1. Expect the unexpected An example of how unanticipated fragmentation behaviour could preclude correct assignment of sites of metabolism Stephen W. Holman 8 th September 2008 swh01@soton.ac.uk

2. 22 Outline Project background Experimental Results and discussion Conclusions Acknowledgements

3. 33 Project background Based upon Wright et al., RCM, 2005, 19, 2005-2014 Radical losses observed from sulfoxides Allows rapid and definitive identification of site and type of metabolism Aim is to identify similar interpretation tools Paper published in RCM, 2008, 22, 2355-2365

4. 4 Experimental Solutions prepared in HCOOH:MeOH (0.1:99.9, v/v) or CH 3 COOD:MeOD (1:99, v/v) –1o µg mL -1 for QIT-MS experiments (LCQ Classic) –1 µg mL -1 for FT-ICR-MS experiments (Apex III) Direct infusion at 3 µL min -1 into ESI source Product ion spectra of [M + H] + or [M + D] + acquired 4

5. 5 Compounds analysed 5 Parent compoundS-oxidised metabolite

6. 66 1 st gen. prod. ion spec. of protonated parent compound 308 261 228

7. 7 323 321 [M + H – 62 m/z units] + 320 322 324 325 338 368 275 260 274 257 242 323 321 [M + H – 62 m/z units] + 1st gen. prod. ion spec. of protonated metabolite

8. 8 Proposed mechanism for the loss of 62 m/z units

9. 9 371 340 325 277 276 262 257 242 323 321 Additional peak 321 322 323 324 325 326 1 st gen. prod. ion spec. of fully exchanged, deuterated metabolite Additional peak Two nominally isobaric ions One loses all the exchangeable hydrogen atoms One loses one exchangeable hydrogen atom

10. 10 Proposed mechanism for the loss of 62 m/z units

11. 11 338 275 374 260 274 257 242 323 321 323 322 324 325 No mass shift 326 All deuterium atoms are lost Ions are nominally isobaric, so both loss the tertiary amine group One loses primary amine  loss is C 2 H 10 N 2 One retains primary amine  loss is C 2 H 8 NO 1 st gen. prod. ion spec. of protonated hard deuterium labelled metabolite analogue No mass shift

12. 12 1 st gen. prod. ion spec. of protonated metabolite using FT-ICR-MS

13. 13 Summary of product ions and losses C 17 H 23 N 2 O 4 S 2 + C 15 H 13 O 4 S 2 + C 15 H 15 NO 3 S 2 + C 2 H 10 N 2 C 2 H 8 NO

14. 14 Proposed mechanism for loss of C 2 H 10 N 2

15. 15 Molecular model of proposed product ion structure at m/z 321.0252

16. 16 Proposed mechanism for loss of C 2 H 8 NO ·

17. 17 Molecular model of proposed product ion structure at m/z 321.0492

18. 18 1st gen. prod. ion spec. of protonated metabolite 338 368 275 260 274 257 242 323 321

19. 19 Protonation at tertiary amine m/z 323 formed via m/z 340 i.e. m/z 321 with two exchangeable hydrogen atoms formed via protonation at tertiary amine and loss of dimethylamine Hydroxyl radical loss involves a non-exchangeable hydrogen atom m/z 321 not formed via m/z 340 i.e. m/z 321 with no exchangeable hydrogen atoms does not protonate at the tertiary amine 2 nd gen. prod. ion spec. of fully exchanged, deuterated metabolite [M + D – 46 m/z units] + * 242 257 262 276 323 340 * = 17 m/z units [M + D – 63 m/z units] + Absence of product ion at m/z 321 m/z 323 m/z 321 340 322 323 325 [M + D – 46 m/z units] + [M + D – 63 m/z units] + Absence of product ion at m/z 321 - 17 m/z units

20. 20 Conclusions S-oxidation can significantly change the fragmentation of a compound Fragmentation under CID conditions difficult to predict Extensive experimentation required to fully understand dissociation Can not assign site of metabolism confidently without rigorous analytical approach HDX experiments particularly useful for determining sites of protonation and elucidating different dissociation pathways

21. 21 Acknowledgements John Langley, University of Southampton Pat Wright, Pfizer Global Research and Development Julie Herniman, University of Southampton Louisa Wronska, University of Southampton 21

22. 22 Thank you for your attention Any questions? swh01@soton.ac.uk 22