Simultaneous Analysis of Beer Components (Xanthohumol, Humulinones, Iso-α-Acids, α-Acids, and β-Acids) Using an Integrated HPLC System

Importance of the Topic

Beer contains bioactive compounds derived from hops, such as xanthohumol and isoxanthohumol, which offer antioxidant, anti-inflammatory and potential anticancer benefits. Bitterness-related α-acids, β-acids and humulinones also influence flavor and quality. Rapid, reliable quantitation of these compounds supports product development, quality control and nutrition research in the brewing industry.

Objectives and Study Overview

This study presents a streamlined method for simultaneous analysis of six hop-derived components (xanthohumol, isoxanthohumol, humulinones, iso-α-acids, α-acids and β-acids) in beer samples. The goals were to:

• Develop a fast HPLC protocol (< 8 minutes) using a large-particle column to mitigate contamination effects.
• Validate calibration linearity, reproducibility and recovery.
• Demonstrate applicability to real beer matrices.

Methodology and Instrumentation

Standard solutions were prepared by dissolving certified reagents in acidic methanol and diluting in mobile phase. Beer samples from three brands underwent degassing with 1-octanol, centrifugation and filtration before injection.

Instrumental setup:

• System: Shimadzu i-Series LC-2050C 3D HPLC.
• Column: Shim-pack Velox™ Biphenyl (100 × 3.0 mm I.D., 2.7 µm).
• Mobile phases: A – 10 mmol/L phosphate buffer (pH 2.6) with EDTA; B – acetonitrile.
• Gradient: 29 % B to 95 % B over 32 min, flow 0.7 mL/min, column at 40 °C.
• Detection wavelengths: 270 nm for iso-α-acids/humulinones, 280 nm for isoxanthohumol, 314 nm for α-acids/β-acids, 370 nm for xanthohumol.

Main Results and Discussion

Calibration curves for all six compounds exhibited excellent linearity (r² > 0.999). Key findings:

• Chromatographic separation achieved in under eight minutes with clear resolution of co-eluting homologs.
• Analysis of three commercial beers yielded concentrations ranging from trace xanthohumol (< 0.01 mg/L) to 34.7 mg/L iso-α-acids.
• Reproducibility tests (n=6) showed relative standard deviations below 3.5 % across all analytes.
• Spike-recovery in Beer 1 ranged from 92 % to 111 %, confirming accuracy in complex matrices.

Benefits and Practical Applications

This integrated HPLC method offers breweries and laboratories a rapid, robust tool for routine monitoring of health-related and bitterness compounds. Reduced column fouling and high throughput support quality control workflows, product differentiation and research into functional beer ingredients.

Future Trends and Potential Applications

Advances may include coupling with mass spectrometry for structural confirmation, miniaturized columns for faster run times, and adaptation to other beverage matrices. Continuous improvement of sample preparation and on-line cleanup will further streamline analytics in industrial settings.

Conclusion

The presented protocol enables fast, accurate quantitation of six key beer components using an integrated HPLC system. High linearity, precision and recovery demonstrate its suitability for quality assurance and research applications in brewing science.

References

• European Brewery Convention, EBC ANALYTICA 7.15, 9.47 and 9.50.
• Vázquez Loureiro P. et al., Determination of Xanthohumol in Hops, Food Supplements and Beers by HPLC, Foods (2019).
• Dieudonné Nimubona et al., Approximate Shelf Life Prediction of Lager Beer via Iso-α-Acids Degradation, Journal of Food Engineering (2012).