DETERMINATION OF 4-METHYL UMBELLIFERONE AND METABOLITES IN WILLIAMS-E-MEDIA AND DOG PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A gradient high performance liquid chromatographic method has been dev eloped for the determination of 4-methylumbelliferone (4MU), 4-methylu mbelliferyl sulfate (4MUS), and 4-methylumbelliferyl beta-D-glucuronid e (4MUG) in dog plasma and William's E media. Samples containing the i nternal standard, umbelliferone were prepared for analysis by precipit ation with acetonitrile prior to injection onto a 150 x 2.1mm C18 Hype rsil reversed-phase column. The compounds were eluted in less than 7 m inutes with a fast linear gradient from 9.5% acetonitrile to 32% aceto nitrile in pH 4.5 acetate buffer with a flow rate of 0.35 mL/min. All compounds were detected at 314 nm. Total sample cycle time was 10 minu tes. Intra-run (n=5) and inter-run (n=18) precision was less than 15% relative standard deviation across the entire calibration range. The l ower limit of quantitiation with a 50 mu L sample size was 2.5 mu M.