High Speed Analysis of Xanthohumolin Beer

Importance of the Topic

Xanthohumol is a prenylated flavonoid in hops with antioxidant, anti-inflammatory and antibacterial effects and potential health benefits. Simultaneous monitoring of xanthohumol and related bitter acids in beer is essential for quality control and research on functional ingredients.

Objectives and Study Overview

The study aimed to develop a rapid high performance liquid chromatography method for simultaneous quantification of xanthohumol, isoxanthohumol, humulinones, iso-α-acids, α-acids, and β-acids in beer within eight minutes per run.

Methodology and Instrumentation

Standard solutions of target compounds were prepared and analyzed under the following conditions:

• Instrument: Nexera X3 high performance liquid chromatograph
• Column: Shim-pack Velox C18, 50 mm × 3.0 mm I.D., 1.8 μm
• Mobile phases: A is sodium phosphate buffer (10 mmol/L, pH 2.6) with 0.2 mmol/L EDTA; B is methanol
• Gradient elution: 50% B to 90% B over six minutes, hold, then return to 50% B by eight minutes
• Flow rate: 0.7 mL/min; column temperature: 40 °C; injection volume: 5 μL
• Detection: photodiode array detector at multiple 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)
• Beer pretreatment: degassing, addition of 1-octanol, methanol extraction, centrifugation, and 0.22 μm filtration

Main Results and Discussion

Calibration curves for each compound showed linearity with coefficients of determination r2 greater than 0.999 across suitable concentration ranges. Analysis of seven commercial beer samples provided concentrations of all target analytes, with xanthohumol levels ranging from 0.012 to 2.458 mg/L. Spike recovery tests on selected beers yielded recovery rates between 91% and 113%, and reproducibility tests indicated relative standard deviations below 9%. The total analysis time per sample was under eight minutes.

Benefits and Practical Applications

• Rapid and simultaneous quantification of health-related flavonoids and bitter acids
• High sensitivity through wavelength optimization for each analyte
• Enhanced laboratory throughput to support quality control in brewing

Future Trends and Applications

The method could be extended to online monitoring in brewing processes, applied to additional hop-derived bioactive compounds, or combined with mass spectrometry for structural characterization. Adaptation to ultrahigh-performance liquid chromatography platforms may further shorten run times and improve resolution.

Conclusion

A fast, precise, and reproducible HPLC method on the Nexera X3 system was validated for simultaneous analysis of xanthohumol, isoxanthohumol, humulinones, iso-α-acids, α-acids, and β-acids in beer. This approach supports efficient quality control and research on functional beer components.

Used Instrumentation

• Nexera X3 high performance liquid chromatograph
• Shim-pack Velox C18 column (50 mm × 3.0 mm I.D., 1.8 μm)
• Photodiode array detector SPD-M40
• 0.22 μm membrane filter for sample preparation
• Sodium phosphate buffer with EDTA and methanol as mobile phases

Reference

1. European Brewery Convention, EBC ANALYTICA 7.15, 9.47, 9.50
2. Vázquez Loureiro P et al. Determination of Xanthohumol in Hops, Food Supplements and Beers by HPLC, Foods, 2019
3. Żołnierczyk AK et al. Isoxanthohumol — Biologically active hop flavonoid, Fitoterapia, 2015
4. Táborský J et al. Dynamics of bitter acids and xanthohumol in hop pellets during storage, Agronomy Research, 2018
5. Gahr A et al. Stability of bitter substances in beer during aging, Brewing Science, 2020
6. Nimubona D et al. Shelf life prediction of lager beer by iso-α-acid degradation, Journal of Food Engineering, 2012