Quantitative Determination of Propranolol in Human Serum by High-Performance Thin-Layer Chromatography

A high-performance thin-layer chromatography (HPTLC) method for the quantification of propranolol in human serum is developed and validated. The method includes a liquid liquid extraction of the analyte from the matrix using n-heptane isoamyl alcohol (98.5:1.5, v/v) as the extraction solvent and verapamil as the internal standard. The HPTLC method employed precoated silica gel F254 HPTLC plates (10 cm x 10 cm and 10 cm x 20 cm; layer thickness 0.2 mm) prewashed with methanol, and a mobile phase comprising chloroform- methanol-ammonia (9:1:0.04, v/v/v). The developing solvent was run up to 80 mm in a vertical CAMAG chamber previously saturated with the solvent mixture for 20 min. Densitometric detection was done at lambda = 290 nm. The method was linear between 5 and 100 ng/band, corresponding to 0.01 and 0.20 ng mu L-1 of propranolol in human serum after the extraction process and applying 50 mu L to the chromatographic plates (correlation coefficient = 0.998). The %RSD (absolute value of the coefficient of variation) of intra-assay and inter-assay precision was in the range 1.84-2.85% (n = 3) and 3.52-3.95% (n = 9), respectively. The limit of detection and limit of quantitation were found to be 3.15 and 4.02 ng/band, respectively. The method proved to be accurate, with a recovery between 96.35% and 98.82%, with an RSD not higher than 4.17%, and was selective for the active substance tested, its major metabolite, and the internal standard. This method was successfully applied to quantify propranolol in patient serum samples. Therefore, the method is useful for the quantitative determination of propranolol in human serum.