Development and validation of high‐performance thin‐layer chromatography method for simultaneous estimation of α‐Amyrin, Betulin, and β‐Sitosterol in Leptadenia reticulata and Marsdenia tenacissima

Abstract High‐performance thin‐layer chromatography (HPTLC), a top‐notch technique in the field of analysis, is quite irreplaceable when it comes to identifying and standardizing medicinal plants . Marsdenia tenacissima and Leptadenia reticulata have claimed traditional uses like anti‐cancer, anti‐inflammatory, anti‐depressant, hepatoprotection, etc., with few similar phytoconstituents. In this study, a precise and validated HPTLC densitometric method was developed for the identification of α‐amyrin, β‐sitosterol, and betulin in the above‐mentioned plants by static maceration with ethanol and aqueous‐ethanol extracts. The developed method was evaluated by linearity, range, accuracy, precision, limit of detection, limit of quantification, robustness, and specificity. The markers were subjected to ascending development with toluene: ethyl acetate: glacial acetic acid at a ratio of 8:1.5:0.5 v/v/v, air‐dried, and derivatized with anisaldehyde‐sulfuric acid. The retardation factor values of the markers were found to be 0.63 (α‐amyrin), 0.52 (β‐sitosterol), and 0.64 (betulin) for L. reticulata , and 0.55 (α‐amyrin), 0.45 (β‐sitosterol), and 0.57 (betulin) for M. tenacissima . The chromatographic results suggest that the developed method suits all three markers and can be simultaneously identified in both plants.