Protocol used to choose analysis methods and analytical qual

In order to obtain reliable composition data, appropriate analytical methods must be used. Further, the laboratory which employs them must have demonstrated its proficiency.

The choice of analytical methods often depends on the matrix in which the constituent to be measured is found.

“Official” methods are preferred.

When there is not official method for a given constituent, or the official methods do not take into account recent developments, we preferred methods which take into account the latest technical developments.

The French Accreditation Committee (COFRAC) suggests a range of methods for which a laboratory may receive accreditation.

Other specific protocols were also used to guarantee the reliability of the analyses.

The fats in aquatic products (particularly in so-called “lean” fish) are characterised by a high proportion of phospholipids. These phospholipids are polar, while the reserve fats, in the form mainly of triglycerides, are neutral. Phospholipids and triglycerides are both partially composed of fatty acids.

In order to measure the fatty acids contained in the whole aquatic product (whether in polar or non-polar molecules), you must first qualitatively extract the fats. You must therefore use a method which combines a polar solvent (to extract the phospholipids) and a non-polar solvent (to extract the triglycerides).

The conventional method used to extract fats with just a non-polar solvent therefore seems an inappropriate way of measuring the fatty acids in aquatic products. We preferred the Folch method, which is much less commonly used because of the precautions needed due to the use of the organic solvents chloroform and methanol (in a 2:1 proportion), in order to measure the fatty acids. This is because it satisfies the specific requirement of the fat composition of aquatic products. The fatty acids extracted using the Folch method are then methylated before gas chromatography analysis.

However, in order to measure total fat content, the conventional method of extraction, using a polar solvent (hydrochloric treatment then hexane extraction, as stated in the COFRAC 80 programme), remains preferable. Indeed, even though this method does not extract non-polar fats satisfactorily, it has the advantage of not extracting the non-lipid polar molecules as the Folch method does.

Referred to in the area of analytical methods:

• AFNOR: The French Association for Standardisation, the official French standardisation body. AFNOR standards are identified by a code preceded by the letters “NF” (e.g.:: moisture was measured according to the AFNOR standard NF V 04-401). The European standards published by AFNOR carry the initials “EN” after the letters “NF” (e.g.:: for vitamin B6, high performance liquid chromatography was used, NF EN 14663). In total, in this project 13 constituents were analysed according to AFNOR standards
• AOAC: Association of Analytical Communities, an international not-for-profit scientific association which establishes validated standard methods. Two constituents were analysed according to AOAC methods: vitamin B3 and vitamin B12. 

Some AFNOR or AOAC analytical methods are cited in legal texts: decrees, directives: for example, for amino acids, the high performance liquid chromatography method according to directive 98/64 is an AFNOR standard, NF V 18-114.

For retinol (also called vitamin A), even though the NF V 18-401 method features on the list of methods which the COFRAC 60 programme proposes for accreditation, high performance liquid chromatography to detect absorption followed by UV detection using AFNOR standard NF EN 12823-1 was preferred because it performs better with the matrices used. Method NF V 18-401 was not suitable for the samples containing more than 10% fat, which may have been the case with some of our samples of fatty fish.

For some analyses, grouped into programmes (such as for example the COFRAC 80 programme “Analysis of meats, meat preparations, products made from meat and products made from aquatic products”, or the COFRAC 60 programme “Analyses of nutritional and diet foods and analyses designed for nutritional food labelling”), COFRAC can give an accreditation to a laboratory. Accreditation is a recognition of analytical skills. In order to gain the accreditation, a laboratory must for example take part in inter-laboratory comparison proficiency tests, which must take place at least twice a year (for methods for which there is a scheme). As part of the project, around twenty constituents are analysed with COFRAC accreditation.

The methodology form “List of analytical methods used” indicates for each constituent the number of test samples and measurements to take per test sample.

When two test samples were performed for a constituent, this indicates that the full analytical protocol (including measurement) was performed successfully for two sampled portions in the batch delivered to the laboratory. For each aquatic product, five batches were analysed. When two test samples were performed for a nutrient, this indicates that the average nutritional value presented on this site for each aquatic product is in fact the result of ten analytical values.

For fatty acid composition and cholesterol content, a reference sample was used per series (the one used for cholesterol came from BIPEA: this was another form of precaution taken to guarantee analytical reliability.)