Quantification of Four Isoflavones in Forages with UPLC®-MS/MS,Using the Box–Behnken Experimental Design to Optimize Sample Preparation

A performant method for the simultaneous quantification of daidzein, genistein, formononetin, and biochanin A in forages using an UPLC^®-MS/MS was developed and fully validated. The ultrasound-assisted extraction and enzymatic hydrolysis used in the sample preparation step were optimized using the Box–Behnken experimental design. The optimal extraction conditions used for a representative mix of forage plants were 80 °C, 10 min, and 55 % methanol, and for hydrolysis, they were 20 °C, 18 h, and pH = 6. The chromatographic separation was achieved using an Acquity UPLC^® HSS T3 column, with a water/methanol linear gradient containing 0.01 % of formic acid at a 0.55 mL min⁻¹ flow rate. The four isoflavones were detected by ESI mass spectrometry in positive ion MRM mode. The method allows high throughput analyses of samples and showed an adequate linear regression model for all isoflavones over a range from 5 to 125 ng mL⁻¹. There were good intra- and inter-day precisions (≤8.2 and ≤7.6 %) and accuracy (≤11.4 and ≤7.1 %). The recovery rates were in an acceptable range of 70–120 %, except for biochanin A, where the rate was about 50 %. Good method repeatability was also observed, and there was no matrix effect or carryover problem. The sample extracts were stable for at least 6 days of storage at -21 and 6 °C. The method proved to be sensitive, precise, and accurate for discriminating a wide variety of forages likely to be grazed by ruminants according to their isoflavone contents and to observe the impact of storage process on isoflavone content in forages.