title: 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 beta-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 degrees 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 degrees C with beta-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 beta-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 beta-glucuronidase hydrolysis to the aglycone preliminary work should involve determining the appropriate enzyme source, buffer pH, and length of time of incubation.
authors.affiliation: College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Canada.
authors.forename: I
authors.initials: I
authors.lastname: Kowalczyk
authors.forename: E M
authors.initials: EM
authors.lastname: Hawes
authors.forename: G
authors.initials: G
authors.lastname: McKay
mesh_terms: D000818:Animals; D002021:Buffers; D002417:Cattle; D004355:Drug Stability; D004926:Escherichia coli / Q000201:enzymology; D005966:Glucuronidase / Q000737:chemistry*; D020719:Glucuronides / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*
chemical_list: D002021:Buffers; D020719:Glucuronides; D004364:Pharmaceutical Preparations; D000644:Quaternary Ammonium Compounds; D005966:Glucuronidase