AQbD Assisted Method Optimization for Simultaneous Quantification of Potential Phytosterols in Novel Polyherbal Formulation by LC-PDA Method

Abstract

Background: Herbal medications are preferred by individuals with cardiovascular disorders (CVDs) worldwide for their distinct benefits in preventing and treating illnesses and lesser side effects. A novel polyherbal formulation compiled of ten plants (Allium sativum, Rubia cordifolia, Garcinia cambogia, Terminalia arjuna, Acacia catechu, Helianthus annus, Vitis vinifera, Commiphora mukul, Linum usitatissimum, and Piper nigrum) was developed for treating cardiovascular diseases. As a part of standardization, marker analysis using high-performance liquid chromatography (HPLC) will be highly encouraged. Materials and Methods: In the present study, we selected lupeol, stigmasterol, and β-sitosterol and went for simultaneous quantification using design of experiments (DoE) software (AQbD-Analytical quality by design) to optimize the experimental parameters. The phenomenex RP C18 column, with 250 mm length, 4.6 mm internal diameter, 5 μm particle size, was preferred for the study. Mobile phase with methanol and 0.01% formic acid solution in water surged with a flow rate of 1.5 mL/min. using a maximum detection wavelength of 208 nm. Results and Discussion: Under the specified circumstances, we determined the retention times of lupeol, stigmasterol, and β-sitosterol to be 14.056, 15.544, and 17.431 minutes, respectively, with the desirability of 0.975. The concentrations of lupeol, stigmasterol, and β-sitosterol in the extract were determined to be 30.72, 370.6, and 212.64 μg/10 mg, respectively. The linear range of 31.25 to 500 μg/mL was found to have an R2 value of 0.9972, 0.9976 and 0.9968, respectively. 0.2534, 0.754, 0.421 μg/mL and 0.7681, 2.29, 1.28 μg/mL were the limit of detection (LoD) and limit of quantitation (LoQ) values, respectively. The RSD for precision was determined to be 0.92, 0.98, and 0.96%, respectively. Recovery experiments were conducted, and the accuracy of the suggested approach was between 99 and 102%. Conclusion: Using the modernized software tool- AQbD (analytical quality by design), the study was first scientifically validated for the quantification of three phytomarkers in the novel polyherbal formulation.