June 1 – 4, 2025

NIST has supported the global food industry with matrix-based Standard Reference Materials (SRMs) since the early 1990s. Early efforts focused on assignment of certified values for nutrients, including elements, vitamins, and fatty acids, as well as for toxic elements in a variety of matrix types across the scope of the AOAC food triangle. Over the last 10 years, stakeholder input has generally guided the NIST food program to a greater focus on materials to support food safety and a more flexible approach to support a constantly changing industry. NIST has developed new reference materials for detection of organic contaminants such as mycotoxins, per- and polyfluoroalkyl substances (PFAS), and pesticide residues in foods, as well as for quality assurance in authenticity and allergen determinations. NIST has also utilized the Food Nutrition and Safety Measurements Quality Assurance Program (FNSQAP) to more rapidly respond to requests for new materials and support stakeholder needs for quality assurance metrics. In these broad and challenging fields, future needs can only be met through a fresh perspective on fitness-for-purpose, value assignment, and unique tools to support accurate measurements that ensure the quality and safety of the global food supply.

Vitamin B9 is an essential nutrient for biological processes, playing a crucial role in nucleotide synthesis, particularly in the form of 5-methyltetrahydrofolate(5-Me-THF).This study focuses on the development of certified reference materials(CRMs), specifically KRISS CRM 108-05-010 and 108-05-011, for 5-Me-THF analysis in spinach and kimchi cabbage, which are important food sources of 5-Me-THF. Fresh spinach and kimchi cabbage underwent washing, freeze-drying, pulverizing, sieving, and V-mixing, followed by bottling into 10 g portions. The analytical method was developed using a tri-enzyme method combined with isotope dilution ultra-performance liquid chromatography-tandem mass spectrometry to certify the CRMs. This method was repeatable and reproducible, with recoveries ranging from 96.5% to 107.2%. Stability studies confirmed that the CRMs remain stable for seven days at room temperature, one month at 20C, and over 13 months at 70C. Consequently, the certified values were determined to be (16.93 0.73) mg/kg for spinach and (11.66 0.25) mg/kg for kimchi cabbage, with homogeneity of 1.85% and 0.91%, respectively. Long-term stability assessments indicated that the CRMs are valid for at least 48 months when stored at 70C. These CRMs will help testing laboratories develop analytical methods for assessing folate content in vegetables.

Algal dietary supplements are frequently prepared using cyanobacterial biomass obtained from open air production lakes. Source materials and final products contaminated with cyanobacterial toxins present serious risks for human health, while also impacting product manufacturers. Reference materials are therefore required to assist in the development and validation of analytical methods for raw material and end product testing for both research and regulatory purposes. A new certified reference material (CRM) for analysis of dietary supplements, CRM-Cyano-T, was prepared by blending biomass of a typically-consumed non-toxic cyanobacteria (Aphanizomenon flos-aquae) with a variety of toxic cyanobacterial species. This created a material with a toxin profile including microcystins, anatoxins, cylindrospermopsins and saxitoxins. The blended matrix was freeze-dried and homogenized, then aliquoted in 0.6g units in amber glass bottles under an inert atmosphere. A liquid chromatography-mass spectrometry (LC-MS/MS) method was developed to comprehensively evaluate the homogeneity and stability of all toxin classes present. Strategies were developed for accurate value assignment of the various toxins, including optimized extraction and class-specific detection methods. Approaches used for matrix effect compensation included isotope dilution and standard addition. Certified values and associated uncertainties were assigned for analytes with available calibration solution CRMs, and concentrations ranged from 1.3 (gonyautoxin-3) to 74.9 (cylindrospermopsin) g/g. Non-target high-resolution mass spectrometry workflows were used to characterize the broader metabolite profile of the CRM. Produced in compliance with international standards on reference materials, CRM-Cyano-T will play an important role in development and validation of methods for nutritional supplement inspection and environmental monitoring.

The growing interest in insect protein as a sustainable alternative to conventional protein sources underscores the need for robust safety and quality assurance measures. While insect-based proteins offer significant nutritional benefits, ensuring their safety requires reliable analytical methods and access to certified reference materials (CRMs) specific to this matrix. However, the limited availability of CRMs for assessing inorganic and organic contaminants in insect proteins presents a critical challenge for the industry.

This poster details the development of an incurred cricket powder CRM for inorganic elements, including toxic elements (cadmium, chromium, mercury, lead, total arsenic, and arsenic species) and selenium. The approach involved tailored feed formulations for crickets reared under controlled farming conditions to achieve specific contaminant levels. The production process adhered to ISO 17034 standards, ensuring the CRM meets stringent quality requirements.

Extensive trials were conducted to ensure sufficient bioaccumulation of contaminants through insect feed and feeding regimens. The resulting material features elevated levels of inorganic elements, making it ideal for use as quality control samples and for method development and validation. Reference values were assigned using high-accuracy analytical methods, ensuring metrological traceability with well-defined measurement uncertainties.

The lack of insect-specific CRMs has hindered efforts to ensure the safety and quality of insect protein products. This CRM development represents a critical advancement, fostering consumer and regulatory confidence in the rapidly expanding insect protein industry while addressing key analytical challenges.

Ergot alkaloids are a class of mycotoxins produced by fungi of the Claviceps genus, which are commonly found in contaminated cereals. Due to their toxic effects, the European Union introduced the first regulatory limit values for ergot alkaloids in food in 2022. Accurate quantification is crucial for food safety monitoring; however, the lack of stable isotopically labeled (SIL) standards has been a major limitation in the development of analytical methods via HPLC-MS/MS.

To overcome this challenge, all 12 priority ergot alkaloids were synthesized as 13CD3-labeled analogs for the first time and used as internal standards for mass spectrometry-based analysis. Using HPLC-MS/MS, we quantified ergot alkaloids in various food matrices, including bread, rye and wheat flour, and bran. The performance of the SIL standards was evaluated by comparing results obtained using external calibration and standard addition approaches. Our study demonstrates that the use of SIL standards significantly improves the accuracy and precision of ergot alkaloid quantification by correcting for matrix effects and signal variations in mass spectrometric analysis. The newly synthesized 13CD3-labeled ergot alkaloids provide a valuable tool for food safety assessments, ensuring more reliable and reproducible data. Furthermore, these findings support the advancement of analytical methods for ergot alkaloid monitoring, contributing to improved food quality control and regulatory compliance.

The National Institutes of Health Office of Dietary Supplements (ODS) Analytical Methods and Reference Materials Program (AMRM) supports the development of research tools that facilitate thorough characterization of dietary supplement ingredients and products, and their metabolites in clinical samples. AMRM goals are accomplished through three program areas: development and validation of quantitative and qualitative methods, production of certified reference materials (CRMs), and support of dietary supplement focused laboratory quality assurance programs. Recent advances in dietary supplement analytical resources supported by AMRM are presented here, including an expanded availability of natural matrix and calibration solution CRMs for key bioactive and marker phytochemical constituents of several popular botanical supplement ingredients, and the deployment of a new online database to identify fit-for-purpose reference materials for dietary supplement research. AMRM supported resources benefit researchers and industry scientists by expanding the analytical toolkit to investigate chemical characterization, biological effects, and safety of dietary supplements more rigorously and reliably. This presentation will provide examples of how the ODS AMRM supported resources have been used for method development and validation, establishing the performance of analytical instruments and methods, and expanding our understanding of dietary supplement health effects.

The rapid expansion of electromobility and energy storage technologies has significantly increased the demand for lithium-ion batteries (LIBs), intensifying the need for reliable certified reference materials (CRMs) to ensure analytical accuracy and comparability throughout the battery life cycle. To address this critical requirement, we recently developed BAM-S014, the first CRM for lithium nickel manganese cobalt oxide cathode material (NMC 111). This established a foundation for quality control and regulatory compliance in LIB manufacturing. Building upon this achievement, our current activities include developing additional CRMs that cover a comprehensive range of materials crucial for the LIB circular economy. These candidate CRMs encompass active battery materials such as advanced cathode formulations (NMC 811) and graphite anode material, raw materials including lithium carbonates from mining at varying purity levels, and recycled battery materials (black mass) derived from NMC and lithium iron phosphate (LFP) battery chemistries.

Certification processes involve international interlaboratory comparisons, rigorous homogeneity and stability testing, and comprehensive uncertainty evaluations. Additionally, informative lithium isotope ratio measurements are conducted, providing valuable insights into raw material provenance and supporting emerging digital battery passport initiatives designed to trace supply chains and enhance transparency. By developing these targeted CRMs, we directly contribute to global efforts towards standardized, sustainable battery-material management practices, enabling reliable material valuation and effective recycling within the circular economy. This work highlights how CRMs support laboratories and industries in meeting evolving analytical and regulatory requirements, promoting efficient resource utilization, and reducing environmental impacts throughout the LIB value chain.

In the 21st century, with the rapid development of biotechnology and bio-industry, coupled with growing challenges to human survival and health, the need for accurate and reliable biological data has become increasingly pronounced. It has not only given rise to the new discipline of biometrology but has also drawn global attention to its standards and biological reference materials (BRMs). Under the new circumstances, the volume of data from bioanalytical measurements is increasing at an exponential rate. Biometrologyincludes nucleic acid metrology, protein metrology, microbial metrology, cell metrology and other areas that fall under the umbrella of biometrology. It plays a particularly crucial role in guaranteeing the validity of biological data. With over 20 years of development, the biometrology systems have been established and continuously enhanced through research efforts in China. A classification study of BRMs was developed. Research and application efforts in biomeasurement technology and standard, along with the development of standard systems, have been initiated for the aim to establish and maintain the measurement standard.

These technologies and BRMs have been used in many fields, including the detection nucleic acids and proteins in transgenic products, in vitro diagnostic, virus detection, and quarantine measures, facilitating the acquisition of accurate values and enabling traceability. Biometrology and BRMs research is also facing with challenges for these demands.

Ciguatoxins (CTXs) are an important class of marine biotoxins linked to the consumption of reef fish harvested from tropical and subtropical regions. There are estimated to be 10 000 to 500 000 ciguatera poisonings globally each year, with increased occurrences emerging in traditionally non-endemic, temperate regions. The lack of reference materials for CTXs has significantly hampered research on pharmacology, environmental monitoring, regulations and long-term management. Ciguatoxins are tasteless, odorless, heat tolerant, large polyether compounds. As mobile vectors, toxicity in fish can be highly variable between individual fish harvested from the same region, making purification of CTXs from fish tissues extremely challenging, impeding the development of calibration reference materials. The recent discovery of an algal produced analogue of Caribbean ciguatoxins (C-CTX5) has facilitated the development of reference materials for this toxin class. Following large-scale culturing of a toxic strain of Gambierdiscus silvae, procedures were developed for isolation of C-CTX5 and subsequent preparation of calibration solutions. Feasibility studies were performed to establish the optimal diluent, assess stability, and to establish the value-assignment. Fish tissues naturally contaminated with C-CTXs were used to conduct small-scale studies and develop protocols for tissue preparation to improve handling and maintain stability of the toxins during storage. These successful feasibility studies provide the basis to prepare large-scale reference materials for ciguatoxins, which are essential to ensure accurate measurements for both research and regulatory frameworks.

Literature suggests that providing shared samples of specific matrices to a community of researchers could be a promising approach for future development of Certified Reference Materials. This presentation describes the components needed to establish such a resource and its expected outcomes. The core elements necessary for this initiative include: 1) a material processing facility, 2) a storage and distribution center, 3) an online database for reporting measurement results.

To illustrate the concept, we present the development of two materials: microplastics and honey for detecting adulteration. The development of new RMs is sometimes connected with novel measurement methods. A challenge arises when characterizing such RMs developments through interlaboratory comparisons, as it creates an RM/Method causality dilemma. New RMs require reliable methods, which in turn need good quality RMs. To overcome this, RM producers must take iterative steps, refining the candidate materials, which in turn allows improving analytical methods. As both the materials and methods evolve, they will eventually reach a point where target parameters can be certified.

When multiple laboratories analyse common samples, clusters of results eventually emerge, providing a network of laboratories capable of performing specific measurements. The materials and database become valuable assets for RM producers. Where, also method developers will benefit, from access to common materials, providing comparability and also enabling them to compare and evaluate the capabilities of different methods. By collaborating, RM producers and method developers can break the cycle of the RM/Method causality dilemma, driving progress in the development of new RMs and measurement methods.

One of the main challenges in validating microplastics detection methods is the lack of suitable reference materials that accurately reflect the properties of interest. To bridge this gap, the EU-funded PlasticTrace project has developed water-soluble tablets containing precisely defined amounts of polyethylene terephthalate (PET) particles. Each tablet includes a specific mass (0.2, 18.5, or 400 g) and particle count (151 or 1764, measured via Raman spectroscopy). Production follows a rigorous solid-phase dilution process, ensuring high reproducibility and quality control. The PET particles used in these tablets have an average size of approximately 40 m, with a range between 10 and 200 m, and an irregular, stone-like shape. All measurements align with ISO/TC 147/SC 2 standardization efforts and adhere to ISO/DIS 16094-2 and ISO/DIS 16094-3. To assess tablet homogeneity, the most widely accepted microplastics detection techniques, i.e. -FTIR, -Raman, Py-GC/MS, and TED-GC/MS, were applied. Different batches were produced, each containing varying particle numbers and masses, reaching the detection limits of the methods used. The results demonstrated excellent consistency between tablets, successfully passing homogeneity tests. Furthermore, no significant changes were observed in the tablets composition or stability after six months of storage. This innovative reference material represents an important advancement in microplastics analysis, supporting laboratories in meeting EU Commission requirements for the monitoring of microplastics in drinking water and wastewater with greater accuracy and reliability. The production process can be transferred for other plastic particles is this size range.

As remarked by the International Panel on Climate Change, the measurement of nutrients in seawater is relevant for understanding large-scale changes in marine biology and monitor the health of marine ecosystems. Although nutrients are among the most measured analytes in seawater, comparability between nutrients data sets remains inadequate for accurate interpretation. The availability and routine use of seawater Certified Reference Materials (CRMs) has been identified by oceanographers as a viable solution for the harmonization of nutrients data. In the past five years, the NRC engaged in the production of two CRMs which could be employed as quality control for nutrients measurements. With the NRC SALT-1 CRM, the main goal was to address one of the most difficult aspect of preparation, namely the stability of the matrix toward biological degradation. The SALT-1 was produced starting from a marine salt spiked with part-per-million levels of nitrate, phosphate, and silicate. Homogenization (u_hom < 1.2%)was obtained by ball milling without the need of further sterilization. When reconstituted in water, the SALT-1 could be effectively used as a seawater standard. Conversely, the NRC MOOS-4 CRM was prepared starting from nearshore seawater collected in Ketch Harbour (Nova Scotia). The MOOS-4 required sterilization by gamma radiation to ensure stability and was certified with high-precision for nitrite (u = 0.4%), nitrate (u = 1.3%), phosphate (u = 0.5%), and silicate (u = 0.9%). In this talk, preparation strategies for nutrients CRMs and advanced analytical methods for value assignment will be discussed.

The increasing applications of stable isotopes in addressing environmental, forensic, and geological highlight the need for more reference materials. There is only one primary reference material in the carbonate matrix, IAEA-603, to anchor the VPDB scale for both carbon (C) and oxygen (⁸O) isotope ratio. This underscores the need for new certified reference materials (CRMs). To address this, we produced a candidate CRM of optical calcite sourced from the Bocaina Formation (Corumb, Brazil). Comprehensive characterization using optical microscopy, X-ray diffraction and X-ray fluorescence confirmed its mineralogical purity. The core of each calcite crystal was fragmented, ground manually using mortar and agate pistil, sieved to 212500 m granulometry, homogenized and packed (1g) under an inert atmosphere into glass vials. For homogeneity assessment, 10 units were randomly chosen from the batch of 120 units and measured in triplicate in aleatory order to evaluate between-unit variability and within-unit variability of the material. Optical calcite showed homogeneity. The propriety values were C = 4.98 and ⁸O = 9.77 (VPDB scale). It also showed stability under transport conditions assessed by keeping two vials in a climatic chamber at 50C and humidity of 80% over 28 days. Long-term stability tests are being performed with monthly measurements of the candidate reference material for at least 4 months. Statistical tests will ensure the material is also stable, with isotopic values within established homogeneity test limits. This new CRM produced according to ISO 17034 will provide reliable calibration material, addressing the shortage of geological and environmental CRMs.

Perfluoroalkyl and Polyfluoroalkyl Substances (PFAS) are persistent, bio accumulative, and also has a risk of carcinogenicity, so these substances are regulated internationally, including the Stockholm Convention. In Japan, the Water Supply Act. have a plan to regulate two types of PFAS, PFOS and PFOA, and as a result, the analysis of these substances will become mandatory for more than 1000 Japanese waterworks laboratories. In order to ensure the reliability of the analysis results for PFAS, it is critical to supply SI-traceable certified reference materials (CRMs) that can be used as standards for quantification. Recently, quantitative analysis with SI-traceability using hydrogen nuclei NMR (1H qNMR) is unitized widely. On the other hand, since many PFASs do not have hydrogen nuclei in the molecule, the application of the 1H qNMR technique to the development of PFAS CRM is not applicable, and therefore, study on new techniques instead of the 1H qNMR is a challenge. Given the above background, we studied the purity determination of PFASs using quantitative analysis with fluorine nuclei NMR (19F qNMR technique) including method validation.

Micro and nanoplastics (MNPs) are novel contaminants found in every environment on earth and have been detected in living organisms, including humans. They are inherently heterogeneous in size, shape and surface chemistry, leading to complex interactions with their environment. To understand the effect of MNPs on their surroundings, we must be able to quantify them in different matrices. While detection technology has improved greatly, validation largely relies upon laboratory synthesized plastics that do not reflect the properties of environmental samples. This is particularly problematic for nanoplastics which can readily cross biological barriers and thus more easily impact living things. To date, there are few candidate nanoplastic materials available for method validation and biological impact assessment. To address this gap, we have developed a new procedure for the fabrication of environmentally relevant nanoscale plastic reference materials. Using multiphoton laser ablation, we break down bulk plastic source materials to produce nanoplastics. We characterize their size and shape using a multimodal approach and find they are highly heterogeneous. Additionally, we find chemical changes consistent with those found in environmentally weathered plastics. We demonstrate the produced nanoplastics are suitable for biological testing, finding that they provoke inflammatory responses consistent with those associated with exposure to environmental MNPs. Taken together, our new procedure and reference materials fulfill the metrological need for reproducible nanoplastics which capture key features of environmental waste. These materials will enable the validation of plastic recovery assays and allow for the biological impact of nanoplastics to be more readily determined.

The accurate measurement of indoor dustfall pollutants, particularly heavy metals which pose a health risk, is hampered by a paucity of certified reference materials (CRMs). In order to address this deficit, we have developed and characterised CRMs from atmospheric dustfall collected via dry deposition in diverse urban and suburban locations (Hebei, Shandong, Shanxi). Samples were characterised for morphology (Scanning Electron Microscopy - SEM) and chemical composition (Inductively Coupled Plasma Mass Spectrometry - ICP-MS; Atomic Fluorescence Spectrometry - AFS; Gas Chromatography-Mass Spectrometry - GC-MS) following appropriate digestion methods. The range of dustfall particles was found to be between 0.054 and 0.074 mm. Elemental composition exhibited significant variations across different locations. Specifically, sodium (Na) levels were found to be enriched in indoor environments, while cadmium (Cd) levels were found to be low. In contrast, industrial and old industrial areas exhibited elevated levels of arsenic (As)/lead (Pb) and mercury (Hg), respectively. Assessments of homogeneity and interchangeability confirmed the suitability of the developed CRM for environmental monitoring and quality assurance. This work establishes a validated atmospheric dustfall CRM, providing a crucial tool for improving the accuracy and comparability of heavy metal pollution analysis in China.

The precise and accurate measurement of cannabinoids in various matrices is essential for quality control, product labelling, and addressing potential health and safety concerns. Laboratories analyzing cannabinoids in any matrix would benefit from certified reference materials (CRMs) suitable for method validation and quality control. The National Research Council of Canada (NRC) has a comprehensive suite of CRMs for cannabinoids, both in pure solutions and matrix. Currently under development are matrix materials for the rapidly developing edibles market including CGUM-1 and CBDO-1, cannabis-infused soft chew and cannabidiol (CBD) oil respectively. A soft chew material was selected following an extensive evaluation of potential products based on cannabinoid content and homogeneity within and between samples. The CBD oil material was purchased and blended to achieve the desired cannabinoid mass fractions. Existing liquid chromatography mass spectrometry (LC-MS) and ultraviolet (LC-UV) methods for cannabinoid quantitation in dried plant material served as a foundation, with chromatographic and mass spectrometric conditions standardized across methods, regardless of matrix. Matrix-specific extractions were developed and validated individually for soft chew and oil samples. CGUM-1 samples were extracted using a QuEChERS method with acetonitrile, while CBDO-1 samples were diluted with methanol. All extracts were further diluted for LC-MS and LC-UV analysis. Up to ten neutral cannabinoids are expected to be certified in CGUM-1 and CBDO-1. These CRMs are unique within the cannabis sector and will contribute to improved measurement comparability and traceable to the International System of Units (SI) for cannabinoids in soft chew and oil-based edible products.

The aims of the multi-year capacity building and knowledge transfer project on Metrology for Safe Food and Feed - Mycotoxins was to support National Metrology Institutes in Member States in the development of mycotoxin metrology capabilities, including the production of certified reference materials and proficiency test materials. These resources are crucial for ensuring the accuracy and reliability of mycotoxin testing worldwide, with particular emphasis on supporting laboratories in developing countries. Key activities of the project included the production and value assignment of mycotoxin calibrants, the provision of training and knowledge transfer through visits to the Bureau International des Poids et Mesures (BIPM), and the coordination of international comparisons to demonstrate the equivalence of mycotoxin calibration solutions. From 2021 to 2025, the focus of these activities was on coordinating CCQM comparisons of calibration solutions for selected key mycotoxins such as aflatoxin B1, deoxynivalenol, patulin, and zearalenone. Four interlaboratory comparisons conducted during the project will be presented, showcasing the advancements made for mycotoxin calibration solution materials as well as guidelines for evaluating the purity and assessing the quality of mycotoxin solution reference materials that were developed and made publicly available. This initiative has resulted in nationally produced mycotoxin reference materials, making them more accessible to food testing laboratories and enhancing food and feed safety standards worldwide.

Mercury is one of the worlds most notorious chemical contaminants and can display such high degrees of toxicity that even ultra-trace levels in food and water can be cause for concern. Therefore, most jurisdictions have strict regulations in place for both environmental compartments and commercially-available foods and beverages in an effort to ensure that exposure is as low as possible. Unfortunately, monitoring for compliance with these regulations can be challenging due to the limitations of common analytical methods used in mercury quantitation. Adequately low detection limits can be difficult to achieve because of mercurys tendency to adhere to surfaces, while analyte losses during sample preparation are not uncommon due to its high volatility. To help support the community in their endeavors to perform more accurate and precise mercury analysis, the National Research Council of Canada has developed a series of Certified Reference Materials (CRMs) ranging from ultra-trace mercury in water and food matrices, to elevated concentrations in fish tissues. These CRMs also cover a wide range of nutritional compositions, including tissues high in protein and fat which tend to be more difficult to analyze than high-carbohydrate foods. We have also developed a number of complimentary methods for the analysis of ultra-trace mercury in these types of matrices. Our hope is that by achieving our overall goal of decreasing detection limits and improving the precision and accuracy of mercury analysis, we will move closer towards meeting regulations and targets for decreasing mercury contamination in the environment and therefore in our food.

The National Institute of Standards and Technology (NIST) and the National Institutes of Health, Office of Dietary Supplements (NIH-ODS), are collaborating to advance non-targeted analytical (NTA) methods for characterizing botanical and other natural product dietary supplement ingredients. While traditional approaches focus on quantifying specific marker compounds, NTA has the potential to capture broader compositional differences that may aid in authentication and quality assessment.

This study investigated the use of LC-QTOF-MS and NMR spectroscopy to authenticate Panax quinquefolius (American ginseng) by evaluating differences across a representative set of samples. The authenticated sample set included 51 American ginseng root samples and 4 leaf samples, 41 P. ginseng (Korean or Asian ginseng) root samples and 20 leaf samples, and 1 P. notoginseng (Chinese or tienchi ginseng) root sample. Additional American and Korean ginseng root and leaf samples, as well as Chinese ginseng root samples, were obtained from commercial sources.

Unless received as a powder, samples were cryo-ground to a fine powder. Extracts were prepared using 70% methanol for LC-QTOF-MS and methanol-d₄ for NMR. Alignment and feature extraction of LC-QTOF-MS data were investigated using MZMine and Progenesis QI. Extracted MS features (ions, areas, retention times) and NMR spectral data were assessed using PCA followed by evaluation with several multivariate classification methods. The results of these investigations will be presented.

Ongoing work focuses on assessing the complementarity of these two analytical techniques in distinguishing American ginseng from related species and selecting representative materials for an interlaboratory study. The long-term goal is to evaluate method reproducibility and robustness across different analytical platforms, contributing to improved strategies for authenticating botanical ingredients in dietary supplements.

Commutability is a property of reference materials (RMs) established on the basis of closeness of results obtained for the RMs and authentic clinical samples when comparing two (or more) measurement procedures (MP). Potential commutability problems are a well-known concern for proteins as the RM production processes might affect their folding or stability. For smaller molecules like 17-estradiol (E2), commutability issues might seem less likely to occur. JRC is producing three new CRMs for E2 in human serum to replace BCR-576, BCR-577 and BCR-578, close to exhaustion. Even though the BCR materials are the only matrix CRMs approved in the JCTLM database for E2, they are not assessed for commutability with immunoassay based in-vitro diagnostic medical devices (IVD-MDs), their intended use being limited to quality control for mass spectrometry (MS) methods. Before production of the new candidate CRMs ERM-DA576, ERM-DA577 and ERM-DA578 (low, mid and high level content of E2, respectively), a feasibility study was conducted with the main aim to secure their commutability. Pilot RMs batches were prepared applying different processing approaches (lyophilisation, freezing, spiking, minimal vs. extensive pre-treatment including delipidation and dialysis) and analysed for the E2 content, together with forty-two native clinical samples, by a reference MP (LC-MS/MS) and three IVD-MDs. The outcome of the study indicated that the processing approach could significantly impact the RMs commutability and was pivotal in deciding the production strategy of the new CRMs with the best chances of being commutable. ERM-DA576, ERM-DA577 and ERM-DA578 were hence produced as frozen serum materials and a follow-up study is on-going to confirm their commutability. When released, these CRMs can be used as trueness control for both MS based reference MP and immunoassay based IVD-MDs targeting the analysis of E2 in patient samples.

In light of the recent advances and anticipated proliferation of lipid nanoparticle (LNP)-delivered vaccines and therapeutics and with no availability of internationally recognized relevant reference materials, National Research Council Canada undertook development of reference materials of LNP systems. Accordingly, we identified multiple LNP and liposome (anionic, neutral, and cationic) candidate formulations and thoroughly characterized their properties focusing initially on the particle size and particle size distribution (PSD), stability at various conditions, and toxicity. In the phase 1 we released anionic LNP and liposome reference materials, ALNP-1 and ALIPO-1, respectively. The -70 C stored formulations have remained stable and homogeneous for over 4 years and are predicted to remain stable for minimum 3 more years. Both ALNP-1 and ALIPO-1 stored at 4 C after thawing remain stable for up to several months. Although their mean particle size very slowly increases linearly with the cube root of the storage time, by up to 3% after 2 months, polydispersity somewhat decreases indicating size distribution narrowing. Both RM have been extensively studied by dynamic light scatterin (DLS), nanoparticle tracking analysis (NTA), and electron microscopy (cryoTEM). PSD have been determined and reconciled between number- and intensity-weighted size measurands. In the phase 2 of the project a new LNP formulation based on custom-synthesized lipids was developed to address some of the deficiencies identified for ALNP-1. The formulation has been thoroughly characterized and the new reference material LNP-2 will be released later this year.

Amphetamines are central nervous system stimulants and some of them are used as drugs of abuse. MDMA (3,4-methylenedioxymethamphetamine) is the most commonly psychoactive substance found in ecstasy, an illicit synthetic drug. A set of four Certified Reference Materials (CRMs) for amphetamine drugs was developed by Inmetro following the requirements of ISO 17034 standard. The certification of batches involved studies of homogeneity, stability under transport and storage conditions, as well as characterization to define the purity value by quantitative nuclear magnetic resonance (NMR) and/or mass balance, providing metrological traceability. The methamphetamine hydrochloride(MA.HCl) wasprovided by the Federal Police, grinded, homogenized and bottled in amber glass vials (50 mg). The certified purity value of MA.HCl is (999 12) mg/g. The 3,4-methylenedioxyamphetamine hydrochloride (MDA.HCl) was synthesized, homogenized and bottled in amber glass vials (10 mg). The certified purity value of MDA.HCl is (99114) mg/g. The 3,4-methylenedioxyethylamphetamine hydrochloride (MDEA.HCl) was also synthesized, homogenized and bottled in amber glass vials (10 mg). The certified purity value of MDEA.HCl is (943 24) mg/g. These batches were homogeneous and stable under transport conditions (50 C) and storage conditions (20-25 C). The 3,4-MDMA.HCl was also synthesized and a calibration solution of 0,4 mg/mL in methanol was produced and ampoulled in 2mL vial. This material is still under certification studies. A new batch of pure MDMA is planned from purification of seized samples. These CRMs are intended for use by forensic laboratories as a calibrant and qualitative reference in chromatography, mass spectrometry and NMR.

Like DNA and proteins, carbohydrates are important bioactive molecules with complex structures and diverse functions. Polysaccharides and oligosaccharides are composed of monosaccharides that are linked with glycosidic bonds. Hydroxy groups at different positions can be modified to form functional domains. Polysaccharide drug has a global market of over 10 billion dollars. Heparin is so far the most widely used anticoagulant, while hyaluronic acid is extensively used in the medical, clinical and cosmetic fields. Several species of oligosaccharide, for instance, the human milk oligosaccharides (HMOs), have been permitted as infant dietary supplements. Despite their huge marketing scale, the study of carbohydrates is far behind that of other biological macromolecules. Standardization of oligo- and poly-saccharides faces great challenges due to their heterogeneity, while related metrological research is still at the preliminary stage. In this study, we will represent research progress in the development of oligosaccharide and polysaccharide reference materials, as well as methods for characterizing their molecular structures. We hope to establish a sweet international collaboration system for the investigation of carbohydrates, and promote the development of glycoscience through a metrological way.

Background: BNP and NT-proBNP are essential biomarkers for diagnosing and predicting the prognosis of heart failure. The absence of corresponding CRMs has led to an incomplete traceability system, resulting in non-comparable clinical measurement results. Key challenges include the low abundance of these biomarkers (ng/L~pg/L range) and molecular heterogeneity in human serum

Objective: A traceable quantitative approach needs to be researched and subsequently applied to the development of primary CRMs. Additionally, serum cleanup strategies need to be carefully characterized, as profiling the molecular forms of these peptides in human serum is vital for defining the measurand.

Methods: Initially, protein hydrolysis (AAA-IDMS) and protein cleavage isotope dilution mass spectrometry (Pep-IDMS) were utilized to develop solution CRMs. High-resolution mass spectrometry was used to identify structurally related impurities, and the results from AAA-IDMS were corrected accordingly. In the Pep-IDMS process, signature peptides were quantified using a separate AAA-IDMS to ensure that the assignment of antigen values is both traceable and accurate. Similarly, IDMS was the core technology employed for the quantification of serum biomarkers. Two serum cleanup strategies were explored: antibody-free and antibody-based methods. Both approaches were characterized and optimized to enhance sensitivity and ensure reliable quantitative results.

Conclusion: Solution CRMs for BNP and NT-proBNP have been developed based on IDMS technology. In the development of serum CRMs, it is crucial to consider not only traceability but also the definition of the measurand and the procedures for matrix pretreatment. These factors are essential in addressing issues related to commutability and clinical application.

Nanomaterials have garnered significant attention in various industries and consumer products, but the small size of their constituent particles poses potential risks for human health and the environment. To address these concerns, the European Union has established a regulatory framework that mandates nanomaterials to undergo thorough risk assessment prior to their use. A key aspect of this framework is distinguishing between conventional materials and nanomaterials, based on the proportion of constituent particles with external dimensions ranging from 1nm to 100nm.

To enable accurate identification of nanomaterials, the European Commissions Joint Research Centre (JRC) has produced ERM-FD104 and ERM-FD105, which are titanium dioxide and barium sulfate powders, respectively. Both CRMs have been characterised by electron microscopy and certified for specific characteristic values of the size distribution of their constituent particles, including those present as single particles and those present in agglomerates and aggregates. These powder CRMs offer a more realistic representation of the challenges encountered during sample preparation, as they introduce an additional layer of complexity compared to traditional nanomaterial suspension CRMs.

By providing a reliable basis for method validation, the CRMs enable laboratories to refine their electron microscopy methods and accurately determine the size of the constituent particles and small particles in the range of 10 nm to 500 nm. The CRMs marks a significant milestone in the JRCs efforts to support the effective implementation of the EUs nanomaterial regulatory framework, ultimately promoting a safe and responsible development of nanomaterial-based products.

Proteins are complex biological molecules and a significant proportion of clinical tests rely on the target protein (antigen) binding to a capture protein, often an antibody. Changes to protein higher-order structure and/or post-translational modification status could affect assay performance. During the recent COVID-19 pandemic numerous manufacturers rapidly responded by producing protein reagents for both serology and antigen testing, however questions regarding comparability remained. It became apparent that well-characterized reference materials (RMs) could be useful for the standardization of testing practices. In response, the National Research Council Canada produced four protein RMs: SARS-CoV-2 spike proteins from both Wuhan and Omicron BA.4/5 strains, SARS-CoV-2 nucleocapsid protein (Np), and human ACE2 receptor. This presentation will highlight significant aspects of the production and characterization of each RM, as well as discuss unique challenges presented by each protein. Key attributes such as measurand definition, purity, homogeneity, and stability will be addressed. Additionally, the results of a bilateral comparison assessing the analytical sensitivities of commercially available rapid antigen detection tests (RADTs) will be presented. Briefly, employing our Np RM and a second Np QC material, we demonstrate order of magnitude differences in analytical sensitivities among distinct RADTs, and find little correlation with the TCID50 assay values reported by manufacturers. Our data highlight the need for readily available protein RMs to insure batch-to-batch quality and accuracy of RADTs across manufacturers. Finally, we will touch on capacity building exercises within the international metrology community to rapidly produce equivalent protein RMs in a future pandemic scenario.

The provision of safe, high-quality food is vital for human health, and innovation in the food sector is needed to protect the environment, ensure sustainability, and respond to future needs. EURAMET, the association of National Metrology Institutes (NMI) in Europe, approved in May 2022 the European Metrology Network (EMN) for Safe and Sustainable Food (EMN Food). The EMN Food aims to foster collaboration and coordination in the measurement science community to meet metrology needs along the food chain, working within the European Unions Farm to Fork Strategy. The network promotes a harmonised approach to food measurements, reference materials and standards, which will allow National Metrology Institutes (NMIs) and Designated Institutes (DIs) across Europe to respond to stakeholders and regulations with confidence and quality. This will afford greater protection to citizens and the environment and accelerate the response to emerging and future metrology needs. The EMN-Food has also promoted scientific activities related to metrological research in food safety and sustainability, and in the framework of national and European projects, for guaranteeing an adequate economical support of the EMN activities. A key objective of the EMN is the definition of a common approach for the production of Certified Reference Materials and Reference Materials for food and food-related matrices and analytesIn this presentation, the SRA and the EMN strategy for reference materials will be presented, together with the national and international projects involving the consortium and aligned with the scope of the EMN.

The performance of testing laboratories can be verified through participation in the Proficiency Testing Scheme (PTS). To evaluate reliability of tests in isotope area, the Institute of Petroleum and Natural Resources (IPR) from PUCRS University organized the comparison in isotope composition between 8 laboratories in Brazil, being carried out based on the ISO/IEC 17043 and ISO 13528 standards, and with impartiality, being organized by the quality sector of the Institute. The carbon isotope-delta value (d13C) was evaluated in all samples and the nitrogen isotope-delta value (d15N) only in the milk powder sample. The designated valuescalculated using the median of the results reported by participantswere: ethanol (d13C= -13.23 ), mineral oil (d13C= -28.78 ) and milk powder (d13C= -22.83 ; d15N= +5.60 ). Data processing was carried out using z-score (to assess accuracy) and RSD% analysis (to assess precision). Regarding performance evaluation, two laboratories showed unsatisfactory results for d15N (3.93 ; z-score= -12.26 and -5.38 ; z-score= -80.77) in milk powder sample, and one laboratory presented an unsatisfactory result for d13C (-28.21 ; z-score= 5.35) in mineral oil sample. The laboratories, in general, presented satisfactory results. Group reproducibility demonstrated concordant results (RSD% group <10 %). The batch of samples produced also became Certified Reference Materials after a complete characterization process, based on ISO 17034, with all the tests performed in IPR-PUCRS labs with ISO/IEC 17025 accreditation. This comparison gave us an overview of the situation of laboratories that work with isotope analysis in the country and supported CRM production.

This work summarizes the results of several interlaboratory comparisons (ILCs) on trace and major elements analysis in marine environmental samples, organized by the International Atomic Energy Agency (IAEA).

These exercises are designed not only to monitor and demonstrate the performance and analytical capabilities of the participating laboratories, but also to identify gaps and problem areas where further development is needed.

The impact of homogeneity verification of ILC samples will be highlighted, alongside strategies to estimate assigned values and associated uncertainties, and general conclusions on the evaluation of reported results.

The ILCs conducted in recent years have provided valuable insights into the consistency and reliability of measurements across different laboratories worldwide, involved in monitoring trace and major elements in marine samples. The strategies employed to estimate assigned values and their associated uncertainties are shown to be critical for correctly evaluating the data accuracy.

The findings indicate biases within the reported results, with several laboratories demonstrating unsatisfactory performance for certain routine analytes. Accurate and quality-assured results in marine environmental monitoring are crucial for robust and meaningful scientific research, as well as for effectively informing policymakers.

The results from the IAEA interlaboratory comparisons emphasize the critical importance of rigorous methodologies and stringent quality assurance practices in laboratories conducting environmental assessments.

When the PT item meets the definition of reference material, it shall be produced under conditions that meet the relevant requirements of ISO 17034 (ISO/IEC 17043:2023 clause 6.1.3). A large majority of PT items are materials that are sufficiently homogeneous and stable with respect to one or more specified properties, and the PT provider has established them as fit for its intended use (as a PT item). Therefore, this requirement will apply across a very wide variety of PT schemes and test materials. How is this to be addressed by PTPs and ABs, and has the requirement had any impact on the providers of PT and RM? The relevant requirements will vary depending on the PT material and any production activities that could affect the validity of PT activities. Accreditation bodies will need policies on what requirements are relevant, and whether other ISO TC334 standards should also be considered as normative, such as ISO 33406 on reference materials with qualitative properties. A second impact is that PTPs can choose to purchase RMs from accredited producers, rather than take on the cost and risk of making their own. In response, some commercial RM producers are offering lines of materials specifically for use in PT. A third potential benefit is a better understanding in the laboratory community of what reference materials are, and how they can be used. This could be new to some areas of testing where PT is being applied, but where RMs are not commonly available.

The Digital Reference Material Document (DRMD) is an XML representation of RM certificates and information sheets, designed to enhance their interoperability and machine readability. It adheres to the requirements of ISO 17034 and ISO 33401.

The DRMD is generated from an XML Schema that builds on the successful Digital Calibration Certificate (DCC) as developed by PTB and others. It is easy to understand and quick to generate while maintaining machine readability for the core essential data, though not for all details.

The DRMD uses the Digital SI (D-SI) to express property values, their uncertainties and units, and QUDT for quantity types. Properties can be linked to existing taxonomies. Multiple digital signatures can be inserted to verify the integrity of the certificate and the validity of the accreditation, if any.

We published the first draft of the Schema in February 2025, along with a set of tools and examples, including an open-source DRMD editor and generator and an XSL style file for the generation of a human-readable representation. Several DRMDs have been uploaded to the CRM database COMAR to demonstrate their feasibility.

As we are continuously improving the DRMD schema and tools, we look forward to discussing and aligning our efforts with others in the field.

Over the last decade, the National Research Council of Canada (NRC) has developed an infrastructure for digital calibration certificates of its reference materials. This initiative aligns with the FAIR data principles findable, accessible, interoperable, and reusable endorsed globally by scientific and political bodies alike. Digital calibration certificates offer significant advantages over traditional PDF formats, which are often unstructured and difficult to interpret for humans and computers alike. By incorporating comprehensive metadata and structured data elements, digital calibration certificates ensure machine-readability and enhance the accessibility of data for researchers worldwide.

Hosted within the National Science Library of Canadas digital repository since 2014, these certificates integrate various data types, including DOIs and InChiKeys, enhancing their visibility and citation in academic literature. The repository supports machine-readability, allowing for data retrieval via programming languages like Python and R, or even by Excel.

The transition to digital certificates not only improves the discoverability of NRC reference materials but also fosters improved interoperability and machine learning applications. Furthermore, digital certificates empower researchers to conduct complex queries, such as identifying compounds with specific chemical properties or retrieving supplementary data from external online databases.

This presentation will outline our decade-long journey and ongoing commitment to digital transformation in chemical reference materials, highlighting the benefits and future potential of this work in creating a more accessible and efficient ecosystem for chemical research.

Society is undergoing the Fourth Industrial Revolution (Industry 4.0) with the development of advanced materials, transformational production processes and the widespread adoption of digital technologies. To keep pace with this rapidly changing environment, the international measurement system must evolve. This need is recognized by various governments and metrology stakeholders including the International Bureau of Weights and Measures (BIPM), National Metrology Institutes (NMIs), and commercial service providers, with efforts ongoing to address the challenges of the digital transformation.

The National Research Council of Canada (NRC) has adopted digital reference material (RM) certificates that comply with the principles of findability, accessibility, interoperability and reusability (FAIR). Using this foundational framework, we developed the Reference Material Explorer (RM Explorer), a web-based application that utilizes digital RM certificate data to present information in an interactive way, and leverages the ability to retrieve additional data from the metadata.

The RM Explorer uses digital certificate data to list bibliographic and scientific information with the ability to interact with the entire catalogue of RMs from the NRC Canada. A key feature of the application lies in its ability to use digital chemical identifiers which enables fetching additional chemical and biological data from external sources, performing calculations of missing physicochemical properties, or searching for chemically similar substances. The RM Explorer also features generation of chemical structures as well as NRC-supplied spectra (NMR and MS). This extensive information on NRC RMs is summarized in a user friendly, publicly accessible interface that is agile and expandable to accommodate the needs of the broader reference material community.

Development of RM Explorer showcases the possibilities when RM certificates are digitalized. The work also has a parallel objective to spur discussion while helping guide future efforts on best-practices for digitalization of RM information, use of identifiers and approaches for improved accessibility of chemical information.

Coming soon.

The qNMR method has rapidly become popular as a traceable quantitative method attributed to its unique measurement principle and has been internationally standardized as ISO 24583 in 2022. Because use of reliable CRMs for qNMR with small uncertainties (primary CRMs for qNMR) is essential to obtain reliable results of qNMR, NMIJ has been developed three kind of primary CRMs for qNMR considering solubility of solvents, chemical shits and ease of handling such as 3,5-bis(trifluoromethyl)benzoic acid (NMIJ CRM 4601) and 1,4-bis(trimethylsilyl)-2,3,5,6-tetrafluorobenzene (NMIJ CRM 4602) for 1H and 19F qNMR, and potassium hydrogen phthalate (NMIJ CRM 4603) for 1H qNMR. To develop these CRMs, we prepared high-purity raw materials to no impurity signals detected on NMR spectra and suppress uncertainty originating from impurity and applied one or two primary methods (the freezing point depression method for NMIJ CRMs 4601 and 4602, and coulometric titmetry for NMIJ CRMs 4601 and 4603) combined with mass balance approach for their characterization. The certified values (valueexpanded uncertainty, k=2, kg/kg) of NMIJ CRMs 4601, 4602 and 4603 are 0.99960.0006, 0.99980.0003 and 0.99980.0003, respectively. The determined certified values were also evaluated by qNMR to confirm their usefulness as CRMs for qNMR. Their uncertainty are sufficiently small to be the primary CRMs for qNMR, and other valuable information for users such as accurate molar mass, solubility in common deuterium solvents as well as their chemical shifts (1H for all CRMs and 19F for CRM 4601 and 4603) are also provided in each certificate.

Quantitative nuclear magnetic resonance (qNMR) spectroscopy has become a standard approach for purity assignment, used by an increasing number of analytical laboratories. This is partly due to its acceptance as a primary reference technique to deliver ISO 17025 accredited analysis and ISO 17034 reference materials, as well as its ability to allow fast and cost-effective material characterisation. One of the challenges for the wider application of qNMR as a high accuracy analytical technique, is the relatively limited scope of analyte/matrix combinations to which it can be applied. Pure, small organic molecules are ideally suited for direct qNMR assays but there is a growing need for similar direct assay approaches for matrix materials. This includes high order reference materials to support comparability and traceability for quantitative magnetic resonance imaging (qMRI). QMRI presents an opportunity to benchmark scanners by characterising their measurement performance and offers a potential step-change in reproducibility for MRI data. This process can be supported by a reference object (phantom) that contains clinically relevant MRI measurands that are traceable to primary standards. This presentation will discuss work undertaken to support the standardisation of qMRI through the provision of improved fat fraction reference standards. It will describe the preparation and qNMR characterisation of fat fractions using a simple fat model (tert-butanol/water) as well as a complex fat fraction (oleic acid/water) that is more representative of human fat. QNMR is used here to assign SI-traceable measurements to underpin qMRI. Further applications of qNMR to more complex materials and associated challenges will also be discussed.

Fungal crop pathogens, including Penicillium and Fusarium, produce mycotoxins that pose significant risks to food safety. Notably, ochratoxin A, produced by Penicillium verrucosum, is highly nephrotoxic and regulated at a threshold of 3 ppb in wheat products. Fusarium graminearum produces several mycotoxins, including the emetic compound 4-deoxynivalenol and the hormone disruptor zearalenone, both of which are also subject to regulatory oversight. Continuous monitoring of these mycotoxins throughout the food supply chain is essential to ensure food safety.

To obtain accurate and reliable results in mycotoxin quantification, the use of Certified Reference Materials (CRMs) as a primary calbrant is strongly recommended. The production of these CRMs necessitates the application of accurate measurement techniques, with quantitative proton NMR spectroscopy recognized for its reliability and accuracy. To further enhance the accuracy of quantitative proton NMR, two 13C-satellite decoupling techniques were investigated: GARP (Globally Optimized, Alternating Phase, Rectangular Pulses) and bi-level adiabatic decoupling. The presence of 13C-satellites can hinder the accuracy of integrals due to signal overlap and mask low-level impurities. Removing these signals from the proton spectrum significantly improves integral accuracy.

GARP decoupling successfully facilitated the production of a 13C-labelled ochratoxin A Certified Reference Material on a 400 MHz instrument. However, GARP is prone to excessive sample heating and is typically limited to narrow decoupling bandwidths, rendering it unsuitable for a 900 MHz spectrometer. To overcome these challenges, bi-level adiabatic decoupling was employed, enabling the successful production of a Certified Reference Material for 4-deoxynivalenol at 900 MHz.

Organic pure materials are the basis of metrological traceability chains for accurate quantification of organic substances across a broad range of sectors including food safety, water quality, clinical chemistry, environmental testing and pharmaceuticals. Over the last decade, quantitative nuclear magnetic resonance (qNMR) has become the method of choice for pure material value assignment due to its versatility and efficiency. Recent applications of quantum chemistry and artificial intelligence (AI) are opening the door to their application in the domain of qNMR and expanding the analytical power of the technique.

qNMR has emerged as a primary technique for purity analysis due to its versatility, nondestructive nature, and rapidity. As part of a collaborative effort started by the BIPM and NMIJ (Japan) and other NMIs, the group has demonstrated the use of an "Octad" of CRMs, for the application of qNMR to the broadest range of organic species 1. Additionally in recent years, the application of quantum chemistry and AI tools to qNMR frequency-time data is enabling advanced post-processing approaches to be developed.

Quantum chemistry has the potential to enrich qNMR analysis by providing molecular-level insights into purity assessments. Techniques such as density functional theory (DFT) and ab initio calculations allow prediction of NMR chemical shifts, coupling constants, and relaxation times, which can then be fitted to qNMR data. Alongside this quantum mechanics structural analysis (QMSA) can provide genotypic data (δ, J and N), instead of the phenotypic spectral data, which when supported by DFT prediction of spectra from compound (C2S) can calculate values from spectra to compound (S2C). This C2S and S2C combination enhances spectral assignment accuracy, particularly for complex organic compounds, while also evaluating spectral quality and user assignments. The potential of computational methods based on quantum chemistry to provide accurate qNMR results is being assessed at the BIPM. We describe initial work on the assessment of one quantum chemistry-based approach, by comparing amount of substance fractions calculated by quantum mechanics with the traditional manual signal integration approach, assessing uncertainty contributions and refining methodologies for trace impurity detection. Two model mixtures of analyte and internal standard (IS) were studied: maleic acid (MA) with potassium hydrogen phthalate (KHP) as IS in deuterated water and dimethyl terephthalate (DMTP) with 3,5-Bis trifluoromethyl benzoic acid (BTFMBA) as IS in hexadeuteroacetone. The purity results obtained using both approaches agreed under specific conditions, although the manual approach provided results with lower uncertainties. Limitations of the quantum methodology are being addressed, such as the spin system recognition and processing of lineshape artifacts. New algorithms, such as the quantum mechanically guided integral, show great promise as they improved the accuracy of the purity determination in the studied MA/KHP model mixture.

The project also highlights the possibilities of applying machine learning algorithms to qNMR data processing, which could be expanded to process large qNMR datasets to identify patterns, predict purity outcomes, enhance sensitivity through noise prediction and suppression and improve spectral quality post-acquisition. AI-driven approaches also hold promise for normalizing and setting new benchmarks for CRMs and international metrology standards.

Digital reference materials (dRMs) can conceptually overcome the intrinsically limited availability and accessibility of identical reference materials (iRMs) that persistently impede chemical analysis of rare and controlled substances, respectively. Quantum mechanics (QM)-driven 1H iterative functionalized spin analysis is digital by nature and capable of referencing molecules with high specificity and full instrumental portability in the form of HifSA profiles by decoding their “nuclear genotype” in the form of the key 1H spin parameters including chemical shifts (δ) and coupling constants (J). This study establishes the proof-of-concept for replacing iRMs with open dRMs for controlled Ephedra alkaloids (ephedrine, pseudoephedrine, methylephedrine) by producing their fully field-scalable HifSA profiles and adopting them for the qualitative and quantitative analysis of E. sinica and traditional Chinese medicines as a E. sinica-containing multi-component mixture. QM-derived qHNMR (QM-qHNMR) results were corroborated via classical integral-based qHNMR (INT-qHNMR) and HPLC-UV methods. Furthermore, extending this method to 12 congeneric phenylethylamines created a starting set of open dRMs within the space of controlled substances. The remarkable congruence and near-identical (within few 10 mHz) coupling constants in the monosubstituted benzenes (AA'BB'C spin systems) cross-validated the trueness of the dRM profiles. In addition, QM-qHNMR analysis of Ephedra alkaloids across a range of magnetic field strength equivalent to 60−600 MHz for 1H confirm the portable nature of the dRMs and highlight their contribution to the sustainability and wider application in analytical chemistry. Practical aspects of HifSA profiling and QM-qHNMR are covered by comparing two software tools, Cosmic Truth (CT) and ChemAdder. Expanding the open dRM concept more broadly within chemical analysis advances the analytical toolbox for complex mixtures, fosters simultaneous identification and quantification of multiple target analytes, and eliminates the need for iRMs.

Engineered nanoparticles have a large application potential in fields such as medicine, sensing, catalysis, energy storage, and opto-electronics 1. The applicability and performance of engineered nanoparticles is largely determined by their surface chemistry, i.e. functional groups and ligands on the particle surface. However, documented standards to quantify functional groups and ligands currently do not exist. Such standards are required to support quality control of nanomaterial production and surface modification processes, and safe-by-design concepts and to meet regulatory requirements.

Here, this issue is addressed by developing and standardizing quantitative Nuclear Magnetic Resonance (qNMR) methods for the characterization of surface functionalized nanoparticles which specifically address the determination of the amount and chemical composition of surface functionalities and coatings.

This work is being developed under the EMP project 23NRM02 SMURFnano - Standardised measurements of surface functionalities on nanoparticles. qNMR competence of 7 qNMR laboratories involved in the project was first tested with a molecular model sample i.e. citrate, to be assessed in terms of purity. Citrate is often used as hydrophilic surface ligand for different nanoparticles.

Then, the first nanoparticle samples, here a set of aminated SiO2 NPs 1 with a particle size of 100 nm and two amino group densities, prepared and characterized by BAM regarding size and surface charge as well as stability, over 21 months with an optical assay and qNMR, were assessed in an international interlaboratory comparison (ILC) on qNMR. Thereby, the amount of surface amino groups introduced by grafting of the silica cores with different amounts of3-aminopropyl) triethoxysilane (APTES) was quantified by each participant following a sample preparation protocol previously developed by BAM and NRC. The results of the ILC were then used to refine the protocol for sample preparation and to identify critical points for qNMR measurement and data analysis.

Biological medicines are inherently heterogeneous due to the nature of their production, this heterogeneity can arise from the chemical composition, molecular weight, higher order structure, etc., of the material. Due to this heterogeneity standards are used to ensure the efficacy and safety of the biological medicines. One class of biological medicine are vaccines against bacterial infections, preventing diseases such as Streptococcus pneumoniae, Haemophilus influenzae B, Salmonella enterica serotype Typhi and Neisseria meningitidis. These vaccines were originally solely composed of capsular polysaccharides and now they come in the form of glycoconjugate vaccines, with the polysaccharide component bound to immunogenic carrier proteins such as Cross-Reactive-Material-197. The National Institute for Biological Standards and Control (NIBSC, now as part of the Medicines and Healthcare products Regulatory Agency (MHRA)) produces polysaccharide standards, initially the unitage of these standards were assigned by traditional methods, there has been a shift over recent years to move to using qNMR to assign the mass unitage (1, 2).

In 2011 the 1st International Standard for Meningococcal Serogroup C Polysaccharide was established by the World Health Organization Expert Committee on Biological Standardization (3). The availability of the standard has aided the consistent manufacture of new and existing vaccine products for over a decade and demand has led to a depletion of stocks of the standard. This necessitated the evaluation of a candidate 2nd International Standard for Meningococcal Capsular Group C polysaccharide material, assigning a value in milligrams Meningococcal Serogroup C Polysaccharide per ampoule. The Meningococcal Capsular Group C polysaccharide is a linear homopolymer comprised of a partly O-acetylated repeating units of sialic acid, linked with α(2→9) glycosidic linkages. In addition to assignment of unitage, the fitness for purpose of the material as a standard for the quantification of Meningococcal Serogroup C Polysaccharide in the manufacture of meningococcal polysaccharide conjugate vaccines was assessed. The process involved twenty laboratories taking part in a collaborative study, twelve of which performed qNMR and based on the qNMR results the candidate material was established as the 2nd International Standard for Meningococcal Serogroup C Polysaccharide in 2023, with assigned content of 0.965 ± 0.024 mg polysaccharide per ampoule (expanded uncertainty with coverage factor k=2.20, corresponding to a 95 % level of confidence) (4).

The use of qNMR is now the expected root for the unitage assignment of candidate World Health Organization Internation Standards for bacterial vaccine polysaccharides, currently the candidate material for the Native Vi polysaccharide (S. Typhi) (2nd International Standard) is going through the collaborative study process and the replacement process for the 1st WHO International Standard Meningococcal group X polysaccharide will start shortly.

A quantitative nuclear magnetic resonance (qNMR) method was developed to determine the relative ethoxy content in ethylcellulose, a polymeric pharmaceutical excipient. This relative qNMR approach employs high-field 1H NMR spectroscopy using a solvent mixture of deuterated chloroform and deuterated trifluoroacetic acid, eliminating the need for internal or external calibrants. Multiple batches of ethylcellulose were analyzed using this method, and the measured ethoxy content values were consistently about 3% higher compared to those reported in Certificates of Analysis (44%–51%) obtained by the standard pharmacopeial method. The developed qNMR method addresses several limitations associated with the current pharmacopeial approach, including complicated sample preparation procedures, chemical reagent pollution, and significant measurement biases. By circumventing uncertainties inherent to the derivatization-based GC-FID method, this 1H qNMR method provides a straightforward, accurate, and reliable alternative for determining the ethoxy content in pharmaceutical-grade ethylcellulose, demonstrating significant practical advantages.

Although solution-state NMR is frequently used in quantification studies, this is less true for solid-state NMR. However, solid-state NMR allows quantification of substances without the need of dissolution, providing a truly non-destructive approach, and extending quantitative NMR (qNMR) to sample classes that are difficult to characterize in solution. Recently, we presented a thorough and rigorous protocol for 19F quantitative solid-state NMR employing a certified reference material (CRM) as external calibrant to provide metrological traceability to absolutely quantify the content of trifluoroacetic acid (TFA) in a sample of the peptide angiotensin II (1). In this contribution we apply our protocol to the quantification of TFA in a sample of human C-peptide (hCP) and compare the results with those obtained by solution NMR as part of the Comité Consultatif pour la Quantité de Matière (CCQM) key comparison CCQM-K115 (2). We used 19F direct-polarization magi-angle spinning solid-state NMR in combination with EASY background suppression (3) and the ERETIC method (4) employing the NRC angiotensin II peptide CRM ANGII-1 (5) as external calibrant. We discuss strategies to determine the experimental uncertainties of the TGA mass fraction using bootstrap resampling.

Nuclear Magnetic Resonance (NMR) spectroscopy is a widely used technique in chemistry and related fields for elucidating chemical structures, determining purity, and analyzing molecular properties. However, processing and analyzing NMR spectra can be challenging and often requires specialized software that necessitates local installation. To address this issue, NMRium (https://www.nmrium.org) is an innovative web-based application that provides a breakthrough solution 1.

In the presentation, we will provide live demonstrations of how NMRium can be used to assign small molecules and load hundreds of spectra from a metabolomics dataset. Additionally, we will introduce the concept of a workspace that allows the definition of integration zones and the creation of mathematical formulas to streamline the quantitative analysis process of a batch of spectra. During the talk, we will also present a new feature developed in collaboration with NMR Solutions: a magnetic field-independent database of reference products.

NMRium supports various NMR formats (including Bruker, Varian, Jeol, and JCAMP-DX), molecule assignment, and superimposition, making it a valuable resource for both researchers and NMR facilities.

Additionally, NMRium can be used for educational purposes, such as e-learning, as it enables the creation of exercise series for students without requiring any software installation 2. It also has a unique feature that provides ‘hints’ to students without giving them the correct answer.

Nonylphenols, which is one of endocrine-disrupting chemicals, is harmful to the environment and human healthy. In order to enhance and expand capability of testing laboratories in the field of nonylphenolsin water, aproficiency testing (PT) fornonylphenolsin water by inter-laboratory comparison was organized bythe Institute for Environmental Reference Materials, Environmental Development Centre of the Ministry of Ecology and Environment, China.The sample comprised a detailed reconstitution protocol and an ampoule of spiking solution. The spiking solution of 4-n-nonylphenolwas prepared in acetonitrile. The solution should be diluted 5 000 times with drinking water prior to analysis. Homogeneity for 4-n-nonylphenol in acetonitrile wasevaluated with one-way analysis of variance, and it was certified to be uniform. Following the sample was randomly distributed to all participating laboratories. 19 laboratories from China participated in the PT. According to statistical methods for use in proficiency testing by interlaboratory comparison (ISO 13528:2022), Z- scores were used to evaluate the performance of laboratories. The assigned values were robust mean from PT results. The results showed 84.2% of PT participants achieved a satisfactory performance (∣Z∣2). Dissatisfactory outcomes may stem from reference materials, laboratory blanks, or sample pretreatment protocols, etc.Detection competence for 4-n-nonylphenol in water from domestic laboratory is generally good, but some laboratories still need to be improved.

Value assignment is a pivotal moment in the development of certified reference materials. This crucial step, however, is often met with frustrations because one can adopt a variety of data analysis methods, many of which require advanced computations. Indeed, modern data analysis cannot be conducted without modern software tools which often necessitate going beyond the Excel Monte Carlo methods, advanced regression analysis, and Bayesian analysis are examples of this.

This presentation will outline decade-long efforts at the NRC Metrology of developing an ecosystem of open-source software tools aimed at facilitating the data analysis necessary for reference material production. At the center of most data reductions relevant to reference material production is the random effects statistical model which recognizes the fact that unaccounted sources of uncertainty (dark uncertainty) remain prevalent in chemical measurement. Indeed, interlaboratory comparisons whereby reliability and competence can be demonstrated rather than just designated relies on a variety of random effects models, same as with the analysis of homogeneity.

This presentation will outline three web applications, with source code provided, all written in R programming language and deployed over the web browser as shinyapps the consensus calculator, homogeneity calculator (Bayesian analysis of variance), and stability calculator (Bayesian kinetic modeling). The development of these tools was inspired from the pioneering work from NIST and together these applications form a framework of computational tools necessary for many reference materials.

COMAR is a database for high-quality CRMs produced by NMIs or developed under ISO 17034 accreditation. Managed by a consortium of NMIs, it is open to RM Producers (RMPs) worldwide, providing them with a free and independent marketing platform for their CRMs.

The February 2025 software release introduced several exciting new features. RMPs can now direct interested visitors straight to the corresponding product in their webshop. Additionally, COMAR is the first platform worldwide to launch the Digital RM Document (DRMD), a new digital format being developed alongside the Digital Calibration Certificate (DCC). Were also working on integrating with the EPTIS PT database and PT calendar, which will significantly increase COMARs visibility within the lab community.

COMAR has a long-standing tradition, and we are committed to building on it and driving growth. Welcome!

Blank control certified reference materials are those characterized as being below method limits of detection (LOD) for target analytes in the relevant sample matrix. They provide a reference for comparison with experimental samples and have utility in method validation and ongoing quality control, with specific applications in spike recovery experiments. From the perspective of reference material providers, production and characterization of blank controls in compliance with ISO17034 requirements presents a unique challenge. For example, homogeneity and stability studies that typically evaluate targeted analytes are not applicable, while certification involves accurate and traceable determination of method LODs.

Liquid chromatography high-resolution mass spectrometry (LCHRMS) enables collection of non-target data on blank control reference materials, providing measurements on a broad range of detectable compounds naturally present in the matrix. This data can then be used to assess homogeneity and stability of the sample matrix, informing decisions on appropriate sampling, storage and transport conditions. Non-target data acquisition methods generate significant amounts of complex data, requiring development and validation of data processing workflows. This presentation will outline a non-target analysis approach, using LCHRMS to detect matrix compounds in a freeze-dried Aphanizomenon flos-aquae reference material. The material was characterized as blank for cyanobacterial toxins including microcystins, anatoxins, saxitoxins, and cylindrospermopsins using spiked extracts for traceable determination of method LODs, presenting unique challenges by LCHRMS. Commercially available metabolomics software was optimized for data analysis, and the results were used to evaluate homogeneity and select conditions for storage and transport.

This poster presents the recent work of the Reference Material Working Group of Eurachem. The working group consists of experts from 14 countries and has undertaken a significant revision of the guide Selection and Use of Reference Materials, originally published in 2002. This revision aims to provide updated and comprehensive guidance to users of reference materials (RMs).

To adapt the revision to the current needs of users, a survey comprising 18 questions was developed and distributed, garnering over 120 responses from a diverse range of stakeholders. The feedback collected has been instrumental in shaping the revised guide, ensuring it addresses current needs and practices. it remains relevant and practical.

The revised guide is designed to assist users in selecting the appropriate reference material for their specific applications and maximizing its benefits. It includes practical guidance from laboratory settings and offers links to more detailed information in related guides, standards, and literature.

The guide aims to consolidate and disseminate the collective knowledge and best practices in the use of reference materials, ultimately enhancing the accuracy and reliability of measurements across various fields.

Stable isotope ratio analysis of a compound at natural abundance, which probes the molecules themselves, is an extremely valuable tool to authenticate and track the origin or metabolic pathways of compounds in the food industry, environmental forensic and geochemistry fields. Isotope ratio mass spectrometry (IRMS) provides the bulk isotope ratios but no-intramolecular isotope composition of the molecule. Quantitative nuclear magnetic resonance (qNMR) provides isotope ratios at individual positions (site-specific), allowing the discrimination of molecules from raw materials to finished products and their geographical origin. To date, several certified reference materials (CRMs) for bulk isotope ratios of organic compounds have been produced, but there are very limited site-specific CRMs available in the market.

Vanillin is a popular flavor in the food and pharmaceutical industries. Here, we present rigorous qNMR workflows to provide metrological traceability and quantification of site-specific carbon and hydrogen isotopes in vanillin, contributing to the certification of site-specific isotope values in two vanillin materials, VANA-1 and VANB-1. The workflows include spectrometer adjustments, a new NMR decoupling sequence and NMR parameters optimized for 13C and 2H-qNMR.Validation of our isotopic qNMR method was conducted by comparison with an independent GC-IRMS approach. We obtained remarkably consistent measurements results from five vanillin samples with difference of 0.6 and 0.9 % for 13C and 2H, respectively.

To the best of our knowledge, the NRC Metrology is the sole National Metrology Institute capable of analyzing the site-specific isotopic composition at natural abundance by qNMR.

Monitoring Angiotensin I levels is crucial for diagnosing and managing cardiovascular diseases, hypertension, and related disorders. However, challenges in analytical measurement, including interlaboratory variability and assay commutability, hinder the standardization of results across different clinical settings. The introduction of an Angiotensin I certified reference material (CRM) can serve as a standard for calibration, method development, and accuracy assessment, ultimately enhancing the reproducibility of assays. In this study, we present an orthogonal approach for the characterization of an Angiotensin I CRM with total peptide content determination by multiple techniques including amino acid analysis with ultraviolet detection (AAA-UV), amino acid analysis with mass spectrometry (AAA-MS), quantitative nuclear magnetic resonance (qNMR) and chromatographic purity assessment by LC-UV/MS. Our findings underscore the importance of well characterized reference materials and advanced analytical methods to improve the reliability of Angiotensin measurements in clinical practice.

The use of amyloid peptide(A)biomarkerscould contribute to an early diagnosisof Alzheimers disease (AD),however there arestill large variations among results from different assays. This variability can be overcome by standardizationof those assays through the use of certified reference materials(CRMs) and the establishment of a traceability chain.In this study, two amyloid peptide(A40and A42) solution certified reference materials (GBW09874~09875) with the certified value of7.580.22and 7.620.22gg-1were developed withcommercially high-purity A as raw materials.For the first time,high performance liquid chromatography-isotope dilutionmass spectrometry (HPLC-IDMS)and high performance liquid chromatography-isotope dilutioninductively coupledplasmamass spectrometry (HPLC-ID-ICP-MS) strategies were employedto certifythe candidate Asolution CRMs, andgood agreement was obtained of the two strategies. The amino acids hydrolysis conditions, Aseparation conditions suitable for ICP-MS detection and influence of species-unspecific isotope dilution were investigated in the method development.It was shown that tenunits with duplicate analysis were enough to demonstrate the homogeneity of thetwocandidate CRMs. The statistical results also showed that the CRMswerestable for at least5 days at -20 ℃and 14months at -70 ℃. The relativeexpanded uncertaintiesof the two CRMsareapproximate 3.0%.These CRMsareprimarily intended to be used as calibrators or quality control materials for the measurement of A, which will do help to early diagnosisof Alzheimers disease (AD).

For almost a century, peptides have been used as therapeutic treatment for various types of diseases including diabetes, cancer, osteoporosis, HIV infection, etc. Teduglutide(TGT), a recombinant glucagon-like peptide 2 (GLP-2) analog, is an approved medication used for the treatment of short bowel syndrome (SBS) patients with chronic intestinal failure (SBS-IF). The development of rigorous and reliable analytical method for teduglutide could help to guarantee drug product quality, efficacy and security, which underlines the necessity to establish traceability chain through the development of relevant certified reference material (CRM) and reference measurement procedure (RMP). In the present study, a teduglutide purity CRM with the certified value and uncertainty of (82.33.8) gg-1 was developed. After purity check and structure confirmation, two independent isotope dilution mass spectrometry (IDMS) strategies based on amino acids (AAs) and sulfur were employed to certify the candidate teduglutide purity CRM. The candidate teduglutide purity CRM showed good homogeneity, and good stability was also demonstrated for at least 14 days at -20 ℃ and 6 months at -70 ℃. The development of teduglutide purity CRM is primarily intended to be used for value assignment to secondary calibrators or CRMs with matrix, which will help to standardize the production, administration and proper usage of teduglutide-relevant drugs.

Analysis of low-level organic contaminants in complex matrices is essential for monitoring global food safety. However, balancing sample throughput with complex experimental designs and/or sample clean-up to best reduce matrix effects is a constant challenge. Multiple strategies exist to mitigate these effects, with internal standard based methods such as isotope dilution mass spectrometry (IDMS) being the most advantageous. Here, multiple calibration strategies were investigated for the quantification of ochratoxin A (OTA) in wheat samples by liquid chromatography mass spectrometry (LC‑MS). Internal standard-based quantitation methods such as single (ID1MS), double (ID2MS), and quintuple (ID5MS) isotope dilution mass spectrometry, as well as external standard calibration were explored and compared. The National Research Council Canada (NRC) has a suite of mycotoxin certified reference materials (CRMs), including calibration solutions and matrix materials, to assist with accurate quantitation of OTA and other mycotoxins. The matrix CRM, MYCO-1, was used to evaluate the accuracy of each method. As expected, external calibration generated results lower than the certified value for MYCO‑1, largely due to matrix suppression effects. Isotope dilution methods overcome matrix effects by instead measuring a ratio of two isotopic forms of OTA. The advantages and limitations of each isotopic method are illustrated, along with the practical considerations such as the level of complexity and sample throughput. Although these calibration strategies were used for the quantification of OTA in wheat, these methods can be applied for the quantitation of a variety of other toxins in food.

Lactose-restricted diet foods are of great importance for managing a condition called lactose intolerance, which affects part of the global population. These products have their lactose content regulated by legislation, where the quantification of this carbohydrate is essential to ensure the safety of these foods. To ensure proper quality assurance for these products, it is necessary to provide appropriate metrological tools such as Certified Reference Materials (CRM). The aim of this work was to evaluate the technical feasibility of producing a zero-lactose CRM in powdered milk, which has not yet been commercially available. Two UPLC-MS/MS analytical methods were developed and validated, one using sucrose as an internal standard and the other using the isotopic dilution technique. Both methods showed satisfactory precision and recovery and were therefore applied to measure the lactose content in the CRM feasibility study. A batch of 60 units containing 2 g of lactose free powdered milk in each bottle was produced and subjected to studies to evaluate homogeneity, stability under transport conditions, and characterization according to the ISO 17034 (2017) and applicable ISO guides. The batch was considered sufficiently homogeneous and could be transported for up to 40 days at a temperature of up to (22 2) C without compromising the lactose content. The informative value for the mass fraction of lactose was (1061.62 193.95) g/g (k=2, 95%) for the powdered milk on a wet basis. Therefore, the production of the proposed CRM was considered initially feasible.

Arsenic occurs in the environment from both natural and manmade sources. It is found in both inorganic and organic forms with the former generally considered to be more toxic. Inorganic arsenic (iAs) has been classed as a Group 1 carcinogen, meaning there is sufficient evidence of carcinogenicity.

The USA, EU and UK have set a limit of 100g/kg for iAs present in rice destined for the production of food for infants and young children in order to minimise long-term exposure, and there are separate limits in place for other rice and cereal based foods.

The LGC National Measurement Laboratory (NML) has produced two food matrix reference materials (7.5 g units) that have been certified for the mass fraction of inorganic arsenic (sum of As(III) and As(V)), total arsenic and total selenium. One material (LGC7503) contains a mass fraction of iAs close to the 100g/kg legislative limit and the other (LGC7504) contains a lower mass fraction of iAs.

The mass fraction of iAs was determined using anion-exchange HPLC-ICP-MS with exact single matched standard calibration. The total arsenic and total selenium mass fractions were determined using ICP-MS with isotope dilution calibration (for selenium) and exact single matched standard calibration (for arsenic).

The intended use of the materials is for the development, validation or quality control of analytical methods for the determination of iAs, total arsenic and total selenium in a rice-based material and will help manufacturers and regulators ensure that food is safe and as low in arsenic as possible.

Laura Carrick-White¹, John Entwisle¹, Christian Ward-Deitrich¹, Ahmad Abukashabeh¹, Julian Rinaldi¹, Sarah Hill¹, Simon Cowen¹, Brooke Preston¹, Stephen Ellison¹, Gill Holcombe¹  ¹LGC

Seafood is one of the most highly-traded international commodities and product price is determined by characteristics which can be potentially falsified at import (i.e. species, provenance, weight), resulting in inflated payouts, encouraging unsustainable practices, and damaging domestic seafood economies. Additionally, species substitutions and the inclusion of additives such as gels, dyes, and antibiotics commonly found in fish feed can negatively impact consumer health. Food regulatory and customs agencies would benefit from authentic seafood materials to aid in identifying cases of import fraud. To address this need, the National Institute of Standards and Technology (NIST) utilized shrimp and salmon, two of the three most consumed seafood products in the US, to develop authenticity reference materials (RMs). Wild-caught and aquacultured seafood materials were procured from verified sources and edible portions, including fish skin, were cryohomogenized and blended from multiple individuals to produce fresh-frozen homogenates. Values for total fat, fatty acids and total protein were provided, as were genetic assessments using targeted PCR-based methods and Next-Generation Sequencing. Data generated from the latter was used to develop a novel method for quantifying confidence in genome-based identifications. Additional untargeted NMR and HRMS-based analyses were conducted to identify global differences in metabolite and lipid profiles based on material source. RM 8256 Wild-caught Coho Salmon, RM 8257 Aquacultured Coho Salmon, RM 8258 Wild-caught Shrimp, and RM 8259 Aquacultured Shrimp are currently available for authentication and seafood safety studies.

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health, Office of Dietary Supplements (NIH ODS) has developed suites of Reference Materials (RMs) and Standard Reference Materials (SRMs) for dietary supplements. SRMs, such as those for Asian ginseng (Panax ginseng, SRMs 3384, 3385, 3389 and RM 8664), Green Tea Camellia sinensis(SRMs 3254 3257), Multivitamin Tablets (SRM 3289), and Fatty Acids in Fish Oil (SRM 3275), include value assignments for targeted organic and/or inorganic compounds with biological activities and/or used for supplement product standardization. SRM use promotes experimental rigor and supports manufacturing quality control efforts. With two-thirds of adults in the U.S. reporting supplement use, RMs are critical for method development and validation for accurate product labeling which assists in ensuring consumer safety. NIST and NIH ODS are working to develop additional reference materials including those focused on botanical identity and safety. Currently available RMs and future RM development plans will be presented.

Cereulide toxin, a cyclic dodecadepsipeptide, is produced by Bacillus cereus and is a causative agent in food-borne outbreaks (FBOs). Several hundred FBOs related to B.c. toxins have been reported in 2023 (European Union One Health Zoonoses Report, EFSA and ECDC). Insufficient hygiene practices are the main reasons for intoxications. Cereulide affects several foods, among others starch-rich foods such as rice. A rice powder candidate CRM was processed which exhibits appropriate homogeneity and short-term stability. A characterisation exercise with ten expert laboratories using LC-MS/MS methods mostly based on ISO 18465 (Microbiology of the food chain quantitative determination of emetic toxin (cereulide) using LC-MS/MS) was carried out. Each laboratory was requested to analyse nine independent sample replicates together with two QC samples (blank rice powder; rice powder contaminated at a very low toxin concentration) under intermediate precision conditions. First, the results were technically scrutinised. A few data sets had to be excluded from further evaluation (e.g. working range of method inadequate, QC results incorrect, non-adherence to the provided instructions). After statistical evaluation, it could be concluded that the laboratory mean results were about normally distributed. No mean and variance outliers were detected. The mean of laboratory means and the uncertainty related to characterisation were calculated. Once long-term stability data will be available, a full uncertainty budget will be drawn up. The CRM will be used for method validation and quality control and thus will enable effective consumer protection.

Milk and milk-based products are vulnerable to adulteration, with milk fat being replaced by cheaper alternatives, compromising product quality and safety. Ensuring milk fat authenticity is essential for meeting regulatory standards and maintaining consumer trust. The European Commissions Joint Research Centre (JRC) has developed a certified reference material (CRM), ERM-BD519, to combat milk fat adulteration. This CRM consists of anhydrous milk fat, certified for its relative mass fraction of triglycerides and cholesterol, supporting the implementation of ISO 17678/IDF 202.

The ISO 17678/IDF 202 method uses gas chromatography-flame ionization detection (GC-FID) to detect adulteration by analysing the unique triglyceride fingerprint of milk fat. This fingerprint is distinct from other fats and oils, allowing for the identification of samples outside the normal range for milk fat. The method applies triglyceride formulae developed in the late 1980s using a large dataset of milk fat samples.

A crucial element of ERM-BD519 is its characterisation, based on an interlaboratory comparison using ISO 17678/IDF 202. The certified values are operationally defined and can only be obtained when tested in accordance with this standard. A common calibrant, comprising a mixture of diverse triglycerides prepared by the JRC, played a key role in this study. This calibrant establishes a direct link to the data-driven triglyceride formulae used in this approach, thereby ensuring its long-term applicability for decades to come.

The regular use of the ERM-BD519 ensures consistent and comparable results across laboratories, which is critical for preventing milk fat adulteration, maintaining consumer trust, and guaranteeing regulatory compliance.

The homogeneity, stability, and availability of candidate materials are critical factors for the development and application of reference materials. The homogeneity and stability of reference materials are intrinsically influenced by specific preparation techniques.

For matrix reference materials of mycotoxin, traditional preparation approaches usually rely on the collection of natural contaminated samples and face the following limitations: (1) limited sample availability ( often requiring screening of a large number of samples from diverse sources), (2) unguaranteed sample homogeneity and stability, (3) undesired mycotoxin concentrations, and (4) difficulties in obtaining multi-concentration series materials essential for method validation, laboratory quality control and proficiency testing across the entire measurement range.

This study presents a novel preparation method for aflatoxin B1 (AFB₁) in high-fat peanut matrices. Through systematic optimization of peanut varieties, sample stabilizers, -ray irradiation, and packaging forms etc., the prepared material (20 g/kg level) demonstrated excellent homogeneity, long-term stability (>5 years at 4 C), as well as transportation stability and post-opening stability.

By precisely blending laboratory-cultivated, sterilized master-batch material (with moderate high AFB₁ concentration) with Aspergillus flavus-resistant blank peanut matrices, the strategy overcomes traditional limitations while enabling customizable aflatoxin B1 concentrations in peanut matrices. The Consistency between formulated concentration and HPLC-FLD quantification result further confirms the methodologys reliability.

There is global concern about cyanobacterial toxins due to animal mortalities, closure of recreational waterbodies and drinking water contamination. Accurate and comprehensive analysis of cyanotoxins is important to manage these issues and support guidelines and regulations in many jurisdictions. Despite the large number of cyanotoxin analogues from distinct classes (e.g. anatoxins, microcystins, saxitoxins), standards are only available for relatively few and not at all for some emerging classes of concern. We present the development of new cyanotoxin calibration solution reference materials. These include several analogues of the anatoxin-class, which have been responsible for animal mortalities worldwide, as well as two cyanotoxins of emerging interest: guanitoxin, a potent neurotoxin first discovered in Canada, and aetokthonotoxin, responsible for mass mortalities of bald eagles in the Eastern USA.

Anatoxins were obtained through bulk-culture of toxic cyanobacteria followed by isolation using preparative and semi-preparative chromatography. Homoanatoxin-a was harvested from culture medium using an adsorbent resin, dihydroanatoxin-a was obtained semi-synthetically from anatoxin-a and 10-hydroxy-dihyroanatoxin-a was isolated directly from cellular extract. Aetokthonotoxin was synthesized using established procedures. Efforts to isolate guanitoxin, which is highly polar and unstable, from cyanobacterial biomass associated with canine and human fatalities, are under way. Calibration solution feasibility studies for these toxins included structural confirmation and purity assessment using multiple analytical techniques, stability evaluations and quantitative measurements using 1H-NMR. Reference materials were prepared as solutions in argon purged, flame sealed glass ampoules have confirmed stability and homogeneity. For homoanatoxin-a, this process has been scaled up to production and certification of NRC CRM-hATX.

Domoic acid (DA), a potent neurotoxin produced by marine diatoms of the Pseudo-nitzschia genus, can accumulate in filter-feeding shellfish, posing health risks for human consumption and uncertainty for seafood producers. As a result, regulations have been established in many countries. Certified Reference Materials (CRMs) for DA have been available in both calibration solution and mussel (Mytilus edulis) matrix format for some time, but additional materials are needed to cover analytical testing requirements for other shellfish species.

This presentation will outline current processes in the production of algal biotoxin reference materials and describe the development of new CRMs for DA in scallop (Pecten maximus) and razor clam (Siliqua patula) matrices. The CRMs were prepared and bottled as wet tissue homogenates and characterized using fully validated methods, including liquid chromatography with tandem mass spectrometry (LCMS/MS) and diode array detection (LCDAD). Studies demonstrated good homogeneity, long-term stability at -12 C as well as a short-term stability at elevated temperatures providing confidence for transportation. Certified values for DA and its epimer (C5-epi-DA) assigned with expanded uncertainties (k = 2) were 3.2 0.1 g/g and 10.6 0.7 g/g for the clam and scallop materials, respectively, traceable to the International System of Units through a DA calibration solution (NRC CRM-DA). The DA isomer profiles of the reference materials were also characterized using liquid chromatography-high resolution mass spectrometry (LC-HRMS/MS). This work broadens the range of DA CRM matrices and concentrations available, providing regulators and research laboratories with essential tools to meet ongoing requirements for DA measurements.

The isotope fingerprint in food and beverage products could differentiate them based on geographical origin, botanical, soil and fertilization processes and fraudulent practices. The availability of Certified Reference Materials (CRM) in a matrix as similar as possible to the analyzed material is essential for obtaining accurate measurements with traceability to the isotopic scale. This work presents the characterization of three new CRMs including ethanol and mineral oil for carbon-isotope delta values (d13CVPDB) and powder milk for both d13C and nitrogen-isotope delta values (d15NAIR). Isotope characterization was performed in an elemental analyzer coupled to isotope ratio mass spectrometer (EA-IRMS) using three-point calibration for traceability. Processing and characterization were performed by the Institute of Petroleum and Natural Resources (IPR) following ISO 17034. The assigned isotope value and associated uncertainties obtained are d13C= -13.36 0.13 for ethanol; d13C= -28.78 0.22 for mineral oil; and d13C= -22.84 0.12 and d15N= +5.77 0.11 for powder milk. All CRMs were homogeneous and stable, proven in transport and long-term storage stability studies. Regarding uncertainty contributions, the main uncertainty source for d13C and d15N values in powder milk was the assigned uncertainty from the reference material used for calibration. In contrast, the main uncertainty source for d13C values in ethanol and mineral oil was transportation stability (comprising 70% of the uncertainty value); this uncertainty contribution was recently included in the ISO 33405:2024 guidance. Stable isotope CRMs in different matrices produced by IPR represent valuable tools for improving food fraud detection and ensuring product integrity.

Food safety is a major concern for both industries and consumers. For the food industry, accurately quantifying contaminants is crucial but often challenging. Certified reference materials (CRMs) are essential as quality controls to ensure metrological traceability, validate analytical methods, and calibrate instruments.

This work is part of the EMP 23IND13 ScreenFood project, which aims to develop reference materials (RMs) for PFAS in relevant food matrices. Here, we present progress towards producing an RM for both legacy and emerging PFAS in a tomato-based matrix.

The selection of the matrix, target analytes, and concentration ranges was guided by current guidelines, recommendations, and stakeholder input, ensuring relevance for the food industry, control laboratories, and consumers. Various industrially produced matrices were provided by suppliers and preliminarily screened for natural PFAS contamination using validated UHPLC-HRMS methods. Spiked samples at different concentrations were then assessed for homogeneity and stability.

The most suitable matrix will be chosen to produce the candidate RM, which will undergo characterization in an interlaboratory study to support the development of a CRM.

In this work, the results of the Supplementary Comparison, EURAMET.QM-S15 PAHs in Protein Matrix are presented and discussed. The EURAMET.QM-S15 was organized by the Federal Institute of Metrology METAS. The target analytes were four PAHs: benzaanthracene (BaA); benzoapyrene (BaP); benzobfluoranthene (BbF); and chrysene (Chr), for which maximum limits are set in the European and Swiss food legislation. The measurands were the mass fractions (g/kg) of these PAHs in a protein-rich food matrix. Six National Metrology Institutes (NMIs) and Designated Institutes (DIs) participated in EURAMET.QM-S15. The study required solvent extraction, separation of the target analytes from interfering matrix components (clean-up), analytical/chromatographic separation and selective detection of the target analytes. Solvent extraction (ASE, PLE, HUPsSE, QuEChERS) followed by SPE cleanup was applied by the participants in the sample pretreatment and GC-IDMS/MS, GC-HR-IDMS and LC-FLD were applied for separation and detection. Due to poor extraction efficiencies resulting from an unfavorable choice of solvent and insufficient demonstration of the metrological traceability of the calibrant, only the results of two participants were included in the evaluation of the Supplementary Comparison Reference Value (SCRV). The SCRV was assigned using the weighted mean of these two participants which agreed well with gravimetrically prepared mass fractions and evidence from an ancillary study performed with external expert laboratories. The study results are highly relevant to the closely related certified reference material (CRM) WP-CBR001 for the determination of PAHs in whey protein powder.

In Uganda, total polyphenol content, antioxidant and cytotoxicity of Canarium schweinfurthii Engl. have not been documented, which may limit its utilization. Henceforth, this study aimed at determining total phenolic content, antioxidant capacity and cytotoxicity of C. schweinfurthii fruit pulp. Following cold and hot water extraction of the fruit pulp extract, Total Polyphenol Content (TPC) was determined using the Folin-Ciocalteu method while the antioxidant capacity on human colon (Caco-2) cell lines was determined using the oxidative stress and 2,7-dichlorodihydrofluorescein diacetate staining model. Cytotoxicity at 1:2, 1:5, 1:10, 1:25, 1:50, 1:100, 1:200 and 1:1000 concentrations was determined using the human colon (Caco-2) cell lines and resazurin assay. The TPC obtained for C.schweinfurthii fruit pulp was 2.480.10 and 2.880.02 mg GAE/g dry weight for cold and hot extracts, respectively. There was no significant antioxidant activity observed after treatment of human colon (Caco-2) cell lines with the different fruit pulp extract concentrations. Zero to very low cytotoxicity was observed in cell lines treated with 1:10, 1:25, 1:50, 1:100, 1:200 and 1:1000 fruit pulp extract. The findings of this study suggest that in addition to its utilization as a food, C.schweinfurthii fruit pulp is a potential therapeutic agent. Keywords Canarium schweinfurthii Engl. Total polyphenol content. Anti-oxidant. Cytotoxicity. Caco-2 cell lines

Since the 1960s, the International Atomic Energy Agency (IAEA) has been assisting its member states in the field of data quality and quality assurance. To support member states in their marine monitoring activities, the Marine Environment Studies Laboratory (MESL) has produced Certified Reference Materials (CRMs) characterized for trace elements and methylmercury using samples of marine origin, including biota and sediments.

This work will present the production process of IAEA-158A marine sediment, characterized for trace elements and rare earth elements (REEs), with an emphasis on the homogeneity study. The between-unit homogeneity is evaluated to ensure that the certified values of the CRM are valid for all produced units within the stated uncertainty. Additionally, within-sample heterogeneity might be significant at the prescribed sample intake and should be considered in the uncertainty evaluation of homogeneity. This is particularly true for environmental solid samples such as marine sediment.

Examples of developed homogeneity designs and data treatment applied for the evaluation of uncertainty arising from sample inhomogeneity in IAEA-158A will be shown. The strategy employed to evaluate collaborating expert laboratories and their reported values, ensuring accurate and traceable certified values, will also be discussed.

A certified reference material (CRM) for PAHs in coastalsediments was developed in accordance withISO Guide 35.The CRM was prepared through rigorous scientific protocols using sediment samples from the Yellow Sea coast of Chinaas the starting material. The key processing steps included drying, milling, homogenization, subpackagingand sterilization.The study focused on the 16 U.S. Environmental Protection Agency (EPA) priority-controlled PAHs, which are of extensive concern due to their environmental hazards. The CRM exhibited homogeneity in assigned property valuesand demonstrated stability for at least one year under room-temperature storage. To determine certified values, a multi-laboratory collaborative measurement wasemployed, utilizing analytical techniques such as gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). The uncertaintiesof the property valueswere comprehensively evaluated incorporating contributions from characterization, homogeneity and long-term stability, and were expressed as expanded uncertainties to ensure data accuracy and credibility.

This CRM could offer broad applicability supporting method validation, quality control, proficiency testing,and deliver critical technical support for environmental quality management of marine sedimentsin China.

Polybrominated diphenyl ethers (PBDEs) are hazardous environmental pollutants regulated in the European Unions Water Framework Directive (WFD, 2000/60/EC). To ensure compliance, (certified) reference materials (C)RMs are required for water quality monitoring. The Environmental Quality Standards (EQS) established in the WFD are set to very low concentrations of PBDEs in surface waters. This presents significant challenges for the development of methods able to detect trace levels of these contaminants and to produce homogeneous test materials that serve as surrogates of surface water samples. The hydrophobic nature of these substances and a non-trivial design of a whole-water CRM often results in issues related to material homogeneity and stability.

The European Commissions Joint Research Centre (JRC-Geel) has developed the first ready-to-use whole-water CRM for the analysis of the six regulated PBDEs. The candidate CRM was prepared by adding 200 mg of jet-milled sediment as a slurry containing known levels of PBDEs into one litre of mineral water. This proved to be a reliable way of preparing an off-the-shelf CRM that closely mimics surface water sample containing suspended particulate matter with a top particle size below 10 m. The transport and storage stabilities were confirmed by isochronous scheme testing over 1 and 12 months at 4 C and -20 C, respectively. The PBDEs mass concentration in the candidate CRM will be assigned according to the standard method EN 16694:2015, which involves an extraction using SPE-disks and PTV-GC-MS/MS analysis. Unfortunately, the standard method does not yet seem to be widely used among monitoring laboratories. This may present a challenge for the adoption and widespread use of the CRM, albeit the WFD stipulates whole-water sample analysis for monitoring purposes. Nevertheless, its existence will certainly support and further improve regulatory compliance.

The presence of nanoplastics, here defined as plastic particles having at least one external dimension smaller than 1 µm, has raised concerns about potential risks to human and ecological health, and further research is needed to fully understand their effects. The small size and complex ageing processes in the environment pose significant challenges to reproduce nanoplastics artificially, hindering the development of reference materials and analytical methods, such as single particle identification techniques. Furthermore, single particle identification techniques also face challenges with respect to detection limits of smaller nanoplastics. To overcome these limitations, this study combines single particle hyperspectral imaging with a representative test material of artificially aged polyethylene terephthalate (PET).

In this study, we demonstrate the ability of an in-house developed hyperspectral microscopy technique to detect and characterise single nanoplastic particles in aqueous matrices. First, a sub-micrometric fraction (< 5 µm) was produced after gravitational sedimentation of 300 µm cryo-milled PET powder, following chemical treatment to increase hydrophilicity. Then, the particles were dispersed in tap water and through centrifugation, five particle fractions were obtained, ranging in particle sizes from 100 to 1000 nm (100-200 nm, 200-400 nm, 400-600 nm, 600-800 nm and 800-1000 nm).

Two different machine-learning algorithms (Random Forest and Neural Network) were trained on the dataset of spectral signatures obtained from the PET nanoparticles in each fraction, ultimately enabling the development of a robust method for accurately identifying nanoplastics in aqueous media. In combination with particle counting algorithms, the number concentration of PET nanoparticles can also be deduced.

Per- and polyfluorinated alkyl substances (PFAS) are a group of synthetic compounds that have been used in various industrial and consumer products for decades. Their wide range of applications is due to water- and oil-repellent properties, as well as their chemical and thermal stability. However, the use of PFAS has raised concerns due to their persistence in environment and their adverse health effects, leading to regulations aimed at controlling their use and minimizing exposure. Substances such as PFOS, PFHxS, and PFOA are listed in the Stockholm Convention on Persistent Organic Pollutants (POP Regulation, EU 2019/1021). Maximum values have been set for PFAS in environmental matrices such as soil and water, but also for textiles as a relevant area of circular economy and part of the current German standardization roadmap Circular Economy.

Reliable PFAS analysis requires an increasing global demand for certified reference materials (CRM). However, CRMs for PFAS in soils and textiles are rare or currently not available. To improve the metrological infrastructure and support PFAS measurements, two CRMs for PFAS in soil (BAM-U027) and PFAS in textiles (BAM-B003) based on ISO17034 and ISO33405 were developed. This poster provides an overview of the different steps for preparation and characterization of the two candidate materials. The assignment of the certified mass fractions for relevant PFAS targets (18 PFAS targets for BAM-B003) is based isotope dilution HPLC-MS/MS at three independent workplaces. The results of the BAM in-house study on BAM-U027 were supported by an interlaboratory comparison study.

Environmental matrix reference materials (RMs)play a significant role in quality control of the environmental monitoring and pollutantanalysis process, ensuring the accuracy and reliability of the analysisresults. The preparation and analysismethod for candidatewater matrixRMof perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) were studied. The effects of sample bottle adsorption and sterilization method tothe target components, as well as the interference of different types of water matrix, were investigated. The homogeneity and stability of the candidate water matrix RMwere also studied. The results showed that the solid-phase extraction/liquid chromatography-triple quadrupole mass spectrometry analysis method established had good linearity in the concentration range of 2-50 ng/ml. The RSDs of six parallel determinations of PFOA and PFOS were 5.05% and 1.86%, respectively, and the recovery rates of the internal standards were 98.5% - 99.9% and 83.3% - 92.3%, respectively. Polyethylene bottles had a certain adsorption effect on PFOS, which could be reduced by ultrasonic treatment for 20 minutesbefore extraction. Thecandidate water matrix RMs were prepared by adding PFOA and PFOSsolutionintopure water. The concentrations of PFOA and PFOS did not change significantly after irradiation sterilization. The samples had good homogeneity and stability insix months storage under both room temperature and 4C. Theprepared matrixRMhas been used for the proficiency testingfordetectionof perfluorinated compounds in water.The preparation technology established in this study will lay the foundation for the development of watermatrixRMsof perfluorinated compounds.

High purity metals are essential not only for industrial applications such as semiconductors and electronics but also play a crucial role in inorganic chemistry by establishing traceability to the International System of Units (SI). The Consultative Committee for Amount of Substance (CCQM) has emphasized the shortage of primary standards in inorganic chemistry. To address this challenge, the National Research Council of Canada (NRC) has been developing a series of high purity metal Certified Reference Materials (CRMs) designed to serve as primary standards.

The chemical purity of each CRM is obtained by the mass balance approach from the mass fraction of over 70 elemental impurities using glow discharge mass spectrometry (GD-MS), a technique that has been recognized by the CCQM as primary method of measurement and has become the industry standard for assessing the elemental impurity profile of high purity materials as it provides extensive elemental coverage. By developing these CRMs, NRC is not only addressing the shortage of primary standards but also strengthening the traceability of measurements in inorganic chemistry.

Since 2002, the NRC GD-MS facility has been accredited by the Standards Council of Canada under ISO/IEC 17025, boasting over 30 years of expertise in analyzing impurities in high purity conductive and semi-conductive materials. NRC has also participated in numerous international CCQM comparisons, such as CCQM-P149, CCQM-K72 and CCQM-K88, using GD-MS analysis to demonstrate its effectiveness and comparability.

The urban wastewater treatment plants (UWWTP) remain an important pathway of pharmaceuticals into the environment. The new EU Urban Wastewater Directive (Directive (EU) 2024/3019) aims to protect the environment and human health from the harmful effects of wastewater discharges, particularly from pharmaceutical (and cosmetics) industry. The Directive recommends Member States to consider upgrading UWWTP with a quaternary purification stage in order to eliminate micropollutants and to enable stronger reuse of treated water. For the proper implementation of this Directive and due to the increasing awareness about antimicrobial resistance, the monitoring of treated urban wastewater should include antimicrobial agents such as antibiotics.

Pharmaceuticals are a diverse group of compounds found in wastewater and subsequently in the environment, primarily due to human and animal use. Antibiotics, steroid compounds, analgesics/nonsteroidal and anti-inflammatory drugs, are among the pharmaceuticals most frequently found in the environment. To address these concerns, the European Commissions Joint Research Centre (JRC) is designing and carrying out a feasibility study to explore the possibility to produce a certified reference material (CRM) for pharmaceuticals in wastewater. Freeze-drying was one approach tested in order to prepare the matrix material. The measurements seems to confirm that the freeze-drying of wastewater do not significantly influence the different classes of pharmaceuticals present in wastewater. The main challenge in producing such material will be to ensure its stability and homogeneity. Providing a CRM will support environmental monitoring laboratories in assessing the performance of the analytical methods by providing a benchmark to evaluate accuracy, repeatability, and reproducibility.

Perfluoroalkyl substances (PFAS) are industrial and consumer product chemicals that pose increasing global concern due to their persistence in the environment and potential risks to public health and ecosystems. Emerging regulations around PFAS testing from the Environmental Protection Agency, the European Union and other organizations specify highly sensitive and specific liquid chromatography-mass spectrometry (LC-MS) methods to detect and quantify trace levels of PFAS in various substrates. PFAS Certified Reference Materials (CRMs) are essential to ensure accurate quantitation. The development of these CRMs considers factors such as material form, hygroscopicityLL1US2, volatility, isomers, and the complexity of mixed formats. This presentation will showcase how we have utilized specialized purification and certification methods, combining preparative techniques, LC-MS and liquid chromatography- charged aerosol detector (LC-CAD) with 19F quantitative nuclear magnetic resonance (qNMR), to produce high-quality PFAS CRMs.

The 14 volatile halohydrocarbons (VHHs) certified reference material (CRM) in nitrogen in high-pressure gas cylinders were successfully prepared by developing a technical route of weighting method with reasonable grouping, respective weighing and final combination preparation. The methodology for determination of the 14 VHHs CRM in nitrogen by directly injecting from high-pressure gas cylinders through six-port valve sampling injection system and detecting using gas chromatography-mass spectrometry (GC-MS) and gas chromatography-electron capture detector (GC-ECD) was developed respectively. The Resolution, asymmetry and sensitivity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision and accuracy were validated. The results showed that both of the methods could effectively separate the 14 VHHs CRM; the LOD and LOQ for GC-MS was within 0.1 pmol/mol-1.51 pmol/mol and 0.4 pmol/mol-6.04 pmol/mol, for GC-ECD was within 0.02 pmol/mol-4.9 pmol/mol and 0.08 pmol/mol-19.6 pmol/mol; the linear correlation coefficients of the fitted straight lines were all greater than 0.995; the repeatability of the system、the method and the intermediate variabilities for GC-MS and GC-ECD was 0.93% - 1.75% and 0.20% - 1.62%、 0.53%-0.89% and 0.30% 0.82%、0.58% 1.09% and 0.29% 0.92%. The results indicated that the developed VHHs CRMs with their GC-MS and GC-ECD analysis methods established in this study were both suitable for the environmental atmospheric monitoring and fixed pollution source exhaust gas monitoring.

Isotopic analysis is fundamental to various scientific disciplines, including geoscience, environmental science, medical and life sciences, and many others 1. Isotopic Certified Reference Materials (iCRMs) are critical for validating isotopic analysis methods, correcting instrumental mass bias, and serving as delta-zero references. Despite extensive efforts by National Metrology Institutes to produce high-quality iCRMs, challenges persist in establishing accurate reference values.

A notable example is zinc. Isotope ratio measurements by Chang et al. (2001) deviated significantly, by 8 sigma, from previous coulometric determinations. Similarly, mass spectrometry measurements of the IRMM-3702 Zn isotopic CRM (Ponzevera et al., 2006) led to a major revision of zincs standard atomic weight, making the current standard atomic weight inconsistent with its predecessor. This situation highlights a broader issue affecting many other elements: the critical need for more independent, reliable measurements as well as high-quality iCRMs to support research that relies on accurate and precise isotope ratio data.

Multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS) is one of the most advanced techniques for isotope ratio measurements, offering exceptional sensitivity and precision. However, achieving accurate results requires effective correction of instrumental mass bias, which remains a significant challenge. In addition to the commonly recognized mass-dependent fractionation (MDF), mass-independent fractionation (MIF) has also been observed in MC-ICP-MS for many elements 1. This complicates the selection of mass bias correction models, as applying MDF-based models to isotopes exhibiting MIF can introduce bias.

To address these challenges, the National Research Council of Canada (NRC) Metrology Research Centre has spent the past two decades developing cutting-edge methodologies for certifying isotopic CRMs using MC-ICP-MS. This lecture will highlight recent advancements in applying the full gravimetric isotope mixture model and optimized regression model for mass bias correction in MC-ICP-MS. Additionally, it will highlight NRCs innovative approaches to producing SI-traceable isotopic reference materials.

To ensure the traceability, comparability, and accuracy of polybrominated diphenyl ethers (PBDEs) detection results in soils, a new environmental certified reference material was developed according to the requirements of ISO Guide 35 by the Institute of Environmental Reference Materials, Environmental Development Center of the Ministry of Ecology and Environment(IERM), China. The whole process of CRMs development including preparation, homogeneity, stability studies, and value assignment. The assignment of the certified mass fractions was based upon interlaboratoryresults via using stable isotope dilution analysis gas chromatography mass spectrometry. The certified values obtained for BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, BDE-183 and BDE-209 were 11428 g/kg, 0.780.20 mg/kg, 0.720.22 mg/kg, 5515 g/kg, 18458 g/kg, 5018 g/kg, 10036 g/kg, 2.010.38 mg/kg, respectively. The expanded measurement uncertainties (coverage factor k=2)were obtained by combining the uncertainty contributions from characterization, between-bottle homogeneity and long term stability. The shelf life of the certified reference material is determined to be 10 years. The developed certified reference material was included into the State Register of the Approved Types of CRMs as GSB 07-4133-2023. This certified reference material can be used for the evaluation of measurement methods and as a quality control sample for environmental monitoring.

Liquid chromatography combined with mass spectrometry (LC-MS) is widely used for the separation and detection of organic compounds in complex mixtures. Mass spectra provide molecular weight and structural information, while retention times (RTs) provide unique identifying characteristics for analytes such as isomers that have similar mass spectra. Unfortunately, RTs can be highly variable between different laboratories and instruments. This generally requires the analysis of chemical reference standards with each batch of samples to allow a good match of RTs for conclusive identification. Not every laboratory can access all standards, so it would be helpful to have a system for reporting retention data in the literature and data bases so that analytes can be more easily identified. One method is to report “retention index (RI)” values. This is accomplished by calibrating the RT scale with a series of homologous reference compounds. This presentation will discuss the development and testing of a reference material (NRC RM-RILC) solution containing twenty unique compounds for measuring RIs in reversed-phase LC-MS. The compounds are a homologous series of N-alkylpyridinesulfonic acids (NAPS), with the alkyl group ranging from 1 to 20 carbons in length. The RM can be co-injected with a sample. The NAPS standards have been designed with permanently ionized functions, a quaternary amine and a sulfonate function, which enhance ionization and sensitivity in mass spectrometry in both the positive and negative ion modes. They have an overall neutral charge state that makes their retention times less sensitive to pH changes in the mobile phase.

It is fascinating to consider that standardisation and measurements are deeply rooted in human culture since the dawn of civilization. The current topical collection on Reference Materials in Analytical Chemistry is dealing with the integrity and realisation of correct measurement results in a modern context. As we all know, the purpose and use of (matrix) Certified Reference Materials (CRMs) is indeed that they are indispensable tools for an objective assessment of the correctness of measurements. Ten years have now passed since Hendrik Emons and Stephen Wise compiled a topical collection on Reference Materials for Chemical Analysis published in this journal. The field, materials/matrices, and analytical techniques are ever evolving and it is therefore about time to present another topical collection on Reference Materials in Analytical Chemistry.

The accuracy and precision of peak area calculation represents an important limit on the utility of qNMR applications. Although this calculation can appear simple at face value, small nuances can have a profound influence on the final results, making it essential to carefully consider the choice of peak fitting software. This presentation breaks down peak area quantification from a programmer’s point of view – focusing on key tradeoffs such as the use of time vs. frequency domain, gobal vs. local algorithms, and sequential vs. parallel processing strategies. A semi-global approach that combines peak processing and fitting into a single step is presented as both a convenient and effective avenue for further software development. The recent release of rnmrfit 2.0 serves as a useful example of this approach. An evaluation of model parameters on precision using 2H and 13C spectra of vanillin standards revealed the span of baseline spline to have a pronounced impact on the results, highlighting the importance of a parallel peak processing/fitting approach. Compared to commercial tools including TopSpin and MestReNova, rnmrfit demonstrated superior precision and trueness, achieving precision as low as 0.05% for 2H and 0.15% for 13C. The new version of rnmrfit is available as an open-source executable, offering a scalable solution for isotopic analysis with minimal user input, paving the way for more reliable isotopic quantification.

Rheo-NMR is used to quantify the rheological properties of complex fluids and flow systems by spatially resolving velocity distributions using pulsed field gradients, which are then interpreted through fluid dynamics models. While effective, conventional Rheo-NMR techniques require large, expensive, and immobile MRI instruments. Recent advancements have led to the development of cost-effective, portable magnetic resonance techniques that use compact permanent magnet arrays. These magnets can be designed to create optimized magnetic field profiles for specific applications. For flow characterization, a constant gradient aligned with the primary flow direction provides motion sensitivity. In this configuration, phase interference effects cause a modulation in signal magnitude when integrating over the finite sensitive region (1). This dependence of signal magnitude on velocity distribution provides valuable insight into dynamic properties of the material, e.g., viscosity and elasticity (2,3). In this work, we apply phase-interference-based portable MR techniques to quantify two different rotating flow systems: Circular Couette flow, used to measure the flow behavior index of non-Newtonian fluids (4), and the spin-up of a rotating fluid to solid-body rotation, used to measure the fluid viscosity. In circular Couette flow, we position a cylindrical sample inside the sensitive region of a magnet array with a constant gradient of 250 G/cm and insert a rotating inner cylinder (rotor) into the center of the fluid. As the rotor spins, a stable velocity distribution forms in the gap between the two cylinders, influenced by the fluid’s flow behavior index and the rotation speed. The resulting velocity distribution affects the MR-sensitive region, leading to varying degrees of phase interference. By acquiring MR signal measurements at different echo times, we fit the signal decay using expressions derived from the analytical equations for circular Couette flow. This approach allowed us to measure the flow behavior index of various shear-thinning fluids, yielding values consistent with expectations. While Couette flow systems are widely used in rheological measurements, some applications may not permit fluid extraction into a Couette cell. This is particularly relevant for hazardous or shear-sensitive solutions, where handling could alter the fluid’s properties. In such cases, a measurement technique capable of assessing fluid properties in situ would be advantageous. This motivated the exploration of a similar approach without a central cylinder, where only the outer cylinder rotates. Given enough time, a rotating fluid will achieve a state of solid body rotation. While this velocity distribution still causes a reduction in MR signal magnitude, it has no dependence on the fluid properties. Instead, we are interested in the time taken for the fluid to reach solid body rotation, which can be monitoring by observing the MR signal magnitude at various points throughout the spin-up process. Thus far, we have tested this method on glycerol-water mixtures with varying viscosities and found that the transition time behaves as predicted by theoretical models and CFD simulations. Our results demonstrate that this approach can be used to characterize the viscosity of an unknown fluid, provided the system is first calibrated with a reference fluid of known viscosity in the same container. These findings establish a strong foundation for extending the method to more complex fluids, such as non-Newtonian fluids. In discussing these methods, we hope to establish the effectiveness and versatility of compact, low-cost portable magnetic resonance techniques that employ simple, easy to implement acquisition schemes to accurately quantify fluid properties in specific, targeted applications.

Quantitative Nuclear Magnetic Resonance (qNMR) has become an accurate and precise method for measuring absolute purity. However, most current methods focus on internal calibration qNMR (IC-qNMR). This can be problematic when analyzing valuable samples that should not be contaminated by internal calibrants or rare natural products where even weighing is difficult. In such cases, external calibration qNMR (EC-qNMR) is a useful alternative. Several EC-qNMR methods exist, such as using coaxial-tube systems or inserting pseudo-FID signals. This presentation focuses on EC-qNMR using the principle of reciprocity, which enables the method to be performed without depending on specific instruments or software (1). This approach, known as PULCON (2), has shown good accuracy in many studies.

However, practical applications of EC-qNMR have often resulted in errors exceeding 5%, highlighting challenges in achieving consistent accuracy. Existing studies mostly emphasize positive outcomes and offer limited guidance on avoiding such errors. As a result, EC-qNMR procedures are not yet standardized, unlike IC-qNMR. In our recent research, we identified critical steps that enable EC-qNMR to achieve accuracy within 1%, comparable to IC-qNMR (3). This presentation will outline the key factors influencing measurement variability and provide practical insights to enhance the reliability and standardization of EC-qNMR.

qNMR technique facilitates measurement traceability of numerous analytes to a single national metrological standard. Therefore, qNMR is well-positioned to serve as a critical enabler for providing traceable organic CRMs.1 ISO 17034 requires validation of the method (or the analytical procedure) used to assign property values (i.e., purity or concentration). ISO 17034 refers to ISO 17025 for method validation and presents inter-laboratory comparison (ILC), evaluation of bias and precision using reference standards or materials as validation techniques, etc. However, ILC has limited versatility because they typically rely upon large-scale collaborative studies involving multiple laboratories. Technique with evaluation of bias and precision using reference standards or reference materials is limited because it requires a corresponding RM or analytical standard for each analyte. To address these issues, we compared the traditional approach and life cycle approach to qNMR method validation, in accordance with the recently published USP Stimuli article 2 and the revisions of the USP NMR-related General Chapters <761> and <1761>. 3,4.

The water proton NMR signal, often regarded as a nuisance, interferes with accurate spectral quantification of critical process parameters and quality attributes in pharmaceutical products. However, as the most abundant component in many drug products, water interacts with the API, excipients, and other ingredients, meaning the dynamics of its NMR signal can provide valuable information for quality control and fingerprinting of liquid aqueous drug formulations. We have developed water proton NMR (wNMR) technology, which enables accurate quantitative assessment of drug products based on the global wNMR parameter—water proton transverse relaxation rate, R₂(¹H₂O). This parameter is sensitive to the aforementioned interactions between water molecules, the API, excipients, and other components. A key advantage of wNMR technology is its ability to perform measurements noninvasively, directly in the original drug product container—such as a vial, syringe, or injection pen—without opening or compromising its contents. In this presentation, we showcase applications of wNMR technology for quantitative analysis, including insulin concentration in injection pens (1), aggregate content in mAb formulations (2), vaccine fingerprinting for counterfeit detection (Figure 1), empty/full ratio assessment in gene therapy products (3), precise fraction estimation in multicomponent pharmaceutical formulations, and in situ cell count monitoring in growing cell culture (Figure 2). We aim to promote the application of wNMR technology within the broader qNMR community throughout the entire lifecycle of pharmaceutical products—from product/process development to formulation, manufacturing, and quality control at both the point-of-release and the point-of-care.

Carbohydrate molecules, known as glycans in a biological context, often have highly complex structures and contain branches, which is unique to this class of biomolecules. They are essential components of living organisms and are, among other things, involved in the storage and transport of energy, cell-cell interactions and regulation of gene expression. Altered glycan patterns can affect inflammatory responses, facilitate cancer cell metastasis, modify apoptosis or promote viral immune escape.

NMR spectroscopy studies of glycans commonly utilize the spin-½ nuclei, 1H and 13C, and when present 15N and 31P, but other nuclei such as 19F and 77Se have also been employed in investigations of carbohydrates and their interactions with proteins. The limited spectral dispersion of oligo- and polysaccharides where most 1H chemical shifts are found in the spectral region ~3 – 4 ppm and most 13C chemical shifts are found in the spectral region ~60 – 80 ppm make resonance assignments a challenging process (1,2).

Concatenation of NMR modules into a single 2D NMR experiment improves efficiency and results in time-saving due to the fact that two or more experiments share a common recovery delay prior to each subsequent scan of the 2D NMR experiment. This concept has been extended to parallel NOAH (NMR by Ordered Acquisition using 1H-detection) supersequences utilizing sequential, parallel and time-shared acquisitions by which ten spectra can acquired in a single measurement, referred to as a p-NOAH-10 (3). A NOAH-5 measurement was tailored to produce NMR data for the computer program CASPER (4), which can be used to determine structure of oligo- and polysaccharides. Specifically, the supersequence (BSCSJT/S) consists of five NMR modules, viz., 1H,13C-HMBC, multiplicity-edited 1H,13C-HSQC-COSY, F2-coupled 1H,13C-HSQC, 1H,1H-TOCSY, and a time-shared multiplicity-edited 1H,13C-HSQC module, covering most of the NMR data used as input to CASPER for structural elucidation or NMR resonance assignments of an oligosaccharide.

Resonance overlap in NMR spectra of oligosaccharides can be greatly reduced, and resolution improved, by utilizing pure shift methods. Even though many correlations are resolved in 1H,13C-HSQC NMR spectra some may still remain, among other things, due to 1H,1H couplings, though these may be refocused and the resulting pure shift 1H,13C-HSQC NMR spectra are thus devoid of the homonuclear proton-proton couplings. However, peak-picking of cross-peaks in 2D NMR spectra is often time-consuming, limiting the potential of CASPER as an efficient analysis tool. Since pure shift methods aim to collapse multiplets into well-resolved singlets, pure shift data are ideal for use in conjunction with CASPER, allowing for efficient analysis by using automated peak-picking routines (5). Further refinement of 1H NMR chemical shifts and nJHH may be carried out by spin-simulation resulting in data useful for conformational analysis of the glycans.

Nuclear magnetic resonance (NMR) has long been used by pharmaceutical industries for the identification and quantification (qNMR) of target bioactive compounds, related impurities, and process contaminants. Recently, the food industry has approached spectroscopy and qNMR has been successfully used, e.g., for product profiling to help detect fraud (1).

Food samples are complex multicomponent matrices that give overcrowded NMR spectra. To deal with the complexity of these samples, acidic hydrolysis combined with high-performance liquid chromatography (HPLC) has historically been employed to analyze sugars and proteins through their constituting monomers (2,3). This total hydrolysis of the sample is also beneficial for qNMR analysis because it helps reduce broad background signals from macromolecules, resulting in sharper NMR signals. Since many parameters must be measured and considered to get precise quantitative NMR results (1,4), analyzing this type of sample entails many challenges for the selection of acquisition parameters and the processing of the spectra. Processing challenges are related to baseline distortions and how to deal with overcrowded spectra with overlapping resonances, but challenges also include pre-acquisition: instrument-related issues such as homogenization of the magnetic field and tuning and matching. Ultimately, all these factors make it difficult to operate the instrument in automation and highlight the necessity for further developments in using a digital platform to manage complex spectra for qNMR (5).

In this study, protein hydrolysates from poultry sidestreams were used as a case study to analyze their amino acid content with 1H-qNMR. The specific aim was to investigate and quantify Hydroxyproline (Hyp), an amino acid characteristic of collagen proteins (3). The advantages and disadvantages of acidic hydrolysis and the use of an acid as a shifting agent for obtaining isolated peaks were studied. The effect of these sample preparation steps on the processing of spectra was evaluated by comparing the quantification obtained using ERETIC2 (Electronic to Access In Vivo Concentration), spreadsheet calculations (4), and a deconvolution approach.

Our findings aim to advance the understanding of qNMR challenges with complex matrices, possibly paving the way for the future development of fully automatic platforms that can assist the industry in routine analysis.

Despite the absence of established regulatory precedence and validation guidelines, quantitative nuclear magnetic resonance (qNMR) spectroscopy is a highly attractive analytical technique in pharmaceutical development. Given this, qNMR occupies a significant portion of our efforts and time as members of the Structure Elucidation Group (SEG). With the ability to validate other analytical methods and/or save analyst time, we have an ongoing initiative to train and educate our analytical and synthetic chemistry colleagues in using qNMR for their processes. In this presentation, we highlight the general external calibration qNMR workflow used for most samples that reach the SEG. We will further discuss some general considerations with this external technique, including the impact of NMR tube quality, a method to compensate for this NMR tube variability (1), the use of heteronuclear qNMR, and showcase the ability to quantify unstable components in neat reagent or reaction solutions. Additionally, we will discuss how qNMR is leveraged to paint a complete picture of samples with multiple components, such as pharmaceutical salts, providing insight into the identity of the species.

Quantitative NMR (qNMR) has become a valuable tool for the chemist and has found applications in areas as diverse as metabolomics1, food analysis2 and certified reference materials3. The major advantage that NMR has over other methods is that under strict conditions the qNMR method has a uniform molar response, including between samples. This makes qNMR using an external calibrant possible in cases where the addition of an internal standard is not desirable4. The large linear dynamic range of the method also means that the calibrant does not need to be in the same range as the analyte. In addition, the dispersion of resonances in an NMR spectrum means that analyte resonances can often be found that do not have interferences from impurities or the solvent resonance. There are applications where it is advantageous where non-deuterated solvents need to be used in the NMR experiment. In these cases some sort of solvent suppression needs to be used in order to reduce the dynamic range requirements of the acquisition. The most common method of solvent suppression is the selective presaturation of the solvent resonance. This can result in transfer of polarization to the analyte changing its molar response. In this work I will discuss the magnitude of the polarization transfer with respect to different solvent systems and analytes and the ramifications to qNMR and the analysis of complex mixtures.

The use of qNMR as a high-accuracy and traceable method to characterize reference materials is well-established in several fields. Purity mass fraction of high-purity materials determined by qNMR using internal standards and deuterated solvents has become one of the methods of choice for establishing metrological traceability in organic analysis. However, the use of external standards corrected by the pulse width has been shown to be an appropriate method for quantitation 20 years ago1 and has been consistently applied by our group to quantitate toxin materials that are only available in limited quantities. By applying such a method, we are able to obtain accurate and traceable measurements of the mass fractions or concentrations of the compounds in solution with deuterated solvent which are in turn diluted to produce Certified Reference Materials (CRMs). When working with compounds that are only available at small amounts, special care has to be taken to ensure that the material will not undergo any undesired transformations. Therefore, minimizing handling of the compounds, including solvent change, is preferable. In recent work it was observed that Caribbean ciguatoxin-5 (C-CTX5)2 undergoes substantial H/D exchanges when dissolved in deuterated solvents, and the rate of back-exchange was suboptimal for use of the same material in final reference material preparation. In such scenarios where use of natural abundance solvents is preferable, performing high-accuracy qNMR can be challenging as the commonly used solvent suppression methods may affect quantitation in ways that are not fully controlled.

In this work, we assessed the quantitative performance of several solvent suppression sequences available in the literature and designed additional sequences using an excitation sculpting scheme with selective 180-degree pulses. We also developed two sequences that enable measurements of the spin-lattice relaxation times (T1) using the inversion-recovery principle along with the binomial-like solvent suppression. This presentation will show how high-accuracy measurements are possible with suppression of solvent signals using binomial-like and WADE pulses3-6, the impact of performing such measurements in different probes/instruments and outline additional allowances for measurement uncertainty estimations when solvent suppression is applied.

Often when determining the concentration of a target molecule in a sample there are signals from impurities which occur in similar regions of the spectrum. For example, in process chemistry, impurities often arise from partially reacted species so share structural similarities with the main component. This means that we have to deal with the problem of peak overlap when determining a sample concentration.

This talk will present a comparison of different approaches to removing the contribution from an impurity close to the main peak of interest. These methods include so-called chromatographic integration, where a local baseline is used to cut off the impurity peak, and an improved version of the “smart sum” approach1 which combines traditional sum integration with peak deconvolution. The advantages and disadvantages of each method will be discussed, including visualization, robustness, sensitivity to noise, dynamic range, and the overall error in the final result.

Hydroperoxides are the primary oxidation products in the degradation of fats. Classical titration methods for determining the Peroxide Value (POV) are unreliable due to their non-specificity and incompatibility with lecithins. We present a novel NMR-based method that employs a redox reaction on a modified phosphine and detects the reaction specifically using 19F NMR spectroscopy. This approach eliminates signal interference from complex lipid mixtures.

The method demonstrates high specificity for hydroperoxides, converting them into alcohol while the reagent is oxidized. All reactants and products can be quantified precisely. The sensitivity of this method is highlighted by the measurable changes in chemical shifts in the 19F NMR spectrum, enabling POV determination even in trace amounts.

A detailed kinetic study of the second-order redox reaction was conducted, and the method was validated using calibrated partially oxidized triolein as a standard. As a primary method, NMR outperforms traditional titrations. Furthermore, this technique allows for the determination of POV in complex lipid systems, such as krill oil and lecithins, regardless of whether they are native or modified.

In addition, the 31P NMR spectrum enables simultaneous quantitative analysis of phospholipids, providing a "one-pot" reaction and analysis approach. Secondary oxidation products such as aldehydes can also be directly identified in the 1H NMR spectrum, along with iodine and acid values, which would otherwise require separate titrations.

This novel method provides a comprehensive, reliable, and efficient alternative to classical POV titration, broadening the scope of lipid oxidation analysis in complex systems.

In general, bioassay-guided fractionation and isolation of bioactive constituents from botanical materials frequently ended up with the reward of a single compound (1). However, botanical materials typically exert their therapeutic actions through multi-pathway effects due to the intrinsic complex nature of chemical constituents. In addition, the content of bioactive compounds in botanical materials is largely dependent on humidity, temperature, soil, especially geographical origins, from which rapid and accurate identification of plant materials is pressingly needed (2,3). These long-standing obstacles collectively impede the deep exploitation and application of these versatile natural sources. To address the challenges, a new paradigm integrating biogravimetric analyses and machine learning-driven origin classification (BAMLOC) was developed. The biogravimetric analyses are based on absolute qHNMR quantification and in vivo zebrafish model-assisted activity index calculation, by which bioactive substance groups jointly responsible for the bioactivities in all fractions are pinpointed before any isolation effort. To differentiate origin-different botanical materials varying in the content of bioactive substance groups, principal component analysis, linear discriminant analysis, and hierarchical cluster analysis in conjunction with supervised support vector machine are employed to classify and predict production areas based on the detection of volatile organic compounds by E-nose and GC-MS. Expanding BAMLOC to Codonopsis Radix enables the identification of polyacetylenes and pyrrolidine alkaloids as the bioactive substance group for immune restoration effect and accurately determines the origins of plants. This study advances the toolbox for the discovery of bioactive compounds from complex mixtures and lays a more definitive foundation for the in-depth utilization of botanical materials.

INTRODUCTION

The Japanese Pharmacopoeia General Test Method <2.46> Residual Solvents outlines the guidelines for controlling, identifying, and quantifying organic solvents present in drug substances, excipients, and drug products. Trifluoroacetic acid (TFA), a solvent, is used in the manufacturing processes of these substances. If it is present in the final products, a reason must be provided for its validation from the perspective of safety. We have previously reported that it is possible to use 19F-qNMR to perform absolute quantification of organic fluorine pharmaceuticals 1. In this study, we quantified the residual TFA in dimethyl sulfoxide (DMSO) using 19F-qNMR. In addition, we quantified TFA using ion chromatography (IC) as a comparison.

METHODS

Samples DMSO solutions containing 0.5, 0.1, 0.05, 0.025, and 0.01 w/w% TFA; qNMR reference standard for 19F-qNMR: 3,5-bis(trifluoromethyl)benzoic acid (3,5-BTFMBA); deuterated solvent: DMSO-d6, Number of preparations: 1–3 preparations for each concentration, number of measurements: 3 non-consecutive measurements, number of measurement institutions: 11, NMR instrument: 400-600 MHz (1H conversion), number of scans: set to the number of scans that gives S/N>100. IC column: Dionex™ IonPac™ AS18, eluent: ultrapure water, detection: electric conductivity detector, number of preparations: 1–3 preparations for each concentration, number of measurement institutions: 1.

RESULTS AND DISCUSSION

The TFA quantities measured by weighing and 19F-qNMR (A and B) were compared for TFA/DMSO solutions at five concentrations, and the values of (B)/(A) ± SD (%) (n = 10) were 0.5 w/w%: 99.62 ± 0.29%, 0.1 w/w%: 99.47 ± 0.38%, 0.05 w/w%: 99.26 ± 0.67%, 0.025 w/w%: 99.41 ± 0.85%, and 0.01 w/w%: 99.52 ± 0.93%, respectively. The variation increased as the concentration decreased; however, the variation remained within 1% for all five concentrations. Furthermore, the ratios (C/A and B/A) of quantities measured by IC (C) and 19F-qNMR (B) to those of weighing (A) were compared at one institution and the results were almost identical among all five TFA concentrations. Therefore, TFA can be quantified using 19F-qNMR at all the concentrations, although slight variations can occur.

Benchtop Nuclear Magnetic Resonance (NMR) spectroscopy has emerged as a powerful analytical tool, offering significant advantages, including portability, user-friendliness, high reproducibility, and precision in quantification.1-3 Benchtop NMR is being incorporated in a wide variety of applications and settings, ranging from R&D labs to process monitoring in industrial manufacturing with the capability to analyze the samples directly, without any sample alteration or minimum sample preparation. The presence of protonated solvents in process monitoring, or manufacturing requires resolving and quantitating the analyte signals of interest from the strong NMR signals of the solvents.1 Additionally, due to the lower field strength available with permanent magnet spectrometers, the NMR spectra of complex mixtures can be more difficult to analyze due to signal overlap. To address some of the challenges of quantitative analysis of components in complex mixtures with protonated solvents, we employed various advanced NMR techniques that allows for the multi-frequency solvent suppression, or filtering out signals by relaxation time (T1, T2), or to separate signal components by molecular self-diffusion.1 In this presentation, we will introduce different approaches to quantitatively analyze complex mixtures including buffered protein solutions and human ophthalmic formulations.1-3

Recent developments in benchtop NMR instrumentation and automation have enabled new applications of quantitative NMR (qNMR) in pharmaceutical development and quality control. Automated benchtop qNMR methods can offer reduced turnaround times and streamlined workflows, providing alternatives to conventional chromatographic techniques that often require extensive sample preparation and specialized detection methods. This is particularly relevant for the control of critical excipients in biologic formulations, such as polysorbates and poloxamers, which are typically monitored using dedicated, lengthy liquid chromatography (LC) with evaporative light scattering or charged aerosol detectors and necessitate specific sample preparation.

In this study we present a new benchtop NMR-based workflow incorporating high levels of automation as an alternative to chromatographic methods. The approach uses an optimized NMR sequence that combines water suppression and T2 filtering, allowing for direct analysis of formulation samples without the need for sample preparation. Automated on-the-fly optimization of water suppression ensures consistent spectral quality across analyses. Quantitative data analysis is performed using the "Advanced Chemical Profiling" software, which enables fully automated processing and reporting based on a single-point external calibration.

The method yields precision and accuracy comparable to traditional LC-based techniques, while simplifying the analytical process and removing the need for NMR expertise in routine operations. This workflow is scalable and can be extended to other organic excipients used in biologic formulations, supporting the replacement of multiple chromatographic assays with a single qNMR analysis. In addition to operational efficiencies, the procedure reduces solvent usage and associated waste, contributing to more sustainable practices in development and quality control laboratories.

Benchtop nuclear magnetic resonance (NMR) technology has greatly increased accessibility, affordability, and automatability for this important analytical technique (1,2). These instruments do not require regular maintenance or the use of cryogens and can easily be used by experts and non-experts alike, making them ideal for use in quality control (QC) environments. Furthermore, a variety of nuclides are available for analysis (e.g., 1H, 7Li, 13C, 19F, 31P, etc.), greatly expanding the possible uses of this instrumentation and enabling many underserved users to incorporate this technique into their workflows.

In this presentation, we will discuss our vision for the use of benchtop quantitative NMR (qNMR) spectroscopy in industry and highlight some key obstacles that could be addressed by the qNMR community to help with further adoption of this technique. Additionally, we will examine some of the hurdles involved in pharmacopeial monograph revision and provide some recommendations to help address some of the relevant pain points.

Furthermore, we will provide some visibility on what we believe is required from a hardware and software perspective for benchtop NMR to be more easily integrated into QC workflows, and will provide some discussion points regarding how the qNMR community can help educate interested parties who could stand to benefit from the incorporation of this important technique into their laboratories, but who might not be aware of the ease with which this could be done. Finally, we will give some concrete examples of key industries where we believe benchtop qNMR technology could have an immediate and powerful impact, such as for excipient and active pharmaceutical ingredient (API) testing, and for the testing of raw materials used to produce polymers, illustrating the wide variety of compounds that can be tested before their use in the final production process.

The gut microbiome plays a vital role in host health by modulating metabolism, supporting immune function, and enhancing disease resistance. Stool sampling provides the most accessible and direct means of assessing the gut microbiome, and NIST has released a human gut microbiome reference material (RM 8048) to support validation of routine metagenomic and metabolomic measurements. 1,2 A companion material to this RM is being developed to improve measurement confidence in gut microbiome metabolomics. Toward this goal, we formulated a complex mixture of 70 metabolites commonly found in human stool samples at biologically relevant relative concentrations. The pilot batch of this synthetic fecal metabolite solution was released as a research-grade test material (RGTM 10212) 3, which is currently under evaluation by stakeholders in the metabolomics community.

Metabolite concentrations were not reported with the release of RGTM 10212. In the next phase of this project, we are investigating methods to improve the accuracy of metabolite quantification for value assignment of a future RM. One approach under evaluation is quantum mechanical spectral analysis (QMSA), which uses quantum chemical simulations to predict NMR spectra based on the molecular structure, enabling more accurate assignment and quantification of overlapping signals in complex mixtures. In parallel, we are exploring other techniques such as reference deconvolution to correct for line shape distortions and increase consistency in NMR spectra, thereby improving the robustness of multivariate analyses used to assess material homogeneity and stability. Progress in these ongoing efforts will be highlighted in this talk. Collectively, this work supports the continued development of high-quality reference materials for metabolomics with the aim of promoting greater measurement consistency across the research community.

Benchtop (BT) NMR spectroscopy has emerged as a practical alternative to high-field NMR systems, offering accessibility and affordability. However, BT NMR presents analytical challenges due to complex spectra, limited resolution, and strong coupling effects. In this study, we present the application of the USP-ID software, which integrates quantum mechanical (QM) spectral models and automated workflows, for the identification and quantification of compounds in mixtures using both 500 MHz and 80 MHz NMR data.

Ten small molecules with challenging ¹H NMR features, such as diastereotopic methylenes and long-range couplings, were selected. Reference QM models were built using high-field spectra and implemented via the ChemAdder interface. These models were then used within USP-ID to analyze both single compounds and mixtures (3–9 components plus TMA) in DMSO-d₆.

Three workflows were explored: (1) compound identification at 80 MHz, (2) mixture identification at 500 and 80 MHz, and (3) absolute quantification at both fields. Despite significant spectral overlap in BT NMR data, USP-ID enabled accurate identification with >95% match scores and no false positives/negatives. Quantification results showed a mean absolute difference of 4.4% between 80 MHz and 500 MHz data, validating the method’s reliability. Notably, consistent underestimation of cinnamyl alcohol across all spectra highlighted sample purity as a critical factor affecting quantification accuracy.

This work demonstrates the viability of applying QM-based analysis for quantitative and qualitative evaluation of complex mixtures using BT NMR. The ability to maintain accuracy in low-field spectra paves the way for broader adoption of BT NMR in routine analysis and supports USP-ID as a robust tool for qNMR applications.

Analysis of biomedical agents by qNMR is an essential tool in collaborative research programs. Investigated materials range from crude natural products (e.g., botanical extracts) to refined mixtures (e.g., dental DESIGNER materials) to highly defined substances (e.g., pharmaceuticals, dietary ingredients, reference standards). This presentation showcases new qNMR applications and methods concepts and highlights new conceptual insights in (q)NMR theory this work has recently led to.

To address conflicting reports about the occurrence and alleged therapeutic role of caffeine in widely consumed Damiana botanical products (Turnera diffusa), we developed a qHNMR assay that can detect caffeine at a level of 500 ng per daily intake equivalent. Outcomes show the small but significant variability of chemical shifts and role of spiking experiments in low-ug threshold qNMR analysis. The results indicate that the caffeine content of damiana leaves is below 10 ppm, eliminating its consideration as a pharmacologically active constituent in this plant.

A new qNMR method was developed for the analysis of phosphatidylserine (PS), another widely used dietary supplement. This method utilizes quantitative 31P-NMR with triphenyl phosphate (TPP) as an internal calibrant (IC). With the newly developed method, two commercial PS preparations (labeled as containing 50% and 70% PS) from two different vendors were analyzed. We studied the differences in the observed quantitative outcomes depending on the processing of the spectra (window functions), criteria used for integration, and selection of reference molecular weight. A deconvolution approach was found to reduce operator bias, especially in integration, and produce more reliable results.

As part of a restorative dentistry project, ECIC qHNMR was employed to investigate the molecular interactions between proanthocyanidins (PACs) as natural dental biomodifiers and methacrylate-based dental resins, enabling high-sensitivity quantification of residual monomers and crosslinking density. Precise 1H qNMR analysis revealed that PACs reduce leaching of bis-GMA and TEGDMA monomers, thereby enhancing polymer network stability. These findings highlight the potential of PACs to improve adhesive performance and longevity at the adhesive-dentin interface.

Regarding qNMR methodologies, we have been developing a new approach termed Quantum Mechanically Guided Integration (QMGI). This new generation of quantitative measuring in qNMR combines the accuracy and stability of classical manual integration with the selectivity, sensitivity, and specificity of automatic quantum mechanical spectral analysis (QMSA) that can utilize HifSA profiles as references for targeted quantitation of analytes.

Finally, regarding theoretical concepts, our recent work contributed to the revision of pharmacopoeial monographs on qNMR that are applicable to the regulatory environment. New insights from the underlying meta analysis of known qNMR concepts as well as processing of experimental data showed the importance of connecting qNMR with its fundamental quantum mechanical background. Among the new insights are implications that this connection of qNMR theory and practice has on the terminology used to describe qNMR outcomes and the entire NMR analytical process.

The ability to assess the identity and composition of complex natural health products is necessary for ensuring reproducible research, establishing product quality, and regulatory compliance with Good Manufacturing Practices. Chemical reference standards play a critical role in measurement quality. Although chemical vendors provide specifications for materials, various factors can influence whether the actual purity aligns with the stated values. Common techniques employed to assign purity, such as TLC or HPLC, only establish purity in reference to impurities observable by the specified detection parameters. To validate the purity of purchased analytical standards, Quantitative Nuclear Magnetic Resonance (qNMR) is a versatile method suited to a wide range of substances, that allows for directly measuring analyte purity by comparing it with an internal standard1, 2. Moreover, qNMR is highly reproducible and traceable to the International System of Units (SI), ensuring reliable and accurate measurements. In this study, we employed a qNMR-based methodology that allows for the rapid and precise purity determination of different chemical standards including alkaloids, flavonoids and chalcones. Enhancing the accuracy and reliability of the qNMR method for purity determination requires careful optimization of key parameters, including solvent selection, relaxation delay, pulse angle, and spectral resolution.

Internal standards play a crucial role and should be chemically inert, highly pure, and yield uncomplicated NMR spectra to prevent signal overlap. Rigorous validation through extensive testing is necessary to ensure reproducibility while reducing uncertainty, making the method robust and reliable for purity analysis3.

Sceletium tortuosum (Kanna) is traditionally used in South African medicine for its mood-enhancing properties, helping to reduce stress, anxiety, and depression. Emerging evidence highlights potential benefits in managing OCD and improving cognitive performance under stress1. Its pharmacological effects are primarily driven by alkaloids including mesembrine, mesembrenone, delta-7-mesembrenone, mesembranol, and mesembrenol. The alkaloid profile is influenced by plant genetics, environmental conditions, harvesting and formulation which significantly impacts the therapeutic efficacy and potency. Precise profiling and quantification of these alkaloids are critical for establishing product quality, regulatory compliance and optimizing the therapeutic potential of products2. Current quantification methods for mesembrine alkaloids rely heavily on analytical standards1 and the lack of available and reliable materials has compromised the reproducibility of results and led to inconsistencies in analytical outcomes. Orthogonal techniques, such as quantitative Nuclear Magnetic Resonance (qNMR), offers a robust alternative for purity verification and alkaloid quantification. In this study we employ a qNMR-based methodology that allows for the rapid and precise quantification of different mesembrine alkaloids in Sceletium tortuosum plant material and commercial products. This technique circumvents the need for preliminary purification steps or reliance on standard compounds, providing a straightforward and efficient approach3, 4. Five alkaloids isolated from Sceletium tortuosum were verified for identity and evaluated for purity by qNMR. 1HNMR chemical shift determination reveals at different regions in the 1H NMR spectrum, the characteristic signals for individual alkaloid are separated well from each other. The quantities of these alkaloids are calculated by the relative ratio of the integral values of the target peak for each compound to the known concentrations of the internal standard. This approach addresses the limitations of traditional techniques and supports quality control for Sceletium tortuosum-derived products.

Nanomaterials (NM) of different size, shape, morphology, composition, and surface chemistry are used in a wide range of applications, including medical diagnostics, and imaging and consumer products. The importance of an adequate and reliable characterization is crucial for quality control during NM production, for ensuring an optimum function for the desired application, and for risk assessment studies. Currently there is a lack of reliable and validated methods and reference materials for quantifying NM surface functional groups, despite the importance of surface chemistry for the production of colloidally stable materials, further processing steps, and the interaction with the environment and biological species.

Following our initial study on the use of qNMR for quantifying the amount of amino groups on surface modified silica (1), we have carried out two bilateral comparisons between NRC and BAM to further develop and optimize a reliable protocol for these measurements (2,3), using aminated silica nanoparticles prepared by multiple methods, both commercial and in-house synthesized, and with varying amine content. Solution qNMR is based on dissolving aminated silica nanoparticles in strong base to release the surface grafted amino silane molecules, followed by the quantification of these molecules by solution qNMR using an internal standard. This method provides the amount of total amino groups present in the sample, which can differ from probe accessible or surface-sensitive measurements performed with X-Ray photoelectron spectroscopy (XPS). Complementary measurements using optical assays, involving a labeling step with a dye reporter, and XPS are employed to assess the probe accessible and surface amine content for representative samples. These measurements, which illustrate the advantages and potential limitations of the different characterization methods, will contribute to establish a basis for testing the protocol in an international inter-laboratory comparison and for standardization at ISO Technical Committee 229 – Nanotechnologies.

Accurate quantification of key phytochemicals in botanical dietary supplements is crucial for product quality and reproducible research. Certified reference materials (CRMs) are needed as calibrants for standardization of quality testing and to better understand the potential health benefits and safety of phytochemicals in dietary supplements. Commercially available solution-based CRMs for phytochemicals in ginger and kava, two popular botanical supplements, were developed in collaboration with the Office of Dietary Supplements at the National Institute of Health (NIH). The kava CRM contained six kavalactones and three flavokawains, while the ginger CRM included three gingerols and three shogaols. Prior to formulation, raw materials were characterized to confirm identity and to assign a mass fraction value. Quantitative 1H NMR (qNMR) was utilized as an orthogonal technique to verify mass fraction assignment by traditional mass balance techniques. Synergies and discrepancies between qNMR and mass balance purity factors of kava and ginger phytochemicals will be discussed. Challenges associated with the qNMR certification of phytochemicals, such as spectral purity, stability, and material availability, will be presented.

Collagen-enriched protein hydrolysates have been widely utilized for pharmaceuticals, medical products, and cosmetics, as well as food and feed products. These protein hydrolysates exhibit varied physicochemical properties and activities based on peptide length and amino acid composition. In our study, aligned with the principles of circular bioeconomy, peptides were produced by enzymatic protein hydrolysis (EPH) using low-value cuts from poultry processing, a protein-rich biomass. To achieve the desired product quality and optimal yield, monitoring and feed-forward control mechanisms for adjusting the enzymatic process are of vital importance (1,2).

Collagen solubilization can be monitored by measuring Hydroxyproline (Hyp), a modified amino acid almost exclusive to collagen (1,3). Traditionally, Hyp has been measured using colorimetric methods or by liquid chromatography, which is the gold standard for total amino acid composition analysis (1,4). Recently, Fourier Transform Infrared spectroscopy (FTIR), a vibrational spectroscopic technique, has been successfully applied to monitor industrial processes, assessing relevant industrial parameters like molecular weight distribution (MWD) and collagen content (2,3). However, while vibrational spectroscopy is not suitable for predicting amino acid composition, Nuclear Magnetic Resonance (NMR) spectroscopy is. NMR provides deeper insights into metabolites and low molecular components, unlike FTIR, offering more reliable quantitative (qNMR) data. For instance, Sarkar et al. have successfully used NMR as a process analytical technology (PAT) to identify, resolve, monitor, and quantify various small molecule components in real-time during an in vitro reaction for producing mRNA-based drugs (5).

In this study, a 1H-qNMR method for quantifying Hyp in hydrolysates was developed and validated. This method is more environmentally friendly than the gold standard for amino acid analysis and safer than the colorimetric methods aforementioned. Subsequently, an FTIR-based partial least squares (PLS) regression model was established to predict the collagen content in EPH samples from poultry by-products. The performance of the model calibrated using a classical colorimetric method (3) was compared with that of the model calibrated using the 1H-qNMR method established.

NMRium1 is an innovative, web-based platform designed to support e-learning in NMR spectroscopy by enabling the creation of custom exercise series for students without requiring software installation. This unique feature allows educators to build exercises using GitHub, providing a seamless experience for both instructors and learners. The platform’s educational capabilities include the ability to offer hints during exercises, guiding students with prompts such as “Have you considered a heteroaromatic ring?” or “What about a tertiary amine?” These hints encourage critical thinking without directly giving away the correct answers.

Additionally, NMRium allows for the simulation and prediction of NMR spectra, enabling students to explore and analyze molecular structures interactively. This combination of flexibility, accessibility, and interactive features makes NMRium an ideal tool for teaching and learning NMR spectroscopy concepts in an online environment2.

Phosphatidylserine (PS) is a phospholipid essential for cell membrane composition and with high concentrations in the brain. It has a key role in neurotransmission, synapse formation, membrane signaling pathways, and neuroinflammation (1). Due to its neuroprotective effect (2), PS supplementation has shown benefits to patients with CNS disorders, which explains the wide use of PS as a dietary supplement.

This work aimed to develop a qNMR method to overcome the lack of analytical compendial methods for this widely used dietary supplement. The qNMR method was inspired by the krill oil monograph, using quantitative 31P-NMR with triphenyl phosphate (TPP) as an internal calibrant (IC). Our findings show that sample preparation is a critical step for successful qNMR analysis: the addition of EDTA 0.2 M (pH 7.2-7.5, Cs2CO3) (3) enhances the lineshape and thus, the resolution of the spectrum by 3.5-fold. The parameters of the acquisition were set as acquisition time (AQ) 3-5 s and relaxation delay (RD) 12-10 s. The molecular weight used for PS quantification is suggested to be based on the free acid form of C17 (C16 and C18 average) acyl chains. As post-acquisition parameters, zero filling to 256k and polynomial baseline correction were used.

Two different PS preparations (labeled 50% and 70% PS) from two different vendors were analyzed with the new method. Slightly different quantitative outcomes (2% differences) will be achieved depending on the processing of the spectra and the criteria used for integration. This could potentially interfere with the exact requirements set forth by pharmacopeial monographs and/or manufacturers. For quantitation, the choices of integration limits and the use of window functions/parameters become part of the convention method. A wider integration range yields a higher percentage of PS and aligns the results more closely with the declared content. A deconvolution approach was found to be a viable means of reducing operator bias, especially in integration. This approach is feasible due to the essential near-singlet nature of the peak patterns. However, all peak patterns still need to be assigned, and molecular weights must be known (or assigned by convention) to obtain reproducible results.

Quantitative NMR (qNMR) has become a key tool in the Pharmaceutical and Chemical Industries for quickly quantifying organic molecules, offering the advantage of estimating purity without specific reference standards. Traditionally, qNMR uses internal or external reference standards. Internal standards, added directly to samples, allow for quick comparisons but require careful selection to avoid interference. External standards, measured separately, extend measurement times.

A novel approach involves digital Reference Materials (dRMs), which eliminate the need for physical standards. By comparing experimental data with a digitally stored reference spectrum of a certified compound, dRMs enable rapid quantification. Calibration of the instrument is necessary for determining instrument sensitivity, requiring six independent measurements and providing one-month validity.

This digital method streamlines quantification, reduces physical handling of standards, and minimizes contamination risks, enhancing consistency and reproducibility in high-through put settings. ChemisTwin offers a sustainable, efficient, and error-proof alternative to traditional methods, and we will present a case study demonstrating its benefits in quickly quantifying organic compounds via 1H NMR spectroscopy.

Coaxial tubes for NMR allow solution NMR by combining two tubes filled with different solutions. As a normal tube, the coaxial tube allows two solutions to be analyzed on a single spectrum. We reported to establish the coaxial-tube qNMR and have similar accuracy to conventional qNMR using various coaxial tubes.1 However, the coaxial-tube qNMR showed a varied quantitative values depending on the tube types. We hypothesized that the cause was the attenuation of excitation efficiency due to pulses in the coaxial tube. Here, we evaluated the following contents in coaxial tube qNMR using each solution of the standard (DSS) and analyte (maleic acid, MA), and these mixtures (A) qNMR comparison of normal and coaxial tubes with the mixture, (B) evaluation of the relationship between peak integral and coaxial tube types by solution-filling position.

The peak areas per 1H number of DSS and MA in the mixture were highly correlated for each mixing ratio. The correlation between both peak areas for coaxial tube qNMR filled with different solutions in inner and outer tubes was lower than mixture qNMR (Fig. 1). The peak area per 1H number relative to the amount of substance was higher in coaxial tube qNMR than in the conventional qNMR. Although the peak area per the product of 1H number and solution volume was constant regardless of the diameter of the coaxial tube, the area was lower for the normal tube than for the coaxial tube (Fig. 2). Those results indicate that the sensitivity to signal detection varies between normal and coaxial tubes.

Quantitative analysis of complex mixtures remains a significant challenge in herbal medicines and biological samples, where numerous structurally analogous compounds are often present (1). The utility of proton nuclear magnetic resonance (1H NMR) in such contexts is limited due to severe signal overlap, which compromises its effectiveness in quantification (2). In contrast, carbon nuclear magnetic resonance (13C NMR) offers superior resolution owing to its broader chemical shift range and ability to detect quaternary carbons, making it a promising tool for analyzing complex mixtures. However, practical limitations, such as long acquisition times and high sample requirements, have restricted its widespread application. In this study, we evaluated the resolving power of 13C NMR for the analysis of saponins. Through complete spectral assignment of 12 common saponins, we identified distinctive carbon signals that enabled the quantification of six saponins in a crude extract of Panax notoginseng, following the exclusion of low-abundance components. To address inherent challenges in 13C NMR, namely sensitivity and time efficiency, we systematically optimized key parameters, including sample concentration, number of scans, and relaxation reagent concentrations. As a result, we established a 150-minute quantitative 13C NMR (13C qNMR) method that balances signal-to-noise ratio (SNR) with experimental throughput. This method demonstrates the potential of 13C qNMR for rapid and reliable quantification of structurally analogous compounds in complex mixtures.

Methacrylate-based dental resins, particularly those composed of bis-GMA and TEGDMA are essential in dentistry.1 It is perceivable that the use of proanthocyanidins (PACs) as dentin biomodifiers and primers for dental restoration influence resin polymerization, crosslinking, and resin leaching properties.2 Understanding how PACs affect these processes is critical for developing more durable, biocompatible dental materials. The ECIC qHNMR method,3 provides an orthogonal and non-destructive approach to investigate the molecular interactions between PACs and the methacrylate network, allowing for precise analysis of polymer network formation, conversion efficiency, and residual monomer content.

This study employed qNMR to systematically evaluate the effects of PACs incorporation on resin structure and performance. The methodology involved preparing a model experimental adhesive, followed by the addition of PACs at a concentration of 1%. Polymer discs (4.8 mm × 1.8 mm) were fabricated by irradiating the resin mixtures. To extract residual monomers, the resin discs were pulverized after 24 hours of polymerization, and each sample (10 mg) was immersed in 5 mL of 75% EtOH containing 0.01% BHT for five days at room temperature. A detailed ¹H qNMR analysis was conducted in CDCl₃, using dimethyl fumarate as an external calibrant to quantify unreacted monomers and extractable fractions. This approach enables the assessment of double bond conversion and crosslinking density, both critical factors in understanding the overall properties of the resin. To ensure the accuracy and reliability of our qNMR analysis, external calibration used the bis-GMA (6.80 ppm) and TEGDMA (3.70 ppm) peak patterns, enabling highly precise quantification of these components at varying concentrations. Using 1, 3, 5, and 10 µg in 200 µL with a 3 mm NMR tube, we demonstrated that 3 µg (15 µg/mL) could be reliably detected on our JEOL 600 MHz NMR (Royal RT probe). This ability to detect such low concentrations of resin monomers makes qNMR a high sensitivity tool for evaluating the efficacy of dental resin formulations.

qNMR serves as an essential analytical tool in this study, providing precise quantification of residual monomers, evaluating crosslinking efficiency, and offering molecular-level insights into PACs-induced changes in methacrylate resins. The findings demonstrate that PACs reduce the extraction rates of bis-GMA and TEGDMA, indicating enhanced polymer stability. Furthermore, these findings provide insight into how PACs contribute to enhancement of adhesive performance and durability at the adhesive-dentin interface. By enabling accurate and reproducible analysis, qNMR plays a critical role in optimizing PACs-modified dental adhesives for better durability and biocompatibility, paving the way for advancements in dentistry.