Development and validation of HPTLC Method For Quantification of Betanin from Peel and Pulp of Hylocereus Polyrhizus Extract

Abstract

This study details the development of validated  a TLC-densitometry method to measure betanin in extracts of Hylocereus spp. (dragon fruit). Chromatographic separation occurred on TLC aluminum plates coated with silica gel 60 F₂₅₄. A mobile phase system was optimized for good resolution of betanin. The method produced sharp, well-defined spots with an Rf value specific to betanin, ensuring clear separation from other components. Densitometric detection was done at a visible wavelength suitable for betanin, in reflection or absorbance mode, allowing for reliable measurement. The method showed good linearity across a concentration range of 10–50 µg/ml, with a high correlation coefficient (R² ≥ 0.99). The limit of detection (LOD) and limit of quantification (LOQ) were at acceptable sensitivity levels, making the method suitable for detecting low amounts of betanin in plant samples. The validated method showed satisfactory accuracy, precision, repeatability, and specificity, in line with ICH guidelines. The measured amount of betanin in the fruit extract was 0.6 ± 0.0032 gm/100 gm, indicating its notable presence as a bioactive compound. In conclusion, the TLC-densitometric method is simple, quick, cost-effective, and reliable for measuring betanin in Hylocereus spp. It can be effectively used for routine quality control, standardization, and screening of betanin in herbal raw materials and products.