Tandem mass spectral library search (MS/MS) is the fastest way to correctly annotate MS/MS spectra from screening small molecules in fields such as environmental analysis, drug screening, lipid ...analysis, and metabolomics. The confidence in MS/MS‐based annotation of chemical structures is impacted by instrumental settings and requirements, data acquisition modes including data‐dependent and data‐independent methods, library scoring algorithms, as well as post‐curation steps. We critically discuss parameters that influence search results, such as mass accuracy, precursor ion isolation width, intensity thresholds, centroiding algorithms, and acquisition speed. A range of publicly and commercially available MS/MS databases such as NIST, MassBank, MoNA, LipidBlast, Wiley MSforID, and METLIN are surveyed. In addition, software tools including NIST MS Search, MS‐DIAL, Mass Frontier, SmileMS, Mass++, and XCMS2 to perform fast MS/MS search are discussed. MS/MS scoring algorithms and challenges during compound annotation are reviewed. Advanced methods such as the in silico generation of tandem mass spectra using quantum chemistry and machine learning methods are covered. Community efforts for curation and sharing of tandem mass spectra that will allow for faster distribution of scientific discoveries are discussed.
In this article, a dataset from a collaborative non-target screening trial organised by the NORMAN Association is used to review the state-of-the-art and discuss future perspectives of non-target ...screening using high-resolution mass spectrometry in water analysis. A total of 18 institutes from 12 European countries analysed an extract of the same water sample collected from the River Danube with either one or both of liquid and gas chromatography coupled with mass spectrometry detection. This article focuses mainly on the use of high resolution screening techniques with target, suspect, and non-target workflows to identify substances in environmental samples. Specific examples are given to emphasise major challenges including isobaric and co-eluting substances, dependence on target and suspect lists, formula assignment, the use of retention information, and the confidence of identification. Approaches and methods applicable to unit resolution data are also discussed. Although most substances were identified using high resolution data with target and suspect-screening approaches, some participants proposed tentative non-target identifications. This comprehensive dataset revealed that non-target analytical techniques are already substantially harmonised between the participants, but the data processing remains time-consuming. Although the objective of a “fully-automated identification workflow” remains elusive in the short term, important steps in this direction have been taken, exemplified by the growing popularity of suspect screening approaches. Major recommendations to improve non-target screening include better integration and connection of desired features into software packages, the exchange of target and suspect lists, and the contribution of more spectra from standard substances into (openly accessible) databases.
Graphical Abstract
Matrix of identification approach versus identification confidence
As a widely used secondary vulcanization accelerator in the rubber industry, 1,3-diphenylguanidine (DPG) poses risks to human health and the environment. To compare and comprehend the disinfection ...process of DPG, this work investigates the reaction kinetics, toxicity, and transformation products (TPs) of DPG during chlorination and monochloramination. It has been revealed that the reactivity of monochloramine is significantly slower compared to chlorination of DPG, with the maximum efficiency observed at pH 7 to pH 8. Cytotoxicity assessment using HepG2 and THP-1 cells reveals that cytotoxicity hierarchy is as follows: chlorine TPs > monochloramine TPs > DPG. Moreover, oxidant-to-DPG molar ratios 10 and 20 lead to higher cytotoxicity in both chlorination and monochloramination compared to ratio 5 and 100. Additionally, cell bioenergetics experiments demonstrate that chlorine and monochloramine TPs induce mitochondrial dysfunction and enhance glycolytic function in HepG2 cells. The genotoxic response from p53 signaling further suggested genotoxic effects of certain TPs. Furthermore, analysis of TPs using high-resolution mass spectrometry (HRMS) identifies ten TPs, with chlorination yielding more TPs than monochloramination. Generally, a chlorine or monochloramine molar ratio to DPG of 10–20 results in an increased formation of TPs and heightened cytotoxicity. Notably, higher oxidant molar ratios increased the formation of monoguanidine TPs and DPG hydroxylation during chlorination, whereas monochloramination lead to DPG substitution predominantly generating chlorinated DPG due to weaker oxidation effects. These findings provide valuable information for the appropriate treatment of DPG and disinfection processes in water facilities to mitigate potential risks to human health and the ecosystem.
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•Maximum monochloramination kinetics efficiency for DPG was observed at pH 7.4.•In-vitro tests revealed cytotoxicity order: chlorine TPs > monochloramine TPs > DPG.•DBPs affected bioenergies by inducing mitochondrial dysfunction, enhancing glycolysis.•Ten TPs were identified, with chlorine TPs more than monochloramine TPs.•DPG treated with chlorine or monochloramine (10–20 ratio) increases TPs, cytotoxicity.
Widespread environmental contamination of legacy long-chain poly- and per-fluoroalkyl substances (PFASs) has triggered chemical regulatory action and a global transitioning to alternative PFASs. More ...than 5000 PFASs are now recognized on various lists, but few have been monitored despite ample evidence of unidentified organic fluorine in human and environmental samples. Nevertheless, our review of the literature indicates that nontarget analytical methods based on high-resolution mass spectrometry have been used to discover more than 750 PFASs, belonging to more than 130 diverse classes, in strategically selected environmental samples, biofluids or commercial products. Among these reports, we summarize the analytical and data-processing strategies for nontarget PFAS discovery, identify knowledge gaps and propose new areas for method development. Discovery of emerging PFASs before they are global contaminants could mitigate future contamination if strategic techniques can be developed to prioritize some of these substances for synthesis and confirmation, further monitoring, source elucidation and hazard characterization.
•Nontarget HRMS is already proven as a powerful tool for discovery of PFASs in strategically selected samples.•Identification of PFAS-features in complex HRMS spectra is a crucial step in the workflow.•Advances in analytical methods and feature identification will likely facilitate further discoveries in background samples.•The many new PFAS discoveries require strategies to prioritize substances for further confirmation, monitoring and research.
Untargeted metabolomics is now widely recognized as a useful tool for exploring metabolic changes taking place in biological systems under different conditions. By its nature, this is a highly ...interdisciplinary field of research, and mastering all of the steps comprised in the pipeline can be a challenging task, especially for those researchers new to the topic. In this tutorial, we aim to provide an overview of the most widely adopted methods of performing LC-HRMS-based untargeted metabolomics of biological samples. A detailed protocol is provided in the Supplementary Information for rapidly implementing a basic screening workflow in a laboratory setting. This tutorial covers experimental design, sample preparation and analysis, signal processing and data treatment, and, finally, data analysis and its biological interpretation. Each section is accompanied by up-to-date literature to guide readers through the preparation and optimization of such a workflow, as well as practical information for avoiding or fixing some of the most frequently encountered pitfalls.
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•A practical tutorial for UHPLC-HRMS untargeted metabolomics.•Preparation of biological samples.•UHPLC-HRMS platform selection.•Data processing and biological hypothesis generation.
Per- and polyfluoroalkyl substances (PFASs) have been manufactured and used for decades. The aquatic environment is a critical transportation and transformation compartment for PFASs. Target analysis ...is the most used method of tracing PFASs in the aquatic environment but is powerless for the large amounts of unknown PFASs. This review summarizes the advantages of three target analysis supplementary approaches, including extractable organofluorine (EOF) analysis, high-resolution mass spectrometry (HRMS) screening, and PFAS precursor oxidative conversion. For known PFASs, more targets, including emerging PFASs, PFAS isomers, ultrashort-chain PFASs, and cationic/zwitterionic PFASs, should be considered. For unknown PFASs and organofluorines, developing comprehensive and low-contaminated sample treatment strategies is essential yet challenging. We propose including PFASs screened via HRMS at confidence level (CL) 3 and above in EOF mass balance analysis and CL 4 for samples collected from a “known source”. Appropriate approaches should be applied to investigate unknown PFASs beyond target analysis comprehensively.
•Optimizing PFAS treatment method is essential for comprehensive analysis in water.•HRMS-screened PFASs at CLs 1−3 shall be included in EOF mass balance analysis.•Mass-labeled standards shall be used to assess oxidative conversion process.•OF mass balance shall be standardized to facilitate result communication.•Combining HRMS screening and precursor oxidative conversion has broad prospects.
Polyphenols comprise a large family of naturally occurring secondary metabolites of plant-derived foods and are among the principal micronutrients associated with the health beneficial effects of our ...diet. Liquid chromatography coupled to mass spectrometry (LC-MS) and, in the last few years, high resolution mass spectrometry (LC-HRMS) is playing an important role in the research of polyphenols, not only for the determination of this family of compounds in food matrices, but also for the characterization and identification of new polyphenols, as well as the classification and authentication of natural extracts in the prevention of frauds. The purpose of this review is to describe recent advances in the LC-MS and LC-HRMS analysis and characterization of polyphenols in food focusing on the most relevant applications published in the last years. Trends regarding sample treatment, chromatographic separation, mass analyzers and chemometric approaches used in the determination and characterization of polyphenols will be addressed.
•Recent advances in LC-MS determination of polyphenols in food are presented.•Sample treatment, separation, ionization and MS detection are addressed.•HRMS relevant applications on polyphenols with Orbitrap and TOF are reviewed.•Chemometrics for optimization and characterization of polyphenols is discussed.
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•Chemicals of Emerging Concern (CECs) are a growing concern in HBM.•Suspect and non-targeted screening (NTS) approaches offer new capabilities for capturing CECs in human ...matrices.•These new approaches applied to HBM come with a number of technical and scientific issues to be addressed.•Harmonisation is required at international level to reinforce NTS methods comparability and performance assessment.
Large-scale suspect and non-targeted screening approaches based on high-resolution mass spectrometry (HRMS) are today available for chemical profiling and holistic characterisation of biological samples. These advanced techniques allow the simultaneous detection of a large number of chemical features, including markers of human chemical exposure. Such markers are of interest for biomonitoring, environmental health studies and support to risk assessment. Furthermore, these screening approaches have the promising capability to detect chemicals of emerging concern (CECs), document the extent of human chemical exposure, generate new research hypotheses and provide early warning support to policy. Whilst of growing importance in the environment and food safety areas, respectively, CECs remain poorly addressed in the field of human biomonitoring. This shortfall is due to several scientific and methodological reasons, including a global lack of harmonisation. In this context, the main aim of this paper is to present an overview of the basic principles, promises and challenges of suspect and non-targeted screening approaches applied to human samples as this specific field introduce major specificities compared to other fields. Focused on liquid chromatography coupled to HRMS-based data acquisition methods, this overview addresses all steps of these new analytical workflows. Beyond this general picture, the main activities carried out on this topic within the particular framework of the European Human Biomonitoring initiative (project HBM4EU, 2017–2021) are described, with an emphasis on harmonisation measures.
Exposure science, in its broadest sense, studies the interactions between stressors (chemical, biological, and physical agents) and receptors (e.g. humans and other living organisms, and non-living ...items like buildings), together with the associated pathways and processes potentially leading to negative effects on human health and the environment. The aquatic environment may contain thousands of compounds, many of them still unknown, that can pose a risk to ecosystems and human health. Due to the unquestionable importance of the aquatic environment, one of the main challenges in the field of exposure science is the comprehensive characterization and evaluation of complex environmental mixtures beyond the classical/priority contaminants to new emerging contaminants.
The role of advanced analytical chemistry to identify and quantify potential chemical risks, that might cause adverse effects to the aquatic environment, is essential. In this paper, we present the strategies and tools that analytical chemistry has nowadays, focused on chromatography hyphenated to (high-resolution) mass spectrometry because of its relevance in this field. Key issues, such as the application of effect direct analysis to reduce the complexity of the sample, the investigation of the huge number of transformation/degradation products that may be present in the aquatic environment, the analysis of urban wastewater as a source of valuable information on our lifestyle and substances we consumed and/or are exposed to, or the monitoring of drinking water, are discussed in this article. The trends and perspectives for the next few years are also highlighted, when it is expected that new developments and tools will allow a better knowledge of chemical composition in the aquatic environment. This will help regulatory authorities to protect water bodies and to advance towards improved regulations that enable practical and efficient abatements for environmental and public health protection.
•The role of modern analytical chemistry is essential in exposure science.•Hyphenation of chromatography and HRMS allows the identification of many organic contaminants in the aquatic environment.•Different strategies (target, suspect, non-target) are used for the broad investigation of emerging chemical risks in water.•Monitoring programs and risks assessments are normally focused on parent contaminants.•Little information exists on occurrence and (eco)toxicological effects of transformation products/metabolites in water.
Liquid chromatography coupled to quadrupole‐based tandem mass spectrometry (QqQ) is termed the “gold standard” for bioanalytical applications because of its unpreceded selectivity, sensitivity, and ...the ruggedness of the technology. More recently, however, high‐resolution mass spectrometry (HRMS) has become increasingly popular for bioanalytical applications. Nonetheless, this technique is still viewed, either as a screening technology or as a research tool. Although HRMS is actively discussed during scientific conferences, it is yet to be widely utilised in routine laboratory settings and there remains a reluctance to use HRMS for quantitative measurements in regulated environments. This paper does not aim to comprehensively describe the potential of the latest HRMS technology, but rather, it focuses on what results can be obtained and outlines the author's experiences over a period of many years of the routine application of various forms of HRMS instrumentation. Fifteen years ago, some nine different QqQ methods were used in the author's laboratory to analyse a variety of different veterinary drug resides. Today, many more analytes are quantified by seven HRMS methods and just three QqQ methods remain in use for the analysis of a small set of compounds yet to be upgraded to HRMS analysis. This continual upgrading and migration of analytical methods were accompanied by regularly participating in laboratory proficiency tests (PTs). The PT reports (covering a range of analytes and analytical methods) were used to compare the accuracy of HRMS‐ versus QqQ‐based measurements. In the second part of this paper, the particular strengths and limitations of HRMS for both method development and routine measurements are critically discussed. This also includes some anecdotal experiences encountered when replacing QqQ assays with HRMS methods.
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