Fast simultaneous determination of free and conjugated isoflavones in soy milk by UHPLC–UV

Abstract

Ultra high pressure liquid chromatography (UHPLC) has become a widely used technique in separation science as it can shorten analysis time while maintaining resolution and increasing sensitivity. Here we developed a UHPLC method with UV detection for the simultaneous determination of isoflavone aglycones (daidzein, glycitein and genistein), their corresponding glycoside forms (daidzin, glycitin and genistin), and acetyl and malonyl conjugate forms of glycosides (malonyldaidzin, malonylglycitin, malonylgenistin, acetyldaidzin, acetylglycitin, acetylgenistin) in soy milk. The method allowed reliable determinations of 12 isoflavones in less than 8 min of chromatographic elution. It provided a satisfactory linearity ranging from 0.1 to 5 mg/L, except for daidzin and genistin, for which it ranged from 0.1 to 10 mg/L. In addition, it showed acceptable chromatographic sensitivity, with a detection limit lower than 0.05 mg/L and a determination limit below 0.2 mg/L. The precision, in terms of relative standard deviation, was lower than 5% and the accuracy, as mean recovery, was greater than 95%. Total isoflavone content in commercial soy milk samples (n = 8) ranged from 1805.22 to 3195.59 mg/kg dw. Genistin and Daidzin were the major isoflavones in all samples. The proportion of aglycones, ß-glucosides, and acetyl and malonyl glucosides differed among the samples. ß-Glucosides were identified as the predominant forms.

The authors would like to thank the Direcció General de Recerca of the Generalitat de Catalunya (2008-00668 SGR) for their support and for a grant from the Ministerio de Educación y Ciencia (Spain) to the Ph.D., student Natalia Toro-Funes. This work was also supported by the Interministerial Commission for Science and Technology (CICYT) of the Ministerio de Educación y Ciencia (Spain) through the Project AGL-2008-05430-C02-02.