Determination of biogenic amines with automated precolumn derivatization

Significance of the Topic

Biogenic amines are low molecular weight organic bases that occur naturally in various foods and function as important metabolic regulators. Their presence and concentration influence food quality, flavor development, and safety. Excessive intake of certain amines, such as histamine and tyramine, can provoke adverse effects including headaches, hypertensive episodes and allergic reactions. Reliable quantification of key amines is therefore critical for enforcing food safety standards and ensuring consumer health.

Aims and Study Overview

This work demonstrates a streamlined analytical approach for the simultaneous determination of histamine, tyramine, putrescine and cadaverine in food matrices. By integrating automated precolumn derivatization with ortho-phthalaldehyde (OPA) into the autosampler workflow, the method seeks to enhance reproducibility, throughput and sensitivity while maintaining compliance with regulatory limits (e.g. EU histamine MRL at 400 mg/kg).

Methodology and Instrumentation

Samples and standards were prepared in deionized water. Derivatization was performed automatically via the “mix method” functionality of the autosampler. Two reagents were employed: a borate buffer (0.5 M sodium tetraborate, pH 9.2) and an OPA mixture (100 mg OPA dissolved in methanol and buffer, plus mercaptoethanol). The protocol entailed sequential addition of buffer and OPA to 350 µL sample, mixing, a 2-minute reaction dwell time, and injection of 1–10 µL.

Used Instrumentation

• HPLC pump (AZURA P6.1L LPG)
• Autosampler with automatic derivatization (AZURA AS 6.1L)
• PDA detector (AZURA DAD 2.1L at 230 nm)
• C18 reversed-phase column (Eurospher 100-5, 250 × 4.0 mm)
• Control software (ClarityChrom 8.1 with PDA and autosampler modules)

Main Results and Discussion

Chromatographic separation achieved baseline resolution (>6) for all derivatives. Limits of detection ranged from 0.10 mg/kg (cadaverine) to 0.28 mg/kg (histamine), while quantification limits spanned 0.33 mg/kg to 0.93 mg/kg, well below regulatory thresholds. Method precision was excellent, with relative standard deviations below 1 % for peak area and below 0.05 % for retention time across five replicates. Blank injections confirmed negligible carry-over and a distinct derivatization peak.

Benefits and Practical Applications

This automated derivatization protocol offers high robustness and repeatability, minimizing manual handling errors. The low detection limits and strong chromatographic performance make the method suited for routine quality control in food and beverage industries, monitoring of spoilage, compliance with legislation, and research into amine-related biochemical pathways.

Future Trends and Opportunities

Emerging opportunities include coupling derivatization workflows with mass spectrometric detection for enhanced specificity, expansion to multi-amine panels in diverse matrices (dairy, meat, fermented products), and integration into high-throughput platforms for real-time process control. Artificial intelligence-driven data analysis may further streamline identification and quantification in complex samples.

Conclusion

The presented method delivers a reliable, fully automated solution for biogenic amine analysis. Its sensitivity, precision and compliance with regulatory requirements position it as a valuable tool for food safety laboratories and academic research. Adoption of automated derivatization reduces operator variability and accelerates sample turnaround.

References

1. ScienceDirect. Biogenic amine. Available at: https://www.sciencedirect.com/topics/neuroscience/biogenic-amine (accessed January 30, 2020).
2. Erdag D, Merhan O, Yildiz B. Biochemical and pharmacological properties of biogenic amines. In: Proestos C, editor. Biogenic amines. IntechOpen; 2018.
3. Austrian Agency for Health and Food Safety (AGES). Biogenic amines. Available at: https://www.ages.at/en/topics/residues-and-contaminants/biogenic-amines/ (accessed January 30, 2020).
4. Commission Regulation (EU) No 1019/2013 of 23 October 2013 amending Annex I to Regulation (EC) No 2073/2005 as regards histamine in fishery products. Official Journal of the EU; 2013.