Simultaneous determination of epimers (+)-catechin and (‒)-epicatechin in Onosma bracteatum Wall. using high-performance thin-layer chromatography‒mass spectrometry method

Abstract

High-performance thin-layer chromatography‒mass spectrometry (HPTLC‒MS) method was developed for the estimation of epimers (+)-catechin (CA) and (‒)-epicatechin (ECA) in Onosma bracteatum Wall. Resolving these epimers is challenging and so method optimization was done for the selection of the stationary phase and the mobile phase to achieve their coherent separation. To further increase the reliability of the obtained densitometric results, HPTLC–MS analysis was performed. The genus Onosma L. is a species-rich genus that exhibits complex patterns of morphological and karyological diversity, and highly debatable taxonomic approaches. Thus, many similar species are described based on morphological differences and often quite ambiguous. To facilitate the identification of O. bracteatum, separation was achieved using pre-coated silica gel 60 F₂₅₄ HPTLC plate as the stationary phase and a mixture of diisopropyl ether–ethyl acetate–formic acid (9.0:0.2:0.7, V/V) as the mobile phase for the separation of epimers CA and ECA. Sample preparation, mobile phase selection and optimization were given importance to manage good resolution (R_F) of these markers. Flavan-3-ols CA and ECA were identified and confirmed on the basis of R_F and in situ UV and MS overlaid spectra with respective standards. The method was validated for linearity, inter-day precision, intra-day precision, repeatability, accuracy, specificity, limit of detection, and limit of quantification. The average recoveries for epimers CA and ECA from ethyl acetate extract fraction (MEF) were found 98.86 and 99.03% indicating the good reproducibility for each marker. The proposed validated HPTLC method is simple, accurate and reproducible and is the first report on the separation and quantification of the epimers CA and ECA in O. bracteatum using HPTLC–MS.