Simultaneous analysis of chlorzoxazone, diclofenac sodium and tramadol hydrochloride in presence of three potential impurities using validated HPLC-DAD and HPTLC methods

Abstract

In recent years, a lot of single-pill combinations (SPC) are manufactured and are used as a promising choice in treatment of signs and symptoms of osteoarthritis and rheumatoid arthritis. However, this trend made a serious challenge to drug analysts because of the difficulty in the analysis of two or more drugs in presence of each other. In this study, two chromatographic methods were developed for the simultaneous analysis of chlorzoxazone (CZ), diclofenac sodium (DIC) and tramadol hydrochloride (TRA) in presence of three of their related substances and potential impurities. Method I involved application of HPLC with diode array detection where Waters Symmetry C8 column was used as stationary phase. Mixture of ortho-phosphoric acid (0.03 M, pH 3) and acetonitrile was used as a mobile system with gradient elution and flow rate 1 mL/min. Peak areas were measured at the wavelengths 218 nm for TRA and 280 nm for CZ and DIC. Peaks eluted at retention times 2.30, 5.75 and 9.74 min for TRA, CZ and DIC respectively. Method II based on HPTLC separation. In this method, HPTLC silica gel 60 F254 plates were used with mobile phase of hexane: ethyl acetate: methanol: acetic acid (12: 6: 2: 0.1, by volume). Densitometry scanning was performed at 280 nm for all drugs. The retardation factor (Rf) values were 0.14, 0.40 and 0.58 for TRA, DIC and CZ respectively. Both methods were validated according to International Conference on Harmonization (ICH) Guidelines with respect to linearity, ranges, accuracy, precision, specificity, robustness and limits of detection and quantitation. Linearity ranges were 15–100 μg/mL for the three drugs in method I. In method II, the obtained data were linear in the ranges 300–1800 ng/spot for TRA and 25–150 ng/spot for DIC and CZ. Both methods showed good specificity by resolution of the three drugs from the related substances and potential impurities 2-amino-4-chlorophenol, 2.6-dichloroaniline and impurity A of DIC. Finally, both methods were successfully applied to the analysis of a real sample of combined tablets containing the three drugs. The suggested methods could be applied for the studied drugs in QC-lab.