Chemische Charakterisierung und Spurenanalytik
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- 1.7 Organische Spuren- und Lebensmittelanalytik (124) (entfernen)
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As part of the "Journées de Chimie Analytique, JCA Dschang 2024", this plenary scientific conference will explore how analytical electrochemistry is fundamentally changing modern chemical analysis methods. We will highlight key innovations such as wearable/handheld sensors, lab-on-a-chip, lab-on-paper, lab-on-a-glove, integrated detection systems with artificial intelligence, and point-of-care (POC) technologies. We will also address the couplings between analytical electrochemistry and various analytical techniques such as mass spectrometry (EC-MS), chromatography (EC-LC), spectroscopy [EC-spectroscopy (UV-VIS, Raman, IR, NMR)], microscopy [EC-microscopy (EM, AFM)], and capillary electrophoresis (EC-CE). These advances enable faster, more accurate, and more accessible analyses, opening new perspectives for research and practical applications in environmental science, food and agriculture, public health, and advanced materials.
The electrochemical behavior of 4-phenylurazole (Ph-Ur) was studied and the latter was used as a molecular anchor for the electrochemical bioconjugation of tyrosine (Y). Cyclic voltammetry (CV) and controlled potential coulometry (CPC) allowed to generate in situ from phenylurazole the PTAD (4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione) species on demand for tyrosine electrolabeling. To evaluate the performance of tyrosine electrolabeling, coulometric analyses at controlled potentials were performed on solutions of phenylurazole and the phenylurazole-tyrosine mixture in different proportions (2:1, 1:1, and 1:2). The electrolysis of the phenylurazole-tyrosine mixture in the ratio (1:2) produced a charge of 2.07 C, very close to the theoretical value (1.93 C) with high reaction kinetics, a result obtained here for the first time. The products obtained were identified and characterized by liquid chromatography coupled to high-resolution electrospray ionization mass spectrometry (LC-HRMS and LC-HRMMS). Two products were formed from the click reactions, one of which was the majority. Another part of this work was to study the electrochemical degradation of the molecular anchor 4-phenylazole (Ph-Ur). Four stable degradation products of phenylurazole were identified (C7H9N2O, C6H8N, C6H8NO, C14H13N4O2) based on chromatographic profiles and mass spectrometry results. The charge generated during the electrolysis of phenylurazole (two-electron process) (2.85 C) is inconsistent with the theoretical or calculated charge (1.93 C), indicating that secondary/parasitic reactions occurred during the electrolysis of the latter. In conclusion, the electrochemically promoted click phenylurazole-tyrosine reactions give rise to click products with high reaction kinetics and yields in the (1:2) phenylurazole-tyrosine ratios, and the presence of side reactions is likely to affect the yield of the click phenylurazole-tyrosine reaction.
Ergot alkaloids, potent mycotoxins produced by Claviceps spp., particularly Claviceps purpurea, pose significant health risks when they contaminate rye and related cereals, leading to ergotism in humans and mammals [1]. In response, the European Union has established Regulation 2023/915, setting maximum residue levels for the sum of 12 principal ergot alkaloids and other mycotoxins including fumonisins (FUM), deoxynivalenol (DON), zearalenone (ZEN), and T2/HT2 toxins in food products. Given the prevalent co-occurrence of mycotoxins, their simultaneous detection is crucial for ensuring the safety of food and feed [2, 3].
Traditionally, chromatographic techniques such as liquid chromatography coupled with (tandem) mass spectrometry (LC-MS/MS) have been employed for multiplex detection of mycotoxins [4, 5]. While effective, these methods require specialized facilities, expensive equipment, and skilled personnel. Immunoassays like ELISA and lateral flow assays offer a more accessible alternative for rapid mycotoxin detection, yet they generally lack the capability for concurrent multi-toxin screening.
This study introduces the SAFIA (Suspension Array Fluorescence Immunoassay), a particle-based immunoassay utilizing fluorescence-encoded microparticles for the simultaneous detection of multiple analytes [6, 7]. The assay's innovative advancement comes with the inclusion of ergot alkaloids, a novel addition to its existing detection capabilities for fusarium toxins and trichothecenes, thereby expanding its scope to a broader range of mycotoxins. The assay employs antibodies targeting the ergoline moiety common to all major ergot alkaloids, facilitated by a synthesized hapten mimicking the ergoline structure. This hapten was conjugated to amino-functionalized beads, and a panel of five monoclonal antibodies was evaluated for hapten recognition, binding specificity, and competitive binding efficiency.
Our findings demonstrate that all antibodies displayed similar affinities towards the hapten and lysergol (a stable and less hazardous analogue of lysergic acid used for calibration), achieving detection limits as low as 2 ppb. Cross-reactivity studies and analysis of round-robin test material indicated a significant underestimation of ergot alkaloid levels in samples. However, accurate detection of ergot alkaloids remains feasible through the application of a correction factor to the results, which compensates for this underestimation and ensures the assay's effectiveness. Despite this adjustment, the necessity for enhancements in antibody specificity to improve assay accuracy is evident. Furthermore, the inclusion of the ergot assay in a multiplexed setup for detecting FUM, DON, ZEN, and T-2 toxins showed no interference, although an unexpected inhibition among four out of five ergot antibodies was observed. This underscores the need for an improved immunogen structure to achieve optimal detection of ergot alkaloids.
In conclusion, our study presents a promising approach for the multiplexed detection of ergot alkaloids alongside other mycotoxins, highlighting the potential of SAFIA in enhancing food and feed safety through improved mycotoxin screening. Future work will focus on refining antibody specificity and assay configurations to overcome current limitations and ensure accurate, comprehensive mycotoxin detection.
Currently, there is a growing interest in the study of environmental degradation pathways of organic contaminants such as pesticides, with the objective to better understand their potential risk for environmental systems and living organisms. In this context, DFT (conceptual density functional theory) and predictive methods may systematically be used to simplify and accelerate the elucidation of environmental degradation. We report herein the electrochemical behavior/degradation of the carbendazim (CBZ) fungicide widely used to treat cereal and fruit crops. Oxidative degradation of CBZ was studied using an electrochemical flow-through cell directly coupled to a mass spectrometer for rapid identification of CBZ degradation products. The structural elucidation of CBZ oxidation products was based on retention time, accurate mass, isotopic distribution and fragmentation pattern by using LC-HRMS an LC-HRMS2. The most important chemical reactions found to occur in the transformation of CBZ were hydrolysis and hydroxylation. EC-LC-MS and EC-MS analysis has made it possible to highlight the identification of degradation products of CBZ. In addition to previously known transformation products common to those observed during environmental degradation (monocarbomethoxyguanidine, benzimidazole-isocyanate, 2-aminobenzimidazole, hydroxy-2-aminobenzimidazole, hydroxycarbendazim, CBZ-CBZ dimer), two new degradation products were identified in this work: a quinone imine and a nitrenium ion. Electrochemistry mass spectrometry hyphenated techniques represent an accessible, rapid and reliable tool to elucidate the oxidative degradation of CBZ, including reactive degradation products and conjugates.
Die Gruppe der PFAS (Per- und polyfluorierte Alkylsubstanzen) mit mehr als 10.000 Substanzen stellt ein zunehmendes Problem bei der Bewertung und Entsorgung belasteter Abfälle dar. Die zuverlässige PFAS-Analytik stellt hier aufgrund der Vielzahl an Einzelparametern mit zum Teil sehr großen Konzentrationsunterschieden eine komplexe Herausforderung dar.
Der Vortrag gibt zunächst einen Überblick über grundlegende Anforderungen an die LC-MS/MS Analytik von PFAS-Targetsubstanzen. Am Beispiel eines von BAM und SenMVKU organisierten Ringversuches 2024 zur Bestimmung von PFAS in Bodenproben (Feststoffe und Eluate) wird auf spezifische Fragestellungen zur PFAS-Analytik eingegangen.
Zur Verbesserung von Richtigkeit und Vergleichbarkeit ist die Einhaltung von Normvorgaben und die Anwendung von Qualitätssicherungsmaßnahmen unerlässlich. Hierzu gehören u.a. die Verwendung zuverlässiger Kalibrierstandards, die Kalibrierung mit definierten n-Isomeren, die Quantifizierung der Summe aus n-/br-Isomeren, der Einsatz isotopenmarkierter interner Standards für die LC-MS/MS Analyse, die Verwendung von Matrix-Referenzmaterialien sowie die Teilnahme an Ringversuchen.
Non‐targeted screenings (NTS) are essential tools in different fields, such as forensics, health and environmental sciences. NTSs often employ mass spectrometry (MS) methods due to their high throughput and sensitivity in comparison to, for example, nuclear magnetic resonance–based methods. As the identification of mass spectral signals, called annotation, is labour intensive, it has been used for developing supporting tools based on machine learning (ML). However, both the diversity of mass spectral signals and the sheer quantity of different ML tools developed for compound annotation present a challenge for researchers in maintaining a comprehensive overview of the field.In this work, we illustrate which ML‐based methods are available for compound annotation in non‐targeted MS experiments and provide a nuanced comparison of the ML models used in MS data analysis, unravelling their unique features and performance metrics. Through this overview we support researchers to judiciously apply these tools in their daily research. This review also offers a detailed exploration of methods and datasets to show gaps in current methods, and promising target areas, offering a starting point for developers intending to improve existing methodologies.
We developed a two-step semi-synthesis for the preparation of isotopically labeled EAs, starting from native EAs. This universal strategy enabled the successful synthesis of all isotopically labeled priority EAs. The structure of the isotopically labeled EAs was confirmed by HPLC-HR-MS/MS using native, unlabeled EAs as a reference standard. The next step will be the implementation of the isotopically labeled standards in the European standard procedure EN 17425 to improve the quantification of EAs in foodstuffs.
A systematic series of industrial-relevant polystyrene-based anion exchange resins that are functionalized with hydro- or fluorocarbon chains are compared regarding their adsorption behavior toward perfluorocarboxylic acids (PFCA) in respect to their charge, chain length, and type of chain. The results clearly show the dominance of electrostatic interactions in the adsorption process as uncharged adsorber materials showed no adsorption at all. In contrast, the charged adsorber materials showed in general a PFCA removal of 80% to 30% over the experiment depending on effluent fraction.
Unexpectedly, for perfluorobutanoic acid (PFBA) the highest removal rate is found with consistently >90%. Despite observing significant benefits in the adsorption of PFCA for fluoroalkylated adsorbers in comparison to their non-fluorinated counterparts, this effect of fluoroalkylation is comparatively small and can not be clearly attributed to fluorophilic interactions between the fluoroalkyl chains. These findings help clarifying that the introduction of fluorocarbon moieties in adsorber materials is not necessary in order to remove fluorocarbon molecules from the environment.
Changes in Black Truffle (Tuber melanosporum) Aroma during Storage under Different Conditions
(2024)
The enticing aroma of truffles is a key factor for their culinary value. Although all truffle species tend to be pricy, the most intensely aromatic species are the most sought after. Research into the aroma of truffles encompasses various disciplines including chemistry, biology, and sensory science. This study focusses on the chemical composition of the aroma of black truffles (Tuber melanosporum) and the changes occurring under different storage conditions. For this, truffle samples were stored under different treatments, at different temperatures, and measured over a total storage time of 12 days. Measurements of the truffle aroma profiles were taken with SPME/GC–MS at regular intervals. To handle the ample data collected, a systematic approach utilizing multivariate data analysis techniques was taken. This approach led to a vast amount of data which we made publicly available for future exploration. Results reveal the complexity of aroma changes, with 695 compounds identified, highlighting the need for a comprehensive understanding. Principal component analyses offer initial insights into truffle composition, while individual compounds may serve as markers for age (formic acid, 1-methylpropyl ester), freshness (2-Methyl-1-propanal; 1-(methylthio)-propane), freezing (tetrahydrofuran), salt treatment (1-chloropentane), or heat exposure (4-hydroxy-3-methyl-2-butanone). This research suggests that heat treatment or salt contact significantly affects truffle aroma, while freezing and cutting have less pronounced effects in comparison. The enrichment of compounds showing significant changes during storage was investigated with a metabolomic pathway analysis. The involvement of some of the enriched compounds on the pyruvate/glycolysis and sulfur pathways was shown.
On-line coupled high performance liquid chromatography-gas chromatography-flame ionisation detection (HPLC-GC-FID) was used to compare the effect of hydrogen, helium and nitrogen as carrier gases on the chromatographic characteristics for the quantification of mineral oil hydrocarbon (MOH) traces in food related matrices. After optimisation of chromatographic parameters nitrogen carrier gas exhibited characteristics equivalent to hydrogen and helium regarding requirements set by current guidelines and standardisation such as linear range, quantification limit and carry over. Though nitrogen expectedly led to greater peak widths, all required separations of standard compounds were sufficient and humps of saturated mineral oil hydrocarbons (MOSH) and aromatic mineral oil hydrocarbons (MOAH) were appropriate to enable quantitation similar to situations where hydrogen or helium had been used. Slightly increased peak widths of individual hump components did not affect shapes and widths of the MOSH and MOAH humps were not significantly affected by the use of nitrogen as carrier gas. Notably, nitrogen carrier gas led to less solvent peak tailing and smaller baseline offset. Overall, nitrogen may be regarded as viable alternative to hydrogen or helium and may even extend the range of quantifiable compounds to highly volatile hydrocarbon eluting directly after the solvent peak.
Ergot alkaloids (EAs) formed by Claviceps fungi are one of the most common food contaminants worldwide, affecting cereals such as rye, wheat, and barley. To accurately determine the level of contamination and to monitor EAs maximum levels set by the European Union, the six most common EAs (so-called priority EAs) and their corresponding epimers are quantified using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The quantification of EAs in complex food matrices without appropriate internal tandards is challenging but currently carried out in the standard method EN 7425:2021 due to their commercial unavailability. To address the need for isotopically labeled EAs, we focus on two semi-synthetic approaches for the synthesis of these reference standards. Therefore, we investigate the feasibility of the N6-demethylation of native ergotamine to yield norergotamine, which can subsequently be remethylated with an isotopically labeled methylating reagent, such as iodomethane (13CD3-I), to yield isotopically labeled ergotamine and its C8-epimer ergotaminine. Testing the isotopically labeled ergotamine/-inine against native ergotamine/-inine with HPLC coupled to high-resolution HR-MS/MS proved the structure of ergotamine-13CD3 and ergotaminine-13CD3. Thus, for the first time, we can describe their synthesis from unlabeled, native ergotamine. Furthermore, this approach is promising as a universal way to synthesize other isotopically labeled EAs.
In this work, the electrochemical behavior of 4-phenylurazole (Ph-Ur) was studied and the latter was used as a molecular anchor for the electrochemical bioconjugation of tyrosine (Y). Cyclic voltammetry (CV) and controlled potential coulometry (CPC) allowed the in-situ generation of the PTAD (4-phenyl-3 H-1,2,4-triazole-3,5(4 H)-dione) species from phenylurazole on demand for tyrosine electrolabeling. The chemoselectivity of the reaction was studied with another amino acid (lysine, Lys) and no changes in Lys were observed. To evaluate the performance of tyrosine electrolabeling, coulometric analyses at controlled potentials were performed on solutions of phenylurazole and the phenylurazole-tyrosine mixture in different proportions (2:1, 1:1, and 1:2). The electrolysis of the phenylurazole-tyrosine mixture in the ratio (1:2) produced a charge of 2.07 C, very close to the theoretical value (1.93 C), with high reaction kinetics, a result obtained here for the first time. The products obtained were identified and characterized by liquid chromatography coupled to high-resolution electrospray ionization mass spectrometry (LC-HRMS and LC- HRMS2). Two products were formed from the click reactions, one of which was the majority. Another part of this work was to study the electrochemical degradation of the molecular anchor 4-phenylazole (Ph-Ur). Four stable degradation products of phenylurazole were identified (C7H9N2O, C6H8N, C6H8NO, C14H13N4O2) based on chromatographic profiles and mass spectrometry results. The charge generated during the electrolysis of phenylurazole (two-electron process) (2.85 C) is inconsistent with the theoretical or calculated charge (1.93 C), indicating that secondary/parasitic reactions occurred during the electrolysis of the latter. In conclusion, the electrochemically promoted click phenylurazole-tyrosine reactions give rise to click products with high reaction kinetics and yields in the (1:2) phenylurazole-tyrosine ratios, and the presence of side reactions is likely to affect the yield of the click phenylurazole-tyrosine reaction.
Metabolomics
(2024)
Oxidative degradation processes of tetrabromobisphenol A (TBBPA), a brominated flame retardant (BFR) in wood, plastics and electronics, were investigated by electrochemistry (EC) coupled online to electrospray ionization mass spectrometry (ESI/MS). Oxidative phase I and II metabolites production was achieved using an electrochemical flow-through cell equipped with a boron doped diamond electrode. Structural elucidation and prediction of oxidative metabolism pathways of TBBPA according to type II ipso-substitution were based on retention time, m/z ratio in negative mode and fragmentation pattern. Using the data obtained through high resolution MS analysis and the identification of single electron transfer (SET) as the initial step of a two-electron oxidation provided the necessary information to propose a mechanism for the electrochemical oxidation of TBBPA. Oxidation reactions involving aromatic hydroxylation and β-scission were the main degradation observed when studying the electrochemical behavior of TBBPA. Computational chemistry experiments using density functional theory (DFT) allowed to identify mono-hydroxylated reaction intermediate and dismissed the mechanism involving two concurrent hydroxylation. TBBPA oxidation products were compared to known metabolites of its biological and environmental degradation confirming the ability of electrochemistry to simulate β-scission reactions.
We report an analytical methodology for the quantification of sulfur in biological molecules via a speciesunspecific postcolumn isotope dilution (online ID) approach using capillary electrophoresis (CE) coupled online with inductively coupled plasma−mass spectrometry (online ID CE/ICP−MS).
The method was optimized using a mixture of standard compounds including sulfate, methionine, cysteine, cystine, and albumin, yielding compound recoveries between 98 and 105%. The quantity of sulfur is further converted to the quantity of the compounds owing to the prior knowledge of the sulfur content in the molecules. The limit of detection and limit of quantification of sulfur in the compounds were 1.3−2.6 and 4.1−8.4 mg L−1, respectively, with a correlation coefficient of 0.99 within the concentration range of sulfur of 5−100 mg L−1. The capability of the method was extended to quantify albumin in its native matrix (i.e., in serum) using experimentally prepared serum spiked with a pure albumin standard for validation. The relative expanded uncertainty of the method for the quantification of albumin was 6.7% (k = 2). Finally, we tested the applicability of the method on real samples by the analysis of albumin in bovine and human sera. For automated data assessment, a software application (IsoCor) which was developed by us in a previous work was developed further for handling of online ID data. The method has several improvements compared to previously published setups: (i) reduced adsorption of proteins onto the capillary wall owing to a special capillary-coating procedure, (ii) baseline separation of the compounds in less than 30 min via CE, (iii) quantification of several sulfur species within one run by means of the online setup, (iv) SI traceability of the quantification results through online ID, and (v) facilitated data processing of the transient signals using the IsoCor application. Our method can be used as an accurate approach for quantification of proteins and other biological molecules via sulfur analysis in complex matrices for various fields, such as environmental, biological, and pharmaceutical studies as well as clinical diagnosis.
Sulfur is an essential element in living organisms, where it plays important roles in various biological processes, such as protein synthesis, enzyme activity, and antioxidant defense. However, the biological effects of different sulfur species can vary widely, and imbalances in sulfur speciation have been observed in a range of diseases, including cancer, Alzheimer’s disease, and diabetes.1−3 The accurate quantification of sulfur and its species in biological samples requires sensitive and selective analytical techniques. In recent years, separation techniques coupled online with inductively coupled plasma−mass spectrometry (ICP−MS) have emerged as powerful online analytical tools complementary to molecular spectrometric methods for speciation analysis of biological compounds.
External calibration4−9 and isotope dilution (ID)10−15 are common calibration approaches applied for online quantification of sulfur species in complex samples. The ID analysis is advantageous over.
Ergot alkaloids form a toxicologically relevant group of mould toxins (mycotoxins) that are among the most common contaminants of foodstuff and animal feed worldwide. Reliable controls are essential to minimise health risks and economic damage. Due to their toxicological relevance, EU limit values for 12 priority ergot alkaloids have been introduced for the first time in 2022 and range from 500 μg/kg in rye milling products down to 20 ug/kg Processed cereal-based foods for infants and young children[1]. High-performance liquid chromatography - mass spectrometry is used to quantify low concentrations of ergots in food, however the European standard analytical procedure cannot be applied due to the lack of isotopically labelled reference standards.
The complex structure of the ergot alkaloids makes a total synthesis extremely challenging, expensive and time-consuming. Therefore, we are focusing on different semi-preparative methods (electrochemistry, organic synthesis, heterogeneous catalysis) to specifically N-demethylate the C8 carbon atom of the lysergic acid moiety. The norergot alkaloid formed is then isotopically labelled using an electrophilic methyl source, i.e. iodomethane or dimethyl sulphate to obtain the specific isotopic labelled ergot alkaloid. Initial experiments have shown that N-demethylation of the ergot alkaloid ergotamine is possible by both electrochemical and wet-chemical organic synthesis. The next step is to improve the previously determined reaction conditions to enable the synthesis of norergotamine on a mg scale for further reactions.
Ergot alkaloids form a toxicologically relevant group of mould toxins (mycotoxins) that are among the most common contaminants of food and animal feed worldwide. Reliable controls are essential to minimise health risks and economic damage. Due to their toxicological relevance, EU limit values for 12 priority ergot alkaloids have been introduced for the first time in 2022 and range from 500 µg/kg in rye milling products down to 20 ug/kg Processed cereal-based foods for infants and young children[1]. High-performance liquid chromatography - mass spectrometry is used to quantify low concentrations of ergots in food, however the European standard analytical procedure cannot be applied due to the lack of isotopically labelled reference standards.
The complex structure of the ergot alkaloids makes a total synthesis extremely challenging, expensive and time-consuming. Therefore, we are focusing on different semi-preparative methods (electrochemistry, organic synthesis, heterogeneous catalysis) to specifically N-demethylate the C8 carbon atom of the lysergic acid moiety. The norergot alkaloid formed is then isotopically labelled using an electrophilic methyl source, i.e. iodomethane or dimethyl sulphate to obtain the specific isotopic labelled ergot alkaloid. Initial experiments have shown that N-demethylation of the ergot alkaloid ergotamine is possible by both electrochemical and wet-chemical organic synthesis. The next step is to adapt the previously determined reaction conditions to enable the synthesis of norergotamine on a mg scale.
Demonstrating competency and equivalence for the measurement capacity of contaminants and nutrients in primary foodstuffs is a priority of the OAWG 10-year strategy for Track A core comparisons. Such measurements have posed significant challenges for reference material producers and calibration service providers. This key comparison (KC), under the topic of “non- polar analyte in high carbohydrate food matrix: trans-Zearalenone (trans-ZEN) in maize powder” , was a sector of the model system selected to align with this class within the OAWG strategy. Evidence of successful participation in formal, relevant international comparisons is needed to demonstrate the Calibration and Measurement Capabilities (CMCs) of national metrology institutes (NMIs) and designated institutes (DIs).
17 NMIs and DIs participated in the Track A KC CCQM- 168 “non-polar analyte in high carbohydrate food matrix: trans-ZEN in maize powder” . Participants were requested to evaluate the mass fraction (μg/kg) of trans-ZEN in maize powder material. Methods like liquid-liquid extraction and SPE were applied in the pre-treatment, and HPLC-MS/MS and HPLC-FLD were used for detection by the participants. The mass fractions for trans-ZEN were in the range of (91.8 to 169) μg/kg with standard uncertainties of (1.5 to 24.7) μg/kg, and corresponding relative standard uncertainties from 1.5% to 14.6%. Two labs, INTI and BAM were excluded from the KCRV evaluation. INTI result was identified as an outlier and confirmed their method had insufficient specificity. For BAM the calibration approach they used does not meet the CIPM traceability requirements. The other 15 labs included in the calculation of the consensus KCRV all agreed within their standard uncertainties. Hierarchical Bayes was used as estimators in calculating KCRV and standard uncertainty.
Successful participation in CCQM-K168 demonstrates the measurement capabilities in determining mass fraction of organic compounds, with molecular mass of 100 g/mol to 500 g/mol, having low polarity pKow < -2, in mass fraction range from 1 μg/kg to 1000 μg/kg in a high carbohydrate food matrix.
Ergot alkaloids are a group of toxic compounds, formed by fungi on infested grasses. In 2022, the European Commission set into effect maximum levels for the sum of the twelve major ergot alkaloids in multiple foods. To facilitate the laborious and costly individual quantification of the twelve major ergot alkaloids by HPLC–MS/MS or -FLD, we recently reported a sum parameter method (SPM) for ergot alkaloid quantification. Here, derivatization to lysergic acid hydrazide—a derivative of the mutual ergoline backbone in all ergot alkaloids—allowed simplified determination of all ergot alkaloids in flour via HPLC-FLD. For the measurement of more complex matrices like processed foods, we now developed a MS/MS-based SPM. Two internal standards (IS), isotopically labelled at different positions of the molecule, were synthesized and employed in the MS/MS-measurements. Method performance using either the 13CD3-labelled or the 15N2-labelled IS was evaluated on naturally contaminated rye and wheat flour samples as well as on processed food matrices. Employing the 13CD3-labelled IS leads to lower variances and better consistency with the reference data (obtained by the FLD-based SPM) in flour samples compared to the 15N2-labelled IS. The novel method significantly improves the measurement of ergot alkaloids in complex food matrices, due to their increased selectivity and thus lower interferences. Furthermore, the application of isotope labelled IS obviates the need for time-consuming steps like the determination of recovery rate based, matrix specific correction factors as described in the MS/MS-based European standard method for ergot alkaloid quantification (EN 17425).
Per- and polyfluoroalkyl substances (PFAS) are a large group of organofluorine surfactants used in the formulations of thousands of consumer goods. The continuous use of PFAS in household products and the discharge of PFAS from industrial plants into the sewer system have been resulted in contaminated effluents and sewage sludge from wastewater treatment plants (WWTPs) which became an important pathway for PFAS into the environment. Because sewage sludge is often used as fertilizer its application on agricultural soils has been observed as significant input path for PFAS into our food chain. To produce high-quality phosphorus fertilizers for a circular economy from sewage sludge, PFAS and other pollutants (e.g. pesticides and pharmaceuticals) must be separated from sewage sludge. Normally, PFAS are analyzed using PFAS protocols typically with time-consuming extraction steps and LC-MS/MS target quantification. However, for screening of PFAS contaminations in wastewater-based fertilizers also the DGT technique can be used for the PFAS extraction. Afterwards, combustion ion chromatography (CIC) can be applied to analyze the “total” amount of PFAS on the DGT binding layer. The DGT method was less sensitive and only comparable to the extractable organic fluorine (EOF) method values of the fertilizers in samples with >150 µg/kg, because of different diffusion properties for various PFAS, but also kinetic exchange limitations. However, the DGT approach has the advantage that almost no sample preparation is necessary. Moreover, the PFAS adsorption on the DGT binding layer was investigated via surface sensitive spectroscopical methods, such as Fourier-transform infrared (FT-IR) and fluorine K-edge X-ray absorption near-edge structure (XANES) spectroscopy.
Per- and polyfluoroalkyl substances (PFAS) are chemicals which were developed to improve humanity’s quality of life. Due to their high chemical stability and resistance to degradation by heat or acids, PFAS were used in a variety of consumer products. The continuous use of PFAS in household products and the discharge of PFAS from industrial plants into the sewer system resulted in the contamination of effluents and sewage sludge from wastewater treatment plants (WWTPs) (Roesch et al. 2022). Since sewage sludge is often used as fertilizer, its application on agricultural soils has been observed as a significant entry path for PFAS into the environment, specifically in our food chain. In Germany the sewage sludge/biosolid application on agricultural land was banned with the amendment of the German Sewage Sludge Ordinance and by 2029 sewage sludge application will be totally prohibited. However, phosphorus (P) from sewage sludge should still be recycled in WWTPs of cities with a population larger than 50,000 residents. To produce high-quality P-fertilizers for a circular economy, PFAS and other pollutants (e.g. pesticides and pharmaceuticals) must be separated from sewage sludge. Due to the strong diversity of industrial PFAS usage it is not clear if a safe application of novel recycled P-fertilizers from WWTPs can be guaranteed. Therefore, we analyzed various sewage sludges and wastewater-based fertilizers. Sewage sludge (SL) samples from various WWTPs in Germany and Switzerland, six sewage sludge ashes (SSA) from Germany, six thermally treated SL and SSA samples with different additives (temperatures: 700-1050 °C), two pyrolyzed SL samples (temperature: 400 °C) and two struvite samples from Germany and Canada were analyzed. The goal was to quantify PFAS in sewage sludges and wastewater-based P-fertilizers with the sum parameter extractable organic fluorine (EOF) by combustion ion chromatography (CIC). The results were compared with data from classical LC-MS/MS target analysis as well as selected samples by HR-MS suspect screening. The EOF values of the SLs mainly range between 154 and 538 µg/kg except for one SL which showed an elevated EOF value of 7209 µg/kg due to high organofluorine contamination. For the SSA samples the EOF values were lower and values between LOQ (approx. 60 µg/kg) and 121 µg/kg could be detected. For the pyrolyzed SLs no EOF values above the LOQ were detected. Moreover, the two wastewater-based struvite fertilizers contain 96 and 112 µg/kg EOF, respectively. In contrast to the EOF values, the sum of PFAS target values were relatively low for all SLs. Additional applied PFAS HR-MS suspect screening aimed to tentatively identify PFAS that could contribute to the hitherto unknown part of the EOF value. The majority of the detected fluorinated compounds are legacy PFAS such as short- and long-chain perfluorocarboxylic acids (PFCA), perfluorosulfonic acids (PFSA), polyfluoroalkyl phosphate esters (PAPs) and perfluorophosphonic acids (PFPA). Moreover, fluorinated pesticides, pharmaceutical as well as aromatic compounds were also identified, which are all included in the EOF parameter. Our research revealed that the current PFAS limit of 100 µg/kg for the sum of PFOS + PFOA in the German Fertilizer Ordinance is no longer up to date. Since the number of known PFAS already exceeds 10,000, the ordinance limit should be updated accordingly. Recent regulations and restrictions on using long-chain PFAS (≥C8) have resulted in a significant shift in the industry towards (ultra-)short-chain alternatives, and other, partly unknown, emerging PFAS. Ultimately, also fluorinated pesticides and pharmaceuticals, which end up as ultrashort PFAS in the WWTPs, have to be considered as possible pollutants in fertilizers from wastewater, too.
Solid wastewater-based fertilizers were screened for per- and polyfluoroalkyl substances (PFAS) by the extractable organic fluorine (EOF) sum parameter method. The EOF values for ten sewage sludges from Germany and Switzerland range from 154 to 7209 mg kg−1. For thermal treated sewage sludge and struvite the EOF were lower with values up to 121 mg kg−1. Moreover, the application of PFAS targeted
and suspect screening analysis of selected sewage sludge samples showed that only a small part of the EOF sum parameter values can be explained by the usually screened legacy PFAS. The hitherto unknown part of EOF sum parameter contains also fluorinated pesticides, pharmaceutical and aromatic compounds. Because these partly fluorinated compounds can degrade to (ultra-)short PFAS in wastewater treatment plants they should be considered as significant sources of organic fluorine in the environment. The combined results of sum parameter analysis and suspect screening reveal the need to update current regulations, such as the German fertilizer ordinance, to focus not solely on a few selected PFAS such as perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) but
consider an additional sum parameter approach as a more holistic alternative. Moreover, diffusion gradient in thin-films (DGT) passive samplers were utilized as an alternative simplified extraction method for PFAS in solid wastewater-based fertilizers and subsequently quantified via combustion ion chromatography. However, the DGT method was less sensitive and only comparable to the EOF values
of the fertilizers in samples with >150 mg kg−1, because of different diffusion properties for various PFAS, but also kinetic exchange limitations.
Ergot alkaloids are a group of mycotoxins occurring in products derived from various grasses (e.g., rye) and have been regulated in the EU recently. The new maximum levels refer to the sum of the six most common ergot alkaloids in their two stereoisomeric forms in different food matrices. Typically, these twelve compounds are individually quantified via HPLC-MS/MS or -FLD and subsequently summed up to evaluate food safety in a time-consuming process. Since all these structures share the same ergoline backbone, we developed a novel sum parameter method (SPM) targeting all ergot alkaloids simultaneously via lysergic acid hydrazide. After extraction and clean-up, in analogy to the current European standard method EN 17425 (ESM) for ergot alkaloid quantitation, the samples were derivatized by an optimized hydrazinolysis protocol, which allowed quantitative conversion after 20 min at 100 °C. The new SPM was evaluated against another established HPLC-FLD-based method (LFGB) and the HPLC-MS/MS-based ESM using six naturally contaminated rye and wheat matrix reference materials. While the SPM provided comparable values to the ESM, LFGB showed deviating results. Determined recovery rates, limits of detection and quantification of all three employed methods confirm that the new SPM is a promising alternative to the classical approaches for ergot alkaloid screening in food.
Thermo-Desorption Gas Chromatography-Mass Spectrometry (TD-GC-MS) was used to investigate the thermal degradation of two different polyurethanes (PU). PU samples were heated at different heating rates and the desorbed products were collected in a cold injection system and thereafter submitted to GC-MS. Prospects and limitations of the detection and quantification of semi-volatile degradation products were investigated. A temperature dependent PU depolymerization was
found at temperatures above 200 °C proved by an increasing release of 1,4-butanediol and methylene diphenyl diisocyanate (MDI) representing the main building blocks of both polymers. Their release was monitored quantitatively based on external calibration with authentic compounds. Size Exclusion Chromatography (SEC) of the residues obtained after thermodesorption confirmed the initial competitive degradation mechanism indicating an equilibrium of crosslinking and depolymerization as previously suggested. Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry of SEC fractions of thermally degraded PUs provided additional hints on degradation mechanism.
Volatile organic compounds (VOCs) are of interest in many different fields. Among them are food and fragrance analysis, environmental and atmospheric research, industrial applications, security or medical and life science. In the past, the characterization of these compounds was mostly performed via sample collection and off-site analysis with gas chromatography coupled to mass spectrometry (GC-MS) as the gold standard. While powerful, this method also has several drawbacks such as being slow, expensive, and demanding on the user. For decades, intense research has been dedicated to find methods for fast VOC analysis on-site with time and spatial resolution. We present the working principles of the most important, utilized, and researched technologies for this purpose and highlight important publications from the last five years. In this overview, non-selective gas sensors, electronic noses, spectroscopic methods, miniaturized gas chromatography, ion mobility spectrometry and direct injection mass spectrometry are covered. The advantages and limitations of the different methods are compared. Finally, we give our outlook into the future progression of this field of research.
Mycotoxins (toxins formed by fungi) in food and have caused problems for mankind since the beginning of time. The group of ergot alkaloids plays a special role in human history. Several tens of thousands of deaths during the middle ages caused by to ergotism (the disease caused by continuous intake of ergot alkaloid contaminated food) underscore the importance of reliable analytical methods to ensure food safety.
More than 50 compounds belong to the group of ergot alkaloids. The 12 most found structures – the major ergot alkaloids – are typically measured, when it comes to ergot alkaloid quantification. High performance liquid chromatography (HPLC) with a fluorescence detector (FLD) is typically used to quantify the ergot alkaloid content. The main disadvantage of this method are the high costs for calibration standards (12 different calibration substances are required). But also the time and effort required for the analysis of 12 peaks and overlapping signals that occur in complex food samples such as bread. As all ergot alkaloids share the ergoline structure and just differ in the substituents attached to this backbone, measurement of all ergot alkaloids in one sum parameter presents a time and cost saving alternative. The most important step for the development of such a sum parameter method is the reaction used to transfer all ergot alkaloids to one uniform structure. In the talk two promising reactions, the acidic esterification to lysergic acid methyl ester and hydrazinolysis to lysergic acid hydrazide, are examined for possible use in a routine analysis method. In addition to yield and reaction rate, factor such as the handling of the reaction and the possibility of parallelization play a role. Next to the current status of the ongoing research project, in this talk, current approaches to ergot alkaloid quantitation will be discussed.
Mycotoxins (toxic compounds formed by fungi) in food and feed have caused problems for mankind since the beginning of time. The group of ergot alkaloids plays a special role in human history. Several tens of thousands of deaths during the middle ages caused by to ergotism (the disease caused by continuous intake of ergot alkaloid contaminated food) underscore the importance of reliable analytical methods to ensure food safety.
More than 50 compounds belong to the group of ergot alkaloids. The 12 most found structures – the major ergot alkaloids – are typically measured, when it comes to ergot alkaloid quantification. High performance liquid chromatography (HPLC) with a fluorescence detector (FLD) is typically used to quantify the ergot alkaloid content. The main disadvantage of this method are the high costs for calibration standards (12 different calibration substances are required). But also, the time and effort required for the analysis of 12 peaks and overlapping signals that occur in complex food samples such as bread. As all ergot alkaloids share the ergoline structure and just differ in the substituents attached to this backbone, measurement of all ergot alkaloids in one sum parameter presents a time and cost saving alternative. The most important step for the development of such a sum parameter method is the reaction used to transfer all ergot alkaloids to one uniform structure. Two promising reactions, the acidic esterification to lysergic acid methyl ester and hydrazinolysis to lysergic acid hydrazide, were examined for possible use in a routine analysis method. In addition to yield and reaction rate, factors such as handling of the reaction and the possibility of parallel sample workup play a role.
The investigation of metabolic fluxes and metabolite distributions within cells by means of tracer molecules is a valuable tool to unravel the complexity of biological systems. Technological advances in mass spectrometry (MS) technology such as atmospheric pressure chemical ionization (APCI) coupled with high resolution (HR), not only allows for highly sensitive analyses but also broadens the usefulness of tracer-based experiments, as interesting signals can be annotated de novo when not yet present in a compound library. However, several effects in the APCI ion source, i.e., fragmentation and rearrangement, lead to superimposed mass isotopologue distributions (MID) within the mass spectra, which need to be corrected during data evaluation as they will impair enrichment calculation otherwise. Here, we present and evaluate a novel software tool to automatically perform such corrections. We discuss the different effects, explain the implemented algorithm, and show its application on several experimental datasets. This adjustable tool is available as an R package from CRAN.
IsoCor
(2022)
Despite numerous advantages offered by hyphenation of chromatography and electrokinetic separation methods with multicollector (MC) ICP-MS for isotope analysis, the main limitation of such systems is the decrease in precision and increase in uncertainty due to generation of short transient signals. To minimize this limitation, most authors compare several isotope ratio calculation methods and establish a multi-step data processing routine based on the precision and accuracy of the methods. However, to the best of our knowledge, there is no universal data processing tool available that incorporates all important steps of the treatment of the transient signals. Thus, we introduce a data processing application (App) IsoCor that facilitates automatic calculation of isotope ratios from transient signals and eases selection of the most suitable method. The IsoCor App performs baseline subtraction, peak detection, mass bias correction, isotope ratio calculation and delta calculation. The feasibility and reliability of the App was proven by reproducing the results from isotope analysis of three elements (neodymium, mercury and sulfur) measured on-line via hyphenated systems. The IsoCor App provides trackability of the results to ensure quality control of the analysis.
Safe food is a prerequisite for good human health, however currently almost one in ten people in the world fall ill after eating contaminated food. National food safety authorities need to be able to manage food safety risks along the entire food chain. The Official Controls Regulation (EU) 2017/625 was put in place to ensure that food and feed law stipulates the need for validated analytical methods. However, whilst method development for food control and proficiency testing are well established by European Union Reference Laboratories and National Reference Laboratories, validation of the measurements involved, certified reference materials and internationally recognised calibration and measurement capabilities are currently often (?) lacking. This may erode trust in the accuracy of the measurements.
This poster presents Food-MetNet, a coordinated preparatory initiative to establish the European Metrology Network for Safe and Sustainable Food, recently approved by the European Association of National Metrology Institutes (EURAMET). Food-MetNet aims to establish an ongoing dialogue between the metrology community, reference laboratories and regulatory bodies, in order to (i) identify stakeholders’ needs, (ii) develop a sustainable knowledge-sharing programme and web-based platform for stakeholders and (iii) develop roadmaps and a strategic research agenda. Improved access to more reliable and accurate food safety measurements will enable reference laboratories to more confidently and effectively compare their measurement results and support accreditation. This is particularly important where national food safety authorities need to assess potential new contaminants, novel food ingredients and newly emerging food risks, and new measurements methods may be required as well as input into new documentary standards. The activities for sustainable food intend to boost the activity within the "EU green deal framework" to reduce food waste and losses, and the development of standards and methods that allow for the reuse of food waste. The support to new business opportunities and innovation will allow increasing the market competitiveness of EU food industries and the valorisation of waste by reducing environmental impacts.
Despite numerous advantages offered by hyphenation of chromatography and electrokinetic separation methods with multicollector (MC) ICP-MS for isotope analysis, the main limitation of such systems is the decrease in precision and increase in uncertainty due to generation of short transient signals. To minimize this limitation, most authors compare several isotope ratio calculation methods and establish a multi-step data processing routine based on the precision and accuracy of the methods. However, to the best of our knowledge, there is no universal data processing tool available that incorporates all important steps of the treatment of the transient signals. Thus, we introduce a data processing application (App) IsoCor that facilitates automatic calculation of isotope ratios from transient signals and eases selection of the most suitable method. The IsoCor App performs baseline subtraction, peak detection, mass bias correction, isotope ratio calculation and delta calculation. The feasibility and reliability of the App was proven by reproducing the results from isotope analysis of three elements (neodymium, mercury and sulfur) measured on-line via hyphenated systems. The IsoCor App provides trackability of the results to ensure quality control of the analysis.
The aim of this study was to identify specific truffle marker substances within the truffle aroma. The aroma profile of different truffle species was analyzed using static headspace sampling with gas chromatography mass spectrometry analysis (SHS/GC-MS). Possible marker substances were identified, taking the additional literature into account. The selected marker substances were tested in an experiment with 19 truffle dogs. The hypothesis “If trained truffle dogs recognize the substances as supposed truffles in the context of an experiment, they can be regarded as specific” was made. As it would be nearly impossible to investigate every other possible emitter of the same compounds to determine their specificity, this hypothesis was a reasonable approximation. We were interested in the question of what it is the dogs actually search for on a chemical level and whether we can link their ability to find truffles to one or more specific marker substances. The results of the dog experiment are not as unambiguous as could have been expected based on the SHS/GC-MS measurements. Presumably, the truffle aroma is mainly characterized and perceived by dogs by dimethyl sulfide and dimethyl disulfide. However, as dogs are living beings and not analytical instruments, it seems unavoidable that one must live with some degree of uncertainty regarding these results.
Rapid, cost-efficient, and eco-friendly methods are desired today for routine analysis of the Fusarium mycotoxin zearalenone (ZEN) in edible vegetable oils. Liquid chromatography with fluorescence detection (HPLC-FLD) is commonly used to reliably control the specified ZEN maximum levels, which requires efficient sample clean-up to avoid matrix interferences. Therefore, a highly selective extraction and clean-up method based on reversible covalent hydrazine chemistry (RCHC) using hydrazine-functionalized silica was developed. This efficient solid-phase extraction (SPE) involves reversible hydrazone formation of ZEN with the hydrazine moiety covalently bound to a solid phase. Optimal conditions were achieved with 1 mL SPE cartridges filled with 400 mg of hydrazine-functionalized silica. The developed RCHC-SPE method was validated in an interlaboratory comparison study (ILC) with twelve participants analyzing six edible vegetable oils with a focus on maize oils. The derived method parameters (ZEN recovery 83%, repeatability 7.0%, and reproducibility 18%) meet the performance criteria of Commission Regulation (EC) No 401/2006. The developed RCHC-SPE-based HPLC-FLD method allows the reliable quantification of ZEN in the range of 47–494 μg/kg for different types of edible vegetable oils, also for matrix-reach native oils. Due to the high efficiency, the significantly reduced matrix load helps to extend the lifetime of analytical equipment. Furthermore, the re-useability of the RCHC-SPE cartridges contributes to an eco-friendly approach and reduced analysis costs. To our knowledge, this is the first report on ZEN quantification in edible vegetable oils based on manual RCHC-SPE cartridges. Due to its high performance, the developed RCHC-SPE method is a promising alternative to the current European standard method EN 16924:2017 (HPLC-FLD part).
Analysis of electrochemical and liver microsomal transformation products of lasalocid by LC/HRMS
(2022)
Rationale: Lasalocid (LAS), an ionophore, is used in cattle and poultry farming as feed additive for its antibiotic and growth-promoting properties. Literature on transformation products (TP) resulting from LAS degradation is limited. So far, only hydroxylation is found to occur as the metabolic reaction during the LAS degradation. To investigate potential TPs of LAS, we used electrochemistry (EC) and liver microsome (LM) assays to synthesize TPs, which were identified using liquid chromatography high-resolution mass spectrometry (LC/HRMS).
Methods: Electrochemically produced TPs were analyzed online by direct coupling of the electrochemical cell to the electrospray ionization (ESI) source of a Sciex Triple-TOF high resolution mass spectrometer. Then, EC-treated LAS solution was collected and analyzed offline using LC/HRMS to confirm stable TPs and improve their annotation with a chemical structure due to informative MS/MS spectra. In a complementary approach, TPs formed by rat and human microsomal incubation were investigated using LC/HRMS. The resulting data were used to investigate LAS modification reactions and elucidate the chemical structure of obtained TPs.
Results: The online measurements identified a broad variety of TPs, resulting from modification reactions like (de-)hydrogenation, hydration, methylation, oxidation as well as adduct formation with methanol. We consistently observed different ion complexations of LAS and LAS-TPs (Na+; 2Na+ K+; NaNH4 +; KNH4 +). Two stable methylated EC-TPs were found, structurally annotated, and assigned to a likely modification reaction. Using LM incubation, seven TPs were formed, mostly by oxidation/hydroxylation. After the identification of LM-TPs as Na+-complexes, we identified LM-TPs as K+-complexes.
Conclusion: We identified and characterized TPs of LAS using EC- and LM-based methods. Moreover, we found different ion complexes of LAS-based TPs. This knowledge, especially the different ion complexes, may help elucidate the metabolic and environmental degradation pathways of LAS.
Lifestyle modifications could prevent almost one‑third to one‑half of all cancer cases. The awareness of cancer risk factors could motivate people to make such changes in their behaviors and lifestyles. This work aims to investigate the cancer awareness level in the Pakistani population. Telephone interviews of 657 individuals in Pakistan were carried out using the Cancer Awareness Measure (CAM) and Cancer Awareness Measure–MYthical Causes Scale (CAM‑MY). We observed that participants scored significantly better on the CAM scale than the CAM‑MY scale, and CAM scores were negatively associated with CAM‑MY scores. Years of formal education or a biology major at undergraduate or graduate level did not affect our population’s cancer awareness levels. Age displayed a weak but statistically significant negative association with CAM scores. Most participants failed to identify modifiable cancer risk factors, e.g., low physical activity. Efforts should be made to improve awareness of modifiable risk factors. We observed that brief training sessions could markedly improve people’s understanding of cancer risk factors and myths.
For more than 110 years, BAM has been producing reference materials for a wide range of application fields. With the development of new analytical methods and new applications as well as continuously emerging more stringent requirements of laboratory accreditation with regard to quality control and metrological traceability, the demand and requirements for reference materials are increasing. This trend article gives an overview of general developments in the field of reference materials as well as developments in selected fields of application in which BAM is active. This includes inorganic and metal analysis, gas analysis, food and consumer products, and geological samples. In addition to these more traditional fields of application, developments in the areas of optical spectroscopy, particulary fluorescence methods, and nanomaterials are considered.
Towards Unbiased Evaluation of Ionization Performance in LC-HRMS Metabolomics Method Development
(2022)
As metabolomics increasingly finds its way from basic science into applied and regulatory environments, analytical demands on nontargeted mass spectrometric detection methods continue to rise. In addition to improved chemical comprehensiveness, current developments aim at enhanced robustness and repeatability to allow long-term, inter-study, and meta-analyses. Comprehensive metabolomics relies on electrospray ionization (ESI) as the most versatile ionization technique, and recent liquid chromatography-high resolution mass spectrometry (LC-HRMS) instrumentation continues to overcome technical limitations that have hindered the adoption of ESI for applications in the past. Still, developing and standardizing nontargeted ESI methods and instrumental setups remains costly in terms of time and required chemicals, as large panels of metabolite standards are needed to reflect biochemical diversity. In this paper, we investigated in how far a nontargeted pilot experiment, consisting only of a few measurements of a test sample dilution series and comprehensive statistical analysis, can replace conventional targeted evaluation procedures. To examine this potential, two instrumental ESI ion source setups were compared, reflecting a common scenario in practical method development. Two types of feature evaluations were performed, (a) summary statistics solely involving feature intensity values, and (b) analyses additionally including chemical interpretation.
Results were compared in detail to a targeted evaluation of a large metabolite standard panel. We reflect on the advantages and shortcomings of both strategies in the context of current harmonization initiatives in the metabolomics field.
MALDI-TOF-MS-Based Identification of Monoclonal Murine Anti-SARS-CoV-2 Antibodies within One Hour
(2022)
During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used 35 monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied to the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 60 min and had a combined sequence coverage of over 80%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 degrees C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0. This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context.
The investigation of metabolic fluxes and metabolite distributions within cells by means of tracer molecules is a valuable tool to unravel the complexity of biological systems. Technological advances in mass spectrometry (MS) technology such as atmospheric pressure chemical ionization (APCI) coupled with high resolution (HR), not only allows for highly sensitive analyses but also broadens the usefulness of tracer‐based experiments, as interesting signals can be annotated de novo when not yet present in a compound library. However, several effects in the APCI ion source, i.e., fragmentation and rearrangement, lead to superimposed mass isotopologue distributions (MID) within the mass spectra, which need to be corrected during data evaluation as they will impair enrichment calculation otherwise. Here, we present and evaluate a novel software tool to automatically perform such corrections. We discuss the different effects, explain the implemented
algorithm, and show its application on several experimental datasets. This adjustable tool is available as an R package from CRAN.
The drug salinomycin (SAL) is a polyether antibiotic and used in veterinary Medicine as coccidiostat and growth promoter. Recently, SAL was suggested as a potential anticancer drug.
However, transformation products (TPs) resulting from metabolic and environmental degradation of SAL are incompletely known and structural information is missing. In this study, we therefore systematically investigated the formation and identification of SAL derived TPs using electrochemistry (EC) in an electrochemical reactor and rat and human liver microsome incubation (RLM and HLM) as TP generating methods. Liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS) was applied to determine accurate masses in a suspected target analysis to identify TPs and to deduce occurring modification reactions of derived TPs. A total of 14 new, structurally different TPs were found (two EC-TPs, five RLM-TPs, and 11 HLM-TPs). The main modification reactions are decarbonylation for EC-TPs and oxidation (hydroxylation) for RLM/HLM-TPs. Of particular interest are potassium-based TPs identified after liver microsome incubation because these might have been overlooked or declared as oxidated sodium adducts in previous, non-HRMS-based studies due to the small mass difference between K and O + Na of 21 mDa. The MS fragmentation pattern of TPs was used to predict the position of identified modifications in the SAL molecule. The obtained knowledge regarding transformation reactions and novel TPs of SAL will contribute to elucidate SAL-metabolites with regards to structural prediction.
The CCQM-K148.a comparison was coordinated by the BIPM on behalf of the CCQM Organic Analysis Working Group for NMIs and DIs which provide measurement services in organic analysis under the CIPM MRA. It was undertaken as a "Track A" comparison within the OAWG strategic plan. CCQM-K148.a demonstrates capabilities for assigning the mass fraction content of a solid organic compound having moderate molecular complexity, where the compound has a molar mass in the range (75 - 500) g/mol and is non-polar (pKow < −2), when present as the primary organic component in a neat organic solid and where the mass fraction content of the primary component in the material is in excess of 950 mg/g.
Participants were required to report the mass fraction of Bisphenol A present in one supplied unit of the comparison material. Participants using a mass balance method for the assignment were also required to report their assignments of the impurity components present in the material. Methods used by the seventeen participating NMIs or DIs were predominantly based on either stand-alone mass balance (summation of impurities) or qNMR approaches, or the combination of data obtained using both methods. The results obtained using thermal methods based on freezing-point depression methods were also reported by a limited number of participants. There was excellent agreement between assignments obtained using all three approaches to assign the BPA content.
The assignment of the values for the mass fraction content of BPA consistent with the KCRV was achieved by most of the comparison participants with an associated relative standard uncertainty in the assigned value in the range (0.1 - 0.5)%.
The ubiquitous Fusarium genus is responsible for the spoilage of vast amounts of cereals and fruits.
Besides the economic damage, the danger to human and animal health by the concomitant exposure to mycotoxins represents a serious problem. A large number of Fusarium species produce a variety of different mycotoxins of which the class of trichothecenes are of particular importance due to their toxicity.
Being identified as the common volatile precursor during the biosynthesis of trichothecenes, (−)-trichodiene (TD) is considered to be a biomarker for the respective mycotoxin content in food samples. We postulated that the development of a non-invasive, on-site GC-IMS method for the quantification of (−)-trichodiene supplemented with a stationary SIDA headspace GC-MS reference method would allow circumventing the laborious and expensive analyses of individual trichothecenes in large cereal samples.
In this work we present the syntheses of the required native calibration standard and an isotope labeled (TD-D3) internal standard.
Abstract Alternariol (AOH) and alternariol-9-monomethyl ether (AME) are two secondary metabolites of Alternaria fungi which can be found in various foodstuff like tomatoes, nuts, and grains. Due to their toxicity and potential mutagenic activity the need for the development of high-throughput methods for the supervision of AOH- and AME-levels is of increasing interest. As the availability of both native and labeled AOH and AME analytical standards is very limited we herein wish to present a novel concise approach towards their synthesis employing a ruthenium-catalyzed ortho-arylation as the key step. Finally, we demonstrate their suitability as internal standards in stable-isotope dilution assay (SIDA)-HPLC-MS/MS analysis commonly used for the quantification of the natural products in food and feed.
Polycyclic aromatic hydrocarbons (PAHs) are a large group of priority organic pollutants, which contaminate environmental compartments, food, and consumer products as well. Due to their frequent occurrence associated with elevated Levels of PAHs, plastic and rubber parts of consumer products and toys are particular sources of exposure. Although European maximum levels exist for eight carcinogenic PAHs in consumer products and toys according to REACH Regulation (EC) No. 1907/2006, certified reference materials (CRM) are still not available. To overcome this lack, the first CRM for the determination of PAHs in rubber toys (BAM-B001) was developed according to the requirements of ISO Guide 35. The whole process of CRM development including preparation, homogeneity and stability studies, and value assignment is presented.
The assignment of the certified mass fractions was based upon in-house study at BAM using stable isotope Dilution analysis (SIDA) gas chromatography mass spectrometry (GC–MS). The obtained values were confirmed by the results of two interlaboratory comparison (ILC) studies with more than 50 expert laboratories from Germany and China. The mass fractions of 14 PAHs including all REACH and GS mark regulated compounds were certified ranging between 0.2 and 15.4 mg/
kg accompanied by expanded uncertainties (coverage factor k = 2). In addition, informative values were determined for 4 PAHs, mainly due to higher uncertainties and/or lack of ILC data for confirmation. BAM-B001 is intended for analytical quality control particularly based on the AfPS GS 2019:01 PAK method and contributes to improve the chemical safety of consumer products including toys.
Given the central role of fatty acids in cancer pathophysiology, the exploitation of fatty acid metabolism as a potential antineoplastic therapy has gained much attention. Several natural and synthetic compounds targeting fatty acid metabolism were hitherto identified, and their effectiveness against cancer cell proliferation and survival was determined. This review will discuss the most clinically viable inhibitors or drugs targeting various proteins or enzymes mapped on nine interconnected fatty acid metabolism-related processes. We will discuss the general significance of each of these processes and the effects of their inhibition on cancer cell progression. Moreover, their mechanisms of action, limitations, and future perspectives will be assessed.
Stir bar sorptive extraction (SBSE) was compared with standardized pump sampling regarding the prospects to assess airborne levels of polycyclic aromatic hydrocarbons (PAHs) in indoor environments. A historic railway water tower, which will be preserved as a technical monument for museum purposes, was sampled with both approaches because the built-in insulationmaterial was suspected to release PAHs to the indoor air. The 16 PAHs on the US EPA list were quantified using gas chromatography with mass spectrometric detection in filters from pump sampling after solvent extraction and on SBSE devices after thermal desorption. SBSEwas seen to sample detectable PAHmasseswith excellent repeatability and a congener pattern largely similar to that observed with pump sampling. Congener patterns were however significantly different from that in the PAH source because release from the insulation material is largely triggered by the respective congener vapor pressures. Absolute masses in the ng range sampled by SBSE corresponded to airborne concentrations in the ng L−1 range determined by pump sampling. Principle differences between SBSE and pump sampling as well as prospects of SBSE as cost-effective and versatile complement of pump sampling are discussed.
This paper describes Food-MetNet, a coordinated preparatory initiative to establish the European Metrology Network on Food Safety (EMN-FS). Food-MetNet aims to establish a long-term ongoing dialogue between the metrology community and relevant stakeholders, in particular, European Union Reference Laboratories (EURLs), National Reference Laboratories (NRLs) and the Joint Research Centre (JRC). This dialogue is meant to support the collection of needs from stakeholders, the take-up of metrological research output and the development of the roadmaps needed to navigate future research.
Polyethylene glycols (PEGs) are widely used in everyday items such as food additives and in personal care products. In addition, they have multiple medical applications: as laxatives, excipients, and covalently coupled to drug molecules leading to improved pharmacokinetics (PEGylation). While generally regarded as biologically inert, the human body is known to produce antibodies against PEGylated molecules. In addition, PEGs have been shown to be part of a biomarker signature to predict colon cancer outcome, suggesting a more complex and yet unknown behavior of PEGs in the human body.
Here, we introduce PEGomics, a retrospective screening approach of publicly available LC-MS data. Using a custom R script to process entire studies, the presence of PEGs was reveled in most human plasma, serum and whole blood samples investigated. Several PEG species and adducts were identified and their correlation with different diseases and health conditions was investigated further.
Blood PEG levels significantly differed between patient groups in multiple clinical studies related to e.g. pregnancy duration, fasting and smoking. We discuss possible causes for these effects in the light of recent reports of allergies against PEGs and outline our further strategies to identify the source of PEGs in the human body as well as possible metabolic transformations.
The general understanding has been that PEGs are biologically inert, meaning they pass the human body without any relevant reaction with anything. But recent studies have shown that migth not be correct. Here a retrospective analysis of LC-MS data was performed to determine correlations between blood PEG levels in humans and different experimental factors.
T cells need to adapt their cellular metabolism for effector cell differentiation. This relies on alterations in mitochondrial physiology. Which signals and molecules regulate those alterations remains unclear. We recently reported, that the mitochondrial protein TCAIM inhibits activation-induced changes in mitochondrial morphology and function and thus, CD effector T cell formation. Using conditional TCAIM knock-in (KI) and knockout (KO) mice, w now show that it also applies to CD8+ T cells and more importantly, delineate the molecular processes in mitochondria by which TCAIM controls effector cell differentiation. TCAIM KI resulted in reduced activation-induced HIF1α protein expression. Metabolomics and transcriptional data in combination with mathematical flux modeling revealed an impaired induction of anabolic pathways, especially of the mevalonate pathway and cholesterol biosynthesis in TCAIM KI CD8+ T cells. Addition of cholesterol completely rescued HIF1α protein expression, activation and proliferation of TCAIM KI CD8+ T cells. At the molecular level, TCAIM delayed activation-induced mitochondria-ER contact (MERC) formation by binding to MERC promoting proteins such as RMD3 and VDAC2. In summary, we demonstrate that TCAIM suppresses effector cell differentiation by inhibiting MERC formation, which induce HIF1α-mediated increase in cellular metabolism and cholesterol biosynthesis.