Reversed Phase - HPLC Method Development and Validation for Simultaneous Estimation of Berberine Hydrochloride, Plumbagin, Conessine in <i>Ayurvedic</i> Formulation

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Background: The objective of this research was to create a dependable RP-HPLC analytical technique for concurrently determining the levels of conessine, berberine HCl, and plumbagin in an ayurvedic formulation. Aim: The main goals were to develop a straightforward, precise, and consistent method for the pharmaceutical analysis of these compounds. Methods: The analytical method was developed using a BDS hypersil C18 tertiary mode column with a mobile phase incorporating 1% perchloric acid in water: methanol: acetonitrile (10:40:65% v/v/v). The analysis utilized a 1 ml/min flow rate, and detection was performed at 212 nm utilizing a PDA detector, SPD-20 A. The retention times for conessine, berberine HCl, and plumbagin were determined to be 3.12 minutes, 6.60 minutes, and 13.64 minutes, respectively. Linearity assessments were conducted in the concentration range of 25–75 ppm for all three phytomarkers. The correlation coefficients for conessine, berberine HCl, and plumbagin were found to be 0.999, 0.999, and 0.997, respectively. Results: The developed RP-HPLC method exhibited excellent linearity for all three compounds. Limit of detection and limit of quantification values were determined, with conessine at 1.36 μg/ml and 1.78 μg/ml, berberine HCl at 3.52 μg/ml and 4.12 μg/ml, and plumbagin at 5.40 μg/ml and 10.67 μg/ml, respectively. Precision studies, including inter-day and intra-day, demonstrated RSD values less than 2.00%. Accuracy was assessed through standard recovery methods, revealing satisfactory recovery values for conessine (100.0%–99.8%), berberine HCl (101.0%–100.9%), and plumbagin (101.4%–99.4%). Conclusion: The developed RP-HPLC method offers a robust and precise tool for the simultaneous estimation of conessine, berberine HCl, and plumbagin in ayurvedic formulations. The method’s accuracy and reproducibility make it suitable for use as a standardization tool in the evaluation of formulations containing these marker compounds. This study contributes to the analytical methods available for quality control in the pharmaceutical industry, particularly in the context of ayurvedic formulations.