Certification of the Deuterium-to-Hydrogen (D/H) amount-of-substance ratio in a 1,1,3,3 - tetramethylurea batch: Certified Reference Material STA-003m

This report describes the production of a tetramethylurea (TMU) reference material (STA-003m), certified for its deuterium-to-hydrogen (D/H) amount-of-substance ratio. The material is to be used as an internal standard in site-specific natural isotope fractionation – nuclear magnetic resonance (SNIF-NMR) spectrometry measurements for determining the D/H amount-of-substance ratios of ethanol distilled from wines, an important measure in wine authenticity testing (Commission Regulation 2676/90). Commercially obtained TMU with a sufficiently high D/H amount-of-substance ratio (>120 x 10-6) was first purified by removing most of the residual water and then filled into amber glass bottles of 500 mL. Between-unit homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. Batch characterisation was accomplished by comparing STA-003m samples with samples of the TMU master batch (IRMM-425) under repeatability conditions and by adhering to the SNIF-NMR technique. The certified value was calculated as the unweighted mean of 18 individual results obtained on 3 different NMR appliances. The uncertainty of the certified value was estimated in compliance with ISO Guide 98-3, Guide to the Expression of Uncertainty in Measurement (GUM); it includes contributions from possible inhomogeneity, instability, and characterisation.JRC.D.2-Standards for Innovation and sustainable Developmen

The Certification of the Mass Fraction of Oxytetracycline in Partially Skimmed Milk - Certified Reference Materials ERM®-BB492 and ERM®-BB493

This report describes the preparation of two milk powder matrix reference materials (ERM-BB492 and ERM-BB493) and the certification of their content (mass fraction) of oxytetracycline. The preparation and processing of the material, homogeneity and stability studies, and the characterisation are described hereafter and the results are discussed. Uncertainties were estimated in compliance with ISO Guide 98-3, Guide to the Expression of Uncertainty in Measurement (GUM) [1]. For ERM-BB492, the uncertainty contains contributions from possible heterogeneity, instability, characterisation, and purity of the common calibrant.JRC.DG.D.2-Reference material

The Certification of the Mass Fraction of Chloramphenicol in Pork Meat - Certified Reference Material ERM®-BB130

This report describes the preparation of the pork meat matrix reference material ERM-BB130 and the certification of the content (mass fraction) of chloramphenicol. The preparation and processing of the material, homogeneity and stability studies, and the characterisation are described hereafter and the results are discussed. Uncertainties were calculated in compliance with ISO Guide 98-3, Guide to the Expression of Uncertainty in Measurement (GUM) [1] and include uncertainties due to possible heterogeneity, instability, and characterisation.JRC.DG.D.2-Reference material

Certification of the crude Protein, Fat, Lactose and Ash Content of whole Milk Powder and the crude Protein and Fat Content of Skim Milk Powder, BCR-380R & BCR-685

This report describes the preparation of two milk powder reference materials and the measurement exercises that led to the certification of the content (mass fraction) of the crude protein (Kjeldahl-N x 6.38), fat, lactose and ash in whole milk powder (BCR-380R) and crude protein (Kjeldahl-N x 6.38) and fat in skim milk powder (BCR-685).JRC.D.2-Reference material

An international proficiency test to detect, identify and quantify ricin in complex matrices

While natural intoxications with seeds of Ricinus communis have long been known, the toxic protein ricin contained in the seeds is of major concern due to its history of criminal, terrorist and military use. 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). Sample materials included highly pure ricin as well as the related R. communis agglutinin spiked into buffer, milk and meat extract; additionally, an organic fertilizer naturally contaminated with R. communis shred was investigated in the proficiency test. The qualitative results showed that either a suitable combination of immunological, MS-based and functional approaches or sophisticated MS-based approaches alone successfully allowed to detect and identify ricin in all samples. In terms of quantification, it was possible to determine a consensus concentration for the highly pure ricin reference material. The results provide a basis for further steps in quality assurance and improve biopreparedness in expert laboratories worldwide.JRC.D.2-Standards for Innovation and sustainable Developmen

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 BoNT was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. A variety of methods was applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo / in vitro approaches (mouse bioassay, hemidiaphrama assay, Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently seen as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need of certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphrama assay delivered quantitative results superior to the mouse bioassay.JRC.D.2-Standards for Innovation and sustainable Developmen

CERTIFICATION REPORT Certified reference materials for testing of the presence/absence of Staphylococcus aureus enterotoxin A (SEA) in cheese: IRMM-359a-c

This report describes the preparation of three cheese powder matrix reference materials (IRMM-359a-c) and their certification for testing of the presence/absence of Staphylococcus aureus enterotoxin A (SEA). Raw milk cheese was decrusted, cut into cubes, chopped in a kitchen-type food processor for a short time, freeze-dried, cryogenically milled, and mixed (blank material IRMM-359a). Moreover, a second portion of raw milk cheese was decrusted and cut into cubes. After addition of water and spiking with a solution of SEA, the sample was homogenised using a high-speed grinder (Ultra-Turrax). The cheese slurry was freeze-dried, cryogenically milled and mixed with blank cheese powder to obtain the two SEA-containing materials at SEA target levels of 0.1 and 0.25 ng/g cheese, respectively (IRMM-359b, IRMM-359c). Between unit-homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. The minimum sample intake is 15.1 g cheese powder (representing 25 g of cheese after reconstitution) per replicate analysis (n=5), as stipulated in Commission Regulation 1441/2007, and therefore no dedicated study on the minimum sample intake was performed. The reference material was characterised in an interlaboratory comparison of laboratories of demonstrated competence and adhering to ISO/IEC 17025 and using the European Screening Method with the VIDAS SET2 and the Ridascreen SET Total for detection (further on named ESM/VIDAS and ESM/Ridascreen, respectively) [4]. Technically invalid results were removed, but no outlier was eliminated on statistical grounds only. Certified values are reported as probability of detection and expressed as either diagnostic specificity (ratio of true negatives divided by the sum of true negatives and false positives) for the blank material, or diagnostic sensitivity (ration of true positives divided by the sum of true positives and false negatives) for the SEA-containing materials. Uncertainties for homogeneity and stability were estimated, but not used for an uncertainty budget due to the nature of the certified values (presence/absence certification). Instead, the certified values are expressed as intervals with a 95% level of confidence. The preparation and processing of the material, homogeneity and stability studies, and the characterisation are described hereafter and the results are discussed.JRC.D.2-Standards for Innovation and sustainable Developmen

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. Expert laboratories currently use differently purified in-house materials, making any comparison of accuracy and sensitivity of different methods nearly impossible. Technically challenging is the discrimination of ricin from R. communis agglutinin (RCA120), a less toxic but highly homologous protein also contained in R. communis. Here, we established both highly pure ricin and RCA120 reference materials which were extensively characterized by gel electrophoresis, liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI MS/MS), and matrix-assisted laser desorption ionization-time of flight approaches as well as immunological and functional techniques. Purity reached >97% for ricin and >99% for RCA120. Different isoforms of ricin and RCA120 were identified unambiguously and distinguished by LC-ESI MS/MS. In terms of function, a real-time cytotoxicity assay showed that ricin is approximately 300-fold more toxic than RCA120. The highly pure ricin and RCA120 reference materials were used to conduct an international proficiency test.Peer reviewe

Influence of the approach to calibration on the accuracy and the traceability of certified values in certified reference materials

The use of calibrants properly characterized with respect to identity and purity is an important requirement to achieve accurate, reliable results in chemical measurements. In particular, in generating data that contribute to assigning a property value to a certified reference material (CRM), compromises often made for routine measurements (non-verification of calibrant purity) are unacceptable, and strict adherence to calibrant quality requirements is indispensable. We compared two approaches to calibration within the framework of laboratory intercomparisons for assigning values to two recently released CRMs in the veterinary drug-residue field, namely chloramphenicol in pork (ERM-BB130) and oxytetracycline in milk (ERM-BB492). First, all laboratories used the same calibrant (common calibrant, a commercially available substance, extensively characterized for purity and identity), and second, each laboratory used its in-house calibrant (commercially available substance, purity as indicated by the provider). The laboratories, mostly accredited to ISO/IEC 17025, used validated methods based on gas or liquid chromatography coupled to mass spectrometry or ultraviolet spectrophotometry. Each measurement series included two calibration curves, one with the common calibrant, and one with the in-house calibrant. For both materials, when the common calibrant was used, the mean of accepted laboratory mean values was 7% and 4% lower for BB130 and BB492, respectively. This shows the impact on accuracy and hints at an overestimation of the purity of the in-house calibrants. Moreover, when the common calibrant was used, the variation among the laboratory mean values in the intercomparisons decreased by 9% (BB130) and 39% (BB492), respectively. The smaller uncertainty from the characterization study resulted in a lower expanded uncertainty of the certified values (4% lower for BB130, and 7% lower for BB492, compared to using in-house calibrants). The use of a common calibrant thus significantly affects and improves the accuracy and provides the basis for a proper metrological traceability statement for the certified values in the CRMs.JRC.D.2-Reference material

Challenges in the Development of Reference Materials for Protein Toxins

High molecular weight protein toxins produced by bacteria, e.g. staphylococcal enterotoxins and botulinum neurotoxins, as well as plant toxins such as ricin and abrin, are relevant analytes in different application areas: food safety, public health, civil security and defense sector, and – in case of botulinum neurotoxins – also in pharmaceutics. For their reliable and accurate detection, identification and quantification, reference materials (RMs), in particular certified reference material (CRM), are required. The present article focuses on challenges in the development (production and certification) of such RMs. Firstly, it highlights the role of RMs and CRMs, what they can be used for, the nature of certified properties, metrological traceability, and uncertainty of certified values, as well as commutability of RMs. Secondly, the molecule-specific technical challenges are highlighted using the example of the mentioned toxins. This includes for instance the choice of a suitable purification strategy (recombinant expression and purification versus the purification of toxin from natural sources), the in-depth characterization of the obtained preparations by a comprehensive set of methods including immunochemical assays, mass spectrometry, and functional assays to verify their identity and establish their purity and activity, and finally, suitable approaches for determining reference values of important toxin properties (protein mass concentration in solution, biological activity). The article summarizes ongoing activities in a new European initiative called EuroBioTox, which aims at the production and certification of RMs for selected protein toxins and the establishment of validated procedures for the detection and identification of biological toxins