Determination of Isoxanthohumol, Xanthohumol, Alpha and Beta Bitter Acids, and trans and cis-Iso-Alpha Acids by HPLC with UV and Electrochemical Detection: Application to Hop and Beer Analysis

Importance of the Topic

The bitterness and flavor stability of beer are driven by hop-derived compounds such as iso-alpha acids, xanthohumol and related polyphenols. Accurate and sensitive measurement of these analytes is critical for quality control, consistency, product differentiation and shelf-life assessment in both brewing and analytical laboratories.

Study Objectives and Overview

This work aimed to establish two gradient HPLC methods combining diode-array (UV) and electrochemical array detection (ECD) for:

• Targeted quantification of isoxanthohumol, xanthohumol, alpha- and beta-bitter acids and their cis/trans iso-alpha isomers in hops and beer
• A metabolomic approach to differentiate beer types based on profiles of known and unknown analytes
• Assessment of iso-alpha acid stability during a two-week storage study

Methodology and Instrumentation

Two LC methods were developed: a polyphenol assay with multi-wavelength diode-array detection and a bitter acid assay using a dual-channel electrochemical cell. Key parameters included:

• C18 column with 3 µm particles, 3 × 150 mm
• Mobile phases of phosphate buffers, acetonitrile, methanol and tetrahydrofuran with pH around 3.4
• Concave gradients up to 97% organic over 45 min
• Injection volumes of 10–20 µL; flow rate 0.65 mL/min

Instrumentation Used

• Thermo Scientific Dionex LPG-3400BM pump and SR-3000 solvent rack
• Dionex WPS-3000TBSL autosampler
• Dionex DAD-3000RS diode-array detector (channels at 218, 240, 254, 275 nm)
• Dionex CoulArray electrochemical detector (16-channel array 0–900 mV; or dual-channel cell at +550 mV and +850 mV)
• Acclaim™ 120 C18 column (3 µm, 3.0 × 150 mm)
• Chromeleon 6.8 and CoulArray software; Pirouette 2.0 for chemometric analysis

Main Results and Discussion

The combined UV/ECD platform delivered high sensitivity and broad dynamic range:

• Limits of detection by ECD of 10–50 pg on column, by UV of 100–500 pg; quantification limits of 200–1000 pg (ECD) and 500–5000 pg (UV)
• Linear calibration (r² > 0.99 for polyphenols; r² > 0.996 for bitter acids); retention time RSD ~0.3–1.2% over 10 days
• Quantification of dozens of polyphenols, phenolic acids, flavonoids and prenylated chalcones in hop and beer samples
• Targeted bitter acid method measured isoxanthohumol, xanthohumol, prenylnaringenin, alpha/beta acids and cis/trans iso-alpha acids in one run without solid-phase extraction
• PCA of EC array data clearly separated matched normal vs. light beers, microbrews, Belgian and Irish styles; UV data lacked similar discrimination
• Stability study in an Ultra IPA showed marked declines in cis-iso-alpha-acid 2 (–61%), trans-iso-humulone (–67%), trans-iso-adhumulone (–56%) and trans-iso-cohumulone (–55%) over two weeks at 4 °C

Benefits and Practical Applications

The dual-detection HPLC approach offers:

• Enhanced sensitivity and selectivity for bitter acids and polyphenols, enabling trace-level monitoring
• Reduced sample preparation by eliminating pre-concentration steps for ECD detection
• Robust quality control tools for brewing, authenticity testing and shelf-life studies
• Metabolomic profiling capabilities for product differentiation, process optimization and fraud detection

Future Trends and Potential Applications

Emerging directions include integration with high-resolution mass spectrometry for structural elucidation, automation of sample throughput, expanded profiling of novel hop metabolites, and real-time process monitoring. Advanced chemometric and machine learning tools may further enhance pattern recognition and authenticity assessment.

Conclusion

The combined HPLC-UV/ECD platform delivers precise, sensitive and comprehensive analysis of hop bitter acids and polyphenols. The targeted methods quantify a broad suite of analytes at picogram levels, while metabolomic profiling distinguishes beer types and tracks compound stability. This versatile approach supports quality assurance, product differentiation and studies of beer aging.

References

• Ullucci PA, Bailey B, Acworth IN, Crafts C, Plante M. The Spectro-Electro Array: A Novel Platform for the Measurement of Secondary Metabolites in Botanicals, Supplements, Food, and Beverages. Pittsburgh Conference; 2012. Poster 1770-1 P.
• De Cooman L, Aerts G, Overmeire H. Alteration of the Profiles of Iso-alpha-Acids During Beer Ageing: Marked Instability of Trans-Iso-alpha-Acids and Implications for Beer Bitterness, Consistency in Relation to Tetrahydroiso-alpha-Acids. Journal of the Institute of Brewing. 2000;106:169–178.