Fast Analysis of Isoflavones in Dietary Supplements – USP Method Transfer onto a UHPLC System

Significance of the topic

Isoflavones are plant-derived phytoestrogens found in soy, red clover, and Kudzu that are used to alleviate menopausal symptoms and are linked to health benefits such as reduced cancer risk and slowed neurodegeneration.
Accurate and rapid quantitation of isoflavones in dietary supplements is essential for quality control, regulatory compliance, and consumer safety.

Study objectives and overview

This study aimed to transfer the USP monograph method for isoflavone analysis from conventional HPLC to a UHPLC platform, reducing analysis time while retaining resolution and accuracy.
Key goals included developing an 18-minute UHPLC method, integrating mass detection for confident peak identification, and validating performance using NIST SRM 3238 and commercial supplement products.

Methodology

• Sample preparation followed USP protocols for defatted soy powder extract and commercial supplement samples; standards and samples were diluted in acetonitrile:water (2:1) with 4 ppm apigenin as internal standard.
• Chromatographic conditions used a Waters ACQUITY Arc UHPLC System with a CORTECS C18 column (2.7 µm, 3.0 × 100 mm); mobile phases were water + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B); flow rate 1.08 mL/min; injection 2 µL; column temperature 30 °C; 18 min gradient including wash and equilibration.
• Detection combined a 2998 photodiode array detector at 260 nm and an ACQUITY QDa mass detector in ESI+ mode (0.8 kV capillary voltage, 600 °C probe, 15 V cone); single ion recording masses were set for 12 isoflavones and apigenin.

Instrumentation

• Waters ACQUITY Arc UHPLC System
• Waters ACQUITY QDa Mass Detector
• Waters 2998 Photodiode Array Detector
• Waters CORTECS C18 Column (2.7 µm, 3.0 × 100 mm)
• Empower 3 Chromatography Data System

Main results and discussion

• Run time was reduced from 74 min to 18 min, yielding a 75% solvent savings and a threefold throughput increase.
• Gradient conditions were recalculated using Waters UPLC Column Calculator and mobile phase additive changed from phosphoric acid to MS-friendly formic acid.
• Mass detection enabled unambiguous assignment of acetyl and malonyl isoflavones without individual standards by monitoring specific SIR masses.
• Calibration curves for all analytes showed excellent linearity (R² > 0.9998) and retention time precision (RSD < 0.12%).
• Comparison with NIST SRM 3238 yielded relative differences below 11% for most compounds; daidzein was 15% above the certified value, consistent with literature.
• Spiking experiments demonstrated recoveries of 98–101% for key isoflavones.
• Analysis of four commercial supplements showed two products in agreement with label claims and one with significantly lower isoflavone content.

Benefits and practical applications

• High-throughput UHPLC method reduces analysis time and solvent consumption, supporting cost-effective QA/QC workflows.
• Mass detection improves confidence in peak identification and streamlines method transfer across platforms.
• Approach is readily applicable to routine analysis of dietary supplements and complex botanical matrices.

Future trends and applications

Continued advances in UHPLC and mass detection will further decrease run times and enhance sensitivity for botanical analyses.
Integration with high-resolution mass spectrometry and automated sample preparation will improve specificity and throughput.
Virtual method transfer tools like column calculators will facilitate seamless migration of pharmacopeial methods to modern instruments.

Conclusion

The USP isoflavone method was successfully migrated to a Waters ACQUITY Arc UHPLC system with PDA and QDa detectors, reducing analysis time to 18 minutes while maintaining chromatographic performance and analytical accuracy.
Mass detection provided robust peak confirmation and expedited method optimization.
Validation with reference materials and commercial supplements confirmed the method’s suitability for routine dietary supplement analysis.

References

1. USP Monograph. Powdered Soy Isoflavones Extract, USP39–NF34 S1 [6841], The United States Pharmacopeial Convention.
2. AOAC Official Method 2008.03. Total soy isoflavones in dietary supplements, supplement ingredients, and soy foods by HPLC-UV. AOAC International, 2008.
3. Fountain K. Transferring USP Compendia HPLC Methods to UPLC Technology for Routine Generic Drug Analysis. Waters Application Note 720004251EN, 2012.
4. Yang J., Benvenuti M., Cleland G. Fast Analysis of Isoflavones – Benefits of Mass Detection in Method Transfer and Sample Analysis. Waters Application Note 720005814EN, 2016.
5. Zhang L.X., Burdette C.Q., Phillips M.M., Rimmer C.A., Marcus R.K. Determination of isoflavone content in SRM 3238 using liquid chromatography–particle beam/electron ionization mass spectrometry. J AOAC Int. 2015;98(6):1483–1490.