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

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

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

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

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

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

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

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

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

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

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

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

Normal phase-liquid chromatography-tandem mass spectrometry with atmospheric pressure photoionization for the purity assessment of 17ß-estradiol.

A normal phase-liquid chromatography-hybrid tandem mass spectrometry (NP-LC-MS/MS) method utilizing atmospheric pressure photoionization (APPI) without dopant has been developed and implemented for the simultaneous determination of several estrogenic steroid hormones. The combination of both NP-LC and APPI-MS/MS tolerates the use of solvents that have the advantages of being self-doping for APPI and, at the same time, inhibit the in situ formation of estrogen dimers as frequently observed for conventional reversed phase (RP)-LC methods. The NP-LC-APPI-MS/MS method has been validated in-house, and its performance characteristics (linearity, repeatability, limits of detection, etc.) were assessed for use in the quantification of estrogens. Moreover, the method was used to characterize and determine the inherent related structure impurities in batches of ß-estradiol, required for the establishment of reference measurement systems for clinical chemistry and laboratory medicine, which served as candidate reference material for an organic purity assessment interlaboratory study (CCQM-K55.a) organized by the International Bureau of Weights and Measures (BIPM) Chemistry Department and carried out within the framework of the Organic Analysis Working Group (OAWG) of the Consultative Committee for Amount of Substance-Metrology in Chemistry (CCQM).

Mass balance method for the SI value assignment of the purity of organic compounds.

A mass balance method is described for determining the mass fraction of the main component of a high purity organic material. The resulting assigned value is established to be traceable to the SI and can be determined with a small associated measurement uncertainty. Pure organic materials with values and uncertainties determined in this way are necessary as primary calibrators of reference measurement systems in order to underpin the metrological traceability of routine measurement results. The method has been applied to materials in which the main components were respectively theophylline, digoxin, 17ß-estradiol, and aldrin. Its performance has been validated in international comparisons coordinated by the BIPM and is in principle applicable to a wide structural range of stable, nonvolatile organic compounds. It has been successfully applied to mass fraction assignments when the main component is present in the range of (950-1000) mg/g and can achieve associated standard uncertainties ranging from 0.5 mg/g (for high purity materials or those containing well-characterized, stable minor components) to 2 mg/g (materials with a significant number or variety of impurities). It is in principle equally applicable to materials with a smaller mass fraction content of the main component.

Simultaneous determination of various cardiac glycosides by liquid chromatography-hybrid mass spectrometry for the purity assessment of the therapeutic monitored drug digoxin.

A high performance liquid chromatography-hybrid tandem mass spectrometry (LC-MS(n)) method utilising electrospray ionisation has been developed and implemented for the simultaneous determination of several cardiac glycosides (CGs) as well as their corresponding aglycones formed by and extracted from herbaceous plants of the genus Digitalis. The method has been validated in-house and its performance characteristics (linearity, repeatability, limits of detection, etc.) were assessed for use in the quantification of CGs and their corresponding aglycones. LODs from 38 to 936pgg(-1) in solution, corresponding to mass fraction impurity levels from 0.0009 (or 0.00008%) to 0.019mgg(-1) (or 0.0019%) detectable in the pure materials have been realized. Moreover, the method was used to characterize and to determine the inherent CG impurities in batches of the therapeutic monitored drug digoxin which served as candidate reference material for an organic purity assessment inter-laboratory study (CCQM-P20.f) organised by the BIPM Chemistry Section and carried out within the framework of the Organic Analysis Working Group (OAWG) of the Consultative Committee for Amount of Substance - Metrology in Chemistry (CCQM). Digoxin was selected from materials required for the establishment of reference measurement systems for clinical chemistry and laboratory medicine.

Determination of molar absorptivity coefficients for major type-B trichothecenes and certification of calibrators for deoxynivalenol and nivalenol.

This paper presents results from the European Commission-funded project Doncalibrant, the objective of which was to produce calibrators with certified mass fractions of the Fusarium toxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-Ac-DON), 15-acetyldeoxynivalenol (15-Ac-DON), and nivalenol (NIV), in acetonitrile. The calibrators, available in ampoules, were sufficiently homogeneous, with between-bottle variations (s (bb)) of less than 2%. Long-term stability studies performed at four different temperatures between -18 and 40 degrees C revealed no significant negative trends (at a confidence level of 95%). Molar absorptivity coefficients (in L mol(-1) cm(-1)) were determined for all four toxins (DON: 6805 +/- 126, NIV: 6955 +/- 205, 3-Ac-DON: 6983 +/- 141, 15-Ac-DON: 6935 +/- 142) on the basis of a mini-interlaboratory exercise. The overall uncertainty of the calibrators' target values for DON and NIV were evaluated on the basis of gravimetric preparation data and include uncertainty contributions from possible heterogeneity, storage, and transport. The Doncalibrant project resulted in the production of calibrators for DON (IRMM-315) and NIV (IRMM-316) in acetonitrile with certified mass fractions of 25.1 +/- 1.2 microg g(-1) and 24.0 +/- 1.1 microg g(-1), respectively. Both CRMs became commercially available from the Institute for Reference Materials and Measurements (IRMM, Geel, Belgium) at the beginning of 2007.

Feasibility study for the production of certified calibrants for the determination of deoxynivalenol and other B-trichothecenes: intercomparison study.

Thirteen European laboratories experienced in the analysis of mycotoxins participated in an intercomparison study within a European Commission-funded project. Goals of the study were to check the fitness for purpose of a small batch of gravimetrically prepared calibrants; to compare individually prepared calibrants with common calibrants; to check the feasibility of toxin mixtures as calibrant solutions; and to give recommendations on the production of future certified reference materials (CRMs) with regard to the nature of the calibrant and the means of certification. Each laboratory received ampules of each common calibrant containing single toxins [solution containing either deoxynivalenol (DON), 3-acetyl-DON (3-Ac-DON), nivalenol (NIV), or 15-acetyl-DON (15-Ac-DON)] and 3 ampules of toxin-mixture (solutions of DON + 3-Ac-DON + NIV in acetonitrile) of known concentrations (about 20 microg/mL). Ampules with single toxins (solution containing either DON, 3-Ac-DON, NIV, or 15-Ac-DON) and a toxin-mixture (solutions of DON + 3-Ac-DON + NIV in acetonitrile) of unknown concentrations were distributed to the participants for quantification. The participating laboratories used mainly high-performance liquid chromatography (HPLC)-diode array detection UV for DON, 3-Ac-DON, NIV, and 15-Ac-DON; gas chromatography-electron capture detection (GC-ECD) and GC-mass spectrometry methods were used sparingly. Linear calibration curves were achieved by >90% of the participants. Relative between-day variation (RSDr) of 26% of the laboratories was greater than the target value of 5% for HPLC, and RSDr of 32% of the laboratories was greater than the desired value of 10% for GC. Relative between-laboratory variation (RSDR) of the GC results obtained with single common calibrants was greater than the target value of 16% for all laboratories. RSDR of the HPLC results for the common unknown single toxin solutions was less than the target value of 8% except for 15-Ac-DON. Generally, better recoveries were observed from common calibrants (102% for mix calibrants and 98% for single calibrants) than from individually prepared calibrants (95%). This international comparison study clearly showed the high scattering of results in the analysis of type-B trichothecenes, particularly when GC was used. Obviously, this intercomparison study was not suited for the certification of B-trichothecenes. A certification of the proposed calibrant material was therefore recommended on the basis of its gravimetrical preparation.

Preparation and certification of zearalenone mass concentration of two low-level maize reference materials.

The contamination of maize by fungi, especially by Fusarium species, is a worldwide problem. One of the most prevalent Fusarium mycotoxins frequently found on European maize is zearalenone (ZON), which has been implicated in a range of human and animal diseases. It shows remarkable estrogenic properties and can cause severe infertility problems in farm animals. Currently, 9 countries have set maximum tolerable levels for ZON in food, ranging from 0 to 1000 microg/kg. This paper describes the preparation of 2 maize reference materials (BCR-716 very low level ZON and BCR-717 low level ZON) and the certification of their individual ZON contents (mass concentration and mass fraction). Uncertainties were calculated in compliance with the Guide to the Expression of Uncertainty in Measurement and include uncertainties that are due to possible inhomogeneity and instability. Finally, BCR-716 was certified at a level of <5 microg/kg and BCR-717 at a level of 83 microg/kg with an expanded uncertainty (k = 2) of 9 microg/kg.

Production of certified reference materials for mycotoxins: IRMM's view on the assessment of uncertainties.

Analytical difficulty and the economic importance of controlling mycotoxin levels in food and feed led the Community Bureau of Reference (BCR) to prepare a series of certified reference materials (CRM) for various mycotoxins. Because of the wide acceptance of these CRM and the need to ensure the comparability and traceability of measurements in the future it is necessary to prepare and certify new batches of mycotoxin reference materials (RM). In the following text two different approaches for evaluation of the characterisation uncertainty of CRM will be compared using the certification of aflatoxin M(1) (AfM(1)) in milk powder as an example. The conventional approach is based on evaluation of characterisation exercise data; the alternative approach is based on measurement uncertainties of the employed analytical methods. Because laboratories are using totally different approaches to estimate the measurement uncertainties, combination of the uncertainties obtained from the participating laboratories was not recommended. Therefore, a new integrated approach for assessment of the measurement uncertainties of the analytical methods on the basis of additional data collected during the characterisation exercise will be described. The conventional approach was found to be the most appropriate and economical approach to evaluate the characterisation uncertainty as a characterisation exercise must be performed anyway to establish the property values of candidate (C)RM, irrespective of whether or not reliable measurement uncertainties can be provided by the laboratories. An integrated approach for assessment of measurement uncertainties based on additional characterisation data as applied here to enable use of an uncertainty-based approach provides more information but is too time-consuming and cost-intensive to become common practice.

Production of a calibrant certified reference material for determination of the estrogenic mycotoxin zearalenone.

Several previous interlaboratory studies in the field of mycotoxin analysis have revealed considerable problems, apparent as high between-laboratory standard deviations, or rather non-comparable and non-traceable results. A major reason is lack of proper calibrants for external calibration. Public awareness of substances that mimic or interfere with the activity of natural hormones (endocrine disrupters) has led to increased interest in mycotoxins with estrogenic potential, e.g. zearalenone (ZON). During a large-scale standard measurement and testing (SMT) project of the European Commission (EC) dealing with the preparation and certification of reference materials for determination of the mycotoxin ZON in maize, a ZON calibrant in acetonitrile was prepared and intensively checked for purity, homogeneity, and stability. Preparation of the material, study of its homogeneity and stability, and characterisation of the calibrant on the basis of its preparation, with discussion of the results obtained, are described in this paper. The certified value of 9.95 micro g mL(-1) for ZON in acetonitrile and its corresponding expanded uncertainty of +/-0.30 micro g mL(-1) were calculated in compliance with the Guide to the Expression of Uncertainty in Measurement (GUM).

Purity assessment of crystalline zearalenone.

Commercially available solid zearalenone (ZON) to be used as a certified liquid calibrant (BCR-699) in a project funded by the European Commission within the Standard Measurement and Testing program was characterized and its purity determined. The degree of purity of the ZON was examined by UV spectrophotometer, liquid chromatography (LC) with diode array and fluorescence detection, 1H and 13C-NMR spectrometry, LC-mass spectrometry (LC/MS/MS), ion chromatography (IC), and differential scanning calorimetry (DSC). The diagrams obtained from DSC analysis and the UV spectrum showed no detectable impurities. Likewise, no impurities were observed by LC analysis with both diode array and fluorescence detection. IC determination revealed negligible contamination of ZON with chloride of 0.020 +/- 0.005% and nitrate of 0.016 +/- 0.006%. Zearalanone (ZAN) was identified as one of 2 minor (0.2%) impurities by LC/MS/MS. The 1H-NMR measurements revealed an additional peak, which has not been previously reported in the literature. It could be identified as part of the ZON spectrum as the signal arising from the phenolic proton attached to C4'. The manufacturer states an additional contamination with 0.2% methylene chloride, which could be confirmed to an extent of 0.1% by 1H-NMR. Minor impurities, whose structures remain unknown, were discovered at 3.5 and < 1 ppm. Total percentage of impurities based on NMR measurement was estimated not to exceed 1%. A purity of 99.5% with a tolerance of +/- 0.5% was finally attributed to the ZON studied in this project.

Preparation of a calibrant as certified reference material for determination of the Fusarium mycotoxin zearalenone.

In the field of mycotoxin analysis, substantial problems shown by high between-laboratory standard deviations and noncomparable and nontraceable results have been caused by the lack of proper calibrants for external calibration. During a large-scale Standard Measurement and Testing project of the European Commission (EC) dealing with preparation and certification of reference materials for determination of the mycotoxin zearalenone (ZON) in maize, a ZON calibrant in acetonitrile was prepared and checked for purity, homogeneity, and stability. Before certification, on the basis of preparation, the calibrant was checked in a mini-interlaboratory study by UV spectrophotometric determination. The molar absorptivities of ZON in acetonitrile at 236, 274, and 314 nm were established, and as a main result, a common reference wavelength of 274 nm with molar absorptivity of 12623 +/- 111 L/mol x cm can be recommended for ZON in acetonitrile. A concentration and expanded uncertainty of the ZON calibrant of 9.95 +/- 0.08 microg/mL was calculated as a preliminary value before final evaluation through the certification panel of the EC.