RP-HPLC–UV method for the quantification of propranolol in rat's serum and krebs buffer using one-step protein precipitation

Krebs buffer is considered one of the most used physiological buffers in biomedical research. In the current work, a rapid reversed-phase high-performance liquid chromatographic (RP-HPLC) method with ultraviolet (UV) detection at 214 nm was developed and validated according to European Medicines Evaluation Agency (EMEA) guidelines for the determination and quantification of propranolol in Sprague–Dawley rat's serum and in Krebs buffer. This method can be applied for both in vivo and in vitro studies with short run time of 7.0 min . Isocratic elution with a flow rate of 1.0 mL/min was employed. BDS Hypersil C-18 column (150 mm × 4.6 mm and 5 μm) was used to obtain satisfactory resolution. The mobile phase used contained a mixture of acetonitrile, methanol, and triethylammonium phosphate solution (15.0:32.5:52.5, v/v). Best separation between propranolol and the internal standard (I. S.) sildenafil was obtained at 4.2 and 5.5 min, respectively. Propranolol was linear over a concentration range of 50.00–3000 ng/mL with acceptable accuracy, and intra- and inter-day precision. Dilution integrity was assessed and was found to be within the acceptable range for both serum and Krebs buffer. Sample stability tests were studied at different storage conditions, and all the analytes were found to be stable. The mean percentage of recovery of propranolol was found to be 97.06% and 98.57% for serum and Krebs buffer, respectively.