Liquid chromatographic determination of the glutathione conjugate and ring-opened metabolites formed from coumarin epoxidation

Species differences in the biotransformation of coumarin are thought to play an important role in its toxicity. Since the putative toxic metabolite is coumarin 3,4-epoxide (CE), methods to measure the metabolites of CE were developed. The glutathione (GSH) conjugate of CE (CE-SG) at the 3-position was purified by reversed-phase (RP)-high performance liquid chromatography (HPLC), and characterized by mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). An RP-HPLC method was developed to quantify CE-SG in hepatic microsomal mixtures and a separate RP-HPLC method was also developed to quantify the three ring-opened coumarin metabolites; o-hydroxyphenylacetic acid (o-HPAA), o-hydroxyphenylethanol (o-HPE) and o-hydroxyphenylacetaldehyde (o-HPA) in hepatic microsomal mixtures. Detection limits for all four products of coumarin epoxidation exceeded 3.5 ng/ml and recovery from hepatic microsomal mixtures was essentially quantitative with RSD values less than 8%. Species differences in o-HPA detoxification were consistent with sensitivity to coumarin, thereby demonstrating that these methods have utility in addressing the fate of CE and its contribution to toxicity.