Evaluation of Isoflavone Aglycon and Glycoside Distribution in Soy Plants and Soybeans by Fast Column High-Performance Liquid Chromatography Coupled with a Diode-Array Detector

An ultrafast HPLC/UV−vis DAD method working at 254 nm was applied for the determination of isoflavone aglycons and glycosides (genistin, genistein, daidzein, daidzin, glycitin, glycitein, ononin, formononetin, sissotrin, and biochanin A) in roots, stems, leaves, and soy pods of soy plants and in soybeans of five varieties (Korada, Quito, Rita, OAC Erin, and OAC Vison). An Atlantis dC18 ultrafast RP chromatographic column (20 mm × 2.1 mm, 3 μm particle size) was applied for separation of the isoflavone aglycons and glycosides. A flow rate of the mobile phase (0.1% (v/v) acetic acid, pH 3.75solvent A and methanolsolvent B) was 0.35 mL min^-1, and the column temperature was 36 °C. A linear gradient profile from 13 up to 22% B (v/v) from zero to 2.5 min, up to 30% B to 3.21 min, up to 35% B to 4 min, up to 40% B to 4.5 min, up to 50% B to 5.14 min, and followed by negative gradient up to 13% B to 7.71 min was used. The absolute limits of detection per sample injection (5 μL) were the highest for biochanin A (166.2 fmol) and the lowest for genistin (17.0 fmol), respectively. An accelerated solvent extraction (ASE) in combination with sonication was applied for isolation of biologically active compounds. A solid-phase extraction procedure was used to purify the extracts in the case of analysis of soy plants parts. The recoveries of 96−106% were obtained for the different concentrations of the isoflavone aglycons and glycosides and the different matrixes (overall RSDs 2−9%). The highest isoflavone concentrations were found in roots (12.5 μg g^-1 dry weight), while the amounts were about 3−1100 μg g^-1 fresh weight in different varieties of soybeans.

Keywords: Isoflavones; liquid chromatography; UV−vis DAD; accelerated solvent extraction; ultrasonic extraction; solid-phase extraction; soy plants; roots; leaves; stems; pods