Simultaneous Quantification of Diarylheptanoids in Alnus nepalensis Using a Validated HPTLC Method

Abstract

Platyphylloside, oregonin and hirsutanonol 5-O-β-D-glucopyranoside are known bioactive metabolites in Alnus nepalensis. In this article, the aforementioned three markers were isolated and simultaneously quantified by the thin-layer chromatography densitometric method. A sensitive, selective and robust qualitative and quantitative densitometric high-performance thin-layer chromatographic method was developed and validated for the determination of above markers in the leaves of A. nepalensis. The separation was performed on silica gel 60F254 high-performance thin-layer chromatography plates using chloroform:methanol:formic acid (75:25:2, v/v) as mobile phase. The quantitation of diarylheptanoids was carried out using the densitometric reflection/absorption mode at 610 nm after post-chromatographic derivatization with vanillin-sulfuric acid reagent. A precise and accurate quantification can be performed in the linear working concentration range of 333-3330 ng/spot with good correlation (r(2) = 0.999). The method was validated for peak purities, precision, robustness, limit of detection and quantitation, etc., as per ICH guidelines. Specificity of quantitation was confirmed using retention factor and spectra correlation of markers in standard and sample tracks.