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Working group activities: collaborative trials
Overview of publications
The WEFTA working group organized and performed a number of collaborative trials and interlaboratory comparison exercises organized by one of the participating laboratories. Beginning in the late 1980s a wide range of application fields (ranging from TVB-N over species identification by IEF and lipid determination in fish and fishery products) was covered. Most final results have been published.
 

Collaborative trials and interlaboratory comparison exercises of the WEFTA Working Group:

 

Fat

Karl, H; Bekaert, K.; Berge, P.; Cadun, A.; Dulfos, G.; Oehlenschläger, J.; Poli, B.M.; Tejada, M.; Testi, S.; Timm-Heinrich, M.(2012):

WEFTA interlaboratory comparison on total lipid determination in fishery products using the Smedes method   

Journal AOAC International 95, 1-5

 

Abstract: Extraction of lipids by cyclohexane and isopropanol. Transfer of lipids to the cyclohexan phase by addition of water. Phase separation by centrifugation.

Gravimetric fat determination after separation of cylcohexane and evaporation to dryness.

 

 

pH

 

Oehlenschläger, J.; Bossier, P.; Bykowski, P.; Etienne, M.; Huidobro, A.; Karl, H.; Luten, J.; Mendes, R.; Palmadottir, H.; Akesson, G. (2002): WEFTA-Laborvergleichsuntersuchung zur pH-Bestimmung in Fischerei-Erzeugnissen. Eine gemeinsame Aktion der Western European Fish Technologists' Association (WEFTA interlaboratory comparison exercise on pH determination in fishery products. A cooperation of the Western European Fish Technologists' Association). Informationen für die Fischwirtschaft aus der Fischereiforschung, 49, 161-167. (in German with English summary) 

 

Abstract: An inter-laboratory trial was conducted on measurement of the pH of salt solutions and fish products using a pH meter. 14 laboratories participated; 2 salt solutions and 4 fish products were analysed with or without dilution 1:1 with water. 1 laboratory consistently gave poor results. The other laboratories gave very good results for most samples, generally ranging only 0.1 pH units from the calculated mean. Some laboratories performed relatively poorly with non-diluted fish products. Regular calibration of the apparatus is recommended, together with interlaboratory trials to detect deficiencies.

 

Full text: http://aquaticcommons.org/3164/

 

Salt

 

Karl, H.; Akesson, G.; Etienne, M.; Huidobro, A.; Luten, J.; Mendes, R.; Tejada, M.; Oehlenschläger, J. (2002): WEFTA interlaboratory comparison on salt determination in fishery products. Journal of Aquatic Food Product Technology, 11, 215-228.  

 

Abstract: 17 laboratories out of 10 countries participated in a WEFTA collaborative trial on salt determination in fish and fishery products. 4 samples containing different salt contents were distributed ( 2 spiked samples and 2 typical commercial products). All laboratories applied a common method involving the precipitation of fish proteins by Carrez I and II followed by titrimetric determination of the salt content according to Mohr. They also carried out salt determination by their own "home" methods. The following results were obtained: the recovery rate of the common method was nearly quantitative and the relative s.d. for reproducibility was between 1.96 and 2.64%, depending on the sample. Larger variations were obtained between different laboratories when applying their home methods. In one sample a significant difference of the measured salt content was found between the average results of the common and the home methods. Previous ashing of the sample always yielded lower salt contents compared to other determination procedures. Results demonstrated that the method is suitable for salt determination in fish and fishery products.

 

Full text: http://www.tandfonline.com/toc/wafp20/11/3-4

 

 

Isoelectric focusing

 

Etienne, M.; Jérôme, M.; Fleurence, J.; Rehbein, H.; Kündiger, R.; Yman, I.; Ferm, M.; Craig, A.; Mackie, I.; Jessen, F.; Smelt, A.; Luten, J. (1999): A standardized method of identification of raw and heat-processed fish by urea isoelectric focusing: a collaborative study. Electrophoresis, 20, 1923-1933.

 

Abstract: A urea-isoelectric focusing method of identifying fish species in processed fishery products was investigated as an interlaboratory collaborative study. The technique was optimized with respect to (i) protein extraction conditions, composition of the extraction solution (urea and SDS solutions), determination of protein concentrations of the fish extracts (five tested methods); (ii) nature of gel (with carrier ampholytes and iImmobilines), conditions of rehydration of commercial dry gels, urea concentration; (iii) staining conditions, Coomassie blue and silver staining. The results of various experiments were compared to select the most appropriate methodology with respect to the discrimination power of differentiating species with minimal influence of heat processing, to reproducibility, speed, and ease of application. The recommended method meets the requirements of food control and customs laboratories.

 

Full text: http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1522-2683(19990701)20:10%3C1923::AID-ELPS1923%3E3.0.CO;2-J/pdf

 

 

Mackie, I.; Craig, A.; Etienne, M.; Jérôme, M.; Fleurence, J.; Jessen, F.; Smelt, A.; Kruijt, A.; Yman, I. M.; Ferm, M.; Martinez, I.; Pérez-Martín, R.; Piñeiro, C.; Rehbein, H. (2000): Species identification of smoked and gravad fish products by sodium dodecyl suphate polyacrylamide gel electrophoresis, urea isoelectic focusing and native isoelectric focusing: a collaborative study. Food Chemistry, 71, 1, 1-7.

 

Abstract: A collaborative study by eight laboratories on the use of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), urea-isoelectric focusing (urea-IEF) and native isoelectric focusing for the identification of species of smoked salmonids, gravad salmonids and smoked eels was carried out. Minor changes took place in the profiles of the processed salmonid species with SDS-PAGE making it impossible or very difficult to identify closely related species. With urea-IEF fewer changes in the profiles due to processing were determined and the system generally had greater species-discriminating power for the processed salmonids than SDS-PAGE. The profiles of the eel species as obtained on SDS-PAGE or urea-IEF were not affected by smoking. Urea-IEF had greater species-discriminating power than SDS-PAGE for the eel species. Native IEF was useful in providing supplementary identification on species difficult to identify by SDS-PAGE or by urea-IEF in the case of cold smoked products.

 

Full text: http://www.sciencedirect.com/science/article/pii/S0308814600001473

 

 

Etienne, M.; Jérôme, M.; Fleurence, J.; Rehbein, H.; Kündiger, R.; Mendes, R.; Costa, H.; Perez-Martin, R.; Pineiro-Gonzalez, C.; Craig, A.; Mackie, I.; Yman, I. M.; Ferm, M.; Martinez, I.; Jessen, F.; Smelt, A.; Luten, J. (2000): Identification of fish species after cooking by SDS-PAGE and urea IEF: a collaborative study. J. Agric. Food Chem., 48, 2653- 2658.

 

Abstract: A collaborative study to validate the use of SDS-PAGE and urea IEF for the identification of fish species after cooking was performed by nine laboratories. By following optimized standard operation procedures 10 commercially important species (Atlantic salmon, sea trout, rainbow trout, turbot, Alaska pollock, pollack, pink salmon, Arctic char, chum salmon, and New Zealand hake) had to be identified by comparison with 22 reference samples. Generally, the urea extraction procedure appeared to be less efficient than the SDS extraction for protein solubilisation. Except for some species belonging to the Salmonidae family, both of the analytical techniques tested (urea IEF, SDS-PAGE) enabled species identification of the samples. SDS-PAGE (ExcelGel homogeneous 15%) appeared to be more suitable for the identification after cooking, of fish such as the tuna and salmon species which are characterized by neutral and basic protein bands, and urea IEF (CleanGel) was better for gadoid species, which are characterized by acid protein bands (parvalbumins). Nevertheless, in contentious cases it is preferable to use both analytical methods.

 

Full text: http://pubs.acs.org/doi/abs/10.1021/jf990907k

 

 

Etienne, M.; Jérôme, M.; Fleurence, J.; Rehbein, H.; Kündiger, R.; Mendes, R.; Costa, H.; Martinez, I. (2001): Species identification of formed fishery products and high pressure-treated fish by electrophoresis: a collaborative study. Food Chem., 72, 105-112.

 

Abstract: The suitability and reliability of three electrophoretic methods of fish species identification (urea isoelectric focusing (IEF), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native IEF) were evaluated on formed fish fillets and high pressure fish flesh by a collaborative study among four institutes. By following optimized standard operation procedures, the protein patterns of processed fish were compared to patterns of raw reference samples. The choice of the best method was influenced by the processing effect on the protein pattern. The proteins obtained from formed products were not denatured and therefore any of the three methods was proved to be adequate with a preference for native IEF which had a better discriminatory power for the analysed species. The high pressure process altered the proteins, and so only urea IEF and SDS-PAGE methods could be used, preferably those with the better discriminating power for the species being examined.

 

Full text: http://www.sciencedirect.com/science/article/pii/S0308814600002053

 

Piñeiro, C.; Barros-Velazquez, J.; Perez-Martin, R. I.; Martinez, I.; Jacobsen, T.; Rehbein, H.; Kündiger, R.; Mendes, R.; Etienne, M.; Jérôme, M.; Craig, A.; Mackie, I. M.; Jessen, F. (1999): Development of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis reference method for the analysis and identification of fish species in raw and heat-processed samples: a collaborative study. Electrophoresis 1999, 20, 1425-1432.

 

Abstract: A collaborative study was carried out in seven European labs with the aim of achieving a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) standard operation procedure to identify fish species in raw and cooked samples. Urea and SDS-containing solutions were evaluated as extractants. Several pre-electrophoretic operations - such as treatment with RNase/DNase, ultrafiltration and desalting - and up to ten types of gels and three SDS-PAGE systems were considered. The SDS-containing solution allowed a higher protein extractability than urea. Unlike urea extraction, SDS extraction seemed not to be influenced so much by the sample (raw, cooked at 60 °C and at 85 °C). Desalting, ultrafiltration or treatment with RNase/DNase did not improve the discriminatory power of the protein patterns. Commercial homogeneous 15% ExcelGels, especially when they were silver stained, yielded good results and afforded higher reproducibility, thus allowing a better matching of results among the laboratories participating in this collaborative study. Under optimized technical conditions all the fish species tested (both raw and cooked) yielded reproducible results and discriminated species-specific protein patterns.

 

Full text: http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1522-2683(19990601)20:7%3C1425::AID-ELPS1425%3E3.0.CO;2-R/abstract

 

Rehbein, H.; Kündiger, R.; Yman, I. M.; Ferm, M.; Etienne, M.; Jérôme, M.; Craig, A.; Mackie, I.; Jessen, F.; Martinez, I.; Mendes, R.; Smelt, A.; Luten, J.; Pineiro, C.; Perez Martin, R. (1999): Species identification of cooked fish by urea isoelectric focusing and sodiumdodecylsulfate polyacrylamide gel electrophoresis: a collaborative study. Food Chem., 67, 333-339.

 

Abstract: The suitability and reliability of urea IEF and SDS-PAGE for the identification of cooked fish flesh was tested by a collaborative study among nine laboratories. Urea IEF was performed with CleanGels as well as with ImmobilineGels, and ExcelGels were used for SDS-PAGE, enabling all three types of gels to be run in the same flat bed electrophoresis chamber. By strictly following optimised standard operation procedures (SOPs), five unknown cooked samples had to be identified with each technique using a set of 10 raw reference samples. With urea IEF only one out of 35 identifications was incorrect, and with SDS-PAGE a similar result was obtained. It was concluded that the applied methods are suitable for checking the species declaration of fishery products.

 

Full text: http://www.sciencedirect.com/science/journal/03088146/67/4

 

 

Nitrogen

 

Oehlenschläger, J. (1997): WEFTA interlaboratory comparison on nitrogen determination by Kjeldahl digestion in fishery products and standard substances. Informationen für die Fischwirtschaft, 44: 31-37 

 

Abstract: Fourteen laboratories in 12 European countries participated in an interlaboratory trial to assess the determination of nitrogen in fish or fish products by the Kjeldahl digestion technique. Standard samples of freeze dried ocean perch (Sebastes mentella) muscle, freeze dried European hake (Merluccius merluccius) muscle, lean white fish meal, l-histidine and urea were analysed. Each laboratory used its own variant of the Kjeldahl procedure. 13 of the 14 laboratories gave results with little scatter about the overall mean values. Coeff. of variation for individual laboratories was generally low (approx. 0.5%). The standard samples were analysed with 98% recovery and good accuracy and precision. Effects of the procedural variants on performance of the Kjeldahl digestion technique for determination of N in fish are discussed; the best results were achieved with a short digestion time (approx. 120 min), approx. 430 °C and selection of an appropriate catalyst.

 

Full text: http://aquaticcommons.org/3784/1/97-1_Seite031-037(engl).pdf

 

 

Sulfite

 

Luten, J.; Bouquet, W.; Oehlenschläger, J.; Meetschen, U.; Etienne, M.; Stroud, G.; Bykowski, P.; Batista, I.;  Vyncke, W.; Stefansson, G. (1997): An intercomparison study of the determination of sulfite in tropical shrimps by the West European Fish Technologists' Association (WEFTA). Food Research and Technology (Zeitschrift für Lebensmitteluntersuchung und - forschung) 204, 237-240.  

 

Abstract: Eight WEFTA laboratories participated in two series of intercomparison exercises on sulfite determination in tropical shrimps. Samples were spiked with sodium metabisulfite (Na2S2O5) and hydroxymethylsulfonate (HMS) at a level of 25-90 mg SO2/kg. Most of the laboratories determined the sulfite content with (modified) methods of Monier-Williams or De Vries et al. The overall mean recovery of sulfite was rather low (47-60%), which may be attributed to an irreversible reaction of sulfite with the tropical shrimps. The repeatability of the methods within the participating laboratories was good. However, reproducibility among the laboratories was poor. It has been shown that the sulfite content in tropical shrimps spiked with Na2S2O5 decreased during storage at -20 °C and at +4 °C. Hydroxymethylsulfonate was stable during storage.

 

Full text: http://rd.springer.com/article/10.1007/s002170050070

 

 

TVB-N

Vyncke, W.; Luten, J.; Moermans, R. (1987): Determination of total volatile bases in fish: a collaborative study by the West European Fish Technologists´ Association (WEFTA). Food Research and Technology (Zeitschrift für Lebensmitteluntersuchung und - forschung) 184, 110-114.

Abstract: Two series of collaborative tests were performed. Nine WEFTA laboratories participated in the first exercise, six in the second one. All laboratories applied a common method involving the precipitation of fish proteins by trichloroacetic acid followed by distillation of the extract after addition of sodium hydroxide ("Codex method”). They also carried out the TVB (total volatile bases) analyses by their own methods ("home methods”). Mackerel and cod were used. The results of both collaborative exercises showed important systematic errors between participating laboratories both with the codex and with the home methods. Better comparability was obtained when pure solutions of ammonia, di- and trimethyl-amine (the main components of TVB) were subjected to the same methods. On the other hand, the addition of TMA showed average recoveries of only 75% with significant variability among collaborators. The analytical methods should be further scrutinized and the effects of minor but possibly important variations should be investigated before proposing a reference TVB method.

Full text: http://rd.springer.com/journal/217/184/2/page/1

Antonacopoulos, N. and Vyncke, W. (1989): Determination of volatile basic nitrogen in fish: a third collaborative study by the West European Fish Technologists´ Association (WEFTA). Food Research and Technology (Zeitschrift für Lebensmitteluntersuchung und - forschung) 189, 309-316.

Abstract: Thirteen laboratories participated in the third WEFTA exercise on determination of volatile base nitrogen (TVB-N) in fish. Plaice and herring, each at three stages of freshness, were distributed. Eleven laboratories applied the direct distillation of fish after addition of magnesium oxide (MgO) (Antona method). Six laboratories carried out analysis by their own methods also, while two laboratories used their own methods only. In five of these own methods, extracts with trichloroacetic or perchloric acid made alkaline with sodium hydroxide (NaOH) were distilled. The results of the third exercise confirm in principle the findings of the second trial, in which participants used an extraction procedure (Codex method). Both Antona and Codex methods show similar systematic errors. The mean recoveries of TVB-N by the Antona method, related to reference values of the test materials, were 94.7±9.4% for plaice and 91.6±8.8% for herring: the mean recoveries of added ammonium sulphate were similar, 94.1% and 88.7%, respectively. Supplementary tests indicated that a pH greater than 11 during distillation promotes secondary generation of ammonia; MgO had an advantage over NaOH in this respect. In order to reduce variations in TVB-N results, a detailed description of all steps of the method is an essential precondition for standardisation: a draft is appended. The TVB-N method is judged to be suitable as a standard method for routine quality control. Being quick and relatively inexpensive to carry out, the direct distillation procedure is preferred for large numbers of samples; extraction methods permit parallel determination of other amines, such as trimethylamine, where confirmation is required.

 

Full text: http://rd.springer.com/article/10.1007/BF01683206

 

 

Fish core content

 

Bon, J.; Brünner, K. K.; Aitken, A. (1986): Determination of fish core content in coated products: Interlaboratory WEFTA studies of three procedures. J. Assoc. Off. Anal.Chem. 69: 75–79.