RP-HPLC with Time Programmed Fluorescence Detection for Quantitation of Avanafil and Dapoxetine Hydrochloride: Application to Pharmaceutical Dosage Form and Biological Fluid
Abstract
Avanafil (AVN) was recently co-formulated with dapoxetine HCl (DAP) for treatment of erectile dysfunction and premature ejaculation. Sensitive and simple reversed-phase (RP) high-performance liquid chromatographic method (HPLC) was developed and validated for their simultaneous determination using tadalafil (TAD) as an internal standard. Isocratic separation was achieved within run time of only 7.0 min on Eclipse C18 column (150 mm × 4.6 mm, 5 µm particle size) using a mobile phase composed of acetonitrile: 0.15% triethylamine (40:60, v/v) at pH = 4.0 adjusted with o-phosphoric acid. The analysis was performed at a flow rate of 1.0 mL/min with fluorescence detection at 236/370 nm for AVN, 236/410 nm for DAP, and 236/330 for TAD using time programming. The analytes were determined by their native fluorescence and the response was linear over concentration ranges of 0.05–40 and 0.01–30 µg/mL for AVN and DAP, respectively, with limits of detection of 0.043 and 0.007 µg/mL in a respective order. The developed method successfully determined AVN and DAP in bulk powder, tablets, and spiked human plasma.
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