title: Liquid chromatographic determination of the glutathione conjugate and ring-opened metabolites formed from coumarin epoxidation.
abstract: 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.
authors.affiliation: Miami Valley Laboratories, The Procter and Gamble Company, Cincinnati, OH 45252, USA. vassalo.jd@PG.com
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Vassallo
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Morrall
authors.forename: Kristi L
authors.initials: KL
authors.lastname: Fliter
authors.forename: Sandra M
authors.initials: SM
authors.lastname: Curry
authors.forename: George P
authors.initials: GP
authors.lastname: Daston
authors.forename: Lois D
authors.initials: LD
authors.lastname: Lehman-McKeeman
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism*; D004852:Epoxy Compounds / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D003374:Coumarins; D004852:Epoxy Compounds; C030123:coumarin; D005978:Glutathione