Expect the unexpected An example of how unanticipated

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Expect the unexpected An example of how unanticipated fragmentation behaviour could preclude correct assignment

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 swh 01@soton. ac. uk

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

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

Project background • Based upon Wright et al. , RCM, 2005, 19, 2005 -2014

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

Experimental • Solutions prepared in HCOOH: Me. OH (0. 1: 99. 9, v/v) or

Experimental • Solutions prepared in HCOOH: Me. OH (0. 1: 99. 9, v/v) or CH 3 COOD: Me. OD (1: 99, v/v) – 1 o µg m. L-1 for QIT-MS experiments (LCQ Classic) – 1 µg m. L-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

Compounds analysed Parent compound S-oxidised metabolite 5

Compounds analysed Parent compound S-oxidised metabolite 5

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

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

1 st gen. prod. ion spec. of protonated metabolite 338 323 [M + H

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

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

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

1 st gen. prod. ion spec. of fully exchanged, deuterated metabolite 340 325 •

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

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

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

1 st gen. prod. ion spec. of protonated hard deuterium labelled metabolite analogue 275

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

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

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

Summary of product ions and losses C 15 H 13 O 4 S 2+

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

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

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

Molecular model of proposed product ion structure at m/z 321. 0252 15

Molecular model of proposed product ion structure at m/z 321. 0252 15

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

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

Molecular model of proposed product ion structure at m/z 321. 0492 17

Molecular model of proposed product ion structure at m/z 321. 0492 17

1 st gen. prod. ion spec. of protonated metabolite 338 275 274 260 323

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

2 nd gen. prod. ion spec. of fully exchanged, deuterated metabolite 257 • Protonation

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

Conclusions • S-oxidation can significantly change the fragmentation of a compound • Fragmentation under

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 20

Acknowledgements • John Langley, University of Southampton • Pat Wright, Pfizer Global Research and

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

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