Assessment of RP-HPLC Method Development and Validation for Simultaneous Estimation of Thymoquinone and Curcumin in Dosage Form

Abstract

Aim: The objectives of this work is to develop and validate a simple, specific, accurate, and precise HPLC method for simultaneously estimating Curcumin (CUR) and Thymoquinone (THQ) in bulk and formulation, in accordance with ICH principles for analytical method development and validation. Study Design: Developing an RP-HPLC method for estimating Thymoquinone and Curcumin in bulk and formulation utilizing a C-18 Inertsil column and optimizing variables. Place and Duration of Study: The current study was conducted at Shri D. D. Vispute College of Pharmacy and Research Center, Panvel in year 2021. Methodology: The RP-HPLC method was developed using an isocratic mobile phase consisting of acetonitrile and water in a ratio of (82:18) v/v, at a flow rate of 0.9 mL/minute over Inertsil ODS, 250× 4.6 mm, 5 µm column, at 30°C column oven temperature. Detection was done using a photodiode array at 256 nm. Results: Curcumin and thymoquinone had retention times of 3.5 and 4.3 minutes, respectively. For thymoquinone and curcumin, the method demonstrated excellent linear response in concentration ranges of 4-18 \(\mu \mathrm{g} / \mathrm{ml}\) and 10-45 \(\mu \mathrm{g} / \mathrm{ml}\), respectively, with correlation coefficient (R2) values of 0.999 for both. System precision and method precision studies were less than the maximum allowable limit percentage of relative standard deviation \(\leq 2.0 \text { i.e., } 1.61 \%\) and 1.62 % for curcumin and 0.47 % and 0.42 % for thymoquinone respectively. Both medications' mean percent recovery was within acceptable ranges. Because all of the results satisfied the acceptance criteria, the designed and validated HPLC method is simple, accurate, precise, and acceptable for analysis. Conclusion: The developed RP-HPLC method at single wavelength was verified according to ICH guidelines for system appropriateness, specificity, linearity, accuracy, precision, and robustness, and can be used for routine quality monitoring of drugs in pharmaceutical dosage forms. This method can be employed for routine quality control analysis of curcumin and thymoquinone samples simultaneously.