Rapid and sensitive high-performance liquid chromatographic assay for 6-hydroxychlorzoxazone and chlorzoxazone in liver microsomes

J Chromatogr B Biomed Sci Appl. 1997 Jun 6;693(2):479-83. doi: 10.1016/s0378-4347(97)00024-8.


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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Chlorzoxazone / analogs & derivatives*
  • Chlorzoxazone / analysis*
  • Chlorzoxazone / pharmacokinetics
  • Chromatography, High Pressure Liquid
  • Male
  • Microsomes, Liver / chemistry*
  • Muscle Relaxants, Central / analysis*
  • Muscle Relaxants, Central / pharmacokinetics
  • Rats
  • Rats, Sprague-Dawley
  • Spectrophotometry, Ultraviolet


  • Muscle Relaxants, Central
  • 6-hydroxychlorzoxazone
  • Chlorzoxazone