Validated liquid chromatographic–ultraviolet method for the quantitation of tadalafil in human plasma using liquid–liquid extraction

A highly selective, sensitive and rapid HPLC method has been developed and validated to quantify tadalafil in human plasma. The tadalafil and internal standard (loratadine, I.S.) were extracted by liquid–liquid extraction technique followed by an aqueous back-extraction allowing injection of an aqueous solvent in the HPLC system. The chromatographic separation was performed on a reverse phase BDS Hypersil C-18 column (250 mm × 4.6 mm, 5 μm, Thermo Separation Co., USA) with a mobile phase of acetonitrile and aqueous solution containing 0.012 M triethylamine + 0.020 M orthophosphoric acid (50/50, v/v). The analytes were detected at 225 nm. The assay exhibited a linear range of 5–600 ng/mL for tadalafil in human plasma. The lower limit of quantitation (LLOQ) was 5 ng/mL. The within- and between batch precision (expressed as coefficient of variation, C.V.) did not exceed 10.3% and the accuracy was within −7.6% deviation of the nominal concentration. The recovery of tadalafil from plasma was greater than 66.1%. Stability of tadalafil in plasma was excellent with no evidence of degradation during sample processing (auto-sampler) and 30 days storage in a freezer. This validated method is applied for the clinical study of the tadalafil in human volunteers.