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-hydroxycyclohexyl)-L-cysteine) in rat urine and plasma using gas chromatography. A mixture of ethyl acetate-acetonitrile (70:30) was used as the extracting solvent for both matrices. This liquid-liquid extraction procedure was followed by the separation of cyclohexene oxide and its metabolites on an HP-FFAP fused-silica capillary column. In order to determine the amount of cyclohexane-1,2-diol-O-glucuronide, samples were incubated at 37 degrees C with beta-glucuronidase and the amount of cyclohexane-1,2-diol formed from the reaction determined. The extraction efficiencies of cyclohexene oxide and cyclohexanol were greater than 90% both in urine and plasma. However, recovery from the plasma and urine for trans-cyclohexane-1,2-diol (60-68%) and N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine (approximately 76%) were considerably less. Long term stability studies showed that urine samples spiked with cyclohexene oxide and trans-cyclohexane-1,2-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 same conditions. The calibration curves for all analytes were linear over the range of 12.5 to 400 micrograms/ml and correlation coefficients (r2) were greater than 0.990. The limit of detection for cyclohexene oxide, cyclohexanol, and N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine is 1.56 micrograms/ml, while the limit of detection for trans-cyclohexane-1,2-diol is 3.12 micrograms/ml. This method has been used for the determination of the disposition and metabolism of cyclohexene oxide, and may be applied in environmental monitoring, as well as in microbiological studies for other epoxide materials.