Rapid and sensitive high-performance liquid chromatographic assay of tiaprofenic acid enantiomers in human plasma and urine.

A sensitive stereospecific high-performance liquid chromatographic assay of tiaprofenic acid (TA) enantiomers in human plasma and urine was developed. The biological specimens are acidified, and the drug and internal standard, (+/-)-2-(4-benzoylphenyl)butyric acid, extracted with an isooctane-isopropanol (95:5) mixture (plasma) or chloroform (urine), followed by sequential reaction of the enantiomers with trichloroethyl chloroformate and L-leucinamide. The reactions were complete at ambient temperature in less than 3 min. The diastereoisomers of TA and internal standard were then extracted into chloroform. The organic layer was evaporated, and the reconstituted residue chromatographed at ambient temperature on a C18 reversed-phase column with a mobile phase consisting of 0.06 M monopotassium phosphate-acetonitrile-triethylamine (65:35:0.02) at a flow-rate of 1 ml/min. The TA diastereoisomers were detected at 310 nm, free of interfering peaks, with a resolution factor of 2.1. Within the examined plasma and urine enantiomeric concentration ranges of 0.2-20 and 10-100 mg/l, respectively, an excellent linear relationship was obtained between the peak-area ratios and the corresponding concentrations. The assay was reproducible and sufficiently accurate to be applied to the stereoselective pharmacokinetic analysis of TA enantiomers in plasma and urine following administration of therapeutic doses of the drug.