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A family of food contaminants belonging to the chemical class of heterocyclic aromatic amines (HAAs) is produced during the heat treatment of protein-rich foods. These compounds have marked genotoxic properties; some of them behave as the most potent bacterial mutagens and were shown to be carcinogenic in rodents and non human primates. Different epidemiological informations seems to indicate that they could be implied as the etiological agents of colorectal cancer and also may be in other forms of tumors.
Therefore, accurate quantification methods are required for the human risk assessment of cooked foods contaminated by these substances. This problem remains a challenge because HAAs are generally found at very low levels (ng/g) in the heated food matrices like cooked meat or fish products
For these reasons, extensive fractionations must be applied, before the analysis. In the past, different solvent extraction procedures were proposed, including complex schemes with subsequent poor recoveries. It is actually recognised that the method developped by G.A. Gross (1990) is the most reliable, with the main advantage of a "on line" working scheme avoiding losses between the usual transfers, whereas the recovery of some compounds like PhlP remains unsatisfactory. The method which is based on a tandem solid phase extraction method with HPLC analysis is simple enough to be widely recommended as a routine procedure. It comprises an efficient sample clean-up through the combination of several modular purification steps using commercially available diatomaceous earth, cation exchange and reversed phase extraction cartridges. Multiple standard addition is necessary because of variable and sample dependent extraction efficiencies of the different HAAs, with TriMelQx as internal standard.
Quantification is done via HPLC with a reverse phase column and UV or electrochemical detection (EC). The most currently used methods are based on UV detection.
The aim of our research which started in 1991, was to develop EC detection in order to improve not only the sensitivity but also the specificity of the analysis.
For these reasons it was necessary to look carefully to a number of analytical parameters which could influence the response of the EC detection after the HPLC separation. The following factors have been investigated:
A particular interest was given to the comparative sensitivity of the EC compared to the UV detection; in the actual state of the project, we obtaineed an increase of sensitivity with a factor of about 5 for EC detection. In a more fundamental point of vue, the mechanism of the electrochemical oxidation was also investigated, in order to explain the mechanism of the oxydation reactions which occurs on the electrode.
On the other hand, parameters which could influence the chromatographic separation were also investigated: the efficiency of the reverse phase column was compared with a cation exchange stationary phases. These results were compared with capillary electrophoresis, which has been proposed as an alternative separation technique.
Finally, the method proposed by G.A. Gross was aplied with different food matrices, in order to validate a procedure which could be recommended as a routine method. The detection limits of four HAs: IQ, MelQ, MelQx and 4,8-DiMelQx were quite similar, with figures around 5 p moles, with our UV detection system. The separation of the compounds is satisfactory, with recoveries between 70 and 80%, after a complete extraction procedure on a mixture of the pure compounds. Different heated meat products have been already tested: beef extract, fried sausage and fried ground beef; it appeared from these assays that the recoveries of the HAs under investigation were quite different.
The UV detection system has been compared with the EC after HPLC analysis with an isocratic solvent on three heat processed meat products:
The dispersion of the data obtained reveals that the reproducibility of the method can be improved; more particularly for IQ which is poorly separated. Under the actual state of these investigation, we can conclude that the EC detection is reliable, compared to the classical UV system, whereas some discrepancies are observed, depending of the sample analysed.
The original HPLC method which has been developed after ion exchange separation (IEC) present different important advantages over the classical reverse phase separation:
Moreover the IEC/EC and IEC/UV methods have been validated by an intercomparison study with a methanolic solution of unknown composition.
These two methods were shown to be suitable for the routine analysis of heat treated foods like commercial beef extract or fried beef.
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