(T0930-09-60) Development, Optimization and Validation of UPLC Method for Determination of Flavonoids from Seed Extracts of Elite Pongamia pinnata

Purpose: Present study reports design of experiment (DoE) based development and validation of a simple, rapid and sensitive ultra performance liquid chromatography (UPLC) method for estimation of Pongamol and Karanjin levels in seed extracts of Elite Pogamia Pinnata.

Methods: Plackett-Burman design, using Minitab statistical software was employed to screen the critical method variables (CMVs) for the UPLC method.¹ DoE was applied to optimize the screened CMVs, flow rate, diluent, injection volume which affect the analytical responses (AR’s) resolution and tailing factor². Acquity UPLC, system with PDA detector was used to perform the trials.

Results: The optimized chromatographic parameters were acetonitrile proportion in mobile phase: 60-75% v/v, pH of buffer-1.5 and flow rate: 0.500 mL/min. Use of these optimized parameters resulted in overall run time of 15min with component retention time of 1.462 min (K) and 3.037min(P), tailing factor < 2 and improved resolution between the separated component peaks of the UPLC method.

Conclusion: The finalised method was capable of the successful separation and estimation of Pongamol and Karanjin levels in seed extracts of Elite Pogamia Pinnata . The study depicts successful development, optimization and validation of a simple UPLC method of Pongamol and Karanjin using DoE approach.

References: 1.Paramita Saha MMP. Design of Experiment (DoE)-Approach Based RP-HPLC Analytical Method Development and Validation for Estimation of Efavirenz in Bulk and Formulations Journal of Chromatographic Science , 2022;60(1), doi:https://doi.org/10.1093/chromsci/bmab029
2. Sadhana P Mutalik PM, Abhijeet Pandey, Smita S Kulkarni, Srinivas Mutalik.
Box-Behnken design aided optimization and validation of developed reverse phase HPLC analytical method for simultaneous quantification of dolutegravir sodium and lamivudine co-loaded in nano-liposomes. Journal of Seperation Science 2021, doi:https://doi.org/10.1002/jssc.202100152

Acknowledgements: I would like to acknowledge University of Bradford, for providing this opportunity and support for the smooth conduct of the research. I would like to extend by gratitude to Phyla earth Limited, for providing the support and their contribution to the research

Fig: Plackett-Burman design for the screening of critical method variables.

Fig: Chromatogram of seed extract indicating the separation of Karanjin(1.462min), Pongamol (3.037min).

Fig: quantitative estimation of falvionids from seed extract, hot oil and cold pressed oil samples.