Determination of Isoflavones in Soybean by LC/MS/MS

Significance of the topic

The analysis of soy isoflavones addresses both nutritional and agronomic concerns. Isoflavones like daidzein, genistein and others exhibit estrogenic activity in human and animal diets, contributing to health benefits and functional food development. In plants, these phytoestrogens influence insect resistance and yield, with implications for crop improvement and food security.

Objectives and study overview

This study aimed to develop and validate a rapid, sensitive LC/MS/MS method for simultaneous quantification of seven soy isoflavones (daidzein, genistein, glycitein, daidzin, genistin, glycitin and rutin) in soybean seed cultivars. The method emphasizes simple sample preparation, high throughput and robust performance in complex food matrices.

Methodology and instrumentation

Sample preparation involved grinding soy seeds into fine powder, extracting with methanol and acidic aqueous solution, sonication, filtration and rotary evaporation to concentrate analytes. The final extract was reconstituted in methanol.

Used instrumentation:

• Liquid Chromatography: Agilent 1290 Infinity II LC with ZORBAX Eclipse Plus C18 column (2.1×100 mm, 1.8 µm).
• Mobile phase: water/0.1 % formic acid (A) and acetonitrile (B) under a gradient from 90:10 to 10:90 over 7 min, flow rate 0.3 mL/min, column temperature 40 °C, injection volume 1 µL.
• Mass Spectrometry: Agilent 6470 triple quadrupole with electrospray ionization in positive MRM mode. Two transitions monitored per compound (quantifier and qualifier), capillary voltage 4 kV, gas temperatures 300 °C, sheath and drying gas flows 10 L/min, nebulizer pressure 20 psi.

Main results and discussion

Calibration curves showed excellent linearity (R²>0.998) over relevant concentration ranges, with limits of detection between 0.7–6.7 ppb and quantification from 2.3–22.5 ppb. Run-to-run precision was confirmed with RSDs below 4 % for standards. Application to two soybean cultivars (Silvania and insect-resistant Dowling) demonstrated reproducible quantification (RSD <10 %) and confirmed lower isoflavone levels in the resistant cultivar.

Benefits and practical applications

• Rapid analysis under nine minutes per sample accelerates screening workflows in food quality and research laboratories.
• Sensitive detection enables monitoring at trace levels, supporting regulatory compliance and nutritional labeling.
• Simple extraction and minimal sample cleanup facilitate routine implementation.

Future trends and applications

Advances may include integration with high-resolution mass spectrometry for broader phytoestrogen profiling, automated sample handling for high-throughput screening, and adoption of greener solvents in extraction. Coupling data with metabolomics and machine learning could reveal new bioactivity correlations and support breeding for enhanced nutritional and resistance traits.

Conclusion

The presented LC/MS/MS method provides a fast, sensitive and reliable tool for quantifying major isoflavones in soybean. Its high precision and low detection limits make it suitable for routine analysis in food chemistry laboratories, supporting nutritional research, crop improvement and quality control.

References

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