Simultaneous determination of Avanafil and Dapoxetine in human plasma using liquid chromatography/tandem mass spectrometry (LC-MS/MS) based on a protein precipitation technique.

A rapid and selective LC-MS/MS method is described for the simultaneous assay of Avanafil and Dapoxetine in human plasma a protein precipitation (PP) sample preparation technique. Tadalafil was chosen as the internal standard reaching good recovery and reproducibility while diminishing the effects of the matrix. An Agilent Zorbax Eclipse XDB C column (4.6 × 50 mm, 1.8 μm) was used for the chromatographic separation and analysis, while 0.1% formic acid : acetonitrile (60 : 40, v/v) was utilized at a flow rate of 0.5 mL min. It was revealed that 6 min stop time accomplished the best separation. The assay was linear over the range of 10-6000 ng mL for both drugs. The established bio-analytical method validation was demonstrated following US-FDA recommendations including sensitivity, selectivity, linearity, accuracy and precision. Furthermore, other validation parameters were assessed such as the dilution integrity, matrix effect, carryover, and analyte stability during both short- and long-term sample processing and storage. The adopted method was efficaciously applied to a clinical study for the concurrent determination of Avanafil and Dapoxetine in human plasma.