Microemulsion liquid chromatographic method for simultaneous separation and determination of five isoflavones in red clover

Green and efficient separation methods are gradually being loved, and microemulsion phase chromatography is used in many fields. This study used a microemulsion liquid chromatographic method for the simultaneous separation of five isoflavones (formononetin, ononin, calycosin, calycosin-7-glucoside, and biochanin A) in red clover within 10 minutes for the first time. Separation and analysis are performed at GS-120-5-C18-AP column with UV detection at 254 nm. The mobile phase was made up of 5% SDS(w/v), 7% n-butyl alcohol (w/v), 0.9% octane (w/v), 87.1% H2O (w/v), pH adjusted to 3.0 with 30 mM phosphoric acid. Under this optimal condition, the linear range of the calibration curve of the five isoflavones is 1–50 μg/ml and the correlation coefficients are greater than 0.9973. Limit of detection (0.18–0.4 μg/ml), limit of quantitation (0.41–0.97 μg/ml), accuracy (82.4–98.6%) and the intra and inter precision (RSD <3.3%, and <5.6%). Method developed in this study is simple, low cost, green, efficient and provides an optional method for the speedy isolation of red clover isoflavones in 10 minutes, and it is not necessary to set a complex gradient method.