A New Simplified and Stability Indicating Liquid Chromatography Method for Routine Analysis of Isoflavones Aglycones in Different Complex Matrices

In this work, a stability indicating method for routine analysis of isoflavones in different matrices was developed and validated. In order to simplify the analytical method, the glycosides were previously hydrolyzed to the corresponding aglycone forms. The separation of all isoflavone aglycones was achieved in 23 min, with a total time of analysis of 30 min, using trifluoroacetic acid 0.1 % (v/v) and acetonitrile:trifluoroacetic acid (100:0.01, v/v) as mobile phase. The LC method specificity was evaluated by the analysis of isoflavone standards submitted to acidic, alkali, neutral, oxidative, and photolytic stress conditions. The isoflavones degraded in alkali at 60 °C or in alkali under ulraviolet C (UVC) radiation, forming, in both conditions, three degradation products D1, D2, and D3 which were analyzed by liquid chromatography mass spectroscopy (LC-MS/MS). The method showed linearity higher than 0.999 with the concentration ranged from 0.1 to 10 μg ml^-1 for all isoflavone aglycones. The limits of quantitation obtained using calibration curves were from 0.28 to 0.37 μg ml^-1 and the intermediary precision at three levels (2, 6, and 10 μg ml^-1) showed RSD values between 0.03 and 0.25 %. After the performed validation, the LC method was applied to compare the isoflavone aglycones content in three different matrices: Glycine max beans, Glycine max dry extract, and isoflavone aglycone loaded nanoemulsion. The repeatability data showed RSD values between 0.02 and 1.41 % and the intermediary precision at three levels showed RSD values between 0.05 and 1.99 %. The recovery data of the isoflavone aglycones standards in the matrices at three levels were between 90.74 and 106.43 %.