Determination of isoflavones in dietary supplements containing soy, Red Clover and kudzu: extraction followed by basic or acid hydrolysis

J Chromatogr A. 2006 Feb 24;1107(1-2):59-69. doi: 10.1016/j.chroma.2005.11.060. Epub 2006 Jan 18.


Isoflavones are phytochemicals found in many plants. Because of their structural similarity to beta-estradiol, health benefits of isoflavones have been evaluated in age-related and hormone-dependent diseases. Dietary supplement preparations contain extracts from soy, Red Clover and kudzu. Soy products contain primarily genistein, daidzein, and glycitein, while Red Clover products contain primarily formononetin and biochanin A. Kudzu extracts contain puerarin and daidzein among other components. Previous methods of analysis focused on the determination of isoflavones from a single botanical source, while dietary supplements are often a blend of extracts from different plants. We developed a method for the analysis of isoflavones in dietary supplements regardless of their botanical composition, using HPLC-PDA because of its applicability to routine analysis. Isoflavones are found as free compounds, glucoside derivatives, 6''-O-malonyl-beta-d-glucoside and 6''-O-acetyl-beta-d-glucoside derivatives. In this study, the samples were extracted at room temperature with 50:50 (v/v) MeCN/water, and then analyzed before and after hydrolyzing the isoflavones by acid or basic digestion. 2'-Methoxy-flavone and 6-methoxy-flavone were used as internal standards and were added together to every sample. Daidzein, glycitein, genistein, puerarin, calycosin, pratensein, pseudobaptigenin, formononetin, biochanin A and prunetin were among the isoflavones determined.

MeSH terms

  • Acids
  • Alkalies
  • Calibration
  • Chromatography, High Pressure Liquid / methods
  • Dietary Supplements / analysis*
  • Hydrolysis
  • Isoflavones / analysis*
  • Mass Spectrometry / methods
  • Pueraria / chemistry*
  • Reference Standards
  • Soybeans / chemistry*
  • Spectrophotometry, Ultraviolet
  • Trifolium / chemistry*


  • Acids
  • Alkalies
  • Isoflavones