Development and Validation of HPTLC Method for the Simultaneous Estimation of Ascorbic Acid and Gallic Acid in Amla Juice Preparation

The aim of this study was to asses a simple, selective, precise, and reproducible high performance thin-layer chromatography (HPTLC) method for the simultaneous estimation analysis of ascorbic acid (AA) and gallic acid (GA) in amla juice preparation. The aluminium-based pre-coated TLC plates (Silica gel G 60F 254) were used for the HPTLC fingerprinting analysis. The chromatograms of samples were developed in twin trough glass chamber pre-saturated with mobile phase (toluene: ethyl acetate: methanol: formic acid; 3:3:2:1, v/v/v/v) at room temperature (25 ± 2°C). The densitometric analysis was carried out in absorbance mode at 254 nm. The optimized mobile phase showed compact spots of AA and GA at 0.59 and 0.86 Rf respectively. The linear regression analysis data for the calibration plots of AA and GA showed good linearity (r2= 0.992 and 0.996 respectively) with respect to peak area in the range of 200-1400 ng/spot. The method was validated as per International Conference on Harmonization (ICH) guidelines. The limits of detection and quantification (40 and 140 ng/spot, respectively) were also established. The proposed method has shown the excellent recovery (98.97–99.89%), which supports the suitability of the method for the analysis of AA and GA in the amla juice and other preparations containing these ingredients. Keywords: Amla juice, Ascorbic acid, Gallic acid, HPTLC, ICH guidelines, Validation.