Method Development and Validation of HPTLC Method for Simultaneous Estimation of Piperine, Bergapten, Plumbagin and Lupeol in <i>Chitrakadi Vati</i>

Background: Chitrakadi Vati is an Ayurvedic formulation containing several phytoconstituents. A reliable method for simultaneous quantification of its key components is essential for quality control and standardisation. Aim: To develop and validate a High-Performance Thin-Layer Chromatography (HPTLC) method for the simultaneous determination of four key phytoconstituents in Chitrakadi Vati: Piperine, Bergapten, Plumbagin and Lupeol. Methods: An HPTLC method was developed using silica gel 60F254 plates with a mobile phase of Petroleum ether: Ethyl acetate: Toluene: Formic acid (7:2:1:0.1 v/v/v/v). The method was validated for linearity, accuracy, precision, specificity, robustness, Limit of Detection (LOD) and Limit of Quantitation (LOQ). Robustness was further evaluated using a 2³ factorial design, examining the effects of mobile phase volume, saturation time, and solvent front on retention factors. Results: The method demonstrated good linearity (R² = 0.9972-0.9989), precision (%RSD <2%), accuracy (97.68-102.80 % recovery) and specificity. LOD and LOQ ranged from 100.8-325 ng/band and 305.5-985 ng/band, respectively. The factorial design revealed that mobile phase volume and solvent front significantly impacted retention factors, while saturation time had minimal effect. Conclusion: The developed and validated HPTLC method proved effective for the simultaneous estimation of Piperine, Bergapten, Plumbagin and Lupeol in Chitrakadi Vati. The comprehensive validation, including the Design of Experiments for robustness evaluation, ensures the method’s reliability for quality control applications.