DEVELOPMENT AND VALIDATION OF HPLC-UV METHOD FOR THE DETERMINATION OF CLOPIDOGREL IN PHARMACEUTICAL DOSAGE FORM AND HUMAN PLASMA

Abstract

A simple, rapid, and sensitive high performance liquid chromatographic method with UV detection has been developed for the quantification of clopidogrel in human plasma and pharmaceutical dosage form using diclofenac sodium as an internal standard and validated according to standard guidelines. The separation of clopidogrel and diclofenac sodium was achieved using Hypersil BDS RP18 (250 × 4.6 mm, 5 µm) at a column oven temperature of 35°C. The methanol and phosphoric acid 25 mM (80:20, v/v) (pH 3.0), used as a mobile phase in the isocratic mode was pumped at a flow rate of 1.2 mL min⁻¹ and eluents were monitored at a wavelength of 220 nm. The retention times for diclofenac sodium and clopidogrel were 4.15 and 4.62 min, respectively. Extraction of drug from plasma was carried out with diethyl ether. The limit of detection (LOD) was 2.0 ng mL⁻¹, while limit of quantification (LOQ) was 5.0 ng mL⁻¹. The calibration curves were linear (r² = 0.999) over the concentration range of 0.005 to 5.0 µg mL⁻¹ for both standard solutions and spiked plasma. The intra-day and inter-days precision (RSDs) was less than 4.08 and 6.38%, respectively, and the accuracy was more than 99%. The method could be applied for the pharmacokinetic study of clopidogrel in healthy human volunteers as well as to pharmaceutical dosage forms.

Keywords:

• clopidogrel
• HPLC-UV
• human plasma
• optimization
• pharmaceutical dosage form
• validation