Optimization of HPTLC densitometric method for curcuminoids and polyphenolics in an ayurvedic Emblica officinalis and Curcuma longa based Nishamalaki formulation by Box-Behnken design

Abstract

The present study focuses on developing a simplified, specific, and accurate high performance thin layer chromatographic (HPTLC) method for the quantitative and qualitative determination of ellagic acid, gallic acid, and curcuminoids (bisdemethoxycurcumin, demethoxycurcumin, and curcumin) in Nishamalaki Ayurvedic formulation. Pre-coated silica gel 60 F₂₅₄ aluminum-backed plates were used as the stationary phase in the chromatographic technique development, and the optimized mobile phase was toluene: dichloromethane: glacial acetic acid: formic acid (6:4:1.6:0.9% v/v/v/v) with double development in linear ascending mode. The detection wavelength for quantification for ellagic and gallic acid was 280 nm, and curcuminoids (bisdemethoxycurcumin, demethoxycurcumin, and curcumin) were 430 nm. The optimized mobile phase showed optimum separation between peaks for ellagic acid, gallic acid, and curcuminoids (bisdemethoxycurcumin, demethoxycurcumin, and curcumin) at R_F of 0.12±0.02, 0.21±0.02, 0.55±0.02, 0.69±0.02 and 0.82±0.02 respectively. Chromatographic conditions were optimized using the Box-Behnken design. Various variables, such as, the volume of formic acid and glacial acetic acid, and chamber saturation time, that are likely to impact R_F were identified for further optimization. The volume of glacial acetic acid may be regarded as a critical method parameter, which caused the greatest change in the R_F value and was the important factor among the three factors. The linear range was 600-1800 ng/band for all markers (r² greater than 0.98). The limit of detection (LOD) and quantification (LOQ) measured indicated the method’s sensitivity. For all markers, the recovery percentage reveal acceptable accuracy, and the method was repeatable and reproducible from precision measurements with less than a 2% relative standard deviation. The optimized method was precise, specific, accurate, robust and reproducible for quantifying ellagic acid, gallic acid, and curcuminoids (bisdemethoxycurcumin, demethoxycurcumin, and curcumin) in the quality-control testing of botanical extract along with Nishamalaki ayurvedic formulation.