Method Validation and Quantification of Lupeol in Hibiscus sabdariffa (Malvaceae) calyx extracts by using High Performance Thin Layer Chromatography

Abstract

The primary objective of the present investigation was to establish and validate a High-Performance Thin-Layer Chromatography (HPTLC) methodology for the precise determination of lupeol content in several extracts derived from the calyx of Hibiscus sabdariffa Linn extracts included ethanol, ethyl acetate, and aqueous extracts. The samples underwent analysis on a thin-layer chromatography (TLC) aluminium precoated plate (60 F254) using a mobile phase composed of toluene and methanol in a volumetric ratio of 8:2. The plate was subjected to derivatization using the Anisaldehyde – Sulphuric Acid reagent, followed by scanning at a wavelength of 550 nm. The procedure of validating the approach included the use of standards set out by the International Council of Harmonisation (ICH). The evaluation of several criteria, such as linearity, precision, accuracy, and robustness, was included by these recommendations. Lupeol was identified in the calyx ethanol extract at a concentration of 0.52% w/w, the ethyl acetate extract at a concentration of 0.47% w/w, and the aqueous extract at a concentration of 0.16% w/w. The ethanol extract exhibited a significant concentration of lupeol, above the levels reported in the other two extracts. As a result, the ethanolic extract was chosen for further validation criteria. The findings of the investigation demonstrated a direct correlation between the concentration of lupeol and the matching band intensity, spanning a range of 20 to 120 ng/band. The obtained correlation coefficient (r²) value of 0.9959 suggests a robust positive relationship between the two variables. The developed technique exhibited a notable degree of precision, as seen by the relatively low values of the interday and intraday precision, with %RSD values of 1.33% and 1.20% respectively. The efficacy of the methodology was assessed by a series of recovery studies carried out at three distinct concentration levels, namely 80%, 100%, and 120%. The findings of the study revealed that the accuracy rates for lupeol were assessed to be 99.93%, 99.36%, and 99.58% respectively. The method used for the validation and quantification of lupeol in Hibiscus sabdariffa Linn was found to be simple, precise, accurate, and robust.