Development and validation of a densitometric high performance thin layer chromatography method for simultaneous analysis of three constituents in Chyavanprash

Abstract

Abstract BACKGROUND: A densitometric High Performance Thin Layer Chromatography (HPTLC) method has been established for simultaneous quantification of Gallic acid, Piperine, and Withaferin A in Chyavanprash. Chyavanprash possesses multiple health benefits including immunity boosting and has been widely used since ancient times. The surge of COVID-19 has further increased its demand resulting lack of monitoring on raw materials to ensure its efficacy. METHODS: Aluminum plates coated with silica gel 60F254 as stationary phase was used with Toluene:Ethyl acetate:Formic acid:Methanol [30:30:8:2] (v/v/v/v) as mobile phase. The plates were developed to a distance of 60 mm at 25 ± 2°C in a chamber previously saturated for 30 min. Densitometric analysis was performed at 254 nm in absorbance mode. RESULTS: Amounts of Gallic acid, Piperine, and Withaferin A are 0.75% (w/w), 0.28 % (w/w), and 0.14% (w/w), respectively, in the formulation. The method was validated for specificity, linearity, precision, repeatability, and accuracy. Calibration plots were linear in the concentration range 0.5–6.0 µg per band for Gallic acid; 0.20–1.00 µg per band for Piperine, and 2.0–8.0 µg per band Withaferin A. The correlation coefficients were 0.990, 0.994, and 0.997 for Gallic acid, Piperine, and Withaferin A, respectively. CONCLUSION: The method is simple, precise, specific, accurate, and economical. It can be used for routine analysis in formulations containing Amalaki, Pippali, and Ashwagandha.