Simultaneous Analysis of Beer Components (Xanthohumol, Isoxanthohumol, Humulinones, Iso-α-Acids, α-Acids, and β-Acids)

Importance of the Topic

Beer quality and functional benefits are strongly influenced by hop-derived compounds such as xanthohumol, isoxanthohumol, humulinones, iso-α-acids, α-acids and β-acids. Simultaneous quantitation of these analytes supports product development, quality control and health-related research.

Objectives and Study Overview

This study aimed to develop and validate a high-performance liquid chromatography method for the simultaneous analysis of six key beer components, referencing EBC 9.47 and adapting extraction and calibration procedures for routine brewery analysis.

Methodology and Used Instrumentation

The procedure combined standard preparation, beer pretreatment and HPLC analysis:

• Standard solutions prepared by dissolving analytes in acidic methanol and diluting with mobile phase mixture.
• Beer samples degassed, spiked with 1-octanol as antifoaming agent, centrifuged and filtered.
• Chromatography on Shim-pack Velox Biphenyl column (100×3.0 mm, 2.7 µm) using a gradient of phosphate buffer (pH 2.6)/acetonitrile.
• Detection wavelengths: 370 nm for xanthohumol, 280 nm for isoxanthohumol, 270 nm for humulinones and iso-α-acids, 314 nm for α-acids and β-acids.

Main Results and Discussion

Calibration curves over five concentration levels achieved coefficients of determination >0.999 for all analytes, demonstrating excellent linearity. Analysis of three commercial beers yielded concentration ranges from non-detectable to tens of mg/L. Reproducibility tests showed relative standard deviations between 0.6% and 3.4%. Spike-and-recovery experiments in Beer 1 returned recoveries between 92% and 111%, confirming method accuracy. Cis/trans isomeric forms of iso-α-acids were observed and appropriately quantified.

Benefits and Practical Applications of the Method

The validated HPLC approach enables precise, reproducible measurement of both health-related flavonoids and bittering acids. It is suitable for:

• Quality control in breweries.
• Research on functional beer ingredients.
• Routine monitoring of raw materials and finished products.

Future Trends and Applications

Advancements may include coupling with mass spectrometry for enhanced sensitivity and selectivity, automation of pretreatment steps for high-throughput analysis, real-time monitoring during brewing and expansion to other polyphenolic and bitter compounds.

Conclusion

The optimized HPLC method on the Shimadzu i-Series LC-2050C 3D system provides a robust, accurate and efficient tool for simultaneous analysis of xanthohumol, isoxanthohumol, humulinones, iso-α-acids, α-acids and β-acids in beer. Its implementation can support quality assurance, research and development in the brewing industry.

References

1. Vázquez Loureiro P. Determination of xanthohumol in hops, food supplements and beers by HPLC. Foods. 2019.
2. Żołnierczyk AK et al. Isoxanthohumol — Biologically active hop flavonoid. Fitoterapia. 2015:71-82.
3. Morita A. High speed analysis of α-acids and β-acids in hops. Shimadzu Co. 01-00025-EN.
4. European Brewery Convention. EBC ANALYTICA, method 9.47.
5. European Brewery Convention. EBC ANALYTICA, method 7.15.
6. European Brewery Convention. EBC ANALYTICA, method 9.50.
7. Nimubona D et al. An approximate shelf life prediction of lager beer by degradation of its iso-α-acids. J Food Eng. 2012:138-143.