A novel approach for predicting acyl glucuronide reactivity via Schiff base formation: development of rapidly formed peptide adducts for LC/MS/MS measurements

Chem Res Toxicol. 2004 Sep;17(9):1206-16. doi: 10.1021/tx049900+.


A novel technique to study the reactivity of acyl glucuronide metabolites to protein has been developed and is described herein. Considered here are acyl glucuronide metabolites, which have undergone the rearrangement of the glucuronic acid moiety at physiological temperature and pH. The investigation of the reactivity of these electrophilic metabolites was carried out by measuring the rate of reaction of rearranged AG metabolites in forming the corresponding acyl glucuronide-peptide adduct in the presence of Lys-Phe. This differs from the parallel technique used in forming AG adducts of proteins that have been previously reported. In the study described here, the Schiff base adduct, diclofenac acyl glucuronide-Lys-Phe product, was generated and structurally elucidated by liquid chromatography tandem mass spectrometry (LC/MS/MS) analysis. The product structure was proved to be a Schiff base adduct by chemical derivatization by nucleophilic addition of HCN and chemical reduction with NaCNBH(3), followed by LC/MS/MS analysis. It is proposed here that the degree of reactivity of acyl glucuronides as measured by covalent binding to protein is proportional to the amount of its peptide adduct generated with the peptide technique described. The application of this technique to the assessment of the degree of reactivity of acyl glucuronide metabolites was validated by developing a reactivity rank of seven carboxylic acid-containing drugs. Consistency was achieved between the ranking of reactivity in the peptide technique for these seven compounds and the rankings found in the literature. In addition, a correlation (R(2) = 0.95) was revealed between the formation of a peptide adduct and the rearrangement rate of the primary acyl glucuronide of seven tested compounds. A structure effect on the degree of reactivity has demonstrated the rate order: acetic acid > propionic acid > benzoic acid derivatives. A rational explanation of this order was proposed, based on the inherent electronic and steric properties of each specific aglycone. In addition, adaptation of this technique to automation in order to more rapidly assess the ranking of reactivity of acyl glucuronide covalent binding to proteins by new chemical entities is proposed.

Publication types

  • Evaluation Study

MeSH terms

  • Acylation
  • Carboxylic Acids / chemistry*
  • Chromatography, Liquid / methods*
  • DNA Adducts / chemistry
  • Glucuronides / chemistry*
  • Lysine / chemistry
  • Mass Spectrometry / methods*
  • Peptides / chemistry*
  • Phenylalanine / chemistry
  • Predictive Value of Tests
  • Schiff Bases / chemistry*


  • Carboxylic Acids
  • DNA Adducts
  • Glucuronides
  • Peptides
  • Schiff Bases
  • Phenylalanine
  • Lysine