and the measured data there may be some mismatch between the two resulting in a compromise in detection performance. Additionally, only the selected target is considered in the detection task. On the other hand, MSF can be used with current data to explore anomalies where the observed anomaly is used as the measurement relevant target. Recent advances in MSF algorithms have improved detection performance, reduced memory and time requirements, and reduced the number of scores images that to be inspected during exploratory analysis. However, the detected anomaly must be compared to library spectra for classification. As a result, it should be seen that target detection and MSF form a complimentary approach to detection and classification. Examples of the two ap-proaches are shown with hyperspectral images.

347 Polymer Gel Permeation Chromatography with Luminescence DetectionMartin Nosowitz, Arkema, 900 First Ave., King of Prussia, PA 19406

Gel permeation chromatography (GPC) is a workhorse tool in the polymer analysis laboratory. Determinations of polymer molar mass distributions are commonly made using GPC with differential RI or evaporative light scattering detectors. Various con-figurations of angle-dependent light scattering detectors (often in conjunction with a viscometer) are used to obtain additional qualitative information such as polymer branching. By exploiting the ability of GPC to separate molecules by molar mass and then applying more specific detection such as UV-Vis absorbance and lumines-cence, compositional information may be extracted from samples. Some polymers exhibit characteristic luminescence derived from their backbone or pendant groups. The presence (and sometimes identity) of additives such as pigments and anti-oxi-dants can be confirmed. It is sometimes possible to elucidate end groups or defects within the polymer structure, given the high sensitivity of luminescence detection. Examples are presented.

348 Fast GC-TOF-MS for High-Throughput Screening of Environmental ContaminantsPete Grosshans, Markes International, 11126-D Kenwood Rd., Cincinnati, OH 45242, Nicola Watson, Charles Haws, Laura McGregor, Nick Bukowski

The introduction of recent legislation, such as the EU Environmental Liabilities Direc-tive 2004/35/EC, has encouraged the development of precise and robust analytical systems for identifying pollutants. However, the sheer number of toxic compounds which may require monitoring at any given time makes this a challenging prospect. Conventional quadrupole gas chromatography mass spectroscopy (GC-MS) meth-ods often employ selective ion monitoring (SIM) for trace-level detection of target compounds. However, this protocol prevents retrospective searching of data for the latest contaminants of emerging concern. The use of time-of-flight mass spectrome-try (TOF MS) can overcome this issue by providing highly sensitive detection whilst acquiring full range mass spectra, to allow both target and unknown identification in a single, rapid analysis. Nevertheless, trace-level identification may become compli-cated for compounds which exhibit weak molecular ions and extreme fragmentation, e.g., the ‘drin’-type pesticides, such as dieldrin and endrin. Select-eV ion source technology aims to combat this problem by allowing both hard and soft electron ion-isation with no inherent loss in sensitivity. Select-eV provides enhanced molecular ions whilst retaining structurally-significant fragment ions, delivering both confident compound identification and lower limits of detection. This work demonstrates the use of fast GC-TOFMS with Select-eV ionisation for accurate identification of a suite of ultra-trace level environmental contaminants.

349 Using Solid-Phase Extraction to Concentrate Human Hormones in Drinking Water and High-Performance Liquid Chromatography to Analyze the RecoveryCarl A. Fisher, Thermo Fisher Scientific, 1214 Oakmead Pkwy., Sunnyvale, CA 94085, Hua Yang, Pranathi Perati

The presence of hormones in drinking water is a human health concern with sev-eral being routinely monitored as part of the United States Environmental Protec-tion Agency (EPA) Unregulated Contaminant Monitoring Rule 3 (UCMR3). Various forms of estrogen are prescribed as a hormonal contraceptive device, for estrogen deficiency syndromes, and to counter the negative effects associated with the nat-ural decline in estrogen levels, such as accelerated bone loss, in postmenopausal

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women. Due to the widespread use of hormone pharmaceuticals, these often end up in the sewage system as a result of excretion and disposal of unwanted quanti-ties. Additionally, hormones from livestock waste can find its way into drinking water sources. There is evidence that hormones may not be effectively removed during wastewater treatment, and as a result, significant amounts of these hormones may be present in drinking water sources. To monitor the levels of the most common hor-mones, EPA method 539 was developed, which describes a procedure for quantify-ing hormones in drinking water using solid phase extraction (SPE) followed by liquid chromatography electrospray ionization tandem mass spectrometry. The present work describes the use of high surface area solid phase extraction cartridges to extract seven sex hormones from drinking water using a SPE instrument. Extracts were subsequently quantified by high performance liquid chromatography to deter-mine the recovery.

350 Low Level Chrysotile in Soil: A Method AssessmentFrank Ehrenfeld, International Asbestos Testing Laboratories, 9000 Commerce Pkwy., Mount Laurel, NJ 08054, Raymond Kennedy, Kristen Goedde, Thomas Barkley

The detection of soil surface asbestos contamination presents particular analytical challenges. This is especially true when the analyte is present at extremely low lev-els. Recently, a new test method for determination of asbestos in soil was approved by the American Society for Testing and Materials (ASTM) Subcommittee D22.07 on sampling and analysis of Asbestos (ASTM D7521-13 Standard Test Method for the Determination of Asbestos in Soil). In this study, commercially available topsoil was used as a blank matrix material. A chrysotile standard reference material was then added to produce known percent laboratory spikes. Multiple spikes were formulat-ed to contain both 0.1% and 0.01% chrysotile by weight. These spikes were then subjected to the test method. The findings are very encouraging for the applicability of this test method.

351 Direct Mercury Analysis of Gaseous Samples Using Sorbent TubesSumedh P. Phatak, Milestone Inc., 25 Controls Dr., Shelton, CT 06484

Direct mercury analysis (DMA) is a technique that is ubiquitous in analytical labs within the environmental industry for analyzing mercury in solid and liquid sam-ples. However, environmental labs are also required to test for mercury in gaseous samples quite frequently. In addition to continuous emission testing and long term testing for relative accuracy test audits (RATA), regulations in the environmental industry have also driven the need for mercury gas testing and analysis. Most con-ventional direct mercury analysis systems do not offer the flexibility of analyzing mercury in gases along with solid and liquid samples, which affects throughput, productivity and costs. To address the need for a highly flexible system, we have studied a configuration with sorbent tubes connected to a mass flow controller for trapping mercury gases and subsequent release and analysis on a DMA-80 Dual Cell (DL – 0.003 ng) or Tri Cell (DL – 0.001 ng) Mercury Analysis System, offering the capability to directly analyze gases along with solid and liquid matrices without sample preparation. The sorbent tubes contain a special mercury adsorbing mate-rial, which traps the mercury from the gases as they flow through the tubes. These mercury containing sorbent tubes can be directly placed on the auto-sampler tray and be directly analyzed along with the regular solid or liquid samples. We present data on the flexibility of analyzing gas samples along with routine operation of mixed samples.

352 Novel QCell Technology for Inference Removal in ICP-MS - Combining Low Mass Filtration with Kinetic Energy DiscriminationFergus Keenan, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA 94085, Craig Seeley

Polyatomic interferences are one of the fundamental challenges in inductively coupled plasma mass spectrometry (ICP-MS). They can be suppressed using a collision/reaction cell with a neutral gas like Helium combined with kinetic energy discrimination (KED) or with a reactive gas like Hydrogen or Ammonia. The iCAP Q ICP-MS features the new QCell which combines KED with mass filtration of interfer-ence precursors. The QCell uses an innovative flatopole design that not only utilizes KED with low mass cutoff but also achieves best in class sensitivity and lowest background in a simple, automated process. The QCell is described along with data from environmental, industrial and food safety applications.

353 Single Particle ICP-MS (SP-ICP-MS) a New Analytical Technique for Counting and Sizing Metal Based Nanomaterials: Theory and ApplicationChady Stephan, PerkinElmer, 25 Nuttall, Brampton, ON, L6S4V7 Canada

Engineered nanomaterials (ENMs) are synthesized by a manufacturing process that produces and controls ENMs to have at least one dimension in the range of 1 to 100nm in size. ENMs often possess different properties than their bulk counter-parts of the same composition, making them of great interest to a broad spectrum of industrial and commercial applications. The widespread use and application of

ENMs will inevitably lead to their release into the environment, which raises concern about their potential adverse effects on the ecosystems and, subsequently, human health. This talk goes over the theory of single particle inductively coupled plasma mass spectrometry (SP-ICP-MS), the importance of data acquisition speed, and describes its use in analyzing metal-based Nanoparticles. The single particle anal-ysis technique allows for the differentiation between ionic and particulate signals, quantitation of each fraction, the measurement of particle concentration (particles / mL), determination of particle size and size distribution. In addition, it allows the user to explore particle agglomeration. SP-ICP-MS is a key analytical instrument in assessing the fate, behavior and distribution of (ENMs) in several types of matrices (environment, food, etc.). It is an essential technique used in evaluating ENMs bio-availability and bioaccumulation in the biota, and improving bio-labeling capabilities as well as furthering advancements in the field of nanomedicine.

354 Quad vs. Magnet, Battle of the ICPMS Instruments for Superiority of Cr Speciation in NJ Drinking Water SamplesElizabeth McCandlish, Rutgers University, 170 Frelinghuysen Rd., Piscataway, NJ 08854, Sanjeev Rai, Brian Buckley, Lee Lippincott

Disinfection of raw water to be used for drinking, generally employs chemical oxi-dation. An Ion chromatography- inductively coupled plasma mass spectrometry (IC-ICPMS) was used to quantify Cr3+ and Cr6+, to measure Cr3+ oxidation to Cr6+ during disinfection. Measurements of NJ drinking water both before and after the disinfection process demonstrated the vast of total chromium found in both raw and finished water were far below (0.02-0.1 ppb) the federal drinking water standard. To measure Cr concentrations at these low levels, significant signal processing of raw data was required. Isotopic ratios of signals of 8.9 to 1 of 52Cr to 53Cr isotopes were used to validate Cr signal vs. isotopic interferences and background signal. Until now this very labor intensive method of post-data processing was the only means for achieving the required sensitivity, even with a great deal of uncertainty. To facilitate Cr measurement for future studies a magnetic sector ICPMS was em-ployed to measure concentrations of both species at much lower levels removing the isobaric interferences of a quadrupole instrument. This presentation focuses on the study results from the drinking water study that demonstrated the need for great-er resolution and sensitivity. In comparison, results using the Nu instrument’s HR magnetic sector ICPMS for quantitation of Cr are presented. The greater sensitivity and resolution were able to overcome many of the difficulties encountered in the original study, opening up the possibility for future studies where these limitations currently restrict progress.

355 Polyol Induced Extraction (PIE) of Water from Organic SolventsMithilesh Deshpande, Seton Hall University, Department of Chemistry and Biochemistry, 400 South Orange Ave., South Orange, NJ 07079

Acetonitrile (ACN) is used in huge quantities for reverse-phase high-performance liquid chromatography (RP-HPLC) experiments. For most part, this results in waste that is only contaminated with large amounts of water (H2O), buffer and trace organ-ic impurities. It would seem worthwhile to purify the ACN from H2O using a mass separating agent (MSA). Our investigation revealed polyols as a new class of MSAs. It was observed that when a polyol such as glycerol or sorbitol or erythritol or xylitol or Isomalt or maltitol was added to a mixture of ACN-H2O, the ACN separates out of mixture to form a new immiscible upper phase. We term the process: Polyol-induced extraction (PIE). The study revealed that glycerol could initiate phase separation of 1:1 (v/v) ratio of ACN-H2O mixture at 20 ºC with a glycerol concentration of 17.5 wt/wt %. The results demonstrate that glycerol can improve purity of ACN up to 96% at lower temperatures. We show that polyols as separating agents are equivalent or better than MSAs such as sugars and salts. Our thermodynamic investigation based on determination of change in free energy (∆ G) and change in enthalpy (∆ H) revealed that glycerol and sorbitol induced phase separation is driven by decrease in free energy of process and is primarily an exothermic process. Our study also demonstrated that solvents in all of the major categories, non-polar, polar aprotic and polar protic can be purified by the PIE process.

356 Biphasic Dissolution Studies of Felodopine: Understanding the Effect of Amorphous Solubility on the Rate of PartitionJon Mole, Sirius Analytical, 100 Cummings Ctr., Beverly, MA 01915, Karl Box, John Comer, Rebeca Ruiz, Robert Taylor, Lynne S. Taylor

A biphasic dissolution assay has been developed to measure the partition rate of active pharmaceutical ingredients (APIs) from an aqueous compartment into an immiscible organic phase to mimic absorption through biological membranes. A stock solution of felodopine, was injected into a chamber containing 45mL of phos-phate buffer adjusted to pH 6.8 and 20 mL of nonanol which formed an immiscible layer above the aqueous compartment to represent a biological sink. A fibre-optic probe was immersed in both the aqueous compartment and organic layer, in order to collect UV spectra in both phases. The partition rate increased proportionally with concentration up to 10 ug/mL. Above this concentration, the rate continued to increase, but the trend was no longer proportional to concentration. The solution in the aqueous compartment was found to be clear at concentrations of 6ug/mL and

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below, but turbid at 10 ug/mL and above. Trend lines were drawn through the data with R2 values >0.98. The concentration at which the trend lines intercepted was close to the reported amorphous solubility. At concentrations below the amorphous solubility of felodopine, the partition rate was directly proportional to concentration. However, above the amorphous solubility, the concentration in solution was expect-ed to remain constant as the precipitation/dissolution kinetics of the amorphous form, were expected to be faster than the partition rate. The observed increase in partition rate above the amorphous solubility was thought to be due to a dissolution event at the interface.

357 Dissolution Performance of Inhaled Product APIs in Simulated Lung Fluid Using Novel Low Volume AssayJon Mole, Sirius Analytical,100 Cummings Ctr., Beverly, MA 01915, Rebeca Ruiz, Karl Box, John Comer, Robert Taylor, Antonio Llinàs, Johan Solandt, Ulrika Tehler

The aim of this study was to measure the dissolution characteristics of active phar-maceutical ingredients (APIs) contained in inhalation products, using novel low vol-ume assays. Samples were compared in a standard phosphate buffer and in a sim-ulated lung fluid (SLF) media. The dissolution performance of six APIs (budesonide, indacaterol, pranlukast, rofleponide, salmeterol and zafirlukast) was measured at 37 °C using two novel technologies. In the first technique, the SiriusT3 was used to run powder dissolution assays in 2mL volumes with quantitation using in-situ UV probes. The second technique used a surface dissolution imaging system (Sir-ius SDI) allowing direct measurement and visualization of dissolution events at the surface of the API in real-time. Dissolution measurements were compared using standard phosphate buffer and a simulated lung fluid media containing 1% sodium dodecyl sulphate (SDS). Powder dissolution results in phosphate buffer ranged be-tween 1% released for pranlukast up to 10% for zafirlukast. There was a marked in-crease in dissolution performance in SLF medium for zafirlukast (up to 20% release) and for salmeterol (up from 12% release in phosphate buffer to 18% in SLF). How-ever, for some APIs there was no little or no increase in dissolution performance. The increase in dissolution in SLF could be attributed to increased wettability in the presence of a surfactant in combination with surface active properties of the API itself. Direct UV imaging of the surface confirmed the increased accessibility of the media at the interface when surfactant was present.

358 Characteristics of Felodipine: HPMCAS Solid Dispersions: Dissolution Performance and Particle Size EffectsJon Mole, Sirius Analytical, 100 Cummings Ctr., Beverly, MA 01915, Karl Box, John Comer, Robert Taylor, Rebeca Ruiz, Brian Stockton, George Butcher, Darren Matthews

To assess the dissolution performance of felodipine: hypromellose acetate succi-nate (HPMCAS) solid dispersions prepared using different spray drying conditions and to characterize the particle properties using laser obscuration time (LOT) and digital image analysis. Nine solid dispersions of felodipine in HPMCAS were pre-pared using different spray drying conditions. Outlet temperature, nozzle pressure and feed concentration were varied. The dissolution profiles of were determined in 20mL volumes of phosphate buffer at pH 7.0, using a SiriusT3 autotitrator with in-si-tu UV-spectroscopy. The particle size distribution of each dispersion was measured using the Sirius Insight using combined techniques of LOT and image analysis. The dispersing medium was isooctane. The dissolution rate from all nine solid disper-sions was greater than that of pure felodopine. The total dissolved mass from the dispersions ranged between 7 and 15 µg/mL in a 60 minute time frame, whereas pure felodopine achieved less than 1 µg/mL in the same period. The LOT and image analysis showed that the batches ranged in particle size distribution from a lowest d50 value of 17-µm up to the largest d50 value of 76-µm. Dissolution performance was found to correlate directly with particle size, where the greatest dissolution en-hancement occurred for the two dispersions with the smallest d50 values. Dissolu-tion performance could be measured in situ from low volume assays using sparing amounts of material and was found to correlate directly with dispersion particle size. LOT and digital image capture enabled fast characterization of the particle size dis-tribution and analysis of particle shape, with minimal application.

359 A PCA Model of Atypical Andersen Cascade DataLauren Seabrooks, Merck, 181 Passaic Ave., Summit, NJ 07901, Jennifer Wylie, Justin Pennington

This research details a novel application of principal component analysis (PCA) to aid in the root-cause determination of atypical Andersen cascade impaction (ACI) data. Andersen cascade impaction is a technique used to measure the aerodynamic particle size distribution of medical inhalers an important indication of drug deposi-tion in the lung. It is one of several methods which provide fractionation of particles based on the differences in inertia. A particle passes through numerous stages each with decreasing nozzle apertures, increasing linear velocity, and either remains entrained in the laminar flow paths or breaks through the stream and impacts on a collection surface located beneath each stage. [1] Although information rich this technique is labor intensive, involving many steps, and ultimately susceptible to

errors. Therefore a prominent goal in the scientific community is to identify whether differences in data are genuine or the result of non-product quality factors causing variability in ACI measurements.[2] For this study, several factors related to analyst error and test design were identified. Next, a design of experiments approach was used to randomize experiments and determine which effects caused statistically significant (p<0.05) changes to the data. Significant effects were then modeled us-ing PCA where each error defined a discrete class and each size fractionated group represented a distinct variable. Two components were sufficient to describe 80% the total variance and separate each class. The model was further successfully validated with three data sets. This research therefore establishes that PCA may aid in the investigation of atypical ACI data.


[1] Mitchell, J. and Nagel, M (2004). Particle size analysis of aerosols from medical inhalers. KONA. 22: 32-65.

[2] Glaab, V., Goodey, A., Lyapustina, S., and Mitchell, J. (2011). Efficient data anal-ysis for MDIs and DPIs: failure mode effect analysis. Respiratory Drug Delivery Europe. 225-236.

360 New Dispersive Technology (Grinder) Sample Preparation Improvements for Disintegration and Dissolution of a Challenging Extended Release Solid Oral Dosage FormPeter I. Tattersal, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903, Nalini Anand, Marlon Rutherford, Shan Xiao, Khanh Ha, John Castoro

A risk assessment was performed on an extended release high-performance liquid chromatography (HPLC) assay method following data trending concerns with impre-cision and accuracy (~3% relative standard deviation with low bias (97%) along with a number of object-oriented analysis investigations) in support of manufacturing. This presented an opportunity to evaluate a new dispersive technology that uses a grinding / blending action for the sample extraction process to efficiently disintegrate tablets in a fraction of the current method time. The approach was optimized through extensive studies on three different formulations / packages (hundreds of assay values) to statistically compare and ensure more consistent / accurate / precise data could be routinely obtained. A robustness study on key sample preparation variables (n=4) was performed as a design of experiment (multi-factorial, 20 ex-periments) based on the optimized conditions. The development studies performed clearly demonstrated that the new sample preparation conditions are effective and robust.

361 NMR Spectroscopy as a Tool in PAT: An Essential Tool in Chemical Process UnderstandingBrian L. Marquez, Bruker Corporation, 15 Fortune Dr., Billerica, MA 01821, Kim Colson, Anna Codina

Process analytical technology (PAT) has become an increasingly more powerful as a tool for the understanding and monitoring of chemical processes. Traditionally these tools vary from pH meters to infrared spectroscopy. However, in recent years, nuclear magnetic resonance (NMR) spectroscopy has become a very powerful tool in the area of process understanding, owing to its capabilities to discern different chemical moieties via unique chemical shift properties (multinuclear) and inherent quantitative capabilities. In this presentation, we will demonstrate the use of a novel flow cell, powerful acquisition and processing software that allows one to monitor chemical reaction in real time providing a tremendous amount of kinetic, quantitative mass balance and mechanistic understanding of example synthetic steps.

362 Optimized Karl Fischer Titration of Lyophilized SubstancesMatt Eby, Mettler Toledo, 1900 Polaris Pkwy, Columbus, OH 43240, David Zavich

The new LabX® Balance Software method optimizes external extraction of lyo-philized, especially when the sample weight is unknown, with a completely guid-ed workflow utilizing an Excellence Analytical balance and Compact Karl Fischer Titrator.

363 Continuous Improvement in Productivity of Pharmaceutical Development through Enhanced Usage of UV Fiber Optic DissolutionLili Lo, Bristol Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903, Xujin Lu

Ultraviolet fiber optic dissolution has been accepted in the pharmaceutical industry since it was endorsed by United States Pharmacopeia in monograph <1092> in 2006. Unlike conventional dissolution testing performed manually or automatically, which requires sample withdrawal during dissolution and HPLC analysis and data processing after the run, the UV fiber optic dissolution system performs in-situ de-tection in the vessel without sample removal and collection, nor post dissolution detection. It saves the costs of sampling, filtration and collection of consumables. It also saves time and labor in set up and data processing of the off-line detection sys-

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tem. Although the benefits of using UV fiber optic dissolution are obvious, the rate of adoption has been modest. HPLC is currently still the primary dissolution finish in some working environments. The resistance to change is in large part because analysts are not comfortable to change working habits and adapt to a new applica-tion. In an environment of continuous improvement within the industry, a proposal to enhance the use of UV fiber optic dissolution has been executed in our depart-ment. In this presentation, we demonstrate the significant savings and productivity improvement achieved by expanded use of UV fiber optic dissolution, with a case study of how the cost and savings were generated and evaluated.

364 Turbidity Measurement of Infusion SolutionsAlex White, Anton Paar USA, 10215 Timber Ridge Dr., Ashland, VA 23005

Turbidity is a parameter that cannot be overlooked when performing quality control checks on infusion solutions for clinical use. Haze or particles in these solutions may result in life-threatening complications. Undesirable reactions may occur when preparing intravenous drugs and solutions. Incompatibility reactions are those which occur inside a fluid container or infusion line and are often visible. These reactions can be either physical or chemical. Physical reactions are those when drugs react and either phase separate or precipitate. Such reactions can increase or decrease drug effectiveness, but can also produce completely different interactions in the body that can often be toxic. Precipitations based on drug incompatibilities are re-sponsible for the most common particle formation seen in infusions. Such reactions can be detected and monitored through turbidity measurement. Chemical reactions that may occur, such as oxidation, reduction, hydrolysis, or decomposition, often re-sult in changes in turbidity. Chemical degradation decreases the amount and/or ef-fectiveness of the active agent and can also form toxic by-products. Turbidity should be measured during production as well as for final quality control checks. Turbidity measurement can also provide critical stability and shelf-life determinations.

365 Material Identification with Advanced Handheld 1064 nm Raman SpectrometersClaire Dentinger, Rigaku Raman Technologies, 14 New England Executive Park, Ste. 102, Burlington, MA 01803, Mark Mabry, Jen Lynch

Portable and handheld Raman spectrometers, especially those with long wave-length excitation lasers, are becoming smaller, lighter, more rugged and easier to use without compromising quality. Long wavelength excitation Raman spectrom-eters expand the range of different materials that can be identified compared to Raman spectrometers using shorter excitation wavelengths. This combined with their small size, ease of use and high spectral quality makes them ideal for mate-rial identification outside the laboratory. Advancements in optical components and electronics have enabled this improvement in long wavelength handheld Raman spectrometers. Applications which benefit from high quality 1064 nm handheld Ra-man spectrometers are presented in the areas of pharmaceutical raw material iden-tification (RMID), finial product verification, biopharmaceuticals, cosmetics, dietary supplements and anti-counterfeiting.

366 In-Situ Monitoring of Form Change as a Function of Relative Humidity in the Solid-State by VTI-RamanCandice Choi, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08904, Duohai Pan, Denette Murphy, Anisha Patel, Roxana Schlam, Shawn Yin, Sruthi Janakiraman

Understanding changes that occur as a function of water activity has become an important aspect of physical characterization of the active pharmaceutical ingredient (API) in all stages of pharmaceutical product development due to the recent in-crease in number of compounds forming both stoichiometric and nonstoichiometric solvates and/or hydrates. Currently, moisture sorption isotherms are acquired to better understand the relationship between water activity and moisture content at a given temperature for an API form/material in the solid state. However, the addition-al complexity of API form/phase change that can occur during a sorption analysis can make the interpretation of the moisture sorption isotherm difficult. Integration of moisture sorption isotherm and Raman spectroscopy provides better understanding of these changes that may be occurring to the API as a function of equilibrated water content and/or %RH in the solid-state. This poster illustrates, via three examples, applications of VTI/Raman in the pharmaceutical field.

367 An Attempt to Increase Hot Melt Extrusion Drug Loading without Compromising DissolutionJulie M. Novak, Merck, 770 Sumneytown Pike, West Point, PA 19486, Karen Pica, Melanie J. Marota, Pavithra Sundararajan, Mary Ann Johnson, Wei Xu, Paul A. Harmon

High drug loaded amorphous solid dispersions (>40%) are desirable in formulation of poorly potent active pharmaceutical ingredients (APIs) requiring higher doses and in formulation of combination products in order to minimize image size. High drug loaded dispersions have been found to have significantly reduced dissolution rate compared to lower drug loads due to the presence of more hydrophobic drug and

less hydrophilic polymer. It was hypothesized that the addition of certain classes of excipients into the dispersion would help to improve hydrophilicity and/or promote disintegration thus maintaining faster dissolution rate at higher drug loads. The fol-lowing classes of additives were evaluated: highly soluble excipients, effervescent salts, superdisintegrants, surfactants, and milling aids. Dispersions were manufac-tured using a small scale hot melt extruder. Dissolution was performed to evaluate the effect on API dissolution rate and nanoparticle formation. The majority of the excipients evaluated did not increase the dissolution rate of the API in high drug loaded extrudates and in some cases even impeded the dissolution. The additives also did not increase nanoparticle formation during dissolution. Only extrudates con-taining high levels of surfactant (10%) provided similar dissolution performance to the lower drug loaded extrudate. However, this level of surfactant is not a viable commercial formulation due to compromised physical stability. Therefore it was con-cluded that the addition of excipients intra-extrudate is not a feasible formulation solution for increasing the dissolution rate of high-drug loaded dispersions.

368 Application of Multivariate Curve Resolution Approach to Complex Thermal Desorption DataJing Zhao, University of Delaware, Department of Chemistry and Biochemistry,102 Brown Laboratory, Newark, DE 19716, Jia-Ming Lin, Andrew V. Teplyakov, Juan Carlos F. Rodriguez-Reyes

The qualitative and quantitative analysis for temperature-programmed desorption (TPD) experiments in surface and catalysis chemistry is often difficult due to over-lapping mass spectral cracking patterns of the compounds produced and complex reaction channels that are difficult to decipher. Empirical methods to analyze TPD results always need a priori knowledge and initial assumptions specific to sys-tems investigated, thus the input of an operator onto the outcome of process of analysis cannot be efficiently eliminated. Multivariate curve resolution (MCR) is a mathematical approach to analyze spectral data in the form of matrix calculation. In this way, spectra of different m/z (mass-to-charge ratio) traces changing with tem-perature can be translated into significant cracking patterns of several desorbates with variations and correlations. This provides a straightforward comparison to the known cracking pattern of every component to understand the reactions followed by desorption. For better understanding of thermal process by MCR approach, three reaction processes were used to demonstrate: 1) the cryogenic desorption of vinyl-trimethylsilane from silicon, an introductory system where the known multilayer and monolayer components are resolved. 2) The thermal reaction of tetrakis(dimethyl-amino)titanium (Ti[N(CH3)2]4) on a Si(100) surface, where the products of surface decomposition are identified and an estimation of the surface composition following thermal reaction is afforded. 3) The thermal reaction of ethylchloride-d5 (C2D5Cl) on a Si(100) surface, where the mechanism is not apparent but the products of thermal reaction can be identified, is followed. The original TPD data and results achieved following MCR process are discussed.

369 Evaluation of a Low-Cost Mass Spectrometer: Waters QDaMichael W. Dong, Genentech, 1 DNA Way, S. San Francisco, CA 94080, Christine C. Gu

A low-cost, compact single-quadrupole mass spectrometer was evaluated for phar-maceutical analysis in supporting method development of stability indicating as-says and cleaning verification testing. The advantages found were: easy to use and maintain, sensitive, and excellent software integration of UV and mass spectrom-etry (MS) data in Acquity ultra high-performance liquid chromatography (UHPLC) Empower systems with automated annotation and reporting functions. Some of limitations found were: not very “portable,” electrospray ionization only, no adjust-ment needed or possible, and “not yet integrated with other vendors’ HPLCs.” The poster includes a description of the system, schematics and empower instrumental method. Case studies shown include: a stability-indicating assay with a peak table containing UV and MS data plus system suitability values for each peak; and a generic cleaning verification method of 12 new chemical entities with automated annotations of UV and MS spectra indexed to each peak and performance data of the UV and MS channels.

370 Performance Comparison of Gel-IEF and cIEF with a Recombinant Model ProteinLeu-Fen Lin, SGS Life Science Services, 75 Passaic Ave., Fairfield, NJ 07004, Jeffrey Hulmes

In this study, we compare the performance of traditional gel-based isoelectric focus-ing (gel-IEF) and capillary-based IEF (cIEF) methods for the detection of impurities and variants of a model protein based upon differences in net charge. The protein in the current study is a proprietary recombinant enzyme of MW ~140 kDa. In tra-ditional gel-IEF analysis, the protein has a major (>90% of total) species having a pI of approximately 5.0 and additional bands at pI’s slightly higher (2-3% of total) and lower (6-7% of total). For gel IEF, Novex pH 3-7, precast IEF Minigel (Life Tech-nologies) was employed with Serva® IEF pH 3-10 protein markers and quantified with densitometer. For cIEF, a Beckman Coulter PA 800 plus system was employed with the vendor’s synthetic peptide marker pI 4.1-10.0 and 280 nm detection. In this

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study, we investigated if any differences exist in the pI assignment for the major species between the two methods. Inherent differences between the two methods include pI markers to generate standard curves (native proteins [gel-IEF] versus synthetic peptides [cIEF]), and the standard curve of pI versus mobility (sigmoidol [gel-IEF] and linear [cIEF]. Additionally, we explored if lyophilization of the liquid pro-tein followed by reconstitution would affect the IEF profile with both methods. The data shows that more species from the sample were clearly resolved with the cIEF method while retaining similar quantitation and precise pI measurement.

371 The Development and Validation of a LC-MS-MS Method for the Determination of CEP-32496 in Human PlasmaCharisse Green, Teva Pharmaceutical, 145 Brandywine Pkwy, West Chester, PA 19380, Edward Hellriegel

B-Raf is an isoform of the RAF serine/threonine kinases family. Mutations in the B-Raf gene are found in approximately 7% of all cancers evaluated. While sever-al mutated forms of B-Raf have been identified, the V600E mutation found in the majority of cases (approximately 80%) is a B-Raf-kinase-activating mutation with approximately 500-fold higher activity than wild-type B-Raf. CEP-32496 is a po-tent orally active B-Raf kinase inhibitor that is currently under clinical development by Teva Branded Pharmaceutical Products R&D, Inc., for the treatment of various cancers. A liquid chromatography tandem mass spectrometry (LC-MS-MS) meth-od has been developed and validated for the rapid determination of CEP-32496 in human plasma. The method was validated over a dynamic range of 1.00-1000 ng/mL. The method utilizes a protein precipitation plate (Biotage) to extract CEP-32496 from 50 μL of human plasma. 13C6-CEP-32496 was used as an internal standard. Chromatographic separation was achieved using a Phenomenex Synergi Fusion™ RP 30.0 mm x 2.00 mm, 4 μm 80Å column with isocratic elution followed by tandem mass spectrometric detection. The run time was 2 minutes. The intra-and inter-day accuracy (%bias) of this method at the quality control (QC) levels tested was <15% (20% at the lower limit of quantitation) of the nominal concentrations. The intra- and inter-day precision (%CV) of this method at the QC levels tested was within 15% (20% at the limit of quantitation). The validated method is currently in use to quan-tify the plasma concentrations of CEP-32496 in patients with advanced carcinoma.

372 Application of Micro Flow Image (MFI), ARCHIMEDES, and Quartz Crystal Microbalance with Dissipation (QCM-D) on the Particle Characterization for a Therapeutic Protein in Formulation DevelopmentSongyan Zheng, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903, Aastha Puri, Vishal Nashine, Jinjiang Li, Monica Adams

During product storage and transportation, proteins can aggregate to form parti-cles either under the influence of shear force or due to interaction with silicon oil in prefilled syringes. This study aims to characterize the particulate matters in the solutions of a therapeutic protein in prefilled syringes after shaking using two ana-lytical methods-micro flow imaging (MFI) and resonant mass measurement (AR-CHIMIDES). Specifically, the quantity of silicon oil particles leached from the syringe was correlated with formation of proteinaceous particles. In addition, the inhibitive effect of polysorbate 80 (PS-80) on particle formation was evaluated. The results from both MFI and ARCHIMIDES indicated that PS-80 can prevent the formation of proteinaceoues particles and the leaching of silicon oil particles from the syringes. Mechanistically, as shown by the QCM-D results, the presence of PS-80 inhibited the adsorption of proteins on the silicone oil through a competitive adsorption. The results suggest that the contact of the protein with hydrophobic silicon caused the formation of proteinaceous particles, and the preferred adsorption of PS-80 on sil-icon oil likely prevented the interaction of protein and silicon particles. Overall, this study also demonstrated the capabilities and utilities of MFI, ARCHIMEDES, and QCM-D for the particle analysis respective to types and sizes.

373 Thromboembolic Disease Discrimination through an Ultrasensitive Assay of Microdosed Synthetic Urinary BiomarkerKevin C. Ngan, Tufts University, Department of Chemistry, 62 Talbot Ave., Medford, MA 02155, Shonda T. Gaylord, Milena Dumont Milutinovic, David R. Walt, Andrew D. Warren, Gabriel A. Kwong, Sangeeta N. Bhatia

The discovery of clinically significant biomarkers has been limited, despite extensive research. It is difficult to resolve endogenous molecules from complex biological matrices, prevent rapid ex vivo degradation, and establish quantification criteria robust enough to account for patient-to-patient variability. An alternative to endoge-nous biomarkers is synthetic exogenous agents. These agents can not only be cus-tomized to exploit specific physiological processes, but also continuously improved upon and optimized through further testing. We describe an ultrasensitive assay to detect thromboembolic disease using microdosed synthetic urinary biomarkers. These synthetic biomarkers are composed of iron oxide nanoworm carriers bound to thrombin-sensitive peptide substrates containing a fluorescein reporter and a bi-otin tag (nanoworm-peptide substrate-fluorescein-PEG-biotin) designed to facilitate immunocomplex formation. After intravenous injection, the nanoworms passively

home to disease sites where proteolytic cleavage of the peptide substrate by throm-bin releases the reporter, which subsequently filters into the urine. For detection, we employ a digital immunoassay with single-molecule resolution. Liberated reporters are captured by microbeads coated with specific capture antibodies and labeled with streptavidin-β-galactosidase. The labeled immunocomplexes are localized into individual microwells and sealed with fluorogenic substrate, generating high local concentrations of fluorescent product. The number of fluorescent wells relative to entrapped beads correlates directly to bulk solution concentration. Conventional ELISA detects these reporters at low picomolar concentrations, but our assay ex-tends into the low femtomolar range, increasing sensitivity by 1000x. This assay discriminates healthy mice from those with thromboembolic disease using doses as low as 3.9 ng, corresponding to a human dose of approximately 8.7 μg.

374 Determination of Latanoprost and Latanoprost Acid by UPLC-MS-MSHemantkumar Naik, Millennium Research Laboratories, 160 New Boston St., Woburn, MA 01801, Monish Chaddha, Teresa Pekol, Ram Bhat

Latanoprost, marketed as a generic and under the brand name Xalatan (Pfizer), is a prostaglandin analog prescribed for reducing intraocular pressure in patients with or at risk for glaucoma or ocular hypertension. The marketed formulations are oph-thalmic solutions (0.005% or 50 µg/mL), and typical dosing is by the application of drops to the eyes, once daily. For many patients, chronic use of the drug is required to control eye pressure, prompting the investigation of controlled/extended release formulations and implants to reduce dosing frequency. In-vitro and in-vivo release testing of latanoprost from these formulations and implants over time requires an analytical method with both specificity and a low limit of detection. Additionally, be-cause latanoprost is an isopropyl ester pro-drug that is quickly hydrolyzed by ester-ases in the cornea to the biologically active latanoprost acid, both the pro-drug and the active drug must be monitored. In this work, a ultra-performance liquid chroma-tography tandem mass spectrometry (UPLC-MS-MS) method for the determination of latanoprost and latanoprost acid was developed and qualified. The LC-MS-MS system comprised a Waters Acquity UPLC with an AB Sciex API 5000 mass spec-trometer. Deuterated (d4) internal standards were included for both latanoprost and latanoprost acid. The total method cycle time was less than 5 minutes. Method de-velopment rationale and the results from qualification assessments (stability, spec-ificity, calibration curve, lower limit of quantitation, accuracy, precision, and matrix effect) will be presented as well as the application of the method to samples from in-vitro release tests.

375 Fluorescence-Based Sugar Sensor on a Gold Nanoparticle PlatformIshan V. Soni, Kutztown University, 15200 Kutztown Rd., Kutztown, PA 19530, Zachary M. Semian, Gary A. Baker, Thomas A. Betts

One of the driving forces behind the development of sugar-sensing platforms has been the need to quantify blood glucose levels for the management of diabetes. As a result a wide variety of spectrophotometric and electrochemical methods are geared towards sugar quantitation. Our sugar sensor relies on boronic acid-capped gold nanoparticles which have a fluorescent dye bound to the boronic acid sugar-binding site. The addition of sugars can displace the fluorescent dye from the proximity of the gold nanoparticle surface resulting in a change in fluorescence intensity.

376 Generic Ligand Binding Immunoassay Method for Rat IgG1-Based Surrogate Bio-PharmaceuticalsWonmi K. Rooney, Bristol-Myers Squibb, Route 206 and Province Line Rd., Lawrenceville, NJ 08543

In antibody drug discovery and development, use of surrogate antibodies, particu-larly rat IgG1-based antibodies, is an advantageous alternative to using humanized antibody therapeutics in expensive non-human primates. As effective bioanalytical support for pharmacokinetic assessment of surrogate antibodies, a generic assay method using commercially available antibody reagents was made by design of experiment on an ELISA platform and was assessed for accuracy and precision. Quantitation of rat IgG1-based surrogate therapeutics in various commonly used preclinical species was with a total error of <20%. The reliability of this method was confirmed orthogonally by, side by side testing using a generic liquid chroma-tography mass spectrometry method. This easy to use method facilitates efficient pharmacokinetic testing of surrogate antibody candidates.

377 Proteomic Investigation of Saliva and Sera from Children with Autism Spectrum Disorder (ASD) and Matched ControlsKelly L. Wormwood, Clarkson University, Box 5810, 8 Clarkson Ave., Potsdam, NY 13699, Armand G. Ngounou Wetie, Izabela Sokolowska, Katherine M. Beglinger, Alisa G. Woods, Costel C. Darie, Jeanne P. Ryan

Approximately 1/88 children are believed to be affected by Autism Spectrum Dis-order (ASD). A more recent study even suggests numbers as high as 1/50. With

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the increased diagnosis of ASD, treatment and understanding of the disorder is a pressing health concern. Protein biomarkers found for ASD may potentially be used for ASD diasgnosis, subtyping, monitoring, treatment and identifying thera-peutic targets and may also provide clues about the causes of ASD. Here, sera and saliva samples from children with ASD and matched controls were analyzed using a combination of gel electrophoresis (Tricine-PAGE, sodium dodecyl sulfate polyacryl-amide gel electrophoresis (SDS-PAGE) and Blue Native PAGE), in gel digestion or insolution digestion and nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS-MS) to investigate difference between the proteomes of ASD patients and matched controls. These results will hopefully shed some light on the possible causes of ASD. In the SDS-PAGE and Tricine-PAGE based experiments using sera, increased levels of Apolipoproteins (Apos) ApoA1 and ApoA4, involved in choles-terol metabolism, and of serum paraoxanase/arylsterase 1, involved in preventing oxidative damage, were discovered in the sera of children with ASD, compared with their matched controls. These three proteins are parts of high density lipoproteins (HDLs) and are presumed to interact with one another. This data suggests that there may be a dysregulation of cholesterol metabolism in children with ASD. Additional dysregulated proteins were also detected, suggesting a dysregulated immunologi-cal response and an enhanced response to environmental toxic factors. This inves-tigation is currently ongoing.

378 Human Breast Milk Proteomics for Cancer Biomarkers DiscoveryRoshanak Aslebagh, Clarkson University, 8 Clarkson Ave., Chemistry Department, Box 5810, Potsdam, NY 13699, Devika Channaveerappa, Armand G. Ngounou Wetie, Costel C. Darie, Sallie S. Schneider, Susan R. Sturgeon, Kathleen F. Arcaro

Early detection and treatment of breast cancer is a challenging subject of study since breast cancer is a major cause of morbidity and mortality in the United States. Mammography has greatly increased the early detection of breast cancer, but has known limitations, especially in younger women with dense breasts. Breast milk can be an appropriate option for identifying breast cancer biomarkers because it provides access to breast tissue, in the form of exfoliated epithelial cells, that when combined with mass spectrometry-based proteomics may offer a novel way to iden-tify young women at increased risk of developing breast cancer. In this study, breast milk samples were separated by sodium dodecyl sulfate polyacrylamide gel electro-phoresis (SDS-PAGE) and stained by Coomassie for qualitative analysis and direct comparison of the samples. Then the gel lanes of cancerous and healthy sample (as a control) were cut into several bands which were digested with trypsin and then analyzed by nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS-MS) using a nanoAcquity ultra-pressure liquid chromatography coupled with quadrupole time-of-flight (Q-TOF) micro mass spectrometer. Data processing and analysis were performed using MassLynx (version 4.1), ProteinLynx Global Served (PLGS 2.4), Mascot and Scaffold 4.0. In the Coomassie-stained SDS-PAGE, al-though the overall protein patterns of most of the major bands are similar, sample specific-bands are also present which make the differences between cancerous and healthy samples. These outcomes are currently being investigated.

379 The Separation and Identification of Omega-3 Fatty Acids in Commercially Available Fish Oil Products Using Capillary ElectrophoresisPasquale W. Carione, Drexel University, 3141 Chestnut St., Philadelphia, PA 19104, Donna M. Blackney, Joe P. Foley

Fish oil supplements are primarily composed of polyunsaturated omega-3 fatty ac-ids that possess multiple double bonds starting at the third carbon from the methyl group. The two main omega-3 fatty acids in fish oil supplements are eicosapentae-noic acid (EPA) and docosahexaenoic acid (DHA). Capillary electrophoresis was used to separate EPA and DHA from each other and from other components found in commercially available oil capsules. The two fish oil supplement brands studied, NatureMade and CVS, do not report the exact quantities of EPA and DHA present in each tablet; only the total amount of EPA and DHA combined is reported. These products also report an amount of “other” omega-3 fatty acids on the product la-bel without reporting the names or individual amounts of each of these fatty acids. Therefore, a quantitative study of the individual amounts of EPA and DHA, as well as the “other” fatty acids was initiated.

380 Simplified Strategy to Develop a Robust UHPLC-MS-MS Assay with Emphasis on Assay Performance and Metabolite Interference AvoidanceAdela Buzescu, Bristol-Myers Squibb, Route 206 and Province Line Rd., Princeton, NJ 08540, Naiyu Zheng, Jianing Zeng, Anne-Francoise Aubry, Mark Arnold

In regulated liquid chromatography tandem mass spectrometry (LC-MS-MS) bio-analysis, the development of a robust assay is very desirable. A simplified method screening strategy was utilized for ultra high-pressure (UHPLC-MS-MS) method development to remove phospholipids, minimize metabolite interference and en-sure assay’s ruggedness. As compared to a systematic screening method, this

strategy only included liquid-liquid extraction (LLE) methodology with three ex-traction solvents under three pH conditions and four sub-2-µm UHPLC columns for method screening. UHPLC separation was optimized to separate metabolites from analyte(s) using extracted incurred plasma samples with selected reaction moni-toring (SRM) transitions of metabolites generated from AB Sciex IDA™ software and ADME knowledge. This methodology has led to the development of a robust assay for Compound X, a Bristol-Myers Squibb development compound with curve range of 50-20000 ng/mL in rat plasma. Plasma samples were extracted into Methyl tert-Butyl Ether (MTBE) by automated LLE in 96-well format. Chromatographic sep-aration was achieved with a gradient elution on an ACQUITY Cortecs™ UPLC C18+ column (2.1 x 50 mm, 1.6-µm). Positive electrospray ionization for Compound X and its isotopically-labeled internal standard was performed using selected reaction monitoring (SRM) mode on an AB Sciex API4000 mass spectrometer. The intra-as-say precision was within 3.9% CV, inter-assay precision was within 1.6% CV, and the assay accuracy was within ±2.7% deviation (% Dev) of the nominal values in rat plasma. An inconsistent internal standard response was overcome by resetting the needle penetration in the autosampler, suggesting that a thorough examination of the hardware settings during method development is essential because ven-dor-to-vendor variations in 96-well plates and other consumables are very common.

381 Electrochemical Sensor Using Redox Polymer as an IndicatorZahilis Mazzochette, Rowan University, 201 Mullica Hill Rd., Glassboro, NJ 08028, Amos Mugweru

DNA hybridization biosensors are important diagnosis of generic diseases including environmental and food safety analysis. Electrochemistry has superior advantages over other existing DNA hybridization detection methods; they provide rapid, easy to use, and inexpensive detection. In this work, a simple electrochemical DNA hybrid-ization sensor was assembled using osmium redox polymer as the electrochemical indicator. First the 3’ thiolated single stranded DNA (ssDNA-SH) probe self-assem-bled on the surface of the gold electrode. Then the hybridization was carried out by immersing the ssDNA-SH/Au system containing the complementary ssDNA strand. The electrochemical behavior of osmium redox polymer on the ssDNA and the hybridized double stranded DNA (dsDNA) was determined using cyclic voltam-metry (CV) and impedance methods. Formation of the hybridized ds-DNA on the gold electrode was indicated by a great increase in the peak currents observed on the voltamograms in comparison with those obtained for the single-stranded DNA modified electrode. These results demonstrate that, osmium redox polymer used in this work can be an excellent hybridization indicator.

382 Withdrawn by the author.

383 Unknown Identification via Vibrational Spectroscopy Coupling with Databases and Analysis AlgorithmsDana Garcia, Arkema, 900 First Ave., King of Prussia, PA 19406, Farrel Borden, Marie Scandone

Identification, classification, and interpretation of vibrational spectra present ongo-ing challenges. The objective of coupling vibrational spectroscopy with databases and analysis algorithms is to minimize experimental work, analyze small/intractable samples and maximize the use of data mining tools. The toolboxes currently avail-able include large cheminformatics spectral and property databases and specialized algorithms for queering optimization based on spectra and property. Additionally chemometrics principal component analysis methods, mixture analysis and ad-vanced visualization tools can be incorporated within the toolbox repertoire. In this presentation we provide examples of solutions to multi-components identification based solely on effective utilization of computational methods. The flowchart ap-proach provides a logical protocol for efficient utilization of available tools.

384 Monmouth County Coastal Lakes: Analysis of Nutrients, Dissolved Oxygen, and SalinityEllen Rubinstein, Monmouth University, 400 Cedar Ave., West Long Branch, NJ 07746, Katlynn Muratore, Payal Patel

Due to concerns based upon 2012 studies, which found higher than minimum eutro-phic level nitrate concentrations in several Monmouth County coastal la-kes, moni-toring of these lakes has been continued and expanded. Potential negative effects on the aquatic ecosystem include damage to fish populations and pollution of the ocean, which is in close proximity to the eastern ends of these lakes. Dissolved ox-ygen and total salinity analysis were incorporated in 2013; our 2014 studies added phosphate measurements. Narrow land masses separate these coastal lakes from the ocean. In October of 2012, breaches occurred in several locations due to the impact of Superstorm Sandy. This year, we returned to a few lakes not studied since the summer of 2012, to examine recovery by comparing current nutrient and pH val-ues in these lakes with pre-storm data. Samples were collected at the eastern ends of the lakes, since the outflow will affect the ocean. Vernier sensors were used, in conjunction with a LabQuest2, to measure dissolved oxygen, pH, and temperature on-site. Vernier electrodes were used in the laboratory, with the LoggerPro comput-

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er program, to determine concentrations of nitrate and phosphate ions and salinity. Contributions to the understanding of the health of Monmouth County lakes are made via these analyses. Future studies may benefit from sample collection and data analysis throughout the year, to examine possible changes in these param-eters with temperature over time. Where continued high levels of nutrients or low levels of dissolved oxygen are measured, efforts at remediation may be indicated.

385 Low Temperature 13C Nuclear Magnetic Resonance to Explore the Formation and Structure of a Carbamoyllithium Anion Used in Diastereoselective Synthesis of Alpha-Amino AmidesScot Campbell, Boehringer Ingelheim Pharmaceuticals, 39 Briar Ridge Rd., Danbury, CT 06810, Nina Gonnella, Jonathan Reeves, Keith Fandrick

Carbamoyl anions, generated from N,N-disubstituted formamides and linear dis-criminant analysis, add with high diastereoselectivity to chiral N-sulfinyl aldimines and ketimines to provide alpha-amino amides. The methodology enables direct highly diastereoselective carbonyl introduction. This asymmetric addition reaction is efficient and gives direct access to a wide range of different substituted amido amides as well as to dipeptides. Low temperature 13C nuclear magnetic resonance and density functional theory computational studies were carried out to investigate the nature of the reactive carbene intermediate. The data support formation of a lithium coordinated oxyanionic carbene structure.

386 Automated Online Desorption and Analysis of DNPH Derivatives of Airborne Aldehydes and KetonesFredrick D. Foster, Gerstel, 701 Digital Dr., Ste. J, Linthicum, MD 21090, John R. Stuff, Kurt C. Thaxton, Edward A. Pfannkoch

The analysis of airborne aldehydes and ketones first involves collection of the analytes by passing air through a cartridge containing 2,4-dinitrophenylhydrazine (DNPH). As the air passes through the cartridge, the analytes react with the DNPH to form hydrazones which are immobilized on the cartridge. The cartridges are then eluted with solvent and the DNPH derivatives can be analyzed using high-per-formance liquid chromatography (HPLC) with UV detection. Using the GERSTEL MPS2-xt robotic sampler with a special tray to hold DNPH cartridges, the entire pro-cess of desorbing the analytes and injection of the samples into the LC-UV system can be easily controlled. Automating the desorption of these cartridges can result in significant improvement in accuracy and reproducibility as well as reducing potential experimental errors by the operator. The intuitive software includes tools that allow the desorption of a cartridge to take place during the chromatographic separation of a previously injected sample to ensure maximum sample throughput. In this report, the automation of the online desorption and analysis of airborne DNPH derivatives by the robotic autosampler is discussed. Examination and calibration for a variety of aldehyde- and ketone-DNPH derivatives is described. Finally, DNPH cartridges collected from representative matrices are desorbed and analyzed online and the resulting precision data are provided.

387 Portable Raman Spectroscopy for Monitoring Polymorphic TransitionsPhilip Zhou, B&W Tek, 19 Shea Way, Ste. 301, Newark, DE 19713

Portable Raman spectroscopy is used as process analytical technology (PAT) tool for online continuous monitoring of chemical reactions as well as polymorph transformations. Citric acid is studied here as a model system because citric acid crystalline powder has two polymorph phases monohydrate and anhydrous, which crystallize from water at different temperatures. With appropriate portable Raman sampling methods, the crystalline forms Raman spectra can be measured and thus by comparing these Raman spectra with standard signatures from pure known sam-ples, the crystalline material formed can be qualified, and even quantified when a chemometric model is well established. Various sampling methods are provided to accord with the real situation showing the versatility of portable Raman for in-situ monitoring.

388 Unique Porous QCM Sensor Coatings: A High Sensitivity Vapor Sensor Based on Porous Poly(Methyl Methacrylate)Ho Yeon Yoo, Stanley Bruckenstein Chemical Consulting, 115 Foxpoint W, Williamsville, NY 14221, Stanley Bruckenstein

A unique way is described on how to create porous films on a solid substrate. This method is advantageous particularly when a quartz crystal microbalance (QCM) is the basis of a sensor. QCMs coated with films having large porosities produce more sensitive sensors than those coated with nonporous films. Here, an initially nonpo-rous Poly(methyl methacrylate) (PMMA) film was used to demonstrate our tech-nique for the model system of water vapor analysis in flowing nitrogen gas. A film of nonporous PMMA on a QCM is a sensor for water vapor in a gas phase. However, a much more sensitive sensor was created by dip coating a 10 MHz AT cut quartz QCM crystal into solutions containing mixtures of PMMA and PDLL(Poly(D,L-Lac-tide)), and then evaporating the solution on the QCM. This forms mixed polymer films of varying PDLL content. The PDLL was then leached by exposure to a NaOH

solution to create PMMA films having various void volumes. A porous PMMA film that originally contained 50% by weight PDLL had a 3.7 times larger QCM sensi-tivity for water vapor than a nonporous PMMA film that contained the same weight of PMMA.

389 Factors that Contribute to the Air Pressure Dependence of Particle Size Analysis by Laser DiffractionZheng (Eric) Li, Ashland Specialty Ingredients, 1005 Route 202/206, Bridgewater, NJ 08807, William Thompson, Guanglou Cheng, Chi-San Wu

Laser diffraction is currently the prevailing technology for particle size analyzing, comparing to its wet measurement method, the dry measurement method is often preferred for simpler sampling procedure and less variables introduced. In the dry method set-up, particle breakage may occur due to the energy introduced by exces-sive air pressure during the sample dispersion process, results in the dependence of particle size and distribution on the amount of air pressure applied. For a certain sample, choosing the proper air pressure becomes the key to establish the standard operating procedure (SOP) to determine the particle size accurately and precisely, which is crucial for batch-to-batch quality control. Ideally, a proper pressure should be sufficient to fully disperse the particles, without breaking them; however, this proves to a difficult task because it largely depends on the property of the samples, including the chemical nature, the size and the morphology, etc. More importantly, it is found in this study that the difference in equipment design plays inevitable role on this dependence particle size analysis on air pressure. The goal of this study is to find the “true” particle size using laser diffraction technique. Materials of different chemical nature, size and morphology were studied, and results by equipment from different vendors (Horiba, Malvern, Sympatec, etc.) were compared and discussed regarding to this issue.

390 Accurate Determination of Moisture Content of Soft Contact Lenses by Near-Infrared (NIR) SpectroscopyKeith Freel, Metrohm USA, 6555 Pelican Creek Cir., Riverview, FL 33578, Hari Narayanan

Moisture content determination is a popular application of near-infrared (NIR) spectroscopy due to the strong absorption bands of water near 1450 nm and 1940 nm. The delicate properties of soft contact lenses require a minimal-contact meth-od for analysis to avoid damage to the lens itself. NIR spectroscopy provides a quick and damage-free solution. Reference values used for calibration were based on gravimetric analysis. To improve the accuracy water content of contact lenses were determined using a KF titration with oven sampling. The water content of the lens samples ranged from 24% to 58%. A quantitative model was developed using chemometric software using the water absorption band at 1450 nm. A partial least squares regression model was developed to correlate the spectra profile with em-bedded moisture percentage. A variety of reflectance and transmission sampling presentations were tested and the accuracy and the precision of the sampling meth-ods were compared. Details of correlation values and the standard error of calibra-tion for each sample presentation are summarized.

391 Combined Headspace Karl Fischer and NIR for Quick, Safe, and Reliable Method Development, Validation, and Routine Analysis as Demonstrated with Lyophilized ProductsKeith Freel, Metrohm USA, 6555 Pelican Creek Cir., Riverview, FL 33578, Hari Narayanan, Denise Root, George E. Porter III

Near-infrared (NIR) spectroscopy has been used for decades as a rapid and cost ef-fective tool for determining the water content in numerous different products across a wide range of industries. The most common challenge of NIR testing is creating a new quantitation model when the matrix or packaging goes through a change. This time-consuming process usually involves creating a number of samples, collecting NIR spectra and then analyzing by Karl Fischer (KF) for water content. In this poster, we show that by mounting a NIR probe on the turntable of a KF oven, we are able to streamline this process. By integrating NIR and headspace KF, there is immense time savings, higher productivity and more reliable results than the former method. This efficiency and accuracy of this method was demonstrated with the quantitative analysis of moisture in lyophilized products. NIR has no sample prep and is fully compatible with glass vials making it the perfect candidate for rapid screening of mass produced freeze dryer vials. The quantitative NIR model for lyophilized prod-ucts was developed and validated based on the KF values. The error of prediction was 0.3% within the range 0.9 – 5.3%. A comparison of volumetric vs. coulometric titration as the primary method is compared, and method details and cost savings are discussed.

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392 Three-Dimensional Solution Structure of Cyclic Antibiotic Teicoplanin Aglycone Using NMR Distance and Dihedral Angle Restraints in a DMSO Solvation ModelNina C. Gonnella, Boehringer Ingelheim, 900 Ridgebury Rd., Ridgefield, CT 06877, Nelu Grinberg, Shengli Ma, Mark Mcloughlin

Teicoplanin is a potent antibiotic existing as a mixture of several glycopeptide like antibiotics which all share a common core system. [1] These compounds are mem-bers of the vancomycin group of glycopeptide antibiotics. A fused ring structure forms the common core of the teicoplanin antibiotic family that is termed teicoplanin aglycone. [2] This work describes the three-dimensional solution structure of teico-planin aglycone in dimethylsulphoxide (DMSO) using nuclear magnetic resonance (NMR) spectroscopy. The structure was derived from NMR inter proton distanc-es and targeted dihedral angle restraints based on measured coupling constants. Structures were generated using systematic searches of conformational space for optimal satisfaction of distance and dihedral angle restraints. Comparison of the NMR derived structure with the X-ray structure of a teicoplanin aglycone analog revealed a common backbone conformation with small deviation of two aromatic side chain substituents. The results revealed flexibility of the core aromatic side chain consistent with the region of the molecule involved in protein binding. Overall the results showed that a combination of NMR derived restraints and a hydrogen bonded DMSO solvation model were needed to generate structures that satisfied experimental data.


[1] Hunt, A. H., et. al. J. Am. Chem. Soc. 1984, 106, 4891.

[2] Barna, J. C. J, et. al. J. Am. Chem. Soc. 1984, 106, 4895.

393 Withdrawn by the author.

394 Quantitative Trace Metals Analysis Using Hydrofluoric Acid AlternativesJane Ramsey, E. I. du Pont de Nemours and Company, PO Box 80228, Wilmington, DE 19880, Thomas Connell, Daniel Iversen, Robert Lockerman, Michael Karney

Hydrofluroic acid (HF) is one of the most dangerous and corrosive acids found in to-day’s chemistry laboratories. Despite the hazards, HF is necessary for the complete dissolution of many samples, most commonly those containing silicon, titanium, or zirconium. Only experienced and trained laboratory personnel should attempt to use HF in their protocols due to the severity of burns. HF will readily penetrate the skin and react with calcium in the blood, leading to cardiac arrest. In previous efforts, researchers from CEM and DuPont worked together to evaluate fluoride surrogates other than HF to provide total dissolution of the sample matrix. Our team used titani-um, silicon and zirconium dioxide as target analytes for this experiment. Ammonium bifluoride (NH4HF2) and fluoroboric acid (HBF4) were chosen as fluoride sources and validated against digestions with HF. The results indicated that the HF, HBF4, and NH4HF2 preparations were statistically the same for the titanium and silicon measurements. Zirconium was less promising as it did not go into solution with the HBF4 and had lower than expected recoveries with the NH4HF2. It was determined that further investigation was required. Our current efforts focus on providing op-timized conditions for the dissolution of titanium and silicon dioxide as well as to provide a better digestion for zirconium. These optimized methods are then applied to reference materials containing trace amounts of our analytes of interest to provide a general procedure for analysis using hydrofluoric acid alternatives.

395 NMR Analysis of Synthetic Polymers Utilized in Medical DevicesRobert A. Schiksnis, Johnson and Johnson Global Surgery Group, PO Box 151, Rte. 22 West, Somerville, NJ 08876, Wendy Feng

Johnson & Johnson Global Surgery Group is a leading producer of surgical care products. It manufactures various medical devices and biologics products such as sutures, meshes, breast implants, hemostasis and antimicrobial devices, etc. These diverse product lines involve wide ranges of materials made using a variety pro-cesses. Suitable analytical techniques and critical in-house expertise are required to support new product development and product life cycle management. Synthet-ic polymers produced for use in our medical devices undergo extensive chemical characterization. Solution nuclear magnetic resonance (NMR) spectroscopy is an enabling technology utilized regularly to analyze a polymer’s chemical composition, sequence distribution, and minor component profile. These parameters, amongst other chemical and physical characteristics, are experimentally determined and evaluated throughout the product development process. We also use NMR to de-tect, identify, and quantify new chemical species that could be produced by specific polymer processing steps. An example is given describing the NMR characterization of a polymeric device component that undergoes laser processing.

396 Comparison of FT-NIR and NMR Spectral AnalysisHerman He, Thermo Fisher Scientific, 4410 Lottsford Vista Rd., Lanham, MD 20706

Fourier transform near-infrared (FT-NIR) and nuclear magnetic resonance (NMR) spectroscopy are the popular analytical tools for many chemists because of its versatility in elucidating molecular structure, optimizing reaction dynamics, mea-suring reaction kinetics, monitoring reaction content and controlling product purity. These two technologies are both nondestructive and complimentary to each other; and provide a wealth of information on physical, chemical and structural aspects of molecular systems. This poster presents spectral data of common liquids and application in wine analysis using a Thermo Antaris FT-NIR analyzer and a Thermo picoSpin Portable NMR spectrometer.

397 Stereochemical Analysis of OrganophosphatesKristi Tami, John Jay College, Science Department, 524 West 59th St., New York, NY 10019, Cristina Kinahan, Gloria Proni

Many commercially available organophosphorus (OP) insecticides are chiral, yet they are sold in racemic mixtures. Since enantiomers possess different levels of toxicity on the various pests they target, it is prudent to isolate each enantiomer and test its relative toxicity in order to find out which enantiomer is most effective. Then, only the more effective enantiomer should be used to eliminate the target pests. High-performance liquid chromatography (HPLC) was used to separate the enantiomers of acephate, methamidophos, and N-methyl methamidophos. The col-umn used to separate the enantiomers was a Chiralcel® OJ-H 250 mm x 20 mm ID. Three complimentary spectroscopic techniques were used to determine the abso-lute configuration of the enantiomers: Optical rotatory dispersion (ORD), electronic circular dichroism (ECD), and vibrational circular dichroism (VCD). Three separate techniques were used in order to ensure a higher degree of confidence. Time-de-pendent density functional theory (TD-DFT) was used to generate theoretical ORD, ECD, and VCD data. Once the absolute configurations were established, biological assays were performed in order to determine the relative toxicity of the enantiomers compared to the racemic mixture.

398 Withdrawn by the author.

399 SERS Detection and Characterization of Gold Nanoparticles on Spinach LeavesZhiyun Zhang, University of Massachusetts-Amherst, Department of Food Science, 240 Chenoweth Laboratory, 100 Holdsworth Way, Amherst, MA 01003, Lili He

The increased prevalence of engineered nanomaterials in environment and their potential toxicity urge researchers to study whether those engineered nanomaterials could possibly contaminate agricultural food products. However, many techniques require invasive and complicated sample preparation procedures to detect and characterize engineered nanomaterials. In this study, we aim to explore the feasibil-ity of surface enhanced Raman spectroscopic (SERS) mapping to detect and char-acterize gold nanoparticles (GNPs), as the model engineered nanomaterials, on spinach leaves. SERS is a technique that studies the enhanced Raman scattering signals on noble metal nanostructures. We prepared various sizes of GNPs (15, 30, 50, 90, and 150 nm) and characterized their background and enhancement signals on chlorophyll, the major pigment in spinach leaves. Results show SERS was able to detect the background and the chlorophyll signals from the GNPs of 30 to 90 nm, and the 50 nm GNPs produced the highest signal. Then, we deposited 10 µl (10-10^5 mg/L) 50 nm GNPs onto the surface of spinach leaves, and detected and char-acterized these GNPs using optical light scattering and SERS mapping before and after washing. The results showed that 1) GNPs were able to attach on the surface of spinach leaves even after washing and their distribution were heterogeneous; 2) characteristic peaks, like the chlorophyll peak, could be observed, which indicates molecular interaction between GNPs and surface leaf cell bio-components. These results demonstrated the feasibility of SERS for detecting and characterizing GNPs contaminates on plant leaves.

400 Wide Field Raman Chemical Imaging for Determination of Content Uniformity in Pharmaceutical BlendsLauren Seabrooks, Merck, 181 Passaic Ave., Summit, NJ 07901, Justin Pennington

Content uniformity is a critical quality attribute measured and monitored throughout the development and commercial supply of pharmaceutical products. Herein we report the successful utilization of wide field Raman chemical imaging to assess content uniformity. This approach is non-destructive, obviates the need for sam-ple preparation and is highly sensitive allowing sample sizes of less than 3 mg. Furthermore, unlike previous reports, a direct sample to sample model approach was leveraged through first scanning the samples for Raman spectra and then di-luting to volume for quantitative analysis. Model blends included caffeine/lactose and albuterol sulfate/lactose. Additionally, each blend included the following active

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to excipient ratios: 1:3; 1:1, and 3:1. Quantitative analysis was achieved using ultra high-performance liquid chromatography-mass spectrometry (UHPLC-MS) as the model comparison method to develop multivariate calibration based models from Raman spectra. In each case a partial least squares (PLS) algorithm provided the most robust and accurate model with r-squared values greater than 0.9 and root-mean-square error (RMSE) for calibration, cross-validation and prediction of ap-proximately 0.1.

401 Chemical Analysis and Characterization of Dried Figs and Dried DatesJerzy Mierzwa, Tennessee State University, Chemistry Department, 3500 John A. Merritt Blvd., Nashville, TN 37209

Dried dates and figs are very popular dried fruits for snacking. The author of this analytical study has carried out chemical analysis, characterization and a com-parison of several samples of dried figs and dates (samples of different origins). The concentration of twelve metals was determined in digested samples by atomic absorption spectrometry (AAS) and inductively coupled plasma – optical emission spectrometry (ICP-OES). Several organic compounds belonging to certain groups (e.g. sugars, vitamins) were analyzed by HPLC (reverse-phase gradient chromatog-raphy, standard C18 column, and UV/Visible absorption detector) and some trace level compounds were also analyzed by gas chromatography mass spectrometry (with ion-trap mass spectrometer). Moreover, these samples were examined by FTIR spectrometry and UV-Visible spectrophotometry. The results of this compar-ative analysis are summarized, and some basic analytical figures-of-merit are pre-sented. A possibility of a reliable geographic fingerprinting of the samples is also discussed.

402 Spectral Studies of Dipicolinic Acid with Bovine Serum AlbuminAnne E. Okafor, St. John’s University, 8000 Utopia Pkwy., Queens, NY 11439, Enju Wang, Neil Jespersen

Dipicolinic acid (DPA) 2,6 pyridinedicarboxylic acid, is a common chemical com-pound of bacterial spores. Endorspores typically contain 2 to 15% of DPA by dry weight. Endospores can germinate into active cell when the appropriate nutrients and environmental conditions suitable for spore germination are present. The presence of spores is sensitively detected through the concentration of DPA. In this present work we investigate whether DPA will alter the conformation of bovine serum albumin, (BSA). BSA is the most abundant protein in the plasma. It has a strong affinity to reversibly bind to large number of endogenous and exogenous substances. It has very many molecular functions. Serum albumins are special car-riers that chaperones important nutrient not soluble in water to hungry cells. BSA is widely used in the laboratory when a generic protein is needed. BSA contribute to colloid osmotic blood pressure and are chiefly responsible for the maintenance of blood pH. BSA has been used as a model protein to study protein aggregation and for biotechnological folding. This present work seeks to investigate the interaction of DPA and its binding mode in BSA. The study is carried out using fluorescence spectroscopy, Fourier transform infrared, and ultraviolet-visible spectroscopies. This study provides insight into the potential toxicity between DPA and the protein. Spec-tral changes of BSA with DPA are looked into. The shift of fluorescence wavelength of BSA with DPA is seen as an interaction of the protein conformational changes.

403 Chiral Analysis by Fourier Transform Rotational SpectroscopyBrooks H. Pate, University of Virginia, McCormick Rd., Charlottesville, VA 22904, Kevin K Lehmann, Simon Lobsiger, Cristobal Perez, Luca Evangelisti

A new spectroscopic method to measure the enantiomeric excess using molec-ular rotational spectroscopy was introduced by Patterson, Schnell, and Doyle in 2013 [Nature 497, 475–477 (2013)]. The technique uses the fact that enantiomers can be distinguished by the way that the dipole moment projects onto the principal axis system for molecular rotation. Three wave mixing experiments are performed that generate a molecular signal proportional to the product of the dipole moment components. The enantiomers differ in the sign of this product and the three wave mixing signal measured using Fourier transform spectroscopy has different signal phases for the enantiomers. A new method for generating the optimal signal using time-separated microwave pulses is demonstrated in measurements of solketal. The measurement uses high-resolution spectroscopy so that chiral analysis can be performed directly in a complex sample matrix. High-resolution spectroscopy also makes it possible to perform chiral analysis on the large number of stereosio-mers that are possible for a molecule with multiple chiral centers without requiring chromatographic separation. The technique has the potential determine the abso-lute configuration of the molecule by determining the absolute signal phase – an experimental challenge that is currently being tackled – and using relatively simple quantum chemistry calculations of the molecular dipole moment.

404 Comprehensive Ion Analysis of Various Water Matrices in the Hydraulic Fracturing ProcessStuart J. Procter, Metrohm USA, 6555 Pelican Creek Cir., Riverview, FL 33578, Jay Gandhi, Anne Shearrow

Oil and natural gas are fossil fuels that are a valuable diminishing resource that are utilized for energy and materials production. These naturally occurring prod-ucts cannot be easily replenished, so to maintain a local and sustainable source, fracking is becoming of increasing importance to the USA’s long term economic stability. Fracking technology represents a doorway to vast untapped reserves, but with it comes environmental concerns and analytical challenges that need to be ad-dressed moving forward. Therefor analytical techniques for the long term monitoring of ground and surface water is important for environmental conservation, as well as the analysis off back waters and discharge water from the fracking process that can have up to 30% sodium chloride as an ionic matrix. In this poster presentation, chemical analysis methodologies using ion analytical instruments are discussed. The new methods enable the simultaneous analysis of anions, cations and organic acids in a 30% sodium chloride matrix that are of environmental interest.

405 Developing Mobile LIBS Solutions for Real World ApplicationsQun Li, B&W Tek, 19 Shea Way, Ste. 301, Newark, DE 19713, Katherine Bakeev, Jing Li, Sean Wang

We present a new type of handheld laser-induced breakdown spectroscopy (LIBS) spectrometer, NanoLIBS for developing mobile solutions for real world applications. A micro diode-pumped passive Q-switched solid-state laser with high repetition rate of well above 1kHz in comparison to 1-10Hz as used in a traditional LIBS instrument is employed to produce a train of laser pulses. The focused laser spot is further fast scanned over a pre-defined area, hence generating several hundreds of micro-plas-mas per second at different locations. Synchronized miniature CCD array spectrom-eter modules collect and generate pre-conditioned LIBS spectra. The NanoLIBS is measured at less than 1.8kg and operable for more than four hours with embedded batteries. The limits of detection are in the range of 1 to 1,000ppm, which is element and matrix dependent. The typical measurement time is within 1-2 second. The handheld LIBS spectrometer has a Linux based operation system, NanoLIBS Op-eration System (NOS). NanoLIBS is an open platform for developing mobile LIBS solutions. For a custom analyzer for certain specific applications, the user will: 1) collect the LIBS data using NanoLIBS; 2) export the collected LIBS data to a PC; 3) using the chemometrics software, BWIQ for model and calibration development; 4) conversion under NOS with custom graphic user interface (GUI). Some examples of successful application development are presented.

406 Localized Surface Plasmon Resonance of Gold Nanoparticle Random ArraysJulie A. Jenkins, University of Connecticut, 55 North Eagleville Rd., Storrs, CT 06238, Sravan Thota, Xiangdong Tian, Xiaowen Zhao, Jing Zhao, Yadong Zhou, Shengli Zou

When electromagnetic radiation interacts with a metal nanoparticle, it excites the particle and causes the conduction electrons to oscillate collectively, known as local-ized surface plasmon resonance (LSPR). The shape and size of the nanoparticle, as well as the local dielectric environment around the nanoparticle will affect the LSPR peak. Therefore, molecular binding with metal nanoparticles can be used as an optical sensing technique. In this work, a highly uniform, LSPR substrate was produced based on the self-assembly of colloidal gold nanoparticles onto a pretreated glass substrate. LSPR wavelength of the gold nanoparticle random array blue-shifts and the LSPR band width narrows compared to that of the single gold nanoparticle on glass substrate as well as the gold nanoparticles in solution, re-sulted from dipole interaction between the nanoparticles. It was also found that not only is the LSPR wavelength sensitive to changes in the dielectric environment, but the LSPR peak width as well. The LSPR substrates are reproducible, uniform and robust with potential applications in LSPR sensing and imaging.

407 Determination of BPA in Aqueous Solution Using FluorescenceSarah J. Pereira, University of Massachusetts Dartmouth, 789 Wilbur Ave., Swansea, MA 02777, Yuegang Zuo

Bisphenol A (BPA) is a compound widely used in plastics and other industrial con-sumer products. However, BPA has been found to show toxicity as well as endocrine disrupting effects and so release of BPA into the aqueous environment has been of great concern. Methods of detection of BPA in aqueous medium have, therefore, be-come of great importance. Current detection methods of BPA are slow, so a quicker method using fluorescence to measure concentration of BPA is being developed. It has been reported that BPA shows fluorescence in organic solvent but is too low to be detected in aqueous solution. (Olmo, 2000). A method for using florescence to measure BPA in aqueous solvent must then be developed. In this study the fluores-cence of BPA in acetonitrile (ACN) and water were compared and solutions of BPA made from solvents with varying percent of acetonitrile to water were measured to determine the effect of water on the fluorescence of BPA in acetonitrile solution. It was found that BPA does show fluorescence in water but is low, especially com-

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pared to ACN. The results also showed that fluorescence increases slightly from pure ACN as water is added but decreases once the solvent is more than 20% water. If fluorescence detection is to be used to detect BPA in real water samples then the method must be able to distinguish between BPA and other compounds with similar structure. To do this constant-wavelength synchronous fluorescence spectroscopy was used.

408 Coupled Exciton and Charge-Transfer Resonances in the Raman Enhancement of Phonon Modes of CdSe Quantum Dots (QDs)Syed K. Islam, City College, City University of New York, 160 Convent Ave., New York, NY 10031, John R. Lombardi, Mohammad A. Sohel

We report the observation of the enhancement of a (TO) phonon mode of e1 sym-metry and a normally forbidden (SO) phonon mode of b1 symmetry in CdSe quan-tum dots (QDs) due to adsorption of 4-mercaptopyridine molecules. The former is observed in 3, 4, and 5 nm diameter particles, while the latter is observed only in the 2 nm particles. Maximum enhancement of the phonon modes is obtained through a coupling of the charge-transfer transition and the exciton transition which are in resonance with the laser energy. Selection rules using Herzberg-Teller (vibronic) coupling are invoked to explain the observed enhancements.

409 New Techniques for the Analysis of Volatiles and Gases: FT-MRR Spectroscopy (Fourier Transform Molecular Rotational Resonance)Justin L. Neill, BrightSpec, 770 Harris St., #104 B, Charlottesville, VA 22903, Robin L. Pulliam, Roger Reynolds, Matthew T. Muckle, Brent J. Harris, Dave McDaniel, Brooks H. Pate

For gas mixture analysis, there is a need for new instruments that offer standalone operation in combination with broad composition analysis capabilities. Infrared based detectors come with constraints in selectivity and chemical coverage, and gas chromatography techniques come with the need for a dedicated analyst. A new commercial molecular spectroscopy technique, FT-MRR, is capable of direct, quan-titative gas mixture analysis without chromatography. The FT-MRR spectral finger-print is specific to the unique mass distribution of a molecule (including its isotopes) and arises from the quantized rotational energy levels. It is also a high resolution technique that offers superior chemical selectivity over infrared spectroscopy for complex mixture analysis. Without the need for optical cavities, a broadband FT-MRR instrument can monitor a wide range of volatile species, and is reprogram-mable in order to incorporate new compounds. The detection limits for small, polar molecules (e.g., hydrogen cyanide, ammonia, hydrogen sulfide, formaldehyde, wa-ter) in direct sampling are in the parts-per-billion (ppb), with parts-per-trillion (ppt) attainable with a pre-concentration sampling system. FT-MRR performance for quantitative analysis using calibration standards in nitrogen will be presented. Sam-pling methods for direct analysis as well as pre-concentration for trace monitoring of volatile organic compounds in air are described.

410 Mid-Infrared Optical Coherence Tomography System Using a High Power, Low Coherence Quantum Cascade Superluminescent SourceDeborah M. Varnell, Princeton University, Department of Electrical Engineering, Equad, Olden St., Princeton, NJ 08544, Ahmed Musse, Nyan Aung, Mei Zheng, Germano Penello, Claire Gmachl

Visible and near-infrared (IR) optical coherence tomography (OCT) imaging systems use light reflected at different depths in a sample to provide high resolution, three-di-mensional images and are used extensively in medicine, research and industry. Using mid-IR light as an OCT source would decrease scattering and give stronger reflections, and therefore better images, from materials with absorption fingerprints in the mid-IR; however no mid-IR OCT systems are commercially available, most likely due to the lack of good sources. Broadband lasers have previously been used as a source but although they have high enough power to give long depth scans, they have poor depth resolution due to the long coherence lengths. We demonstrate progress in developing a mid-IR OCT system using a new high power (3 mW), low coherence (55 µm) super-luminescent emitter to achieve both long depth range and high depth resolution. A free space OCT system has been designed, assembled and tested. This work was supported in part by MIRTHE (NSF-ERC).

411 Optimizing Bio-Energy Production by Imaging Nutrient Exchange in the Plant Root Rhizosphere Using Infrared MicrospectroscopyTiffany Victor, Stony Brook University, 350 Circle Rd., Stony Brook, NY 11790

Plant based biomass is an efficient renewable energy source in the United States, but there is a limited understanding of the nutrient uptake processes that facilitate its availability. By studying the nutrient distribution within the plant rhizosphere, qual-itative and quantitative information can be gathered to spatially resolve nutrients example phosphates and nitrates. This project uses an infrared microscopy method that enables imaging of the nutrient distribution in the rhizosphere to be imaged both in the presence and absence of growth promoting microbes in order to assess how different combinations affect the production of biomass.

412 Effect of Split Ratio on USEPA Method 8260 CompoundsAnne Jurek, EST Analytical, 503 Commercial Dr., Fairfield, OH 45014, Mike Moses, Lindsey Pyron, Kelly Cravenor

United State Environmental Protection Agency (USEPA) Method 8260 involves purging analytes out of a water matrix. During a split injection, the sample volatilizes in the inlet and is swept by the carrier gas through the liner onto the gas chroma-tography (GC) column with a portion of the sample being split off and sent out the split vent line. The amount of sample reaching the detector is dependent upon the split flow rate. Thus, the higher the split rate, the smaller amount of sample on the column. Furthermore, a higher split ratio minimizes column exposure to moisture. During purge and trap sampling moisture control is crucial to decreasing split flow and enhancing detection limits. This application explores the effect split ratios have upon USEPA Method 8260 analytes.

413 Synthesis, Spectroscopic and Electrochemical Characterization and Catalytic Activity of Mn(III) Complexes of Tridentate Schiff Base for the Epoxidation of OlefinsRita N. Egekenze, Howard University, 525 College St. NW, Washington DC 20059, Yilma Gultneh

The Mn(III) complexes of tridentate Schiff-base ligands of 2-aminoethyl-2-pyridine with 5-methoxy-, 4,6- dimethoxy-, 5-bromo-, 3,5-dibromo– and nonsubstituted-sa-licylaldehyde (complexes 1, 2, 3, 4 and 5 respectively) were prepared and charac-terized by Fourier transform infrared, UV-Visible spectroscopy, liquid chromatog-raphy mass spectrometry (LC-MS) and thermal gravimetry.X-ray crystallographic structures showed six-coordinate ML2 (L = tridentate Schiff base) complexes. In their cyclic voltammetry in acetonitrile solution, the Mn(III) complexes of the Schiff bases each showed one reversible redox process in the -1.5 to +1.5 V (vs Ag/AgCl) window (Mn(III) ⇔ Mn(II)). The thermogravimetry show that the decomposition takes place in three steps for all complexes. Evaluation of the catalytic activity of the Mn (III) complexes for the epoxidation of olefins recorded higher yield than in the absence of the complexes. UV-visible absorption studies of the reactions of the complexes with aqueous H2O2 at room temperature and in dichloromethane showed appearance of new peaks at 255 and [323-332] indicating the formation Mn-peroxo and Mn-oxo complexes that are the oxygen atom transfer intermediates leading to the epoxidation of the olefin. We report the crystal structures, LC-MS result, cyclic voltammogram, UV-visible absorption spectra, 1HNMR and gas chro-matography-MS results for the catalytic epoxidation experiment.

414 GCxGC-TOFMS with Variable-Energy Electron Ionization for Enhanced Screening of Pollutants in Complex Environmental MatricesPete Grosshans, Markes International, 11126-D Kenwood Rd., Cincinnati, OH 45242, Laura McGregor, Nick Bukowski, Nicola Watson, Charles Haws

Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GCxGC-TOF MS) offers greatly enhanced peak capacity, through the coupling of two columns of different selectivity, as well as highly sensitive detection and confident mass spectral identification of trace-level analytes. Nevertheless, the identification of individual compounds may be hindered by weak molecular ions or when similar mass spectral characteristics are evident across entire chemical class-es. Select-eV ion source technology aims to combat this problem by allowing both hard and soft electron ionisation with no inherent loss in sensitivity. The availability of full-fragmentation spectra for easy library matching is complemented by the ability of Select-eV to enhance the heavier ions that are critical for confirming compound identity and distinguishing between similar compounds. At low ionisation energies, fragmentation is greatly suppressed, resulting in simplified spectra dominated by structurally-significant ions. Furthermore, the fragment ions that ordinarily dominate high matrix samples are greatly reduced, enhancing selectivity and further improv-ing detection levels. This work shows the suitability of this novel analytical platform for the analysis of both target pollutants and unknown chemicals within various en-vironmental matrices.

415 Residual Solvent Analysis of N-Methylpyrrolidone Using Headspace Sampling and NPD DetectionAmy F. Birch, Boehringer Ingelheim, 5175 Briar Ridge Rd., Danbury, CT 06810, Johanna Ubben

N-Methylpyrrolidone (NMP) is a class 2 solvent commonly used in pharmaceutical synthesis, with an International Conference on Harmonization Q3C alert level of 530 ppm. The high boiling point (204 °C) and low vapor pressure of this compound allows only a small amount to be partitioned into the headspace for residual sol-vent analysis by headspace/gas chromatography (GC) analysis at 140 °C sample incubation temperature. The literature generally recommends that a liquid injection technique be used to quantitate NMP for residual solvent analysis. However, liquid injection of large concentrations of a drug substance can create analyte recovery challenges due to liner contamination. Headspace sampling is preferential in provid-ing a robust method. A nitrogen specific detector (NPD) can be used to quantitate

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NMP, using a headspace injection technique. The NPD detector is specific to nitro-gen compounds and also is more sensitive than the flame ionization (FID) detector in detecting nitrogen compounds by 1-2 orders of magnitude. Thus, the detector provides an increase in sensitivity for NMP, while still using a headspace injection technique to maintain a more robust analytical method. Validation data from a dual detector (FID/NPD) method is presented here to support the use of an NPD/head-space method for the quantitation of NMP in residual solvent analysis.

416 GCxGC Column Kits Offer Choice Plus ConvenienceLen Sidisky, Supelco/Sigma-Aldrich, 595 North Harrison Rd., Bellefonte, PA 16823, Gustavo Serrano, Dan Shollenberger, Jamie Desorcie, Greg Baney, Michael Buchanan

Gas chromatography x gas chromatography (GCxGC) is one of the fastest growing areas in analytical chemistry due to its ability to resolve a large number of com-pounds, even in the most complex samples. It employs two columns in series, sepa-rated by a modulator. One key to the successful operation of GCxGC is that the two columns must have orthogonal selectivity, that is, they must utilize different retention mechanisms. Column selection strategies to achieve orthogonal selectivity include: 1) Non-polar to polar strategy. Analytes are separated on a non-polar column in the first dimension, and on a polar, highly polar, or extremely polar column in the second dimension. 2) Polar to non-polar strategy. Analytes are separated on a polar, highly polar, or extremely polar column in the first dimension, and on a non-polar column in the second dimension. 3) High resolution polar to non-polar strategy. Analytes are separated on a long (100 m) highly polar or extremely polar column in the first dimension, and on a non-polar column in the second dimension. Details, benefits, and GCxGC plots for each of these three column selection strategies are presented.

417 Determination of Natural Gas Components in Drinking Water by Gas Chromatography and Vacuum Ultraviolet DetectionLing Bai, University of Texas-Arlington, 700 Planetarium Pl., Rm. 130, Arlington, TX 76019, Hui Fan, Kevin A. Schug, Jonathan Smuts, Phillip Walsh, Dale Harrison, Zacariah L. Hildenbrand

Natural gas is a complex mixture consisting primarily of low molecular weight hydro-carbons, inert gases, and other impurities including a variety of sulfur compounds. Mixtures of fixed gases are typically difficult to speciate by modern capillary gas chromatography (GC). A new gas chromatography detector based on vacuum ultraviolet (VUV) spectroscopy (GC-VUV) simultaneously collects full scan (120 – 240 nm) VUV and UV absorption of eluting analytes. GC-VUV can be used to provide universal detection in combination with high quality analyte-specific ab-sorption spectra. The vacuum ultraviolet spectrum opens up a wide spectrum of advantages, resolves co-eluting analytes and limits needs to achieve fully resolved analytes chromatographically. Gas chromatography was performed on a Shimad-zu GC-2010, and separations were performed with a HP-PLOT-Q BOND column. Many sample mixtures of gases were tested, ranging from prepared gas mixtures, to natural gas samples collected from a laboratory natural gas supply, as well as sam-ples taken from private water wells in close proximity to unconventional natural gas extraction. Presented will be the ability of VUV detection to analyze natural gas and natural gas contamination samples, including the potential for direct quantification based on absorption cross-section of the measured compounds.

418 Analysis of a PIONA Sample Using Gas Chromatography and Vacuum Ultraviolet SpectroscopyPhillip Walsh, VUV Analytics, 16525 Ennis Trail, Austin, TX 78717, Jonathan Smuts, Daniel Klopp, Dale Harrison

Analysis of a PIONA standard using gas chromatography coupled with a novel vacu-um ultraviolet spectrometer detector are presented. The vacuum ultraviolet detector generates a three-dimensional dataset consisting of time-resolved 125-240nm ab-sorbance spectra. Generally, vacuum ultraviolet absorbance spectra exhibit a high level of uniqueness, providing a selective component to gas chromatography data allowing for deconvolution of co-eluting species. Even in cases where individual species within a given class are harder to distinguish, vacuum ultraviolet absor-bance is often able to distinguish between general classes of analytes. This combi-nation lends itself particularly well to the PIONA analysis, and offers the possibility of reducing the number of separation steps involved in typical hydrocarbon analysis of fuels.

419 Gas Chromatography–Vacuum Ultraviolet Spectroscopy in Fatty Acid Methyl Ester AnalysisHui Fan, University of Texas-Arlington, 700 Planetarium Pl., Arlington, TX 76019, Kevin A. Schug, Jonathan P. Smuts, Phillip Walsh, Dale Harrison

Fatty acid methyl esters (FAMEs) are common transformation products of fatty ac-ids, present in biodiesel, various consumer products, and as a common derivatiza-tion product of fatty acids for gas chromatography (GC). The long chain components of these compounds can contain variable degrees of saturation and unsaturation, the differentiation of which can be critical for assessing health benefits or other bio-

logical functions. Gas chromatography – mass spectrometry is the gold standard for analysis of FAs and FAMEs; however, the differentiation of many isomeric species requires specialized columns, as the mass spectrometry detector does not provide sufficient specificity. A vacuum ultraviolet (VUV) detector was recently developed and tested in fatty acid methyl ester analysis in GC. Here we demonstrate the ability of GC-VUV as a universal detector to classify fatty acids (e.g., unsaturate vs. satu-rate, mono-unsaturate vs poly-unsaturate), and differentiate cis/trans isomers (e.g., elaidic vs. oleic acid) based on their absorption in the 125 – 240 nm wavelength range. The analysis of standards as well as real world samples, such as rapeseed oil demonstrates that less effort is needed to chromatographically separate different species of interest, due to the greater specificity of the VUV detector.

420 Fourier Transform Ion Cyclotron Resonance and Infrared Multiphoton Dissociation Studies of Cadmium Sulfide Metal ClustersDouglas P. Ridge, University of Delaware, Department of Chemistry and Biochemistry, Newark, DE 19716, Kaitlin Papsin, Una Kim, Giel Berden, Jos Oomens

Fourier transform ion cyclotron resonance mass spectrometry involves injecting ions into an electromagnetic trap where they can be detected and mass analyzed on the basis of their cyclotron frequency. The method facilitates manipulating the trapped ions in various informative ways. The reactivity of trapped ions can be ex-amined by exposing the trapped ions to reactive gases. Structure and spectroscopic properties can be examined by subjecting the trapped ions to collisional or photon induced decomposition. The application of these techniques to the study of cadmi-um sulfide metal clusters is described. These metal clusters are important in marine environments both as potentially toxic pollutants and in relation to the exotic biology found around hydrothermal vents in the ocean floor. Although cadmium sulfide is insoluble in water metal sulfide clusters can be prepared by electrospraying soluble metal salts such as cadmium acetate and allowing the resulting cadmium acetate clusters to react with H2S in the ion trap. These results in clusters containing multi-ple Cd atoms and bisulfide and sulfide counter ions. The structure of these clusters have been probed by subjecting them to collision induced decomposition, infrared multiphoton dissociation, and density functional theory calculations. Among the con-clusions is that when excited the clusters tend to lose preferentially metal atoms forming a series of clusters containing hypervalent polysulfides. The energetics of these processes and the consequences for marine environments are discussed.

421 Mass Spectrometry of Airborne Particulate Matter: How it Works and What it Tells Us About the Air We BreatheMurray Johnston, University of Delaware, Chemistry and Biochemistry Department, Newark, DE 19716

Our research group has developed a variety of mass spectrometers for on-line characterization of individual airborne particles. In this presentation, I highlight one particular aspect of this work, particles smaller than 100 nm in diameter. Nanoparti-cles are ubiquitous in the atmosphere and are produced by a variety of natural and anthropogenic processes. These particles represent a potential health hazard to humans when inhaled, and influence climate by acting as seeds for cloud formation. Determining the chemical composition of these particles aids identification of sourc-es and assessment of environmental impacts. The results of several measurement campaigns are summarized, which span both urban and remote locations. These measurements give insight into the air we breathe and why it matters.

422 Mass Spectrometry to Support Industrial FermentationBarbara Larsen, DuPont, Experimental Station E228/120A, 200 Powder Mill Rd., PO Box 8352, Wilmington, DE 19803

A variety of analytical methods are used to support strain development in metabolic engineering of organisms for the production of industrial chemicals. Strain engi-neering requires changes to cell metabolism to direct the carbon flux to produce the industrially valuable chemical. Mass spectrometric methods have been developed to ascertain changes in the protein profile through the fermentation process. Pro-teomics to support the fermentation processes are discussed.

423 Mass Spectrometry Explores Communication in the Tumor EnvironmentCatherine Fenselau, University of Maryland, Department of Chemistry and Biochemistry, Bldg. 091, College Park, MD 20742, Suzanne Ostrand-Rosenberg, Meghan Burke, Rebecca Rose, Lucia Geis-Asteggiante

Communication between cells in the tumor microenvironment is conducted primarily by chemical moieties that are secreted by cells or transported in exosomes shed by cells. We have focused on the potential for communication by proteins carried by exosomes. What proteins are present? How are they modified? What are their biological activities? We have used tandem high resolution mass spectrometry to answer the first two questions, combined with various chromatographies, bioinfor-matics and strategies for sample preparation of proteins from exosomes shed by

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myeloid derived suppressor cells collected from tumor-bearing mice. This talk merg-es the analysis of exosome surface glycoproteins, exosome lysate by top down and bottom up strategies, and ubiquitinated proteins captured by immunoaffinity, to provide insight into the formation and some biological activities of these exosomes.

424 The Incredible Shrinking NIR Instrument; and Why that is Great for the Pharmaceutical IndustryEmil Ciurczak, Doramaxx Consulting, 77 Park Rd., Goldens Bridge, NY 10526

The near-infrared (NIR) instruments available in 1980 were larger than counter-top microwave cookers and had to be used in a laboratory setting. As the decades advanced, not only did the instruments become more rugged, but the sizes also began to shrink. Simultaneous advancements in computers and software allowed spectra to be generated and information generated at an amazing speed. The first qualitative software, for example, in 1984, took hours to generate a usable equation. Today, far more complex equations, based on many hundreds of spectra, take sec-onds to generate and apply. The size of the hardware has also been shrinking (par-tially based on the influx of components from the telecom industry) steadily. By the 1990’s, very good instruments could be purchased with dimensions less than 1/5th those of the 1980’s, equipped with internal power sources and WiFi abilities. This has progressed logically until units are being developed that simply plug into “smart phones,” using the software of the phone for calculations. Why is this so good? Not only can process analytical technology/quality-by-design applications be fulfilled, but field-ready units can be pressed into service to assist in the control of counterfeit products. In short, size does matter; in this case, smaller is better.

425 Validation and Transfer of NIR Methods for Real-Time Release Testing for Continuous ManufactureJianfeng (Frank) Qi, Vertex Pharmaceuticals, 50 Northern Ave., Boston, MA 02210, Kelly A. Swinney, Henrik T. Rasmussen, David E. Nadig

The validation of near-infrared (NIR) methods, or process analytical technology (PAT) methods in general, can follow the general principles in International Con-ference on Harmonization Q2(R1) with some adaptations. For validating a NIR final blend active pharmaceutical ingredient (API) content method, specificity, ro-bustness, accuracy, precision, linearity, and range are assessed. Specificity and robustness are satisfied by demonstrating that the NIR signal (following chemomet-ric treatment) is attributable to the API content and is not impacted by likely interfer-ences across the product design space. Accuracy is determined by comparing the NIR results with results from the reference method (high-performance liquid chro-matography (HPLC)). The precision of the PAT method is assessed using repeat measurements of the same sample at low, middle, and high concentrations across the design space (the range). To facilitate determining only the analytical error (vs. the error associated with both the measurement and sample heterogeneity), NIR precision measurements are made with the powder flow stopped to make sure the same sample is being measured. Linearity is determined by comparing the PAT results to the HPLC results across the range. Since chemometric model develop-ment and validation require a large number of samples to be analyzed to generate reference values, a high-throughput HPLC method, optimized for accuracy, was developed for this purpose. The NIR method is validated on a lab NIR instrument in a PAT stack that is equivalent to the PAT system on the continuous manufacture system (CM-PAT). The method is then transferred to the CM-PAT using a limited method validation approach.

426 Understanding Hot Melt Extrusion via NIRSBrandye Smith-Goettler, Merck, 770 Sumneytown Pike, PO Box 4, West Point, PA 19486

Process analytical technology (PAT) and hot melt extrusion (HME), commonplace in the food and polymer industries, are increasingly being implemented in the phar-maceutical industry. Hot melt extrusion utilizes pharmaceutical grade polymer(s), potentially high temperatures, and mechanical energy to convert drug substance from a crystalline to an amorphous state. This processing platform is ideal for poor-ly soluble drug substances, maximizes production efficiency by combining several pharmaceutical processing steps and enables continuous manufacturing. PAT, in general, yields numerous manufacturing advantages but those specific to HME are highlighted via discussion of in-line, transmission mode, Fourier transform near-in-frared spectroscopy (FT-NIR) as applied to two Merck drug products. In both appli-cations, NIR and partial least squares models were developed for real-time drug loading (%) predictions. These predictions have been successfully used for process development, process fault detection and real-time quality assurance via waste gate automation control.

427 Advances in Applying Near Infrared Measurements to Cell Cultures and Fermentation ProcessesGary Ritchie, Infratrac, Silver Spring Innovation Center, 8070 Georgia Ave., Silver Spring, MD 20910

Near-infrared (NIR) spectroscopy has matured as a useful analytical tool for the measurement and control of biopharmaceutical processes. At the present time, NIR has both a scientific, risk-based regulatory footing and extensive empirical basis for supporting the manufacture of biologic drug substances and drug products. Re-cent publications indicate a surge of interest in the use of this versatile tool for the production of biologics; yet, very little has been said about how biopharmaceuti-cal manufacturers go about adopting NIR for the production of lifesaving drugs. Many factors must be considered before a NIR analyzer is put at, in or on-line for any purpose, but especially in the case of living systems, these considerations are many and extremely complex. This presentation briefly reviews the progress that has been made over the last several years for the use of NIR in the production of biologics and then further presents what should be considered if this tool will have any future role to play in supporting biopharmaceutical manufacturing. Based on recent developments in both the regulatory and industrial arenas, this presentation provides examples that touch upon developments in regulations, equipment, facili-ties and techniques to offer a better understanding of the potential and limitation that NIR spectroscopy may hold for the production of biologics in the twenty-first century.

428 Fast and Efficient Peptide Mapping of Monoclonal Antibodies Using Superficially Porous ParticlesTimothy Rice, Agilent Technologies, 2850 Centerville Rd., Wilmington, DE 019808

Peptide mapping by reverse phase chromatography is the mainstay technique in bio therapeutic analysis, delivering comprehensive characterization of biopharmaceuti-cal products. When interfaced with a mass spectrometer, it can deliver the identifi-cation of proteins and their variants, determine post translational modifications and locations, and confirm protein sequences. However, peptide mapping represents a significant chromatographic challenge due to the inherent complexity of protein digests. Developing robust and reliable peptide maps is a struggle because of low sensitivity, poor peak shapes, and very long separation times. This talk focuses on the AdvanceBio Peptide Mapping column from Agilent as means to overcome these challenges and achieve rapid, highly sensitive, and reproducible peptide maps.

429 Chromatographic Analysis and Separation of Short RNA Oligonucleotides with Novel Liquid Chromatography MethodsMirlinda Biba, Merck, PO Box 2000, MS: RY818-B218, Rahway NJ 07065, Christopher J, Welch, Joe P. Foley

Synthetic oligonucleotides have become increasingly important as part of new de-velopments in the use of antisense and small interfering ribonucleic acid (siRNA) as potential therapies for the treatment of various different diseases. The development of analytical methods for the sensitive and quantitative analysis and separation of oligonucleotides is an essential part for the advancement of this research area. A typical oligonucleotide therapeutic sample is a short RNA-based oligonucleotide with about 21-mer length (~7-8 kDa), and with possible chemical modifications pre-pared by chemical synthesis using an automated synthesizer. Due to their relatively large sizes compared to typical small-molecule compounds, oligonucleotides can be difficult to analyze with traditional analytical methods. There are many different analytical techniques reported for the analysis and separation of oligonucleotides, including capillary gel electrophoresis (CGE), anion-exchange high-performance liquid chromatography (AEX-HPLC), and ion-pair reversed-phase liquid chromatog-raphy (IP-RPLC). In these research studies, existing liquid chromatography meth-ods for the separation of oligonucleotides were evaluated for improved separations. New and novel approaches were also explored for more efficient analysis and sep-aration of oligonucleotides.

430 Case Study of Challenges in ADC Method DevelopmentGirija Krishnamurthy, Bristol-Myers Squibb, 519 Route 173, Bloomsbury, NJ 08804

No abstract submitted by the author.

431 iCE3: A Powerful Analytical Tool for Antibody Drug ConjugateJiaqi Wu, ProteinSimple, 27 Coronet Rd., Toronto, ON M8Z 2L8 Canada

Antibody-drug conjugates (ADCs) are a new class of therapeutic proteins. In an ADC, a targeted monoclonal antibody (mAb) is covalently linked to a cytotoxic small molecule. This allows for targeted drug delivery as the ADCs can selectively bind and kill tumor cells that over express the target antigen while limiting toxicity to normal tissues. Ion exchange chromatography is typically used to characterize the charge heterogeneity of therapeutic mAbs, however analysis of ADCs is problem-atic due to the small molecule drug and linker that increase their hydrophobicity compared to unconjugated mAbs. iCE3 is a powerful tool in the charge character-ization of ADCs. iCE3 performs free solution information exchange forum (IEF) in an open capillary column and the separation of the charge species is purely based

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on overall charge and isoelectric point differences. Because the iCE separation is based on overall charge in free solution it can analyze a wide variety of samples including ADCs. In addition, iCE has the advantages of high speed, high-throughput and quantitation with a typical throughput for ADCs is 4-5 injections per hour with the ability to run 100 analyses in a single batch. In this presentation, the principle of the iCE3 is reviewed and its application to ADCs is discussed.

432 Application of SPME in High-Throughput and In-Vivo Drug QuantificationJanusz Pawliszyn, University of Waterloo, 200 University Ave. W., Waterloo, ON N2L 3G1 Canada

In this presentation, an automated, high-throughput method based on thin-film sol-id-phase microextraction and liquid chromatography mass spectrometry is intro-duced for simultaneous quantitative analysis of 110 drugs chosen from ten classes and varying in physical and chemical properties. The developed solid-phase micro-extraction (SPME) method was optimized in terms of sorbent selection, extraction pH, ionic strength of the sample, washing solution, extraction and desorption times for analysis of urine, blood and saliva samples. Chromatographic separation was obtained in reversed-phase mode and detection was utilized with full scan orbitrap or triple quadrupole mass spectrometer. The developed method was validated ac-cording to the Food and Drug Administration (FDA) criteria, taking into account min-imum required performance level (MRPL) values provided by the World Anti-Doping Agency (WADA). The developed assay offers fast and reliable multi residue anal-ysis as an attractive alternative to the standard methods that are currently used in anti-doping laboratories. We also introduce an in-vivo solid-phase microextraction method, which combines sample preparation, metabolism quenching and extraction as well as eliminates sample collection as an approach to monitor graft function at different stages of the medical procedure related to organ transplantation. To en-sure the best analytical performance of the method, various aspects of the protocol were studied and optimized including selection of coating length, analyte coverage, transportation and storage conditions with particular attention paid to stability of the extracted compounds and convenience of the approach for clinical setup. The applicability of the developed method for determination of metabolic profile of the organs and for monitoring of drug metabolism was verified during lung and liver transplantation in pig models.

433 Investigations into Analytical Extractions Using Green SolventsDouglas Raynie, South Dakota State University, Department of Chemis-try and Biochemistry, Brookings, SD 57007

Extractions and related sample preparation procedures generally represent the larg-est generation of solvent waste in the analytical process. Over the past 15-20 years, solvent minimization as a way of avoiding waste has made great strides through the development of new techniques like supercritical fluid extraction, accelerated solvent extraction, microwave-assisted extraction, solid-phase microextraction, and single-drop extraction. But despite this reduction in solvent use, often the nature of the extracting solvent is overlooked. In this presentation, a variety of solvents and solvent alternatives are reviewed from the perspective of green chemistry. That is, the environmental and health considerations are discussed in four classes of solvent alternatives: 1) Conventional organic solvents. The American Chemical So-ciety’s Green Chemistry Institute Pharmaceutical Roundtable, and member compa-nies, has developed lists of solvent alternatives to conventional organic solvents. For example, ethyl acetate is generally preferred over dichloromethane. These solvent considerations are reviewed. 2) Supercritical fluids. As mentioned, super-critical fluids have become an established solvent alternative and are not thoroughly discussed. 3) Ionic liquids and deep eutectic solvents. These novel solvents have recently found their way into the analytical laboratory. However, because of viscosity and other concerns, their use as traditional solvents is somewhat limited, though utility as stationary phases or in unique experimental set-up is justified. 4) Green solvents. Natural products, such as limonene, or non-peroxide forming ethers are becoming more than a research novelty. As appropriate, these solvent classes are compared for use in the analytical extraction process.

434 Selective Extraction of DNA Using Magnetic Ionic LiquidsJared L. Anderson, University of Toledo, 2801 W. Bancroft St., Toledo, OH 43606, Kevin D. Clark, Omprakash Nacham, Honglian Yu

DNA extraction represents a significant bottleneck in nucleic acid analysis. Tradi-tionally, liquid-liquid extraction with phenol-chloroform was used for the purification of DNA from biological samples. Several adaptations to this method involving the addition of detergents to assist in the removal of proteins and polysaccharides have been made. However, the dependence of these protocols on organic solvents and often time-consuming centrifugation steps has resulted in the development of more environmentally benign techniques that are capable of high-sample throughput. This talk discusses the synthesis of hydrophobic magnetic ionic liquids (MILs) and their implementation as solvents for the rapid and efficient extraction of DNA from aqueous solution. The DNA-enriched MIL microdroplets were manipulated by appli-cation of a magnetic field. The three MILs examined in this study exhibited unique

DNA extraction capabilities when applied towards a variety of DNA samples and matrices. High extraction efficiencies were obtained for smaller single-stranded and double-stranded DNA using the benzyltrioctylammonium bromotrichloroferrate(III) MIL, while the dicationic 1,12-di (3-hexadecylbenzimidazolium) dodecane bis[(triflu-oromethyl)sulfonyl]imide bromotrichloroferrate(III) MIL produced higher extraction efficiencies for larger DNA molecules. The recovery of DNA from the MIL extraction solvent and its implications in downstream analysis including polymerase chain re-action (PCR) is discussed.

435 Advances in Sample Preparation for Dietary Supplements and Other Complex MatricesDerick Lucas, Agilent Technologies, 2850 Centerville Rd., Wilmington, DE 19808, Jerry Zweigenbaum, Bruce Richter, Irina Dioumaeva

Sample preparation is the essential first step of chemical analysis and is increasing-ly important with more chemically complex samples. Laboratories demand methods that can deliver cleaner samples, at the appropriate concentrations, in less time, with minimal cost and method development. The QuEChERS (quick, easy, cheap, effective, rugged, and safe) method addresses many of these sample preparation deficiencies; however, co-extracted matrix components in the form of lipids, sugars, proteins, and pigments can greatly affect instrument cleanliness and analytical re-sults. This work demonstrates the effectiveness QuEChERS provides for the prepa-ration of dietary supplements and advances in selective lipid removal from fatty sample extracts. Yohimbine and its numerous analogues were identified and quan-tified by liquid chromatography quadrupole time-of-flight and liquid chromatography triple quadrupole; providing excellent sensitivity (limit of quantitation = 0.1 ng/g), recovery (99% to 103%), and reproducibility (< 4% relative standard deviation). Re-sults indicated larger than expected amounts of yohimbine in some supplements while others showed undeclared alkaloids and evidence of synthetic adulteration with exogenous Yohimbine. Additionally, advances in selective lipid removal from lipid rich samples such as avocado, oils, and plasma are presented. These results indicated that matrix co-extractives were dramatically reduced over conventional cleanup methods without the loss of target analytes.

436 HPLC Method Development Strategies for Molecules with Multiple Chiral CentersMichael W. Dong, Genentech, 1 DNA Way, S. San Francisco, CA 94080, Kelly Zhang, Meenakshi Goel, Nik P. Chetwyn

Development of new chemical entities (NCE) with high chemical and chiral purity is a regulatory expectation in new drug development. Today, it is not unusual to have complex molecules with three or four stereogenic centers which show high affinity for disease targets. For the process development of these “multi-chiral” molecules, numerous analytical methods capable of separating all stereoisomers (enantiomers and diastereomers) must be developed rapidly to assess and control the stereo-chemistry of raw materials, intermediates and the final active pharmaceutical in-gredient (API). Achiral reversed-phase (RP) methods, used to assess the overall chemical purity assays, can typically be developed to monitor the diastereomeric content in a single run. In many cases, these achiral methods often become the primary quality control (QC) and stability-indicating purity assay methods. This pa-per describes the method development and QC strategies used for these complex multi-chiral drug molecules, which include: 1) adoption of a 3-pronged high-perfor-mance liquid chromatography (HPLC) method development template approach; 2) development of a single RP method using multi-segment gradients for determination of overall purity and diastereomeric content; 3) SFC for rapid initial screening for chi-ral separations; 4) two-dimensional liquid chromatography for peak purity determi-nation and achiral/chiral combinational assays. These strategies are described with actual examples used during clinical development of several new drug candidates.

437 Strategies of Advanced LC-MS-MS Screening and Development for Regulated BioanalysisJian Wang, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ 08543, Ryan Gu, Eliza Fung, Ang Liu, John Lute, Mark Arnold, Anne Aubry

It is important to optimize/select a liquid chromatography tandem mass spectrom-etry (LC-MS-MS) bioanalytical method that provides good sensitivity, accuracy, precision, ruggedness, and selectivity; the latter to avoid potential interference from metabolites or endogenous components. With the advance of technology and changing requirements from regulatory agencies in different regions, the strategy to screen bioanalytical assay conditions is also constantly evolving. This presentation discusses the current strategy of bioanalytical method screening and development in regulated bioanalysis at Bristol-Myers Squibb regarding the optimization of mass spectrometer, chromatography and extraction parameters. Comprehensive, but also fit-for-purpose, method screening and development ensures the data quality and meanwhile avoids unnecessary complexity in execution and data interpreta-tion. Two different LC optimization procedures are applied in various applications: 1) using standards of parent and metabolites to screen mobile phases/columns and optimize gradient followed by confirmation and further refining of the LC method using incurred samples; 2) using pooled incurred samples from the start to screen

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columns and mobile phases, and build a chromatography method. The strategy also considers the impact of mobile phase on MS-MS fragmentation and optimization, as well as including the rationale and procedure to maximize speed and resolution in rapid gradient LC-MS-MS bioanalysis.

438 Selected Ion Flow Tube Mass Spectrometry (SIFT-MS): Real-time, Sensitive Detection of Residual Solvents in PharmaceuticalsBarry J. Prince, Syft Technologies, 3 Craft Place, Christchurch, 8024 New Zealand, Daniel B. Milligan, Vaughan S. Langford, Chuck Renner

Fast analysis, high selectivity, high sensitivity, and elimination of chromatographic separation make selected ion flow tube mass spectrometry (SIFT-MS) an ideal tech-nique for rapid screening for solvent residues in pharmaceuticals. In this presenta-tion, the principles of SIFT-MS are presented together with data that demonstrate very effective analysis, rapid of residual organic solvents. Traditional analytical tech-niques, such as gas chromatography mass spectrometry (GC-MS), usually take 15 to 45 minutes to analyze samples due to use of chromatography. They also struggle to achieve good recoveries for very volatile compounds, including important solvents used in the pharmaceutical industry. SIFT-MS is a direct mass spectro-metric technique [1] that detects and quantifies volatile organic compounds (VOCs) and inorganic gases within seconds to low part-per-trillion levels.[2] Direct sample analysis is achieved without sample preparation, pre-concentration, or chromatog-raphy, because soft chemical ionization is utilized. High selectivity is maintained in SIFT-MS through application of three reagent ions that can be switched in 10 milli-seconds, markedly increasing the selectivity of SIFT-MS versus other direct mass spectrometry techniques. Analytical results compare well with those obtained using a regulatory GC-MS method at an accredited laboratory.[3]


[1] Smith, D., & Spanel, P. (2005). Mass Spec. Rev., 24, 661–700. [2] Prince, B.J., Milligan, D.B., & McEwan, M.J. (2010). Rapid Commun. Mass Spectrom., 24, 1763-1769.

[3] Langford, V.S., Graves, I., & McEwan, M.J. (2014) Rapid Commun. Mass Spec-trom., 28, 10–18.

439 Real-Time Dissolution Behavior of Indomethacin in Spray Dried Dispersion (SDD) in Biorelevant Media Using UV Surface Imaging TechnologySteven Ponce, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903, Xujin Lu, Christopher Zordan

Drugs with low solubility and high permeability are classified as Class II in which dissolution is the rate limiting step to drug absorption according with the Biopharma-ceutics Classification System. In order to increase the oral bioavailability of insoluble drugs several formulation strategies have been developed. One of these strategies includes the utilization of spray dried dispersion (SDD). An SDD formulation con-sists of active pharmaceutical ingredient (API) in a polymer matrix. SDD are formed when a crystalline or amorphous drug is co-dissolved in organic solution with a polymer, sprayed into very fine droplets, and the droplets are rapidly stripped of the solvent. Hydroxypropyl methylcellulose acetate succinate (HPMCAS) is one of the polymers commonly used in formation of SDD. This work uses the UV surface imaging system (ActiPix SDI300) to study the dissolution of SDD in traditional and biorelevant media. Indomethacin co-spray dried with HPMCAS was used as a mod-el drug. Samples with three different drug/polymer ratios were studied. Dissolution rate was measured in a flow cell at a flow rate of 0.2 mL/min and UV detection at 280 nm. UV images were collected across the solid liquid boundary of the samples to examine the drug diffusion. The swelling profiles from the matrix (HMPCAS) were also generated to assess the polymer effect on the drug release. High spatially and temporally resolved mapping of Indomethacin surface gradient concentration were achieved from the UV images. Results obtained from the study of the model drug are beneficial to SDD formulation development.

440 Understanding the Dynamic Process of Dissolution and Hydrolysis of Aspirin by ATR-FTIRVrushali M. Bhawtankar, Seton Hall University, 400 South Orange Ave., South Orange, NJ 07001

Dissolution studies are critical tests for measuring the performance of a drug prod-uct. In the past few years, the importance of the dissolution test has increased. Using in-situ attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy we developed a methodology of analyzing and monitor-ing dissolutions of pharmaceutical active pharmaceutical ingredients (APIs). The accuracy of this technique was found to be ± 3% relative to high-performance liquid chromatography and Ultra-violet. In this presentation we discuss a dynamic analysis of the dissolution and subsequent hydrolysis of aspirin by ATR-FTIR. This technique allows real time analysis of the behavior of aspirin under simulated physiological conditions (pH 1.2, 4.5, 6.8) as aspirin (1205 cm-1) and salicylic acid (1488 cm-1) are detected as separate and distinct peaks in the FTIR. This technique suggests a future potential for real-time studies of dissolution and hydrolysis of other pro-drugs.

441 Extraction of Volatile Organics Using Carbonnanotubes Immobilized Composite MembranesSmruti Ragunath, New Jersey Institute of Technology, 2211 Woodbridge Commons Way, Iselin, NJ 08830, Somenath Mitra

Extraction of volatile organics from air involves a broad range of techniques includ-ing the use of sorbents and cryogenics. Membrane extraction of volatile organics provides a wide range of selectivity and enrichment factor which can be effectively applied towards separation of organics from atmosphere. Carbon nanotubes (CNTs) were immobilized to the existing membrane surface to study the effect of nanotubes to enhance the separation process. Separations of organic species from atmosphere were compared using composite member and carbon nanotubes immobilized mem-branes. The effect of temperature, flow rate and concentration in reference to the flux attained were studied with both the membranes. A maximum enhancement of 94% was obtained for the separation of toluene using carbon nanotubes immobi-lized membrane, followed by 44% for ethanol and 22% for dichloromethane which can be used in effective separation of organics from the atmosphere.

442 The Introduction of PDMS-Overcoated Adsorbent Based Fiber CoatingsLen Sidisky, Supelco/Sigma-Aldrich, 595 North Harrison Rd., Bellefonte, PA 16823, Robert Shirey, Yong Chen

A new line of fibers that contain an overcoating-layer of a proprietary polydimethyl-siloxane (PDMS) over an adsorbent fiber coating has been developed. The primary advantage of adsorbent based fiber coatings is that the porous materials retain an-alytes more tightly than absorbent type fiber coatings. This analyte retention ad-vantage may become a disadvantage if the fiber has to be immersed in a complex matrix sample. The properties of adsorbent coatings that retain analytes tightly also may tightly retain nonvolatile components in the matrix. This can result in fouling of the fiber coating and shorten the lifespan of the fiber. By applying a layer of PDMS coating over the adsorbent coating, many of the large components in the matrix are repelled, while the smaller analytes of interest can pass through the outer PDMS layer and be retained by the adsorbent. This presentation compares the lifespan of overcoated fibers to adsorbent fiber coatings without the PDMS overcoat layer out of complex matrices such as fruit juices. The effect of the matrix on the lifes-pan of the coatings is demonstrated with pictures and data. The effectiveness of the overcoated fibers from a variety of matrices is presented. Because the PDMS overcoating slightly changes analyte uptake kinetics, data is shown that compare the differences. Also, we share some tips on how to use the overcoated fibers more effectively.

443 Comparison of Green Solvents during Chemical Extraction by Diffusion StudiesShanmugapriya Dharmarajan, South Dakota State University, 1424, 8th St., Apt. F, Brookings, SD 57006, Douglas E. Raynie

Safety, health, and environmental concerns of conventional solvents such as hex-ane and related hydrocarbons stimulate the use of green solvents in the extraction processes. Green solvents have been characterized for their low miscibility, high boiling point, and easy biodegradability to nontoxic products under environmental conditions, which makes them safer and less risky to human health and environ-ment. Oils have been extracted from soybeans using green solvents such as d-lim-onene, 2-methyltetrahydrofuran, cyclopentyl methyl ether, ethyl lactate, tert-butyl methyl ether, α–Pinene and deep eutectic solvents. The extractions are carried out using accelerated solvent extraction. We study the extraction kinetics with these green solvents and compare the experimental data to the hot-ball model. The per-formances of these green solvents compared to the conventional hazardous sol-vents in the extraction of soybean oil are validated.

444 Expanding Method Capabilities through Dynamic Sample PreparationKeith Griswold, Pepsico, 100 East Stevens Ave., Valhalla, NY 10595

Dynamic sample preparation is an approach used to provide flexibility and expand the capabilities of an existing validated method. Designing robust chromatographic analysis methods that function across a diverse portfolio of products presents many challenges. Avoiding method proliferation resulting from product development in-novations and new ingredient technologies is among a few. Mode of detection and conditions can be chosen to eliminate most identifiable interferences, but what mea-sures can be taken to prevent an existing method from failing when faced with new ingredients and formulations? Polymeric based mixed-mode solid phase extraction has been investigated as a possible technique for “difficult” sample matrices. The versatility available for exploiting analyte/matrix interactions allow for tunable selec-tivity, creating a great deal of method flexibility and, if required, enhanced sensitivity. To address some of the issues commonly associated with solid-phase extraction, a fully automated version of this process is currently being explored by PepsiCo R&D.

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November 20142014 EAS Abstracts

445 Extraction of Steroids from Water and Herbal Medicines Using QuEChERS Sample PreparationMichelle L. Schmidt, Seton Hall University, 400 South Orange Ave., South Orange, NJ 07079, Nicholas H. Snow

Quick, easy, cheap, effective, rugged, safe (QuEChERS) was an extraction tech-nique originally developed for the extraction of veterinary drugs from animal tissue by Anastassiades; however, its potential for the extraction of basic and polar com-pounds was quickly realized and it has since been primarily used for the extraction of pesticides from food products. QuEChERS is a liquid-liquid microextraction com-bined with a dispersive solid phase extraction cleanup and has been applied to a wide array of pesticides in a multitude of food matrices including fruits, vegeta-bles, teas, fatty food products, etc. Since its inception, different methods have been adapted to enable the analysis of not only basic pesticides, but acidic pesticides as well by the addition of citrate buffer. The overall methodology used including solvent, salts, buffers, and dispersive sorbents depends upon the target analyte(s). This presentation discusses an overview of QuEChERS and its methodology as it pertains to the extraction of steroids from water and herbal medicinal products. Herbal medicinal products are frequently adulterated with steroids in order to speed the healing process and thus it is necessary to develop an analytical method to de-tect the presence of these illegal adulterants. Optimization of the different parame-ters involved in the extraction process is discussed as well as the use of a depletion study for the determination of partition coefficients for the steroids of interest.

446 Single Reaction Chamber Microwave Digestion: Sample Prep for Better Trace AnalysisDavid Gunn, Milestone Inc., 25 Controls Dr., Shelton, CT 06484, Njies Pedjie

The demand for a simplified approach for trace metals analysis is at an all-time high with the development of new regulations, the complexity of new materials and the lower limits of detection. Current sample preparation has challenges with the wide variety of sample types, including United State Pharmacopeia (USP) pharma-ceutical matrices, highly organic samples and difficult inorganic materials. Single reaction chamber (SRC) microwave technology has been utilized in a cross-section of these industries with significant improvements in temperature and pressure ca-pability, digestion quality, and productivity. Trace metal analysis data following new sample preparation techniques for the current USP 232/233 methods along with highly organic samples are discussed.

447 Improved Bonding of Composites, Polymers and Coatings by Monitoring Surface Preparation with Handheld FTIR SpectroscopyJohn Seelenbinder, Agilent Technologies, 14 Commerce Dr., Danbury CT 06795

Surface preparations are key to both adhesive bonding and coating applications. Without the proper preparation, bond strengths deteriorate and performance falls below acceptable levels. Additionally, there are few ways of inspecting the bond after it has cured. These factors lead to a situation where surface testing is re-quire insuring that bonding surfaces have been prepared correctly. Infrared (IR) spectroscopy has long been known to be an effective surface analysis technique. Infrared is sensitive to many of the chemical changes which preparations impart to the surface. Additionally, IR’s specificity allows for easy identification of organic ma-terials, making it especially suitable for detection of surface contamination. Recent advances in the miniaturization of Fourier transform infrared (FTIR) spectrometers have produced a new range of handheld, non-destructive instruments. These instru-ments now allow qualification of surface preparations prior to bonding applications. Handheld use allows these measurements to be truly non-destructive, allowing for increased inspection of the actual surfaces being bonded. We’ll demonstrate meth-ods developed to verify several surface preparations including plasma treatment prior to bonding, evaluation of UV damage prior to coating and evaluation of surface contamination by non-volatile residue. This talk features both new applications and the latest advances in handheld FTIR technology.

448 Non-Contact Material Characterization Using Mid-Infrared Quantum Cascade Laser SpectroscopyMark Norman, Block Engineering, 377 Simarano Dr., Ste. 130, Marlborough, MA 01752, Anish Goyal

We review the applicability of mid-infrared (IR) quantum cascade laser (QCL) spec-troscopy to characterize coatings on a variety of surfaces as well as bulk materi-als. This includes characterization of surface cleanliness prior to coating, chemical composition identification, curing validation, adhesion verification, and coating thick-ness determination. Such testing has traditionally been performed using laborato-ry-based Fourier transform infrared (FTIR) spectrometers, but advances in laser and opto-mechanical technologies are enabling the field testing of coatings using non-contact and non-destructive methods in a hand-held manner. We discuss the pros and cons of various measurement modes including specular reflectance, dif-fuse reflectance, and attenuated-total reflectance.

449 Portable Raman Spectroscopy Opens the Door for Site AnalysisKatherine Bakeev, B&W Tek, 19 Shea Way, Newark, DE 19713, Dawn Yang, Philip Zhou

The portability of Raman spectrometers broadens the reach of this tool for diverse applications in analysis of materials from the ancient past, not to mention through the modern era and even into the future. The ability to perform nondestructive anal-ysis on archaeological samples without removing them from the site of discovery helps to preserve the artifacts while giving us the information that we seek, showing the flexibility of portable Raman. This flexibility can also prove useful in the fields of minerals and mining, such as in the study of the nanomaterials such as carbon nanotubes.

450 Repair of CFRP Composite with Localized Incipient Thermal Damage Guided by Handheld FTIR InspectionTucker Howie, University of Washington, Roberts Hall, Box 352120, Seattle, WA 98195, Ashley Tracey, Brian Flinn, Paul Vahey, Paul Shelley

Incipient thermal damage (ITD) of carbon-fiber-reinforced polymer (CFRP) com-posites cannot be detected by common nondestructive evaluation (NDE) methods, such as ultrasound or visual inspection. ITD can reduce matrix dominated mechan-ical properties such as short beam strength (SBS) of composites and thus requires detection to insure the integrity of composite structures that may be subjected to thermal exposures. Fourier transform infrared (FTIR) spectroscopy is an estab-lished laboratory technique that shows promise for NDE of ITD. The purpose of this research was to determine the effectiveness of using a handheld ExoScanTM FTIR for detecting localized heat damage in a panel. Calibration models were de-veloped by using a partial least squares (PLS) to correlate SBS measurements from coupons with a range of thermal exposures to FTIR spectra. The panels were 24-ply unidirectional 177 °C cure carbon fiber reinforced epoxy laminates. Localized heating was performed using a heat blanket coupled with multiple layers of insula-tion in the center of the panels. Cured panels were subjected to a 1 hour post-cure exposure at peak temperatures of 227, 241, and 254 °C. The FTIR was used to perform ply-by-ply inspection during the scarfed repair process to remove remain-ing ITD material. From the inspection a three-dimensional map was generated to evaluate progression of damage through the thickness direction of the panel. The ExoScanTM FTIR was found capable of guiding a repair inspection for ITD; how-ever, performance could be improved by obtaining a better match of surface finish between the calibration standards and samples.

451 What’s in your “Natural” Supplement? Application of NMR Spectroscopy to Natural ProductsJimmy Yuk, Bruker BioSpin, 15 Fortune Dr., Billerica, MA 01821, Mark Garvey, Christian Fischer, Kim Colson

Natural products such as herbal remedies have been increasing in sales with annual revenue of 5.11 billion dollars in the United States alone. This has raised concerns as surges in adulteration of natural products with other materials have been detect-ed in the market. In some cases, this is due to accidental species misidentification, but in other cases, non-disclosed materials were added to increase product efficacy. This illegal practice can be detrimental to the natural product industry and extremely dangerous to the health of the consumer. Due to the diversity and complexity of chemical compounds within botanical materials, a demand exists for methods to understand the composition of natural products for proper quality control and au-thentication. In this study, a nuclear magnetic resonance (NMR)-based screening tool was applied to natural products. Targeted and non-targeted NMR screenings for adulterated dietary supplements are described. This approach shows the si-multaneous analysis of natural products which involves: 1) Identification of known components, impurities and adulterants in reference spectrum database; 2) quan-tification using a calibrated NMR spectrometer; and 3) multivariate modeling for product classification. With the increasing demand for robust high-throughput an-alytical methods, an automated NMR-based screening tool is a powerful approach for validating the identity, purity, strength, and composition of many natural products.

452 Enabling Strategies and Platforms for NMR Metabolite CharacterizationsJanet Caceres Cortes, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ 08540, Xiaohua Huang, Kim Johnson, Sarah Traeger, Haiying Zhang, Xiaohong Liu, Yue-Zong Shu, Vikram Roongta, Michael Reily

Truly understanding the efficacy and safety profile of drugs and their metabolites places a heavy emphasis on metabolite identification (ID) in the research and de-velopment process, so much so that over time, metabolite ID has garnered attention at earlier stages within the drug discovery-development continuum. Liquid chroma-tography mass spectrometry (LC-MS) based approaches are the front-line analyt-ical tool to analyze metabolites from biological matrices in an efficient time frame. Nuclear magnetic resonance (NMR) spectroscopy has also become an important technique in support of metabolite ID, particularly when challenges arise in precise-ly identifying the exact site and isomeric consequences of metabolic modifications using LC-MS. In this presentation, we discuss two key challenges for metabolite

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November 20142014 EAS Abstracts

identification by NMR. It is well known that low amounts of isolated desired metabo-lites as well as contaminants arising from incubation matrices, coeluting metabolites and solvents are major impediments to NMR metabolite ID analyses. Strategies that address the challenges of isolating sufficient amounts of metabolites with high purity for speedy NMR analysis will be discussed. Another challenge for metabolite ID is the issue of sensitivity since critical characterizations may need to be carried out on high nanogram-low microgram material. Whereas the most common NMR platform utilizes room temperature probes and sample volumes of 180 or 600 mi-croliters, they are not well-suited for the analysis of metabolites in these low mi-crogram quantities. This presentation provides selected examples of the benefit of utilizing nitrogen cryocooled probe technology, as well as reducing sample volumes to between 7 and 40 microliters in specially designed room temperature and helium cryogenic NMR probes.

453 New Methods for the Simultaneous Measurement of Homo- and Heteronuclear Coupling ConstantsJosep Sauri, Merck, 126E. Lincoln Ave., Rahway, NJ 07065, Teodor Parella

A long-range optimized P.E. heteronuclear single quantum multiple bond correlation experiment, named P.E.HSQMBC, is proposed for the simultaneous measurement of a complete set of homo- and heteronuclear coupling constants from a single two-dimensional (2-D) cross-peak. The sign and the magnitude of proton–proton coupling constants are measured along the direct dimension from the relative E.CO-SY-type multiplet pattern displacement generated thanks to the passive one-bond coupling constant; whereas long-range proton–carbon coupling constants are inde-pendently determined in the detected dimension thanks to the In-phase-anti-phase (IPAP) methodology. On the other hand, it will also be shown how using a 13C Boltz-mann polarization instead of the regular insensitive nuclei enhanced by polarization transfer (INEPT)-based 1H Boltzmann polarization in the original P.E.HSQC exper-iment, the C-HA cross-peak corresponding to a diastereotopic CHAHB methylene spin system, which exhibits a characteristic 1:0:1 multiplet pattern along the indirect dimension of a F1-coupled HSQC spectrum, makes visible the central lines of this multiplet pattern. Therefore, a spin-state-selective method is proposed for the ef-ficient measurement of both 1J(CHA) and 1J(CHB) along the indirect dimension of a 2-D spectrum as well as to the magnitude and the sign of the 2J(HAHB) coupling constant from the straightforward analysis of a single four-component E.COSY cross-peak. The extraction of 1J(CH) values for CH and CH3 multiplicities is also performed from the same spectrum. The success of the method is also illustrated for the determination of residual dipolar 1D(CH) and 2D(HH) coupling constants in a small molecule weakly aligned in a polymethyl-methacrylate swollen gel.

454 Experiment Approaches to Probe Long-Range H-X Correlations in 2-D NMR SpectroscopyRonald Crouch, NMR Consultant, 1609 S. Del Norte Ave., Loveland, CO 80537, Craig Butts

An overview of both historical and current sequence techniques for efficient obser-vation of long-range H-X coupling pathways is presented. Both heteronuclear multi-ple-quantum correlation (HMQC) and recent powerful heteronuclear single quantum coherence (HSQC)-derived experimental approaches are discussed and compared. The importance of phased spectra in F1 and in some instances both F1 and F2 when high-resolution is required is a key requirement for quality data. Strategies to obtain high F1 resolution using both F1 band-selection and non-uniform-sampling can afford a tremendous savings of time and some guidance to the use of these strategies are discussed.

455 What can Non-Destructive Micro Multi-Elemental Analyses of Counterfeit Banknotes Reveal? Gene S. Hall, Rutgers University, Department of Chemistry, 610 Taylor Rd., Piscataway, NJ 08854

Banknote counterfeiting in the United States has a long history starting during the issues of Early American currency. This currency had security features such as mica that the papermaker added to the paper pulp. During the Revolutionary War, the British counterfeited this paper currency but did not use the same mica. Energy dis-persive X-ray fluorescence (EDXRF) is a multi-elemental technique that is non-de-structive and provides feature rich qualitative and quantitative information about the bulk. However, if a micro X-ray beam (50-microns) is used to induce X-ray emission, multi-elemental analysis can be used to analyze specific features such as individual printed or written letters, embedded security features, and natural imperfections in the banknote. Then, by operating the EDXRF spectrometer in the scanning mode, elemental imaging can reveal mineral composition and formulations of the printing or written ink. This memorial presentation will provide new insights into multi-ele-mental composition of counterfeit Confederate States of America (CSA) and obso-lete banknotes. Also presented will be elemental imaging of contemporary United States and British counterfeit banknotes to reveal the power of multi-elemental im-aging to uncover printing secrets of the modern-day counterfeiter. Instrumentation,

data processing, and data interpretation are also presented with specific examples from real-world samples from my personal collection. Characterization of colonial currency from New Jersey illustrates the power of micro multi-elemental analyses.

456 TBAJohn Scott, New York Conservation Foundation, 261 Fifth Ave., Rm 2000, New York, NY 10016

No abstract submitted by the author.

457 Emerging Designer Drugs: The New Frontier for Microcrystalline TestingThomas A. Brettell, Cedar Crest College, Forensic Science Program, Department of Chemistry and Physical Sciences, 100 College Dr., Allentown, PA 18104

Microcrystalline testing is based on chemical-precipitation reactions that form crys-tals observed by microscopy. Most of the tests were developed in the late nine-teenth century. Over the years, they have been modified to aid in the identification of controlled substances and offer a low-cost alternative to GC/MS and FTIR. The Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) has estab-lished criteria for their use in the identification of controlled substances. Additionally, the American Society of Testing and Materials (ASTM) have established methods for identifying cocaine, methamphetamine and phencyclidine using microcrystalline testing. A wide variety of new psychotropic substances have emerged over the past few years. These new emerging designer drugs are challenging traditional forensic methodologies that have been in place for many years. Most of these new drugs have never been seen in crime laboratories and hence forensic scientists are in need of new identification methods. Microcrystalline testing can offer the drug ana-lyst an alternative for identification of closely related structures of these substances. The presentation will give an overview of these new emerging drugs and a summary of the present state of microcrystalline testing in the crime laboratories. The talk will highlight some recent developments and the opportunity for new research.

458 Microscopy and Microanalysis Applied to PharmaceuticalsJohn A. Reffner, John Jay College, 524 West 59th St., New York, NY 10019

Hal Ferrari was a member of the greatest generation a pharmacist and an analyt-ical chemist with a devotion to family, baseball, EAS and microanalysis. He joined Lederle Laboratories in the early 1950’s and spent 37 years there as an analytical chemist. Hal was a cherish colleague and friend. When I first met Hal, he was a senior research chemist at Lederle. Later, I joined American Cyanamid I had the opportunity to work with Hal on various projects. We shared our interest in micro-analysis and its application to pharmaceuticals. In Fritz Pregl’s acceptance of the 1923 Nobel Prize in Chemistry he concluded, “I therefore am very hopeful that in the future quantitative organic microanalysis will find many more fields of application and expansion, and that it will make possible much insight and discernment which otherwise might not have been possible, especially following the magnificent rec-ognition that has been awarded to it by the Swedish Academy of Sciences.” (From Nobel Lectures, Chemistry 1922-1941, Elsevier Publishing Company, Amsterdam, 1966) Dr. Pregl’s vision inspired many scientists to follow his lead and it lead to many advances in pharmaceutical science. Thinking small continues to produces large changes in analytical chemistry.

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