Box-Behnken experimental design for optimizing the HPLC method to determine hydrochlorothiazide in pharmaceutical formulations and biological fluid

An isocratic HPLC method was developed to quantify hydrochlorothiazide (HCT) and its impurities (A, B, C). Response surface methodology combined Box-Behnken design was applied to optimize the effects of four independent factors; pH (2-4), acetonitrile (2-15%), methanol (2-15%) and flow rate (0.7-1.3 mL m in(-1)) against the response. The current investigation aimed to choose the utmost significant factors with the maximum influences of chromatographic separation: resolution, tailing factor, and the number of theoretical plates. Statistical analysis of variance (ANOVA) indicated the best conditions for separating the compounds and the most influential parameters. The plots (contour, 3D surface and perturbation) described the statistically significant parameters quantitatively and their interactions. The experimentally derived RSM model was validated using a t-test with observed R-2 value, adjusted R-2, predicted R-2 and F-values, indicating that the developed model was significant. The proposed LC was combined with a PDA detector and detected at 210 nm. The HCT highest separation was achieved on the ODS Hypersil C18 (250 mm x 4.6 mm, 5 lm) column using the following conditions: pH (3.497), acetonitrile (10.6%), methanol (16.2%), flow rate 1 mL/min. The method validated was precise and linear in 1.25-12.75 mg m L-1. The validated method's value of LOD & LOQ was 1.09 and 3.33 mg mL(-1), respectively. Youden and Steiner's factorial design with typical and alternative conditions were performed to verify its robustness study. The % RSD values were within the accepted range of ICH guidelines. Therefore, the proposed method can be applied for routine analysis of HCT and its impurities in pharmaceutical formulation and biological fluid for routine quality control sample analysis. (c) 2022 Elsevier B.V. All rights reserved.

Automated summary

An isocratic HPLC method was developed to quantify hydrochlorothiazide (HCT) and its impurities (A, B, C). Response surface methodology combined with Box-Behnken design was used to optimize the factors affecting chromatographic separation. The experimentally derived model was validated and found to be significant.