Rapid Biofluid Analysis using the Prosolia Velox 360 ... · The world leader in serving science...

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Mari Prieto Conaway, PhD Thermo Fisher Scientific

Product Manager, Ion Sources

Rapid Biofluid Analysis using the Prosolia Velox 360™ PaperSpray® System and Mass Spectrometry

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• Thermo Scientific™ part numbers • Prosolia Velox 360 PaperSpray® system • Clinical research workflow • Basics paper spray technology • Advantages/disadvantages • Quantitative workflow

• Chronograms • Illicit drugs in urine and blood • Drugs of Abuse (DoA) in blood

• Methods developed to date • Cross validation PaperSpray with other

technologies

Outline

Prosolia Velox 360 PaperSpray system

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Part Numbers for the Prosolia PaperSpray source

Thermo Scientific part numbers

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Part Numbers

Example: 00950-01-00468 – Velox 360 for Ion Max mass spectrometers

The flange is specific for the instrument ( New Generation or Ion Max)

The paper spray source does not change with the mass spectrometer

Paper spray source slides over the rods

Magazine holds 40 cartridges; two magazines provided

Used cartridge disposal bucket

Presenter

Presentation Notes

Paper spray can attach to any of Thermo Scientific mass spectrometers. What changes is the flange that couples to the mass spec. When a complete unit is purchased (00950-01-00468 or 00950-01-00450) it comes with a flange for the mass spec of interest. Customer can purchase a different flange if they want to swap the paper spray source between a Q Exactive and an Endura, for example.

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Paper Spray Technology

2013 Dreyfus Prize in Chemical Sciences

R. Graham Cooks Purdue University

Zheng Ouyang Purdue University

Professor Biomedical Engineering-Inventor

Presenter

Presentation Notes

Inventors of PaperSpray technology. PaperSpray® (all together and capitalized is a registered brand of Purdue Research Foundation). Use PaperSpray when referring to the product. Use paper spray (non capitalized, two words) when referring to the technology in general.

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Current Clinical Research Mass Spectrometry Workflow

Thermo Scientific™ TSQ Vantage™ mass

spectrometer

Blood Sample Storage

Plasma Isolation

Solid Phase Extraction

Sample Pretreatment

Protein Precipitation

Liquid-liquid Extraction

Liquid chromatography

For research use only. Not for use in diagnostic procedures.

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Mass spectrometry

Blood Sample Storage

Plasma Isolation

Solid Phase Extraction

Sample Pretreatment

Protein Precipitation

Liquid-liquid Extraction

Liquid chromatography

Current Clinical Research Mass Spectrometry Workflow

• Blood sample storage • Drug extraction • Transfer to MS analysis (drug vaporization and ionization)

Ideally, paper cartridge could act as:


Presenter

Presentation Notes

There is now a technology that can skip sample preparation steps and provide faster answers.

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Charged droplets

Inlet to mass spectrometer

Porous substrate (e.g paper)

Extraction/spray solvent

3.2 kV

He Wang, Jiangjiang Liu, R. Graham Cooks and Zheng Ouyang. Angewandte Chemie 2010, vol 49, p 877.


Paper Spray Ionization Basics

Presenter

Presentation Notes

You need wet paper (organic solvent to allow elecstrospray) and an applied voltage. Signal will last as long as these two are met. Electrospray is in the low uL/min flow rate (~3 uL/min). The photo shows electrospray from the paper tip, the mass spec transfer tube is shown on the right hand side of the photo.

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Blood Analysis – Importance of Dried Blood

Slide courtesy of Nick Manicke, Assistant Professor, IUPUI.

• Fresh blood - mostly lipids are detected in the 700-900 mass range (not shown)

• Dried blood offers a clean baseline in the 100-1000 m/z region, which allows detection of endogenous (and other) small molecules

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309.2

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239.2 185.1 546.4

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Dried blood Small molecules • Amino acids • Creatinine • Urea • Choline • Acylcarnitines • Drugs

Presenter

Presentation Notes

Lipids are mostly detected if fresh (non dried) blood is analyzed from Whatman paper. When dry, the paper serves to retain some of the blood matrix, therefore a cleaner baseline is obtained in the 100-1000 m/z region (shown above). Abundant proteins and lipids suppress small molecular ions (endogenous metabolites and drugs of interest) when fresh blood is analyzed.

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227.1246 144.1017

114.0663

307.0991 189.0730 257.1602 353.0836

Thermo Scientific™ Exactive™ mass spectrometer

Creatinine circled Histidine -156 m/z Caffeine and paraxanthine Urea dimer – 121 m/z ⁞

Slide courtesy of Nick Manicke, Assistant Professor, IUPUI.

Dry Urine Analysis by PaperSpray Ionization

Presenter

Presentation Notes

In this example, a mass spectrum for dry urine sample is shown, Exactive data. Notice the clean baseline that allows small molecules to be detected. These would normally be suppressed in the presence of matrix (wet urine). Common urine endogenous compounds: Creatinine - C4H7N3O, 114.0662 m/z Histidine -C6H9N3O2 , 156 .0768 m/z Caffeine and metabolites (paraxanthine) C7H8N4O2, 181.0720 m/z Urea dimer – 61.0396, 121.0720 m/z

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Prosolia’s Velox 360 System


PaperSpray Source for Thermo Scientific Mass Spectrometers

Presenter

Presentation Notes

The workflow with the Prosolia Velox 360 system. From top left, left to right: the Prosolia source showing hazardous waste disposal bin, spot sample on paper cartridge. Bottom left, stacker automatically drops cartridges which get rewetted by solvent and finally, bottom right, cartridge is placed in front of the mass spectrometer, a high voltage gets applied to the metal contact which generates an electrospray plume.

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Single Use Cartridge – correct way to spot sample

Correct spotting Incorrect spotting For research use only. Not for use in diagnostic procedures.

Presenter

Presentation Notes

The aim in paper spray is for the solvent to extract the whole dry blood spot area (DBS). For that reason, it needs to be spotted so it covers the whole paper area that it’s going to be extracted. The extraction solvent comes from behind the DBS and moves through the cellulose paper through capillarity and possibly also aided by differential voltage when the HV is applied. It is important that the extraction solvent go through the full DBS, in order to extract the whole sample. In the ‘incorrect spotting’ example above (right hand photo), the extraction solvent might go around the DBS and not through it. A DBS becomes hydrophobic upon drying, so the ‘universal’ extraction solvent for DBS is 95/5/0.01, MeOH/H2O/Acetic Acid (by volume). Other solvent combinations for DBS might include dichloromethane and chloroform to assist in rewetting the DBS.

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Positive mode: • Dried Blood Spots (DBS) - 95/5/0.01, • MeOH/H2O/Acetic Acid

• Urine – 90/10/0.01, Acetonitrile/H2O/Acetic acid

Common Solvents Used for Extraction and Spray

Negative mode:

• 100/0.01, MeOH/Acetic Acid

Note: two different solvents might be more appropriate in certain cases: one for rewetting the dry sample and a second one as spray solvent


Presenter

Presentation Notes

Most common solvents for dry blood spots (DBS) and dry urine samples. For DBS use mostly MeOH based solvent mixes, as acetonitrile does not wet the DBS which becomes hydrophobic upon drying. Negative ion mode, as with electrospray in general, requires a low surface tension solvent, so the best solvent is 100% MeOH, with some acid. In general, 0.01 % acetic acid is recommended. Signal suppression might be seen with 0.1% Acetic acid.

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Advantages • No carryover • Small sample volume (12 uL

blood, 6 uL urine or plasma) • Small solvent volume (~100

uL/sample) • No solvent waste • No sample preparation

• Amenable to walk-up analysis • No LC system to maintain • No chromatography to trouble

shoot

Paper Spray Technology

Disadvantages • No separation: relies on exact

mass and/or MS/MS alone for selectivity

• Detection limits are not as good as with LCMS – single digit ng/mL LLOQ are typical


Presenter

Presentation Notes

In spite of the disadvantages mentioned on this slide, paper spray has the sensitivity to satisfy a wide range of applications. All original methods were developed on Thermo triple quads: Access Max, Ultra and Vantage systems. Although the advantages of HRAM instruments is mentioned for method development, it is a great technique for triple quads and ion traps.

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Quantitative Workflow

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Charged droplets

3.2 kV

Inlet to mass spectrometer

Extraction/spray solvent (~100 µL)

1) urine sample spiked with drug

2) Internal standard addition

3) Spot urine onto paper

4) Paper spray MS analysis

Internal standard solution

Sample Processing for PaperSpray Workflow

or


Presenter

Presentation Notes

The steps below correspond to the numbers in the figure: Sample, spiked calibrator or quality control sample is in liquid form. Add Internal Standard either to sample or to paper beforehand (if added to paper, allow it to dry). A. Spot sample on to paper - 6 uL of spiked urine (10-12 uL if blood sample). B. Extraction solvent added in small aliquots of 7 or 10 uL at a time to a total volume ~100 uL. Spot sample onto paper (allow it to dry: urine 10 min, blood 1 hr. at room temperature on benchtop; flowing N2 gas will speed drying time). Apply voltage (3.5-5kV) to generate spray into MS.

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RT: 0.00 - 0.91

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NL: 2.30E5

F:+ c NSI SRM ms2 278.100 8_Drugs_Urine_50ng_mL_014

NL: 3.05E5

F: + c NSI SRM ms2 281.100

8_Drugs_Urine_50ng_mL_014

Amitriptyline 50 ng/mL

Amitriptyline-d3 100 ng/mL

Representative PaperSpray Chronogram

Amitriptyline SRM transitions m/z 278 → 191, 233 (top trace), Amitriptyline-d3 SRM transitions m/z 281 → 91, 233 (bottom trace)

Amitriptyline is a tricyclic antidepressant (TCA)


Presenter

Presentation Notes

The typical signal is ion current intensity changing with time, therefore it’s called a ‘chronogram’ (not chromatogram). Internal standard and analyte signals track each other. Therefore can normalize by IS even if the analyte chronogram is not ideally shaped. Transitions as stated, not a typo.

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0 1 3 5 7 9 11 13 15 Time (min)

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Mass Chronogram of Sitamaquine (m/z 344→271)

Mass Chronogram of 2H10 Sitamaquine (m/z 354→271)

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Manicke et al, JASMS (2011) 22:1501-07


Bottom Trace Indicates Signal Variability of Chronograms

Presenter

Presentation Notes

Sitamaquine – anti parasite (anti Leishmania parasites) Top trace - example of signal from analyte at various concentrations. Bottom trace – deuterated standard (internal standard or IS) at constant concentration to indicate typical variability of signal and why it needs to be normalized by IS for quantitation purposes. Even though signal is variable (see variability of IS at constant concentration), the shape of the chronogram is reproduced in both (analyte and IS), therefore this variability can be corrected if we look at the ratio, ‘analyte/IS’ and allows for quantitation. In screening applications, one IS is added as experimental control.

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Single Compound in Human Urine - Cocaine

Y = -0.000109847+0.00806008*X R^2 = 0.9984 W: 1/X

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1000 <1 2500 6.4

Calibration Curve in Triplicate

%RSD = (Std Dev/Avg) *100

For forensic use only.

Poster 285, ASMS 2014; B Hart et al. Evaluation of a Novel High-Throughput Screening Method for Drugs of Abuse in Urine with Paper-Spray Triple Quadrupole Mass Spectrometry

Presenter

Presentation Notes

Let’s first look at a single compound in urine. Data in triplicate, Quantiva triple quad. Notice the excellent %RSD ((Std Dev/Average) *100) as a measurement of precision. Inset shows linearity at low concentrations. No quality control data available to determine accuracy or % recovery. *Early prototype of paper spray source used.

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• All experiments performed with early prototype source. • Eight compounds from different classes • Human urine • 0.5-2500 ng/mL • Deuterated internal standards for all • 2 SRM transitions for each compound and IS

Multi-Drug Panel – Eight Compounds

Drug m/z SRM 1 SRM 2 Amitriptyline 278.1 191.1 233.0 Amphetamine 136.1 91.2 119.1 Cocaethylene 318.2 150.1 196.1 Cocaine 304.2 105.1 182.2 Codeine 300.2 199.1 215.1 Methamphetamine 150.1 91.1 103.1 PCP 244.2 86.2 91.1 Temazepam 301.1 193.1 255.1



Presenter

Presentation Notes

Table shows the compounds in a single sample spot – 8 compounds, 8 IS, 2 SRM transitions for each, 32 SRMs total. Because the chronogram lasts a long time (compared to HPLC peaks) a lot of compounds can be screened for. *Early prototype of paper spray source used.

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amphetamineY = 0.060314+0.00694195*X R^2 = 0.9098 W: 1/X

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cocaethyleneY = 0.000134274+0.00128168*X R^2 = 0.9958 W: 1/X

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cocaineY = 0.00086511+0.00260058*X R^2 = 0.9923 W: 1/X

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methamphetamineY = 0.00628846+0.010073*X R^2 = 0.9967 W: 1/X

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PCPY = 0.00805257+0.00922576*X R^2 = 0.9954 W: 1/X

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amitriptyline Y = 0.146009+0.0161053*X R^2 = 0.9870 W: 1/X

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temazepam

Y = 0.0262972+0.00528053*X R^2 = 0.9802 W: 1/X

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Results of A Multi-Drug Panel

• LOD 0.5-100 ng/mL • LOQ 0.5-1000 ng/mL • Amphetamine showed high

variability (volatility, matrix effects)

• Codeine was only detected in neat solvent (matrix effects).

• Seven of eight compounds showed good calibration curves

• Further development needed for poor /inconsistent ionizing compounds



Presenter

Presentation Notes

Out of 8 compounds analyzed, there were 6 compounds with good linear correlations. Amphetamine and codeine did not ionize properly or had interferences (matrix effects) in the selected SRMs transitions. To be further investigated. Solvent was 90/10/0.01 acetonitrile/H2O with 100 ppm acetic acid (10uL Ac acid in 100 mL of solvent). Same solvent used to extract the analyte from the urine sample and to generate electrospray. Optimizing solvent conditions can aid in ionization efficiency of compounds. *Early prototype of paper spray source used, collected on a Quantiva.

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• Goal: simultaneous quantitation of illicit drug panel from dry blood spots

• Instrument: Thermo Scientific™ TSQ Quantum Access™ Max MS in SRM mode

• Ratio between quantifier and qualifier transition used to confirm detection

• IS for each analyte spiked into sample

MDA, MW 179 3,4-methylenedioxy-

amphetamine THC, MW 314

(∆9-Tetrahydrocannabinol)

NH2

O

O

Anal Chem. 2014 Aug 5;86(15):7712-8. Slide courtesy of Nick Manicke, PhD, IUPUI

Analyzed Compound Blood Cutoff

Values of Interest (ng/mL)

Amphetamine 50 Methamphetamine 50

MDA 50 MDMA 50 MDEA 50

Morphine 20 Cocaine 50 Δ9-THC 3


Quantitative Analysis of Illicit Drugs - Blood

Presenter

Presentation Notes

Similar work in blood, on a Quantum Access Max triple quad, published in Analytical Chemistry 2014, 86(15): 7712-8. Table shows the cutoff values of interest for some drugs of abuse. Next slide shows paper spray limit of detection. IS= internal standard

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Anal Chem. 2014 Aug 5;86(15):7712-8. Slide courtesy of Nick Manicke, PhD, IUPUI


Compound Blood Cutoff from Clinics

(ng/mL)

Paper Spray LOD from DBS

(ng/mL)

Amphetamine 50 1 Methamphetamine 50 0.3

MDA 50 2 MDMA 50 0.04 MDEA 50 0.3

Morphine 20 12 Cocaine 50 0.05 Δ9-THC 3 4

• Calibration Curve for ∆9-THC (black)

• QC samples in red at 40, 120, 400, and 800 ng/mL (interassay accuracy 90-118 % deviation

Quantitative Analysis of Illicit Drugs - Blood

Presenter

Presentation Notes

Quantum Access Max triple quad, published in Analytical Chemistry 2014, 86(15): 7712-8. Interassay accuracy for delta 9-THC is 90-117%. Cutoff values of interest compared to limits of detection by paper spray. IS= internal standard

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Methadone Donor Samples in Urine – Exactive MS

m/z

Compound Theoretical m/z (M+H)+

Ecgonine methyl ester 200.1281 Benzoylecgonine 290.1387

Cocaine 304.1543

Compound Theoretical m/z (M+H)+

EDDP 278.1903 Methadone 310.2165

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310.2153 227.124 114.066

Base peak intensity = 1.5E8 Mass accuracy 3-4 ppm

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290.1378

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304.1534 114.066

200.1278

Base peak intensity = 2.0E7 Mass accuracy 1-3 ppm

Data courtesy of Joseph Kennedy, Prosolia, Inc. and Nick Manicke, IUPUI For forensic use only.

Presenter

Presentation Notes

Data extracted with a 5 ppm window. Spectrum on the left shows methadone and EDDP. EDDP is a methadone metabolite. Spectrum on the right shows cocaine and metabolites. Cocaine major metabolites: ecgonine methyl ester and benzoylecgonine (liver metabolites; biologically inactive). Other (in minor amounts) cocaine metabolites are norcocaine, and 4'-[18F]fluorococaine. This shows the potential for a method in which methadone use is monitored with or without other drugs present.

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Developed Methods


Presenter

Presentation Notes

Live document from Prosolia which summarizes methods deveped to date.

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Analyte Matrix Extract / Spray Solvent

Reference

Opiates: Morphine, Hydrocodone, 6 acetyl Morphine , Oxycodone, Oxymorphone, Buprenorphine, nor Buprenorphine

Urine 90/10/0.01 Acetonitrile / Water/ acetic acid

“Comparison of Paper Spray -MS/MS and Current Techniques for Screening Drugs of Abuse in Urine”, 62th ASMS Conference on Mass Spectrometry and Allied Topics, Baltimore, MD 2014

Drugs of Abuse: Methadone, EDDP, Cocaine, BZE, Diazepam, nor Diazepam, Alprazolam, Fentanyl, nor Fentanyl


“Comparison of Paper Spray -MS/MS and Current Techniques for Screening Drugs of Abuse in Urine”, 62th ASMS Conference on Mass Spectrometry and Allied Topics, Baltimore, MD 2014

Opiates and Drugs of Abuse Blood Extract: 8/3 Acetonitrile / CHCl3 Spray:90/10/0.1 Acetonitrile / Water/ acetic acid

Unpublished development Prosolia, Inc

Amphetamine, methamphetamine, MDA, MDMA, MDEA, morphine, cocaine

Blood 80/20/0.1 Methanol/CH2Cl2 / hydroxyl amine

“Paper spray and extraction spray mass spectrometry for the direct and simultaneous quantification of eight drugs of abuse in whole blood.” Anal Chem. 2014 Aug 5;86(15):7712-8 2014 Jul 9.

Developed Methods


http://www.ncbi.nlm.nih.gov/pubmed/?term=Paper+Spray+and+Extraction+Spray+Mass+Spectrometry+for+the+Direct+and+Simultaneous+Quantification+of+Eight+Drugs+of+Abuse+in+Whole+Blood

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Reference

Synthetic Bath Salts: Fluoro methamphetamine, Mephedrone, Pentedrone, MDMA, Methylone, MDPV


Unpublished development Prosolia Inc

Amitriptyline, Verapamil, Codeine, Amphetamine, PCP, Cocaine

Blood 95/5/0.01 methanol / Water/Acetic Acid

“Direct Analysis using Paper-Spray Mass Spectrometry: Method Development for the Rapid Screening of Drugs of Abuse for Forensic Toxicology, SOFT Conference, 2013

Pazaponib (Tyrosine kinase inhibitor used in treatment for renal cell carcinoma)

Blood 95/5/0.01 methanol / Water/Acetic Acid

Unpublished Development UTHealth | The University of Texas Health Science Center at Houston | Medical School

Immunosuppressive drugs: Tacrolimus and cyclosporine

Blood 60/40/0.1 Methanol/CHCl3 / Sodium Acetate

“Assessment of paper spray ionization for quantitation of pharmaceuticals in blood spots", International Journal of Mass Spectrometry, 2011, 300, 123-129.

Developed Methods


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Reference

Anti inflammatory dialdehydes: Oleocanthal, Hydroxytyrasol, Tyrosol

Olive Oil 1.5 M Methoxyamine hydrochloride methanol/water (1:1,v/v)

“Evaluation of dialdehydic anti-inflammatory active principles inextra-virgin olive oil by reactive paper spray mass spectrometry “ International Journal of Mass Spectrometry 352 (2013) 87– 91

Free fatty acids, Phosphatidylglycerols, Phosphatidlethanolamines

Bacterial cultures

0.05% CHAPS in Methanol CHAPS is a surfactant

“Rapid discrimination of bacteria by paper spray mass spectrometry” Anal Chem. 2014 Aug 5;86(15):7500-72014 Jul 22.

Paclitaxel (mitotic inhibitor used in cancer chemotherapy)

Blood 60/40 CHCl3 / methanol with 0.1% Sodium Acetate

Unpublished Development UTHealth | The University of Texas Health Science Center at Houston | Medical School

Synthetic Cannabinoids JWH018,JWH073 / 015, JWH200, JWH250, JWH081,JWH122/019, RCS-8, WIN55,212


Unpublished development Prosolia Inc

Developed Methods


http://www.ncbi.nlm.nih.gov/pubmed/?term=Rapid+Discrimination+of+Bacteria+by+Paper+Spray+Mass+Spectrometry

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Cross Validation of Paper Spray Technology

Presenter

Presentation Notes

Data acquisition methods for targeted and unknown screening are the same. Data processing is different.

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• Analysis of chemotherapeutic drugs and pharmacokinetics

• Imatinib

• Pazopanib

• Routine quantitation for immunosuppressant drug monitoring

• Drugs of abuse in urine and blood

Cross Validations – Collaborators Work


Presenter

Presentation Notes

Analysis of chemotherapeutics and immunosuppressant drugs used an early prototype of the source. The last application used the Prosolia Velox 360 source.

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• Analytes • Imatinib, N-Desmethyl Imatinib

• Internal standard • [2H8] – Imatinib for both analytes

• Samples • Incurred plasma samples • 45 total samples from 3 different subjects

• HPLC – MS/MS • Protein precipitation, centrifuge • Inject supernatant

• PaperSpray – MS/MS • Plasma sample spotted onto paper and dried at ambient temperature • MS: Thermo Scientific™ TSQ Ultra™ mass spectrometer

Drug: m/z 494 → 394 IS: m/z 502 → 394

Data courtesy of Jennifer Cunliffe, Novartis


Cross Validation for Imatinib

Presenter

Presentation Notes

Imatinib, marketed by Novartis as Gleevec or Glivec, is a tyrosine-kinase inhibitor used in the treatment of multiple cancers. Instrument is a Thermo Scientific TSQ Ultra.

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Cross Validation against HPLC-MS/MS - Imatinib

slope = 1.0887 R² = 0.9886

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• IS: [2H8] - Imatinib • 95% (40/42) passed acceptance criteria

• Paper Spray was within 20% of HPLC - MS/MS

R² = 0.9996

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Therapeutic Range

Therapeutic range of interest


Data courtesy of Jennifer Cunliffe, Novartis

Presenter

Presentation Notes

Cross validation data is extremely valuable for establishing paper spray technology in markets where the accepted technologies are LC/MS/MS and immunoassays. Please note the therapeutic range of interest on the right hand spectrum is pretty high, therefore not requiring a highly sensitive technique.

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• Collaboration with Stanford Univ. Medical Center • Drug Monitoring of Tacrolimus

• Immunosuppressive drug for organ transplant • Monitoring drug concentrations is required

• Lab currently uses an immunoassay kit • Costs ~$20/sample • Generate gallons of waste/day • Wants to switch to an MS based method

• Validated paper spray MS for quantitation of tacrolimus • Move on to Cyclosporine, Sirolimus (both done by IA) and

Everolimus (currently a send-out)

MW = 804.018

Run Shi et al., Clinica Chimica Acta 441 (2015) 99-104.


Immunosuppressant Drug Monitoring Research

Presenter

Presentation Notes

Samples were in DBS. Instrument is a Vantage TSQ. Immunosuppressive or antirejection drugs inhibit or prevent activity of the immune system.

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Selectivity – Quantifier and Qualifier SRMs

test_run2_Tacro_Cyclo1114201219 #47-299 RT: 0.18-1.04 AV: 50 NL: 9.04E2F: + c ESI SRM ms2 826.510 [443.142-443.152, 616.310-616.320]

443.145 443.150m/z

616.310 616.315 616.320m/z

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test_run2_Tacro_Cyclo1114201212 #39-300 RT: 0.14-1.04 AV: 52 NL: 3.60E3F: + c ESI SRM ms2 826.510 [443.142-443.152, 616.310-616.320]

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616.310 616.315 616.320m/z

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Donor sample Spiked blood

m/z 826 → 616, 443 (tacrolimus, [M + Na]+)

Extraction/spray solvent: 40:40:20 methanol:dichloromethane:chloroform with 0.1% Na acetate MS: Thermo Scientific™ TSQ Vantage™ mass spectrometer



Presenter

Presentation Notes

TSQ Vantage, samples are donor blood. Showing how the SRM transitions look the same in donor vs. spiked blood.

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Tacrolimus Calibration Curves

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calstandards1/x fit

• Calibration curve for Tacrolimus from 0.5 ng/mL to 30 ng/mL

• 2[H]3 – tacrolimus used as IS • Signal at 0.5 ng/mL is ~20X

blank

Tacrolimus, mw = 804.018



Presenter

Presentation Notes

Standards in spiked blood with IS.

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Tacrolimus Cross Validation against HPLC-MS/MS

• Validated against a standard HPLC-MS/MS assay on a Sciex 5500 and an external reference lab

• Paper spray MS/MS on a TSQ Vantage mass spectrometer



Presenter

Presentation Notes

Cross validation data – on the left hand side, paper spray on Thermo Scientific Vantage vs. LC/MS/MS with a Sciex 5500 and on the right hand side, send out analysis to a reference lab and analyzed by LC/MS/MS, unknown mass spec.

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Tacrolimus Cross Validation against Immunoassay System

y = 1.0x - 0.02

R² = 0.99 (n=40)

0

10

20

30

40

0 10 20 30 40Immunoassay (ng/mL)

Pape

r Spr

ay –

MS/

MS

(ng/

mL)

• Tacrolimus paper spray data validated against Architect Immunoassay system, Abbott Diagnostics



Presenter

Presentation Notes

Paper spray validated against the immunoassay used at the Stanford clinical lab.

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Drug Molecular Ion

Internal Standard

Assay range

(ng/mL)

Approximate LOD from DBS

(ng/mL)*

Cyclosporine [M + Na]+ 2[H]3-cyclosporine 25 - 1000 0.05

Tacrolimus [M + Na]+ 2[H]3-tacrolimus 1.5 - 30 0.08

Imatinib [M + H]+ 2[H]8-imatinib 4 - 8000 0.7

Melphalan [M + H]+ 2[H]8-melphalan 100-25000 20

Pazopanib [M + H]+ 2[H]4-pazopanib 100-50000 3

Paclitaxel [M + Na]+ 2[H]5-paclitaxel 10-1000 1

*On a Thermo Scientific™ TSQ triple quad. Model varied


Slide courtesy of Nick Manicke, Assistant Professor, IUPUI

PaperSpray Assays for Drug Monitoring Research

Presenter

Presentation Notes

Summary table for a few of the compounds studied with Thermo triple quads. The point to make is that paper spray limits of detection are much lower than the therapeutic assay ranges, making it not just a suitable technique but a game changer technique considering that there’s no sample preparation and answers are delivered in seconds. Imatinib, melphalan, pazopanib, paclitaxel are chemotherapeutic drugs Cyclosporine and tacrolimus are immnunosuppressants

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• The paper spray technique has been demonstrated as an easy-to-use technique, with no sample preparation, and no prior chromatography.

• The Prosolia Velox 360 PaperSpray system shows extraordinary potential for the screening and quantitative analysis of drugs of abuse and drug monitoring research when coupled to Thermo Scientific mass spectrometers, all in much shorter timeframes than is possible using liquid chromatography techniques.

• As the electrospray signal from paper spray lasts ~40 s, it can accommodate many experiments per sample spot for identification and confirmation in screening applications. • Many SRM transitions per sample when coupled to a Thermo Scientific™ Endura™ or

Thermo Scientific™ Quantiva™ triple quadrupole mass spectrometer. • It can accommodate full scan MS at high resolving power and many MS/MS experiments

when coupled to a Thermo Scientific™ Q Exactive™ mass spectrometer with HRAM capabilities.

• Both positive and negative ion mode available for paper spray ionization

Conclusions


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