Development and Validation of a Simple RP-HPLC Method for Simultaneous Estimation of Three Therapeutically Active Compounds in a Polyherbal Formulation: Habb-e-Irqun-Nisa

Habb-e-Irqun-Nisa (HINa) is a well-documented poly-herbal formulation in Indian System of Medicine (Unani System of Medicine). It is extensively used to treat arthritis, sciatica and gout. A rapid, simple, sensitive, robust, economic and improved RP-HPLC coupled with dual λ absorbance-detector method was developed and validated for estimation of three therapeutically bioactive marker compounds, namely gallic acid, aloin and colchicine in the poly-herbal preparation, HINa. The chromatographic separation was achieved in less than 25 min by RP-HPLC (C18 column, 5.0 µm, 4.6 mm x 250 mm, Waters,) at a column temperature of 30 °C with gradient elution using acetonitrile and water (20 mM ammonium acetate) with a flow rate of 1.0 mL/min and UV-detector at λ_max 287nm for 0 – 7 minutes and 360nm for 8 – 25 minutes. The results were achieved with respect to repeatability (RSD < 3.4%) and recovery (98.1 – 100.8%). The method was validated for linearity, accuracy, repeatability, LOD, and LOQ. The robustness of this method was also evaluated on the small fluctuations of pH in the mobile phase, the mobile phase compositions, temperature of column and the flow rate. The results indicated that the developed analytical RP-HPLC method could be a useful tool for quality control and standardization of HINa. Acknowledgements: The financial support from Central Council for Research in Unani Medicine (CCRUM), Department of AYUSH, Ministry of Health and Family Welfare, Government of India for this work is greatly acknowledged. The Project Fellowships awarded under UGC-SAP and Department of AYUSH to A. Ali is gratefully acknowledged. The research facilities developed from UGC-SAP (DRS-1) grant sanctioned to the Department of Biotechnology and used in this study are also thankfully acknowledged.