Stability and enzymatic hydrolysis of quaternary ammonium-linked glucuronide metabolites of drugs with an aliphatic tertiary amine-implications for analysis

Abstract Quaternary ammonium-linked glucuronide ( N + -glucuronide) metabolites formed at aliphatic tertiary amine functional groups of xenobiotics have not been previously systematically studied with respect to their stability over a wide pH range and the ease of enzymatic hydrolysis by β-glucuronidase from various sources. Three and four N + -glucuronide metabolites were respectively studied regarding their non-enzymatic and enzymatic stabilities where the metabolites were quantified by HPLC procedures. The N + -glucuronide metabolites of clozapine, cyclizine, and doxepin were stored at 18–22°C in buffers at each nominal pH value over the 1–11 pH range. All three metabolites were stable for 3 months over the 4–10 pH range, while two metabolites slowly degraded ( k in the range 0.002–0.01 days −1 ) at each of the other extreme pH values. In the initial enzymatic study the N + -glucuronide metabolites of chlorpromazine, clozapine, cyclizine, and doxepin were each treated in pH 5.0 and 7.4 buffers at 37°C with β-glucuronidase from three different sources, namely commercial brands from bovine liver, mollusks ( Helix pomatia ), and bacteria ( Escherichia coli ). Clozapine N + -glucuronide and the standard phenolphthalein O -glucuronide were susceptible to hydrolysis by the enzyme from all three sources. In contrast, the other three N + -glucuronide metabolites were resistant to hydrolysis, except for the E. coli source of β-glucuronidase at pH 7.4. Also when examined at 50-fold increase in concentration of the enzyme sources from bovine liver and H. pomatia cyclizine N + -glucuronide was still resistant to hydrolysis by the former enzyme preparation. The optimum pH for the hydrolysis of each of the four N + -glucuronide metabolites from the E. coli enzyme source was investigated and was found to be in the pH range 6.5–7.4. These data have important implications with respect to the analysis of N + -glucuronide metabolites formed at an aliphatic tertiary amine: in general, their non-enzymatic stability will not be an important factor in the development of an analytical procedure, and when developing an indirect approach to the analysis of N + -glucuronide metabolites that involves β-glucuronidase hydrolysis to the aglycone preliminary work should involve determining the appropriate enzyme source, buffer pH, and length of time of incubation.