Development, optimisation and validation of a stability-indicating HPLC method of achyrobichalcone quantification using experimental designs.

Introduction: Achyrobichalcone is a new biflavonoid found in Achyrocline satureioides. It is structurally similar to other bioactive bichalcones that were proven to exert anti-cancer activity. Recently we isolated several achyrobichalcone batches on a semi-preparative scale, showing the need to assess the quality and stability of this substance by analytical methods.

Objective: To develop and validate a stability-indicating HPLC method of achyrobichalcone quantification using experimental designs.

Method: The method was developed and optimised by Box-Behnken design using column temperature, flow rate and acetonitrile content in the mobile phase as factors and system suitability parameters as responses. Validation parameters were determined according to official compendiums. Robustness was determined by Plackett-Burman design. Stability of achyrobichalcone was assessed in alkaline, acid, oxidative, thermal and photolytic stress conditions.

Results: The ideal chromatographic conditions were defined from the optimisation: 37 % of acetonitrile, flow rate of 1.2 mL/min and 33°C temperature. All factors were significant for the resolution between achyrobichalcone and impurities peaks and for the retention factor. The mathematical model developed exhibited a good predictive capacity, and the design proved suitable. The HPLC method was successfully validated, being linear, specific, accurate and precise. The robustness test revealed that the flow rate and detection wavelength should be strictly controlled, as they affect achyrobichalcone concentration. The analyte was unstable only in alkaline media.

Conclusion: The new method developed affords evaluation of the quality of achyrobichalcone obtained by isolation, and indicates the stability of the molecule under various stress conditions.