Optimization and validation of high performance liquid chromatography-ultra violet method for quantitation of metoprolol in rabbit plasma: application to pharmacokinetic studies

Purpose: To develop a sensitive, simple and validated high performance liquid chromatography (HPLC) analytical method for the determination of metoprolol tartrate in rabbit plasma.Methods: Mobile phase of methanol and 50 mM ammonium dihydrogen phosphate solution (50:50) at pH 3.05 was used for separation of metoprolol on BDS hypersil C18 column at a wavelength of 223 nm. Flow rate and retention time were 0.6 mL/min and 7.4 min, respectively. For pharmacokinetic study, rabbits were given an oral dose of 8 mg/kg of metoprolol in solution form. Blood samples were taken from jugular vein of the rabbits after drug administration and analysed by HPLC.Results: Separation of metoprolol was not interfered with other components in plasma. The calibration curve was linear in the range of 25 - 1000 ng/mL (r2 = 0.997). Lower limits of detection (LLOD) and quantitation (LLOQ) were 8.87 and 25 ng/mL, respectively. Relative standard deviation (RSD) of intraday and inter-day precision was < 14.27 and 7.61 %, respectively. Relative error of accuracy was between 4.85 and 14.37 %. Maximum plasma concentration (Cmax), time to maximum plasma concentration (Tmax) and half-life (t½) after metoprolol oral administration in rabbits were 186.29 ng/mL, 0.50 h and 2.27 h, respectively.Conclusion: A simple, accurate and precise HPLC-UV method for metoprolol determination in rabbit plasma has been successfully developed and applied to a pharmacokinetic study.Keywords: HPLC-UV, Metoprolol, Pharmacokinetics, Rabbit plasma, Liquid-liquid extraction, Validation