A new, simple, reliable, and validated high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous quantitation of four bioactive markers, ursolic acid (1), betulinic acid (2), β-sitosterol (3), and lupeol (4) in the stem and root barks of Alstonia scholaris. Extraction efficiency of the targeted markers from the bark matrixes with organic solvents using cold percolation, hot extraction, and ultrasonic extraction were studied, which showed that ultrasonic extraction was best for sample preparation. The separation was achieved on silica gel 60F254 HPTLC plates using chloroform-methanol (99:1 v/v) as mobile phase. The quantitation of four markers was carried out using the densitometric reflection/absorption mode at 680 nm after post chromatographic derivatization using vanillin-sulphuric acid reagent. The linear regression analysis data for the calibration plots showed a good linear relationship (r2 ≥ 0.998) in the concentration range 2–10 μg per spot for the ursolic acid and lupeol and 4–20 μg per spot for the betulinic acid and β-sitosterol with respect to area. The method was validated for peak purity, precision, accuracy, robustness, limit of detection (LOD), and quantitation (LOQ). Method specificity was confirmed using retention factor (RF) and visible spectral (post chromatographic scan) correlation of marker compounds in the samples and standard tracks.
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