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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.
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.
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.
Three chromatographic procedures were investigated regarding their potential for the quantification of aniline and 19 of its methylated and chlorinated derivatives in groundwater. These methods were based on liquid-liquid-extraction in combination with gas chromatography and single quadrupole mass spectrometry (GC/MS) according to German standard DIN 38407-16:1999 and its extension using tandem mass spectrometry (GC/MS-MS), both following liquid-liquid extraction, and as third alternative the direct injection of the water sample into a liquid chromatograph coupled to tandem mass spectrometry (LC/MS-MS). Results were compared using fortified water and real-world contaminated groundwater used in an interlaboratory comparison. It could be shown that GC/MS and GC/MS-MS yielded results deviating less than 10% from each other while all three procedure displayed quantification results deviating less than 15% from the intercomparison reference values in case of each analyte in the concentration range between 1 and 45 µg L-1. Though GC/MS-MS displays a ten-fold higher sensitivity than single quadrupole GC/MS, the precision of both methods in the concentration range was similar. LC/MS-MS has the advantage of no further sample preparation due to the direct injection and leads for methylanilines and meta-, para- substituted chloroanilines to results sufficiently equivalent to the standardised GC/MS method. However, LC/MS-MS is not suitable for ortho-chloroaniline derivates due to significantly lower ion yields than meta- and para-substituted chloroanilines.
MACC1 is a prognostic and predictive metastasis biomarker for more than 20 solid Cancer entities. However, its role in cancer metabolism is not sufficiently explored. Here, we report on how MACC1 impacts the use of glucose, glutamine, lactate, pyruvate and fatty acids and show the comprehensive analysis of MACC1-driven metabolic networks. We analyzed concentrationdependent changes in nutrient use, nutrient depletion, metabolic tracing employing 13C-labeled substrates, and in vivo studies. We found that MACC1 permits numerous effects on cancer
metabolism. Most of those effects increased nutrient uptake. Furthermore, MACC1 alters metabolic pathways by affecting metabolite production or turnover from metabolic substrates. MACC1 supports use of glucose, glutamine and pyruvate via their increased depletion or altered distribution within metabolic pathways. In summary, we demonstrate that MACC1 is an important regulator of metabolism in cancer cells.
Background: Prevention of metastatic invasion is one of the main challenges in the treatment of alveolar rhabdomyosarcoma. Still the therapeutic options are limited. Therefore, an anti-tumor screening was initiated focusing on the anti-metastatic and anti-invasion properties of selected medicinal plant extracts and phytoestrogens, already known to be effective in the prevention and treatment of different cancer entities.
Methods: Treatment effects were first evaluated by cell viability, migration, invasion, and colony forming assays on the alveolar rhabdomyosarcoma cell line RH-30 in comparison with healthy primary cells.
Results: Initial anti-tumor screenings of all substances analyzed in this study, identified the plant extract of Vincetoxicum arnottianum (VSM) as the most promising candidate, harboring the highest anti-metastatic potential. Those significant anti-motility properties were proven by a reduced ability for migration (60%), invasion (99%) and colony formation (61%) under 48 h exposure to 25 μg/ml VSM. The restricted motility features were due to an induction of the stabilization of the cytoskeleton – actin fibers were 2.5-fold longer and were spanning the entire cell. Decreased proliferation (PCNA, AMT, GCSH) and altered metastasis (e. g. SGPL1, CXCR4, stathmin) marker expression on transcript and protein level confirmed the significant lowered tumorigenicity under VSM treatment. Finally, significant alterations in the cell metabolism were detected for
25 metabolites, with levels of uracil, N-acetyl serine and propanoyl phosphate harboring the greatest alterations. Compared to the conventional therapy with cisplatin, VSM treated cells demonstrated a similar metabolic shutdown of the primary cell metabolism. Primary control cells were not affected by the VSM treatment.
Conclusions: This study revealed the VSM root extract as a potential, new migrastatic drug candidate for the putative treatment of pediatric alveolar rhabdomyosarcoma with actin filament stabilizing properties and accompanied by a marginal effect on the vitality of primary cells.
Lipid droplets, the dynamic organelles that store Triglycerides (TG) and cholesterol esters (CE), are highly accumulated in colon cancer cells. This work studies the TG and CE subspecies profile in colon carcinoma cell lines, SW480 derived from primary tumor, and SW620 derived from a metastasis of the same tumor. It was previously reported that the total TG and CE content is dramatically higher in SW620 cells; however, TG and CE subspecies profile has not been investigated in detail. The work presented here confirms that the total TG and CE Content is significantly higher in the SW620 cells. Moreover, the fatty acid (FA) composition of TG is significantly altered in the SW620 cells, with significant decrease in the abundance of saturated triglycerides. This resulted in a significantly decreased TG saturation index in the SW620 cells. The saturation index of CE was also significantly decreased in the SW620 cells.
Stormwater from urban areas can transport biocidally active substances and related transformation products from buildings into the environment. The occurrence of these substances in urban runoff depends on the availability of water, and on ultraviolet radiation exposure that causes photolytic reactions. In a systematic laboratory study, painted test specimens were exposed to either ultraviolet radiation, water contact, or a combination of both. Leaching of the biocidally active substances carbendazim, diuron, octylisothiazolinone, terbutryn, and selected transformation products of terbutryn and diuron were observed under various exposure conditions. Remaining concentrations of these substances in the paint were quantified. It was demonstrated that the distribution of active substances and transformation products in eluates and in the coatings themselves differs with exposure conditions. Strategies for environmental monitoring of biocide emissions need to consider the most relevant transformation products. However, environmental concentrations of biocidally active substances and transformation products depend on earlier exposure conditions. As a consequence, monitoring data cannot describe emission processes and predict expected leaching of biocidally active substances from buildings if the data are collected only occasionally.
Zearalenone (ZEN), an estrogenic mycotoxin produced by several species of Fusarium fungi, is a common contaminant of cereal-based food worldwide. Due to frequent occurrences associated with high levels of ZEN, maize oil is a particular source of exposure. Although a European maximumlevel for ZEN in maize oil exists according to Commission Regulation (EC) No. 1126/2007 along with a newly developed international standard method for analysis, certified reference materials (CRM) are still not available. To overcome this lack, the first CRM for the determination of ZEN in contaminated maize germ oil (ERM®-BC715) was developed in the frame of a European Reference Materials (ERM®) project 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 fraction was based upon an in-house study using high-performance liquid chromatography isotope dilution tandem mass spectrometry. Simultaneously, to support the in-house certification study, an interlaboratory comparison study was conducted with 13 expert laboratories using different analytical methods. The certified mass fraction and expanded uncertainty (k=2) of ERM®-BC715 (362± 22) μg kg−1 ZEN are traceable to the SI. This reference material is intended for analytical quality control and contributes to the improvement of consumer protection and food safety.
Ergot alkaloids are mycotoxins formed by fungi of the Claviceps genus, which are some of the most common contaminants of food and feed worldwide. These toxins are a structurally heterogeneous group of compounds, sharing an ergoline backbone. Six structures and their corresponding stereoisomers are typically quantified by either HPLC-FLD or HPLC-MS/MS and the values subsequently summed up to determine the total ergot alkaloid content. For the development of a screening method targeting all ergot alkaloids simultaneously, the alkaloids need to be transferred to one homogeneous structure: a lysergic acid derivative. In this study, two promising cleaving methods—acidic esterification and hydrazinolysis—are compared, using dihydroergocristine as a model compound. While the acidic esterification proved to be unsuitable, due to long reaction times and oxidation sensitivity, hydrazinolysis reached a quantitative yield in 40-60 min. Parallel workup of several samples is possible. An increasing effect on the reaction rate by the addition of ammonium iodide was demonstrated. Application of hydrazinolysis to a major ergot alkaloid mix solution showed that all ergopeptines were cleaved, but ergometrine/-inine was barely affected. Still, hydrazinolysis is a suitable tool for the development of a sum parameter screening method for ergot alkaloids in food and feed.
Mycotoxigenic fungal pathogens Fusarium and Alternaria are a leading cause of loss in cereal production. On wheat-ears, they are confronted by bacterial antagonists such as pseudomonads.
Studies on these groups’ interactions often neglect the infection process’s temporal aspects and the associated priority effects. In the present study, the focus was on how the first colonizer affects the subsequent ones. In a climate chamber experiment, wheat-ears were successively inoculated with two different strains (Alternaria enuissima At625, Fusarium graminearum Fg23, or Pseudomonas simiae Ps9).
Over three weeks, microbial abundances and mycotoxin concentrations were analyzed and visualized via Self Organizing Maps with Sammon Mapping (SOM-SM). All three strains revealed different characteristics and strategies to deal with co-inoculation: Fg23, as the first colonizer, suppressed the establishment of At625 and Ps9. Nevertheless, primary inoculation of At625 reduced all of the Fusarium toxins and stopped Ps9 from establishing. Ps9 showed priority effects in delaying and blocking the production of the fungal mycotoxins. The SOM-SM analysis visualized the competitive strengths: Fg23 ranked first, At625 second, Ps9 third. Our findings of species-specific priority effects in a natural environment and the role of the mycotoxins involved are relevant for developing biocontrol strategies.
Multiple works have studied possible associations between human leukocyte antigen (HLA) alleles and end stage renal disease (ESRD) showing, however, contradictory and inconsistent results. Here, we revisit the association between ESRD and HLA antigens, comparing HLA polymorphism (at HLA-A, -B, -C, -DRB1, -DQB1 and DQA1 loci) in ESRD patients (n = 497) and controls (n = 672). Our data identified several HLA alleles that displayed a significant positive or negative association with ESRD. We also determined whether heterozygosity or homozygosity of the ESRD-associated HLA alleles at different loci could modify the prevalence of the disease. Few HLA allele combinations displayed significant associations with ESRD, among which A*3_26 combination showed the highest strength of association (OR = 4.488, P≤ 0.05) with ESRD. Interestingly, the age of ESRD onset was not affected by HLA allele combinations at different loci. We also performed an extensive literature analysis to determine whether the association of HLA to ESRD can be similar across different ethnic groups. Our analysis showed that at least certain HLA alleles, HLA-A*11, HLA-DRB1*11, and HLA-DRB1*4, display a significant association with ESRD in different ethnic groups. The findings of our study will help in determining possible protective or susceptible roles of various HLA alleles in ESRD.
Simultaneous characterization of poly(acrylic acid) andpolysaccharide polymers and copolymers
(2020)
Copolymer products that result from grafting acrylic acid and other hydrophilicmonomers onto polysaccharides have recently gained significant interest in researchand industry. Originating from renewable sources, these biodegradable, low toxicity,and polar copolymer products exhibit potential to replace polymers from fossil sourcesin several applications and industries. The methods usually employed to character-ize these copolymers are, however, quite limited, especially for the measurement ofbulk properties. With more sophisticated applications, for example, in pharmaceu-tics requiring a more detailed analysis of the chemical structure, we describe a newapproach for this kind of complex polymers. Our approach utilizes chromatographyin combination with several detection methods to separate and characterize reactionproducts of the copolymerization of acrylic acid and chemically hydrolyzed starch.These samples consisted of a mixture of homopolymer poly (acrylic acid), homopoly-mer hydrolyzed starch, and – in a lower amount – the formed copolymers. Several chro-matographic methods exist that are capable of characterizing either poly (acrylic acid)or hydrolyzed starch. In contrast, our approach offers simultaneous characterization ofboth polymers. The combination of LC and UV/RI offered insight into the compositionand copolymer content of the samples. Size exclusion chromatography experimentsrevealed the molar mass distribution of homopolymers and copolymers. FTIR inves-tigations confirmed the formation of copolymers while ESI-MS gave more details onthe end groups of hydrolyzed starches and poly (acrylic acids). Evidence of copolymerstructures was obtained through NMR measurements. Finally, two-dimensional chro-matography led to the separation of the copolymers from both homopolymers as wellas the additional separation of sodium clusters. The methods described in this work area powerful toolset to characterize copolymerization products of hydrolyzed starch andpoly(acrylic acid). Together, our approach successfully correlates the physicochemicalproperties of such complex mixtures with their actual composition.
Since 1 January 2019 a maximum content of 0.6 mg kg−1 cadmium (Cd) in cocoa powder sold to the final consumer or as an ingredient in sweetened cocoa powder sold to the final consumer (drinking chocolate) is set by the Commission Regulation (EU) No. 488/2014. Monitoring compliance with the specified limit value requires analytical measuring methods and reference materials for quality control. However, suitable certified reference materials intended for quality assurance and quality control purposes are still lacking. Therefore, three cocoa reference materials (ERM®-BD513, ERM®-514 and ERM®-515) were developed according to the requirements of ISO 17034 and the recommendations of ISO Guide 35. The whole process of reference material development, including material preparation, assessment of homogeneity and stability, characterisation and value assignment is presented. The assignment of the certified mass fractions was based upon an interlaboratory comparison study involving 19 expert laboratories for Cd and 12 laboratories for acrylamide. The certified mass fractions and expanded uncertainties (k = 2) of the reference materials were (0.181 ± 0.009) mg kg−1 Cd (ERM®-BD513), (0.541 ± 0.024) mg kg−1 Cd (ERM®-BD514) and (0.690 ± 0.029) mg kg−1 Cd (ERM®-BD515). Acrylamide contents are given for information.
N-Myc is a transcription factor that is aberrantly expressed in many tumor types and is often correlated with poor patient prognosis. Recently, several lines of evidence pointed to the fact that oncogenic activation of Myc family proteins is concomitant with reprogramming of tumor cells to cope with an enhanced need for metabolites during cell growth. These adaptions are driven by the ability of Myc proteins to act as transcriptional amplifiers in a tissue-of-origin specific manner. Here, we describe the effects of N-Myc overexpression on metabolic reprogramming in neuroblastoma cells. Ectopic expression of N-Myc induced a glycolytic switch that was concomitant with enhanced sensitivity towards 2-deoxyglucose, an inhibitor of glycolysis. Moreover, global metabolic profiling revealed extensive alterations in the cellular metabolome resulting from overexpression of N-Myc. Limited supply with either of the two main carbon sources, glucose or glutamine, resulted in distinct shifts in steady-state metabolite levels and significant changes in glutathione metabolism. Interestingly, interference with glutamine-glutamate conversion preferentially blocked proliferation of N-Myc overexpressing cells, when glutamine levels were reduced. Thus, our study uncovered N-Myc induction and nutrient levels as important metabolic master switches in neuroblastoma cells and identified critical nodes that restrict tumor cell proliferation.
Metabolites from Alternaria fungi exhibit a variety of biological properties such as phytotoxic, cytotoxic, or antimicrobial activity. Optimization of a literature procedure culminated in an efficient total synthesis of (−)-altenuene as well as a stable isotope-labeled derivative suitable for implementation in a LC-MS/MS method for mycotoxin analysis.