Development and Validation of High-Performance Thin Layer Chromatography Method for Simultaneous Estimation of Catechin, Bergenin and Diosgenin in <i>Varunadi Kvatha Churna</i>

Background: Varunadi Kvatha Churna, an Ayurvedic formulation, is renowned for its efficacy in managing urinary disorders due to the synergistic actions of herbs like Pashanbhed, Varuna, and Gokshura. Aim: This study aims to develop and validate a High-Performance Thin-Layer Chromatography (HPTLC) method for the simultaneous estimation of Catechin, Bergenin, and Diosgenin in Varunadi Kvatha Churna. Methods: The study utilized analytical grade organic solvents and standards of Catechin, Bergenin, and Diosgenin. A marketed formulation of Varunadi Kvatha Churna was procured, and sample solutions were prepared. HPTLC analysis was conducted using a CAMAG system with specific chromatographic conditions and vision CAT Software. Result: The optimal mobile phase was determined to be toluene: ethyl acetate: glacial acetic acid: methanol (7:4:0.5:1v/v/v/v) and Silica gel G60F254 in the stationary phase. Using the absorbance reflectance mode, quantification was done at 494 nm for Catechin, 277 nm for Bergenin, and 449 nm for Diosgenin. It found that the Rf values of Catechin, Bergenin, and Diosgenin were, respectively, 0.31, 0.18, and 0.73. The method exhibited good linearity in the range of 2000–10,000 ng/band for concentration. The LOD was found to be 168, 219.1, and 63.5 ng/band of Catechin, Bergenin, and Diosgenin. Catechin, Bergenin, and Diosgenin were found to have LOQs of 509.1, 654.6, and 192.4 ng/band, respectively. The method exhibited good precision, accuracy, system suitability, and specificity. The robust study confirmed the method’s stability under varying conditions. Conclusion: The developed HPTLC method for simultaneous estimation of Catechin, Bergenin, and Diosgenin was found to be simple, linear, accurate and repeatable. The method can be used further for the estimation of biomarkers in crude drugs, herbal and Ayurvedic formulations.