Use of Cyclic Ion Mobility Spectrometry (cIM)-Mass Spectrometry to Study the Intramolecular Transacylation of Diclofenac Acyl Glucuronide.

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.