Development and Validation of Chromatographic Methods for Resolving Ternary Mixture of Diflunisal, Naproxen and DiflunisalToxic Impurity

Abstract

This search article describes accurate, precise and sensitive TLC-densitometric and HPLC– DAD methods which have been developed and validated for simultaneous determination of Diflunisal (DIF) and Naproxen (NAP) in their binary mixture and in the presence of Diflunisal toxic impurity, Biphenyl-4-ol (BIP). The developed TLC-densitometric method depended on separation of the three components on a stationary phase of silica gel plates 60 F254 using a solvent mixture consisting of methylene chloride: methanol: ethyl acetate: acetic acid (9: 0.5: 0.5: 0.05, by volume) and then scanning at 240 nm. The linearity ranges were found to be 0.2-2, 0.2-1 and 0.2-2 µg/band for DIF, NAP and BIP, respectively. The developed HPLC DAD method depended on using Zorbax Eclipse C18 column as a stationary phase and a mixture of methanol: water (55: 45, by volume, pH=4 with orthophosphoric acid) as a mobile phase, delivered at constant flow rate of 1 mL/min and UV detection at 215 nm. Linear relationships were obtained between the peak area and the concentration in the ranges of 3-40, 3-40 and 3-25 µg/mL for DIF, NAP and BIP, respectively. Factors affecting the developed methods have been studied and optimized. Furthermore, method's validation has been carried out according to International Conference on Harmonization guidelines. The proposed methods were successfully applied for determination of DIF and NAP in Maxipan ® tablets. Also they were statistically compared with the reported method using student's-t and F-tests and no significant difference was obtained. The reported methods were the first developed stability indicating methods for resolving the proposed mixture and their toxic impurity.