Challenges in the indirect quantitation of acyl-glucuronide metabolites of a cardiovascular drug from complex biological mixtures in the absence of reference standards.

This paper describes the quantitation of acyl-glucuronide metabolites (M26 and M5) of a cardiovascular-drug (torcetrapib) from monkey urine, in the absence of their reference standards. LC/MS/MS assays for M1 and M4 (aglycones of M26 and M5, respectively) were characterized from normal and base-treated urine, as their respective reference standards were available. The in vivo study samples containing M26 and M5 were treated with 1 n sodium hydroxide to hydrolyze them to their respective aglycones. The study samples were assayed for M1 and M4 before and after alkaline hydrolysis and the difference in the concentrations provided an estimate of the urinary levels of M26 and M5. Prior to the main sample analysis, conditions for alkaline hydrolysis of the glucuronides were optimized by incubating pooled study samples. During incubations, a prolonged increase in M4 levels over time was observed, which is inconsistent with the base-hydrolysis of an acyl-glucuronide (expected to hydrolyze rapidly). Possible interference of the metabolite M9 (an ether-glucuronide metabolite isobaric to M4) was investigated to explain this observation using chromatographic and wet-chemistry approaches. The strategies adopted herein established that the LC/MS/MS assay and our approach were reliable. The metabolite exposure was then correlated to toxicological observations to gain initial insights into the physiological role of these metabolites.