Improved High Performance Liquid Chromatographic Determination of Chlorzoxazone and its Hydroxy Metabolite in Human Serum and Urine

Methods for the high performance liquid chromatographic determinations of chlorzoxazone and its 6-hydroxy metabolite in human serum and urine are presented. The separation of the analytes is achieved within 15 min on an octadecylsilane column with a mobile phase of 30:70 v/v acetonitrile - aqueous 0.05 M sodium dihydrogen phosphate at a pH 4.4 flow rate of 1 mL/min. The serum procedure utilized an octadecylsilane solid-phase extraction clean-up step with injection of the eluent onto the analytical column and detection at 280nm. The urine method uses a buffer dilution of the urine sample and direct injection onto the analytical column with detection at 295nm. Recoveries of chlorzoxazone and metabolite from serum and urine samples are in the 88–103% range. Drug to internal standard peak height ratios are linear in the 0.5 – 50 μg/mL and 5–100 μg/mL ranges for both analytes in serum and urine, respectively. Limits of quantitation for chlorzoxazone are 0.12 μg/mL in serum and 0.62 μg/mL in urine. Limits of quantitation for the 6-hydroxy metabolite are 0.23 μg/mL in serum and 0.029 μg/mL in urine. Accuracy and precision of both methods are in the 0–5% range, respectively.