Structural elucidation of rat biliary metabolites of corynoxeine and their quantification using LC-MS(n).

Corynoxeine (COR) is one of 4 bioactive oxindole alkaloids in Uncaria species. In this work two phase I metabolites, namely 11-hydroxycorynoxeine (M1) and 10-hydroxycorynoxeine (M2), and two phase II metabolites, namely 11-hydroxycorynoxeine 11-O-β-d-glucuronide (M3) and 10-hydroxycorynoxeine 10-O-β-d-glucuronide (M4), were detected in rat bile after oral dose of COR (0.105 mmol/kg), by optimized high-performance liquid chromatography-tandem mass spectrometry (LC-MS(n) ) with electrospray ionization in positive ion mode. Structures of M1-4 were determined by LC-MS(n) , nuclear magnetic resonance, circular dichroism and high-resolution MS spectra. COR and its metabolites in rat bile were quantified by LC-MS(n) . The LC-MS(n) quantification methods for COR and its metabolites yielded a linearity with coefficient of determination ≥0.995 from 5.0 × 10(-10) to 5.0 × 10(-7)  m. The recoveries of stability tests varied from 96.80 to 103.10%. Accuracy ranged from 91.00 to 105.20%. Relative standard deviation for intra-day and inter-day assay was <5.0%. After the oral dose 0.14% of COR was detected in rat bile from 0 to 8 h, in which in total 97.8% COR biotransformed into M1-4. M1 and M2 yielded 48.1 and 49.7%, which successively glucuronidated to M3 and M4 at 47.2 and 43.8%, respectively.