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Acta Agron Sin ›› 2010, Vol. 36 ›› Issue (12): 2062-2072.doi: 10.3724/SP.J.1006.2010.02062


Establishment of A Rapid HPLC Method for Quantifying Isoflavone Components and Its Application in Tofu Processing

WANG Chun-E,ZHAO Tuan-Jie,GAI Jun-Yi   

  1. 1 National Key Laboratory for Crop Genetics and Germplasm Enhancement / Soybean Research Institute, Nanjing Agricultural University / National Center for Soybean Improvement, Nanjing 210095, China; 2 College of Life Science, Jiujiang University, Jiujiang 332000, China
  • Received:2010-03-04 Revised:2010-08-04 Online:2010-12-12 Published:2010-10-09
  • Contact: GAI Jun-Yi, E-mail: sri@njau.edu.cn, Tel: 025-84395405

Abstract: A rapid, precise, and stable quantifying method of isoflavone components is the key to quality soy–food processing and genetic improvement of quality soybeans. A quick procedure used Agilent 1100 high performance liquid chromatograph (HPLC) system with the diode array detection (DAD) and Zorbax SB-C18 packed column (5 μm, 4.6 ID × 150 mm) using external standards of isoflavone components, including daidzein,daidzin, 6”-O-acetyldaidzin, 6”-O-malonyldaidzin,genistein, genistin,6”-O-acetylgenistin, 6”-O-malonylgenistin, glycitein, glycitin,6”-O-acetylglycitin and 6”-O-malonylglycitin was established for measuring the 12 isoflavones in soybean seeds and its processing products. The procedure includes the following key points: 80% methanol aqueous solution under ultrasonication for 1 h at 50℃ was chosen; for separation of the 12 isoflavone components within 15 min, the mobile phase of 0.1% acetic acid (V/V) aqueous solvent A and 100% methanol solvent B with the flow rate of 2.0 mL min–1, injection volume at 10 μL, column temperature at 36℃ and detection wavelength at 254 nm were selected; and the linear gradient extraction of 0–2 min, 27% B (V/V)→2~3 min, 27~38% B→3–10 min, 38% B→10–12 min, 38–39% B→12–14 min, 39% B→14–15 min, 39%–27% B was adopted. The procedure was linear (R2 = 0.9976–0.9999), precise (CV or RSD ranged from 0.90% to 3.35% for 2 232 samples from NJRIKY), accurate [recoveries were more than 99.00% for the different concentrations of the 12 isoflavones (CV of 0.22%–1.40%)], robust (inter-day CV of 0.24%–3.95%) and rapid (less than 15 min for 12 isoflavones resolved). The procedure was verified to be effective by a large sample determination of isoflavone components in soybean seed, soymilk and tofu using the soybean population of NJRIKY. The data indicated that the isoflavones were 3 695.00 μg g–1 in seed, among them 14.85% (548.88 μg g–1) were transferred to the residual, 85.15% (3 146.12 μg g–1) to soymilk, but only 17.32% (639.89 μg g–1) to tofu while 67.83% (2 506.23 μg g–1) to whey under the traditional tofu processing with CaSO4 as coagulant. The 12 isoflavone components in soymilk was somewhat less than those in seeds with 6”-O-Malonylgenistin the highest in the both, while in tofu 6”-O-Acetylgenistin and 6”-O-Acetylglycitin were deficient but with high contents of genistein and daidzein. In addition, there was an enlarged genetic variation of the 12 isoflavone components among the lines, indicating the increased genetic potential for quality improvement due to recombination between Kefeng 1 and Nannong 1138-2.

Key words: Soybean, Isoflavone component, High performance liquid chromatograph (HPLC), Gradient elution, Tofu, Soymilk

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