A comprehensive Collision Cross Section (CCS) library was obtained via travelling wave ion guide mobility measurements through direct infusion (DI). The library consists of CCS and Mass Spectral (MS) data in negative and positive ElectroSpray Ionisation (ESI) mode for 463 and 479 endogenous metabolites, respectively. For both ionisation modes combined, TW CCSN2 data were obtained for 542 non-redundant metabolites. These data were acquired on two different ion mobility orthogonal acceleration QToF MS systems in two different laboratories, with the majority of the resulting TW CCSN2 values (from detected compounds) found to be within 1% of one another. Validation of these results against two independent, external TW CCSN2 data sources and predicted CCS values indicated be within 1-2% of these other values. The same metabolites were then analysed using a rapid reversed-phase ultra (high) performance liquid chromatographic (U(H)PLC) separation combined with IM and MS (IM-MS) thus providing retention time (tr), m/z and TW CCSN2 values (with the latter compared with the DI-IM-MS data). Analytes for which TW CCSN2 values were obtained by U(H)PLC-IM-MS showed good agreement with the results obtained from DI-IM-MS. The repeatability of the TW CCSN2 values obtained for these metabolites on the different ion mobility QToF systems, using either DI or LC, encouraged the further evaluation of the U(H)PLC-IM-MS approach via the analysis of samples of rat urine, from control and methotrexate-treated animals, in order to assess the potential of the approach for metabolite identification and profiling in metabolic phenotyping studies. Based on the database derived from the standards 63 metabolites were identified in rat urine, using positive ESI, based on the combination of tr, TW CCSN2 and MS data.

Methotrexate (MTX) is a chemotherapeutic agent that can cause a range of toxic side effects including gastrointestinal damage, hepatotoxicity, myelosuppression, and nephrotoxicity and has potentially complex interactions with the gut microbiome. Following untargeted UPLC-qtof-MS analysis of urine and fecal samples from male Sprague–Dawley rats administered at either 0, 10, 40, or 100 mg/kg of MTX, dose-dependent changes in the endogenous metabolite profiles were detected. Semiquantitative targeted UPLC-MS detected MTX excreted in urine as well as MTX and two metabolites, 2,4-diamino- N -10-methylpteroic acid (DAMPA) and 7-hydroxy-MTX, in the feces. DAMPA is produced by the bacterial enzyme carboxypeptidase glutamate 2 (CPDG2) in the gut. Microbiota profiling (16S rRNA gene amplicon sequencing) of fecal samples showed an increase in the relative abundance of Firmicutes over the Bacteroidetes at low doses of MTX but the reverse at high doses. Firmicutes relative abundance was positively correlated with DAMPA excretion in feces at 48 h, which were both lower at 100 mg/kg compared to that seen at 40 mg/kg. Overall, chronic exposure to MTX appears to induce community and functionality changes in the intestinal microbiota, inducing downstream perturbations in CPDG2 activity, and thus may delay MTX detoxication to DAMPA. This reduction in metabolic clearance might be associated with increased gastrointestinal toxicity.

1-β-O-Acyl-glucuronides (AGs) are common metabolites of carboxylic acid-containing xenobiotics, including, e.g., many nonsteroidal anti-inflammatory drugs (NSAIDs). They are of concern to regulatory authorities because of the association of these metabolites with the hepatotoxicity that has resulted in drug withdrawal. One factor in assessing the potential risk posed by AGs is the rate of transacylation of the biosynthetic 1-β-O-acyl form to the 2-, 3-, and 4-O-acyl isomers. While transacylation can be measured using 1H NMR spectroscopy or liquid chromatography-mass spectrometry (LC-MS), the process can be time consuming and involve significant method development. The separation of these positional isomers by ion mobility spectrometry (IMS) has the potential to allow their rapid analysis, but conventional instruments lacked the resolving power to do this. Prediction of the collision cross section (CCS) using a machine learning model suggested that greater IMS resolution might be of use in this area. Cyclic IMS was evaluated for separating mixtures of isomeric AGs of diclofenac and was compared with a conventional ultraperformance liquid chromatography (UPLC)-MS method as a means for studying transacylation kinetics. The resolution of isomeric AGs was not seen using a conventional traveling wave IMS device; however, separation was seen after several passes around a cyclic IMS. The cyclic IMS enabled the degradation of the 1-β-O-acyl-isomer to be analyzed much more rapidly than by LC-MS. The ability of cyclic IMS to monitor the rate of AG transacylation at different pH values, without the need for a prior chromatographic separation, should allow high-throughput, real-time, monitoring of these types of reactions.

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