High-performance liquid chromatographic determination of chlorzoxazone and 6-hydroxychlorzoxazone in serum: a tool for indirect evaluation of cytochrome P4502E1 activity in humans.

Chronic alcohol consumption is known to induce the enzyme cytochrome P4502E1 (CYP2E1), which is involved in the toxicity and carcinogenicity of a number of solvents and xenobiotics. It was recently suggested that in vivo chlorzoxazone metabolism could be a potential tool as a non-invasive probe for measuring CYP2E1 activity in humans. Therefore, a simple and sensitive method was developed for the determination of chlorzoxazone and its major metabolite 6-hydroxychlorzoxazone in both serum and urine. Biological samples were hydrolysed by Helix pomatia juice, deproteinized with perchloric acid, and then extracted using ethyl acetate. The compounds were separated by high-performance liquid chromatography on an octadecylsilane column with a mobile phase of acetonitrile-0.5% acetic acid in water (30:70, v/v) and detected at 287 nm. The linearity of the method was tested in the concentration range 0.5-20 micrograms/ml, and the limit of detection in biological samples was found to be 0.5 microgram/ml. Within- and between-run precision was below 5% and 10%, respectively, for both compounds at three concentrations (0.5, 10 and 20 micrograms/ml). The accuracy of the procedure was in the range 0.3-6%. Serum levels and urinary excretion of chlorzoxazone and its metabolite were studied in five healthy controls and five alcoholic patients, following oral administration of 500 mg of chlorzoxazone. The concentration ratio 6-hydroxychlorzoxazone/chlorzoxazone in blood was shown to be a valuable tool for the evaluation of CYP2E1 activity in humans.