Simultaneous analysis of clivorine and its four microsomal metabolites by high-performance liquid chromatography

A specific high-performance liquid chromatographic assay was developed for a simultaneously qualitative and quantitative determination of clivorine, a hepatotoxic otonecine-type pyrrolizidine alkaloid, and its four putative h epatotoxicity-related metabolites, namely dehydroretronecine, 7-glutathiony ldehydroretronecine, 7,9-diglutathionyldehydroretronecine, and clivoric aci d, generated in rat microsomal incubation. This simultaneous determination was conducted by a direct analysis of aliquots of the supernatant of incuba tes using a specific two-column set-up. Impurities in the supernatant were firstly eluted out from the first PRP-1 guard column (50X4.1 mm) during an initial 5 min washing period with isocratic elution by mobile phase A (0.2% formic acid at pH 3.4 adjusted by ammonia). Subsequently, the guard column was then connected to the second PRP-1 analytical column (250X4.6 mm) and analytes were separated by a gradient elution with mobile phases A and B (a cetonitrile). The assay provided good reproducibility and accuracy for all analytes tested with less than 12% of overall intra- and inter-day variatio ns and higher than 87% of overall accuracy. This developed method was succe ssfully applied to determine the intact clivorine and its four metabolites generated in rat microsomal incubation. (C) 2000 Elsevier Science B.V. All rights reserved.