F +13.9838 Da in comparison to the parent compound, may result from either hydroxylation in combination with desaturation (e.g., di-hydroxylation followed by dehydration) or carbonylation. Even so, the corresponding signals may well also arise from in-source water loss, resulting in the cleavage of aliphatic hydroxyl-groups (e.g., in the 4-methyl-tetrahydropyran- and adamantyl-moiety). In-source water loss was viewed as as probably, where (i) a hydroxylated metabolite was detected, exhibiting a hydroxyl group at a position predestined for in-source water loss, (ii) a co-eluting signal was identified, presenting a dehydration-specific mass shift of -18.0153 Da (-H2 O), and (iii) after fragmentation, when the type and position of biotransformation were identical for the hydroxylated metabolite as well as the alleged artefact. By way of example MC21, a metabolite made by monohydroxylation at the 4-methyl-tetrahydropyran-moiety (i) was detected, but on top of that a signal at the corresponding retention time (Rt) with mass shift of [M + H]+ -18.0153 Da was discovered (ii), which exhibits dehydration in the 4-methyl-tetrahydropyran-moiety (iii). Therefore, this signal was classified as an artefact (MCArt4). The diversity in the hydroxylation patterns of metabolites, particularly in situations of two or three concurrent hydroxylations, tends to make the evaluation of in-source processes very complex. The observed outcomes recommend that the susceptibility for in-source water loss considerably varies among aliphatic structures (e.g., adamantyl α2β1 Inhibitor Formulation versus 4-methyl-tetrahydropyran). This becomes obvious when comparing the peak places of genuine metabolites plus the corresponding in-source artefacts. Within the case of MA2 (hydroxylated in the adamantyl-moiety) the corresponding artefact (MAArt1) showed a 6.8 occasions higher signal than observed for MA2 itself. In comparison, MC21 (hydroxylated in the 4-methyl-tetrahydropyran-moiety) exhibited an in-source dehydration signal of roughly the same intensity as that observed for MC21. Furthermore, positional isomers of hydroxylations inside a moiety led to varying levels of observed water loss. For example, when investigating the metabolite clusterMetabolites 2021, 11,four ofMC8a (consisting of quite a few co-eluting di-hydroxylated metabolites, bearing a hydroxylgroup at the 4-methyl-tetrahydropyran-moiety), in-source water loss varied from excessive (artefact signal [MCArt2a ] metabolite signal) to not detectable. In this study, numerous hydroxylated metabolites of CUMYL-THPINACA and a single of ADAMANTYL-THPINACA were prone to in-source dehydration, in most situations attributable towards the instability on the hydroxylated 4-methyl-tetrahydropyran-moiety. This most likely resulted within the identification of various TrkA Inhibitor Molecular Weight artefacts that happen to be discussed within the corresponding chapters referring to the genuine metabolites. In addition, a number of signals were detected lacking a hydroxylated counterpart, consequently not meeting the above-stated criteria for in-source water loss–they have been thus classified as genuine metabolites produced by hydroxylation and desaturation (MC3, MC6, MC12, MC17, MA3, MA8, MA11) or carbonylation (MC13, MC15, MC18, MC20, MC22, MA13, MA10). Even so, the possibility remains, that the hydroxylated original metabolite was prone to complete in-source water loss, i.e., the original parent ion was no longer detectable. In the context of analytics and also the herein presented aims, the focus of this study lies in the identification of suitable biomarkers, which may possibly.