32 Factorial Design for Optimization of HPLC-UV Method for Quantification of Gallic acid in Lohasava and Pippalyasava

Abstract:

Background: A new, simple, accurate and precise High Performance Liquid Chromatographic method (HPLC-UV) has been developed and validated in accordance with ICH guidelines for the determination of gallic acid in Lohasava and Pippalyasava. The developed method was optimised using 3² full factorial design by evaluating the effect of two independent variables i.e. mobile phase composition and flow rate on the various chromatographic responses such as retention time, area, number of theoretical plates and tailing factor. Materials and Methods: Chromatographic separation was achieved using Sun Chrome C₁₈ column (250 mmX4.6 mm, 5μm) as stationary phase. The optimised mobile phase consisted of 0.05% o-phosphoric acid: Acetonitrile (93:7,v/v) and flow rate was set at 1.5 ml/min with detection at 270 nm. Results: The retention time of gallic acid was found to be 3.71 min. The Limit of Detection (LOD) and Limit of Quantification (LOQ) were found to be 0.0487 µg/ml and 0.1478 µg/ml; respectively. The proposed method was found to be linear in the concentration range of 0.15-6 µg/ml for gallic acid with correlation coefficient of 0.9955. The proposed method was found to be accurate with 99.43%-100.81% recovery. The % RSD for intraday and interday precision was found to be less than 2.0 indicating that the proposed method was precise. The method was found to be robust with respect to % RSD and % w/w of gallic acid. The amount of gallic acid in Lohasava and Pippalyasava was found to be 3.00% and 0.98%; respectively. Conclusion: Factorial design assisted HPLC method was developed and validated for quantitative determination of gallic acid in two commercial formulations of asava. This method can be used for routine quality control of asava using gallic acid as marker compound.

Key words: Gallic acid, Factorial design, Method validation, HPLC-UV, Quality by Design.