HPLC determination of biogenic amines in beer by AQC derivatization

Importance of the Topic

Biogenic amines such as histamine, tyramine and putrescine are common in fermented foods and beverages. In beer they originate from raw materials or microbial activity during fermentation. Monitoring their levels is essential for consumer safety, quality control and detecting microbial contamination.

Objectives and Study Overview

The primary goal of this work was to establish a robust reversed-phase HPLC method for quantifying nine biogenic amines in beer. Pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was used to introduce a fluorescent tag. Ten commercial beers were analyzed to demonstrate method performance.

Methodology

Sample preparation involved:

• Dilution of beer samples followed by filtration and 1:1 dilution.
• Derivatization by mixing borate buffer, AQC reagent in acetonitrile and sample, heating at 55 °C for 10 min.
• Cooling, 1:5 dilution with water and direct injection.

Chromatographic conditions included:

• Accucore aQ C18 column (2.1×100 mm, 2.6 μm).
• Mobile phases: 50 mM ammonium acetate pH 5.0 and acetonitrile.
• Gradient from 4 % to 39 % organic over 26 min at 0.45 mL/min.
• Column temperature 20 °C, injection volume 1 µL.

Instrumentation Used

The analysis was carried out on a Thermo Scientific Vanquish Core Quaternary HPLC system equipped with:

• Diode array detector for UV at 248 nm.
• Fluorescence detector (excitation 248 nm, emission 298 nm).

Main Results and Discussion

All targeted amines produced sharp, baseline-resolved peaks. Fluorescence detection offered excellent sensitivity for most amines, except tryptamine which exhibited quenching and was better monitored by UV. Limits of quantitation were well below levels naturally found in beer. Among ten beers:

• No spermidine or tryptamine was detected.
• Histamine coeluted with a matrix interference, leading to apparent overestimation; true levels were at or below the detection threshold.
• Other amines such as agmatine, putrescine and tyramine were present at low milligram-per-liter concentrations, consistent with literature data.

Benefits and Practical Applications

This method delivers:

• Rapid and simple sample derivatization.
• High sensitivity and selectivity via dual UV/fluorescence detection.
• Robust performance suitable for routine quality control in breweries and food testing laboratories.

Future Trends and Potential Applications

Emerging developments may include:

• Integration with mass spectrometry to improve specificity and reduce interferences.
• Automation of derivatization steps for high-throughput screening.
• Extension to other fermented products such as wine, cheese and fermented vegetables.

Conclusion

AQC derivatization combined with reversed-phase HPLC provides a reliable approach for biogenic amine determination in beer. The method’s sensitivity, simplicity and robustness make it a valuable tool for monitoring product quality and ensuring consumer safety.

References

1. Izquierdo-Pulido M., et al. Biogenic Amines in European Beers. Journal of Agricultural and Food Chemistry. 1996;44(10):3159–3163.