Quantification of SARS-CoV-2 monoclonal IgG mass fraction by isotope dilution mass spectrometry.

The availability of serology assays to measure antibodies against the SARS coronavirus 2 (SARS-CoV-2) expanded rapidly during the Covid-19 pandemic. The interchangeable use of such assays to monitor disease progression and immune protection requires their standardization, for which suitably characterized monoclonal antibody materials can be useful. The methods, based on isotope dilution mass spectrometry, to value assign the mass fraction of such a material in solution within the context of an international interlaboratory comparison study (CCQM-P216) are described. The mass fraction in solution of a humanized IgG monoclonal antibody (mAb) against the SARS-CoV-2 Spike glycoprotein in the study sample has been value assigned through a combination of liquid chromatography, isotope dilution mass spectrometry (LC-ID-MS) methods and size exclusion chromatography with UV detection (SEC-UV). The former were developed for the quantification of amino acids and proteotypic peptides as surrogate analytes of the mAb while the latter was applied for the determination of the relative monomeric mass fraction. High-resolution mass spectrometry (hrMS) allowed the molecular weight evaluation and ruled out the presence of significant impurities. Method trueness was assessed using a subclass homologous IgG1 material value assigned by amino acid analysis. The assigned mass fraction of monomeric SARS-CoV-2 IgG in solution was 390 ± 16 mg/g. The associated expanded uncertainty originated mainly from acid hydrolysis variability and Trypsin/Lys-C digestion variability and efficiency.

Accurate quantification of impurities in pure peptide material - angiotensin I: Comparison of calibration requirements and method performance characteristics of liquid chromatography coupled to hybrid tandem mass spectrometry and linear ion trap high-resolution mass spectrometry.

RATIONALE: The prohormone angiotensin I (ANG I) [amino acid sequence: DRVYIHPFHL] and other structurally related peptide hormones play an essential role in the regulation of the water and electrolyte balance in the human body as well as blood pressure. ANG I is a biomarker for hypertension and diabetes. Therefore, well-characterized pure reference materials and comparable and SI-traceable analytical characterization methods are required to establish reference measurement systems (RMS) for laboratory medicine. METHODS: Two analytical characterization methods based on liquid chromatography/mass spectrometry (LC/MS) systems with electrospray ionization have been developed and validated in-house. Both high-resolution MS (hrMS) and hybrid-tandem MS/MS were used for the identification and quantification of the major structurally related peptide impurities of ANG I. The impurities were quantified by use of external calibrations with original impurity standards. Mass fraction impurity values and corresponding expanded measurement uncertainties were calculated. RESULTS: Five structurally related degradation products were detected as major impurities in a 'pure' ANG I material. The peptides ANG (2-10) [RVYIHPFHL], ANG II [DRVYIHPF] and three ANG I isomers [DRVYLHPFHL, DRVYIHPFHI and DRVYLHPFHI] were identified and corresponding mass fraction values calculated that range from 0.66 to 4.86 mg/g. CONCLUSIONS: The mass fraction values for the major related peptide impurities in the ANG I material obtained with both LC/hrMS and LC/MS/MS systems are in excellent agreement. This study emphasizes the importance of mass spectrometric techniques for application to mass balance approaches for mass fraction value and uncertainty assignment of impurities in 'pure' substance reference materials for peptides.

Impurity identification and determination for the peptide hormone angiotensin I by liquid chromatography-high-resolution tandem mass spectrometry and the metrological impact on value assignments by amino acid analysis.

It is common practice to quantify the mass concentration of a peptide solution through quantitative determination of selected chemically stable amino acids produced following complete hydrolysis of the parent peptide. This is because there is generally an insufficient quantity of material available to allow for the obvious alternative of a direct purity analysis characterization of the parent peptide, and the subsequent constitution of a calibration solution. However, selected accurately characterized pure peptide reference materials are required to establish reference points for the dissemination of metrologically traceable measurements and to develop reference measurement systems for laboratory medicine. In principle, purity assignment of a peptide can be performed by using the so-called mass balance approach, by employing a range of analytical techniques to obtain an estimate of the mass fraction content of all impurities present in the intact peptide, and by utilizing the difference from the theoretical limit value to assign the mass fraction content of the main peptide. Liquid chromatography-high-resolution tandem mass spectrometry (LC-hrMS/MS) is a key technique for the detection, identification, and determination of structurally related impurities present in a peptide material, and experiments characterizing the model peptide hormone angiotensin I (ANG I) are described in the present work. Degradation products that were generated from ANG I after storage at elevated temperatures were screened. The formation of peptide fragments such as ANG II or ANG III was determined by comparison of measured mass values with calculated mass values. The use of a data-dependent acquisition technique enabled the detection and structural characterization of ANG II and other peptide fragments as major impurities in the same LC-hrMS/MS analysis run. Subsequent quantification using external calibration allowed the mass fraction of the major impurities in a candidate reference material to be estimated as 10.4 mg/g. Failure to correct for these impurities would lead to a 1% error in the determination of the concentration of the peptide in solution by amino acid analysis techniques.

Aflatoxin M1 in milk powders: processing, homogeneity and stability testing of certified reference materials.

As part of the certification campaign of three candidate reference materials for the determination of aflatoxin M1 (AfM1) in whole milk powders, homogeneity, short- and long-term stability tests of naturally contaminated milk powders have been performed. The homogeneity of two AfM1-contaminated milk powders was studied by taking samples at regular intervals of the filling sequences and analysing in triplicate for their AfM1 contents by liquid chromatography with fluorescence detection (LC-FLD) using random stratified sampling schemes. The homogeneity testing of an AfM1 'blank' milk powder material was performed by determining the nitrogen content because AfM1 levels were below the limit of detection of the most sensitive determination method. The short-term stability of AfM1-contaminated milk powders was evaluated at three different storage temperatures (4, 18 and 40 degrees C). After storage times of 0, 1, 2 and 4 weeks, samples were investigated using LC-FLD. The long-term stability study comprised of measurements after 0, 6, 12 and 18 months after storage at -20 and 4 degrees C. Analyses were done by LC-FLD. Based on the homogeneity tests, the materials were sufficiently homogenous to serve as certified reference materials. Corresponding uncertainty contributions of 0.23-0.89% were calculated for the homogeneity. The stability measurements showed no significant trends for both short- and long-term stability studies. The long-term stability uncertainties of the AfM1-contaminated milk powders were 7.4 and 6.3%, respectively, for a shelf-life of 6 years and storage at -20 degrees C. Supplementary stability monitoring schemes over a long period of several years are currently ongoing.

Processing and purity assessment of standards for the analysis of type-B trichothecene mycotoxins.

The lack of reliable, certified calibrant solutions for the Fusarium mycotoxins deoxynivalenol (DON), 3-acetyl-DON (3-Ac-DON), 15-acetyl-DON (15-Ac-DON) and nivalenol (NIV) is a serious drawback in the already problematic area of trichothecene analysis. For this reason, purified DON, 3-Ac-DON, 15-Ac-DON and NIV standards were processed, the conditions required for their isolation and purification were optimised, and the crystalline toxins were thoroughly characterised. Several complimentary analytical methods were used to evaluate the identities of the mycotoxins and the types and amounts of impurities; results obtained from 1H and 13C NMR spectra, as well as from IR-spectra, were in agreement with the literature. Elemental analysis revealed that the isolated NIV occurs as monohydrate. If this is not known it results in a weighing error of approximately 5%. Differential scanning calorimetry (DSC) was only successful for 15-Ac-DON, as the other trichothecenes decomposed during measurements. No traces of chloride, nitrate and sulphate were found by means of ion chromatography (IC). As expected UV absorption spectra for DON, NIV, 3-Ac-DON and 15-Ac-DON yielded lambda(max) values of 216, 217, 217 and 219 nm, respectively. Minor peaks due to impurities were observed by high performance liquid chromatography (HPLC) with UV detection. The main impurity peak in the DON sample was identified by LC-tandem mass spectroscopy (LC-MS/MS) as 4,7-dideoxy-NIV (7-deoxy-DON), which occurs at levels of approximately 1.4%. Gas chromatography (GC) was performed, coupled with either an electron capture detector (ECD), a flame ionisation detector (FID), or a mass spectrometric detector (MS); however, derivatisation prior to GC analysis makes the estimation of impurities difficult. LC-MS/MS was found to be unsuitable for quantifying levels of impurities. It can be concluded that high-purity (>97%) B-trichothecene standards were successfully processed and fully characterised for the first time.

DON calibrant: Towards the production of certified B-trichothecene calibrants.

Within an EC-funded project calibrants with certified concentrations of Deoxynivalenol (DON), 3-Acetyl-Deoxynivalenol (3-Ac-DON), 15-Acetyl-Deoxynivalenol (15-Ac-DON) and Nivalenol (NIV) in acetonitrile have been produced. So far the project has led to improved isolation and purification of the solid toxins fromFusarium cultures. In addition, conditions for the production, ampouling and transport of the toxin solutions have been optimised. Further investigations should lead to knowledge about storage conditions and internationally accepted molar absorption coefficients for DON, 3-Ac-DON, 15-Ac-DON and NIV in acetonitrile. The intercomparison study which is currently carried out will also help to support knowledge and experience exchange between laboratories in the field ofFusarium mycotoxin analysis.

Type-B trichothecene calibrants: Comparison of HPLC and GC-results within an intercomparison study.

Within the EC-financed project "Feasibility Study for the Production of Certified Calibrants for the Determination of Deoxynivalenol and other B-Trichothecenes", an intercomparison study was performed with 13 European participants.Main goals of the intercomparison study were to check the feasibility of a small batch of gravimetrically prepared calibrants, to directly compare common and individually prepared calibrants, to test the practicability of toxin mixtures as calibrant solutions and finally to give recommendations for the means of certification. Additionally, it focused on the comparison of gas chromatography (GC) and high performance liquid chromatography (HPLC) for the determination of pure type-B trichothecene solutions, which is described in this publication.The participating laboratories received calibrant solutions as well as toxin solutions of unknown concentration and employed mainly HPLC-UV; GC-ECD (electron capture detection) and GC-MS (mass spectrometry) methods were used less often.The intercomparison study generally suffered from a high rate of outliers (22% of all the data). Throughout the study, 48% of all GC results were classified as outliers and it soon became apparent, that GC results highly infuenced the outcome of the study and that the used GC methods were not robust enough for the certification of type-B trichothecene calibrants. The high discrepancy between HPLC and GC results in the intercomparison study presumably lies in the crucial step of derivatisation.

Trichothecenes: reference materials and method validation.

The frequent contamination of food and feed with trichothecene mycotoxins, the high consumption of these products, and the potential risk associated herewith, has led to an increasing public awareness and therefore to the establishment of measures to control trichothecene contamination. The analytical difficulty and the economic importance of controlling trichothecenes in food and feed support the need for certified reference materials (CRMs) and validated methods. They form invaluable tools to ensure comparability and traceability in analytical measurements and are very useful for the implementation of written standards, legislation/regulations and laboratory accreditation. The present paper provides an overview of previous work, current strategies and prospectives for the production of CRMs and validation of analytical methods in the field of trichothecene analysis. Additional information is given on methodological demands, normative frameworks and commonly accepted procedures.

Development of certified calibrants for the analysis of zearalenone and B-trichothecenes.

Recent intercomparison studies have clearly shown that the use of common calibrants does considerably improve the between laboratory comparability of measurement results. However, there is still a lack of certified calibrants within the field of mycotoxin analysis. Therefore, two EC-funded projects have been carried out with the aim to investigate the feasibility for the production of calibrants with certified concentrations of B-trichothecenes and for the production of a certified calibrant of zearalenone in acetonitrile.

Zearalenone in maize: stability testing and matrix characterisation of a certified reference material.

Within the certification process of a reference material for the determination of the mycotoxin zearalenone (ZON) in maize, short- and long-time stability tests of naturally contaminated maize have been performed. The short-term stability of ZON in the maize was evaluated under four different conditions (4, 25, 40 and 70 degrees C) in preliminary studies. Four storage times of 0, 1, 2 and 4 weeks were investigated using HPLC. The long-term stability study was conducted with measurements after 0, 3, 6, 12, 24 and 36 months under three storage conditions (4, 25 and 40 degrees C) in preliminary studies using HPLC. Stability data gained under two different conditions (4 and 25 degrees C) with five storage times of 0, 1, 6, 12 and 18 months were further evaluated for the contaminated maize in the certification process. Before the certification, the maize matrix had been characterized with respect to dry residue, ash content, fat content, protein content, ergosterol content and total dietary fibre, and the efficiency of gamma-irradiation on the fungal flora was investigated. The stability of the maize matrix was evaluated by monitoring UV absorption and ergosterol content under four different storage conditions (4, 25, 35 and 70 degrees C) with five storage times of 0, 1, 6, 12 and 24 months. Other possibly occurring mycotoxins (deoxynivalenol, nivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, fusarenon X and moniliformin) have been quantified. On the basis of the stability measurements, which showed no significant trends for both short- and long-term stabilities, it can be recommended to store the samples at temperatures < 4 degrees C and ship the samples at ambient temperatures.

International interlaboratory study for the determination of the Fusarium mycotoxins zearalenone and deoxynivalenol in agricultural commodities.

Twenty-eight laboratories from 12 different countries participated in an interlaboratory study for the determination of the Fusarium mycotoxin zearalenone (ZON) in maize and deoxynivalenol (DON) in maize and wheat employing their usual in-house methods. The aim of this study was to obtain information about the state-of-the-art of ZON and DON analysis in cereals and to support a knowledge and experience exchange between the participating laboratories in the field of mycotoxin analysis. Eight different sample types were distributed to the participants, 'blank' materials, spiked samples (102 microg/kg ZON in maize and 475 microg/kg DON in wheat) and naturally-contaminated maize and wheat. For the final separation and quantification either gas chromatography (GC), high performance liquid chromatography (HPLC), thin layer chromatography (TLC) or enzyme linked immunosorbent assays (ELISA) were employed by the participating laboratories. Coefficients of variation (CV) between laboratory mean results (outliers rejected) ranged from 28 to 41% for ZON and from 32 to 38% for DON. The results are close to the between laboratory CV criteria of 40% for DON and ZON at concentration levels of >100 microg/kg established by the CEN in 1999. A good trueness was obtained for the wheat samples spiked at 475 microg/kg DON. However, a significant deviation at p = 0.01 from the respective target value was observed for the maize samples spiked at 102 microg/kg ZON. The high CVs can be traced back to problems occurring by determination of the concentration of the participants' own calibrant solutions. Additionally, the variability of the results is strongly influenced by the use of different final separation and quantification procedures.

Determination of trichothecene mycotoxins in wheat by use of supercritical fluid extraction and high-performance liquid chromatography with diode array detection or gas chromatography with electron capture detection.

The extraction behaviour of the Fusarium mycotoxin deoxynivalenol (DON) and some related type B trichothecenes from spiked seasand, spiked wheat flour and naturally contaminated wheat flour with modified supercritical CO2 has been investigated and optimized under several conditions. The extraction fluid was decompressed over a solid-phase trap and the amount of deposited analytes was determined by HPLC-diode array detection (DAD) or GC-electron capture detection (ECD) without any further clean-up. Recovery rates as high as 90.1 +/- 10.7% were achieved for spiked wheat samples and 53.0 +/- 3.2% for naturally contaminated samples. The performance of the optimized supercritical fluid extraction (SFE) method was compared with an already well established analytical method employing extraction on a rotary shaker in combination with Mycosep clean-up. Moreover, the SFE procedure developed for naturally DON contaminated wheat was employed for the simultaneous extraction of 5 type B trichothecenes by GC-ECD. This work represents the first successful approach in obtaining an SFE-method for the extraction of Fusarium mycotoxins from wheat with reasonable recoveries and good precision.