Simultaneous Quantification of Benznidazole and Posaconazole by HPLC-DAD Using QbD Approach

To develop an analytical method to simultaneous quantification of benznidazole (BNZ) and posaconazole (POS) by high-performance liquid chromatography with diode-array detection (HPLC-DAD) using design of experiments.

Percentages of organic phase, buffer pH and flow rates of mobile phase were selected as independent variables by full factorial design (33), totaling 27 experiments. Significant factors were evaluated using factorial analysis of variance with 95% confidence level. Method optimization was performed using desirability profiles, considering BNZ/POS chromatographic resolution and peak areas. Further, the method was evaluated regarding its suitability and properly validated according to the international compendiums using the parameters: specificity, linearity, accuracy, precision, limit of detection and limit of quantification.

The optimized method was achieved using Discovery® C8 column (250 mm × 4.6 mm; 5 μm particle size), methanol/acetate buffer (pH 3.5)(71:29) and detection at 260 nm. Retention times were 3.6 and 7.6 min for BNZ and POS, respectively, with good suitability of system and it was specific and linear (r2 >0.99) for both drugs, proving the efficiency of the method even in the presence of degradation products of POS.

This new method is a great alternative to perform reliable, faster and cheaper analysis since the simultaneous quantification of the association BZN/POS is not reported yet in the literature.