Expect the unexpected

An example of how unanticipated fragmentation behaviour could preclude correct assignment of

sites of metabolismStephen W. Holman8th September 2008 swh01@soton.ac.uk

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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

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•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

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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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Thank you for your attention

Any questions?

swh01@soton.ac.uk

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