Design of Experiments-assisted Micellar Electrokinetic Chromatography Separation of Phosphodiesterase-5 Inhibitors:...

A micellar electrokinetic chromatography method was developed for the simultaneous separation of the phosphodiesterase-5 inhibitors sildenafil and tadalafil, two therapeutic agents used in the treatment of erectile dysfunction that are frequently identified as illicit adulterants in dietary supplements marketed for sexual enhancement. Method development was supported by a design of experiments strategy. An initial fractional factorial screening design was used to evaluate the influence of selected experimental parameters on resolution (R_s) and migration time. Pareto analysis of standardized effects indicated that cyclodextrin (CD) concentration, methanol (MeOH) content, and separation voltage significantly affected the separation, whereas pH, sodium dodecyl sulfate (SDS) concentration, and temperature did not show a statistically significant impact within the studied range. Based on the screening results, a Box-Behnken response surface design was employed to optimize the significant factors. Optimized separation conditions consisted of a 50 mM phosphate buffer (pH 2.5) containing 50 mM SDS, 10% MeOH, and 5 mM hydroxypropyl-β-CD, with a separation voltage of -20 kV and a capillary temperature of 20°C. Under these conditions, baseline separation was achieved with a R_s of 4.75 and migration times below 6 min. The method was validated in terms of precision, linearity, accuracy, and robustness, showing satisfactory analytical performance. Application to pharmaceutical formulations and dietary supplement samples confirmed the suitability of the proposed method for routine screening and quality control purposes.