Development and Validation of High-Performance Thin-Layer Chromatography Method for Determination of Vicine in Herbal Extract and Formulation

A simple, selective, precise, and reproducible high-performance thin-layer chromatography (HPTLC) method for the analysis of vicine in herbal extracts and formulations containing Momordica charantia was developed. The study was performed on aluminum-based pre-coated TLC plates (silica gel 60F(254)). The chromatograms of samples were developed in twin trough glass chamber pre-saturated with mobile phase comprised of ethyl acetate-methanol-water-formic acid (7.5:3:1:0.1, v/v/v/v) at room temperature (25 +/- 2 degrees C). The densitometric analysis was carried out in absorbance mode at 278 nm. The optimized mobile phase showed a compact spot of vicine (R-F = 0.31 +/- 0.02) from the other constituents present in the samples. The linear regression analysis data for the calibration plots showed good linearity (r(2) = 0.9990) with respect to peak area in the range of 200-1400 ng spot(-1). The method was validated as per International Conference on Harmonization (ICH) guidelines. The limits of detection and quantification (50 and 150 ng spot(-1), respectively) were also established. The proposed method has shown the excellent recovery (98.01-99.33%), which supports the suitability of the method for the analysis. Statistical analysis of the obtained data showed the selectivity of the proposed method for vicine estimation in the herbal samples.