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Stephen W. Holman 8 th September 2008 swh01@soton.ac.uk

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. Outline. Project background Experimental Results and discussion Conclusions

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Stephen W. Holman 8 th September 2008 swh01@soton.ac.uk

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

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

  3. 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 3

  4. Experimental • Solutions prepared in HCOOH:MeOH (0.1:99.9, v/v) or CH3COOD: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. Compounds analysed Parent compound S-oxidised metabolite 5

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

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

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

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

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

  11. 323 No mass shift 275 338 321 274 260 323 324 322 321 325 257 326 374 242 1st gen. prod. ion spec. of protonated hard deuterium labelled metabolite analogue • All deuterium atoms are lost • Ions are nominally isobaric, so both loss the tertiary amine group • One loses primary amine  loss is C2H10N2 • One retains primary amine  loss is C2H8NO• No mass shift

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

  13. Summary of product ions and losses C15H13O4S2+ C2H10N2 C17H23N2O4S2+ C15H15NO3S2+• C2H8NO• 13

  14. Proposed mechanism for loss of C2H10N2 14

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

  16. Proposed mechanism for loss of C2H8NO·

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

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

  19. 340 257 * = 17 m/z units [M + D – 46 m/z units]+ 242 340 276 [M + D – 46 m/z units]+ Absence of product ion at m/z 321 Absence of product ion at m/z321 [M + D – 63 m/z units]+ 323 [M + D – 63 m/z units]+ 262 - 17 m/z units 323 325 322 * 2nd gen. prod. ion spec. of fully exchanged, deuterated metabolite • Protonation at tertiary amine • m/z 323 formed viam/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 viam/z 340 i.e.m/z 321 with no exchangeable hydrogen atoms does not protonate at the tertiary amine m/z 323 m/z 321

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

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