Evaluation of CRM homogeneity in cases of insufficient method repeatability: Comparison of Bayesian analysis with substitutes for ANOVA based estimates.

Insufficient method repeatability is a problem characterising the evaluation of certified reference materials (CRMs). In investigating the homogeneity studies of 216 certified parameters from 36 CRMs released by the European Commission's Joint Research Centre (JRC) over the last four years, it was found that in 1/3 of the cases, the method repeatability (s r) was too high to calculate the standard deviation between units (s bb) by classical analysis of variance (ANOVA). It was also found that the application of the repeatability requirement stated in the ISO Guide 35:2017 is not feasible since it would require unrealistically low repeatability standard deviations or an impossibly high number of replicates per unit. Evaluation of the uncertainty of homogeneity (u bb) as evaluated by ANOVA using both the maximum of s bb and 0, the maximum of s bb and u∗bb, the uncertainty hidden by method repeatability, the maximum of s bb and s bb/√n and Bayesian analysis, using both informative and diffuse priors, as well as the standard deviation of the unit means, were compared using simulated homogeneity studies with repeatabilities of 1-8% and s bb between 0.2 and 2.8%. It was found that using the maximum of s bb and s bb/√n as an estimate of u bb guards against severe underestimation but usually results in a severe overestimation of the between-unit variation. Using the maximum of (s bb, 0) shows the least average bias but results in a severe underestimation of u bb in a high fraction of cases. Using the maximum of (s bb, u∗bb) limits, but does not completely eliminate cases of a severe underestimation. Also, it leads to average results biased towards high values. For the range of s bb and s r investigated, Bayesian analysis performed worse than max (s bb, u∗bb) in limiting severe underestimation of u bb, but limited the average bias towards high results. A risk-based approach to cases of insufficient method repeatability is proposed where, after evaluating the other contributions to the uncertainty of certified values, the effect of severe over- and underestimation of u bb is evaluated, and an appropriate approach is chosen based on this analysis.

Results of an interlaboratory method performance study for the size determination and quantification of silver nanoparticles in chicken meat by single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS).

Single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS) promises fast and selective determination of nanoparticle size and number concentrations. While several studies on practical applications have been published, data on formal, especially interlaboratory validation of sp-ICP-MS, is sparse. An international interlaboratory study was organized to determine repeatability and reproducibility of the determination of the median particle size and particle number concentration of Ag nanoparticles (AgNPs) in chicken meat. Ten laboratories from the European Union, the USA, and Canada determined particle size and particle number concentration of two chicken meat homogenates spiked with polyvinylpyrrolidone (PVP)-stabilized AgNPs. For the determination of the median particle diameter, repeatability standard deviations of 2 and 5% were determined, and reproducibility standard deviations were 15 and 25%, respectively. The equivalent median diameter itself was approximately 60% larger than the diameter of the particles in the spiking solution. Determination of the particle number concentration was significantly less precise, with repeatability standard deviations of 7 and 18% and reproducibility standard deviations of 70 and 90%.

Comparison of total and aqua regia extractability of heavy metals in sewage sludge: The case study of a certified reference material.

A number of different digestion methods, including aqua regia extraction following two ISO guides were used in an inter-laboratory comparison study. The results obtained showed comparable values for the total and aqua regia extractable content of As, Cu, Fe, Hg, Pb and Zn, while Cd, Co and Cr results were about 10% lower when aqua regia was employed. This small difference was covered by the between-laboratory relative standard deviation of the measurements; therefore in this study no difference in the extraction of the elements by the employed methods was found. The high organic matter content, together with low SiO2 and refractory aluminium and iron oxide amount as well as the small particle size of the sewage sludge material was reputed to have an effect on the extracting capacity of a weaker solvent such as aqua regia, bringing its results close to the total content ones.

Improved non-invasive Optical Coherence Tomography detection of different engineered nanoparticles in food-mimicking matrices.

Food industry and regulators require fast and reliable analytical methods for quality control. This especially counts for the detection of engineered nanomaterials (ENMs) in food products. Respective EU regulation is in force, but the development of appropriate methods is still underway. This paper updates the scope of Optical Coherence Tomography (OCT) for ENM/food matrix analysis. A range of nanomaterials and composites - Au@SiO2, Ag, Ag@SiO2 and SiO2 - in a simplified food matrix was investigated. The earlier finding of linear dependencies between concentration in the dispersion and light responses could be reproduced. Being able to analyse non-invasively for a relevant industrial compound such as SiO2, makes OCT an excellent candidate for screening purposes.

In-house validation of a method for determination of silver nanoparticles in chicken meat based on asymmetric flow field-flow fractionation and inductively coupled plasma mass spectrometric detection.

Nanomaterials are increasingly used in food production and packaging, and validated methods for detection of nanoparticles (NPs) in foodstuffs need to be developed both for regulatory purposes and product development. Asymmetric flow field-flow fractionation with inductively coupled plasma mass spectrometric detection (AF(4)-ICP-MS) was applied for quantitative analysis of silver nanoparticles (AgNPs) in a chicken meat matrix following enzymatic sample preparation. For the first time an analytical validation of nanoparticle detection in a food matrix by AF(4)-ICP-MS has been carried out and the results showed repeatable and intermediately reproducible determination of AgNP mass fraction and size. The findings demonstrated the potential of AF(4)-ICP-MS for quantitative analysis of NPs in complex food matrices for use in food monitoring and control. The accurate determination of AgNP size distribution remained challenging due to the lack of certified size standards.

Uncertainties of size measurements in electron microscopy characterization of nanomaterials in foods.

Electron microscopy is a recognized standard tool for nanomaterial characterization, and recommended by the European Food Safety Authority for the size measurement of nanomaterials in food. Despite this, little data have been published assessing the reliability of the method, especially for size measurement of nanomaterials characterized by a broad size distribution and/or added to food matrices. This study is a thorough investigation of the measurement uncertainty when applying electron microscopy for size measurement of engineered nanomaterials in foods. Our results show that the number of measured particles was only a minor source of measurement uncertainty for nanomaterials in food, compared to the combined influence of sampling, sample preparation prior to imaging and the image analysis. The main conclusion is that to improve the measurement reliability, care should be taken to consider replications and matrix removal prior to sample preparation.

Feasibility of non-invasive detection of engineered nanoparticles in food mimicking matrices by Optical Coherence Tomography.

The study was dedicated towards the detection of Engineered Nanoparticles (ENPs) by means of Optical Coherence Tomography (OCT). Polymeric films were produced to mimic complex food matrices whereas gold nanorods (AuNRs) were embedded to act as ENPs. The straightforward coating application resulted in a sufficient film wetting, adhesion and homogenous AuNR distribution. Compared to food samples, these films are simpler and better defined. Such artefacts are therefore promising candidate materials for quality assurance and regulatory matters. The OCT investigations revealed a dependency of the measured signal intensity on the AuNR concentration in the film. The limit of detection for the setup and material was estimated to be -8 dB. This value corresponds to a ppm nanoparticle concentration being well below the concentration used in food additive applications. Thus, the findings indicate the potential of OCT to screen food/feed products for a number of ENPs.

Production of reference materials for the detection and size determination of silica nanoparticles in tomato soup.

A set of four reference materials for the detection and quantification of silica nanoparticles (NPs) in food was produced as a proof of principle exercise. Neat silica suspensions were ampouled, tested for homogeneity and stability, and characterized for total silica content as well as particle diameter by dynamic light scattering (DLS), electron microscopy (EM), gas-phase electrophoretic molecular mobility analysis (GEMMA), and field-flow fractionation coupled with an inductively coupled mass spectrometer (FFF-ICPMS). Tomato soup was prepared from ingredients free of engineered nanoparticles and was spiked at two concentration levels with the silica NP suspension. Homogeneity of these materials was found sufficient to act as reference materials and the materials are sufficiently stable to allow long-term storage and distribution at ambient temperature, providing proof of principle of the feasibility of producing liquid food reference materials for the detection of nanoparticles. The spiked soups were characterized for particle diameter by EM and FFF-ICPMS (one material only), as well as for the total silica content. Although questions regarding the trueness of the results from EM and FFF-ICPMS procedures remain, the data obtained indicate that even assigning values should eventually be feasible. The materials can therefore be regarded as the first step towards certified reference materials for silica nanoparticles in a food matrix.

International interlaboratory study for sizing and quantification of Ag nanoparticles in food simulants by single-particle ICPMS.

This publication describes the first international intercomparison of particle-size determination by single-particle inductively coupled plasma mass spectrometry (sp-ICPMS). Concentrated monodisperse silver nanoparticle suspensions with particle diameters of 20, 40 and 100 nm and a blank solution were sent to 23 laboratories in Europe, the USA and Canada. Laboratories prepared eight nanoparticle preparations in two food simulants (distilled water; 10% ethanol) and reported median particle size, Ag particle mass concentration and Ag particle number concentrations. Average repeatability and reproducibility standard deviation (sr and sR) for the median particle diameter were 1 and 14 nm, respectively. Relative precision was worse for Ag particle number concentrations (RSD r = 11%; RSD R = 78%). While further improvements of the method, especially with respect to software tools for evaluation, hardware options for shorter dwell times, calibration standards for determining nebuliser efficiency and further experience by laboratories are certainly desirable, the results of this study demonstrate the suitability of sp-ICPMS for the detection and quantification of certain kinds of nanoparticles.

Nanoscale reference materials for environmental, health and safety measurements: needs, gaps and opportunities.

The authors critically reviewed published lists of nano-objects and their physico-chemical properties deemed important for risk assessment and discussed metrological challenges associated with the development of nanoscale reference materials (RMs). Five lists were identified that contained 25 (classes of) nano-objects; only four (gold, silicon dioxide, silver, titanium dioxide) appeared on all lists. Twenty-three properties were identified for characterisation; only (specific) surface area appeared on all lists. The key themes that emerged from this review were: 1) various groups have prioritised nano-objects for development as "candidate RMs" with limited consensus; 2) a lack of harmonised terminology hinders accurate description of many nano-object properties; 3) many properties identified for characterisation are ill-defined or qualitative and hence are not metrologically traceable; 4) standardised protocols are critically needed for characterisation of nano-objects as delivered in relevant media and as administered to toxicological models; 5) the measurement processes being used to characterise a nano-object must be understood because instruments may measure a given sample in a different way; 6) appropriate RMs should be used for both accurate instrument calibration and for more general testing purposes (e.g., protocol validation); 7) there is a need to clarify that where RMs are not available, if "(representative) test materials" that lack reference or certified values may be useful for toxicology testing and 8) there is a need for consensus building within the nanotechnology and environmental, health and safety communities to prioritise RM needs and better define the required properties and (physical or chemical) forms of the candidate materials.

Development of the first certified reference materials for several brominated flame retardants in polymers.

The first reference materials certified for several polybrominated flame retardants in polymers were developed. Commercially available polyethylene and polypropylene were fortified with technical mixtures of Pentabrominated diphenylether (Penta-BDE), Octa-BDE, Deca-BDE, and Decabrominated biphenyl (BB) (where the capitalized forms refer to the technical mixtures). Homogeneity was tested on 20 units of each material, and between-unit variation was confirmed to be below 4% for all congeners. Stability was assessed after storage of samples for 1 year at 4, 18, and 60 degrees C. Uncertainty of degradation during transport was found negligible for all congeners, whereas uncertainty of degradation for storage of 24 months at 4 degrees C was estimated between 2% and 11%. A characterization intercomparison involving 16 laboratories was organized. After exclusion of technically doubtful results, between-laboratory standard deviations ranged from 3% to 12%, making this intercomparison the best for this field of analysis so far. Statistical analysis revealed that the use of isotopically labeled internal standards did not improve analytical precision in this study. The good comparability, together with the independent confirmation of the assigned mass fractions via the total bromine content as well as by using non-GC/MS-based methods, allowed for the first time the certification of polymer materials for several brominated flame retardants.

Results of an interlaboratory comparison on the determination of polybrominated flame retardants in poly(ethyleneterephthalate).

An intercomparison to establish the performance of routine laboratories in the determination of polybrominated flame retardants in polymers was organised. Commercial poly(ethyleneterephthalate) was fortified with technical pentabromodiphenyl ether, octabromodiphenyl ether and decabromodiphenyl ether mixtures and with a decabromobiphenyl technical mixture at 0.4-0.8 g/kg. Homogeneity and stability of the total Br content in the material was confirmed. Thirty-seven laboratories from Europe, Asia and the Americas submitted results. Relative repeatability standard deviations for individual congeners ranged from 7 to 17%. Relative between-laboratory standard deviations ranged from 22 to 61%. No significant influence of a common standard, application of a standard method or method parameters could be identified. The quality and uncertainty of the results of this study are significantly worse than those reported in the environmental field and indicate a clear need for a learning process among the laboratories involved. [figure: see text] Mandel's h (between labs): critical level: 1.91.

Development of a certified reference material for genetically modified potato with altered starch composition.

The presence of genetically modified organisms (GMOs) in food and feed products is subject to regulation in the European Union (EU) and elsewhere. As part of the EU authorization procedure for GMOs intended for food and feed use, reference materials must be produced for the quality control of measurements to quantify the GMOs. Certified reference materials (CRMs) are available for a range of herbicide- and insect-resistant genetically modified crops such as corn, soybean, and cotton. Here the development of the first CRM for a GMO that differs from its non-GMO counterpart in a major compositional constituent, that is, starch, is described. It is shown that the modification of the starch composition of potato (Solanum tuberosum L.) tubers, together with other characteristics of the delivered materials, have important consequences for the certification strategy. Moreover, the processing and characterization of the EH92-527-1 potato material required both new and modified procedures, different from those used routinely for CRMs produced from genetically modified seeds.

Determination of tributyltin (TBT) in marine sediment using pressurised liquid extraction-gas chromatography-isotope dilution mass spectrometry (PLE-GC-IDMS) with a hexane-tropolone mixture.

Extraction conditions for the determination of tributyltin (TBT) in sediment samples have been developed further. The analytical procedure is based on spiking with isotopically labelled analyte, pressurised liquid extraction (PLE) with a hexane/tropolone mixture, Grignard derivatization and quantification by GC-MS. It was applied to two unknown sediment samples as part of an intercomparison exercise of the Comité Consultatif pour la Quantité de Matière (CCQM). The detection limit was approximately 1.5 ng/g TBT as Sn, while the repeatability and intermediate precision (as the coefficient of variation) were 1.9% and 3.2%, respectively. The expanded uncertainty was 6.2% (coverage factor k = 2), and the accuracy was confirmed by measurement of a certified reference material.

Toward metrological traceability for DNA fragment ratios in GM quantification. 2. Systematic study of parameters influencing the quantitative determination of MON 810 corn by real-time PCR.

This paper is part of a set of three papers investigating metrological traceability of the quantification of DNA fragments as, for instance, used for quantification of genetic modifications. This paper evaluates the possible impact of several factors on results of real-time Polymerase Chain Reaction (PCR) measurements. It was found that the particle size of the powder samples does not have an influence, whereas the nature of the calibrant (plasmidic or genomic DNA) has a significant effect. Moreover, two real-time PCR detection methods (construct-specific and event-specific) for MON 810 corn were compared. The results obtained in a specifically designed interlaboratory study revealed a significant influence of the DNA extraction method on measurement results when the MON 810 construct-specific real-time PCR detection method was applied. Statistical analyses confirmed the importance of validating DNA extraction methods in conjunction with real-time PCR methods.

Long-term stability of relationships between reference materials for thromboplastins.

Reference materials for thromboplastins are available from the World Health Organization (WHO) and the European Commission (EC). The long-term stability of the reference materials is an essential requirement and must be monitored. The relationship between two reference materials for rabbit thromboplastin, i.e. ERM-AD149 (EC) and RBT/90 (WHO), has been monitored in the period 1996-2002. No significant trend with time was detected. In addition, the relationship between ERM-AD149 and the reference material for bovine thromboplastin (i.e. OBT/79) has been determined in 1994 and in 2005 in multicentre studies (n = 11 and n = 9, respectively). No significant changes were observed in the relationships between these reference materials when all results were included (5% significance level).

Standardization of immunoassays for measurement of myoglobin in serum. Phase I: evaluation of candidate secondary reference materials.

BACKGROUND: Myoglobin is a low-molecular weight protein present in the cytosol of striated muscles. Its concentrations in serum can be measured by immunoassays and are used as an early indicator of myocardial necrosis. Since variability among commercial myoglobin assays exists, standardization of myoglobin assays is needed. METHODS: An international collaborative study was organized with the involvement of seven companies using 12 different automated platforms for measuring myoglobin. Five candidate secondary, i.e., matrixed, reference materials were assayed in relation to linearity, imprecision, recovery rate and commutability to demonstrate a possible identity between the materials and the usual routine serum samples. RESULTS: One lyophilized candidate material (human heart myoglobin in human serum) was selected as the most suitable secondary reference material, based on the criteria examined. Used as a calibrator a posteriori, the bias between the various myoglobin assays for a frozen human serum pool was reduced from 32% to 13%. CONCLUSION: This study provides the basis for the selection of an internationally recognized secondary reference material.

Production of certified reference materials for mycotoxins: IRMM's view on the assessment of uncertainties.

Analytical difficulty and the economic importance of controlling mycotoxin levels in food and feed led the Community Bureau of Reference (BCR) to prepare a series of certified reference materials (CRM) for various mycotoxins. Because of the wide acceptance of these CRM and the need to ensure the comparability and traceability of measurements in the future it is necessary to prepare and certify new batches of mycotoxin reference materials (RM). In the following text two different approaches for evaluation of the characterisation uncertainty of CRM will be compared using the certification of aflatoxin M(1) (AfM(1)) in milk powder as an example. The conventional approach is based on evaluation of characterisation exercise data; the alternative approach is based on measurement uncertainties of the employed analytical methods. Because laboratories are using totally different approaches to estimate the measurement uncertainties, combination of the uncertainties obtained from the participating laboratories was not recommended. Therefore, a new integrated approach for assessment of the measurement uncertainties of the analytical methods on the basis of additional data collected during the characterisation exercise will be described. The conventional approach was found to be the most appropriate and economical approach to evaluate the characterisation uncertainty as a characterisation exercise must be performed anyway to establish the property values of candidate (C)RM, irrespective of whether or not reliable measurement uncertainties can be provided by the laboratories. An integrated approach for assessment of measurement uncertainties based on additional characterisation data as applied here to enable use of an uncertainty-based approach provides more information but is too time-consuming and cost-intensive to become common practice.

IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. Part 1. The concept of reference procedures for the measurement of catalytic activity concentrations of enzymes.

This paper is the first in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C and with the certification of reference preparations. Other parts deal with: Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 4. Reference Procedure for the Measurement of Catalytic Concentration of Alanine Aminotransferase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 6. Reference Procedure for the Measurement of Catalytic fication of Four Reference Materials for the Determination of Enzymatic Activity of y-Glutamyltransferase, Lactate Dehydrogenase, Alanine Aminotransferase and Creatine Kinase at 37 degrees C. A document describing the determination of preliminary reference values is also in preparation.