Analytische Chemie
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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.
Contamination of the environment with antibiotics is of great concern as it promotes the evolution of antimicrobial resistances. In case of amoxicillin (AMX) in the aquatic environment, further risk arises from hydrolysis products (HPs) which can cause allergy. To assess these risks, a comprehensive investigation and understanding of the degradation of AMX is necessary. We investigated the hydrolysis rate of AMX in different types of water as well as the influence of temperature and irradiation. The content of the heavy metal ions copper and zinc was found to be crucial for the hydrolysis rate of AMX and stability of HPs. Eventually, a new degradation pathway for AMX could be elaborated and confirmed by tandem mass spectrometry (LC-MS/MS).
Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is increasingly used to study the distribution of metal-containing drugs, imaging probes and nanomaterials in connection with disease related changes and therapy progress. Additionally, biomolecules can be detected indirectly by using metal-tagged antibodies.
The extracellular matrix (ECM) is, besides the cells, an important component of all body tissues. The macromolecular network of the ECM consists of structural proteins (e.g., collagen, elastin) and proteoglycans composed of highly negatively charged carbohydrates, the glycosaminoglycans (GAGs), which are covalently linked to a protein core. Many diseases, including inflammatory processes and tumors, are associated with characteristic ECM changes at an early stage. Recent studies have shown that contrast agents for magnetic resonance imaging (MRI), which are based on gadolinium containing chelate complexes or iron oxide nanoparticles, can bind themselves to ECM components.
To elucidate the role of GAGs like keratan sulfate (KS) and its modification state in disease, highly specific tools are necessary. As a complement to conventional immunohistochemistry LA-ICP-MS was applied to investigate the distribution of KS in tissue thin sections using a well characterized anti-KS antibody labelled with metal ions. Furthermore, LA-ICP-MS was used for the detection of MRI contrast agents and the identification of their target cells and molecules in tissue samples from animal models, e.g. for cardiovascular diseases. The results show the possibilities of LA-ICP-MS for the elucidation of pathological tissue changes.
Inductively coupled plasma-mass spectrometry (ICP-MS) is a well-established analytical method offering high sensitivity and multi-element analysis. ICP-MS has found acceptance in various application areas ranging from material analysis to applications in the life sciences. Within the last 15 years new strategies for the sensitive detection and accurate quantification of biomolecules in complex biomedical samples have been developed. Recent instrumental improvements have contributed to this progress.
As most of the biomolecules do not contain endogenous metals etectable with ICP-MS, bioconjugation with artificial metal-containing tags based on metal-loaded chelate complexes or nanoparticles is increasingly applied to determine biomolecules indirectly. Especially, the combination of immunohistochemical workflows using lanthanoid-tagged antibodies and ICP-MS detection provides new insights in the complexity and interdependency of cellular processes. Single-cell ICP-MS, also termed as mass cytometry, allows high-dimensional analysis of biomarkers in cell populations at single-cell resolution. For that purpose, lanthanoid isotope labelled antibodies are used to detect their corresponding target molecules. The visualisation of the elemental distribution is possible with laser ablation ICP-MS (LA-ICPMS) at high spatial resolution. Especially, the combination of LA with ICP time-of-flight mass spectrometry, also referred to as imaging mass cytometry (IMC), opens new possibilities for multiparametric tissue imaging at the single-cell level and even below. The lanthanoid localisation and concentration can be linked to their conjugated antibody target providing valuable information about surface markers, intracellular signalling molecules to measure biological function, and the network state of an individual cell in a tissue.
This book chapter focuses on new applications, where the multi-element capabilities of ICP-MS are used for the detection of lanthanoids applied as artificial elemental stains or tags for biomolecules and in particular antibodies.
A new method combining isotope dilution mass spectrometry (IDMS) and standard addition has been developed to determine the mass fractions w of different elements in complex matrices: (a) silicon in aqueous tetramethylammonium hydroxide (TMAH), (b) sulfur in biodiesel fuel, and (c) iron bound to transferrin in human serum. All measurements were carried out using inductively coupled plasma mass spectrometry (ICP–MS). The method requires the gravimetric preparation of several blends (bi)—each consisting of roughly the same masses (mx,i) of the sample solution (x) and my,i of a spike solution (y) plus different masses (mz,i) of a reference solution (z).
Only these masses and the isotope ratios (Rb,i) in the blends and reference and spike solutions have to be measured. The derivation of the underlying equations based on linear regression is presented and compared to a related concept reported by Pagliano and Meija. The uncertainties achievable, e.g., in the case of the Si blank in extremely pure TMAH of urel (w(Si)) = 90% (linear regression method, this work) and urel (w(Si)) = 150% (the method reported by Pagliano and Meija) seem to suggest better applicability of the new method in practical use due to the higher robustness of regression analysis.
Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is nowadays a versatile and powerful analytical method for direct solid sample analysis. The applicability has been demonstrated for a wide variety of samples covering hard and soft materials. In an imaging mode the technique provides quantitative information on the elemental distribution within a sample. LA-ICP-MS imaging is of particular interest in biomedical research as the distribution of an element gives valuable insight on uptake and distribution of essential and toxic trace elements, administered contrast agents as well es nanoparticles. LA-ICP-MS is therefore a powerful complement to other imaging techniques. Recent instrumental improvements, especially in sample chamber design, have contributed to better sensitivity and spatial resolution enabling subcellular imaging. The book chapter provides a comprehensive overview about spatially resolved localisation and quantification of various nanoparticles in cells and tissue thin sections by LA-ICP-MS. Furthermore, different sample preparation strategies and internal standardisation and calibration approaches for bioimaging by LA-ICP-MS are summarized and discussed.
Metal-containing nanomaterials are used in numerous fields ranging from industrial applications to nanomedicine. Several studies have demonstrated that the physicochemical properties of nanoparticles have an impact on their pharmacokinetics, transfer and clearance. The high sensitivity and multielement capability of LA-ICP-MS enables the elucidation of interactions between tissue components and nanomaterials used as imaging probes or drug carriers. Potential toxic effects are investigated as well. Thus, LA imaging significantly supports the clinical translation of safe and efficient nanoparticles for diagnostic and therapeutic purposes.
Nearly all disease processes are associated with variations of components of the extracellular matrix (ECM) that are typically observed during the development of inflammation. This concerns for example proteoglycans and their associated glycosaminoglycans (GAG), which have been shown to bind to cationic metal imaging probes due to their strong complexing activity. The complexing activity largely depends on the degree of GAG sulfation and/or carboxylation as well as on the GAG isomericity. In this central project, we investigate GAG structures from inflammatory disorders (namely cardiovascular diseases, inflammatory intestinal diseases and neuroinflammation) provided by researchers of the Collaborative Research Center at the molecular disaccharidic level using chromatographic and mass spectrometric methods. In parallel, the spatial localization and quantification of metal-based imaging probes are evaluated by LA-ICP-MS imaging.
Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful method for the matrix-independent quantitative analysis of target elements. Developed for the use in inorganic trace analysis, ICP-MS is nowadays a valuable tool for bioanalytical questions. Especially the use of ICP-MS for quantitative proteomics by measuring heteroatoms has gained recognition in the last decade, considering that established quantification methods like organic mass spectrometry depend on labelling of the target protein or the existence of matched protein and peptide standards. The need for reliable quantification of proteins is continuously growing, but only a limited number of well-characterized and quantified protein standards are available so far. Accurately quantified, traceable protein standards are necessary to ensure comparability of measurements between laboratories, not only in basic research but also in a clinical context. One example of this is the Alzheimer’s disease biomarker tau protein. However, existing tau standards lack comparability, emphasizing the need for a well-quantified protein standard.
Therefore, we developed a method for the quantification of pure proteins via sulfur isotope dilution ICP-MS (IDMS). As sulfur is present in two amino acids, cysteine and methionine, it exists in nearly all proteins and can be used for the quantification of proteins of known stoichiometry. We employed simple offline strategies for the separation of non-protein bound sulfur species. Quantification of these contaminations by IDMS allows for correction of the protein content and enables reliable protein quantification. We report the protein mass fractions of a standard reference material and commercially available proteins determined by sulfur IDMS, including the expanded uncertainties. The developed method can be applied for the reliable and traceable quantification of pure proteins for use as in-house standards. Here, we successfully used this method for the quantification of the tau protein.
In den vergangen Jahren hat das Umweltbewusstsein in der Bevölkerung stark zugenommen und somit auch das Interesse an der Vermeidung von anthropogenen (Schad-)Stoffen in der Umwelt. Eine (neue) Substanzklasse, deren Umweltauswirkungen noch nicht vollständig untersucht sind und die in den vergangenen Jahren immer mehr an Bedeutung gewonnen hat, sind (metallbasierte) Nanomaterialien. Im Gegensatz zu bspw. Elementspezies weisen Nanomaterialien eine Vielzahl von Eigenschaften auf und lassen sich nicht über nur ein Merkmal beschreiben - dies stellt eine große analytische Herausforderung dar. Hier haben sich vor allem die Feld-Fluss-Fraktionierung (AF4) und die single-particle-ICP-MS als leistungsstarke analytische Methoden herausgestellt. In (aquatischen) Umweltmatrizes (z.B. Oberflächengewässern) liegen neben artifiziellen auch natürliche Partikel vor, was eine weitere große Herausforderung für den Nachweis von Nanomaterialien darstellt.
Neben dem Nachweis von anthropogenen Stoffen in der Umwelt ist zudem deren ökotoxikologische Bewertung wichtig. In der aquatischen Ökotoxikologie werden hierzu Testorganismen mit den jeweiligen Substanzen über die Wasserphase exponiert. Effektkonzentrationen (EC50) werden dabei auf Basis der Konzentrationen in der Wasserphase abgeleitet - tatsächlich bioakkumulierte Mengen werden hierbei jedoch meist nicht ermittelt; eine weitere große Herausforderung besteht zudem in der Bewertung von Mischungstoxizitäten. Gängige Testorganismen sind u.a. Kieselalgen (Diatomeen). Diatomeen stehen am Anfang der Nahrungskette - toxikologisch relevante Metalle/Nanomaterialien können sich hierüber im Nahrungsnetz der Oberflächengewässer anreichern und ggf. nachhaltig auswirken.
Im ersten Teil des Vortrages werden zunächst neue elementanalytische Methoden zum Nachweis von metallbasierten Nanopartikeln in Umweltmatrizes auf Basis der AF4/ICP-SFMS sowie stabilen Isotopenlabeln am Beispiel von Eisennanopartikeln vorgestellt.
Im zweiten Teil wird eine neue elementanalytische Methode als komplementäre Technik zur ökotoxikologischen Bewertung von (Schad-)Stoffen vorgestellt. Die neue Methode basiert auf der on-line Kopplung von HPLC mit der single-cell-ICP-(ToF)-MS (sc-ICP-(ToF)-MS) [1, 3-5]. Hierüber konnten wir erfolgreich die automatisierte Multielementanalytik einzelner Diatomeen realisieren und zur Analyse von mit Metallen inkubierten Diatomeen (cyclotella meneghiniana) einsetzen. Wir konnten zeigen, dass die sc-ICP-ToF-MS zukünftig eine leistungsstarke, komplementäre Technik in der aquatischen Ökotoxikologie zum z.B. Test von Metallen und Nanomaterialien darstellt.
In this study, we developed a new immunoprecipitation method based on protein-coated nanodiamonds. Performing SDS-PAGE and using the SDS-PAGE buffer as an eluent showed that CER could be successfully enriched from human serum. Based on the copper determination with ICP-MS, the amount of bound CER on the nanodiamonds can be calculated. We could show the fulfilled mass balance of bound CER and CER in the supernatant after incubation with a known amount of CER. For isotope ratio analysis this method can be applied to compare ratios of the total copper content in human serum to copper ratios from CER enrichment.