High‐throughput approaches towards the definitive identification of pharmaceutical drug metabolites. 2. An example of how unexpected dissociation behaviour could preclude correct assignment of sites of metabolism

Abstract

The low-energy collision-induced dissociation product ion spectra of protonated beta-blockers reveal an analogy to fragmentation under electron ionisation conditions", J. Mass Spectrom. 2011Spectrom. , 1182Spectrom. -1185 S.W. Holman, P. Wright, N.J. Wells and G.J. Langley, "Evidence for site-specific intra-ionic hydrogen/deuterium exchange in the low-energy collision-induced dissociation product ion spectra of protonated small molecules generated by electrospray ionisation" J. Mass Spectrom. 2010, 45, 347-357 Abstract S-oxidation is a common metabolic route for sulphur containing compounds. When studying the fragmentation of some chemically synthesised sulphoxides, two unexpected losses of 62 m/z units were observed in the collision-induced dissociation (CID) product ion spectrum protonated 3-dimethylaminomethyl-4-(4-methanesulfinyl-3-methyl-phenoxy)-benzenesulfonamide. A single loss was initially assigned using the low resolution product ion spectrum, acquired by electrospray ionisation-quadrupole ion trap-mass spectrometry (ESI-QIT-MS), as methanethial, S-oxide with the dissociation proceeding via a charge-remote, four-centred rearrangement. This assignment was consistent with well-documented hydrogen rearrangements in the literature. Further, the loss was not observed for the parent compound. Thus, it was inferred that the site of metabolism was involved in the dissociation and the attractive nature of the four-centred rearrangement meant that the loss of methanethial, Soxide was a logical assignment. However, deuterium labelling experiments and accurate mass measurements, performed using electrospray ionisation-Fourier Transform Ion Cyclotron Resonance-mass spectrometry (ESI-FTICR-MS), showed that two distinct losses of 62 m/z units occur, neither of which was of that initially hypothesised from the low resolution product ion spectrum of the protonated molecule. Mechanisms consistent with the experimental findings are postulated. An MS 3 spectrum of the fully exchanged, deuterated species supported the proposed mechanisms by suggesting that 3-dimethylaminomethyl-4-(4-methanesulfinyl-3-methyl-phenoxy)-benzenesulfonamide has multiple sites of protonation in the gas phase. The planar structures of the posited product ions are likely to provide the driving force for the rearrangements. The relevance of the observations with regards to pharmaceutical drug metabolite identification are discussed.

Using density functional theory to rationalise the mass spectral fragmentation of maraviroc and its metabolites

“…Indeed, it is possible that protonation occurs at all suitable sites, but with lower abundance for the less stable protonated species. The inference that protonation may result in a mixture of ionised species is supported in two recent papers both of which report that two isobaric ions23, 24 in the same spectra can only have been formed from precursor ions protonated on different atoms. It should be noted that that the fragmentation reported in the paper by Holman et al ,23 resulting in isobaric product ions, would also be observed if the precursor ion was protonated at a single site during ionisation then underwent proton migration during CID.…”

“…The inference that protonation may result in a mixture of ionised species is supported in two recent papers both of which report that two isobaric ions23, 24 in the same spectra can only have been formed from precursor ions protonated on different atoms. It should be noted that that the fragmentation reported in the paper by Holman et al ,23 resulting in isobaric product ions, would also be observed if the precursor ion was protonated at a single site during ionisation then underwent proton migration during CID. However, the study by Kaufmann et al 24 provides experimental evidence, including differing effects of cone voltage on the sampling of the isobaric [M+H] + ions, that formation of a mixture of singly charged species during ionisation does occur.…”

Predicting collision-induced dissociation spectra: Semi-empirical calculations as a rapid and effective tool in software-aided mass spectral interpretation

“…Other groups have also reported that precursor ions appear to be a mixture of ions protonated at different positions. Two isobaric ions observed in an MS/MS spectrum could only be assigned if they are derived from precursor ions protonated at different sites, giving rise to different product ions . Kaufmann reported that a mixture of singly charged difloxacin species were formed in the source .…”

“…Two isobaric ions observed in an MS/MS spectrum could only be assigned if they are derived from precursor ions protonated at different sites, giving rise to different product ions. [30] Kaufmann reported that a mixture of singly charged difloxacin species were formed in the source. [31] Komaromi et al observed that N-acetyl-O-methoxyproline exhibits two distinct fragmentation pathways indicative of the coexistence of several protonated forms.…”

A review of LC–MS techniques and high-throughput approaches used to investigate drug metabolism by cytochrome P450s

“…A number of recent reviews have discussed in detail the role of bioanalysis in metabolite identification [8,61,[69][70][71]. A consequence of recent advances to address high-throughput technology for screening the metabolic liability of compounds is the demand for more rapid methods for metabolite identification [72][73][74]. The bioanalytical approaches which appear most amenable to support these demands utilise UPLC and HILIC [32,41,51], whose applications have already been presented earlier in the context of metabolic stability studies.…”