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) between parent drugs and their metabolites. Hence, the purpose of the present article was (i) to investigate if such relative shifts in CCS were biotransformation-specific and (ii) to highlight their potential benefits for biotransformation studies. First, mean CCS values of 165 compounds were determined (up to n = 3) using a travelling wave IMS-MS device with nitrogen as drift gas (CCS). Further comparison with their predicted values (CCS, 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 CCS were obtained for demethylation (- 6.5 ± 2.1 Å), oxygenation (hydroxylation + 3.8 ± 1.4 Å, N-oxidation + 3.4 ± 3.3 Å), acetylation (+ 13.5 ± 1.9 Å), sulfation (+ 17.9 ± 4.4 Å), glucuronidation (N-linked: + 41.7 ± 7.5 Å, O-linked: + 38.1 ± 8.9 Å), and glutathione conjugation (+ 49.2 ± 13.2 Å). Consequently, we propose to consider such relative shifts in CCS (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 CCS significantly simplifies the mapping of metabolites into metabolic pathways as demonstrated.