Development and validation of a simple and sensitive HPLC-UV method for the determination of captopril in human plasma using a new derivatizing reagent 2-naphthyl propiolate.

In this study, a simple, sensitive and reliable HPLC-UV method applying rapid sample preparation technique for the determination of captopril in human plasma was developed and validated. The method is based on pre-column derivatization of captopril and 2-propene-1-thiol (internal standard) with a new reagent 2-naphthyl propiolate. Sample clean-up, derivatization and extraction were carried out in two steps, totally less than 30min. The extracts were chromatographed on a C18 column (5μm, 150mm×4.6mmi.d.). The mobile phase consisted of methanol (75%, v/v) and phosphate buffer (25%, pH=8, 0.01M). UV detection was performed at 290nm. To obtain the best reaction yield, the factors that could influence the derivatization process, including the concentration of derivatization reagent, pH of sample solution and temperature were investigated in detail and optimized using Box-Behnken response surface methodology. Under optimized conditions the average extraction recovery of captopril and internal standard were >86%. The achieved lower limit of quantification (LLOQ) was 3ng/mL; the assay exhibited a linear dynamic range of 3-2000ng/mL with correlation coefficient (r(2)) of ≥0.99. The precision was satisfactory in the whole calibration range with RSD of 5.9-12.4% (accuracy: from 97.5% to 93.6%) and of 6.4-12.8% (accuracy: from 97.3% to 95.2%) for intra- and inter-assay, respectively. The method stability was confirmed in a series of experiments including: freeze-thaw, short- and long-term stability testing. Lastly, the developed method was successfully applied to the bioequivalence study of captopril administrated as a single oral dose (50mg) to 12 healthy male volunteers.