Organization and ELISA-Based Results of the First Proficiency Testing to Evaluate the Ability of European Union Laboratories to Detect Staphylococcal Enterotoxin Type B (SEB) in Buffer and Milk.

The aim of this work was to organize the first proficiency test (PT) dedicated to staphylococcal enterotoxin B (SEB) detection in milk and buffer solutions. This paper describes the organization of the PT trial according to EN ISO 17043 requirements. Characterization of the SEB stock solution was performed using SDS-PAGE and SE-specific ELISA, and amino acid analysis was used to assign its protein concentration.

Certified reference materials for testing of the presence/absence of Staphylococcus aureus enterotoxin A (SEA) in cheese.

Staphylococcal enterotoxins (SEs) account for a substantial number of food-poisoning outbreaks. European legislation (Commission Regulation 1441/2007) stipulates the reference procedure for SE analysis in milk and dairy products, which is based on extraction, dialysis concentration and immunochemical detection using one of two approved assays (VIDAS(®) SET2, Ridascreen(®) SET Total). However, certified reference materials (CRMs) are lacking to support laboratories in performing reliable detection of Staphylococcus aureus enterotoxin A (SEA) in relevant matrices at sub-nanogram per gram levels.

Generation and Characterization of Six Recombinant Botulinum Neurotoxins as Reference Material to Serve in an International Proficiency Test.

The detection and identification of botulinum neurotoxins (BoNT) is complex due to the existence of seven serotypes, derived mosaic toxins and more than 40 subtypes. Expert laboratories currently use different technical approaches to detect, identify and quantify BoNT, but due to the lack of (certified) reference materials, analytical results can hardly be compared. In this study, the six BoNT/A1-F1 prototypes were successfully produced by recombinant techniques, facilitating handling, as well as improving purity, yield, reproducibility and biosafety.

An International Proficiency Test to Detect, Identify and Quantify Ricin in Complex Matrices.

While natural intoxications with seeds of Ricinus communis (R. communis) have long been known, the toxic protein ricin contained in the seeds is of major concern since it attracts attention of those intending criminal, terroristic and military misuse. In order to harmonize detection capabilities in expert laboratories, an international proficiency test was organized that aimed at identifying good analytical practices (qualitative measurements) and determining a consensus concentration on a highly pure ricin reference material (quantitative measurements).

Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test.

In the framework of the EU project EQuATox, a first international proficiency test (PT) on the detection and quantification of botulinum neurotoxins (BoNT) was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification.

Characterization of Ricin and R. communis Agglutinin Reference Materials.

Ricinus communis intoxications have been known for centuries and were attributed to the toxic protein ricin. Due to its toxicity, availability, ease of preparation, and the lack of medical countermeasures, ricin attracted interest as a potential biological warfare agent. While different technologies for ricin analysis have been established, hardly any universally agreed-upon "gold standards" are available.

Development of a reference material for Staphylococcus aureus enterotoxin A in cheese: feasibility study, processing, homogeneity and stability assessment.

Staphylococcal food poisoning is caused by enterotoxins excreted into foods by strains of staphylococci. Commission Regulation 1441/2007 specifies thresholds for the presence of these toxins in foods. In this article we report on the progress towards reference materials (RMs) for Staphylococcal enterotoxin A (SEA) in cheese.

Infrared thermography for monitoring of freeze-drying processes: instrumental developments and preliminary results.

Coupling an infrared (IR) camera to a freeze dryer for on-line monitoring of freeze-drying cycles is described for the first time. Normally, product temperature is measured using a few invasive Pt-100 probes, resulting in poor spatial resolution. To overcome this, an IR camera was placed on a process-scale freeze dryer.

Feasibility study for producing a carrot/potato matrix reference material for 11 selected pesticides at EU MRL level: material processing, homogeneity and stability assessment.

The feasibility for producing a matrix reference material for selected pesticides in a carrot/potato matrix was investigated. A commercially available baby food (carrot/potato-based mash) was spiked with 11 pesticides at the respective EU maximum residue limits (MRLs), and further processed by either freezing or freeze-drying. Batches of some 150 units were produced per material type.

Assessment of commutability for candidate certified reference material ERM-BB130 "chloramphenicol in pork".

Chloramphenicol (CAP), an effective antibiotic against many microorganisms, is meanwhile banned in the EU for treatment of food-producing animals due to adverse health effects. The Institute for Reference Materials and Measurements (IRMM) is currently developing a certified reference material (CRM) for CAP in pork, intended for validation and method performance verifications of analytical methods. The material will be certified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) methods and has a target CAP level around the minimum required performance limit (MRPL) of 0.

Evaluation of the state-of-the-art measurement capabilities for selected PBDEs and decaBB in plastic by the international intercomparison CCQM-P114.

An international intercomparison involving eight national metrology institutes (NMIs) was conducted to establish their current measurement capabilities for determining five selected congeners from the brominated flame retardant classes polybrominated diphenyl ethers and polybrominated biphenyls. A candidate reference material consisting of polypropylene fortified with technical mixtures of penta-, octa- and decabromo diphenyl ether and decabromo biphenyl, which was thoroughly assessed for material homogeneity and stability, was used as study material. The analytical procedures applied by the participants differed with regard to sample pre-treatment, extraction, clean-up, employed calibrants and type of calibration procedure as well as regarding analytical methods used for separation, identification and quantification of the flame retardant congeners (gas chromatography coupled to an electron capture detector (GC-ECD), gas chromatography-mass spectrometry in the electron ionisation mode (GC-EI-MS), gas chromatography-mass spectrometry in the electron capture negative ionisation mode (GC-ECNI-MS), and liquid chromatography-inductive coupled plasma-mass spectrometry (LC-ICP-MS)).

Development of a certified reference material for the content of nitroimidazole parent drugs and hydroxy metabolites in pork meat.

Nitroimidazoles have been applied in the past to poultry and pigs to treat protozoan diseases and to combat bacterial infections, but due to adverse health effects their use in food-producing animals has meanwhile been banned in the EU. The request for a certified reference material in a representative matrix was stipulated by the responsible Community Reference Laboratory and is underpinned by the need to improve the accuracy and comparability of measurement data and to establish metrological traceability of analytical results. The Institute for Reference Materials and Measurements (IRMM) has responded to this demand by developing and producing a new certified matrix reference material, ERM-BB124.

Validation of a liquid chromatography-tandem mass spectrometry method for the identification and quantification of 5-nitroimidazole drugs and their corresponding hydroxy metabolites in lyophilised pork meat.

A liquid chromatography-electrospray ionisation tandem mass spectrometry method for the simultaneous detection and quantitation of 5-nitroimidazole veterinary drugs in lyophilised pork meat, the chosen format of a candidate certified reference material, has been developed and validated. Six analytes have been included in the scope of validation, i.e.

Performance evaluation of elemental analysis/isotope ratio mass spectrometry methods for the determination of the D/H ratio in tetramethylurea and other compounds--results of a laboratory inter-comparison.

Tetramethylurea (TMU) with a certified D/H ratio is the internal standard for Site-specific Natural Isotope Fractionation measured by Nuclear Magnetic Resonance (SNIF-NMR) analysis of wine ethanol for detection of possible adulterations (Commission Regulation 2676/90). A new batch of a TMU certified reference material (CRM) is currently being prepared. Whereas SNIF-NMR has been employed up to now, Elemental Analysis/Isotope Ratio Mass Spectrometry ((2)H-EA-IRMS) was envisaged as the method of choice for value assignment of the new CRM, as more precise (better repeatable) data might be obtained, resulting in lower uncertainty of the certified value.

Molecular cloning and characterization of a plant alpha1,3/4-fucosidase based on sequence tags from almond fucosidase I.

Our work with almond peptide N-glycosidase A made us interested also in the alpha1,3/4-fucosidase which is used as a specific reagent for glycoconjugate analysis. The enzyme was purified to presumed homogeneity by a series of chromatographic steps including dye affinity and fast-performance anion exchange chromatography. The 63 kDa band was analyzed by tandem mass spectrometry which yielded several partial sequences.

Sialic acid concentrations in plants are in the range of inadvertent contamination.

The long held but challenged view that plants do not synthesize sialic acids was re-evaluated using two different procedures to isolate putative sialic acid containing material from plant tissues and cells. The extracts were reacted with 1,2-diamino-4,5-methylene dioxybenzene and the fluorescently labelled 2-keto sugar acids analysed by reversed phase and normal phase HPLC and by HPLC-electrospray tandem mass spectrometry. No N-glycolylneuraminic acid was found in the protein fraction from Arabidopsis thaliana MM2d cells.

Evaluation of PCR-based beef sexing methods.

Analysis of the sex of beef meat by fast and reliable molecular methods is an important measure to ensure correct allocation of export refunds, which are considerably higher for male beef meat. Two PCR-based beef sexing methods have been optimized and evaluated. The amelogenin-type method revealed excellent accuracy and robustness, whereas the bovine satellite/Y-chromosome duplex PCR procedure showed more ambiguous results.

Sexing of beef - a survey of possible methods.

The beef trade, amounting to several billion Euro per year, is of great importance in the European Union. Several measures have been introduced to support beef producers, such as intervention buying. However, these payments are only effected for male beef, which represents a temptation for fraud.

Estimation of the minimum uncertainty of DNA concentration in a genetically modified maize sample candidate certified reference material.

Homogeneity testing and the determination of minimum sample mass are an important part of the certification of reference materials. The smallest theoretically achievable uncertainty of certified concentration values is limited by the concentration distribution of analyte in the different particle size fractions of powdered biological samples. This might be of special importance if the reference material is prepared by dry mixing, a dilution technique which is used for the production of the new and third generation of genetically modified (GMO) plant certified reference materials.

Analysis of Asn-linked glycans from vegetable foodstuffs: widespread occurrence of Lewis a, core alpha1,3-linked fucose and xylose substitutions.

The N-glycans from 27 "plant" foodstuffs, including one from a gymnospermic plant and one from a fungus, were prepared by a new procedure and examined by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For several samples, glycan structures were additionally investigated by size-fractionation and reverse-phase high-performance liquid chromatography in conjunction with exoglycosidase digests and finally also (1)H-nuclear magnetic resonance spectroscopy. The glycans found ranged from the typical vacuolar "horseradish peroxidase" type and oligomannose to complex Le(a)-carrying structures.

Development of an ultra-high-temperature process for the enzymatic hydrolysis of lactose: II. Oligosaccharide formation by two thermostable beta-glycosidases.

During lactose conversion at 70 degrees C, when catalyzed by beta-glycosidases from the archea Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB), galactosyl transfer to acceptors other than water competes efficiently with complete hydrolysis of substrate. This process leads to transient formation of a range of new products, mainly disaccharides and trisaccharides, and shows a marked dependence on initial substrate concentration and lactose conversion. Oligosaccharides have been analyzed quantitatively by using capillary electrophoresis and high performance anion-exchange chromatography.

Structural characterization of the N-linked oligosaccharides from tomato fruit.

The primary structures of the N-linked oligosaccharides from tomato fruit (Lycopersicon esculentum) have been elucidated. For the isolation of the protein fraction, two procedures were employed alternatively: a low temperature acetone powder method and ammonium sulfate precipitation of the tomato extract. After peptic digestion, the glycopeptides were purified by cation-exchange chromatography; the oligosaccharides were released by N-glycosidase A and fluorescently labelled with 2-aminopyridine.

A capillary electrophoretic study on the specificity of beta-galactosidases from Aspergillus oryzae, Escherichia coli, Streptococcus pneumoniae, and Canavalia ensiformis (jack bean).

The specificities of the beta-galactosidases from Aspergillus oryzae, Escherichia coli, Streptococcus pneumoniae, and Canavalia ensiformis (jack bean) have been studied by capillary zone electrophoresis. Various di- and oligosaccharides as well as a biantennary asialo N-glycan were used as substrates. Following enzymatic hydrolysis, the mixtures of substrates and products were derivatized with ethyl 4-aminobenzoate and separated by high-performance capillary electrophoresis in a borate buffer system using uv detection.