Simultaneous determination of isoline and its two major metabolites using high-performance liquid chromatography

A simple and reliable high-performance liquid chromatographic (HPLC) assay was developed for a simultaneous determination of isoline, a potent hepatotoxic pyrrolizidine alkaloid, and its two major metabolites, namely M1 (bisline) and M2 (bisline lactone, a new pyrrolizidine alkaloid). The latter two metabolites were produced during in vitro metabolism of isoline by rat and mouse microsomal enzyme systems. The analysis was conducted by a direct injection of aliquots of supernatant of the microsomal reaction mixture treated with the equal volume of ice-cold methanol onto a conventional reversed-phase analytical column (150 x 4.6 mm). The analytes were separated by a gradient elution with mobile phases A (0.01 M dihydro-potassium phosphate, pH 4.8) and B (acetonitrile). The assay has shown excellent precision and accuracy with less than 10% of overall intra- and interday variations and higher than 94% of overall accuracy. The developed HPLC method was successfully applied for the determination of the intact isoline and its two pH- and thermally labile metabolites produced in rat and mouse liver microsomal incubations.