1. Investigation of Oxidative and Conjugative Metabolism Reactions with Liquid Chromatography / Accurate Mass High Resolution Mass Spectrometry Maciej Bromirski Thermo Fisher Scientific (Bremen, Germany) Denver, June 8, 2011

2. 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

3. Methods

4. 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

5. 5 The ROXY potentiostat

6. 6 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

7. 7 Setup with chromatographic separation reaction products are trapped in injection loop content of injection loop is passed to column

8. 8 Resolution 100,000 at 1 scan per second 10,000 at 10 scans per second Mass accuracy ppm mass accuracy Sensitivity 500 fg Buspirone > 10:1 Dynamic Range >4000 within a spectrum Scan speed Up to 10 scans per second Mass Range m/z 50 - 4000 Polarity switching Yes, 1 full cycle <1 sec Exactive™ - specification

9. Metabolites of a pesticide: Thiamethoxam

10. 10 Reaction products of Thiamethoxam C 8 H 10 O 3 N 5 ClS C 8 H 10 O 4 N 5 ClS C 7 H 13 O 5 N 3 C 8 H 10 O 2 N 4 S C 8 H 12 O 3 N 4 S C 8 H 11 ON 4 ClS C 8 H 12 O 1 N 5 ClS C 8 H 10 O 2 N 5 ClS C 8 H 11 O 5 N 5 S -H 2 ? Thiamethoxam +O

11. 11 Voltammogram of selected reaction products 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

12. 12 Reaction product 1: Oxygenated Thiamethoxam?  YES!

13. 13... and it is not Glutathion

14. 14 Reaction product 2: Dehydrogenated Thiamethoxam?  NO!

15. 15... but what else is it??? Dehydrogenated Thiamethoxam: C 8 H 8 O 3 N 5 ClS  exact mass 290.01091 Measured mass: 290.05487  on an Exactive, this is a clear mismatch Proposed elemental composition: C 8 H 11 O 5 N 5 S Proposed structure:

16. Metabolites of a drug: Ticlopidin

17. 17 Reaction products of Ticlopidin 0.0 V EC potential 2.5 V EC potential

18. 18 Metabolites confirmed by microsome incubation 1 264.0594: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

19. 19 A closer look on the oxygenation products C 14 H 12 ONClS Ticlopidin – 2H +O Δm 1.3 ppm C 14 H 14 ONClS Ticlopidin +O Δm 1.1 ppm

20. 20 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

21. 21 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

22. ?