Optimization of chromatographic conditions via Box‒Behnken design in RP-HPLC-PDA method development for the estimation of folic acid and methotrexate in bulk and tablets.

Simultaneous estimation of folic acid and methotrexate in bulk and tablet dosage form by RP-HPLC-PDA was conducted via Box‒Behnken design application. Three-factor numerical values were finalized from the graphical and numerical optimization with built-in ANOVA in BBD. Sharp and symmetric peaks were observed at 4.138 and 6.929 min for folic acid and methotrexate, respectively. The mobile phase composition was methanol and 0.1% formic acid in water with a ratio of 31:69 and a flow rate of 1.1 ml/min. Both drugs were detected at a wavelength of 291 nm. The developed method was validated according to ICH guidelines. The results of the validation parameters were within acceptable limits. Stress stability studies have been performed under acidic, alkali, oxidation, neutral and photolytic conditions. Three different brand-marketed tablets were assessed with the developed method (MGXT, FOLTNAX and TRUXOFOL). In the tablet formulations, chromatogram percentages of folic acid and methotrexate were calculated at 99.13% and 99.50 in MGXT, 99.17% and 99.47 in FOLTNAX, and 99.91 and 100.05 in TRUXOFOL.
Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(© 2023 The Authors. Published by Elsevier Ltd.)