Simultaneous estimation of trans-resveratrol and ε-viniferin in “darakchasava ”by binary gradient reverse phase high performance liquid chromatography

A simple, economic, selective, precise, and stability-indicating HPLC method has been developed and validated for analysis of trans-resveratrol and ε-viniferin, old age Indian “Ayurveda ”formulation “darakchasava”. Reversed-phase chromatography was performed on (LichroCART) ® 250 x 4 mm LiChrospher® (5um) RP-18 column with binary gradient eluent mobile phase (Solvent A, water with 0.0025%TFA and solvent B 80% acetonitrile with 20% solvent A) as mobile phase at a flow rate of 1.00 mL min⁻¹. Detection was performed on fluorescence detector at emission wavelength of 390 nm and exciting wavelength of 300nm and sharp peaks were obtained for trans-resveratrol and ε-viniferin at a retention time of 40 ± 0.01 min and 45± 0.01, respectively. Linear regression analysis data for the calibration plot showed there was a good linear relationship between response and concentration in the range 0.025μg/μl to 0.125μg/μl for trans-resveratrol and 0.0025μg/μl to 0.0125μg/μl for ε-viniferin; the regression coefficient was 0.9953 for trans-resveratrol and 0.9964 for ε-viniferin; the linear regression equation was Y=157051x + 800.2 for trans resveratrol and y = 28772x +330 for ε-viniferin. The detection (Limit of detection-LOD) limits for trans resveratrol and ε-viniferin were 0.0082 and 0.00082 μg μL respectively and the quantification (Limit of quantification-LOQ) limits for trans resveratrol and ε-viniferin were 0.249 μg μL.1 and 0.0025 μg μL⁻¹ respectively. The method was validated for accuracy, precision, specificity, reproducibility, recovery and detection and quantification limits, in accordance with ICH (International Conference on Harmonisation) guidelines. Statistical analysis proved the method was precise, reproducible, specific, and accurate for analysis of trans-resveratrol and ε-viniferin. The wide linearity range, sensitivity, accuracy, short retention time, and simple mobile phase imply the method is suitable for routine quantification of transresveratrol and ε-viniferin with high precision and accuracy.