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A collaborative study was conducted to understand the correlation between pigments purity profile (primary aromatic amine content of the pigments) and the behaviour of these PAAs during cold water extraction (CWE) tests according to EN 645. From a selection of organic pigments based on seven colour indexes (PR122, PR184, PO13, PY74, PY111, PY138 and PY155), the pigment purity profile was established according to European Resolution AP (89) 1, then mono-pigmented inks were prepared and napkins printed with these inks. In a second step, cold water extraction and PAA determination were performed by two independent laboratories. In one laboratory, an analytical method based on LC-MS/MS was used, whereas in the other laboratory a method based on LC-HRMS using Orbitrap technology was developed for the simultaneous analysis of 35 PAAs. Good qualitative results were obtained if we consider that at significant levels the PAAs were positively detected in both laboratories, except for 3-amino-4-methoxybenzanilide and 8-amino-2-methyl-quinoline, for which inter-laboratory differences were observed. It was also shown that no contamination from unexpected PAAs was detected. The comparison between pigment analysis and CWE results shows that if the pigment purity profile is of major importance, other parameters such as pigment surface treatment, ink grinding process or ink formulation could have an important influence on the CWE results. For such sensitive applications, for example napkins or other Food Contact Materials (FCM), it is therefore recommended not only to select a pigment with a good purity profile but also to test the pigment in the final application. Finally, this work highlights the difficulty of validating a product on a single analysis and shows the importance of a multilevel global assessment on worst case application. [ABSTRACT FROM AUTHOR]

BACKGROUND Fluopyram is a pesticide widely used in tomato and cucumber crops cultivation to control fungal diseases that develop especially in environments with moderate temperatures and high humidity, such as in a greenhouse. The pathway of fluopyram dissipation has been monitored in cucumber and cherry tomato under greenhouse conditions. RESULTS: In the greenhouse trials, cherry tomato and cucumber were treated by irrigation water with the commercial product at the manufacturer's recommended dose and double dose. High‐resolution mass spectrometry (HRMS) coupled to ultra‐high‐performance liquid chromatography (UHPLC) has been selected as the technique to obtain the identification of fluopyram and metabolites. The fate of fluopyram in greenhouse tomato and cucumber was investigated over 44 days. The metabolic pathway of fluopyram was: in a first step there was a primary transformation to fluopyram‐7‐hydroxy and fluopyram‐8‐hydroxy, isomeric compounds, and in a second phase to fluopyram‐benzamide and fluopyram‐pyridyl‐carboxylic acid. The behavior of fluopyram does not fit any type of kinetic classical model of degradation. CONCLUSIONS: Greenhouse trials revealed that the fluopyram is a very persistent compound, and their terminal residues do not exceed maximum residue level (MRL) at the end of the study. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Asymmetrical flow field-flow fractionation (AF4) and high-resolution Orbitrap mass spectrometry (HRMS) were used to separate and characterize cellular fractions of the dark- and light-grown Euglena gracilis cellular material. Biological replicates analyzed by HRMS shared 21–73% of commonly detected m/z values. Greater variability in shared features was found in light-grown cellular fractions (p < 0.05), likely due to small variations in growth stage. Significant differences in molecular composition were observed between AF4 cellular fractions, with dark cell fractions showing a propensity towards carbohydrate-like and tannin-like compounds, and higher double-bond equivalent (DBE) and modified aromatic index (AImod) were associated with light-grown cell fractions. Fractionation and high-resolution mass spectrometry aided characterization demonstrated the power of the AF4 to selectively cater to certain compounds/cellular entities with distinct compositional classes and double-bond equivalents and aromaticity index characteristics. [ABSTRACT FROM AUTHOR]

Steroids are essential structural components of cell membranes that organize lipid rafts and modulate membrane fluidity. They can also act as signalling molecules that work through nuclear and G protein–coupled receptors to impact health and disease. Notably, changes in steroid levels have been implicated in metabolic, cardiovascular and neurodegenerative diseases, but how alterations in the steroid pool affect ageing is less well understood. One of the major challenges in steroidomic analysis is the ability to simultaneously detect and distinguish various steroids due to low in vivo concentrations and naturally occurring stereoisomers. Here, we established such a method to study the mass spectrometry behaviour of nine sterols/steroids and related molecules (cholesterol precursors: squalene, lanosterol; sterol metabolites; 7 Dehydrocholesterol, 24, 25 and 27 Hydroxycholesterol; and steroids: progesterone, testosterone, and corticosterone) during ageing in the African turquoise killifish, a new model for studying vertebrate longevity. We find that levels of all tested steroids change significantly with age in multiple tissues, suggesting that specific steroids could be used as biomarkers of ageing. These findings pave the way for use of Nothobranchius furzeri as a novel model organism to unravel the role of sterols/steroids in ageing and age-related diseases. [ABSTRACT FROM AUTHOR]

In the present work, a novel database of drug compounds and a rapid screening method based on ultra‐high performance liquid chromatography coupled to high resolution orbitrap mass spectrometry were developed and applied in the screening and identification of targeted and nontargeted antihypertensive adulterants in dietary supplements and herbal medicines. The established screening database includes retention time, exact mass, fragments, isotopic pattern, and MS2 spectra library of the target compounds and thus provides automated search and identification of the targets with a single injection. The nontargeted compounds in the samples are identified through the full MS scan and MS2 data by using the Chemspider database and the data analysis in XCalibur, MassFrontier and TraceFinder software. In addition, this method possesses excellent quantitative capacity. The novel approach was applied to 65 batches of samples that are claimed as "all‐natural" products having the antihypertensive function, among which nine batches were found to be positive. Multiple targeted and nontargeted antihypertensive adulterants were detected at levels ranging from 2.8 to 27.9 mg/g. The novel database and screening method demonstrated herein will be promising and powerful tools for rapid screening of antihypertensive adulterants in dietary supplements and herbal medicines. [ABSTRACT FROM AUTHOR]

Background: The sensitivities and specificities of C-reactive protein (CRP) and faecal calprotectin (fCal), as recommended for inflammatory bowel diseases (IBD) diagnosis and monitoring, are low. Our aim was to discover new stool protein/peptide biomarkers for diagnosing IBD. Methods: For peptides, MALDI-TOF/MS (m/z 1000–4000) was performed using stools from an exploratory (34 controls; 72 Crohn's disease [CD], 56 ulcerative colitis [UC]) and a validation (28 controls, 27 CD, 15 UC) cohort. For proteins, LTQ-Orbitrap XL MS analysis (6 controls, 5 CD, 5 UC) was performed. Results: MALDI-TOF/MS spectra of IBD patients had numerous features, unlike controls. Overall, 426 features (67 control-associated, 359 IBD-associated) were identified. Spectra were classified as control or IBD (absence or presence of IBD-associated features). In the exploratory cohort, the sensitivity and specificity of this classification algorithm were 81% and 97%, respectively. Blind analysis of the validation cohort confirmed 97% specificity, with a lower sensitivity (55%) paralleling active disease frequency. Following binary logistic regression analysis, IBD was independently correlated with MALDI-TOF/MS spectra (p < 0.0001), outperforming fCal measurements (p = 0.029). The IBD-correlated m/z 1810.8 feature was a fragment of APC2, homologous with APC, over-expressed by infiltrating cells lining the surface in UC or the muscularis-mucosae in CD (assessed by immunohistochemistry). IBD-associated over-expressed proteins included immunoglobulins and neutrophil proteins, while those under-expressed comprised proteins of the nucleic acid assembly or those (OLFM4, ENPP7) related to cancer risk. Conclusions: Our study provides evidence for the clinical utility of a novel proteomic method for diagnosing IBD and insight on the pathogenic role of APC. Moreover, the newly described IBD-associated proteins might become tools for cancer risk assessment in IBD patients. [ABSTRACT FROM AUTHOR]

Aims: Plant-microbial-soil interactions are key to understanding plant community succession, invasion success, patterns of biodiversity and aspects of ecosystem function. Yet root and rhizosphere chemistry is highly complex, and little is known about natural variation across environmental gradients. Variation in tree species root chemical phenotypes should alter how rhizosphere microbes respond, showing a plant conditioning effect on the chemical makeup of the soil. Here, we used metabolomics to assess bulk small molecule profiles addressing the hypothesis that genetic variation across a species range would result in varying metabolic profiles in roots and surrounding soil. Methods: Using UPLC-HRMS we assessed the small molecule profile of root tissue and surrounding rhizosphere soil from 5-year old plant clones collected from six populations of Populus angustifolia across the western U.S., grown in a common environment. Results: Population-level variation was found in over 12,000 root metabolomes and over 5000 soil organic compounds across the populations. Redundancy analysis of over twelve thousand metabolites suggests that plant population origin can account for up to 36% of the variation in roots and 30% of the variation in rhizosphere soil chemistry. Co-inertia analysis indicates that variation in root metabolite profiles explains 15% of the variation in paired soil samples. Conclusion: Distinct populations have evolved different root tissue metabolomes. The difference in root metabolites across populations altered the rhizosphere soil composition, creating variable soil chemical communities from a homogenous starting condition. This suggests that intra-specific plant conditioning of soil varies by plant population. [ABSTRACT FROM AUTHOR]

A multiclass and multiresidue method for screening veterinary drugs and pesticides in infant formula was developed and validated using ultrahigh‐performance liquid chromatography coupled to Quadrupole‐Orbitrap high‐resolution mass spectrometry (UHPLC‐HRMS). A total of 49 veterinary drugs and pesticides investigated belong to 11 classes including antivirals, anticoccidials, macrolides, pyrethroids, insecticides, sulfonamides, beta‐agonists, sedatives, thyreostats, nonsteroidal anti‐inflammatory drugs, and other pharmacologically active substances. A generic sample preparation and highly selective acquisition mode of parallel reaction monitoring (PRM) were deliberately incorporated to perform efficient screening analysis. As a result, the screening target concentrations of the analytes varied from 1 to 500 μg/kg with ≤5% of false compliant rate as specified in Decision 2002/657/EC for screening analysis. The average recoveries ranged from 40.7 to 124.9% as well as the relative standard deviations from 4.2 to 26.6%, respectively. The matrix effects and interferences were effectively controlled by integrated application of dispersive solid phase extraction, PRM scan mode, and matrix‐matched standard calibration. The proposed method will be helpful to provide applicable strategy for screening residues in infant formula with surveillance purpose. [ABSTRACT FROM AUTHOR]

This study aims to assess the influence of growth and culture conditions of the Euglena gracilis protist on the cellular chemical composition. Fourier transform infrared spectroscopy (FT-IR) and direct infusion Orbitrap electrospray ionization mass spectrometry (ESI-MS) were used in tandem for confirmation of functional groups, and to decipher the normalized percentage abundances of compound classes, molecular species, and aromaticity and oxygenation within each growth condition. Abundances of molecular species and compound classes varied with growth and culture conditions. No major differences in the qualitative FT-IR-based screening of functional groups were found between growth conditions. This contrasts with ESI-MS-based results where polyphenols, carbohydrate, and condensed aromatic compounds were the most abundant in dark-grown conditions with glucose supplementation, revealing the differences in physiological states of the cell in different growth conditions. Dark-grown conditions promoted CHO molecular species while light-grown conditions favored CHON, CHOS, and CHONS molecular species. The dark-grown exponential E. gracilis cells grown in glucose-supplemented media showed the greatest normalized abundances of aromatics, carbohydrate, polyphenols, and protein, which are compound classes typically involved in metal binding. [ABSTRACT FROM AUTHOR]

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging is a useful tool for identifying important meta-metabolomic features pertinent for enhancing our understanding of biological systems. Magnaporthe oryzae (M. oryzae) is a filamentous fungus that is the primary cause of rice blast disease. True to its name, M. oryzae primarily destroys rice crops and can also destroy other cereal crops as well. In a previous study, the F-box E3 ligase protein in M. oryzae was noted to be crucial for its growth and pathogenicity. In this study, we inoculated three separate sets of barley with wild-type M. oryzae, an F-box E3 ligase protein knock out of M. oryzae, and a control solution. Over the course of the infection (8 days), we imaged each treatment after development of an advanced polarity switching method, which allowed for the detection of low and high molecular weight compounds that ionize in positive or negative polarities. A set of features from initial experiments were chosen for another analysis using tandem mass spectrometry. Serotonin, a barley defense metabolite, was a compound identified in both positive and negative modes. Serotonin was putatively identified using MS1 data including carbon estimation and sulfur counting then confirmed based on tandem mass spectrometry fragmentation patterns. Metabolites in the melanin pathway, important for infection development of M. oryzae, were also identified using MS1 data but were unable to be confirmed with MS/MS due to their low abundances. [ABSTRACT FROM AUTHOR]

[Display omitted] • Stable products of oxidation measured by FTIR. • Ketohydroperoxides were detected by Orbitrap. • Highly oxidized products were detected by Orbitrap. • Highly unsaturated products were detected by Orbitrap. • Products of Korcek and Waddington mechanisms detected. Limonene-O 2 -N 2 and α-pinene-O 2 -N 2 mixtures were oxidized in a jet-stirred reactor at atmospheric pressure, in the cool flame regime, and fuel-lean conditions. Samples of the reacting mixtures were analyzed by on-line Fourier transform infrared (FTIR) and collected, dissolved in acetonitrile for analysis by flow injection or chromatographic separation by ultra-high performance liquid chromatography and Orbitrap mass spectrometry. OH/OD exchange using D 2 O and reaction with 2,4-dinitrophenylhydrazine were carried out for probing the existence of hydroxyl or hydroperoxyl, and carbonyl functions in the products, respectively. A large number of oxidation products, including highly oxygenated organic products with more than ten oxygen atoms, were observed. Unexpectedly, aromatic and polyunsaturated products with a contribution of 8–10% were detected for both terpenes over the range of temperatures studied. Van Krevelen plots, computed oxidation state of carbon, aromaticity index, and aromaticity equivalent index in products were used to rationalize the results. [ABSTRACT FROM AUTHOR]

[Display omitted] • High-throughput quantitative HRMS method for underivatized amino acid analysis. • Guard column-hydrophilic interaction material for sample introduction into Orbitrap. • Appropriate Orbitrap data acquisition mode was selected. • Guard-column cleaning effect and displacement of analytes from matrix components. • Reliable quantitative analysis of amino acids performed in less than 3 min. Nowadays, there is a great interest in the development of rapid methods for the determination of compounds of clinical interest in biological samples, such as amino acids in urine samples. Mass spectrometry-based methods have been proposed with this objective, although the use of low resolution mass analyzers sometimes cannot provide accurate results. Orbitrap has barely been used with quantitative purposes for the targeted analysis of amino acids in urine although it has been used to a greater extent for non-targeted assays to identify potential biomarkers of different diseases. The use of a guard column for sample introduction into the Orbitrap was evaluated. The coupling of a hydrophilic interaction-guard column significantly improved the MS-signals by producing a matrix cleaning effect with a slight displacement of the target analytes. Possible isobaric interferences were studied and, consequently, different modes of MS data acquisition were tested: Full MS acquisition mode was selected for those analytes for which it was possible their determination by exact mass, and parallel reaction monitoring mode was used for those compounds showing isobaric interferences. Method validation was performed using synthetic urine. Limits of detection between 0.003 and 1.50 mg/L and limits of quantification between 0.10 and 4.95 mg/L were found with recoveries in the 80–130 % range. The runtime was 1.6 min and the total time between injection was less than 3 min. The optimized method was satisfactorily applied to the analysis of urine from healthy volunteers. This work describes for the first time the direct coupling of a hydrophilic interaction-guard column to a Orbitrap high resolution mass spectrometer (HRMS) for the development of a fully validated quantitative method for the determination of underivatized amino acids in urine. The method proposed here represents a high throughput and reliable approach, with an analysis runtime of 1.6 min, much less than the time required when chromatographic techniques were used. [ABSTRACT FROM AUTHOR]

[Display omitted] • Demonstrating the LS-APGD-Orbitrap platform for uranium isotopic determination in the presence of heavy element interferences. • High-resolution Orbitrap MS to resolve interferences which hinder traditional inorganic mass spectrometric platforms. • LS-APGD-Orbitrap platform accurately identified 235U and 238U in the presence (5000×) of heavy elements (e.g., W, Pb). The determination of actinide (e.g., U and Pu) content and isotopics is of importance to the nuclear forensics and safeguards communities. However, in the analysis of environmental samples, such as those collected by the International Atomic Energy Agency, uranium measurements can be complicated by isobaric interferences from polyatomic variants of heavy elements (e.g., Pb). This leads to complex, time-consuming sample manipulations (i.e., separations) before determining isotope ratios. An alternative strategy to sample pretreatment is to use high-resolution mass spectrometric platforms during the analysis to fully resolve the uranium isotopes from potential polyatomic interferences, negating the need for prior chemical separation. The liquid sampling - atmospheric pressure glow discharge (LS-APGD) coupled with an Orbitrap mass spectrometer provides a high-resolution (>70,000 m/Δm at m / z 200) inorganic mass spectrometry platform. As a demonstration of the power of this instrumental platform, and indeed, the high-resolution approach in general, uranium isotope ratios were determined in the presence of elemental impurities (e.g., Pb, Pt, Ta, W) commonly encountered with environmental sample swipe analysis, without any prior treatment. Even at elemental impurity concentrations of 1000–5000× relative to uranium, no interference was observed with the 235U or 238U signal. In addition, the 235U/238U isotope ratio for the samples with the concomitants present are within 2 standard deviations of the values obtained without their addition, indicating that these impurities do not impact the determined uranium isotope ratio. These findings represent a significant first step in leveraging the high resolution of the LS-APGD-Orbitrap-MS to overcome isobaric and molecular interferences instead of relying on chemical separations. [ABSTRACT FROM AUTHOR]

Native mass spectrometry (MS) is a powerful and complementary tool and has been widely used for the characterization of proteins and protein complexes. Over the past decades, several mass analyzers have been developed with extended mass range and increased mass resolution for the analysis of higher-order structures of native macromolecular assemblies. In particular, the Orbitrap mass analyzer has attracted considerable interest due to its superior desolvation and resolution capabilities. In this review, the recent progress of native Orbitrap-MS in the characterization of higher-order structures of proteins is summarized. A brief introduction to the evolution of Orbitrap mass analyzer and commonly used ionization techniques in Orbitrap mass spectrometers is presented, including electrospray ionization (ESI), nanoESI, and desorption ESI (DESI). Then, four activation techniques in tandem MS and their applications in native Orbitrap-MS are examined. The applications of native Orbitrap-MS are emphasized according to three main aspects: membrane proteins, protein assemblies, and proteoform profiling. Finally, future perspectives and challenges are explored regarding the instrumentation and research orientations of Orbitrap-MS. This review provides the scientific community with a comprehensive understanding of native Orbitrap-MS for the analysis of large macromolecular assemblies and encourages the promotion of Orbitrap-MS. [Display omitted] • Instrumentation developments in Orbitrap-based mass spectrometers are reviewed. • Recent applications of native Orbitrap-MS for probing higher-order structures of proteins are summarized. • Current challenges and future perspectives of native Orbitrap mass spectrometry are discussed. [ABSTRACT FROM AUTHOR]

The abundances of different isotopic forms of a compound, or isotopologues, will vary based on its physical and chemical history. The number of isotopologues increases combinatorically with the size of a molecule, and even small molecules such as amino acids have thousands of potentially observable isotopic variants. However, due to the analytical challenges of separating and observing isotopologues, only a few dimensions of isotopic diversity are routinely measured. Overcoming these challenges requires both an experimental method to observe many isotopic properties and a theoretical framework for interpreting these experiments. In Part 1, we presented such a theoretical framework; here, we demonstrate an experimental method, which we apply to methionine. Our approach uses a Q Exactive HF Orbitrap to perform several "M + N experiments", where a sample is ionized, a subset of its isotopologues with cardinal mass N daltons greater than the unsubstituted isotopologue is selected and fragmented, and the proportions of all detectable isotopic forms of those fragment ions are quantified. We perform M + 1, M + 2, M + 3, and M + 4 experiments of a methionine sample and standard where the sample has a 100 ‰ enrichment of 13C at the methyl carbon relative to the natural 13C abundance at that position in the standard, and is otherwise identical to the standard. We observe isotopic forms of 8 fragment ion species for each version of the M + N experiment. With the assistance of a forward model of expected mass spectra, we identify isotopic peaks for each fragment ion based on observed mass and abundance, screen these for data quality, and quantify abundances for 146 unique isotopic peaks at precisions of ≈ 0.3–3 ‰. We present our direct observations and use them to reconstruct the concentrations of 19 individual singly, doubly, and triply-substituted isotopologues; doing so gives fewer constraints and broader error bars than working with the direct observations, but may be more interpretable for some applications. We also examine possibilities for measuring additional peaks, which are primarily limited by the detection limit of the Orbitrap-IRMS method. We then suggest some possible uses of our direct measurements for chemical forensics and hypothesis testing. Our results demonstrate the diversity of isotopic constraints currently observable and interpretable for organic molecules. • Orbitrap-IRMS method to observe high dimensional isotopic data of target compounds. • Observation of 146 isotopic features of methionine at precisions on the order of 1 ‰. • Computation of the enrichment of multiply-, including triply-, substituted isotopologues at specific molecular positions. • Automation aids for experimental design to apply high-dimensional measurements to new compounds. [ABSTRACT FROM AUTHOR]

Oleraceins are a class of indoline amide glycosides found in Portulaca oleracea L. (Portulacaceae), or purslane. These compounds are characterized by 5,6-dihydroxyindoline-2-carboxylic acid N-acylated with cinnamic acid derivatives, and many are glucosylated. Herein, hydromethanolic extracts of the aerial parts of purslane were subjected to UHPLC-Orbitrap-MS analysis, in negative ionization mode. Diagnostic ion filtering (DIF), followed by diagnostic difference filtering (DDF), were utilized to automatically filter out MS data and select plausible oleracein structures. After an in-depth MS2 analysis, a total of 51 oleracein compounds were tentatively identified. Of them, 26 had structures, matching one of the already known oleracein, and the other 25 were new, undescribed in the literature compounds, belonging to the oleracein class. Moreover, based on selected diagnostic fragment ions, clustering algorithms and visualizations were utilized. As we demonstrate, clustering methods provide valuable insights into the mass fragmentation elucidation of natural compounds in complex mixtures.

The analysis of drugs of abuse in hair and other biological matrices of forensic interest requires great selectivity and sensitivity. This has been traditionally achieved through target analysis, using one or more analytical methods that include different preanalytical stages, and more complex procedures followed by toxicological laboratories. There is no exception with 2C-series drugs, such as 2C-B, a new psychoactive substance (NPS), which use has emerged and significantly increased, year by year, in the last decades. Continuously new analytical methods are required to selectively detect and identify these new marketed substances at very low concentrations. In this case report, one former case of a polydrug consumer (charged of a crime against public health in Spain) was reanalyzed in hair matrix. In this reanalysis, 2C-B has been positively detected and identified using liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS/MS). The most selective analytical UHPLC-HRMS/MS method alongside a universal and simpler pretreatment methodology has opened up more possibilities for the detection of substances of different chemical structure and optimization of different HRMS/MS detection approaches allowing the identification of 2-CB in the hair of a real forensic case.

Haloarchaea are extreme halophilic microorganisms belonging to the domain Archaea, phylum Euryarchaeota, and are producers of interesting antioxidant carotenoid compounds. In this study, four new strains of Haloarcula sp., isolated from saline lakes of the Atacama Desert, are reported and studied by high-resolution mass spectrometry (UHPLC-Q-Orbitrap-MS/MS) for the first time. In addition, determination of the carotenoid pigment profile from the new strains of Haloarcula sp., plus two strains of Halorubrum tebenquichense, and their antioxidant activity by means of several methods is reported. The effect of biomass on cellular viability in skin cell lines was also evaluated by MTT assay. The cholinesterase inhibition capacity of six haloarchaea (Haloarcula sp. ALT-23; Haloarcula sp. TeSe-41; Haloarcula sp. TeSe-51; Haloarcula sp. Te Se-89 and Halorubrum tebenquichense strains TeSe-85 and Te Se-86) is also reported for the first time. AChE inhibition IC50 was 2.96 ± 0.08 μg/mL and BuChE inhibition IC50 was 2.39 ± 0.09 μg/mL for the most active strain, Halorubrum tebenquichense Te Se-85, respectively, which is more active in BuCHe than that of the standard galantamine. Docking calculation showed that carotenoids can exert their inhibitory activity fitting into the enzyme pocket by their halves, in the presence of cholinesterase dimers.

Surface ecosystems are rapidly changing on a global scale and it is important to understand how this influences aquifers in the subsurface, as groundwater quality is a major concern for future generations. Dissolved organic matter (DOM) contains molecular and isotopic signals from surface-derived inputs as well as from the biotic and abiotic subsurface environment and is therefore ideal to study the connectivity between both environments. We evaluated a 3-year time series of DOM composition using ultrahigh resolution mass spectrometry and age using 14C accelerator mass spectrometry along a hillslope well transect in the fractured bedrock of the Hainich Critical Zone Exploratory, Germany. We found a wide range of DOM 14C depletion, from Δ14C = −47.9 to Δ14C = −782.4, within different zones of the shallow groundwater. The 14C content of DOM mirrored the connectivity of the aquifers to the surface. The composition of DOM was highly interrelated with its 14C age. The proportions of surface-derived DOM components decreased with DOM age, whereas microorganism-derived DOM components increased. The intensity of surface-sourced DOM signals differed between the wells and likely reflected the hydrological complexity of fractured-rock environments. During recharge, DOM was more enriched in Δ14C, contained more surface-derived molecular components and was more diverse. As a potential response to the varying DOM substrate, bacterial 16S rRNA gene analysis revealed community evolution and increased bacterial diversity during recharge. The influx of diverse, surface-derived DOM potentially fueled evolution within the autochthonous bacterial communities, as in contrast to DOM, the bacterial community did not retreat to the initial diversity and community composition during the recession period. Our results demonstrate on the one hand that combined analyses of the composition and age of groundwater DOM strongly contribute to the understanding of interconnections, community evolution and the functioning of subsurface ecosystems and on the other hand that changes in surface ecosystems have an imprint on subsurface ecosystems.

Optimizing data-independent acquisition methods for proteomics applications often requires balancing spectral resolution and acquisition speed. Here, we describe a real-time full mass range implementation of the phase-constrained spectrum deconvolution method (ΦSDM) for Orbitrap mass spectrometry that increases mass resolving power without increasing scan time. Comparing its performance to the standard enhanced Fourier transformation signal processing revealed that the increased resolving power of ΦSDM is beneficial in areas of high peptide density and comes with a greater ability to resolve low-abundance signals. In a standard 2 h analysis of a 200 ng HeLa digest, this resulted in an increase of 16% in the number of quantified peptides. As the acquisition speed becomes even more important when using fast chromatographic gradients, we further applied ΦSDM methods to a range of shorter gradient lengths (21, 12, and 5 min). While ΦSDM improved identification rates and spectral quality in all tested gradients, it proved particularly advantageous for the 5 min gradient. Here, the number of identified protein groups and peptides increased by >15% in comparison to enhanced Fourier transformation processing. In conclusion, ΦSDM is an alternative signal processing algorithm for processing Orbitrap data that can improve spectral quality and benefit quantitative accuracy in typical proteomics experiments, especially when using short gradients., Competing Interests: Conflict of interest K. L. F., A. K., D. M., K. A., D. G., and A. M. are employees of Thermo Fisher Scientific, the manufacturer of Orbitrap instrumentation used in this research. M. M. is an indirect investor in Evosep Biosystems. All other authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Tyrosine sulfation in the Golgi of secreted and membrane proteins is an important post-translational modification (PTM). However, its labile nature has limited analysis by mass spectrometry (MS), a major reason why no sulfoproteome studies have been previously reported. Here, we show that a phosphoproteomics experimental workflow, which includes serial enrichment followed by high resolution, high mass accuracy MS, and tandem MS (MS/MS) analysis, enables sulfopeptide coenrichment and identification via accurate precursor ion mass shift open MSFragger database search. This approach, supported by manual validation, allows the confident identification of sulfotyrosine-containing peptides in the presence of high levels of phosphorylated peptides, thus enabling these two sterically and ionically similar isobaric PTMs to be distinguished and annotated in a single proteomic analysis. We applied this approach to isolated interphase and mitotic rat liver Golgi membranes and identified 67 tyrosine sulfopeptides, corresponding to 26 different proteins. This work discovered 23 new sulfoproteins with functions related to, for example, Ca 2+ -binding, glycan biosynthesis, and exocytosis. In addition, we report the first preliminary evidence for crosstalk between sulfation and phosphorylation in the Golgi, with implications for functional control.

Parsley is an aromatic herb native to the Mediterranean region and treasured for its phytochemical profile and bioactive properties. Developmental stage at harvest is a factor that modulates the nutritional quality of vegetables, including young greens. Accordingly, an experiment under strictly controlled conditions was carried out to compare the mineral macronutrient and phytochemical composition as well as the antioxidant activity of plain-leaf parsley (Petroselinum crispum cv. Comune 2) at two different harvest maturity stages, microgreens and baby greens. Macronutrients, carotenoids (lutein and β-carotene) and polyphenols were quantified through ion chromatography, high-performance liquid chromatography with a diode-array detector (HPLC-DAD) and UHPLC-Q-Orbitrap high-resolution mass spectrometry (HRMS), respectively. Microgreens accumulated more potassium and phosphorus, whereas baby greens accumulated more calcium and magnesium, and 65.5% less nitrate. In addition, microgreens provided 1.8-fold more lutein and 2.8-fold more β-carotene, whereas baby greens provided 183.6% more total ascorbic acid, 64.2% more total polyphenols and 170.3% higher hydrophilic antioxidant activity. Based on the culinary and phytonutritive scope of the consumers, different harvest maturity stages can be opted for and production schemes designed. Future studies are warranted to appraise the importance of ontogeny as a determinant factor for the composition and bioactive value of additional micro-herb genotypes, including underutilized Mediterranean species.

In this study, we analyzed the proanthocyanidin (PA) composition of 55 plant extracts before and after alkaline oxidation by ultrahigh-resolution UHPLC-MS/MS. We characterized the natural PA structures in detail and studied the sophisticated changes in the modified PA structures and the typical patterns and models of reactions within different PA classes due to the oxidation. The natural PAs were A- and B-type PCs, PDs and PC/PD mixtures. In addition, we detected galloylated PAs. B-type PCs in different plant extracts were rather stable and showed no or minor modification due to the alkaline oxidation. For some samples, we detected the intramolecular reactions of PCs producing A-type ether linkages. A-type PCs were also rather stable with no or minor modification, but in some plants, the formation of additional ether linkages was detected. PAs containing PD units were more reactive. After alkaline oxidation, these PAs or their oxidation products were no longer detected by MS even though a different type and/or delayed PA hump was still detected by UV at 280 nm. Galloylated PAs were rather stable under alkaline oxidation if they were PC-based, but we detected the intramolecular conversion from B-type to A-type. Galloylated PDs were more reactive and reacted similarly to nongalloylated PDs.

Various separation and mass spectrometric (MS) techniques have furthered our ability to study complex mixtures, and the desire to measure every analyte in a system is of continual interest. For many complex mixtures, such as the total molecular content of a cell, it is becoming apparent that no one single separation technique or analysis is likely to achieve this goal. Therefore, having a variety of tools to measure the complexity of these mixtures is prudent. Orbitrap MSs are broadly used in systems biology studies due to their unique performance characteristics. However, GC-Orbitraps have only recently become available, and instruments that can use gas chromatography (GC) cannot use liquid chromatography (LC) and vice versa. This limits small molecule analyses, such as those that would be employed for metabolomics, lipidomics, or toxicological studies. Thus, a simple, temporary interface was designed for a GC and Thermo Scientific™ Ion Max housing unit. This interface enables either GC or LC separation to be used on the same MS, an Exactive™ Plus Orbitrap, and utilizes an atmospheric pressure chemical ionization (APCI) source. The GC-APCI interface was tested against a commercially available atmospheric pressure photoionization (APPI) interface for three types of analytes that span the breadth of typical GC analyses: fatty acid methyl esters (FAMEs), polyaromatic hydrocarbons (PAHs), and saturated hydrocarbons. The GC-APCI-Orbitrap had similar or improved performance to the APPI and other reported methods in that it had a lower limit of quantitation, better signal to noise, and lower tendency to fragment analytes. [ABSTRACT FROM AUTHOR]

Charge detection mass spectrometry (CDMS) of low-level signals is currently limited to the analysis of individual ions that generate a persistent signal during the entire observation period. Ions that disintegrate during the observation period produce reduced frequency domain signal amplitudes, which lead to an underestimation of the ion charge state, and thus the ion mass. The charge assignment can only be corrected through an accurate determination of the time of ion disintegration. The traditional mechanisms for temporal signal analysis have severe limitations for temporal resolution, spectral resolution, and signal-to-noise ratios. Selective Temporal Overview of Resonant Ions (STORI) plots provide a new framework to accurately analyze low-level time domain signals of individual ions. STORI plots allow for complete correction of intermittent signals, the differentiation of single and multiple ions at the same frequency, and the association of signals that spontaneously change frequency. [ABSTRACT FROM AUTHOR]

• Pesticide metabolites have been determined in urine samples by LC-Orbitrap. • Automated TurboFlow method has been compared with conventional off-line procedures. • A post-targeted analysis was performed using a database with 1500 contaminants. • An untargeted analysis was also carried out, detecting two nicotine metabolites. • TurboFlow-LC-Orbitrap allows the detection of a wide scope of compounds in urine. There is a growing concern to pesticide exposure in humans, being the organophosphate (OP), carbamate and pyrethroid the most commonly used insecticides. Therefore, a fast, simple and automated screening method based on ultra-high performance liquid chromatography coupled to Orbitrap high resolution mass spectrometry (UHPLC-Orbitrap-MS) has been developed to determine six OPs metabolites (3,5,6-trichloro-2-pyridinol "TCPy", 2-isopropyl-6-methyl-4-pyrimidinol "IMPY", 4-nitrophenol "PNP", 4-nitrophenol-d4 "PNP-d4″, 3-chloro-7-hydroxy-4-methylcoumarin "CMHC" and 2-diethylamino-6-methylpyrimidin-4ol "DEAMPY") and three pyrethroid metabolites (3-phenoxybenzoic acid "3-PBA", 4-fluoro-3-phenoxybenzoic acid "4-F-3-PBA" and 3-(2,2-dichlorovinyl)-2,2-dimethyl-1-cyclopropanecarboxylic acid "cis/trans-DCCA") in human urine samples. Off-line solid phase extraction (SPE), on-line SPE and turbulent flow chromatography (TurboFlow™) extraction methodologies were compared, obtaining better results when TurboFlow™ was applied, achieving a total analysis time of 13.83 min. Several validation parameters were tested, obtaining suitable results for all studied compounds. Recoveries ranged from 70 to 116%, meanwhile repeatability and reproducibility were lower than or equal to 7% and 13%, respectively. The limits of quantification (LOQs) were set in the range from 1 μg/L to 5 μg/L (except for DCCA at 10 μg/L). Finally, thirty seven urine samples from women and men living near the agricultural areas of Almeria (Spain) were analyzed to know and control the contamination on the human health. PNP, DEAMPY and IMPY were detected in six urine samples at trace levels except IMPY, which was detected at 3.44 μg/L in one of the samples. Additionally, a post-targeted and untargeted analysis were carried out in the samples, identifying eleven insecticides such as bensulide or cinerin II, one insecticide metabolite, fipronil sulfone, one drug metabolite, carboxyibuprofen and two nicotine metabolites, cotinine and 2-(1-pyrrolidinyl)isonicotinic acid, in several samples. [ABSTRACT FROM AUTHOR]

• Discovery strategy: unscheduled LC-TripleQuad + neutral loss + screening LC-HR MS/MS Orbitrap. • Reliable quantification of glycine-dihydroxy sulfate bile acids in serum. • Different fasting and postprandial bile acid profiles between males and females. • Existence of possible metabotypes amongst population. An ultrahigh performance liquid chromatography tandem mass spectrometry method (UHPLC-MS/MS) was developed for the determination of 41 target and 8 additional bile acids isomers (BAs) in biological fluids. BAs were analysed by solid-phase extraction on 50 μL biofluid-aliquots, followed by a properly optimised 27 min-chromatographic run. The method provided high sensitivity (limits of detection 0.0002–0.03 μM, limits of quantitation 0.0007–0.11 μM), linearity (R2>0.99) and precision (relative standard deviations ≤16%). A strategy of scheduled/ unscheduled injections of real samples together with neutral loss (80 Da and 176 Da) scans allowed us to find additional bile acid isomers not a priori included in the method, while high resolution full scan and MS/MS fragmentation analysis confirmed their structural adherence to the bile acid family. Moreover this is the first study quantifying four sulfate glycine conjugated-dihydroxy bile acid isomers, independently of the diet and postprandial time. Application to a dietary intervention kinetic study confirmed the existence of possible metabotypes amongst the study population (n = 20). A trend differentiating males from females was observed suggesting that serum samples from women contained smaller amounts of certain bile acids. [ABSTRACT FROM AUTHOR]

This review is focused on the application of Orbitrap™ mass spectrometry for pesticide residue analysis. The importance of mass accuracy and resolution is explained. Also, the most typical acquisition modes available in QOrbitrap™ are discussed. • Application of Orbitrap mass spectrometry for pesticide residue analysis. • Importance of mass resolution and mass accuracy. • Targeted and non-targeted data acquisition. [ABSTRACT FROM AUTHOR]

We describe an instrumental configuration for the structural characterization of fragment ions generated by collisional dissociation of peptide ions in the typical MS2 scheme widely used for peptide sequencing. Structures are determined by comparing the vibrational band patterns displayed by cryogenically cooled ions with calculated spectra for candidate structural isomers. These spectra were obtained in a linear action mode by photodissociation of weakly bound D2 molecules. This is accomplished by interfacing a Thermo Fisher Scientific Orbitrap Velos Pro to a cryogenic, triple focusing time-of-flight photofragmentation mass spectrometer (the Yale TOF spectrometer). The interface involves replacement of the Orbitrap's higher-energy collisional dissociation cell with a voltage-gated aperture that maintains the commercial instrument's standard capabilities while enabling bidirectional transfer of ions between the high-resolution FT analyzer and external ion sources. The performance of this hybrid instrument is demonstrated by its application to the a1, y1 and z1 fragment ions generated by CID of a prototypical dipeptide precursor, protonated L-phenylalanyl-L-tyrosine (H+-Phe-Tyr-OH or FY-H+). The structure of the unusual z1 ion, nominally formed after NH3 is ejected from the protonated tyrosine (y1) product, is identified as the cyclopropane-based product is tentatively identified as a cyclopropane-based product. [ABSTRACT FROM AUTHOR]

Black pepper, though commonly employed as a spice, has many medicinal properties. It consists of volatile oils, alkaloids, pungent resins, etc., of which piperine is a major constituent. Though safe at low doses, piperine causes alteration in the activity of drug metabolising enzymes and transporters at high dose and is known to precipitate liver toxicity. It has a potential to form reactive metabolite(s) (RM) owing to the presence of structural alerts, such as methylenedioxyphenyl (MDP), α, β‐unsaturated carbonyl group (Michael acceptor), and piperidine. The present study was designed to detect and characterize stable and RM(s) of piperine formed on in vitro incubation with human liver microsomes. The investigation of RMs was done with the aid of trapping agents, viz, glutathione (GSH) and N‐acetylcysteine (NAC). The samples were analysed by ultra‐high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC‐HRMS) using Thermo Scientific Q Exactive Plus Orbitrap. Full scan MS followed by data‐dependent MS2 (Full MS‐ddMS2) mode was used to establish mass spectrometric fragmentation pathways of protonated piperine and its metabolites. In total, four stable metabolites and their isomers (M1a‐c, M2a‐b, M3a‐c, and M4a‐b) were detected. Their formation involved removal of carbon (3, M1a‐c), hydroxylation (2, M2a‐b), hydroxylation with hydrogenation (3, M3a‐c), and dehydrogenation (2, M4a‐b). Out of these metabolites, M1, M2, and M3 are reported earlier in the literature, but their isomers and two M4 variants are novel. In addition, six novel conjugates of RMs, including three GSH conjugates of m/z 579 and three NAC conjugates of m/z 435, were also observed. [ABSTRACT FROM AUTHOR]

Abstract An untargeted screening method for the rapid identification of veterinary drug residues in incurred animal tissues using liquid microjunction surface sampling probe mass spectrometry (LMJSSP-MS) was developed. Current analytical methods for veterinary drug residue screening involve lengthy sample preparation, extraction, and instrumental analysis steps. This method identifies veterinary drug residues in several different incurred animal tissues more quickly than conventional analytical methods. This LMJSSP-MS method uses an ambient ionization technology called liquid microjunction surface sampling probe along with a data dependent scan function of a quadrupole orbitrap mass spectrometer. Collected product ion spectra are searched against the mzCloud™ online mass spectral database to identify veterinary drug residues found in incurred animal tissue samples. Examples of veterinary drugs identified with this method include flunixin, tilmicosin, pentobarbital, xylazine, and ketamine. Optimization of method parameters is described and discussed. The limit of identification (LOI) of this method is estimated to be approximately 1 μg g−1 for xylazine and ketamine. Graphical abstract Image 1 Highlights • Method for fast untargeted screening and identification of drug residues in tissues. • Liquid microjunction surface sampling probe combined with data dependent MS/MS. • Product ion spectra search and matching with online high-resolution MS/MS database. • Multiple classes of veterinary drugs identified in a variety of species and tissue types. • Enables unknown drug residue identification in less than one hour. [ABSTRACT FROM AUTHOR]

Chlorinated paraffins (CPs) are complex mixtures of persistent contaminants present throughout the aquatic food web. In this study 122 farmed and 11 wild salmon samples were collected over the course of four years (2014–2017). The ratio of short-chain CP and medium-chain CP and the corresponding homologue patterns were determined by means of gas chromatography (GC) with high resolution, accurate mass Orbitrap mass spectrometry (MS) technology. Characteristic patterns were observed, enabling differentiation between European and non-European (Chilean) samples. Concentration ranges of short-chain CPs (0.97–170 ng/g ww) and medium-chain CPs (1.1–79 ng/g ww) were similarly widespread over three orders of magnitude. Yet, both the mean and median concentrations of MCCPs were usually higher than those of the SCCP. CP levels were generally higher than those of marker polychlorinated biphenyls (PCBs) and hexabromocyclododecanes (HBCDDs). An age- and gender-dependent estimated intake range of 4.6–35 ng/kg bw/week for short and medium-chain CPs via the consumption of salmon was calculated for adults in Germany. Image 1 • 133 salmon samples (2014–2017, German market) were analysed on chloroparaffins (CPs). • CP homologue patterns showed clear differences between sample origins. • CP levels were higher than those of marker PCBs and HBCDDs. • SCCP and MCCP levels indicated shift towards MCCPs in Europe. • The weekly CP intake for German adults via salmon consumption was estimated. [ABSTRACT FROM AUTHOR]

Many fundamental questions of astrophysics, biochemistry, and geology rely on the ability to accurately and precisely measure the mass and abundance of isotopes. Taken a step further, the capacity to perform such measurements on intact molecules provides insights into processes in diverse biological systems. Described here is the coupling of a combined atomic and molecular (CAM) ionization source, the liquid sampling-atmospheric pressure glow discharge (LS-APGD) microplasma, with a commercially available ThermoScientific Fusion Lumos mass spectrometer. Demonstrated for the first time is the ionization and isotopically resolved fingerprinting of a long-postulated, but never mass-spectrometrically observed, bi-metallic complex Hg:Se-cysteine. Such a complex has been implicated as having a role in observations of Hg detoxification by selenoproteins/amino acids. Demonstrated as well is the ability to mass spectrometrically-resolve the geochronologically important isobaric 87Sr and 87Rb species (Δm ~ 0.3 mDa, mass resolution m/Δm ≈ 1,700,000). The mass difference in this case reflects the beta-decay of the 87Rb to the stable Sr isotope. These two demonstrations highlight what may be a significant change in bioinorganic and atomic mass spectrometry, with impact expected across a broad spectrum of the physical, biological, and geological sciences. [ABSTRACT FROM AUTHOR]

Abstract N -nitrosodimethylamine (NDMA) is a disinfection byproduct that has been classified as probable human carcinogen by the US Environmental Protection Agency. According to the published literature, natural dissolved organic matter (DOM) can be a source of NDMA precursors in drinking water. New advances in chemical characterization of DOM with high resolution mass spectrometry (HRMS) are allowing researchers to understand these ultra-complex mixtures. The objective of this study is to investigate analytical methodologies based on HRMS to explore NDMA formation from natural waters. To this aim, different waters from drinking water reservoirs in Spain containing NDMA precursors (quantified by means of NDMA formation potential) in concentrations between 17 and 60 ng/L have been studied. The workflow includes DOM solid-phase extraction and Orbitrap analysis with and without chromatographic separation. Here, we show that the molecular composition of DOM across the studied drinking water reservoirs is correlated with the NDMA formation potential. In particular, we found that NDMA formation potential is associated with compounds with high hydrogen saturation (H/C ≥ 1.5), corresponding also to reservoirs with higher background nutrient concentrations and wastewater indicators. Further chromatographic fractionation did not allow better definition of these possible precursors as they were present in different fractions of the chromatogram, suggesting that they were isomerically complex. Graphical abstract Unlabelled Image Highlights • Orbitrap is used for DOM fingerprinting and data is visualized with Van Krevelen plots. • PCoA is used to investigate relationship between NDMA formation potential and DOM. • Compounds correlated with NDMA formation potential appear in the H/C ≥ 1.5 region. • Chromatographic separation does not provide better results than direct infusion. [ABSTRACT FROM AUTHOR]

Abstract Water samples from 29 small waterways located in 10 different countries in the European Union were screened for the presence of a large number of pesticides (275) and veterinary drugs (101). Solid phase extraction was combined with liquid chromatography coupled to Orbitrap high resolution tandem mass spectrometry to quantify the levels of pesticides in the samples and to detect the presence of veterinary drugs. All the sampled European rivers and canals included in this investigation were contaminated with mixtures of pesticides and, in most of the cases, with several veterinary drugs at the time of sampling, without a clear national or regional pattern. In total, 103 different pesticides, 24 of them banned in the EU, and 21 veterinary drugs were found in the analysed samples. Herbicides were the main contributor to the total amount of pesticides found in the samples, with terbuthylazine present in all the samples. The maximum individual concentration recorded was of dimethenamid at 59.85 μg L−1. The maximum combined pesticide concentration was found in a sample from the Wulfdambeek canal, Belgium, with 94.02 μg L−1 comprised of a mixture of 70 different pesticides. European regulatory standards defining acceptable concentration levels were exceeded for at least one pesticide in 13 of the 29 samples analysed, with the neonicotinoid insecticides imidacloprid and clothianidin most frequently present above such limits. The majority of the veterinary drugs detected were antimicrobials, most being antibiotics. The β-lactam antibiotic dicloxacillin was present in two thirds of the analysed samples. The application of this consistent research approach across Europe allowed the identification of a significant threat to the aquatic environment associated with pesticide contamination, and in some cases veterinary drugs, at the time of sampling in the water bodies tested. Graphical abstract Unlabelled Image Highlights • Comprehensive determination of 275 pesticides and 101 veterinary drugs by SPE-LC- Orbitrap-MS. • Analysis of 29 selected small waterways across 10 countries in the European Union. • All the sampled streams were contaminated with pesticides and, in most of the cases, veterinary drugs. • The maximum combined pesticide concentration in one sample was 94.02 μg L-1, comprised of a mixture of 70 pesticides. • European regulatory standards were exceeded for at least one substance in 13 samples. [ABSTRACT FROM AUTHOR]

Recombinant human erythropoietin (rHuEPO) is the first cloned hematopoietic growth factor available for pharmaceutical treatment, especially anemia, since 1988. Unfortunately, the ability of rHuEPO in boosting erythropoiesis, and thus aerobic capacity has led to its abuse in endurance sport. Besides, the expiry of the original rHuEPO patent has resulted in many biosimilars being produced which led to special requirements regarding quality control. As a consequence, there is a huge demand for all rHuEPOs to be well characterized for the ease of identification, differentiation, and detection. Glycoproteomic mass spectrometry, the most current promising approach for rHuEPOs analysis, was employed to characterize 4 rHuEPOs including epoetin-α, epoetin-β, darbepoetin-α and Mircera. Via nanoLC-ESI-MS/MS, distinct glycoproteomic profiles of each rHuEPO have been achieved for differential analysis. With two different fragmentation methods, collision-induced dissociation (CID) and higher-energy collision dissociation (HCD), maximum of 75%, 70%, 70%, and 77% protein sequence coverages were attained for epoetin-α, epoetin-β, darbepoetin-α and Mircera, respectively. From the peptides/glycopeptides mixture, similar and unique peptides/glycopeptides (biomarkers) for each rHuEPO have been identified. With the discovery of high quality and unique biomarkers, a more standardized and efficient method for quality control and rHuEPO abuse detection can be developed. [ABSTRACT FROM AUTHOR]

The structures of individual molecules in crude oil remain largely unknown despite the considerable amount of research dedicated to this topic. The extreme complexity of crude oil (recently Marshall reported the observation of more than 400,000 unique compounds in one sample) makes it impossible to separate crude oil into individual compounds and determine their structure by NMR or X-ray spectroscopy. Recently, isotope exchange, performed both in solution and in the gas phase, combined with high-resolution mass spectrometry was used for speciation of certain structural fragments of individual molecules in crude oil and humic substances. 16O/18O exchange allows enumeration of =O groups and speciation of furans, whereas H/D exchange allows enumeration of –OH groups, –NH groups, aromatic hydrogens, alpha hydrogens, etc. Unfortunately, crude oil is insoluble in water (the most available and cleanest source of isotopes), so performance of the exchange in solution requires harsh conditions, such as concentrated acids or bases, which could considerably modify the sample. Here we describe the use of a cheap and simple analytical approach for performing both H/D and 16O/18O exchange in crude oil using only water as the source of the isotopes. Crude oil was incubated in near-critical water and the reaction was monitored by high-resolution Fourier transform mass spectrometry. Although isotope exchange results in complication of the spectrum, the resolving power of modern mass spectrometers is sufficient to determine the number of exchanges for each molecule simultaneously. We determined the number of 16O/18O exchanges in 276 species and the number of H/D exchanges in 150 species. Our results allow deeper investigation of crude oil and other nonpolar samples on the molecular level. [ABSTRACT FROM AUTHOR]

In the Solar System, extra-terrestrial organic molecules have been found on cometary primitive objects, on Titan and Enceladus icy moons and on Mars. Identification could be achieved for simple organic species by remote sensing based on spectroscopic methods. However in situ mass spectrometry is a key technology to determine the nature of more complex organic matter. A large panel of mass spectrometers has already been developed for space exploration combining different types of analysers and ion sources. Up to now the highest mass resolution reached with a space instrument is 9000 at m/z 28 and corresponds to the DFMS-ROSINA instrument (Balsiger et al., 2007) dedicated to the study of the comet 67P/Churyumov-Gerasimenko's atmosphere and ionosphere, in a low pressure environment. A new concept of mass analyser offering ultra-high mass resolving power of more than 50,000 at m/z 56 (under high vacuum condition about 10−9 mbar) is currently being developed for space applications: the CosmOrbitrap (Briois et al., 2016), based on the Orbitrap™ technology. This work challenges the use of LAb-CosmOrbitrap, a space instrument prototype combining Laser Ablation ionisation and the CosmOrbitrap mass analyser, to identify solid organic molecules of relevance to the future space exploration. For this purpose a blind test was jointly organised by the JAXA-HRMS team (Japan Aerospace Exploration Agency-High Resolution Mass Spectrometry) and the CosmOrbitrap consortium. The JAXA team provided two organic samples, whereas the CosmOrbitrap consortium analysed them without prior information. Thanks to the high analytical performances of the prototype and our HRMS data post-processing, we successfully identified the two molecules as HOBt, hydroxybenzotriazole (C 6 H 5 N 3 O) and BBOT, 2,5-Bis(5-tert-butyl-benzoxazol-2-yl)thiophene (C 26 H 26 N 2 O 2 S), with a mass resolving power of, respectively, 123 540 and 69 219. The success of this blind test on complex organic molecules shows the strong potential of LAb-CosmOrbitrap for future space applications. • Efficient ionisation of solid sample by nano-pulsed single laser shot. • Powerful analytical performances of CosmOrbitrap mass analyser. • Successful blind test identification of organics ionised by Laser-CosmOrbitrap. • Laser-CosmOrbitrap relevant technique for space exploration of organic rich worlds. [ABSTRACT FROM AUTHOR]

The official methodologies used for the identification and comparison of vine cultivars are ampelography and ampelometry. These methodologies are essentially based on qualitative assessments or biometric dependent morphological features of the plant. The heterogeneity of cultivars and consequently the increasing demand for a more detailed product typization, led to the introduction of new methodologies for the varietal characterization. In this scenario, proteomics has already proved to be a very useful discipline for the typization of many kinds of edible products. In this paper, we present a proteomic study carried out on three cultivars of Vitis vinifera peculiar of south Italy (Campania) used for white wine production (Fiano, Greco and Falanghina) by advanced biomolecular mass spectrometry approach. Our data highlight variations in the proteomic profiles during ripening for each cultivar and between analyzed cultivars, thus suggesting a new way to outline the biomolecular signature of vines. Unlabelled Image • The increasing heterogeneity of cultivars requires a new and more detailed product molecular characterization. • The proteome represents probably the best way to specifically identify and characterize a cultivar. • Our results show an overall variation in terms of proteins profile for each cultivar during ripening and among different cultivars. [ABSTRACT FROM AUTHOR]

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Vacuum-assisted evaporative concentration (VEC) was successfully applied and validated for the enrichment of 590 organic substances from river water and wastewater. Different volumes of water samples (6 mL wastewater influent, 15 mL wastewater effluent, and 60 mL river water) were evaporated to 0.3 mL and finally adjusted to 0.4 mL. 0.1 mL of the concentrate were injected into a polar reversed-phase C18 liquid chromatography column coupled with electrospray ionization to high-resolution tandem mass spectrometry. Analyte recoveries were determined for VEC and compared against a mixed-bed multilayer solid-phase extraction (SPE). Both approaches performed equally well (≥ 70% recovery) for a vast number of analytes (n = 327), whereas certain substances were especially amenable to enrichment by either SPE (e.g., 4-chlorobenzophenone, logDow,pH7 4) or VEC (e.g., TRIS, logDow,pH7 − 4.6). Overall, VEC was more suitable for the enrichment of polar analytes, albeit considerable signal suppression (up to 74% in river water) was observed for the VEC-enriched sample matrix. Nevertheless, VEC allowed for accurate and precise quantification down to the sub-nanogram per liter level and required no more than 60 mL of the sample, as demonstrated by its application to several environmental water matrices. By contrast, SPE is typically constrained by high sample volumes ranging from 100 mL (wastewater influent) to 1000 mL (river water). The developed VEC workflow not only requires low labor cost and minimum supervision but is also a rapid, convenient, and environmentally safe alternative to SPE and highly suitable for target and non-target analysis. [ABSTRACT FROM AUTHOR]

Background: Carboxymethyl lysine is an advanced glycation end product of interest as a potential biomarker of cardiovascular and other diseases. Available methods involve ELISA, with potential interference, or isotope dilution mass spectrometry (IDMS), with low-throughput sample preparation.Methods: A high-throughput sample preparation method based on 96-well plates was developed. Protein-bound carboxymethyl lysine and lysine were quantified by IDMS using reversed phase chromatography coupled to a high-resolution accurate mass Orbitrap Exactive mass spectrometer. The carboxymethyl lysine concentration (normalized to lysine concentration) was measured in 1714 plasma samples from the British Regional Heart Study (BRHS).Results: For carboxymethyl lysine, the lower limit of quantification (LLOQ) was estimated at 0.16 μM and the assay was linear between 0.25 and 10 μM. For lysine, the LLOQ was estimated at 3.79 mM, and the assay was linear between 2.5 and 100 mM. The intra-assay coefficient of variation was 17.2% for carboxymethyl lysine, 9.3% for lysine and 10.5% for normalized carboxymethyl lysine. The inter-assay coefficient of variation was 18.1% for carboxymethyl lysine, 14.8 for lysine and 16.2% for normalized carboxymethyl lysine. The median and inter-quartile range of all study samples in each batch were monitored. A mean carboxymethyl lysine concentration of 2.7 μM (IQR 2.0-3.2 μM, range 0.2-17.4 μM) and a mean normalized carboxymethyl lysine concentration of 69 μM/M lysine (IQR 54-76 μM/M, range 19-453 μM/M) were measured in the BRHS.Conclusion: This high-throughput sample preparation method makes it possible to analyse large cohorts required to determine the potential of carboxymethyl lysine as a biomarker. [ABSTRACT FROM AUTHOR]