expect the unexpected an example of how unanticipated fragmentation behaviour could preclude correct...
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Expect the unexpected
An example of how unanticipated fragmentation behaviour could preclude correct assignment of
sites of metabolismStephen W. Holman8th September 2008 [email protected]
22
Outline
• Project background
• Experimental
• Results and discussion
• Conclusions
• Acknowledgements
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
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
5
Parent compound
S-oxidised metabolite
66
1st gen. prod. ion spec. of protonated parent compound
308
261228
7
323
321
[M + H – 62 m/z units]+
320
322 324325
338
368
275
260
274
257
242
323
321
[M + H – 62 m/z units]+
1st gen. prod. ion spec. of protonated metabolite
8
Proposed mechanism for the loss of 62 m/z units
9
371
340
325
277
276
262
257
242
323
321
Additional peak
321
322
323
324
325
326
1st 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
Proposed mechanism for the loss of 62 m/z units
11
338275
374
260
274
257
242
323321
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 C2H10N2
• One retains primary amine loss is C2H8NO•
1st gen. prod. ion spec. of protonated hard deuterium labelled metabolite analogue
No mass shift
1212
1st gen. prod. ion spec. of protonated metabolite using FT-ICR-MS
1313
Summary of product ions and losses
C17H23N2O4S2+
C15H13O4S2+
C15H15NO3S2+
•
C2H10N2
C2H8NO•
1414
Proposed mechanism for loss of C2H10N2
1515
Molecular model of proposed product ion structure at m/z 321.0252
16
Proposed mechanism for loss of C2H8NO·
NH
SO
O
H2N
O
S+
HO-
O+
S+O-
SO
O
H2N
HOH
SO
O
H2N S+O-
SH2N S+
O-
O
O
O
N
SO
O
H2N S+O-
H+
1717
Molecular model of proposed product ion structure at m/z 321.0492
18
1st gen. prod. ion spec. of protonated metabolite
338
368
275
260
274
257
242
323
321
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
2nd 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 323m/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
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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
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