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Concept: Certified reference materials

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BACKGROUND: Despite considerable global investigation over several decades, the roles of vitamin D in health and disease development remains convoluted. One recognised issue is the difficulty of accurately measuring the active forms of vitamin D. Advances made include some new methods addressing the potential interference by excluding epimers and isobars. However, there is no evidence that epimers are without function. Therefore, the aim of this study was to develop and validate, for the first time, a new assay to simultaneously measure levels of 6 forms of vitamin D along with two epimers. The assay was applied to multilevel certified reference material calibrators and 25 pooled human sera samples obtained from the Vitamin D External Quality Assessment Scheme (DEQAS) to demonstrate its efficiency. RESULTS: The assay is capable of simultaneously measuring 8 vitamin D analogues over the calibration ranges and LODs (in nmol/L) of: 1alpha25(OH)2D2 [0.015-1; 0.01], 1alpha25(OH)2D3 [0.1-100; 0.01], 25OHD3 [0.5-100, 0.025], 3-epi-25OHD3 [0.1-100, 0.05], 25OHD2 [0.5-100, 0.025], 3-epi-25OHD2 [0.1-100, 0.05], vitamin D3 [0.5-100, 0.05] and vitamin D2 [0.5-100, 0.05], using stanozolol-d3 as internal standard. Certified reference material calibrators and external quality control samples (DEQAS) were analysed to meet the standards outlined by National Institute of Standards and Technology (NIST). Validation steps included recovery and both precision and accuracy under inter- and intra-day variation limit of detection, and analysis of each analyte over a linear range. All validation parameters were in line with acceptable Food and Drug Administration (FDA) guidelines and the standards of the National Institute of Standards and Technology (NIST). All eight analogues were quantified with the 25OHD levels being commensurate with DEQAS data. CONCLUSIONS: This report details the application of a new LC-MS/MS based assay for the efficient analysis of eight analogues of vitamin D over a range of samples, which is a significant advance over the existing methods. Simultaneous measure of 8 vitamin D analogues does not compromise the analytical capability of the assay to quantify the commonly used biomarker (25OHD) for vitamin D status. The results demonstrate the feasibility of applying the assay in research and clinical practice that i) excludes misleading measures owing to epimers and isobars and ii) is able to quantify the excluded component to facilitate further in vivo investigation into the roles of ubiquitous epimers.

Concepts: Vitamin D, Mass spectrometry, Measurement, Validation, Analytical chemistry, Standard, Calibration, Certified reference materials

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The importance of hemoglobin A2 (HbA2) as an indicator of the presence of β-thalassemia was established many years ago. However, clinical application of recommended HbA2 cut off values is often hampered due to poor equivalence of HbA2 results among methods and laboratories. Thus, the IFCC Standardization program for HbA2 was initiated in 2004 with the goal of achieving a complete reference system for this measurand. HbA2 standardization efforts are still in progress, including the development of a higher-order HbA2 reference measurement procedure and the preparation of a certified reference material in collaboration with the IRMM. Here, we review the past, present and future of HbA2 standardization and describe the current status of HbA2 testing.

Concepts: Present, Time, Future, Work in progress, The Current, Past, Certified reference materials, Standards

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The certification of a new standard reference material for small-angle scattering [NIST Standard Reference Material (SRM) 3600: Absolute Intensity Calibration Standard for Small-Angle X-ray Scattering (SAXS)], based on glassy carbon, is presented. Creation of this SRM relies on the intrinsic primary calibration capabilities of the ultra-small-angle X-ray scattering technique. This article describes how the intensity calibration has been achieved and validated in the certified Q range, Q = 0.008-0.25 Å(-1), together with the purpose, use and availability of the SRM. The intensity calibration afforded by this robust and stable SRM should be applicable universally to all SAXS instruments that employ a transmission measurement geometry, working with a wide range of X-ray energies or wavelengths. The validation of the SRM SAXS intensity calibration using small-angle neutron scattering (SANS) is discussed, together with the prospects for including SANS in a future renewal certification.

Concepts: Diffraction, Scattering, Standard, Calibration, Certified reference materials, X-rays, Small-angle X-ray scattering, Small-angle scattering

1

We have developed a highly sensitive and selective analytical method capable of quantifying a total of 15 polybrominated and polychlorinated biphenyls (11 PBBs and 4 PCBs) in human serum. Samples were subjected to liquid-liquid extraction followed by solid-phase extraction prior to measurement using gas chromatography-tandem mass spectrometry in multiple reaction monitoring mode. Quantification was performed using isotope-dilution calibration covering a concentration range of 0.005-12.5 ng/mL. Limits of detection for all target compounds were in the low range (0.7-6.5 pg/mL). The method was validated using in-house pooled human serum fortified at two concentrations (0.5 ng/mL and 1.0 ng/mL), whole semen fortified at one concentration (0.25 ng/mL), and NIST Standard Reference Material (SRM) 1958, which includes five of the target compounds. Method accuracies for all target compounds ranged from 84 to 119% with relative standard deviations (RSDs) of <19%. The measured values for the five target compounds present in the SRM agreed with the certified reference values (89-119% accuracy with RSDs <9%). As this method was developed to support ongoing epidemiologic investigations, we evaluated its suitability by analyzing subsets of serum and whole semen samples from the Michigan PBB Registry cohort. PBB-153, PCB-118, PCB-138, PCB-153 and PCB-180 were detected in all serum samples analyzed, with PBB-77 and PBB-101 detected less frequently in serum. PBB-153, PCB-118, PCB-138, PCB-153 and PCB-180 were detected in at least one whole semen sample.

Concepts: Mass spectrometry, Analytical chemistry, Standard, Calibration, Certified reference materials, Polychlorinated biphenyl, Biphenyl, Polychlorinated dibenzodioxins

1

From measurements over the last two years we have demonstrated that the charge collection system based on Faraday cups can robustly give near-1% absolute implantation fluence accuracy for our electrostatically scanned 200 kV Danfysik ion implanter, using four-point-probe mapping with a demonstrated accuracy of 2%, and accurate Rutherford backscattering spectrometry (RBS) of test implants from our quality assurance programme. The RBS is traceable to the certified reference material IRMM-ERM-EG001/BAM-L001, and involves convenient calibrations both of the electronic gain of the spectrometry system (at about 0.1% accuracy) and of the RBS beam energy (at 0.06% accuracy). We demonstrate that accurate RBS is a definitive method to determine quantity of material. It is therefore useful for certifying high quality reference standards, and is also extensible to other kinds of samples such as thin self-supporting films of pure elements. The more powerful technique of Total-IBA may inherit the accuracy of RBS.

Concepts: Measurement, Quality assurance, Test method, Ion source, Standard, Calibration, Certified reference materials, Standards

1

Nucleic acid amplification technique (NAT)-based assays are increasingly used for the detection of mycoplasma contamination of cell cultures, as an alternative to culture-based approaches. Assay features, like limit of detection or quantification vary widely between different mycoplasma NAT assays. Biological reference materials may be useful for harmonization of mycoplasma NAT assays. An international feasibility study included lyophilized preparations of four distantly related mycoplasma species (Acholeplasma (A.) laidlawii, Mycoplasma (M.) fermentans, M. orale, M. pneumoniae) at different concentrations which were analysed by 21 laboratories using 26 NAT assays of qualitative, semi-quantitative or quantitative design. An M. fermentans preparation was shown to decrease interassay variation when used as a common reference material. The preparation was re-manufactured, characterized in a comparability study and its potency determined in “NAT detectable units” across different NATs. The WHO Expert Committee on Biological Standardization (ECBS) established this preparation as “1st WHO International Standard (IS) for mycoplasma DNA for NAT assays designed for generic mycoplasma detection” with a potency of 200,000 International Units/ml. This WHO IS is now available as a reference preparation for characterization of NAT assays e.g determination of analytic sensitivity, for calibration of quantitative assays in a common unitage and for defining regulatory requirements in the field of mycoplasma testing.

Concepts: DNA, Nucleic acid, Analytical chemistry, Biochemistry, Mycoplasma, World Health Organization, Certified reference materials, International Organization for Standardization

0

We propose an improved method for precise sulfur isotopic measurements by multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS) in conjunction with a membrane desolvation nebulization system. The problems of sulfur loss through the membrane desolvation apparatus are carefully quantified and resolved. The method overcomes low intrinsic sulfur transmission through the instrument, which was initially 1% when operating at a desolvation temperature of 160 °C. Sulfur loss through the membrane desolvation apparatus was resolved by doping with sodium. A Na/S ratio of 2 mol mol(-1) produced sulfur transmissions with 98% recovery. Samples of 3 nmol (100 ng) sulfur achieved an external precision of ±0.18‰ (2 SD) for δ(34)S and ±0.10‰ (2 SD) for Δ(33)S (uppercase delta expresses the extent of mass-independent isotopic fractionation). Measurements made on certified reference materials and in-house standards demonstrate analytical accuracy and reproducibility. We applied the method to examine microbial-induced sulfur transformation in marine sediment pore waters from the sulfate-methane transition zone. The technique is quite versatile, and can be applied to a range of materials, including natural waters and minerals.

Concepts: Scientific method, Sediment, Method acting, Accuracy and precision, Isotope, Certified reference materials, Standards, Transmission

0

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950 Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each lab using a different lipidomics workflow. A total of 1527 unique lipids were measured across all laboratories, and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and inter-laboratory quality control and method validation. These analyses were performed using non-standardized laboratory-independent workflows. The consensus locations were also compared to a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

Concepts: Metabolism, Metrology, Chemistry, Management, Lipids, Standard, Uncertainty, Certified reference materials

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After environmental and occupational exposure to naphthalene, 1,2-dihydroxynaphthalene (1,2-DHN) was shown to be one major metabolite in human naphthalene metabolism. However, the instability of free 1,2-DHN complicates the reliable determination of this promising biomarker in urine. To solve this stability problem, glucuronide conjugates of 1,2-DHN and the corresponding isotopically labelled D6-1,2-dihydroxynaphthalene (D6-1,2-DHN) were synthesised and applied as reference material and internal standard in a gas chromatographic-tandem mass spectrometric (GC-MS/MS) method. The determination of 1- and 2-naphthol (1-MHN, 2-MHN) was included in the procedure to enable a comprehensive assessment of naphthalene metabolism and exposure. The results of the validation showed a high reliability and sensitivity of the method. The detection limits range from 0.05 to 0.16 μg/L. Precision and repeatability were determined to range from 1.4 to 6.6% for all parameters. The simultaneous determination of 1- and 2-MHN as additional parameters besides 1,2-DHN enables the application of the method for further metabolism and kinetic studies on naphthalene. The use of glucuronide-derivative reference substances and the application of structurally matched isotopic-labelled internal standards for each substance guarantee a reliable quantification of the main naphthalene metabolites 1,2-DHN and 1- and 2-MHN. Graphical abstract Reliable quantification of 1,2-dihydroxynaphthalene in urine using a conjugated reference compound for calibration.

Concepts: Scientific method, Metabolism, Metrology, Reliability, Standard, Certified reference materials, Metabolomics, Metabolome

0

The absolute quantification of five toxins involved in ciguatera fish poisoning (CFP) in the Pacific was carried out by quantitative ¹H-NMR. The targeted toxins were ciguatoxin-1B (CTX1B), 52-epi-54-deoxyciguatoxin-1B (epideoxyCTX1B), ciguatoxin-3C (CTX3C), 51-hydroxyciguatoxin-3C (51OHCTX3C), and ciguatoxin-4A (CTX4A). We first calibrated the residual protons of pyridine-d₅ using certified reference material, 1,4-BTMSB-d₄, prepared the toxin solutions with the calibrated pyridin-d₅, measured the ¹H-NMR spectra, and quantified the toxin using the calibrated residual protons as the internal standard. The absolute quantification was carried out by comparing the signal intensities between the selected protons of the target toxin and the residual protons of the calibrated pyridine-d₅. The proton signals residing on the ciguatoxins (CTXs) to be used for quantification were carefully selected for those that were well separated from adjacent signals including impurities and that exhibited an effective intensity. To quantify CTX1B and its congeners, the olefin protons in the side chain were judged appropriate for use. The quantification was achievable with nano-molar solutions. The probable errors for uncertainty, calculated on respective toxins, ranged between 3% and 16%. The contamination of the precious toxins with nonvolatile internal standards was thus avoided. After the evaporation of pyridine-d₅, the calibrated CTXs were ready for use as the reference standard in the quantitative analysis of ciguatoxins by LC/MS.

Concepts: Spin, Metrology, Nuclear magnetic resonance, Standard, In vivo magnetic resonance spectroscopy, Certified reference materials, Standards, Ciguatera