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Validated high-performance thin-layer chromatographic—densitometric method for the isolation and standardization of ayapanin in Ayapana triplinervis

A simple, rapid, quantitative, and validated high-performance thin-layer chromatographic (HPTLC) method has been developed for the determination of ayapanin in the extract of Ayapana triplinervis leaves. Densitometric analysis of ayapanin was carried out in the absorbance mode at 254 nm. The method gave spot at RF = 0.56, corresponding to ayapanin. The limit of detection (54.32 ng) and limit of quantification (168.85 ng) per spot were confirmed with the mobile phase petroleum ether and ethyl acetate in a ratio of 60:40 v/v. Linear regression analysis data for the calibration plot for ayapanin showed good linear relationship with a correlation coefficient (r) of 0.997 in the concentration range of 200 ng to 1000 ng per spot. The method was validated for sensitivity, linearity, accuracy, precision, and specificity as per the International Conference on Harmonization (ICH) guidelines. After development and validation, the method was applied for the isolation of ayapanin from the extract. The isolated ayapanin was characterized by Fourier-transform infrared, nuclear magnetic resonance, and mass spectroscopy techniques.