Expect the unexpected An example of how unanticipated
- Slides: 22
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 2
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 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
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 – 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
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
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
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
Molecular model of proposed product ion structure at m/z 321. 0252 15
Proposed mechanism for loss of C 2 H 8 NO· 16
Molecular model of proposed product ion structure at m/z 321. 0492 17
1 st gen. prod. ion spec. of protonated metabolite 338 275 274 260 323 321 257 242 368 18
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 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 Development • Julie Herniman, University of Southampton • Louisa Wronska, University of Southampton 21
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