Simultaneous Determination of Six Phosphodiesterase-5 Enzyme Inhibitors in Traditional Herbal Products by HPLC–UV and Verification with LC–MS/MS

An HPLC–UV method was developed for the simultaneous determination of six phosphodiesterase-5 enzyme inhibitors, namely sildenafil, tadalafil, avanafil, vardenafil, udenafil and mirodenafil, in traditional herbal products and an energy drink. Chromatographic separation was achieved on an ODS3 column with gradient elution at a flow rate of 1.0 mL/min. The mobile phase consisted of a mixture of 0.01 M formic acid/formate buffer at pH 3.2 in water and acetonitrile. The identification of the phosphodiesterase-5 enzyme inhibitor peaks was based on the comparison of retention times in HPLC–UV and mass spectra in HPLC–MS/MS. Calibration curves were constructed in the range of 0.50 to 50 µg/mL in a matrix-matched solution. The method exhibited high linearity with a correlation coefficient greater than 0.9981. Recovery ranged from 87.33 to 101.67% with an RSD ranging from 0.17 to 8.42%. Accuracy and precision of the method were expressed in terms of RE and RSD which were lower than 5.81 and 4.21% at 1 µg/mL and 3.30 and 1.11% at 25 µg/mL, respectively. Reproducibility of the method was better than 2.67% RSD for peak areas and 1.10% RSD for retention times. The LODs for the six PDE-5 enzyme inhibitors ranged from 0.03 to 0.11 µg/mL and while LOQs ranged from 0.12 to 0.38 µg/mL. The results of the validation and LC–MS/MS verification results indicate that the developed HPLC–UV method is accurate, precise and reproducible, and applicable for the simultaneous determination of six phosphodiesterase-5 enzyme inhibitors in herbal products and energy drinks.