Liquid Chromatographic Validation of a Quantitation Method for Phytoestrogens, Biochanin‐A, Coumestrol, Daidzein, Formononetin, and Genistein, in Lucerne

Abstract

A methodology was validated to evaluate biochanin‐A, coumestrol, daidzein, formononetin, and genistein in lucerne (Medicago sativa). Extraction done through acid hydrolysis at 80°C, followed by extraction with HLB cartridges. A C₁₈ column (4.6×250 mm) with a 0.8 mL/min flow of methanol solution (isocratic) and detection at 260 nm was applied. Separation factors were greater than 1.1 and resolutions were superior to 2.4. Linearity was established and correlation coefficients of responses were greater than 0.995. The detection and quantification limits were between 0.12–0.17 µg · mL⁻¹ and 0.37–0.53 µg · mL⁻¹, respectively. The coefficients of variation were between 5 and 15%. The accurate results in lucerne were 78, 35, 20, 14, and 11 mg (kgDM)⁻¹ for coumestrol, formononetin, daidzein, genistein, and biochanin‐A.

Keywords:

• Lucerne
• Isoflavones
• Coumestrol
• LC
• Validation
• Quantification

Acknowledgments

This work was supported financially by the Portuguese programme PRODEP III, Medida 5/Acção 5.3 Concurso 4/5.3 de 2000.