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SUPPLEMENT TO

THE

APPLICATION NOTEBOOK

February 2012

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THE APPLICATION

NOTEBOOK

Biological

10 Separation of Heat-Degraded Recombinant Human

EPO Protein

Phu Duong, Agilent Technologies, Inc.

11 Scherzo SS-C18’s Novel Multi-Mode Stationary Phase

Shows Improved Retention and Resolution

of Neurotransmitters

Bhavana Verma and Itaru Yazawa,

Imtakt USA (formerly Silvertone Sciences)

12 Improving Intact IgG Separations with Aeris WIDEPORE

Core-Shell HPLC–UHPLC Columns

Michael McGinley, Deborah Jarrett, and Jeff Layne, Phenomenex Inc.

13 Using Longer Aeris PEPTIDE Core-Shell HPLC–UHPLC

Columns for Improved Peptide Mapping

Michael McGinley, Deborah Jarrett, and Jeff Layne , Phenomenex Inc.

14 Analysis of Saccharides by Hydrophilic Interaction

Chromatography (HILIC) Using TSKgel® NH2-100 Columns

Atis Chakrabarti, Tosoh Bioscience

16 Guidelines for Routine Use and Maintenance of

Ultra Performance Size Exclusion and Ion Exchange

Chromatography Systems

Paula Hong and Kenneth J. Fountain, Waters Corporation

THE APPLICATION NOTEBOOK – FEBRUARY 20124 Table of Contents

18 Analysis of Paralytic Shellfish Toxins

Saji George, Maria Ofitserova, and David Mazawa,

Pickering Laboratories, Inc.

19 Agarose-Heated Transfer Lines

Wyatt Technology Corporation

Chiral

20 Use of Amine Additive in the Sample Diluent in a

Supercritical Fluid Chromatography (SFC) Purification

Natascha Bezdenejnih-Snyder, Valerie Hoesch, and Nancy DeGrace,

AstraZeneca R&D Boston, Infection IMED

Environmental

21 One-Step Extraction and Concentration for Identifying

Pharmaceuticals and Personal Care Products in Water,

Biosolids, and Solids

FMS, Inc.

22 Screening for Pyrethroid Insecticides in Sediment

Samples by GC–MS-MS

Ed George, Bruker Daltonics, Chemical Analysis Division

24 Enhancing Recoveries and Maximizing Throughput

of Semi-Volatile Organics Using the Direct-to-GC-Vial

Concentrator Tube and DryVap® Concentrator System

Michael Ebitson and David Gallagher, Horizon Technology, Inc.

25 Microwave Assisted Extraction of Dioxins and Furans

Compared to Soxhlet

Melissa Lightner, Milestone Inc.

26 Purge-and-Trap GC Analysis of Methane in Water Samples

Associated with Hydraulic Fracturing

Laura Chambers, Gary Engelhart, and Scott Hazard, OI Analytical

THE APPLICATION NOTEBOOK – FEBRUARY 2012 Table of Contents 5

27 Fast Cap IC Determinations of Inorganic Anions and

Cations in Drinking Water

Fei Pang, Terri Christison, and Khalil Divan, Thermo Fisher Scientific

28 Rapid On-Site Screening of Environmental VOCs in Soil

Using Solid Phase Microextraction and a Person-Portable

GC–MS

Tiffany C. Wirth, Charles S. Sadowski, and Douglas W. Later, Torion

Technologies Inc.

Food and Beverage

29 Comparison of BTEXS in Olive Oils by Static and

Dynamic Headspace

Roger Bardsley, Teledyne Tekmar

30 A Variety of Agilent ZORBAX RRHD Phases Offers

Selectivity Options for the Determination of

Anthocyanins in Blueberries with UHPLC/MS

Anne E. Mack, Agilent Technologies, Inc.

32 Chromatography Methods for Determining

Carbohydrates in Coffee

Lipika Basumallick and Jeffrey S. Rohrer, Thermo Fisher Scientific

33 Impurities in Wines by GC–MS

Benedicte Gauriat-Desroy, Eric Phillips, Stacy Crain, and Trisa Robarge (with

special thanks to members of OEnologic Center of Grezillac),

Thermo Fisher Scientific

34 Quantitation of Omega Fatty Acids by HPLC and

Charged Aerosol Detection

Marc Plante, Bruce Bailey, and Ian Acworth, Thermo Fisher Scientific

35 Faster, More Sensitive Determination of Carbamates in

Drinking Water

Chen Jing, Xu Qun, and Jeffrey S. Rohrer, Thermo Fisher Scientific


36 Determination of Dyes in Fish Tissue by HPLC/UV

Daniel A. Fonseca, Brian Kinsella, Thomas August, and Craig A. Perman,

UCT, LLC

Pharmaceutical

37 New Generation Monolithic Silica Columns for Fast, High

Resolution Drug Separations without High Pressures

Karin Cabrera and Egidijus Machtejevas, Merck Millipore

38 New HPLC/UHPLC Assay Methods for Impurities

in Tetracycline

Suparerk Tukkeeree and Jeffrey S. Rohrer Thermo Fisher Scientific

39 LC–MS-MS Method for the Determination of Enalapril and

Enalaprilat from Human Plasma Using SOLA

Ken Meadows, Thermo Fisher Scientific

40 Liposome Characterization by FFF–MALS–QELS

Wyatt Technology Corporation

Polymer

41 Analysis of Over 15 Million Dalton Polymers by Aqueous

SEC Method

Kanna Ito, ShodexTM/Showa Denko America Inc.

General

42 Analysis of Multiple Classes of Cigarette Smoke

Constituents by GC×GC-TOFMS

Elizabeth Humston-Fulmer and Joe Binkley, LECO Corporation

43 UHPLC Column Protection Dramatically Extends UHPLC

Column Performance and Lifetime

J.T. Presley III, Tom Cleveland, and Jeff Layne, Phenomenex, Inc.

THE APPLICATION NOTEBOOK – FEBRUARY 2012 Table of Contents 7

44 Resistive Glass Inlet Tubes Increase Ion Throughput

Paula Holmes, PhD and Bruce N. Laprade, Photonis USA

45 Improved Internal Diameter Control of Tubing Used for

Gas Chromatography

Joe Macomber and Roland Fischer,

Polymicro Technologies, a Subsidiary of Molex

46 HPLC Method for the Separation of Nine Key Components

of Milk Thistle

Dave Bell, Supelco/Sigma-Aldrich

Articles

47 Trace Metabolic Profiling and Pathway Analysis of

Clomazone Using LC–MS-MS and High-Resolution MS

Wei Zou, Hagai Yasuor, Albert J. Fischer, and Vladimir V. Tolstikov

55 Establishing USP Rebaudioside A and Stevioside

Reference Standards for the Food Chemicals Codex

Yi Dang, Jeffrey Moore, Gloria Huang, Markus Lipp, Barbara Jones, and

James C. Griffiths

Departments

59 Call for Application Notes

Cover Photography: Getty Images


THE APPLICATION NOTEBOOK – FEBRUARY 2012 9

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Director of Content Peter Houston

THE APPLICATION NOTEBOOK – FEBRUARY 201210 Biological

Erythropoietin protein (EPO) is a glycoprotein hormone

found in plasma. It is a cytokine for erythrocyte (red blood

cell) precursors in the bone marrow. EPO controls red blood cell

production, and has neuroprotective activity against a variety of

potential brain injuries and antiapoptotic functions in several tis-

sue types. Recombinant human EPO protein (rEPO) is produced

by Chinese hamster ovary (CHO) cells using recombinant DNA

technology. rEPO is one of the most widely produced proteins

worldwide for therapeutic agents.

Te HPLC separation of EPO protein from its impurities can

be achieved by using a variety of chemistries, including reversed-

phase chromatography. In this example, we separated heat-de-

graded, CHO-derived EPO protein using an Agilent ZORBAX

300SB-C18 1.8 μm column on an Agilent 1290 Infinity LC

system.

Experimental

Recombinant human EPO protein was heated at 60 °C over-

night (16 h) at neutral pH (pH 7.0) and acidic pH (pH 4.0).

Due to its nature, rEPO protein will be degraded or form other

isoforms when heated at high temperatures such as 60 °C at

different pH. At neutral pH, rEPO forms limited isoforms,

but when acidic pH conditions are used, the structure of rEPO

protein will be altered significantly. Te lower the pH, the greater

the change (1).

Column: Agilent ZORBAX RRHD 300SB-C18,

2.1 × 50 mm, 1.8 μm

Sample: Recombinant human EPO protein (rEPO)

Sample conc: 1.0 mg/mL

Injection vol: 3 μL

Flow rate: 1.0 mL/min

Pressure: 650 bar

Mobile phase: A 0.1% TFA in deonized water; B 0.01%

TFA in 100% ACN

Gradient: 5 to 100% B from 0 to 2.5 min

Detector: UV, 280 nm

System: Agilent 1290 Infinity HPLC

Results and Discussion

Figure 1 shows separations obtained at the same temperature but

different pH. Panel A shows data from a sample heated at neu-

tral pH. Te column resolved the main peak of rEPO from its

degraded products or isoforms very well.

Panel B shows the separation of heat-treated rEPO at pH 6.0.

Te separation conditions were the same as for the neutral pH

trial but the chromatogram was drastically changed. Heating with

acidic conditions can greatly alter and degrade the conforma-

tion of the rEPO protein. Te retention time of the major peak

changed from 1.49 min to 1.69 min and the peak also contained a

shoulder peak on the right. In addition, more impurity peaks also

were observed. Tis clearly indicated that the ZORBAX RRHD

300SB-C18, 1.8 μm column could resolve rEPO impurities and

degradation products very well, and can be used for monitoring

the stability of rEPO.

Reference

(1) Yoshiyuki Endo et al., J. Biochem. 112(5), 700–706 (1992).

Agilent Technologies, Inc.5301 Stevens Creek Blvd., Santa Clara, CA 95051

(800) 227-9770 (Directory), fax (866) 497-1134

Email: [email protected]

Website: www.agilent.com/chem/bioHPLCproteins

0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 min

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A Heated at 60 °Cat pH 7.0

Heated at 60 °Cat pH 6.0

1.492

0.464

1.838

1.688

1.787

1.864 2.568

Figure 1: Heat-treated rEPO protein well resolved by the Agilent ZORBAX RRHD 300SB-C18, 2.1 × 50 mm, 1.8 μm column.

Separation of Heat-Degraded Recombinant Human EPO Protein Phu Duong, Agilent Technologies, Inc.

THE APPLICATION NOTEBOOK – FEBRUARY 2012 Biological 11

S imultaneous HPLC analysis of neurotransmitters can be challeng-

ing due to the similarity of their structures. Furthermore, since

neurotransmitters are small polar molecules, they can be diTcult to re-

tain on a standard C18 column. Imtakt’s multi-mode Scherzo SS-C18

column overcomes these challenges through the innovative nature of its

stationary phase. fie Scherzo SS-C18 stationary phase contains a large

amount of strong ionic ligands, which enables the column to retain po-

lar ionic compounds better than a standard C18 column (see Figure 1).

Experimental

All data was generated with a semi-micro HPLC system equipped with UV or ELS detection.

Separation of 13 key neurotransmitters was performed using gradi-

ent elutions on Scherzo SS-C18 (150 × 3 mm) and Unison UK-C18

(150 × 3 mm) HPLC columns (see Figure 2). fie Scherzo SS-C18

multi-mode column separated the neurotransmitters in well resolved

peaks within a run time of 25 min. fie more traditional C18 col-

umn, Unison UK-C18 column, could not retain these compounds

suTciently, as the peaks were clustered together and eluted close to the

void volume. fie Scherzo SS-C18 multi-mode column outperformed

the more traditional C18 column.

More targeted separation of monoamine/amino acid neurotrans-

mitters was performed using isocratic elution on a Scherzo SS-C18

column of dimensions 250 × 3 mm (see Figure 3). fiis data shows

baseline separation of neurotransmitters that have similar structures,

which would be diTcult to accomplish on a traditional C18 column.

All peaks were well resolved, and the use of ammonium phosphate buf-

fer resulted in sharper peaks than did the use of phosphoric acid buffer.

Conclusions

The new multi-mode Scherzo SS-C18 column showed better sep-

aration of neurotransmitters than a standard C18 column. Thir-

teen key neurotransmitters can be separated simultaneously with

this column without any coelution. Moreover, neurotransmitters

of similar structure, such as L-adrenaline and L-noradrenaline,

are well resolved on this column. The unique ionic/hydropho-

bic stationary phase on the Scherzo SS-C18 provides a better

alternative to scientists seeking to retain and resolve small polar

neurotransmitters.

Imtakt USA

6703 Germantown Avenue, Suite 240, Philadelphia, PA 19119

tel. (888) 456-HPLC, (215) 665-8902, fax (501) 646-3497

Website: www.imtaktusa.com; Email: [email protected]

Figure 2: Scherzo SS-C18 simultaneously separates 13 key neu-rotransmitters

Figure 3: Scherzo SS-C18 retains monoamine/amino acid neu-rotransmitters.

Figure 1: Three novel multi-mode stationary phases, SS-C18 far left.

Scherzo SS-C18’s Novel Multi-Mode Stationary Phase Shows Improved Retention and Reso-lution of NeurotransmittersBhavana Verma and Itaru Yazawa, Imtakt USA (formerly Silvertone Sciences)


A new widepore core-shell HPLC–UHPLC column (Aeris WIDEPORE) has been introduced that is speciTcally de-

signed to improve protein separations. Rather than utilize a similar morphology of small molecule core-shell columns with larg-er pores, a completely difierent particle was developed that takes into account the slower difiusion of proteins into porous particles. When one looks at difierent intact protein separa-tions, the resolution of difierent glycoforms of therapeutic IgG antibodies probably stands out as one of the more diffcult due to the large size and structure of IgG. In this article improved separation of IgG is shown using Aeris core-shell HPLC–UHPLC columns.

Materials and Methods

All chemical, standards, and antibodies were obtained from Sigma Chemical (St. Louis, Missouri). Solvents were purchased from EMD (San Diego, California). Fully porous 5 μ 300 Å C18 columns and core-shell Aeris 3.6 μm WIDEPORE XB-C18 columns (100 × 4.6 mm) were obtained from Phenomenex (Torrance, California). Mouse Immunoglobulin IgG samples were analyzed on an Agilent 1200 HPLC system with autosam-pler, column oven, solvent degasser, and UV detector set at 214 nm. Data was collected using Chemstation software (Agilent, Santa Clara, California). Mobile phases used were 0.1% TFA in water (A) and 0.1% TFA in acetonitrile and a gradient from 10 to 40% B in 15 min was used at 1 mL/min. Column was main-tained at 80 °C.


Aeris was designed to maximize resolution of proteins greater than 10 kilodaltons molecular weight regardless of HPLC (or UHPLC) system used. A thin porous shell minimizes protein peak band spreading due to difiusion in and out of the porous layer and a larger particle size reduces column backpressure allow-ing for the use of longer columns for increased resolution. �e re-sult is a column with performance on par or better than sub 2 μm widepore fully porous media at backpressures signiTcantly lower than 3 μm fully porous columns. �e performance advantage of Aeris WIDEPORE core-shell columns is demonstrated in Figure 1 where an Aeris column is compared to a 5 μm fully porous widepore column. IgG immunoglobulins are considered diffcult proteins to separate by reversed phase LC due to their large size (150 KDa) and hydrophobicity. Typically, elevated column tem-peratures and isopropanol mobile phase are required to improve

recovery and resolution. In this example mouse immunoglobulin IgG is compared on each column using an acetonitrile-only mo-bile phase at 80 °C (Aeris is stable to 90 °C). Note the signiT-cantly narrower peak width for the Aeris WIDEPORE core-shell column resulting in the resolution of the three main glycoforms of IgG versus the fully porous columns where only two compo-nents are seen. Of additional note is the greater recovery for the Aeris column; low hydrophobicity and good inertness result in greater recovery for hydrophobic proteins. �e good resolution and recovery for this application demonstrate the utility of using core-shell Aeris WIDEPORE columns for immunoglobulin and large protein separations.

Improving Intact IgG Separations with Aeris WIDEPORE Core-Shell HPLC–UHPLC Columns Michael McGinley, Deborah Jarrett, and Jeff Layne, Phenomenex Inc.

Phenomenex Inc.411 Madrid Avenue, Torrance, CA 90501

tel. (310) 212-0555; (310) 328-7768

Website: www.phenomenex.com

Figure 1: Comparison between a fully-porous 5 µm 300 Å C18 column (top chromatogram) to an Aeris 3.6 µm WIDE-PORE XB-C18 column (bottom chromatogram) for a mouse Ig-G sample. Note the improved resolution and greater pro-tein recovery for the Aeris wide-pore core-shell column.

mAU20

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Anew 3.6 μm 100 Å HPLC–UHPLC column (Aeris PEP-TIDE) has been introduced that is speciTcally designed to

improve separations of peptide and peptide mapping applications. fie Aeris PEPTIDE XB-C18 column was developed to comple-ment Aeris WIDEPORE XB-C18 core-shell columns for protein characterization. When one looks at peptide mapping applica-tions, performance requirements are signiTcantly different versus intact protein separations, as increased retention and selectivity are required to separate the large number of peptides generated in pep-tide mapping applications. Because increased resolution is a higher priority versus speed, a larger particle (3.6 μm) core-shell particle was developed allowing the use of longer columns at lower back-pressures. In this application the increased resolution that longer Aeris PEPTIDE 3.6 μm XB-C18 provide will be demonstrated.

Materials and Methods

All chemicals, standards and antibodies were obtained from Sigma Chemical (St. Louis, Missouri). Solvents were purchased from EMD (San Diego, California). Core-shell Aeris PEPTIDE 3.6 μm XB-C18 columns (150 × 4.6 mm and 250 × 4.6 mm) were obtained from Phenomenex (Torrance, California). Bovine serum albumin was digested with trypsin and analyzed on an Agi-lent 1200 HPLC system with autosampler, column oven, solvent degasser, and UV detector set at 214 nm. Data was collected using Chemstation software (Agilent, Santa Clara, California). Mobile phases used were 0.1% TFA in water (A) and 0.1% TFA in acetonitrile and a gradient from 3 to 65% B in 15 min was used at 1.2 mL/min. Column was maintained at 40 °C.


Aeris PEPTIDE 3.6 μm XB-C18 core-shell particles demonstrate similar or better performance as sub-2 μm fully-porous columns at a fraction of the backpressure, allowing the use of longer columns at backpressures compatible with existing HPLC systems. fie 3.6 μm core-shell media is of particular utility for peptide map applications where the increased resolution of longer columns is desired (for high-speed UHPLC applications the Aeris PEPTIDE 1.7 μm XB-C18 can be used instead). An example of the utility is demonstrated in Figure 1 where 150 × 4.6 mm and 250 × 4.6 mm Aeris PEPTIDE 3.6 μm XB-C18 columns were com-pared for a peptide map of BSA. fie 150 × 4.6 mm column pro-vides excellent separation of the peptide mixture at a low column backpressure (140 bar at 1.2 mL/min) such that a longer column could be used to achieve additional resolution if required. When the 250 × 4.6 mm Aeris PEPTIDE 3.6 μm XB-C18 column was used for the separation, additional peptides were resolved while still at a backpressure amenable to using standard HPLC systems (200 bar at 1.2 mL/min). fiese results demonstrate the perfor-mance advantage and utility of the Aeris PEPTIDE 3.6 μm XB-C18 media for highly complex peptide mapping mixtures where one can utilize different column lengths to optimize resolution and separation time based on the needs of a speciTc application.

Using Longer Aeris PEPTIDE Core-Shell HPLC–UHPLC Columns for Improved Peptide Mapping Michael McGinley, Deborah Jarrett, and Jeff Layne, Phenomenex Inc.

Phenomenex Inc.411 Madrid Avenue, Torrance, CA 90501

tel. (310) 212-0555; (310) 328-7768


Figure 1: BSA Tryptic map separated on different length Aeris PEPTIDE 3.6 µm XB-C18 columns (150 × 4.6 mm left, 250 × 4.6 mm right). Note the good separation on the shorter Aeris PEPTIDE column and the increased resolution provided by the longer Aeris PEPTIDE (250 × 4.6 mm) column. Because backpressure for the Aeris 3.6 µm column is so low, one can optimize column lengths based on their separation time and resolution requirements.

7.0 8.0 9.0 10.0 11.0 12.0 13.0 min

010

20

mAU

12 14 16 18 20 22 min

10

20

30

mAU


Saccharides are fundamental substances that express various bioactivities and may exist independently or form complexes

with proteins or lipids. Tey can be classified into monosaccha-rides, disaccharides, oligosaccharides, polysaccharides, etc., based upon the degrees of polymerization and condensation. Sugar alcohols are a class of polyols. A polyol is an alcohol containing multiple hydroxyl groups. Sugar alcohols are commonly added to foods since they are of lower caloric content than the correspond-ing sugars. Te analysis of saccharides and sugar alcohols provides valuable information for the medical research, food industries and regulatory agencies.

In the past, various analytical techniques have been used to analyze saccharides, including all modes of high performance liquid chromatography (HPLC). Normal phase chromatogra-phy, in tandem with a differential refractometer as a detec-tor, has long been used for the analysis of saccharides, as it provides good selectivity with relatively short analysis times. Hydrophilic interaction liquid chromatography (HILIC) selectively retains saccharides and polyhydric alcohols, such as sugar alcohols, while most of the substances with low polarity, as well as monohydric alcohols, elute in or very close to the void volume of the column.

We report the successful analysis of different kinds of sac-charides and sugar alcohols using a TSKgel NH2-100 HILIC HPLC column. TSKgel NH2-100 columns are packed with 3 µm silica particles containing 100-Å pores. A novel bond-ing strategy was adopted to improve chemical stability of the bonded phase. First the silica is reacted with a trimethylsi-lane endcapping reagent at a low stoichiometric ratio, before reacting residual and accessible silanol groups with trifunc-tional alkylaminosilane reagent. The resulting bonded phase provides a better safeguard against hydrolysis of the underly-ing silica. TSKgel NH2-100 columns are unique in that the bonded phase ligand not only, as expected, has a terminal pri-mary amino group, but that the spacer also incorporates sec-ondary as well as tertiary amino groups (see Figure 1). TSKgel NH2-100 columns are well suited for the analysis of all types of hydrophilic compounds, including saccharides and sugar alcohols.

Experimental ConditionsLC system: HP-1100 HPLC with Chemstation (ver B.03.01)Column: TSKgel NH2-100, 3 µm, 2.0 mm ID × 5 cmMobile phase (Isocratic): 80% ACN in H20Flow rate: 0.2 mL/minDetection: RITemperature: 50 ∘CInjection vol.: 2 µL

Te following commercially available compounds were used to prepare standard solutions:

Sucrose (Fisher S2-500)Mannitol (Sigma M-4125, Lot 22K0111)All the standards and samples were filtered through a

0.45 µm PVDF filter (Gelman) before injecting onto the column. High purity chemicals and HPLC grade solvents were used for the preparation of stock standards, samples, and mobile phases.

Analysis of Saccharides by Hydrophilic Interaction Chromatography (HILIC) Using TSKgel® NH2-100 Columns

Atis Chakrabarti, Tosoh Bioscience

Si

O

CH3

H3C

H3CSi

O

CH3

H3C

H3C Si

O O O

R

NH2

CH2

CH2R = Spacer

Aminoalkylmoiety

Figure 1: Structure of TSKgel NH2-100.

Table I: Preparation of standards

Saccharides WeightStock Standard (mg/mL)

Sucrose0.1023 g in 10.0 mL of 50% ACN in H2O

10.23

Polyols* WeightStock Standard (mg/mL)

Working Standard (mg/mL)

Mannitol0.1011 g in 10.0 mL of 50% ACN in H2O

10.11Same as stock

* Mannitol was readily soluble in 50% ACN in H2O.



The symmetry and efficiency of the eluted sucrose standard, a

disaccharide, from the TSKgel NH2-100 column is shown in

Figure 2.

To determine the suitability of the system and method, three

consecutive injections of sucrose were loaded onto the TSKgel

NH2-100 column. Te study yielded a very consistent result

across all peak parameters (see Table II). Tis column shows very

high reproducibility for the analysis of sucrose.

Tosoh Bioscience LLC

3604 Horizon Drive, Suite 100, King of Prussia, PA 19406

tel. (484) 805-1219; fax (610) 272-3028

Website: www.tosohbioscience.com

-10000

-5000

0

5000

10000

15000

20000

25000

30000

35000

40000

0 2 4 6 8 10Retention time (min)

nR

IU

Sucrose (Disaccharide)

Column: TSKgel NH2-100, 3µm, 2.0mm ID x 5cm

Mobile phase: 80% ACN in H2O

Flow rate: 0.2mL/min Detection: RITemperature: 50°CInjection Vol.: 2µL

Figure 2: Analysis of sucrose standard.

Mannitol, a sugar alcohol, was successfully analyzed on a

TSKgel NH2-100 column within 5 min (Figure 3).

As in the case of sucrose, the analysis of mannitol could be

reproduced with a high degree of consistency (Table III).

Conclusions

Tis study shows that a TSKgel NH2-100 column is suitable for the

analysis of sucrose, a disaccharide, and mannitol, a sugar alcohol.

Both standards separated on this column with good symmetry

and eficiency. System suitability studies show that the analyses of

sucrose and mannitol can be reproduced with very low %RSD in

peak parameters using the TSKgel NH2-100 column.

Tosoh Bioscience and TSKgel are registered trademarks of Tosoh Corporation.

Table II: System suitability results — sucrose analysis

Sucrose

Run RT k Area AF N

1 7.275 10.58 0.863 1.40 2732

2 7.280 10.59 1.070 1.40 2408

3 7.277 10.59 0.842 1.40 2734

Average 7.277 10.59 0.925 1.40 2625

Stdev 0.003 0.01 0.126 0.01 187.6

%RSD 0.03% 0.05% 13.6% 0.71% 7.1%

Column: TSKgel NH2-100, 3µm, 2.0mm ID x 5cm

Mobile phase: 80% ACN in H20

Flow rate: 0.2mL/min Detection: RITemperature: 50°CInjection vol.: 2µL

-20000

-10000

0

10000

20000

30000

40000

50000

60000

0 1 2 3 4 5 6 7

Retention time (min)

nR

IU

Mannitol

OH OH

OH

OH OH

HO

Figure 3: Analysis of mannitol standard.

Table III: System suitability results — mannitol analysis

Mannitol

Run RT k Area AF N

1 4.135 5.58 0.829 1.58 2124

2 4.135 5.58 0.923 1.63 2030

3 4.131 5.58 0.950 1.58 2027

4 4.134 5.58 0.915 1.69 2029

5 4.133 5.58 0.886 1.66 2028

Average 4.134 5.580 0.901 1.63 2048

Stdev 0.002 0.01 0.046 0.049 42.72

%RSD 0.04% 0.18% 5.12% 2.99% 2.09%


For the analysis of biotherapeutics, size-exclusion and ion-exchange

chromatography are typically conducted under native separation

conditions, requiring high ionic strength, 100% aqueous eluents. For

high performance liquid chromatography systems, these conditions

can be problematic: in the absence of bactericides, lack of proper

maintenance can lead to bacterial contamination within hours. Te

presence of high salt concentrations increases the potential of particu-

lates in the mobile phases. However, with proper set-up and care of

a chromatographic system, robust and reproducible chromatography

can be achieved with minimal down time (1,2).

Discussion

Te care and use of a size-exclusion and/or ion-exchange chro-

matographic system requires many of the same standard practices

as any other system. However, there are some additional protocols

that are required for high salt, aqueous mobile phases. While the

practices outlined in this document are described for ACQUITY

UPLC® Systems, the principles apply to any chromatographic sys-

tem. Overall system recommendations include:

• If using a steel system, modify according to manufacturer’s

recommendations. For use with a Waters UPLC® System,

detailed guidelines are available.

• Clean laboratory glassware properly.

• If possible, use mobile phases containing a bacteriostat

(i.e., 0.02% sodium azide) to prevent microbial growth.

Additional recommendations are listed below by component.

Tese considerations are for microbial growth, system suitability

and/or protein stability.

Solvent Delivery System:

The buffers used in SEC and IEX can favor microbial growth lead-

ing to contamination of the column and system. Other recom-

mendations include:

• Always filter aqueous mobile phase through compatible

0.22 μm or smaller membrane filters. The use of sterile

filters and containers is also recommended.

• Use only high purity water (18.2 MΩ cm). Bottled water should

be opened the day of use.

Guidelines for Routine Use and Maintenance of Ultra Performance Size Exclusion and Ion Exchange Chromatography SystemsPaula Hong and Kenneth J. Fountain, Waters Corporation

Figure 1: SEC-PDA chromatogram of bovine serum album (BSA) (5 mg/mL in water) shows the effect of �ow cell material on peak shape. BSA monomer exhibits extensive peak tailing.


• Never “top-off” mobile-phase bottles. Always change bottles

when replacing mobile phase.

• Replace eluents on a regular basis.

• All eluent bottles should be visually inspected daily for microbial

growth and/or particulates. Microbial growth can be a film on the

bottle surface or may be observed by swirling the bottle.

• If microbial growth has occurred in the eluent bottle, replace the

mobile-phase filter or flush it with a 70% isopropanol solution.

Microbes can contaminate mobile-phase filters.

• The solvent manager should never be left idle in either high salt

mobile phases or 100% water.

Sample Manager:

SEC and IEX conditions often require high-ionic-strength solu-

tions in the sample manager wash lines (wash/purge, strong and

weak needle). However, these eluents may have a detrimental effect

on the sample syringe and/or needle. Recommendations include:

• Ensure the sample is soluble in the mobile-phase and sample

manager washes.

• Follow the manufacturer’s recommendations for wash solvents.

• If the sample manager is idle, purge needle and/or wash lines

with high purity water preferably containing a bacteriostat.

UV Detectors:

Waters recommends titanium or stainless steel optical Tow cells

when performing SEC or IEX under aqueous conditions. fie

standard ACQUITY® optical Tow cell contains TeTon AF in the

Tuidic path. Some proteins, under native conditions, may inter-

act with the Tow cell surface, resulting in peak tailing and sloped

baseline. Recommendations for detectors include:

• Use titanium or stainless steel Tow cells to reduce protein-

surface interactions. Other Tow cell material (i.e., TeTon) may

cause peak tailing.

• Never leave the detector idle in high salt eluents. Flush thor-

oughly with water followed by higher organic eluent.

Column Storage:

To maintain long column lifetimes and minimize the risk of microbial

contamination, the following recommendations should be followed:

• Flush and store columns following the manufacturer’s recom-

mendations. Typical recommendations are 10–20% methanol

or with a bactericide (i.e., 0.1% sodium azide).

• Size-exclusion columns can typically be stored at 4–8 °C to

reduce microbial growth. Ion-exchange columns are usually

stored at room temperature. Check the manufacturer’s recom-

mendations for details.

Summary:

SEC and IEX chromatography are performed under native con-

ditions, requiring high-ionic strength, 100% aqueous eluents. To

minimize protein-surface interactions these conditions may re-

quire the use of a bio-compatible chromatographic system specifi-

cally designed for these applications. Precautions must be taken

to prevent and minimize bacterial contamination. Signs of such

contamination (3), which can occur within hours include: deterio-

rating peak shape, resolution and column lifetime. Unfortunately,

once the column has been contaminated, regeneration is difficult.

To decrease the frequency of system repairs and contamination,

a series of steps have been outlined for maintenance and care of

a chromatographic system and columns used for the analysis of

biomolecules. These recommendations include maintenance for

the solvent manager, sample manager, detector and column. Using

these procedures in combination with good laboratory practices

ensures a robust, reproducible system for ultra-performance-size-

exclusion and ion-exchange chromatography.

References

(1) “Size-Exclusion and Ion-Exchange Chromatography of Proteins using the

ACQUITY UPLC System”, Waters User Manual (2010), Rev A, Part

Number 715002147.

(2) “Size-Exclusion and Ion-Exchange Chromatography of Proteins using the

ACQUITY UPLC H-Class System”, Waters User Manual (2010), Rev

A, Part Number 715002909.

(3) “Controlling Contamination in LC/MS and HPLC/MS Systems”, Waters

User Manual, Part Number 715001307.

©2012 Waters. Waters, ACQUITY UPLC, ACQUITY, UPLC and fie Sci-

ence of What’s Possible are registered trademarks of Waters Corporation. All

other trademarks are the property of their respective owners.

Waters Corporation

34 Maple Street, Milford, MA 01757

tel. (508) 478-2000, fax (508) 478-1990

Website: www.waters.com


The paralytic shellTsh toxins are a group of 18 secondary me-

tabolites deposited in bivalve mollusks by dinofiagelates. Di-

nofiagelate blooms are seasonal, occurring during warm months.

Since it is unpredictable whether the infestation will occur, the

shellTsh population should be regularly monitored for toxins. In-

gestion of contaminated shellTsh can lead to paralytic shellTsh

poisoning: a life-threatening illness.

The Mouse Bioassay method used to detect dinoflagelate-

derived neurotoxins has major drawbacks, which led to ex-

ploration of chromatographic methods of analysis. Recently,

a HPLC method that utilizes post-column oxidation of the

toxins under alkaline conditions has been approved as a new

official AOAC method — OMA 2011.02. The products of

post-column derivatization of the toxins can be detected with

high sensitivity using a fluorescence detector, leading to the

determination of toxin type and concentration. We describe

the use of Pickering Laboratories post-column derivatization

system for analysis of paralytic shellfish toxins according to

AOAC Method 2011.02

Analytical Conditions

Column: Zorbax Bonus RP column, 3.5 μm, 4.6 × 150 mm

(Agilent Technologies)


Mobile Phase: A. 11 mM heptane sulfonate, 5.5 mM

phosphoric acid, adjusted to pH 7.1 with

ammonium hydroxide

B. 11 mM heptane sulfonate, 16.5 mM

phosphoric acid, 11.5% acetonitrile, adjusted

to pH 7.1 with ammonium hydroxide

Injection volume: 10 μL

Post-Column Conditions

Post-Column system: Pinnacle PCX or Vector PCX

Reactor Volume: 1.0 mL

Reactor Temperature: 85 °C

Reagent 1: 100 mM phosphoric acid, 5 mM periodic acid,

adjusted to pH 7.8 with 5 M sodium hydroxide

Reagent 2: 0.75 M nitric acid

Reagent Flow Rates: 0.4 mL/min

Detection: Fluorescence detector, λEX: 330 nm,

λEM: 390 nm

References

(1) J.M. van de Riet, R.S. Gibbs, F.W. Chou, P.M. Muggah, W.A. Rourke, G.

Burns, K. Thomas, and M.A. Quilliam, J. AOAC Int. 92, 1690–1704 (2009).

(2) AOAC Official Method 2011.02. Paralytic Shellfish Toxins in Mussels,

Clams, Oysters, and Scallops. Post-Column Oxidation (PCOX) Method.

Analysis of Paralytic Shell�sh Toxins Saji George, Maria O�tserova, and David Mazawa, Pickering Laboratories, Inc.

Pickering Laboratories, Inc.1280 Space Park Way, Mountain View, CA 94043

tel. (800) 654-3330; fax (408) 694-6700

Website: www.pickeringlabs.com

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

GTX4

GTX1

dcGTX3

GTX3

STX

neoSTX

GTX2

dcGTX2

GTX5

dcSTX

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

neo

STX

GTX

1

GTX

4

GTX

3

STX

Matrix

Peak

Figure 1: Chromatogram of GTX and STX mixed toxins standard.

Figure 2: Chromatogram of mussels sample naturally contami-nated with paralytic shell�sh toxins.


Agarose is a polysaccharide produced from seaweed that

forms a gel at room temperature. Te characteristics of

agarose gels depend on the agarose concentration, solvent,

weight-average molar mass, and molar mass distribution. Pow-

dered agarose, or agarose gel, needs to be heated in order to

dissolve properly. Reheating a sample as well as other process-

ing steps can change the agarose gel characteristics. Tis note

describes the characterization of agarose by size-exclusion chro-

matography (SEC) and multi-angle light scattering (MALS) at

an elevated temperature.

Te agarose sample was dissolved by boiling for 10 s and then

storing the solution at 50 °C. A second sample was dissolved,

allowed to gel at 25 °C, then reheated to dissolution and main-

tained at 50 °C prior to analysis. All components of the SEC-

MALS system were heated, operating at 50 °C. Te solvent was

heated to 50 °C by a coiled tube in the column oven and the

injector and column were also housed in an oven. Te tubing

between the DAWN EOS and oven employed WTC’s heated

transfer lines. Te fiow cells of the MALS detector and the Opti-

lab DRI detector were temperature-controlled at 50 °C as well. A

schematic is shown in Figure 1.

Te results for the fresh and reheated samples are overlaid

in a cumulative distribution plot in Figure 2. It is clear from

the graph that cooling and reheating degrades the sample. Te

molar mass results for the two freshly prepared samples overlay

well, whereas the molar mass of the reheated agarose sample has

decreased. Tis establishes a clear explanation for the differences

in the performance of products from freshly prepared versus

reheated agarose samples.

Tis note shows that reliable results can be obtained even for

delicate samples such as agarose. Te thermostatic control options

of the DAWN, coupled with its heated transfer lines, prove ex-

tremely useful in applications where sample solubility is required

at elevated temperatures.

Agarose-Heated Transfer Lines Wyatt Technology Corporation

Wyatt Technology Corporation6300 Hollister Avenue, Santa Barbara, CA 93117

tel. (805) 681-9009; fax (805) 681-0123

E-mail: [email protected]; Website: www.wyatt.com

Figure 2: Cumulative molar mass plot of two agarose samples. Reheating of the sample results in a decrease of the molar mass.

Figure 1: HPLC system with MALS and DRI detectors at 50 °C. The heated lines prevent the agarose from gelling as it is analyzed.

THE APPLICATION NOTEBOOK – FEBRUARY 201220 Chiral

AstraZeneca R&D Boston, Infection IMED 35 Gatehouse Drive, Waltham, MA 02451

tel. (781) 839-4000, Fax (781) 839-4500

Website: www.astrazeneca.com

Use of Amine Additive in the Sample Diluent in a Supercritical Fluid Chromatography (SFC) PurificationNatascha Bezdenejnih-Snyder, Valerie Hoesch, and Nancy DeGrace, AstraZeneca R&D Boston, Infection IMED

Basic compounds often require basic additives in the modifier to

improve peak shape and resolution in SFC (1). Tese additives

can be difficult to remove post-purification (2). In this application

note, we present a chiral SFC purification of a proprietary Astra-

Zeneca zwitterionic compound, containing a functionalized py-

rimidone and other aromatic substituents, known to retain amine

additives after isolation. Dimethylethylamine was added to the

sample diluent to improve the chromatography and to avoid fur-

ther purification of the desired stereoisomer to remove the amine.

Experimental Conditions

SFC System: Tar MultiGram III Preparative SFC

Column: Chiralpak AD-H, 5 µm, 30 × 250 mm

Modifier: Isopropanol

Mobile phase: 75% Carbon dioxide, 25% isopropanol

Flow rate: 120 mL/min

Detection: UV at 254 nm

Column temperature: 40 °C

Outlet pressure: 100 bar

Results

Figure 1 shows the chiral separation of an AZ compound in

neutral modifier and sample diluent. Tree peaks eluted off the

semi-preparative column. Te first peak contained a mixture of

both stereoisomers. Te second peak was primarily Stereoisomer

1, while the third peak was pure Stereoisomer 2. Previous batches

of this compound were purified by high performance liquid chro-

matography (HPLC) using an amine additive in the modifier, and

the NMR data showed that the isolated stereoisomers contained

the amine after dry-down.

Figure 2 shows the same separation with the addition of one

percent of dimethylethylamine, by volume, to the sample diluent

instead of the modifier. Te amine eluted at the beginning of the

run and the two stereoisomers were well-resolved.

Since Stereoisomer 2 was the desired compound, the stacking con-

ditions were programmed to collect dimethylethylamine with Stereo-

isomer 1. Stereoisomer 2 was isolated and did not contain dimethyl-

ethlamine after dry-down.

Conclusions

Te addition of dimethylethylamine to the sample diluent improved

chromatography, and the lack of the amine in the modifier ensured its

absence in the desired fraction. Tis eliminated the need for a subse-

quent purification for amine removal.

References

(1) M. Maftouh, C. Granier-Loyaux, E. Chavana, J. Marini, A. Pradines,

Y.V. Heyden, and C. Picard, J. Chrom. A 1088, 67–81 (2005).

(2) C. Hamman, D.E. Schmidt Jr., M. Wong, and M. Hayes, J. Chrom. A 1218,

7886–7894 (2011).

Figure 1: Semi-preparative chromatogram, 64 mg injection (loading: 4 mL of a 16 mg/mL sample solution). The sample diluent and the modifier were neutral.

Figure 2: Semi-preparative chromatogram, 128 mg injection (loading: 8 mL of a 16 mg/mL sample solution). Dimethylethyl-amine (1% by volume) was added to the sample diluent. The modifier was neutral.

THE APPLICATION NOTEBOOK – FEBRUARY 2012 Environmental 21

Over the last decade the use of Pharmaceuticals and Personal Care

Products (PPCPs) has doubled in the United States. As a result,

PPCPs have entered the environment through both human activity

and as by-products from manufacturing, agricultural activities, medi-

cal use and veterinarian facilities. PPCPs are usually introduced into

the environment through the disposal of unused medications into

sewer systems and trash. PPCPs tend to be water soluble and do not

evaporate under normal temperatures, which is why they end up in

soil and water. Te full effects of PPCPs on the environment are not

fully understood and there is concern about the potential threat they

pose to the food chain. Because of the high solubility of most PPCPs,

aquatic organisms are most vulnerable. Te classes of pharmaceuticals

found in these organisms have been linked to slow growth in frogs and

the increased feminization of exposed fish. Te scope of human ex-

posure to PPCPs from the environment is complicated and increased

monitoring is occurring to determine the effect on humans of long-

term, low-level exposure to PPCPs.

Due to their persistent nature and toxicity, monitoring water sourc-

es for PPCPs is a growing priority for both government agencies and

consumers. Te following procedure outlines the fully automated,

sample-to-vial extraction and concentration of water matrices for the

detection of these compounds in one rapid and efficient process.

Instrumentation and Consumables

FMS, Inc. PowerPrep™ SPE system (Solid Phase Extraction)

FMS, Inc. PowerVap™ Concentrator

FMS, Direct-to-Vial concentrator tubes

1 g Waters Oasis™ HLB cartridge

UPLC, LC–MS

Procedure: Sample Prep Extraction and Concentration

PowerPrep SPE- for conditioning, sample loading and elution of

the extract directly to the concentrator.

PowerVap Direct to Vial Concentrator- for concentrating the

extraction to final volume in a vial to be loaded on the LC–MS.

Conclusions

Analysis of the LC–MS data demonstrates excellent recoveries

and reproducibility from a traditionally difficult sample matrix.

Adding to the efficiency was the use of nitrogen and vacuum to

dry the cartridge producing a water free extract that enables a fast

concentration step with no loss of analytes. The extract takes 45

min to concentrate using the PowerPrep™ SPE and PowerVap

Direct to Vial concentration system when compared to all other

drying methods. Using the automated, one step SPE and Direct-

to-Vial Concentration tubes from FMS, Inc. eliminates error-prone

manual or semi-automated steps from the sample prep process. No

sample transfer was necessary, the sample was extracted and au-

tomatically sent to the PowerVap Concentrator where the final

extract is concentrated directly in a vial for LC–MS analysis. This

capability eliminated human error, saved time and increased ef-

ficiency while producing reproducible and consistent recoveries.

One-Step Extraction and Concentration for Identifying Pharmaceuticals and Personal Care Products in Water, Biosolids, and SolidsFMS, Inc.

FMS, Inc.

580 Pleasant Street, Watertown, MA 02472

tel. (617) 393-2396, fax (617) 393-0194

Website: www.fmsenvironmental.com

Table I: Mean recoveries from five extracts

Compound Average Recovery

Atenolol 88%

Atorvastatin 81%

Avobenzone - A 97%

Avobenzone - B 92%

Ciprofloxacin 99%

Benzophenone-1 98%

Benzophenone-3 94%

DEET 90%

4,4-Dihydroxybenzophenone 86%

Estradiol 81%

Estrone 84%

Naproxen 95%

Methylparaben 85%

Propanolol 80%

Ranitidine 99%

Sulfamethoxazole 98%

Sucralose 97%

TCEP 86%

Trimethoprim 83%

Thiabendazole 92%

Warfarin 87%

Xanthine 92%

THE APPLICATION NOTEBOOK – FEBRUARY 201222 Environmental

A simple screening method for Pyrethroid insecticides

in sediment samples is described. Electron impact ion-

ization (EI) with tandem MS-MS in combination with

programmed temperature vaporization injection (PTV)

were used in order to obtain excellent speci�city and

low limits of detection.

Pyrethroids are synthetic chemical insecticides whose chemical

structures were adapted from the chemical structures of the py-

rethrins. Pyrethroids have been modiTed to increase their stability in

sunlight. ffey are toxic to aquatic organisms at extremely low con-

centrations, especially to invertebrates that are at the bottom of the

food chain (1). Many of the compounds end up in wastewater outfalls

due to inefiective water treatment. As a result, state and federal agen-

cies have initiated monitoring programs to characterize the extent of

contamination in water supplies and their potential impact on aquatic

life. In general, there are no set requirements for minimum detection

or reporting levels. However, the lowest reporting level that can be

achieved by a given technique is desired.

ffe Scion TQ triple quadrupole mass spectrometer in EI mode

with PTV injection is ideal for screening sediment extracts at low to

sub-part-per-billion concentrations.

Experimental

451-GC Gas Chromatographic Conditions

Column: BR-5ms, 30 m × 0.25 mm × 0.25 µm

Column Flow Rate: 1.0 mL/min constant ffiow

Injector: Bruker PTV injector, with 3.4 mm ID Siltek Fritted Liner

Injector Conditions: 60 °C hold 0.4 min to 310 °C at 200 °C/min,

hold 30 min

Column Temperature Program: 55 °C, hold 3 min, ramp to 200 °C at

40 °C/min, hold 1 min; ramp to

310 °C at 5 °C/min, hold 1 min

Injection: 8.0 µL using Bruker 8400 auto sampler

Screening for Pyrethroid Insecticides in Sediment Samples by GC–MS-MS Ed George, Bruker Daltonics, Chemical Analysis Division

Table I: MS-MS parameters for pyretroids insecticides

Compound Name RT (min) RT Window Precursor Product Collision Energy

Lambda Cyhalothrin_Ep1+2 19.150 1.000 208 181 -10

19.150 1.000 181 152 -20

Fenvalerate+Esfenvalerate 24.230 1.000 167 125 -15

24.230 1.000 225 91 -25

24.230 1.000 225 119 -10

Bifenthrin 17.560 0.500 181 165 -18

17.560 0.500 181 166 -10

17.560 0.500 181 115 -40

trans+cis-Permethrin 20.880 1.000 183 168 -18

20.880 1.000 183 128 -20

20.880 1.000 183 152 -20

Cypermethrin-Isomers 22.580 1.200 181 152 -20

22.580 1.200 181 127 -30

Cy�uthrin-Isomers 21.980 1.200 226 206 -15

21.980 1.200 206 151 -15

Resmethrin_1+2 16.690 1.000 171 128 -12

16.690 1.000 171 143 -12

Fenpropathrin 17.860 0.500 265 181 -20

17.860 0.500 265 210 -10

Deltamethrin-1+2 25.200 1.000 253 174 -10

25.200 1.000 253 172 -10


Scion TQ MS Conditions

Scion TQ MS Conditions

Ion Source Temperature: 250 °C

Transferline Temperature: 280 °C

Filament Delay time: 12 min

Filament Emission Current: 80 µA

Dwell Time: 100 ms each transition

Calibration standards in hexane were prepared at 0.1, 1, 5, 10, 25,

50, 100, and 200 ppb. In addition, a blank sediment matrix was

spiked at concentrations of 0.5, 5, 10, 25, 50, 100, and 200 ppb.

Sample preparation: 5 g copper powder/magnesium sulfate was

added to 20 g wet sediment. The mixture was extracted with 2 ×

75 mL 50:50 acetone:hexane using a shaker at 185 rpm (2 × 15

min). The extracts were concentrated to a final volume of 2 mL

and cleaned up on Florisil® Sep Pak.

Results

Te Scion TQ has a unique software feature known as Compound

Based Scanning (CBS) that was used to easily set up the MRMs re-

quired for the analysis. Te pyrethroids were found in the Factory Li-

brary within the CBS software, and were directly downloaded into

the scan acquisition method (Figure 1). In addition, the data han-

dling table is automatically created and linked to the scan acquisition

method. Tis saves time because the operator does not have to create

additional tables and manage changes to the analytical method.

Te use of MS-MS provided excellent sensitivity and speciffc-

ity for the analysis. Compounds detected in the sediment sample

spiked at 0.5 ppb are shown in Figure 2.

Calibration curves prepared in pure solvent (hexane) and in

blank sediment extracts were linear indicating no matrix interfer-

ence from the sediment extract.

Conclusion

PTV injection combined with the sensitivity and speciffcity of

MS-MS result in excellent low-level screening of pyrethroids in

river and lake sediments.

Te Scion TQ with Compound Based Scanning allows easy set-

up and management of the pyrethroid MRM parameters. Com-

pound MRM information is loaded directly into the method by

choosing them from a factory or user created library. It links the

mass spectrometer acquisition table directly with data handling

parameters, streamlining the data process.

Acknowledgements

California Department of Food and Agriculture

(http://www.cdfa.ca.gov/)

Reference

(1) http://www.beyondpesticides.org/pesticides/factsheets/Synthetic%20

Pyrethroids.pdf

Bruker Daltonics, Chemical Analysis DivisionBillerica, MA

tel. (978) 663-3660, fax (978) 667-5993

Website: www.bruker.com

Figure 2: Calibration curves for Bifenthrin prepared in hexane from 0.1 to 200 ppb (plot at left) and those prepared in a spiked sediment extract (plot at right) calibrated from 0.5 ppb to 200 ppb.

Figure 1: MS-MS for Bifenthrin (top), two epimers of lamda-cyhalotrin (mid), and cyfluthrin isomers (bottom) in the sedi-ment matrix.

Bifenthrin80

70

60

50

40

30

10.0

7.5

5.0

2.5

17.25 17.50 17.75 18.00

Lambda cyhalothrin_Ep1+2; 208.0>181.0; 0110_0.5ppb_MTX.xms

Lambda-cyhalothrin Epimers 1 and 2

minutes

KO

ps

KO

ps

Bifenthrin; 181.0>165.0; 0110_0.5ppb_MTX.xms

2.0

1.5

1.0

0.5

21.50 21.75 22.00 22.25 minutes

cy�uthrin-Isomers; 226.0>206.0; 0110_0.5ppb_MTX.xms

cy�uthrin isomers

KO

ps


The purpose of this study is to determine how the Direct-to-GC vial

concentrator tube and the DryVap® Concentrator System can op-

timize the recoveries for both low and high boiling point semi-volatile

compounds. A spiking mixture from EPA method 8270D was chosen

due to the more volatile compounds in the mix that are diTcult to re-

tain during the concentration and subsequent rinsing process; while the

higher boiling compounds are more likely to absorb onto the glass walls.

ffe DryVap® Concentrator System was intentionally designed to

provide enhanced recoveries of semi-volatile organic compounds such

as those found in EPA Method 8270D. ffe unique design of the

DryVap® system seals the Direct-to-GC vial concentrator tube induc-

ing condensation of the evaporating solvent on the inside walls of the

concentrator tube and forcing the concentrated extract towards the

GC vial tip. ffis condensation, similar to the Kuderna-Danish ap-

paratus, allows condensed solvent to recapture the more volatile com-

pounds so they are not lost to the vapor phase. While this design is

beneficial for more volatile compounds, solvent condensation on glass

surfaces is a likely source for the adsorption of higher boiling point

compounds, especially on the crest and tip of the concentrator tube.

ffe Direct-to-GC vial concentrating tube eliminates the need to

transfer an extract from a concentration tube to a GC vial. ffis added

feature will help you enhance your recoveries by keeping the original

glassware from the concentration step to the analysis step. It also re-

duces the chance of a potential spill or loss of a sample by eliminating

the need for traditional pipette transfer techniques to GC vials.

Instrumentation

• Horizon Technology

- DryVap® Concentrator System

- Reclaimer™ Solvent Recovery System

- Direct-to-GC Vial Concentrator Tubes

• 8270D Spiking Mix

• Agilent

- 6890 GC

- 5973 Mass Selective Detector

Method Summary

1. Place 125 mL of methylene chloride into a DryVap® Direct-to-

GC concentrator tube.

2. Add 50 µg of 8270D Spiking Mix into the 125 mL of methy-

lene chloride.

3. Place the concentration tube on one of the six positions and

lower the lid.

4. Configure heater power to 5, nitrogen 20 psi and vacuum to -7 in Hg.

5. Press start and gently push down on the lid to seal the

concentration tube.

6. When the sample is done, its position will light up “FINISH”,

and the DryVap® will beep.

7. Raise the lid and rinse the lower heater coil with 0.25 mL of

methylene chloride. ffe rinse should drip into the bottom of

the concentrator tube (this step can be automated by selecting

“ Auto Rinse Mode 1” on the control panel before starting the

concentration process).

8. Using a pipette, rinse the concentrated sample along the

bottom ffiat part of the concentration tube several times.

9. Dispense the concentrated sample back into the GC vial tip

of the concentrator tube and read the meniscus to ensure its

final volume is at 1.0 mL.

10. Unscrew the GC vial and cap.

11. Run extract on GC–MS.

Results

ffe recoveries for the 114 semi-volatile compounds were very

good, averaging from 86–113%, with the majority in the 90th

percentile. Standard deviation and RSD were low showing great

reproducibility throughout the concentration process.

Conclusions

ffe purpose of this study was to demonstrate enhanced semi-

volatile recoveries for an extract while truly automating the entire

concentration process. Using the Direct-to-GC vial concentrator

tube with the DryVap® eliminates the transfer step done in tradi-

tional processes allowing for greater eTciency and reproducibility.

Enhancing Recoveries and Maximizing Throughput of Semi-Volatile Organics Using the Direct-to-GC-Vial Concentrator Tube and DryVap® Concentrator SystemMichael Ebitson and David Gallagher, Horizon Technology, Inc.

Horizon Technology, Inc.,45 Northwestern Dr., Salem, NH, 03079

tel: (603) 893-3663, fax: (603) 893-4994

Website: www.horizontechinc.com


Microwave assisted extraction (MAE) offers signi�-

cant bene�ts over traditional soxhlet extraction for

dioxins and furans determination from environmen-

tal samples including higher sample throughput, time

savings, reduced solvent and great reproducibility.

Polychlorinated dibenzodioxins (PCDDs) and polychlorinated

dibenzofurans (PCDFs) are groups of polyhalogenated com-

pounds found as environmental pollutants resulting from a num-

ber of manufacturing processes, and have been linked to a number

of human biological disorders through accumulation in fat tissue.

PCDDs and PCDFs can be formed by pyrolysis, or incineration at

high temperatures, of chlorine containing products such as PVC,

PCBs, and other organochlorides.

Soxhlet is the traditional approach for extracting PCDDs and

PCDFs from environmental soils, sludges, and other matrices.

Although effective, soxhlet extraction requires large amounts of sol-

vent and takes hours to complete. Alternatively, microwave extrac-

tion, using approved EPA method 3546, provides a fast and repro-

ducible method without the high solvent consumption. PCDD/

PCDF recoveries by MAE are equivalent or better than soxhlet.


MAE using Milestone’s Ethos EX was compared to soxhlet extrac-

tion for dioxins and furans extraction from fiy ash. ffie Ethos EX

labstation can extract 24 samples simultaneously in a closed vessel

system. With increased pressure capabilities, temperatures above

the boiling point of the solvent are used to decrease the extraction

time to 45 min with 30 mL of solvent per sample. ffie closed vessel

system along with magnetic stirring for constant agitation, direct

temperature monitoring, and the ability to reuse stored methods

allows for great reproducibility of dioxins and furans recoveries.

For the MAEs, 2 g of fiy ash were weighed into a microwave

vessel. A stir bar, a carbon coated wefion button (for absorbing mi-

crowaves when using non-polar solvents), and 30-mL of toluene

were added to the vessel. Extractions were completed at 140 °C

with a total program time of 45 min. For soxhlet, the extractions

took 24 h to complete using 150 mL of solvent. Both extracts were

analyzed by GC–MS using an Agilent J&W DB-5ms column.

Results

Extraction rates with Milestone’s Ethos EX are 32 times faster,

with recoveries comparable or better than traditional soxhlet, and

500% or greater reduction in solvent required.

Conclusions

Milestone’s Ethos EX successfully extracted dioxins and furans

from fly ash in just 45 min with very little solvent. As the results

show, MAE yields comparable or better results than soxhlet ex-

traction. The ability to perform 24 samples simultaneously is ideal

for high throughput labs looking to save space, time, and money

while getting great recoveries of dioxins and furans.

Microwave Assisted Extraction of Dioxins and Furans Compared to SoxhletMelissa Lightner, Milestone Inc.

Milestone Inc.25 Controls Drive, Shelton, CT 06484

tel. (866) 995-5100, fax: (203) 925-4241

Website: www.milestonesci.com

Table I: GC-MS recovery results from microwave assisted and soxhlet extraction

CompoundSoxhlet Extraction Recovery (ng/g)

Microwave Extraction Recovery (ng/g)

2,3,7,8-TCDD 0.180 0.170

1,2,3,7,8-PCDD 0.769 0.715

1,2,3,4,7,8-HxCDD 0.814 0.767

1,2,3,7,8,9-HxCDD 2.742 2.357

1,2,3,6,7,8-HxCDD 4.628 4.010

2,3,7,8-TCDF 0.879 0.919

1,2,3,7,8-PCDF 1.716 1.788

2,3,4,7,8-PCDF 2.608 2.461

1,2,3,4,7,8-HxCDF 2.279 2.363

1,2,3,6,7,8-HxCDF 2.633 2.668

2,3,4,6,7,8-HxCDF 2.924 2.992


Shale gas reservoirs, such as the Marcellus shale reserve in

Pennsylvania and Barnett shale reserve in Texas are a grow-

ing source of natural gas in the United States. Hydraulic frac-

turing or “fracking” involves pumping water, sand, and chemi-

cals at extremely high pressure into deep underground wells to

crack open hydrocarbon-rich shale formations and extract natural

gas. Chemicals used for hydraulic fracturing include potentially

toxic substances such as diesel fuel and disinfectants which can

contaminate underground sources of drinking water (USDW).

In some cases methane has been detected in household drinking

water from wells (1).

Tis application note describes the use of a purge-and-trap gas

chromatography system to analyze methane, ethane, ethene, and

propane hydrocarbons (C1-C3) in drinking water samples.


Instrumentation used for this study was an OI Analytical Eclipse

4660 Purge-and-Trap sample concentrator with a proprietary

trap speciffcally designed to trap methane. Te P&T was inter-

faced to an Agilent 7890 GC/FID with a split/splitless injector

and a SUPEL-QPLOT column (30-meter × 0.32-mm i.d.).

Results

Four unique 9-point calibration curves (0.01 to 39.6 ppm) were

prepared and analyzed on the optimized system. Methane peak

area counts were plotted as a function of concentration to gener-

ate an external calibration curve for each set of analyses. Te four

resulting curves are plotted together in Figure 1, along with their

linear correlation coeficients (R2) and illustrate the overall reli-

ability and stability of the analytical system.

Figure 2 is the FID chromatogram from analysis of a 5-mL

water sample saturated with methane (~ 40 ppm) and containing

single-digit ppm levels of ethane, ethene, and propane. Baseline

resolution was obtained for all four peaks.

For complete results of this study, refer to OI Analytical

Application Note # 3792 (2).

Conclusions

Te results of this study demonstrate that purge-and-trap GC

analysis can be used to measure methane contamination in wa-

ter samples. Te system yielded linear calibrations and complete

baseline separation of methane, ethane, ethene, and propane.

References

(1) S.G. Osborn, A. Vengosh, N.R. Warner, and R.B. Jackson, Methane con-

tamination of drinking water accompanying gas-well drilling and hydrau-

lic fracturing. Proceedings of the National Academy of Sciences, 109(20),

8172–8176 (2011).

(2) OI Analytical Application Note # 3792, “Purge-and-Trap GC Analysis of

Methane in Water Samples Associated with Hydraulic Fracturing”.

Purge-and-Trap GC Analysis of Methane in Water Samples Associated with Hydraulic FracturingLaura Chambers, Gary Engelhart, and Scott Hazard, OI Analytical

OI AnalyticalP.O. Box 9010, College Station, TX 77842

tel. (800) 653-1711, or (979) 690-1711, fax (979) 690-0440

Email: [email protected], Website: www.oico.com

Four 9-Point Calibration Curves

5.0E+09

4.5E+09

4.0E+09

3.5E+09

3.0E+09

2.5E+09

2.0E+09

1.5E+09

1.0E+09

5.0E+08

0.0E+00

0.0 5.0 10.0

Calibration #1 Calibration #2 Calibration #3 Calibration #4

15.0 20.0 25.0 30.0 35.0 40.0

R2 =0.9998

R2 =0.9994

R2 =0.9998

R2 =0.9998

45.0

Concentration (ppm)M

eth

an

e R

esp

on

se

0.01 to 39.6 ppm Methane in 5mL Water

Response

Methane

Ethane

Ethane

1.00 2.00 3.00 4.00

Propane

2.6e+08

2.4e+08

2.2e+08

1.8e+08

1.6e+08

1.4e+08

1.2e+08

1e+08

8e+07

6e+07

4e+07

2e+07

Time 0.00 5.00

2e+08

Figure 1: Nine-point calibration curves of methane in water with linear correlation coef�cients.

Figure 2: FID chromatogram of methane saturated sample (~40 ppm) with single-digit ppm levels of ethane, ethene, and propane.


The determination of common inorganic anions and cations

in drinking water is important due to the toxicity of anions

(e.g., Tuoride, nitrite, and nitrate) and secondary contaminants

(e.g., chloride and sulfate) which can affect the water’s aesthet-

ics. fierefore, these secondary contaminants are monitored and

primary contaminants regulated for compliance by the U.S. EPA

and other agencies around the world.

Ion-exchange chromatographic determination of dissolved

alkali and alkaline earth metals and ammonia in drinking water

is another important application. Sodium is monitored under

the U.S. EPA Safe Drinking Water Act, whereas ammonium is

a required target analyte for wastewater discharge permits and is

monitored in process wastewaters.

Capillary IC requires µL/min Tow rates. Due to its low con-

sumption of eluent, the system can remain on continuously,

thereby eliminating the need for calibration prior to each use to

provide a true walk-up system. fie low Tow rate leads to longer

lifetime of consumables and smaller amount of waste, thereby re-

ducing the overall cost of ownership.

Conditions and Sample Preparation

fie experimental setup and the sample preparation procedures

are described in Application Brief 133, fiermo Fisher Scientiffic,

Inc. (formerly Dionex Corp.).

Conclusion

All anions were separated and eluted within 13 min. fie peak

area relative standard deviations for each analyte was 0.6% when

60 injections were evaluated within 24 h. Capillary Reagent-

Free™ IC redeffines the workTow for IC analysis of inorganic an-

ions and cations, providing enhanced mass sensitivity and ease of

use. It is a fast and accurate solution for routine characterization

of different water samples.

Scan to receive complete application note.

Fast Cap IC Determinations of Inorganic Anions and Cations in Drinking WaterFei Pang, Terri Christison, and Khalil Divan Thermo Fisher Scienti�c

Thermo Fisher Scienti�c, Inc. (formerly Dionex Corp.) 1228 Titan Way, P.O. Box 3603, Sunnyvale, CA 94088

tel. (408) 737-0700, fax (408) 730-9403

Website: www.thermoscienti�c.com/dionex

10 200

µS

–1

16

Minutes

1

2

3

10 200

µS

–1

10

Minutes

1

2 3

4

5

Figure 1: Anions in undiluted municipal drinking water. Column: Thermo Scienti�c Dionex IonPac AG19, AS19, Capillary, 0.4 mm. El-uent: KOH (RFIC-EG). Gradient: 15 mM (0–7 min), 15–60 mM (7–18 min). Flow Rate: 10 µL/min. Inj. Volume: 0.4 µL. Temp: 30 °C. Peaks: 1) Chloride 105.7 mg/L; 2) Nitrate 2.8 mg/L; 3) Sulfate 30.4 mg/L.

Figure 2: Cations in undiluted municipal drinking water. Col-umn: Dionex IonPac™ CG12A, CS12A, Capillary, 0.4 mm. Eluent: MSA (RFIC-EG). Gradient: 6–65 mM (0–30 min). Flow Rate: 10 µL/min. Inj. Volume: 0.4 µL Temp: 40 °C. Peaks: 1) Sodium 3.25 mg/L; 2) Ammonium 0.11 mg/L; 3) Potassium 0.25 mg/L; 4) Magnesium 0.37 mg/L; 5) Calcium 2.89 mg/L.


Volatile organic compounds (VOCs) in soil were

rapidly extracted and concentrated using solid phase

microextraction (SPME) followed by on-site analysis

and identi�cation with the TRIDION™-9 portable gas

chromatograph – toroidal ion trap mass spectrome-

ter (GC-TMS). 37 VOCs were resolved and analyzed

quickly in under 3 min.

The ability to screen soil samples in the Teld by identifying

volatile environmental contaminants is a valuable tool.

Results of the screening procedure may be used to guide

other sample collection activities and will help identify which

EPA extraction methods to use. In combination with the

CUSTODION™ SPME syringe, the TRIDION-9 portable

GC-TMS can positively identify unknown VOCs in the Teld and

prescreen samples similar to EPA method 3815.


A CUSTODION SPME syringe with a 65 µm polydimethyl-

siloxane/divinylbenzene (PDMS/DVB) Tber was used to extract

analytes from a soil sample spiked with VOCs. Each ranging in

concentration from ~15,000 µg/kg to ~2400 µg/kg. 5 g of soil

sample were added to 5 mL of H2O with 25% NaCl (w/v).

Sample vial was shaken vigorously by hand for 10 s, after which

the SPME Tber was exposed to the headspace for 50 s. ffis was

repeated Tve times for a total sampling time of ~5 min.

Following sample extraction, the SPME syringe was inserted

into the TRIDION-9 GC-TMS injection port where the target

analytes were desorbed into a split-splitless injector (280 °C) cou-

pled with a low thermal mass capillary GC column (MXT-5, 5 m

× 0.1 mm, 0.4 µm df ). After an initial 10 s hold at 40 °C, the GC

temperature was increased at 2 °C/s to 280 °C for a total run time

of ~2 min. ffe capillary GC is coupled to a TMS detector. ffe

target analytes were identiTed with a target compound library and

advanced custom deconvolution algorithm.

Results

Figure 1 shows the GC-TMS separation of VOCs spiked into a soil sample. 37 VOCs were detected and positively identified by the TRIDION-9.

Conclusions

ffe CUSTODION SPME syringe and TRIDION-9 GC-TMS

are uniquely suited for Teld screening of soil samples to support

rapid decision making in the Teld. ffe short cycle time between

injections allows the user to quickly analyze samples on-site with

high sensitivity and speciTcity.

Acknowledgements

Torion®, CUSTODION® and TRIDION™ are trademarks of

Torion Technologies Inc. The CUSTODION SPME Syringes

are manufactured and sold under license from SUPELCO under

US Patent 5,691,206, and/or any divisions, continuations, or

revisions thereof.

Rapid On-Site Screening of Environmental VOCs in Soil Using Solid Phase Microextraction and a Person-Portable GC–MSTiffany C. Wirth, Charles S. Sadowski, and Douglas W. Later, Torion Technologies Inc.

Torion Technologies Inc.796 East Utah Valley Dr., Suite 200, American Fork, UT 84003

tel. (801) 705-6600, email [email protected]

Websie: www.torion.com

78

23

22 25

2829

30

32

34

36

37

35

2021

19

15

1814

1312

119

10

20 40

Retention Times (s)60

2426

1617

80

33

65

4231

Figure 1: Chromatograph of 37 VOCs extracted from a soil sample. Peak identi�cations: 1) Trichlorofiuoromethane; 2) 1,1-dichloroeth-ylene; 3) Dichloromethane; 4) MTBE; 5) 1,2-dicholoroethylene (z); 6) Chloroform; 7) 1,1,1-trichloroethane; 8) 1,2-dichloroethane; 9) Car-bon Tetrachloride; 10) Benzene; 11) 2-butanone; 12) Trichloroethyl-ene; 13) Bromodichloromethane; 14) 1,3-dichloro-1-propene (z); 15) 1,3-dichloro-1-propene (e); 16) Toluene; 17) 1,1,2-trichloroethane; 18) 4-methyl-2-pentanone; 19) Tetrachloroethylene; 20) Chloroben-zene; 21) 1,1,1,2-tetrachloroethane; 22) Ethylbenzene; 23) Xylene; 24) Bromoform; 25) Styrene; 26) 1,1,2,2-tetrachloroethane; 27) Iso-propylbenzene; 28) Bromobenzene; 29) 1,2,3-trichloropropane; 30) 1,3,5-trimethylbenzene; 31) 1,2,4-trimethylbenzene; 32) 1,3-di-chlorobenzene; 33) 1,4-dichlorobenzene; 34) 1,2-dichlorobenzene; 35) 1,2-dibromo-3-chloropropane; 36) 1,2,4-trichlorobenzene; 37) Naphthalene.

THE APPLICATION NOTEBOOK – FEBRUARY 2012 Food & Beverage 29

The benefits of olive oil as part of a healthy diet reaches back

to the 1950’s. Methods were developed to determine benzene,

toluene, ethylbenzene, xylenes and styrene (BTEXS) by static head-

space methods. Both static and dynamic capabilities allow labora-

tories to characterize the volatile flavor components of olive oils and

accurately quantify low levels of compounds like BTEXS.

Seven olive oil samples were obtained consisting of light flavor,

pure, and extra virgin olive oils. Six were in glass containers, one in a

plastic container. Te countries of origin were indicated on the labels.

Experimental

Te HT3 headspace instrument, to a Termo Focus GC/DSQII MS

and a Zebron ZB-624 30 m × 0.32 mm × 1.8 µm column was used.

A Vocarb 3000 trap was used in the dynamic mode. Te platen tem-

perature was 90 °C for both methods.

Te sample size was 10 g for the static analysis and 2 g for the

dynamic analysis. BTEXS standards were used for identification.

Results

Te BTEXS peak areas were determined from their primary

quantitation ions. Te peak areas were normalized for the olive oils

and are presented in Table I. Figure 1 compares the static and dynamic

headspace TIC for an olive oil samples.

Conclusions

Te data indicated similar results for all of these compounds except

benzene. Te dynamic mode detected higher concentrations of

benzene in six of the seven olive oil samples.

References

(1) Teledyne Tekmar, Comparison of BTEXS in Olive Oils by Static and

Dynamic HT3 Headspace.

Comparison of BTEXS in Olive Oils by Static and Dynamic HeadspaceRoger Bardsley, Teledyne Tekmar

Teledyne Tekmar4736 Socialville Foster Rd., Mason, OH 45040

tel. (800) 874-2004

Website: www.tekmar.com.

NL: 5.50E710.57

11.13

10.42

8.54

6.37

100

90

80

70

60

50

40

30

20

10

0

6

6.29

8.52

10.42

10.58

11.13

7 8 9 10 11

11.53

m/z=77.50-78.50+90.50-91.50+103.50-104.50+105.50-106.50

NL: 5.50E5

Benzene

Dynamic

Static

Toluene

Ethylbenzene

Time (min)

RT: 5.64-11.72

Re

lati

ve

Ab

un

da

nce

m-, p-Xylene

o-XyleneStyrene

m/z=77.50-78.50+90.50-91.50+103.50-104.50+105.50-106.50

Figure 1: Summed ion chromatograms of BTEXS ions for olive oil. Note the scale difference (NL Dynamic 5.5e7, NL Static 5.50e5).

Table I: Normalized data of seven olive oil samples

Compound Method S1 Light S1 Pure S1 Ex Vir S2 S3 S4 S5

BenzeneStatic 100.0 35.8 13.5 14.4 18.3 12.8 15.7

Dynamic 100.0 67.4 51.7 57.7 77.7 42.4 73.5

TolueneStatic 11.6 25.7 68.6 37.0 100.0 48.2 28.4

Dynamic 6.9 22.5 65.8 42.2 100.0 48.0 32.0

EthylbenzeneStatic 0.0 34.0 36.5 23.0 100.0 50.1 42.1

Dynamic 7.0 43.2 45.3 34.9 100.0 57.4 19.6

m,- p-XyleneStatic 1.8 21.0 46.4 32.5 100.0 55.7 14.1

Dynamic 4.0 29.6 46.9 32.9 100.0 57.4 18.3

o-XyleneStatic 0.0 26.0 45.2 29.8 100.0 54.4 15.8

Dynamic 1.7 31.0 41.2 29.1 100.0 52.0 20.3

StyreneStatic 0.0 4.4 100.0 17.1 15.3 51.8 10.7

Dynamic 0.8 11.0 100.0 19.2 16.8 54.2 14.8

THE APPLICATION NOTEBOOK – FEBRUARY 201230 Food & Beverage

Blueberries are extracted into acidified methanol and

analyzed for anthocyanins with a methanol/formic

acid gradient via UHPLC/MS; four different Agilent

ZORBAX RRHD stationary phases are quickly evalu-

ated. The Phenyl-Hexyl column separates the most

anthocyanin peaks from the blueberry sample, while

the SB-Phenyl is the most orthogonal of the columns.

Advancements in liquid chromatography have led to sig-

nificantly improved sample throughput. fle Agilent 1290

Infinity UHPLC and Agilent ZORBAX Rapid Resolution High

Definition (RRHD) columns are manufactured to withstand

pressures up to 1200 bar. flis allows the use of faster Tow rates

and rapid column screening to easily take advantage of stationary

phase selectivity differences during method development.

Advancements in liquid chromatography have led to signifi-

cantly improved sample throughput. fle Agilent 1290 Infinity

LC System and Agilent ZORBAX Rapid Resolution High Defini-

tion (RRHD) columns are manufactured to withstand pressures

up to 1200 bar. flis allows the use of faster Tow rates and rapid

column screening to easily take advantage of stationary phase se-

lectivity differences during method development.

Two different phenyl columns are currently available within the

ZORBAX RRHD family, Eclipse Plus Phenyl-Hexyl and StableBond

SB-Phenyl. Both phases excel with the analysis of anthocyanins due

to the compounds’ abundant conjugation. fle π electrons in double

bonds in these compounds interact with the π electrons in the phenyl

stationary phase, providing a unique selectivity mechanism over tradi-

tional alkyl phases, such as C18 (3). While the π-π interactions are not

as strong as the hydrophobic interactions responsible for retention with

alkyl phases, they may provide slight selectivity advantages for phenyl

columns when analyzing closely related conjugated compounds.

Experimental

An Agilent 1290 Infinity LC System with an Agilent G6410A

Triple Quadrupole Mass Spectrometer was used in this experiment.

Mobile Phase A – 5% formic acid in water

B – methanol

Flow rate 0.65 mL/min

Gradient 10–50% B in 15 min

Sample 5 µL injection of blueberry extract

TCC 30 °C

A Variety of Agilent ZORBAX RRHD Phases Offers Selectivity Options for the Determination of Anthocyanins in Blueberries with UHPLC/MSAnne E. Mack, Agilent Technologies, Inc.

HO

OH

OH

R3

R2

R1

O

+

Figure 1: Chemical structures of five common anthocyanidins found in blueberry extract.

Table I: Total number of blueberry anthocyanin peaks resolved by four Agilent ZORBAX RRHD columns with a methanol/formic acid gradient

CompoundAgilent ZORBAX Eclipse Plus C18

Agilent ZORBAX Eclipse Plus Phenyl-Hexyl

Agilent ZORBAX StableBond SB-Aq

Agilent ZORBAX StableBond SB-Phenyl

Cyanidin, m/z 286 11 13 10 10

Peonidin, m/z 300 9 8 9 10

Delphinidin, m/z 302 12 13 12 11

Petunidin, m/z 316 9 12 9 9

Malvidin, m/z 330 6 6 6 6

Total number of resolved peaks 47 52 46 46

Anthocyanidin R1 R2 R3 MW

Cyanidin OH OH H 287

Delphinidin OH OH OH 303

Peonidin OCH3 OH H 301

Petunidin OCH3 OH OH 317

Malvidin OCH3 OH OCH3 331


Agilent Technologies, Inc.2850 Centerville Road, Wilmington, DE 19808

tel. (800) 227-9770, fax (302) 633-8901

Website: www.agilent.com

12

Eclipse Plus C18

Eclipse Plus phenyl-Hexyl

StableBond SB-Aq

StableBond SB-Phenyl

x10

0.95

0.9

0.85

0.8

0.75

0.7

0.65

0.6

0.55

0.5

0.45

0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0

0.5 1.5 2.5 3.5 4.5 5.5 8.5

Counts (%)vs.Acquisition Time (min)




7.5 8.5 9.5 10.5 11.5 12.5 13.5 14.52 3 4 5 8 7 8 9 10 11 12 13 141

0.5 1.5 2.5 3.5 4.5 5.5 8.5 7.5 8.5 9.5 10.5 11.5 12.5 13.5 14.52 3 4 5 8 7 8 9 10 11 12 13 141

0.5 1.5 2.5 3.5 4.5 5.5 8.5 7.5 8.5 9.5 10.5 11.5 12.5 13.5 14.52 3 4 5 8 7 8 9 10 11 12 13 141

0.5 1.5 2.5 3.5 4.5 5.5 8.5 7.5 8.5 9.5 10.5 11.5 12.5 13.5 14.52 3 4 5 8 7 8 9 10 11 12 13 141

0.95 1

2

21

1

x10

x10

0.9

0.85

0.8

0.75

0.7

0.65

0.6

0.55

0.5

0.45

0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0

0.95

0.9

0.85

0.8

0.75

0.7

0.65

0.6

0.55

0.5

0.45

0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0

0.95

x1012

1

0.9

0.85

0.8

0.75

0.7

0.65

0.6

0.55

0.5

0.45

0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0

1

Figure 2: Extracted ion chromatograms from LC–MS scan data of blueberry anthocyanins.

MS MS2 Scan: 200–1000, ESI posi-

tive mode

Scan time 100 ms, 0.2 amu step

Fragmentor 180 V

Drying gas 10 L/min, 350 °C

Nebulizer pressure 50 psig

Capillary voltage 3500

EICs for anthocynadins Cyanidin m/z 286

Delphinidin m/z 302

Peonidin m/z 300

Petunidin m/z 316

Malvidin m/z 330

Four Agilent ZORBAX RRHD 2.1 × 100 mm, 1.8 µm columns

were used:

Eclipse Plus C18 (p/n 959758-902)

Eclipse Plus Phenyl-Hexyl (p/n 959758-912)

StableBond SB-Aq (p/n 858700-914)

StableBond SB-Phenyl (p/n 858700-912)


Ten grams of blueberries are extracted into acidified methanol

and analyzed by LC–MS. A rapid screening of these four columns

is possible within this 1200 bar system and column pressure limit.

With this methanol/formic acid gradient at 0.65 mL/min, the

maximum pressure generated is 1020 bar, and each analysis is ac-

complished in only 15 min. Total ion chromatograms (TIC) are

generated from a scan from 200–1000 with the blueberry extract

on each column. Extracted ion chromatograms (EIC) reveal the

glycosides and acylglocysides of five common anthocyanidins.

fle EICs in Figure 1 clearly show the distinct glycosides and ac-

ylglycosides of the five diTerent anthocyandins, each marked with

a unique color. By looking at the EICs it is easy to see many of the

smaller peaks in the chromatogram, several of which coelute with

larger peaks which would not be recognizeable with UV or TIC

chromatograms. From these EICs, the total number of resolved an-

thocyanin peaks is determined and summarized in Table I. flis data

shows that the Eclipse Plus Phenyl-Hexyl column resolves a few more

anthocyanin peaks with this methanol/formic acid gradient than the

other three phases. To see more details and exhibits, reference the full

application note on www.agilent.com (publication 5990-8470EN).

References

(1) R.L. Prior, S.A. Lazarus, G. Cao, et al., “Identification of procyanidins

and anthocyanins in blueberries and cranberries (vaccinium spp.) using

high performance liquid chromatography/mass spectrometry” J Agric.

Food. Chem. 49(3), 1270–6 (2001).


Cofiee carbohydrates constitute the major part (at least 50% of

the dry weight) of raw cofiee beans, and are good tracers for

assessing the authenticity of instant cofiee.

Currently, the Association of Oflcial Analytical Chemists (AOAC)

Method 995.13 — which is based on high-performance anion-

exchange chromatography with pulsed amperometric detection

(HPAE-PAD) — is used to determine carbohydrates in instant cofiee.

Tis study ffrst tested Method 995.13 on a Termo Scientiffc Di-

onex ICS-3000 system. A few modiffcations to the oflcial method

are proposed to achieve separation of two pairs of sugars that are oth-

erwise diflcult to resolve. A fast method using the Termo Scientiffc

Dionex CarboPac SA10 column was also tested.

Both methods described provide good sensitivity and consis-

tent response. Te fast method is recommended when rapid sepa-

ration is desired, keeping in mind that two pairs of sugars are not

resolved. In applications where all 11 common cofiee carbohy-

drates need to be resolved, Method 995.13 (with minor modiffca-

tions) is recommended.

Equipment

Te experimental setup and the sample preparation procedures

are described in Dionex Application Note 280 (now part of Ter-

mo Fisher Scientiffc, Inc.).

Results

Figure 1A shows the separation of the carbohydrates present in a mix

of standards. Rhamnose-arabinose and sucrose-xylose are not com-

pletely resolved. Te oflcial method suggests excluding rhamnose

from the mixed standard solution and performing 2–3 injections of

the standard solution, or increasing the re-equilibrium time in order

to achieve separation of the above-mentioned pairs. As an alternative,

the column temperature may be lowered (15 °C) to achieve separation

of all 11 carbohydrates (Figure 1B), albeit with increased run time.

Te ffrst set of coeluting peaks (rhamnose and arabinose) was re-

solved by eluting with 10 mM hydroxide for the ffrst 6 min, then

switching to DI water (Figure 1C). Only the mobile phase was modi-

ffed; all other chromatography conditions were the same as in Method

995.13.

Using the fast method, the mixture of cofiee carbohydrate stan-

dards separated on a Dionex CarboPac™ SA10 column (Figure

1D) in 8 min. Note that two pairs of sugars coelute.

For both methods, precisions ranged from 0.13–1.8% for retention

time, 1.05–5.4% for peak area, and 70-127% for the average recovery

of the sugars.

Summary

Both methods are sensitive, accurate, reliable, and difier primarily

in their total analysis time and peak resolutions for cofiee carbo-

hydrate determinations.


Chromatography Methods for Determining Carbohydrates in Coffee Lipika Basumallick and Jeffrey S. Rohrer, Thermo Fisher Scienti�c

Thermo Fisher Scienti�c, Inc. (formerly Dionex Corp.)1228 Titan Way, P.O. Box 3603, Sunnyvale, CA 94088

tel. (408) 737-0700, fax (408) 730-9403


0 1 2 3 4 5 6 7 8 Minutes

47

nC 1

2

7 4

3, 5

68

9

10, 11

0 10 20 30 40 50 60 70 -55

250

nC

1

2 3

4 6

8 9

Column Wash

C

A

B

D

5

3, 4

10 11

9 10 11 8 7

6 5 3 4

5 6 7

8 9 10

11

55

Figure 1: Chromatogram of mixed coffee carbohydrate stan-dards: (A) Method 995.13; (B) Method 995.13, but with column temperature 15 °C; (C) Method 995.13, but with 10 mM hydrox-ide for 6 min, and sucrose not included in mix of standards (su-crose usually not present in instant coffee extracts); (D) the fast method. Chromatograms A, B, and C were collected with a Di-onex CarboPac PA1 column set, and D with a Dionex CarboPac SA10. Peaks: 1. Mannitol; 2. Fucose; 3. Rhamnose; 4. Arabinose; 5. Galactose; 6. Glucose; 7. Sucrose; 8. Xylose; 9. Mannose; 10. Fructose; 11. Ribose.


By identifying maturation tracers and molecules commonly

responsible for taste defects, gas chromatography–mass spec-

trometry (GC–MS) augments expert opinion with objective and

quantitative information. When using a solid phase micro extrac-

tion (SPME) method, GC–MS requires very small sample sizes and

a minimum of sample preparation while providing rapid analysis of

target molecules. GC–MS can provide an automated technique with

repeatable results for detecting all of these compounds.

Extracted wine samples were analyzed by a sequential full-scan/

SIM acquisition on a GC–MS system consisting of a fiermo Scien-

tiflc ISQ Single-Quadrupole GC–MS and a TRACE GC Ultra Gas

Chromatograph. fie results were compared to the sensitivity limits

of human tasters. fiis method allows wine makers to obtain precise

measurements on the organoleptic parameters that determine wine

purity on site rather than having to send samples for expensive,

external analysis.

Method Conditions and Sample Preparation

fie experimental conditions and sample preparation are de-

scribed in Application Note 52242, fiermo Fisher Scientiflc, Inc.

Conclusion

GC–MS detects several contaminants in wine at lower concen-

trations than the limit of human tasters, and its ease of use with

single-step, 2-min sample preparation make it a useful tool for the

wine industry. fie sequential full-scan/SIM acquisition method

for detecting the impurities requires minimal training of person-

nel to provide accurate results. Also, this general method may be

improved or customized to particular wines by incorporating new

parameters.

fie wine, champagne, and spirit market can be well served by

analytical chemistry tools such as GC–MS. By conducting their

product analysis on site, wine and other spirit producers avoid

their recipes being compromised when outsourcing this analysis.

Also, analyzing competitors’ products using a GC–MS can help

producers quantify what makes one wine superior to another.


Impurities in Wines by GC–MS Benedicte Gauriat-Desroy, Eric Phillips, Stacy Crain, and Trisa Robarge (with special thanks to members of OEnologic Center of Grezillac), Thermo Fisher Scienti�c

Thermo Fisher Scienti�c, Inc. (formerly Dionex Corp.)2215 Grand Avenue Parkway, Austin, TX

tel. (800) 532-4752, fax (561) 688-8731

Website: www.thermoscienti�c.com

Figure 1: 4-Ethylgaiacol from 50 to 100 μg/L: Y = 1.31e2X2 + 8.069e5X – 3.862e6; R2: 0.9967; Origin: Ignore; W: 1/X; Area.

Figure 2: Geosmine 112 from 10 to 50 ng/L: Y = -2.591e0X2 + 2.543e4X + 2.694e5; R2: 0.9995; Origin: Ignore; W: 1/X; Area.


Omega-3 and -6 fatty acids are essential compounds required

for normal growth. Omega-3 consumption is purported to

have a number of health benefits, e.g., cancer prevention, car-

diovascular disease prevention, and improved immune function.

Although both omega-3 and -6 fatty acids can give rise to eico-

sanoid-signaling molecules (prostaglandins, prostacyclins, throm-

boxanes, and leukotrienes), the omega-6 eicosanoids are generally

proinflammatory. A person’s diet requires a balance of omega fatty

acids at suitable levels.

Omega lipids found in foods are commonly determined after

extraction, hydrolysis, and derivatization for measurement by gas

chromatography (GC). Analyte derivatization can adversely af-

fect temperature-sensitive functional groups on specialized lipids.

High-performance liquid chromatography (HPLC) with ultra-

violet detection at low wavelengths limits solvent selection and

increases the likelihood of matrix interference.

Here is a direct inverse-gradient HPLC method with charged

aerosol detection. Fourteen underivatized standard omega acids

were identified — along with a number of other analytes —

with near-baseline resolution. Tis method was used to measure

omega-3, -6, and -9 fatty acids in traditional and commercially

produced meat, fish, oils, and over-the-counter supplements.

Charged aerosol detection is mass sensitive and provides the most

consistent response for nonvolatile and some semivolatile ana-

lytes of all HPLC detection techniques. It works by measuring

the charge induced on analyte particles and is not dependent on

light scattering, which has large variability and generally lower

sensitivity.

Charged aerosol detection has been successfully used to charac-

terize lipids of all classification, including phospholipids (reversed

and normal phase), acylglycerides, phytosterols, and free fatty

alcohols. Tis method complements the free-fatty acids method

using higher specificity for these analytes on a newly developed

Termo Scientific Acclaim C30 reversed-phase column.


Te experimental setup and sample preparation procedures are

described in Poster Note LPN 2931-02, Termo Fisher Scientific,

Inc. (formerly Dionex Corp.).

Conclusions

After saponification to release the fatty acids, samples were neutral-

ized and directly analyzed for omega-3, -6, and -9 fatty acids using

this HPLC method with charged aerosol detection. The method

can be used to analyze omega fatty acids from a variety of sources.

The mobile phase is compatible with mass spectrometry, allowing

the possibility of identifying unknown analytes.


Quantitation of Omega Fatty Acids by HPLC and Charged Aerosol Detection Marc Plante, Bruce Bailey, and Ian Acworth, Thermo Fisher Scientific

Thermo Fisher Scientific, Inc. (formerly Dionex Corp.)1228 Titan Way, P.O. Box 3603, Sunnyvale, CA 94088

tel. (408) 737-0700, fax (408) 730-9403

Website: www.thermoscientific.com/dionex

6.1 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 -2.1

10

20

30

40

50

61.5

pA

1 2 3

4

56 7

8

9

101112

13

14

15

1617

18

19

202122

2324

2526

27

28

29

30

31

32

3334

38

3735

36

Minutes

Figure 1: HPLC/charged aerosol detection of fish oil, prepared as described with addition of 200 µL Isopropanol to aid in solubility. A total of 38 peaks were identified, with all 14 standards present. Column: Acclaim™ 120 C30, 3 µm, 3 × 250 mm, 30 °C. Mobile Phase A: Water/Acetic Acid/Mobile Phase B (900:3.6:100). Mobile Phase B: Acetone/Acetonitrile/Tetrahydrofuran/Acetic Acid (675:225:100:4). Gradient: 0–1 min 0–60% B; 13 min 70% B; 22 min 95% B; 24 min 95–0% B; 29 min 0% B. Inverse Gradient: 0–0.8 min 0% A; 0.8–1.8 min 0–60% A; 13.8 min 70% A; 22.8 min 95% A; 24.8 min 95–0% A; 29.8 min 100% A. Flow Rates: 1 mL/min. Inj. Volume: 20 µL. Nebulizer Temp: 15 °C. Detection: Thermo Scientific Dionex Corona Ultra RS Charged Aerosol Detector.


U.S. EPA Method 531.2 describes the determination of widely

used carbamate pesticides in raw surface water using HPLC

with fiuorescence detection following postcolumn derivatization,

which enhances method sensitivity and selectivity as compared to

UV absorbance detection. Per European Union (EU) regulation

98/83/EC for pesticides in drinking water, the maximum admissible

concentration of each individual pesticide component is 0.1 µg/L,

and the total concentration is not to exceed 0.5 µg/L. Detection of

these regulated compounds at such low concentrations poses a chal-

lenge for many water testing laboratories using EPA Method 531.2.

flis work describes a faster and more sensitive method for the

determination of the carbamates speciTed in EPA Method 531.2.

Separation is performed on a flermo ScientiTc Acclaim Carba-

mate column with detection using a flermo ScientiTc Dionex

FLD-3400RS Fluorescence Detector. Following separation, the

carbamates are derivatized with by o-phthalaldehyde (OPA) and

detected by fiuorescence. Baseline separation of these carbamate

compounds is achieved within 20 min with resolutions (Rs) ≥1.5.

Equipment

fle experimental setup and sample preparation procedures are de-

scribed in Application Update 177, flermo Fisher ScientiTc, Inc.

(formerly Dionex Corp.).


Chromatography

Figure 1 trace C illustrates good separation of the carbamates listed

in EPA Method 531.2 using the Acclaim™ Carbamate column, de-

signed for the baseline separation of these carbamates. Resolution

(Rs) for all peaks is ≥1.5, exceeding the EPA Method required values

(≥1.0). fle mixture of carbamates was added to a tap water sample

at a concentration of 0.4 µg/L each, demonstrating method sensitiv-

ity. Traces A and B show deionized water and the tap water sample,

respectively. No detectable carbamates were found in the tap water.

Minimum Detection Limits (MDLs)

Per EPA Method 531.2, nine replicate injections of reagent water

fortiTed with 0.2 µg/L of carbamate standard mixture were used to

determine the MDLs. fley ranged from 0.004 to 0.010 µg/L, four

to ten-fold lower than reported the 531.2. fle improved MDLs may

be attributed to the improvements in fiuorescence detector sensitiv-

ity and reversed-phase column technology since the original EPA

work was completed. fle EPA method uses a 4 µm, 3.9 × 150 mm

column, while this method used a 3 µm, 3.0 × 150 mm column to

yield more effcient peaks. flese improved detection limits easily

allow the analyst to reach the minimum reporting limits of the

original method.

Conclusion

Described here is an optimized method for determining carbamates in

drinking water on a Dionex HPLC system with a 3 µm Acclaim Car-

bamate column. Improvements in MDLs (≤0.01 µg/L) and quantiT-

cation limit (0.05 µg/L) signiTcantly exceed the values in EPA Method

531.2 and meet the requirements of EU 98/83/EC, making it ideally

suited for determining carbamates in drinking water.


Faster, More Sensitive Determination of Carbamates in Drinking Water Chen Jing, Xu Qun, and Jeffrey S. Rohrer, Thermo Fisher Scienti�c

Thermo Fisher Scienti�c, Inc. (formerly Dionex Corp.)1228 Titan Way, P.O. Box 3603, Sunnyvale, CA 94088

tel. (408) 737-0700, fax (408) 730-9403


0

1

2

3 4

5

6 7

8

9

10

11

12

13

14

15

16

-1500

0

3500

counts

a

1

2

3

4

5

6

7 8

9

10

11

12

b

c

Minutes

Figure 1: Chromatograms of (A) deionized water, (B) tap water, and (C) sample B forti�ed with a carbamate standard mixture with 0.4 μg/L for each carbamate. Column: Acclaim Carbamate, 3.0 × 150 mm, 3 μm. Mobile Phase: Methanol–H2O. Fluorescence Detection: Excitation/330 nm and Emission/465 nm. Peaks: 1. Al-dicarb sulfoxide; 2. Aldicarb sulfone; 3. Oxamyl; 4. Methomyl; 5. 3-Hydroxy carbofuran; 6. Aldicarb; 7. Propoxur; 8. Carbofuran; 9. Carbaryl; 10. 1-Naphthol; 11. Methiocarb; 12. BDMC (I.S.).


Various triarylmethane and fiuorescent dyes are used in aquacul-

ture as antibacterial, antifungal, and anthelmintic agents for the

treatment of flsh disease. Te FDA has banned the importation of cer-

tain flsh from several countries due to possible contamination of these

food products. Most published methods only detect malachite green,

crystal violet and their metabolites. UCT has developed a procedure

for the extraction and analysis of nine dyes used in aquaculture using

a carboxylic acid ion-exchange SPE cartridge.

1) Sample Preparation

a. Prepare tilapia fish tissue (purchased at a local grocery store) by homogenization with dry ice.

b. Add 100 µL of each dye standard (50 µg/mL) to 1 g of fish tissue.

c. Vortex 30 s.d. Add 10 mL of acetonitrile/100 mM ammonium acetate buf-

fer at pH 3 (90:10, v/v).e. Vortex for 30 s.f. Centrifuge at 4500 rpm for 5 min and use the supernatant

for analysis.g. Add 10 mL of 100 mM phosphate buffer (pH 7.0) to the

supernatant (1:1 dilution).h. Prepare the vacuum manifold or automated SPE system using

EUCCX156 cartridge(s).

2) Condition Cartridges (no vacuum used)

a. Add 3 mL of methanol to each cartridge.

b. Add 3 mL of reagent grade water to each cartridge.

c. Add 1 mL of 100 mM phosphate buffer (pH 7.0).

3) Extract Sample (no vacuum used)

a. Extract sample using 1–2 mL/min flow rate.

4) Wash Cartridge

a. Add 3 mL of 100 mM phosphate buffer solution to cartridge.

b. Slowly draw through.

c. Dry cartridge for 10–15 min at full vacuum.

5) Elute Dye Analytes

a. Add 3 mL of (95:5) methanol/acetic acid solution (adjusted

to pH 2.5).

b. Collect eluate using 1–2 mL/min fiow rate.

c. Evaporate eluate to dryness under N2 at <40 °C.

d. Redissolve residue using 500 µL of methanol:H2O (50:50, v/v).

LC/UV Analysis

Instruments

Waters 1525 Binary HPLC Pump, 717plus autosampler and 487

UV Detector (other instrumentation may be used).

LC/UV Parameters:

Column: Selectra C18, 150 × 2.1 mm, 3 µm particle size

(or equivalent)

Injection: 20 µL, Oven temp: 20 °C.

Data Mode: Dual absorbance: 265 and 600 nm.

Pump Mode: Gradient

Mobile Phase:

(A) DI H20 with 10 mM ammonium acetate and 5 mM octanesulfonic acid

(B) Methanol with 10 mM ammonium acetate and 5 mM octanesulfonic acid

Conclusion

Water soluble dyes used in fish aquaculture can be readily ana-

lyzed with good recovery using UCT’s EUCCX156 carboxylic

acid cation exchange SPE cartridge. No oxidation to the leuco

metabolites is required prior to LC/UV detection. An LC–

MS-MS method is being developed to detect the dyes at lower

concentrations according to FDA and EU criteria and will be

published shortly.

Determination of Dyes in Fish Tissue by HPLC/UVDaniel A. Fonseca, Brian Kinsella, Thomas August, and Craig A. Perman, UCT, LLC

UCT, LLC 2731 Bartram Road, Bristol, PA 19007

tel. (800) 385-3153; Email: [email protected]

Website: www.unitedchem.com

Figure 1: % Recovery of dyes from �sh.

Table I: Dyes included in study

Malachite Green Azure B

Leucomalachite Green Nile Blue A

Crystal Violet Victoria Blue B

Leucocrystal Violet Methylene Blue

Brilliant Green

0.022

0.020

0.018

0.016

0.014

0.012

0.010

0.008

0.006

0.004

0.002

0.000

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

Time (min)

AB LCV CV NB BG VBMB MG LMG

Ab

sorb

an

ce (

Au

)

22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

THE APPLICATION NOTEBOOK – FEBRUARY 2012 Pharmaceutical 37

Using a novel new-design monolithic silica column (Chromo-

lith® HighResolution RP-18e), fast, high resolution separations

were achieved for various drug mixtures without the high

pressures characteristic of modern particulate technology.

Monolithic silica technology has evolved as a significant development

in fast HPLC analysis over the last decade. As the true alternative

to conventional particle-packed column technology, the columns are made

of a continuous single piece of high purity porous silica. A sol-gel synthesis

process is used, leading to single-particle rod columns, which possess a bi-

modal pore structure with macro- (flow path) and meso- (active site) pores

in the 1 µm and 140 Angstrom range. High performance results from the

permeability efficiency of the fixed flow pathways in the highly-controlled

porosity of the synthesized rigid silica skeleton. Te resulting very low op-

erating backpressures compared to closely-packed spherical particles allow

for more flexible flow rates and solvent choice and enable high throughput

analysis without loss of separation efficiency and peak capacity. Monolithic

silica columns are increasingly being used for quality control of drugs. Easy

and cost-effective method transfer offers it great advantages over other trends

toward fast HPLC analysis. Flow programming with monolithic columns

provides additional reduction of analysis time. Since there is no particle bed

to disturb, pressure shocks do not affect its performance or lifetime. Te pos-

sibility for direct injection of biological fluids without pretreatment has made

it more applicable for bioanalysis (1). To date, hundreds of papers have been

published describing the use of monolithic columns in various analytical

fields which include routine drug analysis and QC, food and environmental

analysis, natural products analysis and bioanalysis (2).

In this application note a separation of drug substances is presented. Seven

beta-blockers are baseline separated in 4 min at 2 mL/min flow rate with just

76 bar backpressure (Figure 1). Even short columns have sufficient resolu-

tion to separate five drugs in < 3 min (Figure 2). Both separations possess

high efficiency and high peak symmetry. Both of these applications can easily

be adapted to MS detection, by simply adding a post-column flow splitter to

a MS detector for further characterization of the API.

Conclusions

Separation of drugs is easily achieved on a newly designed monolithic silica

Chromolith® HighResolution columns. Tis application note illustrates

that ultra-high efficiencies could be reached without ultra-high pressures.

References

(1) E. Machtejevas and K.K. Unger, in Proteomics Sample Preparation, Sample prepa-

ration for HPLC – based proteome analysis, Jörg von Hagen, Ed. (Wiley-VCH,

Weinheim, Germany, 2008), pp. 245–264.

(2) M. Taha, A. Abed, and S. El Deeb, in Monolithic silicas in separation science, Quality control of drugs, K.K. Unger, N. Tanaka, and E. Machtejevas, Eds. (Wiley-VCH, Weinheim, Germany, 2011), pp. 189–206.

New Generation Monolithic Silica Columns for Fast, High Resolution Drug Separations without High PressuresKarin Cabrera and Egidijus Machtejevas, Merck Millipore

Merck KGaAFrankfurter Str. 250

D 64293 Darmstadt, Germany

Figure 1: 1 µL injection of seven beta-blockers (atenolol 100 µg/mL, pindolol 300 µg/mL, metoprolol 155 µg/mL, bisoprolol 100 µg/mL, labetalol 200 µg/mL, propanolol 20 µg/mL, alprenolol 150 µg/mL) in mobile phase on a 100 × 4.6 mm Chromolith® HighResolution RP-18e column using a 23:77 (v/v) acetonitrile and 20 mM potassium dihydrophosphate buffer (pH adjusted to 2.5 with phosphoric acid) mobile phase at 2 mL/min flow rate. UV detection at 230 nm, column temperature 25 °C.

Figure 2: Separation of five drugs on Chromolith® HighResolution RP-18e 25-4.6 mm column. Chromatographic conditions: 2 μL injec-tion of 100 ppm of actaminophen (98 µg/mL), quinine (152 µg/mL), salicylic acid (140 µg/mL), diltiazem (81.2 µg/mL), verapamil (98.4 µg/mL) in mobile phase; linear acetonitrile gradient from 1% to 60% water with 0.1% TFA in 1.8 min; flow rate 2 mL/min; column back-pressure 31 bar; UV detection at 230 nm, column temperature 25 °C.

THE APPLICATION NOTEBOOK – FEBRUARY 201238 Pharmaceutical

Tetracycline (TC), a common antibiotic used to treat urinary

tract infections, acne, gonorrhea, and other conditions, yields

a toxic degradation product, 4-epianhydrotetracycline (EATC).

General Chapter 226 of the U.S. Pharmacopeia and National

Formulary (USP-NF), referred to by monographs for epitetra-

cycline and drug products containing tetracycline hydrochloride

(TC-HCl), prescribes an antiquated assay for EATC impu-

rity in TC. fiis method uses a self-packed column eluted with

chloroform. fie EATC impurity elutes as a yellow band and is

detected by its visible absorbance.

fie TC-HCl drug substance monograph and some TC-

HCl-containing drug products use a high-performance liquid

chromatography (HPLC) method. fiis antiquated method, used

to determine the amount of EATC and to assay TC, uses a large

resin bead size HPLC column and the undesirable mobile phase

component dimethylformamide.

fiis study reports HPLC and ultra HPLC (UHPLC) methods

for assay of EATC in TC-containing drug products. After devel-

oping the HPLC method, it was transferred to a UHPLC system,

which reduced the run time from 8 to 2 min. Both methods were

evaluated using a TC-HCl drug product (TC-HCl capsules). fie

results from both methods exceed the speciflcations of the HPLC

method in the USP TC-HCl monograph.


fie experimental setup and sample preparation procedures are

described in Dionex Application Note 288 (now part of fiermo

Fisher Scientiflc, Inc.).


Separation

The Thermo Scientif ic Acclaim PA2 column was chosen

for this study because it contains a polar-embedded station-

ary phase with similar selectivity to a standard C8 or C18

stationary phase, but can be used under both lower and higher

pH conditions and with highly aqueous conditions. Using the

Acclaim™ PA2 and a gradient separation with an ammonium

dihydrogen phosphate pH 2.2/acetonitrile mobile phase, TC and

EATC were well resolved, but in the opposite elution order com-

pared to the L7 column used in the TC-HCl monograph (Figure

1). After first developing the separation on a 3 µm, 4.6 × 150 mm

PA2 column, the method was transferred to a 2.2 µm, 2.1 × 100

mm Acclaim PA2 column.

Conclusion

This study describes HPLC and UHPLC methods that can be

used to assay TC and EATC in a TC-HCl capsule. Both methods

meet or exceed the criteria in the appropriate USP monographs,

are faster and show higher resolution, generate less waste, and use

less hazardous organic solvents. These methods may be applicable

to other TC-HCl-containing drug products.


New HPLC/UHPLC Assay Methods for Impurities in Tetracycline

Suparerk Tukkeeree and Jeffrey S. Rohrer Thermo Fisher Scienti�c

Thermo Fisher Scienti�c (formerly Dionex Corp.)1228 Titan Way, P.O. Box 3603, Sunnyvale, CA 94088

tel. (408) 737-0700, fax (408) 730-9403


-100

200

400

600

800

1,000

12

Ab

sorb

an

ce (

mA

U)

A1,200

Minutes

-100

200

400

600

800

1,000

1,200

Ab

sorb

an

ce (

mA

U)

2

1

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

B

Figure 1: UHPLC Determination of EATC in a TC-HCl capsule. Panel A shows the system suitablility solution and Panel B shows the prepared TC-HCl capsule sample. Column: Acclaim PA2, 2.2 μm, 2.1 × 100 mm. Eluent: A: 20 mM NH4H2PO4, pH 2.2. B: 50% CH3CN in 20 mM NH4H2PO4, pH 2.2. Gradient: 30% B -1–0 min, ramp to 100% B in 1.4 min, hold 100% B 0.2 min, and return to 30% B in 0.1 min. Flow Rate: 0.6 mL/min. Inj. Volume: 1 μL. Tem-perature: 40 °C. Tray Temp.: 10 °C. Detection: UV 280 nm, data collection rate 25 Hz, response time 0.5 s. Sample A: Mixture of TC and EATC standards. Sample A Peaks: 1. TC 100 conc. (mg/L); 2. EATC 25 conc. (mg/L). Sample B: TC-HCI capsule. Sample B Peaks: 1. TC 497 conc. (mg/L); 2. EATC 0.98 conc. (mg/L).

THE APPLICATION NOTEBOOK – FEBRUARY 2012 Pharmaceutical 39

A liquid chromatography tandem mass spectrometry

method for enalapril and enalaprilat from human

plasma has been developed using Thermo Scientific

SOLA cartridges. Sample preparation is fast, efficient,

and reproducible giving excellent recovery levels for

each compound.

SOLA™ is a revolutionary Solid Phase Extraction (SPE) product.

Tis first in class SPE product range introduces next-generation,

innovative technological advancements, giving unparalleled perfor-

mance characteristics compared to conventional sample preparation

techniques

SOLA products have significant advantages for the analyst when

processing compounds in complex matrices particularly in high

throughput bioanalytical and clinical laboratories where reduced fail-

ure rate, higher analysis speed and lower sample/solvent requirements

are critical. SOLAs’ increased performance gives higher confidence in

analytical results and lowers cost without compromising ease of use or

requiring complex method development.

Advantages of SOLA products ensure good recoveries, accuracy,

linearity and precision with a reduction in elution solvent volume,

hence reduced solvent costs and subsequently reduced drying times.

Experimental Details

Sample Pretreatment

Prepare a mixed standard spiking solutions in methanol (enalapril and

enalaprilat).

Prepare a working internal standard solution in water (benazepril).

Take 200 µL of blank human plasma or sample.

For standards and quality control (QC) samples add 10 µL of stan-

dard spiking solution for all other samples add 10 µL of methanol.

For standards and QC’s and unkown’s add 10 µL of working

internal standard solution for blanks add 10 µL of water.

Add 4 µL of formic acid.

Mix well.

Sample Preparation

Compound(s): enalapril, enalaprilat, benazepril (IS)

Matrix: Human plasma

Cartridge type: Thermo Scientific SOLA 10 mg/1mL

p/n 60109-001

Conditioning stage: 1 mL methanol, 1 mL water

Application stage: Load and allow to flow under gravity

Washing stage: 200 µL 0.1% formic acid in water

Elution stage: 2 × 200 µL 2% ammonia solution in methanol

Additional stage: Dry and reconstitute in 200 µL 90:10 (v/v)

water/methanol. Sonicate for 5 min.

Separation Conditions

Instrumentation: Termo Scientific Accela 600

Column: Hypersil GOLD, 1.9 µm, 50 × 2.1 mm

p/n 25002-052130

Mobile phase A: water + 0.1% formic acid

Mobile phase B: acetonitrile + 0.1% formic acid

Gradient: 10–100%B in 1 min


Column temperature: 70 °C

Injection vol: 2.5 µL

Results

Extracted enalapril and enalaprilat standards from human plasma

were linear over the dynamic range of 1 and 100 ng/mL with an

r2 correlation of 0.9986 and 0.9974 . QC samples analyzed at a

concentration of 50 ng/mL (n=6). Precision and recovery data is

shown in Table I.

Conclusions

SOLA SPE cartridges and Hypersil GOLD HPLC allow robust

and efficient analysis of enalapril and enalaprilat from human

plasma to be carried out simply and quickly with excellent

reproducibility and recovery being obtained.

LC–MS-MS Method for the Determination of Enalapril and Enalaprilat from Human Plasma Using SOLA

Ken Meadows, Thermo Fisher Scientific, Runcorn, Cheshire, UK

Thermo Fisher Scientific (formerly Dionex Corp.)1228 Titan Way, P.O. Box 3603, Sunnyvale, CA 94088

tel. (408) 737-0700, fax (408) 730-9403

Website: www.thermoscientific.com/dionex

Table I: Precision and recovery data

Standard AnalyteAverage

Calculated Concentration

Average %Diff

Precision (%CV)

% Recovery

Enalapril 49 -15 6.6 81

QC 50ng/mL Enalapril 53 5.9 3.5

QC 50ng/mL Enalaprilat 46 -7.3 6.6 85

QC 50ng/mL Enalaprilat 48 -4.5 0.5

THE APPLICATION NOTEBOOK – FEBRUARY 201240 Pharmaceutical

Liposomes are made of lipid bilayers and are often used in drug

delivery by encapsulating the core with therapeutic drugs.

During liposome research, formulation, manufacturing, and

quality control, it is of great importance to monitor liposome size

and encapsulation. Field fiow fractionation (FFF) with the con-

comitant use of multi-angle light scattering (MALS) and quasi-

elastic light scattering (QELS, aka dynamic light scattering) is an

ideal tool for such characterization.

Here, we report the analytical results of two liposome samples,

one empty, and one fllled. Using the Eclipse FFF system followed

by a DAWN HELEOS (with embedded WyattQELS instrumen-

tation), the FFF method was optimized with the aid of Wyatt

ISIS FFF simulation software. ffie online QELS directly mea-

sures the hydrodynamic radius, Rh, whereas the HELEOS mea-

sures the root-mean square radius, Rg.

ffie WyattQELS detector was placed at approximately 143° in

order to extend the Rh measurement up to 300 nm. Both Rg and Rh

are plotted against elution time in Figure 1. ffie results from dupli-

cate runs demonstrate excellent reproducibility of the FFF–MALS–

QELS method. Figure 1 also shows that the Rh values for both empty

and fllled liposomes are well overlaid, suggesting the separation is

based on hydrodynamic radius as expected from an FFF separation.

However, Rg values for these two liposomes do not overlay, which

indicates these two liposomes have diTerent degrees of encapsulation.

Root-mean square radii, Rg, were then plotted against hydro-

dynamic radii, Rh, for these two liposomes. ffie slope of Rg vs.

Rh plot yields the internal structure of the liposomes. ffie empty

liposome sample has a slope of 1.0, consistent with a spherical

shell structure. ffie fllled liposome sample, on the other hand,

has a slope of 0.75, in good agreement with that of a solid sphere

structure of uniform density.

For liposomes or other nanoparticles, FFF–MALS–QELS

provides an easily adaptable yet powerful characterization tool

to obtain information on particle size, size distribution, particle

count, as well as structure — all without making assumptions

about the particles or their composition.

DAWN®, miniDAWN®, ASTRA®, Optilab® and the Wyatt Technology logo

are registered trademarks of Wyatt Technology Corporation. ©2011 Wyatt

Technology Corporation.

Liposome Characterization by FFF–MALS–QELSWyatt Technology Corporation

Wyatt Technology Corporation6300 Hollister Avenue, Santa Barbara, CA 93117

tel. (805) 681-9009, fax (805) 681-0123

Email: [email protected], Website: www.wyatt.com

60.0

20.0 30.0 40.0

hydrodynamic radius (nm)

RMS radius vs. Rh Plot

rms

rad

ius

(nm

)

50.0 60.0

1.01±0.02

1.03±0.02

0.76±0.04

0.74±0.03

50.0

40.0

30.0

20.0

100.0

hydrodynamic radius vs. time

hyd

rod

yn

am

ic r

ad

ius

(nm

)

rms

rad

ius

(nm

)

80.0a b

60.0

40.0

20.0

0.0

100.0

rms radius vs. time

80.0

60.0

40.0

20.0

0.0

14.0 16.0 18.0

time (min)

20.0 22.0 24.0

LS LS

14.0 16.0 18.0

time (min)

20.0 22.0 24.0

Figure 2: Root-mean square radius, Rg, plotted against hydrody-namic radius, Rh, for empty liposome sample (red) and fllled lipo-some sample (green). The slopes for empty and fllled liposomes are 1.0 and 0.75, respectively.

Figure 1: Hydrodynamic radius (a) and root-mean square radius (b) plotted against elution time overlaid with 90° LS signals for empty liposome sample (red) and fllled liposome sample (green). The Rh and Rg values are determined by the respective QELS and MALS detectors. The results from duplicate runs of each sample are shown here to demonstrate the reproducibility of the FFF–MALS–QELS analysis.

THE APPLICATION NOTEBOOK – FEBRUARY 2012 Polymer 41

Size exclusion chromatography (SEC) is a simple HPLC tech-nique used for the molecular weight (MW) distribution analy-

sis of polymers. Several manufacturers provide SEC columns with different base materials and various pore sizes to cover a wide range of polymer analysis. Commonly available aqueous SEC column al-lows analysis of polymers with maximum MW (i.e., exclusion lim-it) around the range of 8–15 million dalton (Da). Obviously, tar-get polymers that have a larger MW than SEC columns’ exclusion limit cannot be determined accurately. Shodex™ OHpak SB-807 HQ is the only aqueous SEC column that offers exclusion limit of 500 million Da. Tis is advantageous for the MW determination of very large polymers. Below are examples showing analysis of poly-acrylamide and hyaluronic acids using SB-807 HQ.


1. A comparison analysis was carried between OHpak SB-807 HQ (8.0 × 300 mm, 35 µm, 30,000 Å) and OHpak SB-806 HQ (8.0 × 300 mm, 13 µm, 15,000 Å). Sample tested for the com-parison was polyacrylamide, having weight-average (Mw) of 5.5 million Da. Te following conditions were used: Mobile phase, 0.2 M NaCl (aq); column temperature, 30 °C; flow rate, 0.5 mL/min; detector, MALS and RI; and injection volume 100 µL.

2. Analysis of hyaluronic acid. Hyaluronic acids having Mw great-er than 1 million Da were obtained from three different sources. Tey were analyzed by SB-807 HQ under following conditions: Mobile phase, 0.2 M NaCl (aq); column temperature, 30 °C; flow rate, 0.5 mL/min; detector, RI; and injection volume 100 µL.

Results

Te RI chromatogram obtained by SB-806 HQ showed an unsymmetrical peak that people may consider a result of tailing (Figure 1). However, this peak shape is a result of high-MW end polyacrylamide being eluted all at the same time; as it was over the exclusion limit of SB-806 HQ (~20 million Da). Tis is also confirmed by the MALS plot showing no elution prior to 6 mL, but rather a rapid elution just after 6 mL. Meanwhile, the peak ob-tained by SB-807 HQ was symmetrical showing an accurate MW distribution of the sample. Also, the larger particle size of SB-807 HQ prevented potential shear degradation of macro polymers.

Figure 2 shows analysis of three different hyaluronic acid sam-ples. All of them showed symmetrical peaks indicating that even the largest part of the sample was within the exclusion limit of SB-807 HQ analysis range.

In both applications, sodium salt in the mobile phase helped prevent association and ionic repulsion of the sample. Type and concentration of salt should be optimized for each analyte. Recommended sample concentration for analysis is <0.02% and <0.05% for compounds having MW >3 million Da and >2 million Da, respectively.

Conclusions

Shodex™ OHpak SB-807 HQ is ideal for the MW distribution analysis of macro polymers with a MW over 15 million Da that commonly available-SEC columns from other manufacturers cannot resolve. Te solvent durability of SB-807 HQ (maximum 30% methanol and acetonitrile with a working pH range 3–10) is also beneficial for the analysis at different conditions.

Analysis of Over 15 Million Dalton Polymers by Aqueous SEC Method

Kanna Ito, Shodex™/Showa Denko America Inc.

Shodex™/Showa Denko America Inc.

420 Lexington Avenue Suite 2850, New York, NY, 10170

tel. (212) 370-0033 x109, Fax: (212) 370-4566

Website: www.shodex.net

1.0×103

1.0×107

1.0×106

1.0×105

4.0 6.0 8.0Volume (mL)

SB-806 HQSB-807 HQ

10.0 12.0M

ola

r M

ass

(g

/mo

l)

0.04

0.02

LS,

Au

x (

V)

0.00

-0.024.0 8.0 12.0 Volume (mL)

Sample ASample BSample C

Figure 1: SB-807 HQ shows an accurate MW distribution of polyacrylamide sample having Mw 5.5 million Da. For conditions refer to the text.

Figure 2: Analysis of three hyaluronic acids having Mw over 1 million Da by SB-807 HQ. For conditions refer to the text.

TM

THE APPLICATION NOTEBOOK – FEBRUARY 201242 General

GC×GC-TOFMS provided a comprehensive analysis of

tobacco smoke. Individual smoke constituents across

several target compound classes were extracted,

chromatographically resolved, then identified and

quantified with mass spectral detection.

Tobacco pyrolysis produces harmful vapors that contain toxic

and carcinogenic components. Characterization of these

components is challenging because tobacco smoke is a complex

mixture containing compounds from several chemical classes.

Tis complexity has traditionally required multiple analysis meth-

ods along with considerable sample clean-up to target compound

classes individually. GC×GC provides improved peak capacity

and low level detection to measure multiple compound classes

simultaneously, thus minimizing sample clean-up and the need

for multiple analyses.

Experimental

Smoke was generated from Kentucky 3R4F reference cigarettes

with an automated smoking machine per ISO smoking condi-

tions (Arista Laboratories, Richmond, Virginia). Smoke con-

stituents were collected on Cambridge filter pads, extracted into

methanol, and analyzed with LECO’s Pegasus® 4D as follows:

Injection: 1.5 µL splitless with inlet at 250 °C

Carrier Gas: He at 1.0 ml/min, corrected constant flow

Columns: 30 m Rtx-5Sil MS (0.25 mm × 0.25 µm) with 1.5 m

Rtx-200 (0.18 mm × 0.20 µm) (Restek, Bellefonte, Pennsylvania)

Temperature Program: 3 min at 45 °C, ramped 8 °C/min to

300 °C, held 10 min; secondary oven +10 °C

Modulation: 3 s (temperature +25 °C from main oven)

Transfer Line Temperature: 280 °C

MS Acquisition: 33–400 m/z at 200 spectra/s with source at 250 °C

Data processing: ChromaTOF® software

Results

A TIC contour plot of tobacco smoke extract is shown in

Figure 1A. Deconvolution via ChromaTOF isolated individual

analytes from the matrix for identification and quantification by

mass spectral matching and peak area/height, respectively. Several

target compounds were identified and analytes were grouped by

compound classes through ChromaTOF’s Classification tools.

Peak marker color, in Figure 1B, indicates approximate class

assignment. Representative reference standards were also analyzed

to generate calibration information for quantification. Linear

calibrations ranged from 1 ppb to 50 ppm, analyte dependent.

Te calibrations were applied to quantify compounds extracted

from the filter in the smoke extract data. Typical masses were in

the µg range.

Discussion

GC×GC-TOFMS provided a comprehensive analysis of tobacco

smoke across several compound classes. Characterization was

accomplished with a single separation lasting under 45 min.

Individual constituents of smoke were efficiently isolated from the

complex matrix with sufficient resolution to identify and quan-

tify representative analytes from many of the target compound

classes. Full mass range TOFMS acquisition allowed for positive

identification of both target and non-targeted compounds

through mass spectral matching to data base standards. TOFMS

detection also offered quantitative calibration of representative

analytes from the target compound classes, using ChromaTOF’s

Calibration Feature. Tis methodology reduces the need for

time consuming sample clean-up and/or repeat injections that

individually target each compound class.

Analysis of Multiple Classes of Cigarette Smoke Constituents by GC×GC-TOFMS

Elizabeth Humston-Fulmer and Joe Binkley, LECO Corporation

LECO Corporation3000 Lakeview Avenue, St. Joseph, MI 49085

tel. (269) 985-5496, Fax (269) 982-8977

Website: www.leco.com

Figure 1: Representative chromatogram (A) with analytes grouped based on their compound class (B), using ChromaTOF’s Classification Feature.

THE APPLICATION NOTEBOOK – FEBRUARY 2012 General 43

UHPLC columns can significantly improve chromatographic

separations, but they also present unique challenges. Once

the UHPLC system components are optimized, perhaps your

greatest concern is protecting the column from the damaging ef-

fects of microparticulates and sample contaminants.

An ultra-high performance column protection system, specifi-

cally designed for UHPLC systems using sub-2 µm and core-shell

particle columns, can be used to extend column lifetime (sav-

ing both money and time through less frequent column replace-

ment), while minimizing system troubleshooting and downtime.


It is well known that UHPLC systems and columns require higher

levels of care and attention than traditional HPLC in order to reap

their full ultra-high chromatographic performance benefits. Once

system components are optimized, chromatographic cleanliness is

vital to maintain UHPLC performance, and column protection

is compulsory. Unprotected columns may suTer from reduced

performance, lifetime, and may lead to an increased need for

system troubleshooting and/or downtime.

UHPLC columns packed with sub-2 µm particles tend to clog

much more rapidly than traditional HPLC columns packed with

larger 3 µm and 5 µm particles. flis may be due to the fact that, not

only is the interstitial space between the particles much smaller, but

columns packed with sub-2 µm particles also use frits with a much

smaller porosity compared to conventional HPLC columns. With a

“tighter”, more restricted ffiow path, any undissolved matter or par-

ticulates from the sample, the mobile phase, or the system (such as

micro-particulates shedding from piston seals and injection valve ro-

tors), will quickly and irreversibly foul your UHPLC column.

An easy way to extend the performance and lifetime of your

UHPLC columns (either sub-2 µm fully-porous or core-shell media)

is to prevent any contaminants from reaching the column by using

the SecurityGuard™ ULTRA guard cartridge system (Figure 1).

Presented in Figure 2 is an accelerated lifetime test using an en-

dogenous biological matrix injected onto a core-shell column (Ki-

netex 2.6 µm C18 50 × 4.6 mm column). With the unprotected

column (grey dots), sequential injections of the matrix lead to a

steady and irreversible increase in backpressure. Without Securi-

tyGuard ULTRA column protection, the increase in backpressure

becomes exponential. flis increase in backpressure will eventual-

ly lead to degraded chromatography, including band broadening

and possibly peak splitting. As a result, method sensitivity, quan-

titation, and peak identification may also be adversely aTected.

However, column lifetime is greatly extended by using the Se-

curityGuard ULTRA (red boxes). In this case, sequential injec-

tions of the matrix will still lead to an increase in pressure, but

this is due to the particulates being captured in the SecurityGuard

ULTRA itself, rather than in the UHPLC/HPLC column. flus,

by simply replacing the SecurityGuard ULTRA cartridge at regu-

lar intervals, backpressure returns to starting levels and eTective

column lifetime can be greatly extended.

UHPLC Column Protection Dramatically Extends UHPLC Column Performance and LifetimeJ.T. Presley III, Tom Cleveland, and Jeff Layne, Phenomenex, Inc.

Phenomenex, Inc.411 Madrid Avenue Torrance, CA 90501

tel. (310) 212-0555; (310) 328-7768


Figure 1: Scanning electron microscopy of non-contaminated and contaminated column inlet frit.

Figure 2: SecurityGuard ULTRA cartridges protect UHPLC, sub-2 µm and core-shell columns from microparticulates and chemical contamination, as well as sample matrix fouling. The result is in-creased column lifetime, longer performance, and more repro-ducible chromatography.


Resistive glass tubes and plates are designed to guide

ions by generating a uniform electric field. PHOTONIS

resistive glass products are composed of a proprietary

lead silicate glass that has been specially processed

to create a resistive layer at the surface. The resistiv-

ity can be varied over several orders of magnitude to

suit the specific application.

Resistive glass is manufactured by using a hydrogen firing pro-

cess to create an integral semi-conductive layer on the sur-

face. Tis reduced lead silicate layer is typically several hundred

angstroms thick. Resistive glass can be formed into plates, tubes,

cylinders, sheets, washers, or other shapes. Te products are resis-

tant to scratches from light to moderate abrasions, and can easily

be cleaned ultrasonically with water, acetone, methanol, or IPA

without degrading the performance.

Application

One application of resistive glass is capillary inlet tubes for at-

mospheric pressure ionization sources. Single capillary inlet tubes

made from resistive glass significantly improve ion transfer efl-

ciency when compared to conventional quartz inlet tubes. Volt-

age applied across nickel-chromium electrodes at each end of the

inlet tube creates an electric field that preferentially attracts either

positive or negative ions. Polarity switching can also be accom-

plished more quickly than with conventional inlet tubes.

Te properties of resistive glass help prevent ions from collid-

ing with the tube walls and with each other, reducing ion loss and

resulting in a more eflcient sample transfer by forcing more ions

into the mass spectrometer.

PHOTONIS has also developed a multicapillary resistive glass

inlet tube. A proprietary multibore extrusion process creates a cir-

cular array of six individual channels in the same footprint as a

single capillary inlet tube (see Figure 1).

Multicapillary resistive glass inlet tubes provide increased sen-

sitivity by further improving ion transmission when compared to

single capillary inlet tubes.

Tubes made from resistive glass can also be used in other mass

spectrometry applications, such as for drift tubes, collision cells,

ion mirrors, voltage dividers, or reffiectron lenses.

Another application of resistive glass is for use in ion mobility

spectrometry drift tubes. Resistive glass drift tubes operate on the

same principle as capillary inlet tubes, and demonstrate a similar

improvement in ion transmission. The solid tube body also

provides containment for counter-ffiow gas, eliminating the need

for an additional enclosure.

Results

An increase in ion transfer eflciency by a factor of 100 has been

reported from using PHOTONIS single capillary inlet tubes.

An increase in ion transmission of up to 10× using multicap-

illary tubes when compared to single capillary inlet tubes has been

achieved by a leading mass spectrometer manufacturer, dramati-

cally enhancing instrument sensitivity. Resistive glass multicapil-

lary tubes therefore provide an increase in ion transfer eflciency

of up to 1000× when compared to conventional quartz tubes.

A demonstrated improvement in ion transmission is also real-

ized with the use of single-piece construction resistive glass IMS

drift tubes when compared to traditional multipiece lens and

ring assemblies.

Resistive glass reffiectron tubes provided equal or better perfor-

mance in an orthogonal TOF system. Tis comparison showed

superior resolution, indicating better energy focusing, while spec-

tra between the two were nearly identical.

Overall, resistive glass tubes offer benefits to a variety of mass

spectrometer applications, many of which can be realized by re-

placing an existing tube with one made from resistive glass.

Resistive Glass Inlet Tubes Increase Ion Throughput

Paula Holmes, PhD and Bruce N. Laprade, Photonis USA

Photonis USA660 Main Street, Sturbridge, MA 01566

tel. (508) 347-4000, Fax: (508) 347-3849

Website: www.photonis.com

Figure 1: PHOTONIS’ multi-capillary resistive glass inlet tubes of-fer six individual channels in a standard footprint.

THE APPLICATION NOTEBOOK – FEBRUARY 2012 General 45

Synthetic fused silica capillary tubing is used in the

manufacture of most gas chromatography (GC) col-

umns. In this note, we discuss recent advances in the

dimensional control of the most popular tubing sizes.

Ahistorical review of the dimensional specifi cations for T ex-

ible synthetic fused silica capillary reveals a clear efl ort to

reduce both i.d. and o.d. tolerances. Previous work summarized

the changes in published specifi cations and presented empirical

data on i.d and o.d. variability (1). Additional studies examined

in-draw stability (2). Efl orts to further improve the dimensional

stability of tubing used to manufacture GC columns is warranted

by the potential for improved chromatographic performance, as

well as by ease of column use as the industry moves toward plug

and play concepts. ffi is application note provides a comparative

review of these critical dimensional parameters, with a focus on

the i.d.

Experimental

Capillary tubing employed in this study included TSP250350

(0.25 mm i.d.), TSP320450 (0.32 mm i.d.), and TSP530660

(0.53 mm i.d.). ffi e tubing was manufactured using current

draw conditions and operating parameters. Of noted difl erence

to previous work, improvements have been implemented in the

integration of new control limit concepts and trend monitoring

software algorithms.

Measurements were collected using an Olympus PME3 inverted

microscope, which was calibrated and confi gured as reported previ-

ously (1). Gauge R&R studies continue to be conducted so that op-

erator contributions to variability are understood.

Over the period of the study samples were collected and measured

from both ends of every spool of material produced. ffi e study time

frame ensured a minimum of 1400 sample values (N >1400).

Results

A summary of both past and present statistical data for the capil-

lary i.d. can be found in Table I. Mean values are on target for

all products studied. ffi e standard deviation on all products has

decreased, indicating an improvement in process control. ffi e

Cpk values for i.d. obtained in the past study ranged from 1.02

for 0.53 mm tubing to 1.23 for 0.25 mm tubing. ffi ese have

improved to 1.12 and 1.55, respectively. Recall that Cpk quanti-

fi es the process capability and is defi ned as the ratio of tolerance

to 6σ. A Cpk value of 1.0 or better suggests that the process is

in control. ffi e reader is reminded that both ends of every spool

produced is physically measured and must be within specifi cation

for release to the market. For comparative purposed, a histogram

of TSP320450 is shown in Figure 1.

Data on o.d. has shown similar improvements, although they

have not been as dramatic. It should be noted that primary efl orts

have focused on reducing i.d. variability.

Conclusion

Continued efl orts by Polymicro’s production team have resulted

in an overall improvement in both i.d. and o.d. variation, with i.d.

standard deviations being reduced by as much as 20%. Polymicro

continues to evaluate its manufacturing and metrology processes

in an efl ort to insure the tightest tolerances in the industry.

References

(1) J.Macomber, P.Nico, and G.Nelson, LCGC App.Ntbk. June 2004, p. 71.

(2) J.Macomber and P.Nico, LCGC App. Ntbk. Feb. 2005, p. 55.

Improved Internal Diameter Control of Tubing Used for Gas ChromatographyJoe Macomber and Roland Fischer, Polymicro Technologies, a Subsidiary of Molex

Polymicro Technologies, a Subsidiary of Molex18019 North 25th Avenue, Phoenix, AZ 85023

tel. (602) 375-4100, Fax: (602) 375-4110

Website: www.polymicro.com

315 317 319 321 323 325

USLLSL

Figure 1: Histogram of TSP320450 i.d. data, N = 2094.

Table I: Summary of GC tubing study results, past and present

Product Current Specification 2004 i.d. Statistical Data 2011 i.d. Statistical Data

ID Mean Std Dev Cpk Mean Std Dev Cpk

TSP250350 250 ± 6 250.4 1.62 1.23 250.5 1.29 1.55

TSP320450 320 ± 6 320.5 1.76 1.14 320.9 1.44 1.39

TSP530660 536 ± 6 536.4 1.97 1.02 536.3 1.78 1.12


0 10 20

Min

2

3 4

5

6

1

7

8

9

Gradient: Min %A %B

0 65 35

3 65 35

13 55 45

15 55 45

Supelco/Sigma-Aldrich595 North Harrison Road, Bellefonte, PA 16823

tel. (800) 359-3041, (814) 359-3041

Website: sigma-aldrich.com/analytical

HPLC Method for the Separation of Nine Key Components of Milk Thistle

Dave Bell, Supelco/Sigma-Aldrich

Silymarin, derived from the milk thistle plant, has been used as

a natural remedy for the treatment of a number of liver diseases

as well as in the protection of the liver from potential toxins. More

recently, silymarin has been connected to antitumor promoting ac-

tivity (1). It is therefore of interest to have available analytical meth-

ods for the analysis of silymarin components in various matrices.

fie silymarin complex consists of several closely related, biologi-

cally active components. fie objective of this study was to investigate

several modern stationary phase chemistries for optimal selectivity

toward the development of an eTcient method for the analysis of

silymarin. fie method would optimally provide baseline resolution

for nine of the known components of silymarin in less than 15 min.

Initial screening experiments were used to select a single com-

bination of stationary phase and organic modifler that demon-

strates the greatest potential for the separation. fie information

from subsequent optimization experiments was processed with

simulation software to predict an optimized set of conditions. Fi-

nally the predicted conditions were conflrmed using the analysis

of an herbal supplement obtained from a local drug store.

Experimental

fie screening protocol utilized flve Ascentis Express stationary phases

including C18, C8, RP-Amide, Phenyl-Hexyl, and pentaffiuorophen-

ylpropyl (F5). Gradient elution with two distinct organic modiflers

was performed. Injections of a standard containing nine of the known

constituents of silymarin were made under each condition.

fie HPLC screening method employed gradient elution from

5–95% of either methanol or acetonitrile. fie aqueous compo-

nent of the mobile phase was 0.1% formic acid (pH 2.6).


Using a simple peak counting approach, the combination of the

C18 stationary phase along with methanol as the organic modi-

fler resulted in the most visible peaks with nine. Other combina-

tions of stationary phase and organic modifler produced between

six and eight peak responses.

Four separate chromatographic runs using the C18 stationary

phase and methanol modifler were conducted where the gradient

slopes were varied (5% and 10% ramp) at two different tempera-

tures (30 °C and 60 °C). fie data was then analyzed using ACD

LC-Simulator to predict the most suitable optimized conditions.

Because the suggested conditions ran to just 45% organic, an ad-

ditional step to 100% organic was included to elute potential hy-

drophobic components from a natural sample.

Finally an herbal supplement labeled as containing milk thistle along

with dandelion, fennel, and licorice was extracted using water:ethanol

50:50, v/v and analyzed. As shown, all nine of the targeted compo-

nents could be observed in the herbal supplement material.

Conclusions

Stationary phase screening at the onset of method development, es-

pecially when dealing with a complex set of analytes, provides a fac-

ile means of analytical method development. As shown in the study,

a few short experiments, coupled with powerful prediction software

provided chromatographic conditions suitable for the analysis of

biologically active milk thistle components. fie developed condi-

tions should prove useful in natural supplement, raw material and/

or biological monitoring of silymarin complex components.

Reference

(1) J.I. Lee, B.H. Hsu, D. Wu, and J.S. Barrett, J. of Chromatog. A 1116,

57–68 (2006).

Figure 1: Identi�cation of silymarin components from a commer-cial natural supplement. Column: Ascentis Express C18, 10 cm × 3.0 mm I.D., 2.7 µm particle size; Mobile Phase A: water with 0.1% formic acid; Mobile Phase B: methanol; Flow Rate: 0.6 mL/min; Temp.: 35 °C; Det.: UV, 254 nm; Injection: 20 µL; Sample: herbal supplement extracted in 50:50, water:methanol. Peak IDs: 1. Taxi-folin, 2. Silychristin, 3. Apigenin-7-D-glucose, 4. Silydianin, 5. Quer-citrin, 6. Silybin A, 7. Silybin B, 8. Isosilybin A, 9. Isosilybin B.


Wei Zou*, Hagai Yasuor†, Albert J. Fischer†, and Vladimir V. Tolstikov‡

*California State Department of Toxic Substances Control and †Department of Plant Sciences, University of California. ‡UC Davis Genome Center. Direct correspondence to: [email protected].

Trace Metabolic Profiling and Pathway Analysis of Clomazone Using LC–MS-MS and High-Resolution MS

Detection, analysis, and characterization of low-abundant metabolites

remain an unresolved problem in metabolic studies. In this study,

we report a novel approach to address this challenge. The current

methodology is derived from the predictive multiple reaction monitoring

(pMRM) mode available on triple-quadrupole linear ion trap mass

spectrometry (MS) systems. The pMRM mode offers the highest

sensitivity among various acquisition modes for studying trace levels

of metabolites of the herbicide clomazone in plants. Additionally,

this method allows for the identification of positional isomers of

metabolites. Unknown metabolites were further identified and validated

by obtaining accurate masses and isotopic ratios using selected ion

monitoring (SIM) and data-dependent MS-MS scans on high-resolution

liquid chromatography (LC)–MS. During structural assignment of its

metabolites, the unique chlorine isotopic signature of clomazone was

used as a naturally occurring label to study MS-MS fragmentations.

Clomazone (2-[(2-chlorophenyl)

methyl]-4,4-dimethyl-3-isoxa-

zolidinone), a carotenoid biosyn-

thesis inhibitor, has been used as an her-

bicide for the last four years in California

to control late watergrass (Echinochloa phyl-

lopogon [Stapf] Koss). Clomazone detoxi-

fication is known in other species and soil

microorganisms (1,2). Biotransformation

of clomazone in rice and early watergrass

has also been investigated with radiolabeled

clomazone, which resulted in schemas of

biotransformations and lists of metabolites

(3). However, the radiolabeling technique

is not sensitive enough to reveal physiologi-

cal important metabolites and cannot offer

structural information on detected entities.

Currently, the analysis of low-abun-

dant metabolites represents an unre-

solved problem in metabolic profiling.

Although multiple techniques have

been used to conduct metabolic profil-

ing (4,5), LC–MS is considered the most

promising platform (6,7) because of its

superior sensitivity, selectivity, flexibility,

and wide-range of metabolite detection.

However, LC–MS profiling performed

in full scan mode, in spite of deliver-

ing overwhelming numbers of metabo-

lites detected, is not sensitive enough

to detect and characterize metabolites

at trace levels. In contrast, liquid chro-

matography–tandem mass spectrometry

(LC–MS-MS) provides excellent sen-

sitivity in multiple reaction monitoring

(MRM) mode, but suffers from a lack

of structural information and metabolite

coverage.

Recently, a hybrid MS system com-

bining a triple-quadrupole (QQQ)

scanning functionality with sensitive

linear ion trap (LIT) scans was made

commercially available (see Experimen-

tal). Working in LIT mode, the system

provides improved performance and

enhanced sensitivity in full scan (EMS)

THE APPLICATION NOTEBOOK – FEBRUARY 201148

and product ion scan (EPI) modes.

Additionally, the instrument can operate

under all the triple-quadrupole scanning

modes including MRM, precursor, and

constant neutral loss scanning. There-

fore, accurate quantification and addi-

tional structural information can be

obtained simultaneously in a single run

by combing MRM and EPI scanning

via the built-in information-dependent

acquisition (IDA) functionality.

MRM-IDA-EPI methods have recently

been applied in drug discovery (9–13).

In addition to previously described meth-

ods, predictive multiple reaction moni-

toring (pMRM) technology utilizing

new predicting algorithms has been used

in the present study (14–17). The pMRM

algorithms generate theoretical metabo-

lite MRMs based on the latest updated

database sets of well-known Phase I and

Phase II biotransformations. More than

80 biotransformations of exogenous

metabolites were described (15). Inter-

estingly, the new pMRM approach can

identify positional isomers that are help-

ful in identification of positional changes

in metabolism. For example, clomazone

has a molecular ion [M+H]+ mass charge

ratio of m/z 240, and the fingerprint frag-

ment is m/z 125, representing the phenyl

ring substructure. Di-oxidation adds two

oxygen atoms on the parent molecule.

After the collision of the parent ion in

Q2, the fingerprint fragment may be m/z

125 + 2 × 16, m/z 125 + 1 × 16, and m/z

125, corresponding to di-OH-clomazone

isomers with two, one, and zero oxygen

atoms on the phenyl ring substructure of

the clomazone.

Detection and characterization of

metabolites are not straightforward pro-

cesses. In many cases, LC–MS-MS meth-

odology is insufficient to fully describe

the molecule of interest. The MS system

operates at unit mass resolution, which is

not sufficient to deduce unique elemen-

tal composition of unknown metabo-

lites. Accurate mass measurements and

MS-MS fragmentation patterns may help

with structural assignments. Accurate

mass measurement using high-resolution

MS is necessary for validating newly

identified secondary metabolites. High

mass accuracy and high resolving power

can be obtained (for example, <5 ppm;

maximum resolving power 100,000 at

m/z 400, full width at half maximum

oN

o

+H+

+H+

+H+

+NH

π

π

N

CI

100 24007973

24207618

125.01580

127.01285

128.01621

126.01916

128.07115

129.07451

130.07540

130.02292

24008249

2430795424503318

75

50

25

0

100

75

50

100

75

50

25

0

123 124 125 126 127128 129 130131132133134 135136137138 139140141 142143

25

0

235

120 121122 123124 125126 127128129130 131132 133 134 135136 137138139140

236 237238 239 240241242 243 244245246247 248249250251252253 254255

CI

CI

CI

CI

m/z 125.01525

i

i

rHR

rHc

rHc

+

+H+

¬H+

CI

CI

¬H+

OH+

+

o

oN

o

o

o

Cl

+NH

o

N

o o

o

++

o

iN N

o

o

oN

o

ClH+

o

m/z 240.07858

m/z 240.07858

m/z 240.07858

m/z 240.07858

m/z 240.07858

m/z 240.07858

m/z 240.07858

m/z 128.07060m/z 240.07858

Figure 1: Clomazone and its theoretical fragmentation pattern depicted with the assistance of the Mass Frontier software.

100

90

80

70

60

50

40

30

20

10

0

130000

120000

110000

100000

90000

80000

70000

60000

50000

40000

30000

20000

10000

0

130000

120000

110000

100000

90000

80000

70000

60000

50000

40000

30000

20000

10000

0

1.10 2.88

14.41

13.30

10Time (min)

010Time (min)

0

120 125 130m/z

122.273 130.273 237.455

m/z

240 245

244.000

242.091

240.091

243.000

128.091

127.091

125.091

0.52

Rela

tive a

bu

nd

an

ce

Inte

nsi

ty

Rela

tive a

bu

nd

an

ce

100

90

80

70

60

50

40

30

20

10

0

14.41

Inte

nsi

ty

Figure 2: LC–MS chromatograms of the in-source clomazone fragmentation experi-ment demonstrating unique chlorine signature found in clomazone M0 parent and daughter ions.


[FWHM]); however, the data acquired

with high-resolution MS are insufficient

for assigning unique elemental composi-

tions without supporting information on

isotope ratios (18,19).

The isotopic abundance pattern serves

as a powerful additional constraint for

identification of candidates with very

similar elemental composition. Previous

studies demonstrated that interpretation

of isotopic abundance patterns removes

more than 95% of false candidate for-

mulas for molecules smaller than 500

Da (18,19). It was concluded that instru-

ments with 3 ppm mass accuracy and

2% relative error for isotopic abundance

pattern outperformed those with <1 ppm

accuracy that do not include isotope

information in the calculation of molec-

ular formulas. Self calibrated lineshape

isotope profile search (sCLIPS) algo-

rithms corrected the instrument line-

shape and enabled exact isotope model-

ing when comparing the MS response

of an unknown ion against theoretically

calculated responses for all possible can-

didate formulas for data acquired in

continuous mode. As long as the isoto-

pic pattern can be measured accurately,

sCLIPS algorithms can be used to pre-

dict elemental composition of any mol-

ecule. Actually, our recent experimental

data supported the use of exact isotope

modeling as a key filter for the unique

elemental formula assignment (20).

In the present study, we developed a

workflow that takes advantage of sensi-

tive MS hybrid hardware and feature-rich

software, successfully identifies several

low-abundant secondary metabolites,

and constructs metabolic biotransforma-

tion pathways of the herbicide clomazone

in E. phyllopogon plants (21,22).

Experimental

Chemicals and Materials

LC–MS-grade acetonitrile was purchased

from Burdick and Jackson (VWR Inter-

national, West Chester, Pennsylvania);

the purity of each lot was investigated

by LC–MS infusion. Extrapure formic

acid was purchased from Fluka (Sigma-

Adrich, St. Louis, Missouri). The herbi-

cide clomazone and 5-hydroxy clomazone

were obtained from FMC (Princeton,

New Jersey). Pure water was supplied by

a Milli-Q gradient A10 unit (Millipore

Corporation, Billerica, Massachusetts)

with a residual total organic carbon con-

tent of <5 ppb. Fresh aqueous buffers

for LC–electrospray ionization (ESI)-

MS were prepared on the working day.

Also, on each working day, a clomazone

stock solution (0.1 mg/mL) for tuning

was prepared freshly in a solvent system

with identical composition to the initial

LC mobile phase. A 0.2 mg/mL reserpine

stock solution was made in methanol.

Sample Preparation

Fresh leaf samples (100 mg) from E. phyl-

lopogon plants hydroponically treated at

the two- to three-leaf stage with 50 µM

clomazone for 96 h were mixed with 0.5

mL of 4:3:1 (v/v/v) water–acetonitrile–

isopropanol, and the mixture was placed

into a 2-mL microcentrifuge tube. Tis-

sues were homogenized for 0.5 min at 30

Hz with a stainless steel ball mill (Retsch

MM 301, Retsch GmbH & Co., Ger-

many), the tube holder of which had been

prechilled to –80 °C; the homogenate was

then sonicated in an ultrasonic bath for 1

min, and extracted at 4 °C on an orbital

shaker at 750 Hz for 5 min in the dark.

The mixture was centrifuged at 16,100

rcf for 2 min and the supernatant was

transferred to a clean tube. The pellets

were resuspended in 0.5 mL of the extrac-

tion solvent and mixture, centrifuged at

16,100 rcf for 2 min, and the supernatant

was combined with the initial extracted

volume. Extracts were injected (10 µL) in

the LC instrument.

Reversed-Phase LC–MS

Reversed-phase LC–ESI-MS analysis was

conducted using an Acquity UPLC system

composed of a binary solvent manager, a

sample manager, a column manager, and

a TUV detector (Waters Corporation,

Milford, Massachusetts). The system

was operated in high performance liquid

chromatography (HPLC) mode. Because

clomazone has a phenyl group in its struc-

ture, a 150 mm × 3 mm, 3-µm dp Luna

phenyl-hexyl column (Phenomenex, Tor-

rance, California) was chosen to separate

clomazone and its metabolites assuming

that π-π interactions may provide effi-

cient selectivity. Mobile phases used were

as follows: A, 0.1% formic acid in water;

B, 0.1% formic acid in acetonitrile. After a

2-min isocratic run at 3% B, a gradient to

60% B was concluded at 6 min, and then

ramped to 95% B for up to 15 min. Col-

umn wash with 100% B was followed by

equilibration with 3% B starting at 21 min

100

100

Rela

tive a

bu

nd

an

ce

100

Rela

tive a

bu

nd

an

ce

100

Rela

tive a

bu

nd

an

ce

Rela

tive a

bu

nd

an

ce

7.918.168.27

8.82

9.199.33

9.5 7.9 8.0

8.00

8.18

8.1 8.2 8.3 8.49.08.5

256.07298

258.07001

257.07630

255.17378

255 256 257 258m/z m/z

259 260 271 272 273 274 275 2760

259.07330271.15551

274.06487

272.06786

273.07113

275.06809260.07372

Time (min) Time (min)

8.0

8.56

0

Figure 3: A high-resolution base peak chromatogram and spectrum of the second-ary metabolites of clomazone, illustrating the isotopic pattern at 100,000 resolution obtained using an FT-ICR–MS system.


and maintained until 25 min. All injection

volumes were 10 µL, the column tempera-

ture was 50 °C, and the flow rate was 0.3

mL/min. The weak-wash solvent was 1:1

(v/v) water–methanol and the strong-wash

solvent was 3:1 (v/v) acetonitrile–isopro-

panol. The entire effluent from the LC

column was directed into the ESI source

of an API 4000 QTrap hybrid triple-quad-

rupole linear ion trap mass spectrometer

(Applied Biosystems/MDS Sciex, Foster

City, California) equipped with a Tur-

boIonSpray source (a heated electrospray

source with an orthogonal source of heated

gas to help desolvate the spray). Ion-source

parameters were set manually. The scales

of the horizontal and vertical axes of the

ion source were set at 6. Final TurboIon-

Source parameters were as follows: curtain

gas (CUR), 20 psi; collision gas (CAD),

high; ionSpray voltage (IS), 5.2 kV; tem-

perature (TEM), 300 °C; ion source gas 1

(GS1), 50 psi; ion source gas 2 (GS2), 50

psi; and interface heater (IHE), on.

Acquisition Methods

Using clomazone as the reference, com-

pound-specific parameters were automati-

cally optimized using LightSight software

(V2.0, Applied Biosystems/MDS Sciex).

The automatic method creation tool in

the software was used to generate the

following data: positive mode: predictive

multiple reaction monitoring (pMRM-

IDA), EMS full scan, precursor scan of

125 (the fingerprint fragment of cloma-

zone), precursor scan of 141 (hydroxyl

group added on the fingerprint fragment

of clomazone), neutral loss of 115 scan (the

molecular ion of clomazone is 240, minus

the fingerprint fragment of 125), neutral

loss scan of 129 for unknown glutathione

conjugates; and 2); negative mode: neutral

loss scan of 176 for unknown O-glucuro-

nides, precursor scan of 272 for unknown

glutathione conjugates.

The pMRM-IDA method was com-

posed of one positive MRM scan, one

IDA criteria, and one enhanced product

ion (EPI) scan. The known and described

biotransformations (15) were used to

generate an MRM list. Table I shows a

truncated list of relevant transitions. The

MRM scan had a list of 122 transitions

calculated from the predictive algorithm

embedded in LightSight 2.0 (Table I).

Parameters for the MRM scan were as

follows: declustering potential (DP), 61

V; entrance potential (EP), 10 V; colli-

sion energy (CE), 29 V; and collision cell

exit potential (CXP), 20 V. Q1 was set as

unit resolution and Q3 as low resolution.

Dwell time of each MRM channel was 5

ms and pause time was 2.5 ms. IDA crite-

ria were set as the most intense ion exceed-

ing 500 counts triggering an EPI scan to

confirm charge state and isotope pattern

selection. Parameters for EPI were as fol-

lows: scan mode, profile; scan rate, 4000

amu/s; LIT fill time, 5 ms; dynamic fill

time, on; DP, 40 V; CES, 25 V; CE, 60 V;

CXP, 20 V. Q1 was set as unit resolution.

High-Resolution LC–MS

The HPLC method was the same as

described earlier. The entire effluent from

the HPLC column was directed into the

ESI source of an LTQ-Orbitrap MS sys-

tem (Thermo Fisher Scientific, San Jose,

California) operated under Xcalibur soft-

ware (V2.07, Thermo Fisher Scientific) or

an LTQ-FT Ultra hybrid linear ion trap

7.0 T Fourier-transform ion cyclotron res-

onance (FT-ICR) system (Thermo Fisher

Scientific) operated under Xcalibur soft-

ware (V2.07, Thermo Fisher Scientific).

It was realized by our group that the

Orbitrap system was 10–100 times more

sensitive than the FT-ICR system, but the

latter system could achieve a resolution of

1,000,000 at m/z 400 and had better mass

accuracy (routinely reached to the range

Table I: The predictive MRM (pMRM) transitions list

Q1 Mass (amu) Q3 Mass (amu) Dwell (ms)

Parent Ions Daughter Ions

240.11 125.20 5.00

204.11 125.20 5.00

204.11 89.20 5.00

214.11 125.20 5.00

214.11 99.20 5.00

223.11 125.20 5.00

223.11 108.20 5.00

225.11 125.20 5.00

225.11 110.20 5.00

226.11 125.20 5.00

226.11 111.20 5.00

242.00 125.20 5.00

254.11 125.20 5.00

254.11 139.20 5.00

256.11 125.20 5.00

256.11 141.20 5.00

268.11 125.20 5.00

268.11 153.20 5.00

272.11 125.20 5.00

272.11 141.20 5.00

272.11 157.20 5.00

320.11 125.20 5.00

320.11 205.20 5.00

336.11 125.20 5.00


of 0.1–2 ppm). Therefore, the Orbitrap

system was used to identify low abun-

dant unknown metabolites, whereas the

FT-ICR system was used when maximum

mass accuracy and resolution were needed.

Both systems used identical source and

scanning parameters. The ion source volt-

age was 5 kV. The nitrogen sheath and

auxiliary gas flow were 60 and 20 units,

respectively. Nitrogen was produced by

a nitrogen generator system (Peak Scien-

tific, Billerica, Massachusetts). The ion

Table II: Detected secondary metabolites of clomazone

Name Measured [M+H]+ Transition (Q1/Q3) Metabolite References

M0 240.07858 240/125 23

M1 242.09423 242/125 2

M2 254.05785 254/125 2

M3 256.07350 256/125 2, 24

M4 256.07350 256/141 2, 24

M5 272.06843 272/125 Present study

M6 272.06843 272/141 2

M7 272.06843 272/157 2

M8 268.037112* 268/125 Present study

M9 320.053156* 320/125 Present study

*Calculated


transfer capillary temperature was 300

°C. Typical ion gauge pressure was 0.90

× 10-5. Briefly, the mass spectrometer

was operated in the data dependent mode

to automatically switch between MS and

MS-MS acquisition. Survey full scan

MS spectra (from m/z 200–600) were

acquired with the resolution R = 50,000

(FWHM) in the Orbitrap system and R =

100,000 (FWHM) in the FT-ICR system

at m/z 400 (after accumulation to a “target

value” of 1,000,000 in the linear ion trap

using the automatic gain control (AGC).

The most intense ions were isolated and

fragmented in the linear ion trap using

collisionally induced dissociation (CID) at

a target value of 100,000. All scan events

were acquired with one microscan. Full-

scan spectra were acquired with a 200 ms

maximum ionization time. Parameters

applied in MS-MS scan events were an

isolation width of 2 Da, an activation time

of 30 ms, a normalized collision energy

of 40%, and an activation Q of 0.250.

Dynamic exclusion was used with the fol-

lowing parameters: repeat count, 2; repeat

duration, 15 s; exclusion duration, 45 s.

Structure Elucidation

Based on Mass Spectral Data

Mass spectra of the feature peaks were

spectrally corrected using MassWorks

software (version 2, Cerno Bioscience,

Danbury, Connecticut) to achieve high

mass and spectral accuracy after data

acquisition. MassWorks sCLIPS algo-

rithms corrected the instrument’s line-

shape and enabled exact isotope modeling

when comparing the MS response of an

unknown ion against theoretically calcu-

lated responses for all possible candidate

formulas. The use of exact isotope model-

ing with MassWorks was a key feature to

the unique elemental formula identifica-

tion. The unique elemental formula was

searched against the CAS database (Amer-

ican Chemical Society, Washington, D.C.)

using the strategy of “Explore Substances –

Chemical Structure.” The chemical struc-

tures that corresponded to the elemental

formula were saved and imported to Mass

Frontier 5.0 (HighChem Ltd, Bratislava,

Slovakia) for the MS-MS fragmentation

modeling analysis. The Mass Frontier

“Fragments and Mechanisms” module is

an expert system providing information

about basic fragmentation and rearrange-

ment processes based on literature, start-

ing from a user-supplied chemical struc-

ture. The theoretical fragments generated

by Mass Frontier were compared to those

acquired from LC–MS providing assis-

tance with elucidation of the structures of

feature peaks.


Fragmentation Mechanism of

Clomazone in ESI Positive Mode

Clomazone has one chlorine atom

attached to its phenyl ring and shows

the unique chlorine signature in its mass

spectrum (Figure 1) that can be used to

track clomazone metabolites. During an

LC–MS experiment using an MS system

at unit mass resolution, we demonstrated

that in-source fragmentation of cloma-

zone ([M+H]+ m/z 240) produced the

fragment of m/z 125, which retained the

chlorine signature, suggesting the cleav-

age of the substructure of phenyl ring of

clomazone (Figures 1 and 2). In addition,

it was previously shown in well-estab-

lished environmental LC–MS-MS stud-

ies on water contamination of clomazone

that m/z 240/125 is a typical MRM tran-

sition (21,22). Extracted ion chromato-

grams illustrated that clomazone M0 m/z

240 and its major fragment m/z 125 had

the same retention time, confirming the

origin of the fragment m/z 125 was from

M0 m/z 240 (Figure 2). The theoretical

fragmentation mechanism simulated in

Mass Frontier confirmed the above men-

tioned results. In addition, the theoreti-

cal fragmentation mechanism suggested a

possibility to produce fragment m/z 128

without the chlorine atom in this frag-

ment’s structure (Figure 1). However, in

the clomazone molecule, the C-N bond

was easier to break down than the C-C

bond so that m/z 240/128 was a minor

fragmentation pathway. This minor frag-

mentation pathway explained the inten-

sity elevation in m/z 128 in the lower left

panel of Figure 2. Finally, the fragment

of m/z 125 was used as the fingerprint of

clomazone and its secondary metabolites

during automatic method creation.

Data Acquisition

The following methods were used for

data acquisition: positive mode: pre-

dictive multiple reaction monitoring

(pMRM-IDA), EMS full scan, precur-

sor scan of 125 (the chlorobenzyl ring of

the clomazone molecule), precursor scan

of 141 (the hydroxyl chlorobenzyl ring,

specifically for M4 searching), neutral

loss of 115 scan (the alkyl chain of the

clomazone molecule), neutral loss scan of

129 for unknown glutathione conjugates;

and negative mode: neutral loss scan of

176 for unknown O-glucuronides, pre-

cursor scan of 272 for unknown glutathi-

one conjugates. Among all of the above

methods, pMRM-IDA-EPI methods

detected the greatest number of cloma-

zone-related secondary metabolites, sug-

gesting the highest sensitivity is achieved

with the pMRM technique (21,22). With

HO

HOO

¬

N

O

Cl+

+

+

H¬ +

H

H

H

††

+H

†

+H

+NH

†

†

+H

†

π

HO

HO

HOO

N

O

ClH+

O

O

+NH

Cl

O

O

OOH

ON

0

O

OH

OH

OH

Cl

Cl

Cl

+

OH

O

O

OH

OH+

O

N

N

π

N

N

O

+

Cl

rH

rH

i

rH

rH

+ Cl

Cl

Cl

i

i

O

ClH+

ON

Cl

OH

+H+

+

i

m/z 256.07350

m/z 256.07350

m/z 256.07350

m/z 141.01017m/z 125.01525

m/z 256.07350

m/z 256.07350

m/z 256.07350

m/z 256.07350m/z 256.07350

m/z 256.07350

m/z 256.07350

Figure 4: Theoretical fragmentation mechanism of the secondary metabolites of clomazone, m/z 256, either with a fingerprint fragment of 125 (the hydroxy group on the left ring of the parent compound, M3) or with a fingerprint fragment of 141 (the hydroxy group on the right ring of the parent compound, M4) depicted with Mass Frontier assistance.


prior knowledge of the parent compound

and its fragmentation pathway, the theo-

retical MRM transitions for metabo-

lites were determined, thus generating

a pMRM-IDA method. A complete set

of MRM transitions was generated for

metabolites that are the result of modi-

fications (Table I). Two critical compo-

nents in generating a pMRM method

were the algorithm that generated the

comprehensive MRM transitions and the

new comprehensive biotransformation

sets according to the possible clomazone

biotransformation known from the liter-

ature. These new biotransformation sets

had more than 100 metabolic pathways,

thus enabling better prediction of MRM

transitions of potential metabolites.

Metabolite Detection

and Characterization

To better understand complex and

overlapping metabolic pathways, high-

throughput de novo metabolite identifi-

cation was applied. Although the pMRM

approach was good at finding compounds

at trace levels because of the high sensi-

tivity of the hybrid triple-quadrupole lin-

ear ion trap system in MRM mode, the

unit resolution and low mass accuracy of

the system warranted further metabolite

identification and validation using high-

resolution high-accuracy MS. For further

confirmation, the MS-MS fragmentation

pattern was analyzed based on informa-

tion about basic fragmentation and rear-

rangement processes in the literature.

Several secondary metabolites were

detected, analyzed, and characterized

using the current methodology (Table

II). For example, metabolite M3, the oxi-

dation product of clomazone, m/z 256,

was found using pMRM transition of

256/125. High-resolution MS spectral

data showed that M3 had an accurate

mass of 256.073 with the single-chlorine-

atom isotopic signature. Isotopic model-

ing in MassWorks suggested the unique

elemental formulae of C12H15NO3Cl.

Fragmentation pattern modeling in Mass

Frontier indicated that the fragmentation

mechanism of M3 was the same as M4

MS-MS spectra, but the fingerprint frag-

ments were different because of the dif-

ferent position of the hydroxyl group in

the parent molecule (Figure 4). Using the

hybrid triple-quadrupole linear ion trap

system, enhanced product ion scans (EPI)

of M3 in clomazone-treated plant sam-

ples and pure standard solutions showed

a high degree of similarity, suggesting

the existence of M3 in plant samples. In

addition, time course data on M3 forma-

tion proposed another physiological line

of evidence (Figure 5). Therefore, it was

concluded that the unknown secondary

metabolite M3 was 5-hydroxy-clomazone.

Compared to M3, metabolite M4 might

have a different location of the hydroxyl

group attached to the benzene ring, sup-

ported by the facts that M3 and M4 had

different retention times and different fin-

gerprint fragments (Figure 5). Metabolite

M5, having a transition of m/z 272/125,

with an accurate monoisotopomer mass of

272.06843, had a clear single-chlorine sig-

nature (Figure 3). Elemental composition

assignment of M5 was offered by best-fit

formulas at delta of 2 ppm. However,

there were a few isomers with fingerprint

fragment m/z 125 possessing chlorine iso-

topic patterns. Finally, the most possible

candidate (with the highest probability

score) was suggested (Table II). Keep in

mind though, M3, M4, and M5 could

be found at different retention times due

to different positional isomers (Figure 5).

Other interesting metabolites, M8 and

5.7e6

5.0e6

4.0e6

3.0e6

2.0e6

1.0e6

0.0

5.7e6

5.0e6

4.0e6

3.0e6

2.0e6

1.0e6

0.0

5.7e6

6 8 10 12

6 8 10 12 6 8 10 12

6 8

None

9648

0

S

Retention time (min)

R

10 12

6 8 10 126 8 10 12

6 8

MO:240g125(10.20)

MO:240g125 (10.20)

MO:256g141 (9.40)

MO:240g125 (10.20)

MO:240g125 (10.20)MO:240g125 (10.20)

MO:240g125 (10.20)

MO:240g125 (10.20)

M3:256g125 (9.01)

M3:256g125 (9.01)

M4:256g141 (9.40)

M4:256g141 (9.10)

M4:256g141 (9.10)

10 12

5.0e6

4.0e6

3.0e6

2.0e6

1.0e6

0.0

5.6e6

Inte

nsi

ty (

cps)

5.0e6

4.0e6

3.0e6

2.0e6

1.0e6

0.0

5.7e6

5.0e6

4.0e6

3.0e6

2.0e6

1.0e6

0.0

5.6e6

5.0e6

4.0e6

3.0e6

2.0e6

1.0e6

0.0

5.6e6

5.0e6

4.0e6

3.0e6

2.0e6

1.0e6

0.0

Figure 5: Representative ion intensities (cps) and retention times (RT) of major cloma-zone metabolites obtained from reversed-phase LC–MS-MS analysis of growth me-dium (50% Hoagland solution containing 50 µM clomazone) samples, which were taken at 0 h, 48 h, and 96 h after adding the herbicide to growth media with or without (none) herbicide-resistant (R) or -susceptible (S) E. phyllopogon plants. Com-pounds are labeled as MRM transitions with their retention times. The transforma-tion of parent clomazone (M0: m/z 240/125, RT 10.20 min) in the growth medium in absence of plant roots resulted in the accumulation of two major metabolites with monohydroxylation on the aromatic moiety of the clomazone molecule (M4: m/z 256/141, RTs 9.10 and 9.40 min); when roots were present, these compounds were absent and a metabolite with monohydroxylation on the isoxazolidinone ring moiety (M3: m/z 256/125, RT 9.01 min) accumulated.


M9, were found using pMRM transitions

of 268/125 and 320/125. Unfortunately,

these metabolites could not be detected

on hybrid triple-quadrupole linear ion

trap system with any other modes of

acquisition. Attempts to detect M8 and

M9 with the Orbitrap or FT-ICR MS sys-

tems also failed. On the other hand, this

result actually demonstrated the higher

selectivity of pMRM mode compared

to that of the full-scan profiling mode.

Regardless, we suggested putative chemi-

cal structures to be validated (Table II). In

the future, after HPLC fractionation and

concentration, offline nuclear magnetic

resonance (NMR) and accurate mass

measurement will be applied to confirm

the suggested structures of M5, M8, and

M9. However, it is known that isolation

of unstable metabolites is a challenging

process. Therefore, there is no guarantee

that the above mentioned plan will be

successful in our future endeavors. Other

metabolites, M1 (m/z 242/125), M2 (m/z 254/125), and M7 (m/z 272/125) were of

less physiological importance and were

not pursued as diligently as those metabo-

lites mentioned above.

In summary, we demonstrated a novel

workflow that was successfully applied

in complex time-course experiments of

the weed watergrass treated with differ-

ent levels of clomazone, revealing more

metabolites as well as assigning meta-

bolic pathways (21,22).

Conclusions

In biological bodies, exogenous com-

pounds go through all kinds of bio-

transformations; however, most of

these biotransformations could not be

detected by previous technology (15). In

the present study, we demonstrated for

the first time that low-abundant cloma-

zone metabolites that were buried under

dominating abundant metabolites could

be successfully detected, analyzed, and

characterized. Chemical structures of

these detected metabolites were pro-

posed, based on MS and MS-MS spectra.

The described methodology was applied

in complex experiments in which plants

were treated with clomazone at different

levels in a time-course manner, and was

effective in elucidating biotransforma-

tions of clomazone. We believe that the

current methodology can be extended to

study endogenous metabolic pathways.

This approach opens an exciting oppor-

tunity for metabolic pathways elucida-

tion, thus providing a powerful tool for

small-molecule biomarker screening and

drug discovery.

Acknowledgments

This work was financially supported by

the UC Davis Genome Center Pilot Proj-

ect and partially funded by grants from

the California Rice Research Board. Our

appreciation was give to Professor Scott D.

Stanley from California Animal Health &

Food Safety Laboratory for access to the

LTQ-Orbitrap mass spectrometer.

References

(1) H. Yasuor, P.L. TenBrook, R.S. Tjeerdema,

and A.J. Fischer, Pest. Manag. Sci. 64, 1031–

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141–158.

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C.L. Brummel, Rapid Commun. Mass Spec-

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multiple reactions monitoring (pMRM)

in Metabolomics,” presented at the 5th

Annual Metabolomics Society International

Conference, Edmonton, Alberta, Canada,

August, 2009.

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M.A. Zern, Differentiation and Character-

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Allied Topics, Denver, Colorado, June,

2008.

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Tjeerdema, J. Agric. Food Chem. 58, 3674–

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Wei Zou is with the California State Department of Toxic Substances Control in Berkeley, Califonia. Direct correspondence to: [email protected]. Hagai Yasuor and Albert J. Fischer are with the Depart-ment of Plant Sciences at the University of California in Davis, California. Vladimir V. Tolstikov is with the UC Davis Genome Center at the University of California in Davis, Califonia. ◾

For more information on this topic,

please visit



Yi Dang*, Jeffrey Moore*, Gloria Huang †, Markus Lipp*, Barbara Jones*, and James C. Griffiths*

*U.S. Pharmacopeial Convention, Rockville, Maryland; †U.S. Food and Drug Administra-tion. Direct correspondence to: [email protected] for reference standards and [email protected] for monograph information.

Establishing USP Rebaudioside A and Stevioside Reference Standards for the Food Chemicals Codex

The United States Pharmacopeial Convention (USP) publishes the Food

Chemicals Codex (FCC), which is a compendium of food ingredient

documentary standards (monographs) that provide tests, procedures,

and acceptance criteria to indicate the safety and quality of a food

ingredient. Physical reference standards can be associated with

the procedures of an FCC monograph and the reference standards

are needed for conducting the procedures. This article describes

characterization and collaborative testing of new USP rebaudioside A

and stevioside reference standards and their suitability for intended uses

in the FCC rebaudioside A monograph.

The stevia plant contains rebaudio-

side A and seven or more related

steviol glycosides. As a family of

related compounds with similar chemi-

cal structures and properties, the steviol

glycosides form and coexist naturally

with different contents in the same

plant and have similar but not identical

organoleptic features. For example, they

are all sweet but to different degrees and

with different f lavor profiles. Various

manufacturers follow different extrac-

tion and purification steps, yielding

varying amounts of the most prevalent

compound, rebaudioside A, or the whole

family of steviol glycosides. Figure 1

shows the chemical structure of rebaudi-

oside A, a glycoside of the ent-kaurenoid

diterpenoid aglycone family. Analysts

require sophisticated tests and special-

ized knowledge to separate such large

and complex molecules from each other

or from other plant-derived and similarly

large, complex molecules for analysis

and characterization. The compositional

complexities and analytical challenges

also require physical reference stan-

dards for comparative test procedures

such as identification, assay, and impu-

rity limits to establish the authenticity,

purity, and quality of the rebaudioside

A. For these intended purposes, USP has

released rebaudioside A and stevioside

(an impurity) reference standards for ste-

via products containing no less than 95%

rebaudioside A. This article describes the

results of characterization and collabora-

tive studies that USP conducted to make

these reference standards available.

Experimental

As stated in USP–NF General Chapter

USP Reference Standards <11> (1), the

establishment and release of USP refer-

ence standards are “under the authority

of the USPC [United States Pharma-

copeial Convention] Board of Trustees

upon recommendation of the USP Refer-

ence Standards Expert Committee, which

approves each lot as being suitable for use

in its compendial applications.” To sup-

port these stipulations, USP scientists

generate a set of documents including

procurement specifications and a testing

protocol and obtain a bulk material, gen-

erally from a major manufacturer of the

article. The material is tested and charac-

terized in an interlaboratory collaborative

study organized according to the testing

protocol. The results are evaluated by


USP staff, who may conduct additional

testing or investigations when necessary.

USP staff then compile a report for review

and approval by the relevant USP Expert

Committee — in this instance, the Food

Ingredients Expert Committee. After its

approval the material is subdivided as

appropriate, quality checked, labeled, and

made available for distribution (1).

Liquid Chromatography

Table I summarizes the chromatographic

conditions given in the monograph.

To meet specified HPLC system suit-

ability requirements for retention time

and tailing factor of the rebaudioside A

peak from rebaudioside A standard solu-

tion, the monograph allows analysts to

adjust the ratio of ammonium acetate to

acetic acid in preparing the acetate buf-

fer solution. The adjustment changes

the solution pH that affects the reten-

tion times of rebaudioside A and other

peaks (that is, decreasing the pH of the

buffer will decrease the retention time

and affect the tailing of the rebaudio-

side A peak). Other modifications of the

chromatographic conditions also may be

allowed in a given range.

Characterizing and Establishing

Rebaudioside A and Stevioside

Materials as Suitable Reference

Standards

The collaborative study followed these

steps and procedures: We first selected

suitable bulk materials of rebaudioside A

and stevioside as reference standard candi-

dates that fulfill, at a minimum, all quality

requirements of the rebaudioside A mono-

graph. Then we sent samples of these ref-

erence standard candidates to three col-

laborative laboratories that had already

received the testing protocols. During the

collaborative testing of the two reference

standard candidates, all laboratories per-

formed most of the monograph-specified

tests whether or not a test required the ref-

erence standard. In addition, all laborato-

ries performed additional general tests to

characterize and to confirm the chemical

structures of the rebaudioside A and ste-

vioside candidate materials, respectively.

Rebaudioside A: The rebaudioside

A reference standard is required in two

monograph comparative identification

(ID) tests: infrared (IR) spectroscopy

and high performance liquid chromatog-

raphy (HPLC) retention time. To com-

ply with the IR test requirement, the IR

spectrum of the rebaudioside A sample

(product) must exhibit maxima at the

same wavelengths as those in the spec-

trum of the reference standard. To meet

the acceptance criteria for the HPLC

ID test, the rebaudioside A peak in the

sample solution should elute at the same

retention time as the rebaudioside A peak

obtained from the rebaudioside A (refer-

ence standard) standard solution.

The IR spectra of this reference stan-

dard candidate by attenuated total

reflectance (ATR) analysis are consistent

among collaborators, and characteristic

IR absorption peaks are observed for

functional groups O–H (~3500 cm–1,

broad), C–H for CH2 and CH groups

(~2900 cm–1, multiples), C=C (vinyli-

dine, 1650 cm–1, weak), C=O (1720

cm–1, medium), and C–O (1040–1070

cm–1, strong). The IR data support the

chemical identity, and this standard is

suitable for its intended monograph com-

parative IR test.

The rebaudioside A reference standard

also is required to verify the HPLC system

suitability for the rebaudioside A assay test

and for the limit test of organic impuri-

ties–related steviol glycosides. Based on

the 5000-mg/L rebaudioside A standard

solution prepared from rebaudioside A

reference standard candidate, the system

suitability requirements include specifica-

tions that the rebaudioside A peak area

and retention time do not vary more than

2.0% relative standard deviation each and

that the retention time of the rebaudioside

A peak is less than 15.0 min.

Figure 2 shows a typical scaled HPLC

chromatogram of rebaudioside A stan-

dard solution provided by one of the

collaborators. In the chromatogram the

retention time of the rebaudioside A peak

is about 12 min obtained by the mono-

graph procedure.

Quantitatively, the rebaudioside A

reference standard is used in the HPLC

assay to determine the rebaudioside A

content in rebaudioside A product (sam-

ple). To comply with the FCC purity

requirement, the rebaudioside A content

(purity) determined by the assay against

the rebaudioside A reference standard

must not be less than (NLT) 95.0% on

the anhydrous and solvent-free basis. The

volatiles, including water and residual

solvents (methanol and ethanol), in the

material can be determined as directed

in the monograph tests.

For qualitative uses such as the ID

tests and HPLC system suitability

requirements, a labeled purity value for

the reference standard may not apply.

However, for quantitative applications

such as assay and impurity limit tests,

users do need to use the purity value of

the standard for their calculations. To

meet their needs and according to the

rule set in USP general chapter <11>,

rebaudioside A reference standard as an

OH

OH

CH3

CH3

HO

O

OO

O

O

O

O

O

O

HOHO

HO

HO

HO

HO

OH

OH

OH

OH

HO

CH2

H

H

Figure 1: Chemical structure of rebaudioside A (13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] kaur-16-en-18-oic acid β-D-glucopyranosyl ester).


assay standard is labeled to three signifi-

cant figures. This reference standard was

determined to contain 0.969 mg (96.9%)

of rebaudioside A in 1.000 mg of the ref-

erence standard material on the anhy-

drous basis. This value was determined

by a standard mass-balance approach by

which organic impurities, steviol glyco-

sides, residual solvents, and inorganic

impurities are subtracted from 1.000 mg

of the reference standard material. Ana-

lysts should use this labeled purity value

for their assay and related steviol glyco-

sides test calculations as directed in the

monograph procedures.

The 96.9% purity is in compliance

with the rebaudioside A monograph

assay requirement of NLT 95.0%. This

lot reference standard also meets the

monograph acceptance criteria for impu-

rity limits: ethanol NMT 0.50% (found:

0.1%), methanol NMT 0.02% (not pres-

ent), related steviol glycosides NMT 5%

(found: 3%), and residue on ignition

NMT 1% (found: <0.01%).

To determine the hygroscopicity of

the rebaudioside A material and the basis

for the purity assignment and for refer-

ence standard label instructions, vapor

sorption and desorption analyses were

performed. The analytical data indi-

cated that rebaudioside A is hygroscopic,

an observation that was supported by

the variation in water content (1–3%)

among three collaborators. As a result,

for quantitative applications (assay and

limit tests), the reference standard label

instructs users to determine the water

content titrimetrically at the time of use

and to use the purity value on the anhy-

drous basis.

In addition to the IR and HPLC sup-

porting data, the identity and chemical

structure (Figure 1) of the rebaudio-

side A reference standard also has been

fully characterized by specific rotation, 1H and 13C nuclear magnetic resonance

(NMR) spectroscopy, mass spectrometry

(MS), and elemental analysis.

The average specific rotation by two

determinations was –29.4° (anhydrous),

and the monograph acceptance crite-

rion was [α]D25 between –29° and –31°

in the initially proposed rebaudioside A

monograph. The 1H and 13C NMR spec-

tra and their correlations are consistent

between two collaborators. The NMR

data confirm the chemical structure,

and the chemical shift assignments are

in agreement with those reported in the

literature (2). The MS analysis by direct

infusion electrospray ionization (ESI)

showed the base peak at 989.64 m/z as

the sodium salt adduct of the molecular

ion. With the correction of about 2%

water content for elemental analysis, the

results for carbon and hydrogen contents

are consistent with the corresponding

theoretical values, which support the

chemical composition of this standard.

Stevioside: The stevioside reference

standard is used as a qualitative standard

for the HPLC system suitability require-

ments for detector response in the assay

and related steviol glycosides tests. It also

is used as a quantitative standard for the

limit test for stevioside content in the

sample.

Based on the monograph assay accep-

tance criteria for a stevioside reference

standard working standard solution at

0.5 mg/L, the detector response by the

peak-to-noise ratio (peak height/base-

line noise) of the stevioside peak must be

NLT 3. This standard met the require-

ment, and a minimal value of 4 was

reported by the collaborators.

For the limit test of related steviol

glycosides, we requested collaborators to

use the monograph method to analyze

the contents of related steviol glycoside

impurities in this stevioside reference

standard candidate material. The candi-

date was tested at rebaudioside A sample

0.28

0.26

0.24

0.22

0.20

0.18

0.16

0.14

0.12

0.10

0.08

0.06

0.04

0.02

0.00

0.30

Ab

sorb

an

ce (

AU

)

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

Time (min)

Figure 2: Chromatogram of rebaudioside A reference standard solution at 5000 mg/L.

Table I: Chromatographic conditions

Mode HPLC

Detection UV at 210 nm

Column15 cm × 4.6 mm, packed with a propyl-ami-no silane phase bonded to silica gel (5-µm particle diameter)

Column temperature 30 °C

Flow rate 1.5 mL/min

Injection 15 µL

Diluent25% (v/v) acetate buffer (1.46 mM ammo-nium acetate adjusted to pH 4.3 with glacial acetic acid) in acetonitrile

Mobile phase13% (v/v) acetate buffer (see above) in acetonitrile

Rebaudioside A/stevioside sample concentration

5000 mg/L in diluent


concentration of 500 mg/L, twice as high

as the monograph-specified 250 mg/L

for stevioside standard stock solution.

Such a high concentration allows more

accurate detection of all possible impu-

rities in stevioside material. The average

total HPLC impurities are approximately

2% by this analysis.

Figure 3 shows a typical HPLC chro-

matogram of stevioside reference stan-

dard solution with the retention time

of the stevioside peak at 6.6 min as sug-

gested in the monograph. Based on the

monograph suggested retention times,

the two small peaks at 10 min and 12

min were probably because of the pres-

ence of rebaudioside F and rebaudioside

A, respectively.

As a limit standard (1), the purity of

the stevioside standard for the current

Lot F0I080 was established to be 0.97

mg of stevioside per 1.00 nominal mg

of the reference standard material on an

as-is basis. The assignment was deter-

mined by the mass-balance approach

that subtracts all HPLC impurities and

volatiles (water and residual solvents).

Unlike the rebaudioside A material,

the vapor sorption and desorption analy-

sis data indicate the stevioside material

does not appear to be hygroscopic at 50%

or less relative humidity (RH). Collabo-

rators also reported consistent water con-

tents. All the data support as-is testing

for quantitative uses of stevioside refer-

ence standard.

We also requested the collaborators

who participated in the rebaudioside A

project to perform other similar tests

for the stevioside project. In addition to

the HPLC data, the identity and chemi-

cal structure have been fully character-

ized by IR, specific rotation, 1H and 13C

NMR, MS, and elemental analysis. The

IR spectra of the stevioside reference

standards are very similar to those of the

rebaudioside A reference standard, and

small differences in peak intensity were

observed for the C–O stretches in the

1040–1070 cm–1 region (two or possibly

more peaks). Structurally, the stevioside

molecule contains one less six-member

glucose ring.

The average specific rotation by two

determinations was –34.2° (as is), which

is different from the value of –29.4°

reported for the rebaudioside A. The 1H

and 13C NMR spectra and their corre-

lations are consistent between two col-

laborators. The NMR data confirm the

chemical structure, and the chemical

shift assignments are also in agreement

with those reported in the literature (2).

The MS analysis by direct infusion ESI

(+) showed the base peak at 827.43 m/z

as the sodium salt adduct of the molecu-

lar ion. With the correction of about 1%

water content for elemental analysis, the

results for carbon and hydrogen contents

are consistent with the corresponding

theoretical values, which support the

chemical composition of this standard.

Conclusion

The USP reference standards for rebau-

dioside A and stevioside have been fully

characterized by IR, NMR, MS, and ele-

mental analysis, and have been established

by collaborative studies for suitability as

new USP reference standards. These two

new reference standards support the FCC

rebaudioside A monograph test proce-

dures for identity (authenticity) and purity

(rebaudioside A content and other steviol

glycoside impurities). Both the docu-

mentary standard in FCC (rebaudioside

A monograph) and the newly available

rebaudioside A and stevioside USP refer-

ence standards support manufacturers,

purchasers, and regulators in ensuring

the identity, purity, and quality of stevia-

containing food and beverage ingredients.

These standards help ensure the safety

and quality of those ingredients and prod-

ucts throughout the supply chain, which

ends with use by a consumer.

Acknowledgments

The authors thank William Koch, Susan

S. de Mars, and Roger L. Williams for

constructive reviews and Stefan Schuber

for editorial assistance. All are colleagues

at USP.

References

(1) USP, USP Reference Standards <11>, USP

32–NF 27 (USP, Rockville, Maryland,

2009), pp. 35–58.

(2) A.S. Dacome, C.C. da Silva, C.E.M. da

Costa, J.D. Fontana, and S.C. da Costa,

Process Biochem. 40, 3587–3594 (2005).

Yi Dang, Jeffrey Moore, Markus Lipp, Barbara Jones, and James C. Griffiths are with the U.S. Pharmacopeial Convention, Rockville, Maryland. Direct correspondence to: [email protected] for reference standards and [email protected] for monograph information.

Gloria Huang is currently at the U.S. Food and Drug Administration. The opinions expressed here are those of the authors and do not represent the official position of the U.S. Food and Drug Administration or the U.S. government. ◾

0.70

0.60

0.50

0.40

0.30

0.20

0.10

0.00

Ab

sorb

an

ce (

AU

)

0.00 5.00 10.00 15.00 20.00 25.00

Time (min)

30.00 35.00 40.00 45.00 50.00 55.00

Figure 3: Chromatogram of stevioside reference standard solution at 5000 mg/L.

For more information on this topic,

please visit


THE APPLICATION NOTEBOOKCall for Application Notes

LCGC is planning to publish the next issue

of Te Application Notebook special supple-

ment in June. Te publication will include

vendor application notes that describe

techniques and applications of all forms of

chromatography and capillary electrophore-

sis that are of immediate interest to users in

industry, academia, and government. If your

company is interested in participating in

these special supplements, contact:

Michael J. Tessalone, Group Publisher,

(732) 346-3016

Edward Fantuzzi, Associate Publisher,

(732) 346-3015

Stephanie Shaffer, East Coast Sales

Manager,

(508) 481-5885

Application Note Preparation

It is important that each company’s mate-

rial fit within the allotted space. Te edi-

tors cannot be responsible for substantial

editing or handling of application notes

that deviate from the following guidelines:

Each application note page should be no

more than 500 words in length and should

follow the following format.

Format

• Title: short, specific, and clear

• Abstract: brief, one- or two-sentence

abstract

• Introduction

• Experimental Conditions

• Results

• Conclusions

• References

• Two graphic elements: one is the company

logo; the other may be a sample chromato-

gram, figure, or table

• Te company’s full mailing address, tele-

phone number, fax number, and Internet

address

All text will be published in accordance with

LCGC ’s style to maintain uniformity through-

out the book. It also will be checked for gram-

matical accuracy, although the content will

not be edited. Text should be sent in electronic

format, preferably using Microsoft Word.

Figures

Refer to photographs, line drawings, and

graphs in the text using arabic numerals in

consecutive order (Figure 1, etc.). Company

logos, line drawings, graphs, and charts must

be professionally rendered and submitted as

.TIF or .EPS files with a minimum resolution

of 300 dpi. Lines of chromatograms must be

heavy enough to remain legible after reduc-

tion. Provide peak labels and identification.

Provide figure captions as part of the text,

each identified by its proper number and title.

If you wish to submit a figure or chromato-

gram, please follow the format of the sample

provided below.

Tables

Each table should be typed as part of the main

text document. Refer to tables in the text by

roman numerals in consecutive order (Table

I, etc.). Every table and each column within

the table must have an appropriate heading.

Table number and title must be placed in a

continuous heading above the data presented.

If you wish to submit a table, please follow

the format of the sample provided below.

References

Literature citations must be indicated by arabic

numerals in parentheses. List cited references

at the end in the order of their appearance.

Use the following format for references:

(1) T.L. Einmann and C. Champaign, Science

387, 922–930 (1981).

Te deadline for submitting application notes for the

June issue of Te Application Notebook is:

April 16, 2012

Tis opportunity is limited to advertisers in LCGC North America.

For more information, contact:

Mike Tessalone at (732) 346-3016, Ed Fantuzzi at (732) 346-3015,

or Stephanie Shaffer at (508) 481-5885.

Table I: Factor levels used in the designs

Factor Nominal value Lower level (−1) Upper level (+1)

Gradient profile 1 0 2

Column temperature (°C) 40 38 42

Buffer concentration 40 36 44

Mobile-phase buffer pH 5 4.8 5.2

Detection wavelength (nm) 446 441 451

Triethylamine (%) 0.23 0.21 0.25

Dimethylformamide 10 9.5 10.5

Figure 1: Chromatograms obtained using the conditions under which the ion sup-pression problem was originally discov-ered. The ion suppression trace is shown on the bottom. Column: 75 mm × 4.6 mm ODS-3; mobile-phase A: 0.05% heptafluo-robutyric acid in water; mobile-phase B: 0.05% heptafluorobutyric acid in aceto-nitrile; gradient: 5–30% B in 4 min. Peaks: 1 = metabolite, 2 = internal standard, 3 = parent drug.

APPLICATION NOTES – FEBRUARY 2012 59

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