Development and Validation of a Novel, Rapid Gradient HPLC Method for Simultaneous Estimation of Bioactive Marker Compounds in a Mixture of Convolvulus pluricaulis, Withania somnifera and Bacopa monnieri Extracts.

Abstract

The use of herbs as medicine is an ancient form of healthcare known to mankind. Standardization of herbal medicines is however a challenging task and is the major bottleneck in their acceptance as the primary therapeutic option. The aim of this study was to develop and validate a simple, rapid HPLC method for standardizing the mixture of extracts of three Medhya Rasayanas (neurotonic), Convolvulus pluricaulis, Withania somnifera and Bacopa monnieri. Simultaneous estimation of the respective bioactive markers of these plants viz., scopoletin, withaferin A, bacoside A 3, bacopaside II, jujubogenin and bacosaponin C has been reported for the first time. The method was developed using Waters Hybrid X-Bridge shield with BEH technology 2.5μm, 4.6 × 75mm column and validated according to ICH guidelines. The 20minutes run time makes the method eco-friendly. The method was linear over a range of 12.5-400ng/10μL for scopoletin and 62.5-2,000ng/10μL for withaferin A, bacoside A 3, bacopaside II, jujubogenin and bacosaponin C with detection limits of 8.0, 48.3, 30.4, 40.7, 15.6 and 18.9ng/10μL and quantification limits of 24.5, 146.5, 92.2, 123.4, 47.4 and 57.4ng/10μL, respectively. The correlation coefficient for each analyte was >0.999. The intra-day and inter-day precision was <2%. These results confirmed the precision, accuracy and robustness of the proposed method.