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Reference material for heterocyclic amines quantification in heat processed food


The heat treatment of protein-rich food like meat and fish products generates potent mutagenic compounds like heterocyclic aromatic amines (HAAs). These genotoxic food contaminants are strongly suspected to be involved in human colon and breast cancer.
Due to the very low levels (ng/g) of these molecules present in broiled/fried/baked foods, the identification and quantitative estimation of HAAs remains a challenge, which needs extensive fractionations, in multiple step procedures. Actually, about 20 structures have been identified, but complex industrial preparations like processed meat like flavors seems to contain isomers with extraction and chromatographic profiles very similar to HAAs. In order to estimate the level of human consumption of HAAs and to elaborate a reliable risk assesment it is necessary to develop strong routine methods in order to quantify correctly these HAAs, in different food matrices. In the past, several complicated methods have been proposed, but the results were not reproducible, leading to confusion in the calculated amounts ingested.

A first objective of the project was to establish and optimize the accuracy, repetability and reproducibility of the available end-methods for the quantification of four HAAs (IQ, MeIQx, 4,8-DiMeIQx and PhIP), during a preliminary intercomparison with methanolic solutions .
A second intercomparison was organized on a common batch of a commercial sample of beef extract , in order to evaluate the influence of the complete extraction and clean up procedure on the final recovery of the four HAAs.
The results of the first intercomparison revealed a good agremment between the results obtained by the participants and the target content; no discrepancies linked to the analytical method were detected.No statistical difference was found regarding the day of work, the use of an automatic sampler or the calculation method used to determine the unknown content.
Based on the results obtained, the participants have agreed the within-laboratory and between-day reproducibility, for the four HAAs considered.
The study of the stability of the four HAAs in beef extract revealed a significant loss at +25°C and +60°C. A recommend solid phase extraction procedure was used by most of the participants followed by HPLC analysis. The comparison of the results obtained revealed important variations not only between but also within laboratories. This led to the conclusions that whereas the analytical methods used were satisfactory, the extraction and clean up procedure could be improved.
In a first phase, a common batch of four methanolic solutions of the most representative HAAs was prepared and checked for stability and homogeneity. When the results were found satisfactory, an intercomparison was organized between height European laboratories, which received a metanolic solution of identity and content both unknown to participants, together with the reference solutions. Whereas the choice of the end-method was left free, all the participants used HPLC with various detection systems.
In a second step, a batch of commercial beef extract was selected, spiked with the four HAAs, tested for stability and homogeneity and finally distributed to the participants.


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Université Catholique de Louvain
73,Avenue E. Mounier
1200 Bruxelles

Participants (2)

Rue Saint Martin 292
75141 Paris
647,Marti I Franques, 1-11
08028 Barcelona