Method Development and Validation of HPTLC Method for Simultaneous Estimation of Atropine, Piperine and Vasicine in <i>Ayurvedic</i> Formulation

Background: Dhumayoga, an Ayurvedic formulation is used for the management of respiratory disorders, including dyspnea and asthma. Aim: To develop and validate a High-Performance Thin-Layer Chromatography (HPTLC) method for the simultaneous estimation of atropine, vaccine and piperine in Dhumayoga Ayurvedic formulation. Methods: The three biomarkers were resolved using precoated silica gel 60F254 as stationary phase, dioxane: toluene: methanol: ethyl acetate: Aqueous ammonia (4:7:3:5:1v/v/v/v/v) as mobile phase and detected and quantified at 254nm. Results: The Rf values of atropine, vasicine, and piperine were found to be 0.406 ± 0.02, 0.56 ± 0.02, 0.87 ± 0.02, respectively. The calibration plot was linear in the range of 500-2500ng per zone with a correlation coefficient (r2) of atropine, vasicine and piperine was 0.994, 0.9986 and 0.9935, respectively. Conclusion: The developed method was applied for the quantitation of biomarkers in Dhumayoga. The method was validated in terms of linearity, precision, specificity, accuracy and robustness as per International Council for Harmonisation (ICH) guidelines.