Simultaneous determination of loxoprofen and its diastereomeric alcohol metabolites in human plasma and urine by a simple HPLC-UV detection method.

A simple, reliable HPLC-UV detection method was developed for the simultaneous determination of loxoprofen and its metabolites (i.e. trans- and cis-alcohol metabolites), in human plasma and urine samples. The method involves the addition of a ketoprofen (internal standard) solution in methanol, zinc sulfate solution and acetonitrile to plasma and urine samples, followed by centrifugation. An aliquot of the supernatant was evaporated to dryness, and the residue reconstituted in a mobile phase (acetonitrile:water=35:65 v/v, pH 3.0). An aliquot of the solution was then directly injected into the HPLC system. Separations were performed on octadecylsilica column (250x4.5 mm, 5 microm) with a guard column (3.2x1.5 cm, 7 microm) at ambient temperature. Loxoprofen and the metabolites in the eluent were monitored at 220 nm (a.u.f.s. 0.005). Coefficients of variations (CV%) and recoveries for loxoprofen and its metabolites were below 10 and over 96%, respectively, in the 200 approximately 15000 ng ml(-1) range for plasma and 500 approximately 50000 ng ml(-1) range for urine. Calibration curves for all the compounds in the plasma and urine were linear over the above-mentioned concentration ranges with a common correlation coefficient of 0.999. The detection limit of the present method was 100 ng for all the compounds. These results indicate that the present method is very simple and readily applicable to routine bioavailability studies of these compounds with an acceptable sensitivity.