Determination of Biogenic Amines in Fermented Foods: Analytical Methods, Standards, and Applications

Determination of biogenic amines in fermented foods is a critical food safety and quality control task, as compounds such as histamine and tyramine can trigger adverse reactions even at low concentrations. This technical article reviews the target analytes, sample types, extraction and derivatization procedures, chromatographic methods, and international standards used by accredited third-party laboratories for reliable biogenic amine testing.

Technical Overview

Biogenic amines (BAs) are low-molecular-weight organic bases formed in fermented foods primarily through decarboxylation of free amino acids by microbial enzymes. While the controlled microbial activity in fermentation develops desirable flavor, aroma, and texture, it also promotes the growth of decarboxylase-positive organisms such as certain lactic acid bacteria, Enterobacteriaceae, staphylococci, and yeasts, which convert precursor amino acids into amines. Histidine yields histamine, tyrosine yields tyramine, ornithine yields putrescine, lysine yields cadaverine, and tryptophan yields tryptamine and tryamine-related indole amines.

From a toxicological perspective, histamine is the most significant amine, responsible for histamine (scombroid-type) poisoning characterized by flushing, headache, palpitations, and hypotension. Tyramine can provoke hypertensive crises in patients taking monoamine oxidase inhibitors, while putrescine and cadaverine potentiate histamine toxicity by inhibiting intestinal detoxification enzymes such as diamine oxidase. Because fermentation inherently carries a risk of amine accumulation, quantitative determination of biogenic amines is essential for risk assessment, regulatory compliance, and process optimization in the fermented food industry.

Testing Samples

Fermented Alcoholic Beverages

Red and white wine, beer, cider, rice wine, and sake frequently contain elevated tyramine, histamine, and putrescine derived from malolactic fermentation and yeast autolysis. These liquid matrices are typically filtered, degassed, and diluted prior to extraction.

Fermented Dairy Products

Ripened cheeses, yogurt, kefir, and fermented milk are classic high-risk matrices, since long ripening periods allow extensive proteolysis to release free amino acids and enable amine formation by non-starter lactic acid bacteria. Tyramine and histamine often dominate in aged cheeses.

Fermented Vegetable Products

Sauerkraut, kimchi, and traditional pickled vegetables may accumulate tyramine, putrescine, and cadaverine when fermentation is poorly controlled or raw material hygiene is inadequate. Acidic brine matrices require neutralization during sample preparation.

Fermented Soybean Products

Soy sauce, miso, tempeh, fermented bean curd, and broad bean chili paste contain abundant free amino acids and high microbial loads, resulting in diverse amine profiles including histamine, tyramine, putrescine, and phenylethylamine. High-salt and high-protein matrices demand careful extraction and cleanup.

Fermented Meat Products

Dry-fermented sausages, cured hams, and fermented mince products can accumulate tyramine, putrescine, and cadaverine, where amine levels serve as indicators of raw material quality and hygienic conditions during ripening.

Fermented Seafood Products

Fish sauce, fermented shrimp paste, salted anchovies, and traditional fermented fish carry the highest histamine risk due to the high histidine content of dark-fleshed fish species and the activity of histidine decarboxylase-producing bacteria during long enzymatic maturation.

Testing Items

Accredited third-party laboratories typically quantify the following analytes individually and as a sum total:

• Histamine (primary safety indicator, regulatory limits typically 100–400 mg/kg depending on product and jurisdiction)
• Tyramine (migraine trigger and MAOI interaction risk)
• Putrescine (hygiene indicator, histamine toxicity enhancer)
• Cadaverine (indicator of Enterobacteriaceae contamination)
• 2-Phenylethylamine (vasoactive amine)
• Tryptamine
• Spermidine and spermine (naturally occurring polyamines)
• Agmatine (arginine-derived amine)
• Total biogenic amine content and calculated quality indices, such as the biogenic amine index used to evaluate hygienic quality

Testing Methods

Biogenic amines are highly polar and lack strong chromophores, so chromatographic determination after derivatization is the industry standard approach. The general analytical workflow comprises homogenization, acid extraction, derivatization, separation, and quantification.

Sample Preparation and Extraction

Homogenized samples are extracted with perchloric acid (commonly 0.4–0.6 mol/L) or trichloroacetic acid (5–10%) to precipitate proteins and liberate amines. An internal standard such as 1,7-diaminoheptane is added at the extraction stage to correct for matrix effects and volumetric losses. Extracts are centrifuged and filtered through 0.22–0.45 micrometer membranes before derivatization.

Derivatization Techniques

Dansyl chloride is the most widely used reagent because it reacts with primary and secondary amines simultaneously, enabling single-run quantification of eight to ten amines. Alternatives include benzoyl chloride (ultraviolet detection), o-phthalaldehyde (fluorescence detection, primarily for histamine), dabsyl chloride, and fluorenylmethyl chloroformate (FMOC-Cl). Reaction pH, temperature, and time are tightly controlled to ensure complete and reproducible derivatization.

HPLC with UV or Fluorescence Detection

Reversed-phase HPLC on C18 columns with gradient elution of acetonitrile–water or acetonitrile–ammonium acetate mobile phases separates dansylated derivatives within 25–40 minutes. Ultraviolet detection at 254 nm or fluorescence detection provides detection limits at the sub-mg/kg level, suitable for most regulatory purposes. This approach aligns with the Chinese national food safety standard GB 5009.208, which covers the determination of biogenic amines in foods by HPLC.

LC-MS/MS and UHPLC-MS/MS

Tandem mass spectrometry delivers superior selectivity and sensitivity in complex fermented matrices, with detection limits in the microgram-per-kilogram range. Isotope-labeled internal standards and multiple reaction monitoring minimize matrix interferences, making LC-MS/MS the preferred confirmatory technique for dispute resolution and low-level surveillance.

Ion Chromatography and Direct Determination

High-performance anion-exchange chromatography with integrated pulsed amperometric detection allows direct quantification of underivatized amines, eliminating derivatization variability and shortening total analysis time.

Rapid Screening Methods

Enzymatic biosensors based on diamine oxidase, colorimetric test kits, and enzyme-linked immunosorbent assay (ELISA) provide cost-effective screening for histamine in high-throughput production environments. Positive or borderline results are confirmed chromatographically.

Reference Standards and Validation

• ISO 19343 specifies methods for histamine determination in fish and fishery products using HPLC, capillary electrophoresis, or fluorometry, and is widely applied to fermented fish products
• AOAC official fluorometric methods for histamine are accepted for screening and compliance testing
• EU microbiological criteria establish histamine limits of 100–400 mg/kg for fishery products including enzyme-ripened and fish-sauce categories
• GB 5009.208 defines the HPLC procedure for eight to nine biogenic amines in food matrices in China
• All methods are validated for linearity, recovery, repeatability, limits of detection and quantification, and measurement uncertainty under ISO/IEC 17025 quality systems

Applications

Biogenic amine testing supports multiple practical objectives across the fermented food supply chain:

• Regulatory compliance and export clearance, demonstrating conformity with histamine limits in destination markets
• Raw material and incoming inspection for fish, meat, and vegetable ingredients with high amine formation potential
• Fermentation process control, comparing starter cultures and selecting decarboxylase-negative strains to minimize amine accumulation
• Shelf-life and storage studies evaluating temperature abuse, packaging atmospheres, and time-temperature profiles
• Hygienic quality assessment, using putrescine and cadaverine as markers of undesirable microbial contamination
• Product development support for low-histamine and reduced-amine claims on fermented food labels
• Scientific research, including screening of amine-producing microorganisms and detection of decarboxylase genes such as hdc, tdc, and odc
• Epidemiological and dietary exposure assessment for risk characterization by public health authorities

FAQ

What level of biogenic amines is considered unsafe?

No universal threshold exists, but histamine above 100 mg/kg in a food is generally regarded as a health concern, and regulatory limits range from 100 to 400 mg/kg depending on product category and jurisdiction. Total biogenic amine intakes above approximately 1000 mg per meal have been associated with adverse effects, while susceptible individuals may react at much lower levels.

Why do fermented foods contain more biogenic amines than fresh foods?

Fermentation involves dense microbial populations, extended processing times, and proteolysis that releases abundant free amino acid precursors, all of which favor enzymatic decarboxylation and amine accumulation.

How should samples be collected and transported?

Samples should be collected in clean, inert containers, kept chilled during short transport, and stored frozen at minus 18 degrees Celsius or below for longer periods. Repeated freeze-thaw cycles must be avoided because they cause uneven amine distribution and matrix degradation.

How much sample is required for testing?

Most laboratories request 100–200 grams of solid product or 100–200 milliliters of liquid product to accommodate extraction replicate analysis and confirmatory testing.

How long does a biogenic amine analysis take?

Standard HPLC determination with dansyl derivatization is typically completed within five to seven working days, while LC-MS/MS confirmation and full validation-grade reports may require additional time.

Can all biogenic amines be quantified in a single run?

Yes. Using dansyl chloride derivatization with reversed-phase HPLC or LC-MS/MS, laboratories routinely quantify eight to ten amines simultaneously, including histamine, tyramine, putrescine, cadaverine, phenylethylamine, tryptamine, agmatine, spermidine, and spermine.