Effect of sample pre-treatment on isoflavones quantification in soybean

High performance liquid chromatography is the most common technique for analysing isoflavones in soy; however, the appropriate sample pre-treatment is required, including extraction and, often, hydrolysis. In the present work, the kinetics of extraction was investigated with regard to the number of extraction steps and their duration for the exhaustive extraction of bonded forms of isoflavones and aglycones. In addition, a systematic experimental study of hydrolysis depending on the temperature and acidity of the medium was conducted. The results showed that the assisted methods were more effective for the isolation of isoflavones. Moreover, ^the differences between the results obtained using ultrasound-assisted extraction (UAE), microwave-assisted extraction and pressurised liquid extraction were not statistically significant; however, the appropriate number of extraction steps and their duration for UAE were required. The investigation also revealed that, due to the varying susceptibility of glycosides on conversion to free forms and the stability of the aglycones obtained, two parallel hydrolysis procedures should be conducted to obtain the highest hydrolysis efficiency. The optimised conditions, taking into account the shortest time combined with the highest efficiency, were as follows: 4 M HCl and 60 min for genistein; 8 M HCl and 30 min for glycitein and daidzein.