Validated high performance liquid chromatography (HPLC) method for the determination of sumatriptan in rabbit plasma: Application to pharmacokinetic study

Abstract

A new, sensitive and specific isocratic reverse phase-high performance liquid chromatography (RP-HPLC) method with fluorescence detection was developed and validated for the determination of sumatriptan in rabbit plasma using sulpiride as an internal standard (IS). Sumatriptan was extracted from plasma by a liquid-liquid extraction with a mixture of tert-butyl methyl ether, dichloromethane and ethyl acetate (2:2:3, v/v). Chromatographic separation of the analyte and internal standard was achieved on a Phenomenex C4 (250 × 4.6 mm, 5 µm) analytical column maintained at 40°C. The mobile phase was composed of 25 mM ammonium acetate (pH 6.5) and acetonitrile (85:15, v/v), pumped isocratically at a flow rate of 0.9 ml/min. Column eluent was monitored at excitation and emission wavelengths of 225 and 350 nm. The calibration curve was linear over a concentration range of 1 to 300 ng/ml (r2 = 0.9999) with a limit of quantification, 1 ng/ml. The intra-day and inter-day precision and accuracy were between 2.24 and 4.28% and -1.10 and 2.86%, respectively. The mean recoveries of sumatriptan and sulpiride were 89.92 and 91.03%, respectively. Sumatriptan containing plasma samples were stable at -20°C for 14 days. The validated method was successfully applied for pharmacokinetic study after a single oral administration of sumatriptan (50 mg) to rabbits. Key words: Sumatriptan, rabbit plasma, reverse phase-high performance liquid chromatography (RP-HPLC), liquid-liquid extraction, pharmacokinetics.