Analysis of Isoflavones

Significance of the Topic

Isoflavones are plant derived compounds with notable biological activities including antioxidant effects and hormone modulation. Accurate analysis of these molecules is essential for quality control of soybean products and dietary supplements, as well as for research into their health benefits.

Objectives and Study Overview

The goal of this application note is to establish a robust reversed phase liquid chromatography method for simultaneous separation and quantification of eight key isoflavones. Target analytes include glycoside forms such as daidzin and genistin, their malonyl derivatives, and corresponding aglycones.

Methodology and Instrumentation

A gradient elution program was developed using aqueous acetic acid (0.1 percent) and acetonitrile with 0.1 percent acetic acid. The gradient ramped mobile phase B from 15 percent to 35 percent over 20 minutes, followed by a high organic wash and reconditioning. Separation was achieved at 1.0 mL per minute, 40 °C column temperature, and UV detection at 254 nm.

Instrumentation Used

• Liquid chromatograph: Nexera X3 system
• Column: Shim-pack GIST-HP C18 (150 mm × 4.6 mm I.D., 3 μm)
• Detection: UV detector set at 254 nm

Main Findings and Discussion

The optimized method provided baseline resolution of eight compounds within a 30 minute runtime. Representative retention order was daidzin, malonyl daidzin, malonyl glycitin, genistin, malonyl genistin, daidzein, glycitein, and genistein. Peak shapes were sharp and symmetric, with consistent retention times and no carryover after high organic wash.

Benefits and Practical Applications

• High throughput screening of soybean extracts and supplement formulations
• Reliable quantification of glycoside and aglycone isoflavones for regulatory compliance
• Applicability to research laboratories investigating isoflavone bioactivity

Future Trends and Potential Applications

Advancements may include coupling this method with mass spectrometry for enhanced sensitivity and selectivity, miniaturized column formats to reduce solvent consumption, and use of greener mobile phase modifiers to decrease environmental impact.

Conclusion

The presented reversed phase LC method using Shim-pack GIST-HP C18 and Nexera X3 offers a fast, reliable, and reproducible approach for comprehensive isoflavone analysis. Its robustness and clarity of separation make it valuable for both industrial and research settings.

Reference

Shimadzu Corporation. Application News 01-00165. First Edition December 2021.