Fast Analysis of Isoflavones in Dietary Supplements – Method Transfer to an ACQUITY UPLC H-Class System

Importance of the Topic

Isoflavones are biologically active compounds found in soy, red clover, and kudzu. They are widely used in dietary supplements for their potential health benefits, including hormonal balance and antioxidant activity. Rapid and accurate quantitation of these components is critical for quality control, regulatory compliance, and consumer safety.

Objectives and Study Overview

This application note describes the transfer of a standard USP isoflavone HPLC-UV method onto a high-throughput UPLC platform. The primary goals were to:

• Reduce analysis time from 74 minutes (HPLC) and 18 minutes (UHPLC) to under 10 minutes per injection.
• Demonstrate method performance in terms of accuracy, precision, and linearity.
• Showcase the advantages of integrating a compact mass detector for peak identification and interference reduction.

Methodology and Instrumentation

Sample preparation followed USP guidelines, with defatted soy powder and commercial supplement samples extracted and diluted in an acetonitrile–water mixture containing 0.1% formic acid. Apigenin served as the internal standard at 4 ppm.

• Chromatographic system: ACQUITY UPLC H-Class with CORTECS UPLC C18 column (2.1 × 75 mm, 1.6 µm).
• Detectors: ACQUITY PDA (UV–Vis at 260 nm) and ACQUITY QDa mass detector using single ion recording (SIR) for target masses.
• Mobile phases: water/0.1% formic acid (A) and acetonitrile/0.1% formic acid (B).
• Gradient: 90% A to 10% A over 6.5 min, then re-equilibration to initial conditions by 8.10 min.
• Flow rate and temperature: 0.55 mL/min at 30 °C.

Main Results and Discussion

The method transfer utilized a column calculator to adapt USP gradient parameters to UPLC conditions. The QDa detector enabled unambiguous peak assignment of acetyl and malonyl derivatives without pure standards. Calibration curves for six major isoflavones showed R² > 0.998 over 0.05–10 ppm, and retention time RSDs < 0.27%.

Accuracy assessment with NIST SRM 3238 yielded relative differences below 14%. Spiking experiments returned 95–102% recoveries for daidzein, genistein, and glycitein. Analysis of three commercial supplements demonstrated agreement with label claims for two products; one sample exhibited significantly lower total isoflavone content than stated.

Benefits and Practical Applications

The UPLC-UV/MS method delivers:

• Eight-minute run time, doubling throughput compared to 18 min UHPLC and offering ninefold higher throughput versus 74 min HPLC.
• Over 90% reduction in solvent consumption relative to HPLC methods.
• High selectivity through MS detection, minimizing interference from co-eluting compounds.
• Efficient method development with default QDa parameters and rapid optimization of flow rate and temperature.

Future Trends and Opportunities

Advancements in small-footprint mass detectors and core-shell column technology will continue to accelerate method development and reduce analysis costs. Integration of automated sample preparation and data processing workflows could further enhance laboratory efficiency. Expanding the method to other classes of natural products and implementing multi-analyte panels are promising directions.

Conclusion

A standard USP isoflavone assay was successfully transferred to an ACQUITY UPLC H-Class system with PDA and QDa detection, achieving an eight-minute analysis with excellent linearity, precision, and accuracy. The approach combines high throughput, reduced solvent use, and strong selectivity, making it well suited for routine quality control of dietary supplements.

References

• USP Monograph. Powdered Soy Isoflavones Extract, USP39–NF34 S1 [6841].
• AOAC Official Method 2008.03. Total Soy Isoflavones in Dietary Supplements by HPLC-UV.
• Fountain K. Transferring USP Compendial HPLC Methods to UPLC Technology, Waters Application Note, 2012.
• Yang J., Benvenuti M., Cleland G. Fast Analysis of Isoflavones – Benefits of Mass Detection in Method Transfer and Sample Analysis, Waters Application Note, 2016.