Optimization and Validation of HPLC-UV Method for the Determination of Vardenafil, Sildenafil, and Tadalafil in Honey-Mixed Herbal Sachets Using a Design of Experiment

A method was developed for the simultaneous determination and analysis of sildenafil, vardenafil, and tadalafil in honey-mixed herbal sachets using high-performance liquid chromatography with a UV detector (HPLC–UV). This method eliminates the employment of complex procedures and abolishes time-consuming and labour-intensive pre-treatment processes. In ten minutes, the separation process (at 25oC) of sildenafil, vardenafil, and tadalafil using a C18 150 mm × 4.6 mm x 5 μm column (Shim-pack GIST) was successful with high selectivity and sensitivity. The mobile phase was a 60:40 (v/v) mixture of 0.1 percent formic acid in water and 0.1 percent formic acid in acetonitrile. Using the mobile phase as an extraction mixture, it gave recoveries in the range of 93.0-103.3% at spike levels of 50–150 mg/kg with relative standard deviations (RSDs) lower than 10%. The intra-day and inter-day precision results were in the range of 0.4–0.8% and 1.0–1.7%. Furthermore, the retention times for sildenafil acid, vardenafil acid, and tadalafil were 1.93, 2.47, and 9.62 minutes, respectively, and the limits of detection (LOD) were 1.70, 2.16, and 1.03 mg/L, while the limits of quantification (LOQ) were 5.65, 7.21, and 3.42 mg/L.