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

J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Sep 5;794(2):257-71. doi: 10.1016/s1570-0232(03)00473-2.


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.

MeSH terms

  • Animals
  • Chromatography, High Pressure Liquid / methods*
  • Coumarins / chemistry
  • Coumarins / metabolism*
  • Epoxy Compounds / metabolism*
  • Glutathione / metabolism*
  • Magnetic Resonance Spectroscopy
  • Male
  • Microsomes, Liver / metabolism
  • Rats
  • Rats, Inbred F344
  • Reproducibility of Results
  • Sensitivity and Specificity


  • Coumarins
  • Epoxy Compounds
  • coumarin
  • Glutathione