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Evaluation and development of methods for the determination of histamine in food

Project Code: E01045

31/03/2002

LGC
Lawrance, P

Histamine (2-(4- imidazolyl) ethylamine, CAS Number: 51-45-6) is a biogenic amine that is
produced post-mortem in the muscle of Scombroid fish, such as tuna, by the action of certain
bacteria. It is also found in red wine, and fermented foods such as cheese and processed meats.
The level of histamine produced in Scombroid or other histidine-containing fish serves as an
indicator of the state of freshness of the fish and more widely, when present at higher levels,
histamine represents a health hazard.

Universally accepted or harmonised methods of analysis are essential for monitoring histamine
levels whether for product control or for the enforcement of food and animal feeding stuffs law
under both EU and UK legislation. Anecdotal evidence has suggested that the variety of existing
methods of analysis for histamine may give incomparable results and may not be rugged. This
work was undertaken to evaluate the equivalence, accuracy, precision and suitability of the most
commonly used procedures for the determination of histamine in fish and other foods.

Histamine has been analysed as part of the FAPAS^®; proficiency scheme. Data from two recent
FAPAS^®; Rounds were critically reviewed to assess the effect of methodological differences on
results reported. The findings are presented and discussed.

Six analytical approaches were then selected from the literature. These were identified as the
most commonly used procedures and were developed by workers in Netherlands, Spain, France,
Germany and the USA. The techniques evaluated included HPLC, fluorimetry and ELISA.

Test materials were prepared and tested for homogeneity. These test materials included
unprocessed and processed fish, meat and cheese containing two different, known concentrations
of histamine. The test materials were then analysed in duplicate by each of the analytical
procedures and the results were compared.

All of the methods evaluated were suitable, under appropriate conditions, for the analysis of
histamine in fish products. The HPLC methods have the advantage of being able to quantify
other biogenic amines that are also present in fish, which may enable a more reliable assessment
of the fish quality.

All of the HPLC methods were suitable for the determination of histamine but two of the
procedures (Methods 2 and 6 based on HPLC with o-phthaldialdehyde post-column
derivatisation) had superior overall performance in all foodstuffs examined. The use of either
perchloric acid or trichloroacetic acid for the extraction of histamine from food had little effect
on the concentration determined.

Fluorimetry was reliable for the determination of histamine in fish whilst colorimetric assays
used in the FAPAS^®; Rounds produced high results and are not recommended.

The ELISA method gave acceptable results for histamine in cheese, pork and fish but had a
tendency to over recovery (mean 112%;) and had a high variability (24%; CV).

Variability between analytical procedures does not appear to be a significant problem when
methods are under analytical control but it is apparent from the FAPAS^®; data that some
laboratories still have difficulties in this area. Analytical errors, lack of experience with the
method used and incorrect selection of the appropriate method are all likely factors in this
variability. From the work carried out, any of the methods studied, when properly validated, will
give acceptable results for the determination of histamine. For ease of use, precision, reliability
and general applicability to a range of foods, HPLC with ion-pairing, gradient elution and postcolumn
detection using o-phthaldialdehyde is recommended.

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