Rapid and sensitive high-performance liquid chromatographic assay for 6-hydroxychlorzoxazone and chlorzoxazone in liver microsomes.
A high-performance liquid chromatographic assay was developed for the quantitation of chlorzoxazone and its major metabolite 6-hydroxychlorzoxazone. These compounds along with phenacetin, the internal standard, were extracted from incubation mixtures using ether extraction. The extracts were analyzed on a Brownlee Spheri-5 C8 column with a mobile-phase of acetonitrile-0.5% phosphoric acid (30:70, v/v). The assay utilized UV detection at 287 nm which provided sensitivity and specificity to simultaneously quantify chlorzoxazone and 6-hydroxychlorzoxazone from liver microsomal samples at amounts of 10 ng and greater. The mean correlation coefficient of the standard curves for 6-hydroxychlorzoxazone and chlorzoxazone was 0.998 and 0.993, respectively, over the range of 25-400 ng, and the regression curves were found to be linear at least through 1600 ng. All components eluted within 7 min, resulting in a total analysis time of 8 min. The inter-day and intra-day coefficients of variation were <7 and <3%, respectively. This method provides a rapid, sensitive and cost-effective assay for 6-hydroxychlorzoxazone and chlorzoxazone in liver microsomal incubations.
Chittur
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Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown 26506, USA.
Tracy
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D000818:Animals; D002753:Chlorzoxazone / Q000031:analogs & derivatives* / Q000032:analysis* / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid; D008297:Male; D008862:Microsomes, Liver / Q000737:chemistry*; D009125:Muscle Relaxants, Central / Q000032:analysis* / Q000493:pharmacokinetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013056:Spectrophotometry, Ultraviolet
D009125:Muscle Relaxants, Central; C039289:6-hydroxychlorzoxazone; D002753:Chlorzoxazone