Investigation of the Presence of Sildenafil in Herbal Dietary Supplements by Validated HPLC Method

Abstract

Objectives: As the first FDA-approved phosphodiesterase type 5 inhibitor, sildenafil (SDF) is widely used in the treatment of erectile dysfunction due to its strong pharmacodynamic activity. Since many food supplements are now involved in illegal adulteration, the presence of SDF in food supplements is very important because of their toxicological risks. In this study a simple fast, reliable high-performance liquid chromatography method with ultraviolet (UV) detector has been developed and validated for SDF analysis in herbal dietary supplements (HDSs).

Materials and methods: 10 mM phosphate buffer containing 0.1% triethylamine (pH 3.5) and acetonitrile (65:35, v/v), as mobile phase was applied isocratically to a reverse phase C₁₈ analytical (4.6×250 mm, 5 μm) column. Chromatographic separation was achieved by a C₁₈ reverse-phase analytical column 4.6×250 mm, 5 μm particle size, using acetonitrile, with 10 mM phosphate buffer containing 0.1% triethylamine (65:35, v/v, pH 3.5) as a mobile phase. The mobile phase flow rate was 1 mL min^-1 and the column temperature was 35°C. The UV detector was set at 293 nm. The liquid-liquid extraction method used in the study provided a simple and practical method for the recovery of SDF in HDSs and their obtained values ranged from 87.6 to 111.7%.

Results: The method showed linearity with an excellent correlation coefficient (r²>0.999). Moreover, it was specific and sensitive with the limit of quantification, 6.5 ng mL^-1. Intraday and interday method precision was ≤8.2 (relative standard deviation %). Intraday and interday method accuracy was between -4.0 and 7.1 (RE%). The method was strong according to the robustness test results obtained from UV detection, mobile phase buffer pH, column temperature, and flow rate changes. The described procedure was simple, fast, precise, and feasible for routine adulteration analysis of SDF, especially in food control or toxicology laboratories. This method was successfully applied to 50 individual solid and liquid form HDSs.

Conclusion: The results showed that 37 out of 50 samples of HDSs (represented 74.0%) examined contained SDF between 0.01 and 465.47 mg/g, 150.87±127.48 (mean ± standard deviation), which could lead to serious health problems and might even be fatal for consumers. The described procedure was found to be simple, rapid, precise and feasible for routine adulteration analysis of SDF, especially in food control or toxicology laboratories.

Keywords: Sildenafil; adulteration; herbal dietary supplements; high-performance liquid chromatographic-ultraviolet detection; validation.

Figure 1

Chemical structure of SDF (A) and CZP (B) used as an internal standard SDF: Sildenafil, CZP: Clozapine

Figure 2

A. The chromatogram of the SDF blank sample that belongs to HDS extract used as a quality control sample in validation tests. B. The chromatogram of an HDS extract that was prepared by standard addition method containing 1000 ng mL-1 SDF. C. The chromatogram of a real HDS sample extract determined to be adulterated with SDF. SDF: Sildenafil, HDS: Herbal dietary supplements, CLZ: Clozapine

Figure 3

Real HDS sample chromatograms containing SDF (A=sample 50, B=sample 49, and C=sample 51) HDS: Herbal dietary supplements, SDF: Sildenafil