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

Quaternary ammonium-linked glucuronide (N+-glucuronide) metabolites formed at aliphatic tertiary amine functional groups of xenobiotics have nor, been previously systematically studied with respect to their stability over a w ide pH range and the ease of enzymatic hydrolysis by beta-glucuronidase fro m various sources. Three and four N+-glucuronide metabolites were respectiv ely studied regarding their non-enzymatic and enzymatic stabilities where t he metabolites were quantified by HPLC procedures. The N+-glucuronide metab olites of clozapine, cyclizine, and doxepin were stored at 18-22 degrees C in buffers at each nominal pH value over the 1-11 pH range. All three metab olites were stable for 3 months over the 4-10 pH range, while two metabolit es slowly degraded (k in the range 0.002-0.01 days(-1)) at each of the othe r extreme pH values. In the initial enzymatic study the N+-glucuronide meta bolites of chlorpromazine. clozapine, cyclizine and doxepin were each treat ed in pH 5.0 and 7.4 buffers at 37 degrees C with beta-glucuronidase from t hree different sources, namely commercial brands from bovine liver, mollusk s (Helix pomatia), and bacteria (Escherichia coli). Clozapine N+-glucuronid e and the standard phenolphthalein O-glucuronide were susceptible to hydrol ysis by the enzyme from all three sources. In contrast, the other three N+- glucuronide metabolites were resistant to hydrolysis, except for the E. col i source of beta-glucuronidase at pH 7.4. Also when examined at 50-fold inc rease in concentration of the enzyme sources from bovine liver and H. pomat ia 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 impl ications with respect to the analysis of N+-glucuronide metabolites formed at an aliphatic tertiary amine: in general, their non-enzymatic stability w ill not be an important factor in the development of an analytical procedur e, and when developing an indirect approach to the analysis of N+-glucuroni de metabolites that involves beta-glucuronidase hydrolysis to the aglycone preliminary work should involve determining the appropriate enzyme source, buffer pH, and length of time of incubation. (C) 2000 Elsevier Science B.V. All rights reserved.