SIMULTANEOUS DETERMINATION OF CYCLOHEXENE OXIDE AND ITS METABOLITES IN RAT PLASMA AND URINE BY GAS-CHROMATOGRAPHY

An assay was developed for the simultaneous measurement of cyclohexene oxide and its metabolites (cyclohexanol, trans-cyclohexane-1,2-diol, cyclohexane-1,2-diol-O-glucuronide, and N-acetyl-S-(2-hydroxycyclohexy l)-L-cyste in rat urine and plasma using gas chromatography. A mixture of ethyl acetate-acetonitrile (70:30) was used as the extracting solv ent for both matrices. This liquid-liquid extraction procedure was fol lowed by the separation of cyclohexene oxide and its metabolites on an HP-FFAP fused-silica capillary column. In order to determine the amou nt of cyclohexane-1,2-diol-O-glucuronide, samples were incubated at 37 degrees C with beta-glucuronidase and the amount of cyclohexane-l,2-d iol formed from the reaction determined. The extraction efficiencies o f cyclohexene oxide and cyclohexanol were greater than 90% both in uri ne and plasma. However, recovery from the plasma and urine for trans-c yclohexane-l,2-diol (60-68%) and N-acetyl-S-(2-hydroxycyclohexyl)-L-cy steine (similar to 76%) were considerably less. Long term stability st udies showed that urine samples spiked with cyclohexene oxide and tran s-cyclohexane-l,2-diol are stable at -20 degrees C for up to 9 weeks. However, plasma samples are only stable for up to 2 weeks under the sa me conditions. The calibration curves for all analytes were linear ove r the range of 12.5 to 400 mu g/ml and correlation coefficients (r(2)) were greater than 0.990. The limit of detection for cyclohexene oxide , cyclohexanol, and N-acetyl-S-(2-hydroxycyclohexyl)-L-cyste is 1.56 m u g/ml, while the limit of detection for trans-cyclohexane-1 ,2-diol i s 3.12 mu g/ml. This method has been used for the determination of the disposition and metabolism of cyclohexene oxide, and may be applied i n environmental monitoring, as well as in microbiological studies for other epoxide materials. (C) 1997 Elsevier Science B.V.