A novel validated instrumental thin-layer chromatographic method and marker-based standardization of liquid herbal formulation using atropine, rutin and vanillin as biomarkers

As Ayurvedic herbal medicines have grown more commercialized, it has become crucial to standardize these products using modern techniques. Mansyadi Kwatha is an Ayurvedic formulation indicated for insomnia, epilepsy, hysteria and anxiety. It consists of three potent drugs, i.e., Nardostachys jatamansi rhizome, Withania somnifera L. root and Hyoscyamus niger L. seed. In this study, a novel instrumental thin-layer chromatography (TLC) method was developed for the simultaneous estimation of atropine, rutin and vanillin. It was then employed for marker-based standardization of Mansyadi Kwatha for detection of these constituents and quantifying the same in the formulation. A pre-coated silica gel TLC plate 60 F₂₅₄ was used as the stationary phase with tetrahydrofuran‒toluene‒methanol‒formic acid (5:3.5:2:0.5, V/V) as the mobile phase for chromatographic separation with densitometric scanning at 206 nm. The developed method gave the desired separation with optimum R_F values of 0.090 ± 0.0039, 0.290 ± 0.0099 and 0.679 ± 0.0056 for atropine, rutin and vanillin, respectively. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2(R1) guidelines were followed for method validation. The method was found to be specific with comparable R_F and no interference of the mobile phase. It showed a good linear relation with correlation coefficient greater than 0.99. The method was also precise, robust with percentage relative standard deviation less than 2% and accurate with percent recovery between 95‒105%.