Maciej Bromirski Thermo Fisher Scientific (Bremen, Germany) Denver, June 8, 2011

Investigation of Oxidative and Conjugative Metabolism Reactions with Liquid Chromatography / AccurateMass High Resolution Mass Spectrometry

Maciej Bromirski

Thermo Fisher Scientific (Bremen, Germany)

Denver, June 8, 2011

2

Why?

• In metabolic studies, the identification of metabolites in blood or urine samples as well as liver microsome incubations is relatively easy, but the detection of reactive species turns out to be tricky

• Trapping experiments deliver only indirect proof of identitiy

• Electrochemical simulations of metabolic processes, coupled to high performance liquid chromatography (HPLC), have been shown to be a simple and fast alternative to classical approaches

• Combination of electrochemistry with high resolution accurate mass (HRAM) mass spectrometry may deliver enhanced sensitivity and confidence in metabolite detection, especially for reactive metabolites

Methods

4

Experimental Setup

• For the shown experiments a ROXY™ potentiostat equipped with a Reactor Cell™ thin layer flowcell from Antec B. V. (Zoeterwoude, The Netherlands) was used, coupled to a Thermo Scientific Exactive Benchtop Orbitrap mass spectrometer

• Analyte solutions were provided in water / acetonitrile 1:1 with 0.1% formic acid

• Protein solutions for conjugation experiments were provided in 10 mM ammonium formiate solution, adjusted to pH 7.8

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The ROXY potentiostat

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Infusion experiments for Phase I and Phase II studies

Phase I studies

Phase II studies

only analyte passing EC cell is analysed

conjugation of reaction products with protein after EC cell

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Setup with chromatographic separation

reaction products are trapped in injection loop

content of injection loop is passed to column

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• Resolution100,000 at 1 scan per second 10,000 at 10 scans per second

• Mass accuracy ppm mass accuracy

• Sensitivity500 fg Buspirone > 10:1

• Dynamic Range>4000 within a spectrum

• Scan speedUp to 10 scans per second

• Mass Rangem/z 50 - 4000

• Polarity switchingYes, 1 full cycle <1 sec

Exactive™ - specification

Metabolites of a pesticide:Thiamethoxam

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Reaction products of Thiamethoxam

Voltammogramm Thiamethoxam 20110428 #625 RT: 7.85 AV: 1 NL: 1.88E8T: FTMS + p ESI Full ms [100.00-750.00]

200 210 220 230 240 250 260 270 280 290 300 310m/z

0

5

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Re

lativ

e A

bu

nd

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ce

247.04140

292.02628

220.09268

245.07022

249.03836 262.05215

294.02322

227.05960204.09781276.03137

230.01485309.05267264.04916

213.09799

243.05437

232.03033303.05478259.10352 267.07803 290.05493278.02841234.02750

274.04871251.03394 296.01865209.10304

218.07700

C8H10O3N5ClS

C8H10O4N5ClS

C7H13O5N3

C8H10O2N4S

C8H12O3N4S

C8H11ON4ClS

C8H12O1N5ClS

C8H10O2N5ClS

C8H11O5N5S

-H2?

Thiamethoxam

+O

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Voltammogram of selected reaction products

RT: 0.00 - 11.00

0 1 2 3 4 5 6 7 8 9 10 11Time (min)

0

50

1000

50

1000

50

1000

50

1000

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100

Re

lativ

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ce 0

50

1000

50

1000

50

10010.604.983.52 4.642.57 5.054.193.161.01 5.662.08 10.216.320.63 1.31 9.217.10 9.158.717.12

7.65 7.857.51 8.136.47 7.33

6.43 8.376.31 8.536.125.58 8.723.43 3.97 4.242.431.200.62 3.181.94 9.38

7.897.617.45 8.10 8.296.926.53

8.466.328.62 8.876.16 9.505.49

8.09 8.147.85

7.707.46 8.41

8.59 8.777.29 9.28 9.642.29 10.646.600.08 4.213.31 4.691.49 5.990.47 2.92 5.763.858.338.27

8.377.828.577.51 8.79

9.227.409.70 10.217.261.540.44 1.22 1.94 2.64 4.023.22 4.37 7.015.00 5.76

8.298.14 8.337.65

8.587.518.78 9.217.36 9.69 10.261.520.15 7.220.54 2.00 3.472.90 3.962.68 4.37 4.84 5.14 6.955.57

7.94 8.027.757.438.46

7.197.06 8.636.60 8.82

9.289.816.35 10.631.550.52 6.110.85 1.88 2.73 5.453.42 3.66 4.39

8.148.09 8.247.74

8.417.51 8.57

7.43 8.93 9.21 9.84 10.207.251.360.81 1.570.04

NL: 2.32E8m/z= 292.02479-292.02771 F: FTMS + p ESI Full ms [100.00-750.00] MS Voltammogramm Thiamethoxam 20110428








Thiamethoxam

M1 m/z 308

M2 m/z 290

M3 m/z 276

M4 m/z 262

M5 m/z 247

M6 m/z 245

M7 m/z 230

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Reaction product 1: Oxygenated Thiamethoxam?

YES!

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... and it is not Glutathion

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Reaction product 2: Dehydrogenated Thiamethoxam?

NO!

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... but what else is it???

• Dehydrogenated Thiamethoxam: C8H8O3N5ClS

• exact mass 290.01091

• Measured mass: 290.05487• on an Exactive, this is a clear mismatch

• Proposed elemental composition: C8H11O5N5S

• Proposed structure:N

SNO

N N

NO O

O

OH

N

SNO

N N

NO O

Cl

Metabolites of a drug:Ticlopidin

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Reaction products of Ticlopidin

260 262 264 266 268 270 272 274 276 278 280 282m/z

0

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0

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Re

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264.0598

266.0567

265.0632

267.0600

262.0441

264.0594

260.0290

278.0390266.0566263.0477

265.0630 280.0359261.0324

279.0423

267.0601

NL: 2.49E6data06#130-145 RT: 1.14-1.27 AV: 16 T: FTMS {1,1} + p ESI Full ms [50.00-1000.00]

NL: 1.46E6data06#663 RT: 5.76 AV: 1 T: FTMS {1,1} + p ESI Full ms [50.00-1000.00]

0.0 V EC potential

2.5 V EC potential

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Metabolites confirmed by microsome incubation1

• 264.0594: Ticlopidin• 262.0441: Ticlopidin – 2H• 260.0290: Ticlopidin – 4H

• 278.0390: Ticlopidin – 2H + O

• 280.0544: Ticlopidin + O

1 Davie et al., DMD 32:49–57, 2004

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A closer look on the oxygenation products

data06 #663 RT: 5.76 AV: 1 NL: 3.17E5T: FTMS {1,1} + p ESI Full ms [50.00-1000.00]

278.0 278.5 279.0 279.5 280.0 280.5 281.0 281.5 282.0m/z

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Re

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278.0390

280.0359

279.0423

282.0515281.0392

C14H12ONClSTiclopidin – 2H +OΔm 1.3 ppm

C14H14ONClSTiclopidin +OΔm 1.1 ppm

data06 #663 RT: 5.76 AV: 1 NL: 1.03E5T: FTMS {1,1} + p ESI Full ms [50.00-1000.00]

279.9 280.0 280.1 280.2m/z

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110

Re

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280.0359

280.0544

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Summary

• Metabolites can be detected with high sensitivity and specificity

• Ultra high resolution provides clear separation from interferences

• Unique scan-to-scan mass accuracy provides clear distinction of false positives from correct signals

• High confidence in identification is provided

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Acknowlegements

• Antec B. V., Zouterwoude, The Netherlands• Agnieszka Kraj

• University of Muenster, Germany• Uwe Karst

• Anne Baumann

• Thorsten Vielhaber

• Thermo Fisher Scientific, Germany• Olaf Scheibner

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Documents

Maciej Bromirski Thermo Fisher Scientific (Bremen, Germany) Denver, June 8, 2011