HPLC determination of biogenic amines in beer

Importance of the Topic

A reliable analytical method for determining biogenic amines in beer is essential for food safety and quality control. Biogenic amines arise from microbial or enzymatic decarboxylation of amino acids during fermentation or spoilage. High levels of compounds such as histamine, tyramine and cadaverine may indicate poor hygiene or overfermentation and pose health risks to sensitive individuals. Monitoring these amines helps ensure product freshness, compliance with safety standards and consumer protection.

Objectives and Overview of the Study

The primary goal was to develop and validate a reversed-phase HPLC method with pre-column derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) for quantifying nine biogenic amines in beer. The method aimed to combine high sensitivity, precision and reproducibility while maintaining a simple sample preparation workflow. Application to a series of commercial lager and wheat beers was demonstrated to illustrate the method’s practical utility.

Methodology and Instrumentation

Samples were degassed, filtered and diluted prior to derivatization. AQC reagent in borate buffer was mixed with beer aliquots and incubated at 55 °C for 10 minutes. Derivatized amines were separated on a polar endcapped C18 column using a gradient of ammonium acetate buffer (pH 5.0) and acetonitrile. Detection was performed by fluorescence (excitation 248 nm, emission 298 nm) for eight amines and by UV absorbance at 248 nm for tryptamine. Calibration curves were constructed using six standard levels and external standard calibration. Limits of quantitation ranged from 0.004 to 0.05 mg/L depending on the amine.

Instrumentation Used

• Vanquish Core quaternary HPLC system
• Accucore aQ C18 polar endcapped column (2.1 × 100 mm, 2.6 µm)
• Fluorescence detector with micro flow cell
• Diode array UV detector for complementary detection
• Standard laboratory equipment for sample heating, filtration and pH measurement

Main Results and Discussion

Chromatographic separation achieved baseline resolution for seven of nine targeted amines. Histamine coeluted with a matrix interference peak in most samples, resulting in overestimated values. Calibration curves exhibited excellent linearity (R2 > 0.996) and method precision with retention time RSD below 0.6 % and area repeatability within 5 %. Recovery experiments yielded values between 86 % and 126 %. In ten Bavarian beers (five lager, five wheat), agmatine, tyramine, putrescine, cadaverine and spermine were detected at low concentrations, generally below 6 mg/L. Spermidine and tryptamine remained below detection. Phenylethylamine was consistently below its LOQ. Cadaverine appeared above LOQ in only one lager. Histamine quantification was compromised by a coeluting component, except in one wheat beer where interference was minimal.

Benefits and Practical Applications

• Sensitive quantitation of multiple biogenic amines relevant to beer quality and safety
• Simple and reproducible sample preparation compatible with routine QA/QC
• High chromatographic precision enabling confident peak identification
• Applicability to a range of fermented beverages and foods following minor adjustments

Future Trends and Opportunities

Advances in derivatization chemistry and ultra-high-performance chromatography could further reduce analysis time and improve resolution of interfering components. Integration with high-resolution mass spectrometry may offer enhanced selectivity for histamine and other critical amines. Online sample preparation and automation could streamline workflow in high-throughput laboratories. Development of rapid screening sensors or biosensors for on-site monitoring presents a complementary approach to conventional HPLC analysis.

Conclusion

The described HPLC-FLD method with AQC derivatization provides a robust tool for monitoring biogenic amines in beer. It delivers high sensitivity, precision and reproducibility for seven amines and reliable quantitation across a relevant concentration range. While histamine analysis requires further optimization to resolve matrix interferences, the method is well suited for routine quality control and safety assessment in the brewing industry.

References

1. Nuñez M, del Olmo A, Calzada J. Biogenic Amines. In Encyclopedia of Food and Health; Caballero B, Finglas P, Toldrá F, editors. Academic Press; 2015. p. 416–423.
2. Matissek R. Lebensmittelsicherheit: Kontaminanten – Rückstände – Biotoxine. Springer; 2020.
3. Lovejoy K, Fabel S, Pietsch M, Park SH, Steiner F. Amino acid analysis of mammalian cell culture medium by liquid chromatography with UV and fluorescence detection and derivatization with AQC. Thermo Fisher Scientific Application Note. 2019.
4. Lakowicz JR. Principles of Fluorescence Spectroscopy. Springer; 2010.
5. Izquierdo-Pulido M, Hernández-Jover T, Mariné-Font A, Vidal-Carou MC. Biogenic Amines in European Beers. J Agric Food Chem. 1996;44(10):3159–3163.