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

  • Anal Chem. 2019 Sep 3;91(17):11388-11396. doi: 10.1021/acs.analchem.9b02717.
Edouard Niyonsaba  1 ,  McKay W Easton  1 ,  Erlu Feng  1 ,  Zaikuan Yu  1 ,  Zhoupeng Zhang  2 ,  Huaming Sheng  3 ,  John Kong  3 ,  Leah F Easterling  1 ,  Jacob Milton  1 ,  Harry R Chobanian  2 ,  Nicholas R Deprez  4 ,  Mark T Cancilla  2 ,  Gozdem Kilaz  5 ,  Hilkka I Kenttämaa  1
Affiliations
  • 1. Department of Chemistry , Purdue University , West Lafayette , Indiana 47907 , United States.
  • 2. Department of Pharmacokinetics, Pharmacodynamics, & Drug Metabolism , Merck & Co., Inc. , West Point , Pennsylvania 19486 , United States.
  • 3. Analytical Research & Development , Merck & Co., Inc. , Rahway , New Jersey 07065 , United States.
  • 4. Process Chemistry , Merck & Co., Inc. , Rahway , New Jersey 07065 , United States.
  • 5. Purdue University , School of Engineering Technology , West Lafayette , Indiana 47907 , United States.
PMID: 31381321 DOI: 10.1021/acs.analchem.9b02717
Abstract

Glucuronidation, a common phase II biotransformation reaction, is one of the major in vitro and in vivo 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-, O-, and/or N-glucuronide metabolites, respectively. This process is traditionally thought to be a detoxification pathway. However, some acyl-glucuronides react with biomolecules in vivo, 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-, N-, and O-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-, N-, and O-glucuronides. Only deprotonated N-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 O-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-, N-, and O-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.

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