Differentiation of Deprotonated Acyl-, <i>N</i>-, and <i>O</i>-Glucuronide Drug Metabolites by Using Tandem Mass Spectrometry Based on Gas-Phase Ion–Molecule Reactions Followed by Collision-Activated Dissociation

Abstract

Glucuronidation, a common phase II biotransformation reaction, is one of the major <i>in vitro</i> and <i>in vivo</i> metabolism pathways of xenobiotics. In this process, glucuronic acid is conjugated to a drug or a drug metabolite via a carboxylic acid, a hydroxy, or an amino group to form acyl-, <i>O</i>-, and/or <i>N</i>-glucuronide metabolites, respectively. This process is traditionally thought to be a detoxification pathway. However, some acyl-glucuronides react with biomolecules <i>in vivo</i>, which may result in immune-mediated idiosyncratic drug toxicity (IDT). In order to avoid this, one may attempt in early drug discovery to modify the lead compounds in such a manner that they then have a lower probability of forming reactive acyl-glucuronide metabolites. Because most drugs or drug candidates bear multiple functionalities, e.g., hydroxy, amino, and carboxylic acid groups, glucuronidation can occur at any of those. However, differentiation of isomeric acyl-, <i>N-</i>, and <i>O</i>-glucuronide derivatives of drugs is challenging. In this study, gas-phase ion-molecule reactions between deprotonated glucuronide metabolites and BF₃ followed by collision-activated dissociation (CAD) in a linear quadrupole ion trap mass spectrometer were demonstrated to enable the differentiation of acyl-, <i>N-</i>, and <i>O</i>-glucuronides. Only deprotonated <i>N</i>-glucuronides and deprotonated, migrated acyl-glucuronides form the two diagnostic product ions: a BF₃ adduct that has lost two HF molecules, [M - H + BF₃ - 2HF]^-, and an adduct formed with two BF₃ molecules that has lost three HF molecules, [M - H + 2BF₃ - 3HF]^-. These product ions were not observed for deprotonated <i>O</i>-glucuronides and unmigrated, deprotonated acyl-glucuronides. Upon CAD of the [M - H + 2BF₃ - 3HF]^- product ion, a diagnostic fragment ion is formed via the loss of 2-fluoro-1,3,2-dioxaborale (MW of 88 Da) only in the case of deprotonated, migrated acyl-glucuronides. Therefore, this method can be used to unambiguously differentiate acyl-, <i>N-</i>, and <i>O</i>-glucuronides. Further, coupling this methodology with HPLC enables the differentiation of unmigrated 1-β-acyl-glucuronides from the isomeric acyl-glucuronides formed upon acyl migration. Quantum chemical calculations at the M06-2X/6-311++G(d,p) level of theory were employed to probe the mechanisms of the reactions of interest.