Qualitative and quantitative analysis of PDE-5 inhibitors in counterfeit medicines and dietary supplements by HPLC-UV using sildenafil as a sole reference.

Due to their popularity, medicinal products containing the phophodiesterase type 5 enzyme (PDE-5) inhibitors sildenafil, vardenafil and tadalafil are often subject to counterfeiting. In addition, illicit herbal dietary supplements adulterated with these substances or their analogs have appeared on the market offering an easy and anonymous sale. This paper describes an analytical method for qualitative and quantitative screening of sildenafil, vardenafil, tadalafil and 11 of their designer analogs in illegal erectile dysfunction products by high-performance liquid chromatography with UV detection (HPLC-UV). Sildenafil served as a single external standard for both identification and quantification of all analytes. Relative retentions and reference UV spectra were used for qualitative, and correction factors for quantitative analyses, respectively. The separation was performed on a Kinetex C18 reverse-phased column at 25°C using gradient elution. Mobile phase A consisted of 200mM ammonium acetate solution while mobile phase B was a 1:1 (v/v) mixture of methanol and acetonitrile with a flow rate of 0.5ml/min and injection volume of 5µl. Detection wavelength was set to 290nm. The method was validated in accordance with the appropriate guideline of the International Conference on Harmonization (ICH) in terms of specificity, selectivity, precision, linearity, limit of quantitation, limit of detection, accuracy, robustness and stability, and was successfully applied to the analysis of natural dietary supplements and herbal remedies with an indication for enhanced male sexual potency. The proposed method offers a cheap and simple alternative to LC-MS screening used by control laboratories for routine analysis of suspicious products.

Chiral recognition of dapoxetine enantiomers with methylated-gamma-cyclodextrin: a validated capillary electrophoresis method.

The enantiomers of dapoxetine, a serotonin transporter inhibitor for the treatment of premature ejaculation have been separated by cyclodextrin modified capillary zone electrophoresis using uncoated fused-silica capillary. Over 20 cyclodextrins were screened as chiral selectors, investigating the stability of the inclusion complexes and enantioseparating properties. According to the preliminary experiments as chiral selector randomly methylated-γ-cyclodextrin was chosen. The basic chemical and instrumental parameters of enantioseparation as concentration of buffer, chiral selector and organic additive, pH, temperature and applied voltage were optimized afterwards using an orthogonal experimental design. Using this methodology not only the optimal parameter values for chiral separation (15 °C, +15 kV, 70 mM acetate, 20 v/v% MeOH, pH*=4.5, 3 mM methylated-γ-CyD) but also the significance order of factors on resolution was determined. Applying these parameters an optimal resolution of 7.01 was achieved. The optimized method was then validated according to the ICH guideline Q2 (R1) with regard to repeatability, linearity range, LOD, LOQ, accuracy and robustness.

Separation and characterization of modified pregabalins in terms of cyclodextrin complexation, using capillary electrophoresis and nuclear magnetic resonance.

The (S)-(+)-isomer of 3-isobutyl-GABA (pregabalin), the blockbuster drug in the treatment of neuropathic pain has been separated from its R isomer by cyclodextrin modified capillary zone electrophoresis (CZE) using uncoated fused-silica capillary. Derivatization of the single isomer and the racemate with tosyl- and dansyl-chloride was carried out to introduce strong UV chromophores of different size. CE-pH titrations were performed to determine the dissociation constants for both derivatives. 30 cyclodextrin (CD) derivatives as chiral agents were used at four different pH values to study the enantioseparation of the differently protonated guest molecules. The separation was optimized as a function of CD concentration, buffer type and concentration, pH and applied voltage. For the tosylated derivate the best resolution (R(s)=2.76) was found with 6-monodeoxy-6-mono-(3-hydroxy)-propylamino-beta-cyclodextrin hydrochloride (PA-beta-CD) at pH 6.8, while with the same selector at pH 7.2 enantioseparation with an R(s) value of 4.32 could be achieved for the dansylated pregabalin. At pH 2.5 for the dansylated derivative trimethylated alpha- and beta-CD systems resulted the most significant separation (R(s)=7.38 and R(s)=7.74, respectively). Experiments with dual CD systems were carried out as well. The stoichiometry of the complexes was determined using the Job plot method and resulted in a 1:1 complex in both cases. The structures of the inclusion complexes were elucidated using 2D ROESY NMR experiments.