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Concept: Certified reference materials


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


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


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


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


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


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


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


Digestion techniques for ICP analysis have been poorly studied for biological samples. This report describes an optimized method for analysis of trace metals that can be used across a variety of sample types. Digestion methods were tested and optimized with the analysis of trace metals in cancerous as compared to normal tissue as the end goal. Anthropological, forensic, oncological and environmental research groups can employ this method reasonably cheaply and safely whilst still being able to compare between laboratories. We examined combined HNO3 and H2O2 digestion at 170 °C for human, porcine and bovine samples whether they are frozen, fresh or lyophilized powder. Little discrepancy is found between microwave digestion and PFA Teflon pressure vessels. The elements of interest (Cu, Zn, Fe and Ni) yielded consistently higher and more accurate values on standard reference material than samples heated to 75 °C or samples that utilized HNO3 alone. Use of H2SO4 does not improve homogeneity of the sample and lowers precision during ICP analysis. High temperature digestions (>165 °C) using a combination of HNO3 and H2O2 as outlined are proposed as a standard technique for all mammalian tissues, specifically, human tissues and yield greater than 300% higher values than samples digested at 75 °C regardless of the acid or acid combinations used. The proposed standardized technique is designed to accurately quantify potential discrepancies in metal loads between cancerous and healthy tissues and applies to numerous tissue studies requiring quick, effective and safe digestions.

Concepts: Iron, Metal, Zinc, Copper, Standard, Digestion, Certified reference materials, Nickel


Multi-isotope calibration (MICal) is a novel approach to calibration for inductively coupled plasma mass spectrometry (ICP-MS). In MICal, only two calibration solutions are required: solution A, composed of 50% v v(-1) of sample and 50% v v(-1) of a standard solution containing the analytes, and solution B, composed of 50% v v(-1) of sample and 50% v v(-1) of a blank solution. MICal is based on monitoring the signal intensities of several isotopes of the same analyte in solutions A and B. By plotting the analytical signals from solution A in the x-axis, and from solution B in the y-axis, the analyte concentration in the sample is calculated using the slope of that graph and the concentration of the reference standard added to solution A. As both solutions contain the same amount of sample, matrix-matching is easily achieved. In this proof-of-concept study, MICal was applied to the determination of Ba, Cd, Se, Sn, and Zn in seven certified reference materials with different matrices (e.g., plant materials, flours, and water). In most cases, MICal results presented no statistical difference from the certified values at a 95% confidence level. The new strategy was also compared with traditional calibration methods such as external calibration, internal standardization and standard additions, and recoveries were generally better for MICal. This is a simple, accurate, and fast alternative method for matrix-matching calibration in ICP-MS. Graphical abstract Multi-isotope calibration: fast and innovative matrix-matching calibration for ICP-MS.

Concepts: Mass spectrometry, Chemistry, Analytical chemistry, Standard, Solution, Calibration, Certified reference materials, Standards


The synthesis and characterization of a series of donor-π-acceptor-π-donor (D-A-D) curcuminoid molecules are here presented incorporating π-extended aryl and electron-donating amino terminal functionalization. Computational evaluation shows these molecules possess quadrupolar character with the lowest energy transitions displaying high molar extinction coefficients with broad tunability through manipulation of terminal donating groups. Consistent with their quadrupolar nature, these molecules show varying degrees of solvatochromic behavior in both their absorption and emission spectra which has been analyzed by Lippert-Mataga and Kamlet-Taft analysis. Photophysical and photoacoustic (PA) properties of these molecules have been investigated by the optical photoacoustic z-scan (OPAZ) method, which found select curcuminoid molecules display nonlinear behavior at a high laser fluence through excited state absorption that translates to the production of an enhanced photoacoustic emission. A relative comparison of ‘molar PA emission’ has been presented with the crystal violet linear optical absorbing/linear PA emitting (LA) system being utilized as a standard reference material for OPAZ experiments. Furthermore, PA tomography experiments are also presented to illustrate the enhanced PA contrast obtainable via an excited state absorption.

Concepts: Photon, Stimulated emission, Chemistry, Atom, Absorption spectroscopy, Certified reference materials, Mass attenuation coefficient, Molar absorptivity