Development of reference materials for paralytic shellfish poisoning toxins.

A project was undertaken to develop mussel reference materials that were certified for their mass fractions of saxitoxin and decarbamoyl-saxitoxin. Fifteen laboratories from various European countries participated. Three of these had major responsibility for substantial parts of the work and overall coordination of the project. The project involved 4 main activities: (1) procurement and characterization of calibrants; (2) improvement of analytical methodology; (3) preparation of reference materials, including homogeneity and stability studies; (4) 2 interlaboratory studies and a certification exercise. The joint activities resulted in 3 homogeneous and stable reference materials: 2 lyophilized mussel materials with and without naturally incurred paralytic shellfish poisoning (PSP) toxins, and a saxitoxin enrichment solution. The reference materials were certified with respect to their saxitoxin and decarbamoyl-saxitoxin content. The lyophilized mussel material with PSP toxins (CRM 542) contained <0.07 mg saxitoxin x 2HCl/kg and 1.59 +/- 0.20 mg decarbamoyl-saxitoxin x 2HCl/kg. The lyophilized mussel material without PSP toxins (CRM 543) contained <0.07 mg saxitoxin x 2HCl/kg and <0.04 mg decarbamoyl-saxitoxin x 2HCl/kg. The certified value of the saxitoxin mass fraction in the saxitoxin enrichment solution (CRM 663) was 9.8 +/- 1.2 microg/g.

The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. II: Certification study.

This paper describes the second part of a project undertaken to develop certified mussel reference materials for paralytic shellfish poisoning toxins. In the first part two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin and decarbamoyl-saxitoxin in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the certification exercise. Fifteen laboratories participated in this certification study and were asked to measure saxitoxin and decarbamoyl-saxitoxin in rehydrated lyophilized mussel material and in a saxitoxin-enriched mussel material. The participants were allowed to use a method of their choice but with an extraction procedure to be strictly followed. The study included extra experiments to verify the detection limits for both saxitoxin and decarbamoyl-saxitoxin. Most participants (13 of 15) were able to meet all the criteria set for the certification study. Results for saxitoxin.2HCl yielded a certified mass fraction of <0.07 mg/kg in the rehydrated lyophilized mussels. Results obtained for decarbamoyl-saxitoxin.2HCl yielded a certified mass fraction of 1.59+/-0.20 mg/kg. The results for saxitoxin.2HCl in enriched blank mussel yielded a certified mass fraction of 0.48 +/- 0.06 mg/kg. These certified reference materials for paralytic shellfish poisoning toxins in lyophilized mussel material are the first available for laboratories to test their method for accuracy and performance.

Quality of Life Programme--food, nutrition, and health--projects promotion.

The EC Quality of Life Programme (QoL), Key Action 1--Food, Nutrition & Health aims at providing a healthy, safe, and high-quality food supply leading to reinforced consumer's confidence in the safety of the European food. Key Action 1 is currently supporting several European projects investigating analytical methods for food control including sensors, risk analysis, and food safety standardisation. Their objectives range from the development and validation of prevention strategies for mycotoxin formation via the development of a communication platform for Genetically Modified Organisms (GMO), validation and standardisation of diagnostic Polymerase Chain Reaction (PCR) for food-borne pathogens, up to the evaluation of the potential cancer-preventing activity of pro- and pre-biotic ("SYNBIOTIC") combinations in human volunteers. This paper also informs on future research needs in food safety.

Contribution of European research to risk analysis.

The European Commission's, Quality of Life Research Programme, Key Action 1-Health, Food & Nutrition is mission-oriented and aims, amongst other things, at providing a healthy, safe and high-quality food supply leading to reinforced consumer confidence in the safety, of European food. Its objectives also include the enhancing of the competitiveness of the European food supply. Key Action 1 is currently supporting a number of different types of European collaborative projects in the area of risk analysis. The objectives of these projects range from the development and validation of prevention strategies including the reduction of consumers risks; development and validation of new modelling approaches, harmonization of risk assessment principles methodologies and terminology; standardization of methods and systems used for the safety evaluation of transgenic food; providing of tools for the evaluation of human viral contamination of shellfish and quality control; new methodologies for assessing the potential of unintended effects of genetically modified (genetically modified) foods; development of a risk assessment model for Cryptosporidium parvum related to the food and water industries, to the development of a communication platform for genetically modified organism, producers, retailers, regulatory authorities and consumer groups to improve safety assessment procedures, risk management strategies and risk communication; development and validation of new methods for safety testing of transgenic food; evaluation of the safety and efficacy of iron supplementation in pregnant women, evaluation of the potential cancer-preventing activity of pro- and pre-biotic ('synbiotic') combinations in human volunteers. An overview of these projects is presented here.

An Internet compendium of analytical methods and spectroscopic information for monomers and additives used in food packaging plastics.

An internet website (http:¿cpf.jrc.it/smt/) has been produced as a means of dissemination of methods of analysis and supporting spectroscopic information on monomers and additives used for food contact materials (principally packaging). The site which is aimed primarily at assisting food control laboratories in the European Union contains analytical information on monomers, starting substances and additives used in the manufacture of plastics materials. A searchable index is provided giving PM and CAS numbers for each of 255 substances. For each substance a data sheet gives regulatory information, chemical structures, physico-chemical information and background information on the use of the substance in particular plastics, and the food packaging applications. For monomers and starting substances (155 compounds) the infra-red and mass spectra are provided, and for additives (100 compounds); additionally proton NMR are available for about 50% of the entries. Where analytical methods have been developed for determining these substances as residual amounts in plastics or as trace amounts in food simulants these methods are also on the website. All information is provided in portable document file (PDF) format which means that high quality copies can be readily printed, using freely available Adobe Acrobat Reader software. The website will in future be maintained and up-dated by the European Commission's Joint Research Centre (JRC) as new substances are authorized for use by the European Commission (DG-ENTR formerly DGIII). Where analytical laboratories (food control or other) require reference substances these can be obtained free-of-charge from a reference collection housed at the JRC and maintained in conjunction with this website compendium.

Sampling plans for the determination of aflatoxin B1 in large shipments of animal feedstuffs.

Incremental samples (50, 100, and 500 g) were systematically collected from large shipments of copra meal pellets, copra cake, and palm kernel cake to study the distribution of aflatoxin B1 and evaluate adherence of distribution to the model, CV(2)is (EQ) = A + B/Mis (where CVis = coefficient of variation of the true concentration of aflatoxin B1 within the incremental samples; Mis = mass of the incremental samples; and A and B are constants). Also evaluated was the distribution of aflatoxin B1 among 1 kg composite samples, produced both by random combination of existing incremental samples and by collection of 1 kg composite samples (composed of 10 x 100 g increments) from additional batches of copra meal pellets and cottonseed cake. The efficiency of selected sample preparation (grinding and subdivision) procedures was compared, culminating in the development and description of a variety of sampling plans. The coefficient of variation (CV) among incremental samples varied from 0 to 38%, and was independent of incremental sample size. No significant difference (F-test, 5% significance level) was found between the efficacy of 4 sample preparation methods when these methods were applied to the commodities described above. Various sampling plans were evaluated with estimated CVs from 4.0 to 12.5%, for the aflatoxin B1 content of the composite samples.

The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. I: Interlaboratory studies of methods of analysis.

This paper describes the first part of a project undertaken to develop mussel reference materials for Paralytic Shellfish Poisoning (PSP) toxins. Two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin (STX) and decarbamoyl-saxitoxin (dc-STX) in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the second part of the project: the certification exercise. In the first study, 18 laboratories were asked to measure STX and dc-STX in rehydrated lyophilized mussel material and to identify as many other PSP toxins as possible with a method of their choice. In the second interlaboratory study, 15 laboratories were additionally asked to determine quantitatively STX and dc-STX in rehydrated lyophilized mussel and in a saxitoxin-enriched mussel material. The first study revealed that three out of four post-column derivatization methods and one pre-column derivatization method sufficed in principle to determine STX and dc-STX. Most participants (13 of 18) obtained acceptable calibration curves and recoveries. Saxitoxin was hardly detected in the rehydrated lyophilized mussels and results obtained for dc-STX yielded a CV of 58% at a mass fraction of 1.86 mg/kg. Most participants (14 out of 18) identified gonyautoxin-5 (GTX-5) in a hydrolysed extract provided. The first study led to provisional criteria for linearity, recovery and separation. The second study revealed that 6 out of 15 laboratories were able to meet these criteria. Results obtained for dc-STX yielded a CV of 19% at a mass fraction of 3.49 mg/kg. Results obtained for STX in the saxitoxin-enriched material yielded a CV of 19% at a mass fraction of 0.34 mg/kg. Saxitoxin could not be detected in the PSP-positive material. Hydrolysis was useful to confirm the identity of GTX-5 and provided indicative information about C1 and C2 toxins in the PSP-positive material. The methods used in the second interlaboratory study showed sufficiently consistent analysis results to undertake a certification exercise to assign certified values for STX and dc-STX in lyophilized mussel.

Establishment of a reference collection of additives and an analytical handbook of reference data to support enforcement of EU regulations on food contact plastics.

A collection has been made of additives that are required as analytical standards for enforcement of European Union legislation on food contact plastics. The 100 additives have been characterized by mass spectrometry, infra-red spectroscopy and proton nuclear magnetic resonance spectroscopy to provide reference spectra. Gas chromatographic retention times have been recorded to facilitate identification by retention index. This information has been further supplemented by physico-chemical data. Finally, chromatographic methods have been used to indicate the presence of any impurities in the commercial chemicals. Samples of the reference substances are available on request and the collection of spectra and other information will be made available in printed format and on-line through the Internet. This paper gives an overview of the work done to establish the reference collection and the spectral atlas, which together will assist enforcement laboratories in the characterization of plastics and the selection of analytical methods for additives that may migrate.

Activities and current research from the EC, standards, measurements and testing programme (SMT) in the area of food contact materials.

The EC, Standards, Measurements and Testing Programme (SMT) is the successor of the Measurements and Testing Programme (M&T) and the Community Bureau of Reference (BCR). Its objectives include the provision of research and technical support to standardization and health of the society when it is required to improve the competitive position of European industry, and for the development or implementation of Community policy. The SMT-Programme is currently supporting a number of different types of collaborative projects in the food packaging sphere. Their objectives range from method development via preparation and certification of reference materials, preparation of a handbook and the update of a spectral atlas to pre-normative research providing information on a number of sources and wide range of packed products being transported throughout Europe.

An intercomparison of methods for the determination of ochratoxin A in pig kidney.

The preparation of two pig kidney materials is described, together with a report on the results of an intercomparison study of methods to determine ochratoxin A levels in these materials. The materials were prepared, and the intercomparison study carried out within the European Commission. Measurements and Testing Programme, which is the successor of the Community Bureau of Reference (BCR). The materials were prepared from blank and naturally-contaminated pig kidneys and were freeze-dried. Details are given on the freeze-drying and packaging procedure, and the checks to ensure homogeneity and stability of the freeze-dried materials. The intercomparison study involved 20 European laboratories, which analysed the naturally-contaminated (freeze-dried) sample (ochratoxin A content approximately 10 micrograms/ kg based on fresh weight) and the 'blank' sample (ochratoxin A content < 0.6 microgram/kg based on fresh weight) using a variety of procedures for extraction and clean-up. All laboratories used HPLC as the determinative step. Recoveries were found to range from 43 to 128%. The study highlighted problems with recovery of spiked ochratoxin A from freeze-dried pig kidney material. There is a clear need to improve analytical performance, particularly with respect to the extraction efficiency from this type of matrix.

Ochratoxin A in wheat: certification of two reference materials.

The preparation of two wheat reference materials and the certification of their ochratoxin A content is described. The materials were prepared and certified within the European Commission. Measurements and Testing Programme (M&T). The first and second phases of this project, two intercomparisons of procedures for the determination of ochratoxin A in wheat, at levels of approximately 13 micrograms/kg, and 7 micrograms/kg, respectively have already been reported. This paper describes the work carried out in certification of the ochratoxin A content (mass fraction) of two wheat reference materials: a blank wheat CRM 471, and a contaminated wheat, CRM 472. These materials were prepared for use in the second intercomparison referred to above and reported previously. Reference material CRM 472 was prepared from naturally-contaminated wheat blended with the blank wheat (CRM 471). Details are given of the milling, blending and packaging procedure, and the checks to ensure homogeneity and stability of the material. The certification exercise was carried out by 15 laboratories using a variety of extraction and clean-up procedures, and the certified ochratoxin A content (mass fraction) of CRM 471 was < 0.6 microgram/kg. The value for CRM 472 was 8.2 micrograms/kg with an uncertainty of 1.0 microgram/kg.

Stability of fumonisins at different storage periods and temperatures in gamma-irradiated maize.

The effect of gamma-irradiation on fumonisin B1 (FB1) and fumonisin B2 (FB2) occurring in maize materials has been investigated together with the stability of fumonisins in gamma-irradiated maize stored at different temperatures (-18 to +40 degrees C) for different periods (2, 4, 13 and 26 weeks). Fifteen KGy gamma-irradiation was required to sterilize efficiently maize flour. This process caused a decrease in fumonisin content of about 20%. The stability studies showed that fumonisins are stable in gamma-irradiated maize for at least 6 months at 25 degrees C or at least 4 weeks at 40 degrees C. These data indicate that gamma-irradiation is an appropriate technique for obtaining sterilized maize materials to be used for intercomparison studies on analytical procedures for the measurement of the fumonisin content.

European intercomparison study for the determination of the fumonisins content in two maize materials.

The results of an intercomparison study for the analysis of fumonisin B1 (FB1) and fumonisin B2 (FB2) in a contaminated maize material (Maize B, containing approximately 2 micrograms/g FB1 and 1 micrograms/g FB2) and its 'blank' counterpart (Maize A, containing less than 20 ng/g FB1 and FB2) are reported. Maize materials were distributed in 60 g sachets, submitted to gamma-irradiation at 15 kGy, and distributed to participating laboratories. The study was carried out by 24 European laboratories, most of which have national or international responsibilities for food/feed quality control. Participants used basically one method with some modifications, based on clean-up through SAX minicolumn and reversed phase HPLC with fluorescence detection of the OPA-fumonisin derivatives. The intercomparison study generated data (after correction for recoveries) with repeatability and reproducibility levels of: RSDr = 10% and RSDR = 11% for FB1; RSDr = 11% and RDSR = 13% for FB2. The recoveries obtained by most participants were considered low (70% for FB1 and 69% for FB2), being considerably affected by the extraction mode. Average recoveries for laboratories using blending were 62% and 60%, whereas for laboratories using shaking they were 85% and 86% for FB1 and FB2, respectively. The day-to-day- data showed a between-day, within-laboratory repeatability lower than 15% (CV < 15%) for FB1 for all laboratories, whereas only three laboratories obtained a between-day, within-laboratory reproducibility higher than 15% (CV > 15%) for FB2.

Beta-agonists in animal feed. IV: Intercomparison study of a candidate reference confirmatory method.

The objective of this intercomparison study was to evaluate the qualitative aspects and the interlaboratory performance of the method selected to be recommended as the official Community reference confirmatory method for the analysis of beta-agonists in animal feed. This method contains three possible options, i.e. a narrow range method for clenbuterol-type compounds based either on HPLC or on GCMS as the end-determination step and a broad range GCMS method for clenbuterol-type and salbutamol-type-beta-agonists. Three types of animal feed materials were provided: a series of blank materials and two series of materials contaminated with clenbuterol and salbutamol at a low and a high level, respectively. The results showed that the majority of the laboratories were able to identify blank, low and high level materials both for clenbuterol and salbutamol. For clenbuterol the narrow range GCMS method has been shown to be the most satisfactory. Although the participants had comments on the purity of the extracts obtained by means of the broad range method it was found appropriate as a multi-residue method which is able to measure simultaneously clenbuterol-type and salbutamol-type beta-agonists. A statistical evaluation of the quantitative measurement was also performed.

beta-agonists in animal feed. III: Optimization of the clean-up and the end-determination step.

This study represents the second part of an interlaboratory study intended to develop an official modular Community confirmatory method for the detection of beta-agonists in animal feed. Homogeneous pools of primary extracts were prepared by means of an extraction module based on the conclusions of a previous part of this work. The primary extracts were further processed by four laboratories each using a different clean-up scheme. The final extracts thus obtained were cross-distributed between the same laboratories and measured either by GCMS or HPLC. Two laboratories (B and D) applied separate clean-up schemes for clenbuterol and salbutamol. All clean-up schemes for clenbuterol were found to be compatible with all end-determination steps. In contrast, for salbutamol clean-up method D was found not to be compatible with the end-determination steps applied by laboratories B and C. The results of this study have clearly demonstrated that the clean-up methods for both clenbuterol and salbutamol applied by laboratory B yielded superior recoveries with an acceptable standard deviation. Therefore, in conclusion to this study, the participating laboratories recommend the clean-up schemes applied by laboratory B to serve as part of the official Community confirmatory method.

Beta-agonists in animal feed. I: First intercomparison of methods of analysis for clenbuterol in animal feed materials.

The European Commission, Measurement and Testing Programme (BCR) has initiated a project to improve the methodology for analysis of beta-agonists in animal feeding stuffs. An intercomparison of methods for clenbuterol in animal feed is described. The study involved 13 European laboratories which analysed a blank feed and three feed samples with three different levels of clenbuterol contamination. The participants used a variety of extraction (organic or aqueous solvents), clean-up (liquid-liquid, silica and C-18 solid phase and immuno-affinity chromatography) and end-point detection (HPLC, GC-MS and TLC) steps. The purpose of this study was to identify and to quantify clenbuterol. The coefficient of variation from all the results for the low level (25 micrograms/kg) was 39%, for the intermediate level (100 micrograms/kg) 52% and for the high level (1000 micrograms/kg) 35%. The study showed that the initial extraction, the modular clean-up step and their compatibility to the HPLC and the GC-MS determination step were critical steps.

Beta-agonists in animal feed. II: Optimization of the extraction.

New projects of the European Commission, Measurement and Testing Programme (BCR) were set up in order to develop a modular sample preparation system for the determination of beta-agonists and animal feeds. Three phases are included: an extraction study, a clean-up study and finally a Second Intercomparison. This paper describes the extraction study in which four laboratories were involved. A total of 33 extraction conditions were tested regarding their yield on clenbuterol and salbutamol, their compatibility towards several clean-up and chromatographic end-methods and the influence of undesired coextractives. The conditions differed with respect to five factors: with or without organic solvent, temperature, pH, agitation and centrifugation. Their influence was examined via a ruggedness-test approach. A unique set-up allowed the combination of individual results in a complete factorial design. The addition of an organic solvent was found to be the most important factor. Interactions between factors were also studied. The best combinations of factors regarding the extraction are given. Finally limits for applicability and influence of organic solvents, pH and temperature were evaluated in a fifth laboratory towards enzyme immunoassay as detection method.

Ochratoxin A in wheat: a second intercomparison of procedures.

The European Commission, Measurements and Testing Programme (BCR) has undertaken a project to improve methodology and to prepare certified reference materials for ochratoxin A determination. The first phase of this project, an intercomparison of procedures for the determination of ochratoxin A in wheat, at a content of approximately 13 micrograms/kg, has already been reported. The second intercomparison study, described in this paper, involved 26 European laboratories, from 11 countries, which analysed wheat naturally contaminated at a level of approximately 7 micrograms/kg, and a 'blank' wheat sample (ochratoxin A content < 0.2 microgram/kg). The participants used a variety of procedures which involved different extraction solvents and clean-up procedures. All laboratories used HPLC as the determinative step. Some laboratories also used immunoaffinity column clean-up in comparison with their normal method. Recoveries of the normal methods used by laboratories ranged from 58 to 114%; only three laboratories obtained recoveries outside the accepted range of 70 to 110%. Recoveries of the immunoaffinity column methods, using two sources of column, ranged from 58 to 114% for one and from 4 to 86% for the other. The between-laboratory reproducibility coefficient of variation for all results was 34% for the normal methods, and 34 and 42% for the two types of immunoaffinity columns. It was noted that, after the results were corrected for spike recovery, some laboratories became outliers owing to low spike recoveries. Further investigations of the spiking protocols used by each laboratory showed that the time left for evaporation of the spiking solvent was crucial to the recovery obtained.

BCR- and M&T-activities in the area of mycotoxin analysis in food and feedstuffs.

In order to avoid undesirable side effects such as high contents of mycotoxins in food and feed, measurements at critical steps of the food and feed production chain are performed. These measurements need to be accurate and precise to satisfy these aims. The accuracy and precision requirements to measurements form the basis of the Bureau Communautaire de Référence (BCR)-and Measurements and Testing (M&T)-Projects. Such projects aim to improve and/or develop analytical methodology, sampling plans, to harmonise agreement of results between European Union Member States and to prepare suitable certified reference materials (CRMs). The selection of the matrix CRMs and their corresponding level of mycotoxin contamination is made on the basis of requirements of the applied analytical procedures, stability requirements, regulations, norms, and inter-or intra-trade agreements which are in relation to quality specifications of imported and/or exported goods. The availability of appropriate matrix CRMs is not only a prerequisite for the implementation of directives and norms. It allows the validation of new methods and provides a possible solution to trade disputes, means for the statistical control of analytical results, tools for laboratory accreditation, and it is the basis for harmonisation and traceability of proficiency schemes.

Occurrence of fumonisins in Europe and the BCR--measurements and testing projects.

Fumonisins are mycotoxins mainly produced by Fusarium moniliforme, one of the most prevalent seed-borne fungi of maize. Strains of Fusarium moniliforme isolated from cereals in Europe produced in cultures high levels (up to 4 mg/g) of fumonisins B1 and B2 (FB1 and FB2). FB1 and FB2 have been found in maize and maize-based foods and feeds in most European countries. In Italy these mycotoxins have been directly involved in a fatal outbreak of equine leukoencephalomalacia, and have been found at different levels in most maize-based food products, including polenta--a staple food in a region with high incidence of esophageal cancer. The European Commission, Community Bureau of Reference (BCR) and Measurements and Testing Programme (M&T), has undertaken two consecutive intercomparison studies, involving over 20 laboratories from 12 European countries, to improve the quality of fumonisins analysis at European level. The first study consisted in the evaluation of the determination of fumonisins in an unknown solution, while the second one involved the analytical methodology for fumonisins in contaminated and blank maize samples. A definite improvement in the performance of the different laboratories has been observed in the second study. In view of the production of reference materials certified for their fumonisins contents, new analytical procedures need to be developed.