Validated stability-indicating high-performance liquid chromatography and thin-layer chromatography methods for the determination of zopiclone in pharmaceutical formulation

Abstract

Two novel, sensitive, and selective stability-indicating chromatographic methods were described for the analysis of zopiclone (ZOP) in the presence of its degradation products, namely, 7-oxo-6,7-dihydro-5H-pyrrolo[3,4-b]pyrazin-5-yl-4-methylpiperazine-1-carboxylate (hydrolytic DEG) and 5H-pyrrolo[3,4-b]pyrazine-5,7(6H)-dione (oxidative DEG), in drug substance and product. The first method was an isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) using Inertsil ODS3 (250 × 4 mm, 5 μm) column. Upon using HPLC, the run time could be reduced, and actually, the solvents consumption decreased. Quantification was achieved by detection wavelength at 237 nm, based on peak area. Chromatographic separation was performed over the range of 1–10 μg mL−1 with limits of detection (LOD) and quantification (LOQ) of 0.18 and 0.55 μg mL−1 and a mean recovery of 99.98 ± 0.55. The analysis was achieved at 30°C using a mixture of acetonitrile and water (50:50 v/v) as the mobile phase. The second method was thin-layer chromatography (TLC) applied for the separation and analysis of zopiclone in the presence of its alkaline, acidic, and oxidative degradation products. Chromatography was performed on silica gel 60 F254 plates with ethyl acetate–methanol–ammonia 33% (17:2:1 v/v) as the mobile phase. Successful resolution was observed with significant difference in the RF values, followed by densitometric measurement at 303 nm. Evaluation was carried out over the range of 0.1–2 μg per spot with a mean recovery of 100.52% ± 0.24. The developed methods were successfully applied to the analysis of ZOP in bulk powder, laboratory-prepared mixtures containing different percentages of its degradation products, and pharmaceutical dosage form. The degradation products were separated by HPLC as well as identified by TLC, infrared (IR), and mass spectrometry (MS) to confirm its structures and elucidate degradation pathway. The developed methods were validated as per the International Conference on Harmonization (ICH) guidelines. The results obtained by the proposed methods were statistically compared with the reported methods revealing high accuracy and good precision.