- Development and validation of analytical methods for several families of compounds in food matrices: antibiotics, selenium species, sugar analysis in industrial syrup, radio nuclide determination;
- Analytical method development and validation for selected proteins and metal-binding proteins of high interest;
- Support for the implementation of cosmetic Directives: European reference method for oxidative hair dyes.
- Specific Deliverables to DGs;
- Validated method for sugars in industrial syrups (DG AGRI);
- Validated method for oxidative hair dyes in cosmetic formulations (DG ENTR);
- Measurement of selenium species in reference materials (DG RTD, IRMM Reference Materials unit);
- Deliverables as a result of the research;
- Validation of methods for quinolone antibiotics in meat samples;
- Participation to the certification of selected proteins in clinical samples.
Summary of deliverables made by: 31/12/2001
Oxidative Hair Dyes EC Directive: 76/768/EEC DG ENTR.
This research is required for the implementation of the Cosmetic Directive through harmonisation of analytical methods. At the 17th plenary meeting on 12.06.01 of the Scientific Committee on Cosmetic and Non-food products, a population based control study showed a significantly increased risk of bladder cancer among long-term regular users of hair dyes.
On discussions with the working group "Methods of Chemical Analysis of Cosmetics", 5 commercial formulations (3 shampoos, 2 creams, blond and brown shades) and 16 oxidative hair dyes were proposed for complete validation with a maximum of 8 in a formulation. Change of the sample preparation and re-optimisation of the LC-DAD method was completed as well as quantifying the maximum deviation in concentration for each formulation.
Final in-house validation was completed with the 16 compounds fully identified and quantified in the 5 selected commercial formulations. The maximum deviation obtained on the concentration was less than 10% in most cases. For the latter, a complete and detailed standard operating procedure was also written. The method was presented in details to the expert working group of DG ENTR.
Se in Feed Stuff EC Directives 70/524/CEE, 89/398/CEE DG RTD.
Scientific Committee on Food (SCF/CS/ADD/NUT/20/FINAL)
This is a key issue for consumer health and greatly impinges upon the quality of food (Se enriched yeast is, for example, used as a food supplement in cancer prevention).
For the SCA MULSPOT (MULti SPecies in Oyster Tissue) results of the inter-comparison exercise for Se determination in oyster tissue (T 38 material) have been obtained and were sent to the coordinator in Rome. Furthermore, a new sample preparation procedure (enzymatic hydrolysis in two steps) has been developed for Se extraction in oyster tissue and applied to BCR 710 candidate reference material. Experiments in anion-exchange HPLC-ICP-MS show that selenomethionine and selenocyctine represent about 90% of the total Se present in oyster tissue. Results will be discussed in October 2001 in a meeting organised by DG RTD (M. Soren Bowadt).
For the SCA SEAS (Feasibility Studies for Speciated CRMs for Arsenic in Chicken, Rice, Fish, Soil and Selenium in Yeast and Cereal), following the first meeting in Madrid, (March 2001), base materials were selected and delivered for certification of Se and speciation analysis in selenised yeast. Preliminary studies for the determination of various Se-containing proteins in selenised yeast extracts started and first results are promising and allow detecting at least four Se-containing proteins in soluble fractions in aqueous solution.
A method has been developed for the determination of low-molecular mass Se-containing compounds in yeast and this method was applied to the candidate reference material (selenised yeast). It allows the identification and quantitation of selenomethionine, selenocystine and inorganic Se. The study revealed the presence of unidentified Se species that account for more than 60% of the total Se present in yeast.
Sugar in industrial syrups Dir. 79/796/CEE, Reg. 394/70 DG AGRI
On direct request of DG AGRI a modern method for sugar in industrial syrups was needed to replace an outdated official method.
IRMM started to organise an inter-laboratory comparison for the validation of an analytical method for the determination of sugars in industrial syrups. The protocol and the samples (2 reference materials 351J and 351K and 3 industrial syrups) were sent to the 17 participants. The results are expected by October 2001.
Proteins/metal-binding Proteins Directives 83/417/CEE, 91/32/CEE -La and the two genetic variants ß-LgB and ß-LgA. This has led to overall improvements in the separation and quantification (new calibrations curves established) of the seven major proteins in bovine milk. A complete uncertainty budget as well as detailed statistical "fitness for purpose" evaluations (e.g. ANOVA) was established for the method. The method was presented to the expert group on dairy products of DG AGRI and an Expression of Interest was submitted with 6 partners.
For Metal-Binding Proteins (MBPs) a Capillary Electrophoresis method was optimised for the separation of 11 selected proteins. Anion exchange chromatography was applied to the determination of transferrin in fresh human serum (non-lyophilized). Separation of some glycosylated sub-isoforms of transferrin was also achieved in both standard solutions and human serum.
In the frame of the certification of a reference material for haemoglobin isoform analysis, experiments were carried out on the determination of the glycated fraction of haemoglobin HbA1c.(DGs AGRI and SANCO.
Reference method for milk proteins are required in order to control potential fraudulent manipulations.
For milk authentication major improvements were obtained in the separation between the major whey proteins, A method based on cation exchange chromatography has been developed. Pre-treatment of the blood samples and the chromatographic parameters affecting the separation were optimised. Furthermore, the method proposed by IFCC is being implemented using both LC-MS and LC-CE.
Antibiotics in Food Directive 70/524/CEE, Regulation 2821/98 DG ENTR
Analytical methods for antibiotics in food is a key issue for the control of safety and quality of food.
The MS detection of Deuterated Norfloxacine was optimised and the LC-MS-MS analysis of 11 other quinolones in aqueous solution was performed. The full LC-MS-MS analysis of quinolones was then validated (in terms of selectivity, linearity, accuracy, repeatability and estimated intermediate precision) for pig kidney samples.
For the LC-ECD method for macrolides, an additional compound (Roxithromycin) was added so a reasonable separation of the four major components of a solution containing four commercial (low purity) macrolides has been performed. A systematic pH study for a total of nine macrolides was carried out individually using both detectors e.g. DAD and ECD on line.
For the CE-MS method, investigations were conducted to solve the memory effect caused by quinolone antibiotics adhering to the capillary.
Database for Consumer Protection (CPS) 76/768/EEC and 93/35/EEC DG SANCO
The creation of a thematic network to develop a platform for consumer protection in Europe based on the CPS database was deemed the best possible solution for redeploying the database. The study and arrangements made by CARTC (Consumers' Association Research and Testing Centre, UK) on this respect were followed up and an expression of interest was submitted. CARTC will formally apply (September 2001) for the creation of a FP5 Thematic Network. Redeployment of the CPS dB for industrial use (labelling of food products) is presently being considered. This task has been officially finalised at IRMM.
Output Indicators and Impact
For the characterisation of clinical diagnostic materials, the identification and quantitative determination of complex proteins like haemoglobin or myoglobin that are used as new diagnostic markers and produced as global CRMs (in support of the implementation of the IVD Directive 98/79/EC) will be performed. The characterisation of bio-molecules (like proteins in support to the production of CRMs for clinical diagnosis) will figure strongly in the future. The development of standardised control methods to be used in the fight against fraud by custom laboratories or national competent authorities (e.g. to determine antibiotics in meat and proteins in milk (DGs AGRI and TAXUD) will also be expanded. Proficiency Testing Programmes will be developed to provide support to the Community Reference Laboratories (CRLs) and National Reference Laboratories (NRLs) for the control of contaminants and residues in foods stuff (e.g. heavy metals and antibiotics in meat).
Summary of the project
With globalisation, EU expansion and an increased importance attached to world trade issues, more scientific reference methods are needed to enact EU policies and provide hands-on scientific support towards EU harmonisation and interaction with the EU's trading partners, particularly future Member States.
Reference analytical measurements are developed to provide cornerstones for ensuring the quality and safety of food, agricultural and consumer products, since they allow the unambiguous characterisation of a product in respect to its chemical composition. The reference analytical measurements performed at IRMM are fully validated, meaning that the performance characteristics have been extensively established. They thus fulfil the requirements for reliability and accuracy to the highest State-of-the-Art degree. Reference analytical measurements are developed at IRMM on behalf of Directorates General (DGs) to support the implementation of Community legislation and for the IRMM production programme of Reference and Proficiency Testing Materials (CRMs, PTMs).
An in-house potential for certification of reference materials is indispensable and serves to provide credibility for the assignment of reference values for CRMs and PTMs as required by internationally agreed concepts for CRM production (e.g. very strongly emphasised by the no. 1 provider of CRMs, the NIST in the USA) and in metrological concepts for CRM certification. The mutual recognition of CRMs between the US and EU providers (IRMM being globally the second largest provider) is however a goal foreseen in the EU-US Mutual Recognition Agreement developed under the Transatlantic Economic Partnership.
The continuous compilation of appropriate analytical methods and know-how will also form an important asset for the scheduled take-over of the production of the BCR-CRMs from the Research DG (in the new Framework Programme), because then a substantial amount of certification work will be performed in-house, which will often not only be more reliable but also usually more cost effective.
The Analytical Laboratory at IRMM hosts a consortium of high-performance instrumentation including HPLC-UV, HPLC-MS-MS, LC-ICP-MS, LC-HG-AAS, CE-UV, CE-MSn, ICP-MS, AAS, ICP-OES, NAA, XRF. With this set-up the IRMM is one of the few Metrology Institutes, which has a large potential for chemical measurements.