Simultaneous determination of triflusal and its major active metabolite, 2-hydroxy-4-trifluoromethyl benzoic acid, in rat and human plasma by high-performance liquid chromatography

A rapid, selective and sensitive high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of triflusal and its major active metabolite, 2-hydroxy-4-trifluoromethyl benzoic acid (HTB), in rat and human plasma. HPLC analysis was carried out using a 5-μm particle size, C18-bonded silica column and acetonitrile–methanol–water (25:10:65, v/v/v) as the mobile phase and UV detection at 234 nm. Furosemide was used as the internal standard. The method involved extraction with an acetonitrile–chloroform mixture (60:40, v/v) and evaporation to dryness with nitrogen stream. The chromatograms showed good resolution and sensitivity and no interferences by plasma constituents. The mean absolute recovery for human plasma was 93.5±4.2% for triflusal and 98.5±3.1% for HTB. The lower limits of quantification of triflusal and HTB in human plasma were 20 and 100 ng/ml, respectively. The calibration curves in human plasma were linear over the concentration range 0.02–5.0 μg/ml for triflusal and 0.1–200.0 μg/ml for HTB with correlation coefficients greater than 0.999 and with inter- or intra-day coefficients of variation (CV) not exceeding 10.0%. This assay procedure was applied to the study of metabolite pharmacokinetics of triflusal and HTB in rat and human.