Objective and Scope of the method
Histamine (or scombrotoxin), putrescine, cadaverine, tyramine and agmatine biogenic amines are produced from the decarboxylation of histidine, ornithine, lysine tyrosine and arginine respectively by decarboxylase enzymes from some bacteria present in spoiling fish.
Histamine Fish Poisoning (HFP) is often referred to as scombrotoxin poisoning because of the frequent association of the illness with the consumption of spoiled scombroid fish such as tuna and mackerel.
The formation of histamine in fish products is directly correlated with the concentration of histidine in the tissue and the level of micro-organisms present in the product, due to bacterial histidine decarboxilase reaction.
Biogenic amines have been proposed as indicators of spoilage of fish. It is interesting to note that most of the biogenic amines are stable to thermal processing, so their presence in finished canned products is a good indication that the raw material was spoiled prior to heating.Law regulations for histamine
Regulation (EC) 2073/2005 establishes histamine maximum levels for species especially rich in histidine:
"Nine samples must be taken from each batch. These must fulfil the following requirements :
- the mean value must not exceed 100 ppm;
- two samples may have a value of more than 100 ppm but less than 200 ppm;
- no sample may have a value exceeding 200 ppm.”
Those species belong to the families: Scombridae, Clupeidae, Engraulidae, Coryfenidae, Pomatomidae and Scombresosidae.
However, fish belonging to these families which have undergone enzyme ripening treatment in brine may have higher histamine levels but not more than twice the above values.
The FDA guidelines 1998c for tuna, mahi mahi and related fish advised histamine levels "500 ppm set based on toxicity, 50 ppm set as defect action level, because histamine is generally not uniformly distributed in a decomposed fish. Therefore, if 50 ppm is found in one section, there is the possibility that other units may exceed 500 ppm".Analytical methods for histamine and biogenic amines
Biological methods were the first methods of evaluation of scombroid toxine, they measured such parameters as amount of contraction of a histamine sensitive organ as guinea pig ileum and the first AOAC method of determination of histamine in food was a biological method (AOAC Official method 954.04), but these techniques have been supplanted by simpler and more convenient methods.
- Quantitative methods: Fluorimetric techniques have been developed for an accurate measurement of histamine and with the evolution of the techniques new procedures were tested. Now, the chromatographic techniques, such as gas chromatography (GC), high performance liquid chromatography (HPLC), and high performance thin layer chromatography (HPTLC), as well as capillary electrophoresis, offer one major advantage: they allow the simultaneous analysis of histamine and others biogenic amines in fish and fishery products. HPLC techniques are the most used today.
Methods for routine testing in QC laboratories
- Semi-quantitative methods: they are suitable for routine screening of histamine. Some convenient immuno-enzymatic kits are commercialised and other techniques such as colorimetric, thin layer chromatography (TLC) and enzymatic methods are used for many years.
With regard to quality control methods for scombrotoxin: To ensure the safety regarding histamine it is preferable to use a rapid method to do a screening, even if the method is semi-quantitative, because it allow checking a great quantity of samples. However it is necessary to define the limits of the method and to validate its reliability in comparison with a reference method; and in case of doubt regarding the results or in the event of dispute, the official method must be used.
The colorimetric, TLC and enzymatic methods require in general a small commitment of equipment and inexpensive reagents, however they are qualitative or at best semi-quantitative.
The immuno-enzymatic test kits giving quantitative results require little equipment, a photometer, and some reagents that are sold ready to use. Several ELISA procedures have been developed by companies which produce qualitative and/or quantitative analysis kits for the measure of histamine. They are able to distinguish between products that contained less than 50 ppm and those that contained more than 50 ppm.
HPTLC can be used as screening test in quality control laboratories; it can replace old TLC methods, improving the quality of the results, is not expensive and doesn't require sophisticated instrumentation. It is also a sensitive and reproducible method.Methods for validation
The AOAC fluorimetric method (977.13) is used to compare the performance of new methods in comparative studies. It is recommended by Codex Alimentarius as reference method.
An HPLC method based on extraction of biogenic amines with perchloric acid, followed by direct HPLC analysis of the extract on a reverse phase column with on-line derivatization with ophthaldialdehyde and fluorescence detection at 365/418 nm was studied in a European project, tested on several food matrixes. It is a German reference method (Amtliche-Sammlung-von-Untersuchungsverfahren-nach-Paragraph-35-LMBG., L 10.00-5).
Quantitative precise methods often need relative sophisticated equipment and skilled technicians. HPLC technique being sophisticated, the method can be used in specialized laboratories to check some samples after a first screening test. Standards
Histamine.2HCl, Putrescine.2HC1, Cadaverine.2HC1 and other biogenic amines are commercially available. Recommendations
Sample management: Histamine and biogenic amines are produced by enzymatic reaction, their level increases in the chain, even under chilling condition, so it is important to perform the analysis very quickly after the sampling or when it is possible, depending on the method, to prepare the acidic extract that can be kept about one week at +4°C. Histamine and biogenic amines are heat and acid stable and they cannot be destroyed by cooking or freezing.
Expression of Analytical Results: The analysis result should be expressed clearly, i.e.histamine in mg/kg with the reference of the used method and details about the sampling (nature, date, place). Official methods of reference
HPLC method (separation of dansyl-derivatives) is the reference method of the Commission Regulation (EC) N° 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. It is a technique published by Malle et al. (1996) followed by an internal validation (Duflos et al., 1999).Technical references
Commission Regulation (EC) N° 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs.
Malle P., Valle M., Bouclet S. Assay of biogenic amines involved in fish decomposition. J. AOAC Internat., 1996, 79, 43-49.
Duflos G., Dervin C., Malle P., Bouquelet S. Relevance of matrix effect in determination of biogenic amines in plaice (Pleuronectes platessa) and withing (Merlangus merlangus). AOAC Internat., 1999, 82, 1097-1101.