Overcoming challenges regarding reference materials and regulations that influence global standardization of medical laboratory testing results.

BACKGROUND: Standardized results for laboratory tests are particularly important when their interpretation depends on fixed medical practice guidelines or common reference intervals. The medical laboratory community has developed a roadmap for an infrastructure to achieve standardized test results described in the International Organization for Standardization standard 17511:2020 In vitro diagnostic medical devices - Requirements for establishing metrological traceability of values assigned to calibrators, trueness control materials and human samples. Among the challenges to implementing metrological traceability are the availability of fit-for-purpose matrix-based certified reference materials (CRMs) and requirements for regulatory review that differ among countries. A workshop in December 2021 focused on these two challenges and developed recommendations for improved practices. DISCUSSION: The participants agreed that prioritization of measurands for standardization should be based on their impact on medical decisions in a clinical pathway. Ensuring that matrix-based CRMs are globally available for more measurands will enable fit-for-purpose calibration hierarchies for more laboratory tests. Regulation of laboratory tests is important to ensure safety and effectiveness for the populations served. Because regulations are country or region specific, manufacturers must submit recalibration changes intended to standardize results for regulatory review to all areas in which a measuring system is marketed. RECOMMENDATIONS: A standardization initiative requires collaboration and planning among all interested stakeholders. Global collaboration should be further developed for prioritization of measurands for standardization, and for coordinating the production and supply of CRMs worldwide. More uniform regulatory submission requirements are desirable when recalibration is implemented to achieve internationally standardized results.

An SI-traceable reference material for virus-like particles.

A reference material for virus-like particles traceable to the International System of Units (Système International d'Unités - the SI) is reported. The material addresses the need for developing reference standards to benchmark virus-like gene delivery systems and help harmonize measurement approaches for characterization and testing. The material is a major component of synthetic polypeptide virus-like particles produced by the state-of-the-art synthetic and analytical chemistry methods used to generate gene delivery systems. The purity profile of the material is evaluated to the highest metrological order demonstrating traceability to the SI. The material adds to the emerging toolkit of reference standards for quantitative biology.

Metrological framework to support accurate, reliable, and reproducible nucleic acid measurements.

Nucleic acid analysis is used in many areas of life sciences such as medicine, food safety, and environmental monitoring. Accurate, reliable measurements of nucleic acids are crucial for maximum impact, yet users are often unaware of the global metrological infrastructure that exists to support these measurements. In this work, we describe international efforts to improve nucleic acid analysis, with a focus on the Nucleic Acid Analysis Working Group (NAWG) of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM). The NAWG is an international group dedicated to improving the global comparability of nucleic acid measurements; its primary focus is to support the development and maintenance of measurement capabilities and the dissemination of measurement services from its members: the National Metrology Institutes (NMIs) and Designated Institutes (DIs). These NMIs and DIs provide DNA and RNA measurement services developed in response to the needs of their stakeholders. The NAWG members have conducted cutting edge work over the last 20 years, demonstrating the ability to support the reliability, comparability, and traceability of nucleic acid measurement results in a variety of sectors.

Structurally related peptide impurity identification and accurate quantification for synthetic oxytocin by liquid chromatography-high-resolution mass spectrometry.

Oxytocin (OXT) is an important peptide that is mainly used as a therapeutic drug to induce labor or strengthen uterine contractions, or to control bleeding after childbirth. OXT has also been reported as a biomarker linked to emotion, and as a potential biomarker for cancer diagnosis. The accurate purity characterization of OXT calibrators is critical for quality control of pharmaceuticals and the development of reference measurement systems for this analyte in laboratory medicine. OXT possesses the particular analytical measurement challenge of a disulfide bond. Accurate value assignment of the purity of oxytocin calibrators can be carried out by applying the mass balance approach or alternative approaches such as amino acid analysis, quantitative nuclear magnetic resonance spectrometry, and nitrogen determination. In order to avoid biases, all these approaches require a correction for structurally related peptide impurities. Structurally related peptide impurities present in a synthetic OXT material have been identified and quantified by a newly developed and in-house-validated liquid chromatography-high-resolution mass spectrometry (LC-hrMS) method. This method was adopted for the measurement of the study material used for an international comparison evaluating the competencies of laboratories to perform peptide characterization. Eighteen structurally related impurities were identified, confirmed, and accurately quantified in the OXT study material by using LC-hrMS. The study material contained a total mass fraction of 31.1 mg/g structurally related OXT impurities with an associated expanded uncertainty of 1.7 mg/g.

The BIPM Mycotoxin Metrology Capacity Building and Knowledge Transfer Program: Accurate Characterization of a Pure Aflatoxin B1 Material to Avoid Calibration Errors.

Background: The contamination of food and feed by mycotoxins, Aflatoxin B1 (AfB1) being one of the most prominent examples, is of imminent concern to many countries. Regulatory limits for mycotoxins have been implemented, and these need to be supported by a sound measurement infrastructure for mycotoxin analysis in order to enforce and verify products, protect populations, and avoid technical barriers to trade in food stuffs. Objective: A Capability Building and Knowledge Transfer program on Metrology for Safe Food and Feed in Developing Economies was started at the International Bureau of Weights and Measures to allow National Metrology Institutes or Designated Institutes to work together to strengthen their national mycotoxin metrology infrastructure. Methods: Knowledge transfer to scientists is provided to enable the characterization of selected pure mycotoxin materials and the production of corresponding certified reference material solutions. Results: This higher-order measurement capability can in turn support mycotoxin testing laboratories within their countries through the provision, for example, of standard solutions of critical analytes that are traceable to the International System of Units (SI). Conclusions and Highlights: The purity characterization and value assignment for a high-purity AfB1 material (979.6 ± 2.3 mg/g, k = 2) intended to be used for the gravimetric production of SI traceable calibration solutions for AfB1 is described using an approach combining quantitative NMR and LC-diode array detection-tandem MS for the correction of the mycotoxin-related impurity content.

Identification and accurate quantification of structurally related peptide impurities in synthetic human C-peptide by liquid chromatography-high resolution mass spectrometry.

Peptides are an increasingly important group of biomarkers and pharmaceuticals. The accurate purity characterization of peptide calibrators is critical for the development of reference measurement systems for laboratory medicine and quality control of pharmaceuticals. The peptides used for these purposes are increasingly produced through peptide synthesis. Various approaches (for example mass balance, amino acid analysis, qNMR, and nitrogen determination) can be applied to accurately value assign the purity of peptide calibrators. However, all purity assessment approaches require a correction for structurally related peptide impurities in order to avoid biases. Liquid chromatography coupled to high resolution mass spectrometry (LC-hrMS) has become the key technique for the identification and accurate quantification of structurally related peptide impurities in intact peptide calibrator materials. In this study, LC-hrMS-based methods were developed and validated in-house for the identification and quantification of structurally related peptide impurities in a synthetic human C-peptide (hCP) material, which served as a study material for an international comparison looking at the competencies of laboratories to perform peptide purity mass fraction assignments. More than 65 impurities were identified, confirmed, and accurately quantified by using LC-hrMS. The total mass fraction of all structurally related peptide impurities in the hCP study material was estimated to be 83.3 mg/g with an associated expanded uncertainty of 3.0 mg/g (k = 2). The calibration hierarchy concept used for the quantification of individual impurities is described in detail. Graphical abstract ᅟ.

Implementing a Reference Measurement System for C-Peptide: Successes and Lessons Learned.

BACKGROUND: Assessment of endogenous insulin secretion by measuring C-peptide concentrations is widely accepted. Recent studies have shown that preservation of even small amounts of endogenous C-peptide production in patients with type 1 diabetes reduces risks for diabetic complications. Harmonization of C-peptide results will facilitate comparison of data from different research studies and later among clinical laboratory results at different sites using different assay methods. CONTENT: This review provides an overview of the general process of harmonization and standardization and the challenges encountered with implementing a reference measurement system for C-peptide. SUMMARY: Efforts to harmonize C-peptide results are described, including those by the National Institute of Diabetes and Digestive and Kidney Diseases-led C-peptide Standardization Committee in the US, activities in Japan, efforts by the National Institute for Biological Standards and Control in the UK, as well as activities led by the Bureau International des Poids et Mesures and the National Metrology Institute in China. A traceability scheme is proposed along with the next steps for implementation. Suggestions are made for better collaboration to optimize the harmonization process for other measurands.

Impurity determination for hepcidin by liquid chromatography-high resolution and ion mobility mass spectrometry for the value assignment of candidate primary calibrators.

In metrology institutes, the state-of-the-art for purity analysis of peptides/proteins mainly addresses short and unfolded peptides. Important developments are anticipated for the characterization of nonlinear peptides or proteins. Hepcidin 1-25 is an interesting model system because this small protein contains four disulfide bridges with a particular connectivity that is difficult to reproduce and could induce a bias in quantification. Hepcidin 1-25 is involved in iron-related disorders and anemia, in an inflammatory context, and its clinical relevance in neurodegenerative disorders is under investigation. It is also an emerging biomarker. Recent inter-laboratory studies showed a need for standardization of hepcidin assay and the need to produce certified reference materials. This paper discusses two hepcidin standards from different synthesis pathways that have been characterized by high-resolution mass spectrometry and ion mobility mass spectrometry.

Ozone Cross-Section Measurement by Gas Phase Titration.

Elevated values of ground-level ozone damage health, vegetation, and building materials and are the subject of air quality regulations. Levels are monitored by networks using mostly ultraviolet (UV) absorption instruments, with traceability to standard reference photometers, relying on the UV absorption of ozone at the 253.65 nm line of mercury. We have redetermined the ozone cross-section at this wavelength based on gas phase titration (GPT) measurements. This is a well-known chemical method using the reaction of ozone (O3) with nitrogen monoxide (NO) resulting in nitrogen dioxide (NO2) and oxygen (O2). The BIPM GPT facility uses state-of-the-art flow measurement, chemiluminescence for NO concentration measurements, a cavity phase shift analyzer (CAPS) for NO2 measurements, and a UV ozone analyzer. The titration experiment is performed over the concentration range 100-500 nmol/mol, with NO and NO2 reactants/calibrants diluted down from standards with nominal mole fractions of 50 µmol/mol. Accurate measurements of NO, NO2, and O3 mole fractions allow the calculation of ozone absorption cross section values at 253.65 nm, and we report a value of 11.24 × 10-18 cm2 molecule-1 with a relative expanded uncertainty of 1.8% (coverage factor k = 2) based on nitrogen monoxide titration values and a value of 11.22 × 10-18 cm2 molecule-1 with a relative expanded uncertainty of 1.4% (coverage factor k = 2) based on nitrogen dioxide titration values. The excellent agreement between these values and recently published absorption cross-section measurements directly on pure ozone provide strong evidence for revising the conventionally accepted value of ozone cross section at 253.65 nm.

Use of the international system of units (SI) in isotope ratio mass spectrometry.

In this communication, we show how the established principles of the International System of Units (SI) can be used to develop a basis for stable, comparable and coherent measurements of isotope ratios by mass spectrometry. We show that there are no important differences in the traceability of quantities that represent ratios of the abundance of isotopes and those that represent fractions. However, it is important to distinguish between results expressed in 'absolute' terms and those expressed relative to stated references such as those expressed in terms of the commonly used quantity 'delta'.