A selective and sensitive procedure for quantitation of a new antithrombotic drug (GRS) in rat plasma was developed and validated using an HPLC-UV. The method was validated according to recommendations of the FDA, EMA in terms of selectivity, linearity, accuracy, precision, recovery, matrix effect, stability, and carry-over. The preparation of the biological sample included liquid-liquid extraction with acetonitrile, separation of water-organic mixture with inorganic salts, organic phase clean-up with a sorbent (QuEChERS method), its evaporation to dryness and reconstitution of the residue with A:B eluents mixture (1:1). The chromatographic separations were performed on a micro-column 75 × 2 mm, 5 μm particle size sorbent ProntoSIL 120-5-C18 AQ. The flowrate was of 0.15 ml/min, detector wavelength was set at 360 nm for GRS and at 230 nm for papaverine (IS). It was found that GRS recovery from rat plasma is 94%, the response linearity is in the range of 10 to 1000 ng ml-1. The accuracy values for intra-day determination were of 93.2 to 101.8%, for inter-day determination were of 91.2 to 102.2%, coefficient of variation for intra- and inter-day precision did not exceed 4.1 to 9.3%. The application of the method was shown in pharmacokinetic studies of GRS in rats at a dose of 20 mg kg-1.
Keywords: Antithrombotic drug; HPLC-UV; Pharmacokinetic studies; Rat plasma.
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