Potential of measured relative shifts in collision cross section values for biotransformation studies

Ion mobility spectrometry-mass spectrometry (IMS-MS) separates gas phase ions due to differences in drift time from which reproducible and analyte-specific collision cross section (CCS) values can be derived. Internally conducted in vitro and in vivo metabolism (biotransformation) studies indicated repetitive shifts in measured CCS values (CCS_meas) between parent drugs and their metabolites. Hence, the purpose of the present article was (i) to investigate if such relative shifts in CCS_meas were biotransformation-specific and (ii) to highlight their potential benefits for biotransformation studies. First, mean CCS_meas values of 165 compounds were determined (up to n = 3) using a travelling wave IMS-MS device with nitrogen as drift gas (^TWCCS_{N2, meas}). Further comparison with their predicted values (^TWCCS_{N2, pred}, Waters CCSonDemand) resulted in a mean absolute error of 5.1%. Second, a reduced data set (n = 139) was utilized to create compound pairs (n = 86) covering eight common types of phase I and II biotransformations. Constant, discriminative, and almost non-overlapping relative shifts in mean ^TWCCS_N2, meas were obtained for demethylation (- 6.5 ± 2.1 Ų), oxygenation (hydroxylation + 3.8 ± 1.4 Ų, N-oxidation + 3.4 ± 3.3 Ų), acetylation (+ 13.5 ± 1.9 Ų), sulfation (+ 17.9 ± 4.4 Ų), glucuronidation (N-linked: + 41.7 ± 7.5 Ų, O-linked: + 38.1 ± 8.9 Ų), and glutathione conjugation (+ 49.2 ± 13.2 Ų). Consequently, we propose to consider such relative shifts in ^TWCCS_{N2, meas} (rather than absolute values) as well for metabolite assignment/confirmation complementing the conventional approach to associate changes in mass-to-charge (m/z) values between a parent drug and its metabolite(s). Moreover, the comparison of relative shifts in ^TWCCS_{N2, meas} significantly simplifies the mapping of metabolites into metabolic pathways as demonstrated.