Gas chromatography-isotope dilution mass spectrometry method validation for target pesticides in soybeans.

The production of certified reference materials requires the application of highly accurate methods for characterisation. A gas chromatography-isotope dilution mass spectrometry method, setting ambitious performance criteria, was developed for eight selected pesticides in soybeans. Pressurised liquid extraction was followed by automated gel-permeation chromatography and solid-phase extraction clean-up. Pesticides identification respected a Commission Decision and guidelines of the Directorate General for Health and Food Safety (DG SANTE). Reliable quantification involved stable isotopically labelled analogues as internal standards. Validation, according to ISO/IEC 17,025 and DG SANTE guidelines, assessed linearity, LOD/LOQ, trueness, selectivity, precision, stability and robustness. Mean recoveries ranges (83-109%, relative standard deviations < 3%), repeatability (2.2-4.8%), day-to-day variation (0.6-4.2%) and combined uncertainty (1.2-4.2%) were fit for purpose. The method is highly accurate and suitable for certification of the selected pesticides in soybean matrix reference material. Chemical compounds studied in this article: Diazinon (PubChem CID: 3017); malathion (PubChem CID: 4004); chlorpyrifos (PubChem CID: 2730); captan (PubChem CID: 8606); endosulfan (PubChem CID: 3224); tebuconazole (PubChem CID: 86,102); iprodione (PubChem CID: 37,517); cypermethrin (PubChem CID: 2912).

First steps in the standardization of immunoglobulin IgG myeloperoxidase-anti-neutrophil cytoplasmic antibody measurements.

The standardization of immunoassays for immunoglobulin (Ig)G myeloperoxidase-anti-neutrophil cytoplasmic antibodies (MPO-ANCA) could contribute to a more accurate diagnosis and follow-up of small vessels-associated vasculitis, a systemic autoimmune disorder that leads to necrosis of blood vessel walls. Despite significant efforts by different groups, the level of comparability of results from commercially available immunoassays used for IgG MPO-ANCA detection is still poor. Therefore, the potential for improvement using reference materials was assessed. The evaluation of a set of 30 patient samples with 11 assays showed that differences between assays result in different interpretations for individual patients. Only 10 of 30 patient samples had the same clinical interpretation among 11 assays applying the cut-off values provided by each respective manufacturer. The correlation between results from 13 different assays was assessed in a pairwise manner. The correlation between results from patient samples was systematically very good for combinations of seven of those assays. The correlation of results ranged from reasonable to good for combinations with four other assays, therefore it should be possible to improve the comparability of results using a commutable reference material for calibration. Feasibility studies were conducted in order to find a reference material format most suitable for a calibrator. Two sets of candidate reference materials were produced from different raw materials, and assessed according to their suitability. A final format was selected, and a candidate reference material was produced.

Accurate determination of selected pesticides in soya beans by liquid chromatography coupled to isotope dilution mass spectrometry.

A sensitive, accurate and simple liquid chromatography coupled with mass spectrometry method for the determination of 10 selected pesticides in soya beans has been developed and validated. The method is intended for use during the characterization of selected pesticides in a reference material. In this process, high accuracy and appropriate uncertainty levels associated to the analytical measurements are of utmost importance. The analytical procedure is based on sample extraction by the use of a modified QuEChERS (quick, easy, cheap, effective, rugged, safe) extraction and subsequent clean-up of the extract with C18, PSA and Florisil. Analytes were separated on a C18 column using gradient elution with water-methanol/2.5 mM ammonium acetate mobile phase, and finally identified and quantified by triple quadrupole mass spectrometry in the multiple reaction monitoring mode (MRM). Reliable and accurate quantification of the analytes was achieved by means of stable isotope-labelled analogues employed as internal standards (IS) and calibration with pure substance solutions containing both, the isotopically labelled and native compounds. Exceptions were made for thiodicarb and malaoxon where the isotopically labelled congeners were not commercially available at the time of analysis. For the quantification of those compounds methomyl-(13)C2(15)N and malathion-D10 were used respectively. The method was validated according to the general principles covered by DG SANCO guidelines. However, validation criteria were set more stringently. Mean recoveries were in the range of 86-103% with RSDs lower than 8.1%. Repeatability and intermediate precision were in the range of 3.9-7.6% and 1.9-8.7% respectively. LODs were theoretically estimated and experimentally confirmed to be in the range 0.001-0.005 mg kg(-1) in the matrix, while LOQs established as the lowest spiking mass fractionation level were in the range 0.01-0.05 mg kg(-1). The method reliably identifies and quantifies the selected pesticides in soya beans at appropriate uncertainty levels, making it suitable for the characterization of candidate reference materials.

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. RMs are crucial to enforce legislation and to implement and safeguard reliable measurements. First, a feasibility study revealed a suitable processing procedure for cheese powders: the blank material was prepared by cutting, grinding, freeze-drying and milling. For the spiked material, a cheese-water slurry was spiked with SEA solution, freeze-dried and diluted with blank material to the desired SEA concentration. Thereafter, batches of three materials (blank; two SEA concentrations) were processed. The materials were shown to be sufficiently homogeneous, and storage at ambient temperature for 4weeks did not indicate degradation. These results provide the basis for the development of a RM for SEA in cheese.

Quantification of protein calibrants by amino acid analysis using isotope dilution mass spectrometry.

This work demonstrates that amino acid analysis based on isotope dilution mass spectrometry (IDMS) can be applied to quantify proteins having different complexities and natures. Five proteins and one decapeptide were selected for the study: C-reactive protein (CRP), beta-2-microglobulin (B2M), cystatine C (CysC), human serum albumin (HSA), Ara h1, and angiotensin I. The quantification was based on the determination of four amino acids, proline (Pro), isoleucine (Ile), valine (Val), and phenylalanine (Phe) within a working range between 5 and 100 pmol/injection of each amino acid, after 60 min digestion with HCl at 150°C. The amino acids were selected taking into account their abundance in the protein sequence and to include the more difficult to break peptide bonds. Quantification of the protein amounts calculated from each amino acid is consistent, indicating that the method is working reliably. This consistency points to a complete hydrolysis of the proteins. The trueness of the method was proven when dry mass determination after dialysis was applied to HSA and CRP and the results were compared to those from amino acid analysis. Traceability to SI was assured by extensive characterisation of the amino acid calibrants by nuclear magnetic resonance, neutron activation analysis, and Karl Fischer titration.

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. This incurred lyophilised pork meat material was certified according to ISO guides 34 and 35 for the mass fractions of six nitroimidazole compounds. Processing of the frozen muscle tissue to the final material was accomplished by application of cutting, freeze-drying, mixing and milling techniques. Homogeneity and stability measurements were performed using liquid chromatography tandem mass spectrometry. The relative standard uncertainty due to possible heterogeneity showed to be below 1.8% for all analytes. Potential degradation during transport and storage was assessed by isochronous stability studies. No significant instability was detected at a storage temperature of -20 degrees C for a shelf-life of 2 years. The certified mass fraction values were assigned upon evaluation of the data acquired in an international laboratory inter-comparison involving 12 expert laboratories using different sample preparation procedures, but exclusively LC-MS/MS methods. Relative standard uncertainty contributions for the characterisation (between-lab variation of mean values) were found to be between 1.6 and 4.8%. Certified values for five analytes were in the range of 0.7 to 6.2 microg kg(-1), with expanded relative uncertainties ranging between 7 and 14%. Dimetridazole could be certified as "<0.25 microg kg(-1) with a probability of 95%". All values are traceable to the International System of Units (SI). The material is intended to be used for method validation purposes (including trueness estimation) and for method performance assessment.

First certified reference materials for molecular fingerprinting of two approved probiotic Bacillus strains.

At present probiotic bacteria are widely used in human and animal nutrition because they beneficially influence the balance of the intestinal flora of the host. Positive effects related to probiotics are various and include enhancement of digestion, strengthening of the immune system and stimulation of vitamin production. Moreover, implementation of probiotics is intended to reduce the use of antibiotics and improve animal growth and feed conversion. To protect human and animal health and to improve consumer confidence, a strict legislation on the use of probiotics exists within the European Union (EU). Official controls by national authorities are performed to ensure verification of compliance with feed and food law. Apart from the risk of using unauthorized strains, mislabelling is a known problem, partly because of the use of phenotyping or genotyping methods with a lack of discriminative power. In addition to official controls, private controls by food and feed producing companies are important in the frame of protection of patented strains and industrial property rights. To support these applications, IRMM has developed certified reference materials (CRMs) consisting of genomic DNA inserts of B. subtilis DSM 5749 and B. licheniformis DSM 5750, two strains that received EU approval. In this study we investigated the use of these CRMs, IRMM-311 and IRMM-312, for the detection and unambiguous discrimination of Bacillus strains by pulsed-field gel electrophoresis (PFGE). Identical fingerprints were obtained for the CRMs and control strains isolated from the feed additive Bioplus 2B. On the other hand a distinction could be made from other not approved B. licheniformis and B. subtilis strains. The reference materials discussed in this study are the first CRMs based on a whole bacterial genome and suitable for PFGE. They offer perspectives for applications in other domains such as analysis of foodborne pathogens in outbreaks or routine analysis.

Towards the certification of the purity of calibrant reference materials for thyroid hormones: a chicken and egg dilemma.

The certification of the purity of CRMs intended for calibration, where no other certified material already exists for comparison, raises principle questions on how to determine the purity of a "first" calibrant in the calibration hierarchy. We developed and certified two calibration CRMs for their purity in thyroid hormones taking into consideration inorganic residues, residual solvents and organic impurities detectable by HPLC-UV and HPLC-MS. IRMM-468 was certified for a thyroxine (T(4)) mass fraction of 98.6+/-0.7% and IRMM-469 was certified for a 3,3',5-triiodothyronine (T(3)) mass fraction of 97.1+/-0.7%. The approach we used aims to determine the purity of these two CRMs to the best of our knowledge and taking all scientific aspects properly into account for the estimation of an uncertainty related to the stated purity.

Disseminated mucormycosis in haematological patients: CT and MRI findings with pathological correlation.

Disseminated mucormycosis is a rare, mostly fatal infectious complication in immunocompromised haematological patients. The purpose of our study was to describe the multiorgan manifestations of disseminated mucormycosis documented at CT and MRI in four patients and correlate these with the pathological findings and patient outcome. Irrespective of the site of infection, infarction or haemorrhage are the constant features of invasive mycosis. Identification of one or both of these two major imaging findings in immunocompromised patients should be regarded as an indicator of possible infection by angiotropic fungi, including the genre Mucorales.

Multiple skeletal metastases from a giant cell tumour of the distal fibula with fatal outcome.

A giant cell tumour is a primary lesion of bone of intermediate severity. Its histogenesis is unclear. In a few cases pulmonary metastases have been described. Multiple skeletal metastases in the absence of sarcomatous change have been observed. We present a case report of a 25-year-old woman with a recurrent giant cell tumour of the distal fibula. After a second recurrence and six years after the initial diagnosis, she rapidly developed multiple bony metastases. The outcome was fatal.

Hydrogen cycling of niobium and vanadium catalyzed nanostructured magnesium.

The reaction of hydrogen gas with magnesium metal, which is important for hydrogen storage purposes, is enhanced significantly by the addition of catalysts such as Nb and V and by using nanostructured powders. In situ neutron diffraction on MgNb(0.05) and MgV(0.05) powders give a detailed insight on the magnesium and catalyst phases that exist during the various stages of hydrogen cycling. During the early stage of hydriding (and deuteriding), a MgH(1< x < 2) phase is observed, which does not occur in bulk MgH(2) and, thus, appears characteristic for the small particles. The abundant H vacancies will cause this phase to have a much larger hydrogen diffusion coefficient, partly explaining the enhanced kinetics of nanostructured magnesium. It is shown that under relevant experimental conditions, the niobium catalyst is present as NbH(1). Second, a hitherto unknown Mg-Nb perovskite phase could be identified that has to result from mechanical alloying of Nb and the MgO layer of the particles. Vanadium is not visible in the diffraction patterns, but electron micrographs show that the V particle size becomes very small, 2-20 nm. Nanostructuring and catalyzing the Mg enhance the adsorption speed that much that now temperature variations effectively limit the absorption speed and not, as for bulk, the slow kinetics through bulk MgH(2) layers.

Detection and traceability of genetically modified organisms in the food production chain.

Both labelling and traceability of genetically modified organisms are current issues that are considered in trade and regulation. Currently, labelling of genetically modified foods containing detectable transgenic material is required by EU legislation. A proposed package of legislation would extend this labelling to foods without any traces of transgenics. These new legislations would also impose labelling and a traceability system based on documentation throughout the food and feed manufacture system. The regulatory issues of risk analysis and labelling are currently harmonised by Codex Alimentarius. The implementation and maintenance of the regulations necessitates sampling protocols and analytical methodologies that allow for accurate determination of the content of genetically modified organisms within a food and feed sample. Current methodologies for the analysis of genetically modified organisms are focused on either one of two targets, the transgenic DNA inserted- or the novel protein(s) expressed- in a genetically modified product. For most DNA-based detection methods, the polymerase chain reaction is employed. Items that need consideration in the use of DNA-based detection methods include the specificity, sensitivity, matrix effects, internal reference DNA, availability of external reference materials, hemizygosity versus homozygosity, extrachromosomal DNA, and international harmonisation. For most protein-based methods, enzyme-linked immunosorbent assays with antibodies binding the novel protein are employed. Consideration should be given to the selection of the antigen bound by the antibody, accuracy, validation, and matrix effects. Currently, validation of detection methods for analysis of genetically modified organisms is taking place. In addition, new methodologies are developed, including the use of microarrays, mass spectrometry, and surface plasmon resonance. Challenges for GMO detection include the detection of transgenic material in materials with varying chromosome numbers. The existing and proposed regulatory EU requirements for traceability of genetically modified products fit within a broader tendency towards traceability of foods in general and, commercially, towards products that can be distinguished from each other. Traceability systems document the history of a product and may serve the purpose of both marketing and health protection. In this framework, segregation and identity preservation systems allow for the separation of genetically modified and non-modified products from "farm to fork". Implementation of these systems comes with specific technical requirements for each particular step of the food processing chain. In addition, the feasibility of traceability systems depends on a number of factors, including unique identifiers for each genetically modified product, detection methods, permissible levels of contamination, and financial costs. In conclusion, progress has been achieved in the field of sampling, detection, and traceability of genetically modified products, while some issues remain to be solved. For success, much will depend on the threshold level for adventitious contamination set by legislation.

Hydrogen adsorption in carbon nanostructures: comparison of nanotubes, fibers, and coals.

Single-walled carbon nanotubes (SWNT) were reported to have record high hydrogen storage capacities at room temperature, indicating an interaction between hydrogen and carbon matrix that is stronger than known before. Here we present a study of the interaction of hydrogen with activated charcoal, carbon nanofibers, and SWNT that disproves these earlier reports. The hydrogen storage capacity of these materials correlates with the surface area of the material, the activated charcoal having the largest. The SWNT appear to have a relatively low accessible surface area due to bundling of the tubes; the hydrogen does not enter the voids between the tubes in the bundles. Pressure-temperature curves were used to estimate the interaction potential, which was found to be 580+/-60 K. Hydrogen gas was adsorbed in amounts up to 2 wt % only at low temperatures. Molecular rotations observed with neutron scattering indicate that molecular hydrogen is present, and no significant difference was found between the hydrogen molecules adsorbed in the different investigated materials. Results from density functional calculations show molecular hydrogen bonding to an aromatic C[bond]C that is present in the materials investigated. The claims of high storage capacities of SWNT related to their characteristic morphology are unjustified.

Determination of tributyltin in marine sediment: Comité Consultatif pour la Quantité de Matière (CCQM) pilot study P-18 international intercomparison.

The capabilities of National Metrology Institutes (NMIs-those which are members of the Comité Consultatif pour la Quantité de Matière (CCQM)of the CIPM) and selected outside "expert" laboratories to quantitate (C(4)H(9))(3)Sn(+) (TBT) in a prepared marine sediment were assessed. This exercise was sanctioned by the 7th CCQM meeting, April 4-6, 2001, as an activity of the Inorganic Analysis Working Group and was jointly piloted by the Institute for National Measurement Standards of the National Research Council of Canada (NRC) and the Laboratory of the Government Chemist (LGC), UK. A total of 11 laboratories submitted results (7 NMIs, and 4 external labs). Two external laboratories utilized a standard calibration approach based on a natural abundance TBT standard, whereas all NMIs relied upon isotope dilution mass spectrometry for quantitation. For this purpose, a species specific (117)Sn-enriched TBT standard was supplied by the LGC. No sample preparation methodology was prescribed by the piloting laboratories and, by consequence, a variety of approaches was adopted by the participants, including mechanical shaking, sonication, accelerated solvent extraction, microwave assisted extraction and heating in combination with Grignard derivatization, ethylation and direct sampling. Detection techniques included ICP-MS (with GC and HPLC sample introduction), GC-MS, GC-AED and GC-FPD. Recovery of TBT from a control standard (NRCC CRM PACS-2 marine sediment) averaged 93.5+/-2.4% ( n=14). Results for the pilot material averaged 0.680+/-0.015 micro mol kg(-1) ( n=14; 80.7+/-1.8 micro g kg(-1)) with a median value of 0.676 micro mol kg(-1). Overall, performance was substantially better than state-of-the-art expectations and the satisfactory agreement amongst participants permitted scheduling of a follow-up Key comparison for TBT (K-28), a Pilot intercomparison for DBT (P-43), and certification of the test sediment for TBT content and its release as a new Certified Reference Material (HIPA-1) with a TBT content of 0.679+/-0.089 micro mol kg(-1) (expanded uncertainty, k=2, as Sn) (80.5+/-10.6 micro g kg(-1)).

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. Consequently, reliable methods for sexing of beef are required. This report summarises existing methods in EU countries as well as possible alternatives deduced from the literature. Individual methods are discussed for their advantages and disadvantages as well as their general applicability.

Evaluation of measurement uncertainties of an analytical method for the determination of aflatoxin M1 in milk and milk powder.

The mycotoxin aflatoxin M1 (AfM1) is a serious food safety hazard for which the European Commission has already established a maximum permissible level of 0.05 µg/kg AfM1 in milk and products thereof. For control analysis laboratories are increasingly asked to submit full uncertainties of their analytical results.The evaluation of measurement uncertainties of an analytical method for the determination of AfM1 in milk and milk powder on the basis of 'in-house' validation data in compliance with the 'Guide to the Expression of Uncertainty in Measurement (GUM)' [1] and the 'EURACHEM Guide' [2] is described. A similar approach will be used to assess the performance of methods employed by laboratories participating in the certification of reference materials for AfM1 in milk powder.