Determination of phosphodiesterase 5 enzyme inhibitors in natural products

Erectile dysfunction is defined as a persistent inability to achieve and/or maintain an erection sufficient for sexual activity. Synthetic phosphodiesterase 5 (PDE5) enzyme inhibitors such as vardenafil, sildenafil, avanafil, udenafil, mirodenafil and tadalafil are used to treat erectile dysfunction. This thesis consists of three parts: spectrophotometric, electroanalytical, and chromatographic studies. The first part of this thesis aimed to identify natural inhibitors of the PDE5 enzyme. Since, at the end of the spectrophotometric studies, no chemical interaction between the PDE5 enzyme and the sildenafil citrate inhibitor could be detected, it was not possible to develop a method for detecting the presence of PDE5 enzyme inhibitors, therefore the determination of the species with PDE5 enzyme inhibitor properties in plants could not be carried out. In the electroanalytical experimental studies carried out in the second part of the thesis, an adsorptive stripping linear scanning voltammetric method was developed for the simultaneous determination of sildenafil citrate and tadalafil. A glassy carbon electrode modified with carboxylic multi walled carbon nanotubes was used as the voltammetric working electrode. Both the sildenafil citrate and tadalafil analytes spontaneously accumulated on the modified surface owing to their adsorption affinity. Cyclic voltammetry (CV) was used to evaluate the electrochemical behaviors of sildenafil citrate and tadalafil. The developed method is linear in the range of 0.01-0.50 µM with a correlation coefficient of 0.9997 and 0.9985 for sildenafil citrate and tadalafil, respectively. The LOQ of the developed method was determined to be 0.010 µM for both the analytes. The intra day and inter day repeatability of the method was less than 10% RSD in all cases. The recoveries ranged between 83% and 94%. The applicability of the proposed method was investigated by determining the analytes in two different adulterated samples (an energy drink and a traditional herbal product). The chromatographic experimental studies carried out in the third part of this thesis presented a method for the simultaneous determination of vardenafil, sildenafil, avanafil, udenafil, mirodenafil, and tadalafil using an HPLC-UV system. At the end of the optimisation studies, chromatographic conditions were defined as mobile phase A: 0.01 M pH:3.2 formic acid/formate buffer, mobile phase B: acetonitrile, gradient program (A:B): 0-8.5 min 72:28, 8.5-20 min 50:50 (v:v) at a flow rate of 1.0 mL/min, injection volume 50 μL and absorbance measurements were performed at 230 nm. It was observed that the matrix effect on the analyte signals in the range of 2.00-49.10% enhanced the signals of vardenafil, udenafil, mirodenafil, tadalafil and suppressed the signals of sildenafil and avanafil. Therefore, all chromatographic studies were performed in a matrix medium to minimize the matrix effects. The developed chromatographic method was found to be highly linear in the range of 0.5-50 mg/L with correlation coefficients of 0.9981; 0.9982; 0.9995; 0.9995; 0.9984; 0.9997 and the LOD values of the method were 0.10; 0.07; 0.07; 0.09; 0.11; 0.03 mg/L and the LOQ values were 0.32; 0.23; 0.23; 0.30; 0.38; 0.12 mg/L for vardenafil, sildenafil, avanafil, udenafil, mirodenafil and tadalafil respectively. The intra day and inter day repeatability of the method was less than 2.67 % RSD in all cases. The recoveries ranged between 87.66% and 101.47%. In the precision study, the highest percentage relative errors were 5.81 and 3.30 for concentrations of 1 and 25 mg/L, respectively. The determination of these synthetic PDE5 inhibitors in traditional herbal products purchased from herbalists and energy drinks purchased from supermarkets in Manisa and Izmir was carried out using the developed HPLC-UV method. It was found that significant amounts of sildenafil citrate were added to all herbal products to induce erection. In order to compare the results obtained, some of these products were analysed by LC-MS/MS method and it was observed that the results obtained by HPLC-UV and LC-MS/MS methods were very close to each other.