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A new laboratory OrbitrapTM cell-based mass spectrometer, OLYMPIA (Orbitrap anaLYseur MultiPle IonisAtion), without a C-trap module, has been developed and constructed. The first operation of the OrbitrapTM cell-based device with the continuous ion source and without the C-trap module is reported. OLYMPIA is being developed and used as a workbench platform to test and develop technologies for the next generation of spaceborne mass spectrometers and as a laboratory instrument to perform high-resolution studies of space-relevant chemical processes. This instrument has been used to measure the quantitative composition of CO/N2/C2H4 mixtures of the same nominal mass using an electron ionization ion source. The relative abundance of ions has been measured using a short acquisition time (up to 250 ms) with a precision of better than 10% (for most abundant ions) and a mass resolution of 30,000–50,000 (full width at half maximum) over the mass range of m/z 28–86. The achieved mass accuracy of measurements is better than 20 ppm. This performance level is sufficient to resolve and identify the CO/N2/C2H4 components of the mixtures. The dynamic range and relative ion abundance measurements have been evaluated using a reference normal isotopic distribution of krypton gas. The measurement accuracy is about 10% for the 4 most abundant isotopes; 6 isotopes are detectable.

Producers of drinking water (DW) occasionally require chemical identification of new or unexpected contamination, e.g., caused by an incident. The state-of-the-art technique for the identification of organic compounds is High-Resolution Mass Spectrometry (HRMS). The ability to discover unexpected compounds at low concentrations in any sample by HRMS is facilitated by comparison to non-contaminated reference samples. Samples of raw and drinking water were collected regularly over one whole year from six Swedish drinking water treatment plants (DWTPs). The samples were analyzed by LC-HRMS together with spiked samples mimicking an incident. This setup enabled evaluation of the significance of having access to a collection of matrix-matched reference samples. The main variation in the organic compound profile in the data set was explained by the uniqueness of the raw water and purification steps in the individual DWTPs. Seasonal variations were also significant but subordinate. This subject was further explored by the analysis of drinking water sampled once from 90 of the 1750 Swedish DWTPs, where a similarity between DW originating from rock aquifers was observed. No other significant correlation between samples was observed—e.g., other types of raw water, which types of purification steps were involved, or which additives or process chemicals were added—which could aid in the selection of relevant reference samples. The conclusion from the study is that it would be imperative for the DWTPs to have access to their individual reference samples for use in the investigation of an incident. A library of such reference samples, e.g., collected monthly and covering the last 12 months, could be stored and used together with a fresh "suspected sample" for non-target HRMS investigations as described. [ABSTRACT FROM AUTHOR]

A method for the simultaneous screening of 258 pesticide residues in silage using modified QuEChERS combined with liquid chromatography (LC)- and gas chromatography (GC)- quadrupole-Orbitrap mass spectrometry (Q-Orbitrap/MS) has been developed. After hydration, the silage was homogenized with a 1% acetic acid–acetonitrile solution, and the extract was purified using C18, PSA, and anhydrous magnesium sulfate. Finally, the sample was detected using LC/GC-Q-Orbitrap/MS, and quantified using an external standard method. The results showed that 258 pesticides had an excellent linear relationship in the range of 0.1–50 μg L−1, and that the coefficients of determination (R2) were more than 0.99. The screening detection limit (SDL) of silage was in the range of 0.5–50 μg kg−1, and the limit of quantitation (LOQ) was in the range of 1–50 μg kg−1. The accuracy and precision of the method were verified at the spiked levels of 1-, 2- and 10-times LOQ, and the recovery of 258 pesticides was in the range of 66.5–119.8%, with relative standard deviations (RSDs) of less than 20% (n = 6). This method was simple, rapid, and reliable, and could be applied to screen and quantify multi-pesticide residues in silage. [ABSTRACT FROM AUTHOR]

The principal purpose of this study was to determine antioxidant and ACE-inhibitory (angiotensin converting enzyme) activities of 5 ripened Turkish cheese varieties (Kashar, Erzincan Tulum, Izmir Tulum Gruyere, Mihalic). Among the water-soluble extracts with a molecular weight smaller than 3 kDa Gruyere and Erzincan Tulum exhibited the highest free radical scavenger activity in FRAP (Ferric Reducing Ability of Plasma) and ABTS [2,20-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)] assays, respectively. For the inhibition of ACE, Mihalic was more effective than others. The water-soluble extracts of five cheese were analyzed with liquid chromatography-tandem mass spectrometry to identify the peptide sequences. Totally 394 peptides were sequenced; of these, 134 from αs1-casein, 44 from αs2-casein, 198 from β-casein, and 18 from κ-casein. Among these sequences, 48 peptides were reported as bioactive with the activities including ACE-inhibitory, antioxidant, antimicrobial, DPP-IV inhibitory, antidiabetic, immunomodulating, antithrombotic, anti-inflammatory, and inhibition of cholesterol solubility. These results showed that Turkish cheeses are good sources of bioactive peptides. [ABSTRACT FROM AUTHOR]

A novel method for detecting pesticide multi‐residue in grass forage (alfalfa and oat) was established based on the one‐step automatic extraction and purification technology of quick, easy, cheap, effective, rugged, and safe combined with ultrahigh‐performance liquid chromatography quadrupole Orbitrap high‐resolution mass spectrometry. The crushed sample was extracted with acetonitrile with 1% acetate, followed by a cleanup step with a primary‐secondary amine, octadecylsilane, and graphitized carbon black. The extraction and purification were carried out using the one‐step automatic pretreatment equipment. The target pesticides were acquired in positive ion electrospray ionization mode and full scan/data dependent secondary scan mode. The calibration curve shows good linearity over the corresponding concentration range, with the coefficient of determination greater than 0.99. The screening detection limits were 0.5–50 μg/kg, and the limit of quantification for the 206 pesticides was set at 1–50 μg/kg. At the spiking levels of one, two, and 10 times of limit of quantification, more than 95% of pesticides had recovery between 70–120%, with a relative standard deviation ≤20%. The method was proved to be simple, rapid, high‐sensitivity, and could be routinely used for the high throughput screening and quantitative analysis of pesticide residues in alfalfa and oat. [ABSTRACT FROM AUTHOR]

The time-domain transients in the Fourier transform mass spectrometry (FTMS) analysis of monoclonal antibodies (mAbs) are known to exhibit characteristic isotopic beat patterns. These patterns are defined by the isotopic distributions of all gaseous mAb ions present in the FTMS mass analyzer, originating from single or multiple charge states, and from single or multiple proteoforms. For an isolated charge state of a single proteoform, the mAb isotopic beat pattern resembles narrow splashes of signal amplitude (beats), spaced periodically in the time-domain transient, with broad (often exceeding 1 s) "valleys" between them. Here, we reinforce the importance of isotopic beat patterns for the accurate interpretation and presentation of FTMS data in the analysis of mAbs and other large biopolymers. An updated, mAb-grade version of the transient-mediated FTMS data simulation and visualization tool, FTMS Simulator is introduced and benchmarked. We then apply this tool to evaluate the charge-state dependent characteristics of isotopic beats in mAbs analyses with modern models of Orbitrap and ion cyclotron resonance (ICR) FTMS instruments, including detection of higher-order harmonics. We demonstrate the impact of the isotopic beat patterns on the analytical characteristics of the resulting mass spectra of individual and overlapping mAb proteoforms. The results reported here detail highly nonlinear dependences of resolution and signal-to-noise ratio on the time-domain transient period, absorption or magnitude mode spectra representation, and apodization functions. The provided description and the demonstrated ability to routinely conduct accurate simulations of FTMS data for large biopolymers should aid the end-users of Orbitrap and ICR FTMS instruments in the analysis of mAbs and other biopolymers, including viruses. [ABSTRACT FROM AUTHOR]

Ranibizumab is an FDA-approved drug used to treat wet age-related macular degeneration (AMD), diabetic retinopathy, macular edema, and myopic choroidal neovascularization. Bevacizumab is another drug often used off-label to treat wet AMD. In order to reduce unwanted angiogenesis, ranibizumab and bevacizumab target circulating VEGF-A in the eye. Concentration levels in human vitreous and aqueous humor can be used to provide valuable efficacy information. However, vitreous and aqueous humor's aqueous environment, and vitreous humor's viscosity, as well as the stickiness of the analytes can provide bioanalytical challenges. In this manuscript, we describe the development, optimization, and fit-for-purpose validation of an LC-HRMS method designed for intact quantitative bioanalysis of ranibizumab and bevacizumab in human vitreous and aqueous humor following intravitreal administration. In order to fully develop this method, evaluations were conducted to optimize the conditions, including the data processing model (extracted ion chromatograms (XICs) vs deconvolution), carryover mitigation, sample preparation scheme optimization for surrogate and primary matrices, use of internal standard/immunocapture/deglycosylation, and optimization of the extraction and dilution procedure, as well as optimization of the liquid chromatography and mass spectrometry conditions. Once the method was fully optimized, a fit-for-purpose validation was conducted, including matrix parallelism, with a linear calibration range of 10 to 200 µg/mL. The development of this intact quantitative method using LC-HRMS provides a proof-of-concept template for challenging, but valuable new and exciting bioanalytical techniques. [ABSTRACT FROM AUTHOR]

The main objectives of this study were to determine antioxidant, angiotensin-converting enzyme-inhibitory (ACE-I), and antibacterial activities of traditional Turkish white cheeses. The bioactivities of the cheeses tested in the low-molecular-weight fractions of water-soluble extracts. The effect of in vitro digestion on the functional potential of white cheeses was also considered. The antioxidant activity of water-soluble extracts of white cheeses was tested with FRAP (Ferric Reducing Ability of Plasma) and ABTS [2,20-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid)] assays. Cow cheese and goat cheese showed better antioxidant activities in the FRAP and ABTS assays, respectively. However, after in vitro digestion, goat cheese displayed higher radical scavenging activity than its counterparts. Goat cheese also exhibited better ACE-I activity, although ACE-I activity was inhibited by the in vitro digestion, probably due to the further proteolysis of ACE-I peptides. All samples showed an antibacterial effect on Salmonella typhimurium ATCC 14028 and Staphylococcus aureus ATCC 25923. The fractions were analyzed with orbitrap liquid chromatography–tandem mass spectrometry to determine the sequences of peptides. White cheeses contained 42 sequences previously reported as bioactive peptides with the functions of ACE-I, antioxidant, antimicrobial, inhibition of cholesterol solubility, osteoanabolic, and antidiabetic. White cheese is a rich source of bioactive peptides with various properties. [ABSTRACT FROM AUTHOR]

The removal of contaminants of emerging concern (CECs) occurring in wastewater effluents, such as pharmaceutically active substances (PhACs) and personal care products, pose a big research challenge since they can be a major source of pollution for water bodies and a danger to public health. The objective of this work was to perform a comprehensive monitoring of a broad set of PhACs (>130) in a wastewater treatment plant (WWTP) close to Thessaloniki (Greece), as well as to evaluate the potential of heterogeneous catalytic ozonation for the removal of CECs from wastewater through a continuous flow system. The high-resolution mass spectrometry analysis revealed the highest average concentrations for irbesartan (1817 ng/L). Antihypertensives along with antibiotics, psychiatrics, and β-blockers were found to aggravate the effluents. Removal efficiency after conventional treatment was >30%. The results from catalytic ozonation unit operation indicate that the introduction of a proper solid material that acts as catalyst can enhance the removal of CECs. A preliminary risk assessment using the risk quotient (RQ) revealed that irbesartan and telmisartan entail high acute risk. The overall results underline the urgent need to incessantly monitor PhACs and expand the toxicological studies to establish the sublethal and chronic effects on aquatic organisms. [ABSTRACT FROM AUTHOR]

We present a novel, scan-centric method for characterizing peaks from direct injection multi-scan Fourier transform mass spectra of complex samples that utilizes frequency values derived directly from the spacing of raw m/z points in spectral scans. Our peak characterization method utilizes intensity-independent noise removal and normalization of scan-level data to provide a much better fit of relative intensity to natural abundance probabilities for low abundance isotopologues that are not present in all of the acquired scans. Moreover, our method calculates both peak- and scan-specific statistics incorporated within a series of quality control steps that are designed to robustly derive peak centers, intensities, and intensity ratios with their scan-level variances. These cross-scan characterized peaks are suitable for use in our previously published peak assignment methodology, Small Molecule Isotope Resolved Formula Enumeration (SMIRFE). [ABSTRACT FROM AUTHOR]

Introduction: Ravulizumab (RAVUL) is a new complement inhibitor, with a difference of 4 amino acids in the heavy chain from a predecessor compound, eculizumab (ECUL). Objectives: First, to utilize mass spectrometry (MS) to characterize RAVUL and verify differences from its predecessor and, second, to validate and implement a lab developed test (LDT) for RAVUL that will allow for quantitative therapeutic monitoring. Methods: A time-of-flight mass spectrometer (TOF-MS) was used to characterize and differentiate the molecular weight differences between RAVUL and ECUL by both digest and reduction experiments. In parallel, an LDT for RAVUL was validated and implemented utilizing IgG4 enrichment with light chain detection and quantitation on a high throughput orbitrap MS platform. Results: The TOF-MS platform allowed for the mass difference between RAVUL and ECUL to be verified along with providing a proof of concept for a new intact protein quantitation software. An LDT on an orbitrap MS was validated and implemented using intact light chain quantitation, with the limitation that it cannot differentiate between ECUL and RAVUL. The LDT has an analytical measuring range from 5 to 600 mcg/mL, inter-assay imprecision of ≤13% CV (n = 13) and accuracy with

Honey represents a valuable food commodity, known since ancient times for its delicate taste and health benefits due to its specific compositional characteristics, mainly the phenolic compound content. “Anama” honey is a monofloral honey produced from the nectar of Erica manipuliflora plant, a heather bush of the Greek island of Ikaria, one of the Mediterranean’s longevity regions. “Anama” is characterized by a unique aroma and taste, with a growing demand for consumption and the potential to be included in the list of products with a protected designation of origin. The aim of this study was to determine the chemical and botanical profile of authentic Anama honey samples and find similarities and differences with honey samples of a different botanical origin from the same geographical area. Untargeted Ultrahigh-Performance Liquid Chromatography-Hybrid Quadrupole-Orbitrap High-Resolution Mass Spectrometry (UHPLC-HRMS) metabolomics study was conducted on authentic heather, pine, and thyme honey samples from Ikaria and neighboring islands. The Principal Component Analysis (PCA), Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA), and differential analysis were performed using the entire metabolic profile of the samples and allowed the identification of chemical markers for sample discrimination. Thirty-two characteristic secondary metabolites (cinnamic acids, phenolic acids, flavonoids, terpenes) and other bioactive phenolic compounds, some of them not previously reported in a heather honey (aucubin, catalpol, domesticoside, leonuriside A, picein among others), emerged as potential chemical indicators of Anama honey. Melissopalynological analysis was also carried out to decipher the botanical and geographical origin of Anama honey. The relative frequency of the pollen of dominant plants of the Ericaceae family and a multitude of nectariferous and nectarless plants contributing to the botanical profile of Anama was evaluated. The identification of the pollen sources enabled a potential correlation of differentially increased secondary metabolites and chemicals with their botanical origin. The physicochemical profile of Anama was also determined, including the parameters of pH, color, electrical conductivity, diastase, moisture, as well as sugars, supporting the high quality of this heather honey.

Objective To study the changes of metabolites in serum and tissues （kidney, liver and heart） of mice died of acute tetracaine poisoning by metabolomics, to search for potential biomarkers and related metabolic pathways, and to provide new ideas for the identification of cause of death and research on toxicological mechanism of acute tetracaine poisoning. Methods Forty ICR mice were randomly divided into control group and acute tetracaine poisoning death group. The model of death from acute poisoning was established by intraperitoneal injection of tetracaine, and the metabolic profile of serum and tissues of mice was obtained by ultra-high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry （UPLC-Orbitrap HRMS）. Multivariate statistical principal component analysis （PCA） and orthogonal partial least square-discriminant analysis （OPLS-DA） were used, combined with t-test and fold change to identify the differential metabolites associated with death from acute tetracaine poisoning. Results Compared with the control group, the metabolic profiles of serum and tissues in the mice from acute tetracaine poisoning death group were significantly different. Eleven differential metabolites were identified in serum, including xanthine, spermine, 3-hydroxybutylamine, etc.; twenty-five differential metabolites were identified in liver, including adenylate, adenosine, citric acid, etc.; twelve differential metabolites were identified in heart, including hypoxanthine, guanine, guanosine, etc; four differential metabolites were identified in kidney, including taurochenodeoxycholic acid, 11, 12-epoxyeicosatrienoic acid, dimethylethanolamine and indole. Acute tetracaine poisoning mainly affected purine metabolism, tricarboxylic acid cycle, as well as metabolism of alanine, aspartic acid and glutamic acid. Conclusion The differential metabolites in serum and tissues of mice died of acute tetracaine poisoning are expected to be candidate biomarkers for this cause of death. The results can provide research basis for the mechanism and identification of acute tetracaine poisoning.

• Pixel-based chemometrics using a mass spectrum-oriented approach. • OPLS-DA models achieve high accuracy in coffee property estimation. • Proposed method arises as a promising quality assurance tool for the coffee industry. This paper presents a study that assesses the application of chemometrics for classifying coffee samples in a quality control context. High-resolution and accurate mass measurements were utilized as input for pixel-based orthogonal partial least squares discriminant analysis (OPLS-DA) models. The compositional data were acquired through a fully automated workflow combining headspace solid-phase microextraction and gas chromatography-high-resolution mass spectrometry (GC-HRMS) using an FT-Orbitrap® mass analyzer. A workflow centered on accurate mass measurements was successfully utilized for group-type analysis, offering an alternative to methods relying solely on MS similarity searches. The predictive models underwent thorough evaluation, demonstrating robust multivariate classification performance. Five key coffee attributes, bitterness, acidity, body, intensity, and roasting level were successfully predicted using GC-HRMS data. The results revealed strong predictive accuracy across all models, ranging from 88.9 % (bitterness) to 94.4 % (roasting level). This study represents a significant advancement in automating methods for coffee quality control, notably increasing the predictive ability of the models compared to existing literature. [ABSTRACT FROM AUTHOR]

Food safety and quality issues are becoming increasingly important and attract much attention, requiring the development of better analytical platforms. For example, high-resolution (especially Orbitrap) mass spectrometry simultaneously offers versatile functions such as targeted/non-targeted screening while providing qualitative and quantitative information on an almost unlimited number of analytes to facilitate routine analysis and even allows for official surveillance in the food field. This review covers the current state of Orbitrap mass spectrometry (OMS) usage in food analysis based on research reported in 2012–2019, particularly highlighting the technical aspects of OMS application and the achievement of OMS-based screening and quantitative analysis in the food field. The gained insights enhance our understanding of state-of-the-art high-resolution mass spectrometry and highlight the challenges and directions of future research. [ABSTRACT FROM AUTHOR]

Natural biomass magnetic porous carbon was successfully prepared via a cost‐effective and green route using mangosteen shells as raw material. The prepared magnetic porous carbon was used as a magnetic solid‐phase extraction adsorbent for bisphenols enrichment from beverages followed by high‐performance liquid chromatography‐quadrupole‐Orbitrap high‐resolution mass spectrometry. Parameters affecting extraction efficiency including sample solution pH, adsorbent amount, extraction time, eluent type, and volume were optimized. Results showed that biomass magnetic porous carbon had excellent adsorption properties for bisphenols due to its large specific surface area and abundant functional groups, which could form hydrogen bonding and π‐π stacking with bisphenols. The enrichment factor of 3 bisphenols was in the range of 15–19. Under optimum conditions, favorable linearity for all analytes was obtained with correlation coefficients higher than 0.998. Recoveries of spiked samples were in the range of 88.5–105.1% with a relative standard deviation of 3.4–5.5%. These results demonstrated that magnetic porous carbon may be a promising adsorbent for the enrichment of aromatic compounds. [ABSTRACT FROM AUTHOR]

This paper reports the non-targeted analysis of unknown volatile chemicals in medical masks through headspace gas chromatography-Orbitrap high-resolution mass spectrometry. In view of the difficulties that may be encountered in the qualitative analysis of unknown substances, several typical cases and the corresponding reliable solutions are given from the perspective of comprehensive score and retention index, chemical ionization identification molecular formula, fragment ion detail comparison for distinguishing isomers, and identification of alkanes. With this method, 69 volatile substances were identified in 60 masks. The identified substances were divided into nine categories. Alkanes, esters, benzenes, and alcohols were the top four groups of substances identified in masks and accounted for 34.8%, 15.9%, 10.1%, and 7.2% of the total substances, respectively. In addition, ketones, ethers, phenolics, amides, and other substances were identified. Ethanol, 1,4-dichlorobenzene, toluene, m-xylene, dimethyl glutarate, and N,N-dimethylacetamide had high detection rates. The identified substances were further filtered and screened according to their detection rate, toxicity, and response intensity. Finally, 12 high-risk volatile chemicals in medical masks were listed. This study could serve as a reference for identifying unknown substances and a guide for monitoring volatile chemicals in masks and promoting chemical safety improvements in products.

Endothelial cells (ECs) comprise the lumenal lining of all blood vessels and are critical for the functioning of the cardiovascular system. Their phenotypes can be modulated by alternative splicing of RNA to produce distinct protein isoforms. To characterize the RNA and protein isoform landscape within ECs, we applied a long read proteogenomics approach to analyse human umbilical vein endothelial cells (HUVECs). Transcripts delineated from PacBio sequencing serve as the basis for a sample-specific protein database used for downstream mass-spectrometry (MS) analysis to infer protein isoform expression. We detected 53,863 transcript isoforms from 10,426 genes, with 22,195 of those transcripts being novel. Furthermore, the predominant isoform in HUVECs does not correspond with the accepted "reference isoform" 25% of the time, with vascular pathway-related genes among this group. We found 2,597 protein isoforms supported through unique peptides, with an additional 2,280 isoforms nominated upon incorporation of long-read transcript evidence. We characterized a novel alternative acceptor for endothelial-related gene CDH5, suggesting potential changes in its associated signalling pathways. Finally, we identified novel protein isoforms arising from a diversity of RNA splicing mechanisms supported by uniquely mapped novel peptides. Our results represent a high-resolution atlas of known and novel isoforms of potential relevance to endothelial phenotypes and function. [ABSTRACT FROM AUTHOR]

There is continued interest in the occurrence and monitoring of emerging contaminants (ECs) in wastewater treatment plants (WWTPs) to assess their potential impact when released into the environment. The objective of this study was to determine the occurrence of pharmaceutical and personal care products (PPCPs) in WWTP influents and effluents and receiving river waters in Pretoria, South Africa. Targeted screening and identification of PPCPs in the samples was done using liquid chromatography (LC) coupled with high resolution mass spectrometry (HRMS) Orbitrap™ HRMS, after clean-up and preconcentration with Waters Oasis® hydrophilic-lipophilic-balanced (HLB) solid phase extraction (SPE) cartridges. Of the 156 target analytes screened in the study, 120 could be satisfactorily determined under the study conditions, with 71 and 73 compounds being detected and quantified in the influent and effluent samples, respectively. Concentrations in the influents ranged from < ILOQ-14.2 μg L−1 and those in the effluent from < ILOQ - 2.45 μg L−1. It was found that 84% of the quantified compounds belonged to various classes of pharmaceuticals. Ibuprofen, caffeine, paracetamol and estradiol were the major individual contaminants in influent samples with average concentrations of 14.2, 8.68, 4.79 and 1.02 μg L−1, respectively. In the effluent samples, the highest concentrations were recorded for estradiol (2.45 μg L−1), ibuprofen (1.03 μg L−1) and efavirenz (0.58 μg L−1). The highest mean concentrations (above 0.5 μg L−1) recorded in the river water samples were for ibuprofen, (4.14 μg L−1downstream; 3.19 μg L−1 upstream), caffeine (2.98 μg L−1 downstream; 1.42 μg L−1 upstream), paraxanthine (1.22 μg L−1 downstream; 0.798 μg L−1 upstream). Analysis of river water samples also revealed that PPCP contamination in the river system cannot be solely attributed to the WWTP effluents. Twenty-four compounds presented in this study are reported for the first time in South African surface waters.

Monoclonal antibodies (mAbs) are protein biotherapeutics with a proven efficacy toward fighting life-threatening diseases. Their exceptional healing potential drives the annual increase in the number of novel mAbs and other antibody-like molecules entering clinical trials and the number of approved mAb-based drugs. Mass spectrometry (MS) offers high selectivity and specificity for the potentially unambiguous identification and comprehensive structural characterization of proteins, including at the proteoform level. It is thus not surprising that MS-based approaches are playing a central role in the biopharma laboratories, complementing and advancing traditional biotherapeutics characterization workflows. A combination of MS approaches is required to comprehensively characterize mAbs’ structures: the commonly employed bottom-up MS approaches are efficiently complemented with mass measurements at the intact and subunit (middle-up) levels, together with product ion analysis following gas-phase fragmentation of precursor ions performed at the intact (top-down) and subunit (middle-down) levels. Here we overview our group’s contribution to increasing the efficiency of these approaches and the development of the novel strategies over the past decade. Our particular focus has been on the top-down and middle-down MS methods that utilize electron transfer dissociation (ETD) for gas-phase protein ion fragmentation. Several approaches pioneered by our group, particularly an ETD-based middle-down approach, constitute a part of commercial software solutions for the mAb’s characterization workflows.

Pharmaceutical analysis refers to an area of analytical chemistry that deals with active compounds either by themselves (drug substance) or when formulated with excipients (drug product). In a less simplistic way, it can be defined as a complex science involving various disciplines, e.g., drug development, pharmacokinetics, drug metabolism, tissue distribution studies, and environmental contamination analyses. As such, the pharmaceutical analysis covers drug development to its impact on health and the environment. Moreover, due to the need for safe and effective medications, the pharmaceutical industry is one of the most heavily regulated sectors of the global economy. For this reason, powerful analytical instrumentation and efficient methods are required. In the last decades, mass spectrometry has been increasingly used in pharmaceutical analysis both for research aims and routine quality controls. Among different instrumental setups, ultra-high-resolution mass spectrometry with Fourier transform instruments, i.e., Fourier transform ion cyclotron resonance (FTICR) and Orbitrap, gives access to valuable molecular information for pharmaceutical analysis. In fact, thanks to their high resolving power, mass accuracy, and dynamic range, reliable molecular formula assignments or trace analysis in complex mixtures can be obtained. This review summarizes the principles of the two main types of Fourier transform mass spectrometers, and it highlights applications, developments, and future perspectives in pharmaceutical analysis.

(1) Background: In toxicological laboratories, various screening methods can be used to identify compounds involved in intoxication. High-resolution mass spectrometry has been increasingly used in this context for the last years, because of its sensitivity and reliability. Here, we present the development and validation of a screening method that uses liquid chromatography coupled with a high-resolution mass spectrometer. (2) Methods: This method required only 100 µL of whole blood or plasma sample. Pretreatment consisted of a rapid and simple deproteinisation with methanol/acetonitrile and zinc sulphate. This new assay was validated according to international guidelines. (3) Results: To perform the method validation, 53 compounds were selected. The selection criteria were as follows: various chemical structures and therapeutic families (>15), large m/z distribution, positive or negative ionisation mode, and various elution times. The assays showed high selectivity and specificity, with optimal process efficiency. The identification limits, determined using predefined criteria, were established at sub-therapeutic or therapeutic concentrations. Applicability was evaluated using spiked plasma controls and external quality controls. (4) Conclusions: The new method was then successfully applied to routine clinical and forensic samples.

Herein, a chemophenetic significance, based on the phenolic metabolite profiling of three Senecio (S. hercynicus, S. ovatus, and S. rupestris) and two Jacobaea species (J. pancicii and J. maritima), coupled to morphometric data, is presented. A set of twelve morphometric characters were recorded from each plant species and used as predictor variables in a linear discriminant analysis (LDA) model. From a total 75 observations (15 from each of the five species), the model correctly assumed their species’ membership, except for 2 observations. Among the studied species, S. hercynicus and S. ovatus presented the greatest morphological similarity. A phytochemical profiling of phenolic specialized metabolites by UHPLC-Orbitrap-MS revealed 46 hydroxybenzoic, hydroxycinnamic, and acylquinic acids and their derivatives, 1 coumarin and 21 flavonoids. Hierarchical and PCA clustering applied to the phytochemical data corroborated the similarity of S. hercynicus and S. ovatus, observed in the morphometric analysis. This study contributes to the phylogenetic relationships between the tribe Senecioneae taxa and highlights the chemophenetic similarity/dissimilarity of the studied species belonging to Senecio and Jacobaea genera.

The water-based extract of broad bean hulls contains several bioactive molecules, including polyphenols well-known to exert antioxidant activity, which could justify its use in nutraceutical formulations. Hence, the current investigation aimed to establish the polyphenolic profile of water-based extracts from broad bean hulls through UHPLC–Q-Orbitrap HRMS analysis. The findings highlighted that p-coumaric acid, chlorogenic acid, and epicatechin were the most common compounds found in the tested extracts, being quantified at a mean concentration of 42.1, 32.6, and 31.2 mg/100 g, respectively. Moreover, broad bean hull extracts were encapsulated into a nutraceutical formulation, after which the antioxidant properties and the bioaccessibility of phenolic compounds during the simulated gastrointestinal (GI) process were investigated and compared with the digested non-encapsulated extract. The data highlighted that following the GI process, the capsules were able to preserve active compounds from the adverse effects of digestion, resulting in a greater antioxidant capacity and polyphenol bioaccessibility in the duodenal and colonic phases, compared with the non-encapsulated extract. Our results showed that the water extract from broad bean hulls may be considered a valuable source of natural polyphenolic compounds; in addition, the use of a gastric-resistant capsule could be a suitable alternative to transport these bioactive compounds to the target tissues.

Deep proteomic profiling of complex biological and medical samples available at low nanogram and subnanogram levels is still challenging. Thorough optimization of settings, parameters, and conditions in nanoflow liquid chromatography-tandem mass spectrometry (MS)-based proteomic profiling is crucial for generating informative data using amount-limited samples. This study demonstrates that by adjusting selected instrument parameters, e.g., ion injection time, automated gain control, and minimally altering the conditions for resuspending or storing the sample in solvents of different compositions, up to 15-fold more thorough proteomic profiling can be achieved compared to conventionally used settings. More specifically, the analysis of 1 ng of the HeLa protein digest standard by Q Exactive HF-X Hybrid Quadrupole-Orbitrap and Orbitrap Fusion Lumos Tribrid mass spectrometers yielded an increase from 1758 to 5477 (3-fold) and 281 to 4276 (15-fold) peptides, respectively, demonstrating that higher protein identification results can be obtained using the optimized methods. While the instruments applied in this study do not belong to the latest generation of mass spectrometers, they are broadly used worldwide, which makes the guidelines for improving performance desirable to a wide range of proteomics practitioners.

Skin secretion represents the only means of defense for the majority of frog species. That phenomenon is based on the fact that the main components of the secretion are peptides demonstrating greatly varying types of bioactivity. They fulfill regulatory functions, fight microorganisms and may be even helpful against predators. These peptides are considered to be rather promising pharmaceuticals of future generation as according to the present knowledge microorganisms are unlikely to develop resistance to them. Mass spectrometry sequencing of these peptides is the most efficient first step of their study providing reliably their primary structures, i.e., amino acids sequence and S-S bond motif. Besides discovering new bioactive peptides, mass spectrometry appears to be an efficient tool of taxonomy studies, allowing for distinguishing not only between closely related species, but also between populations of the same species. Application of several tandem mass spectrometry tools (CID, HCD, ETD, EThcD) available with Orbitrap mass analyzer allowed us to obtain full sequence of about 60 peptides in the secretion of Slovenian population of brown ranid frog Rana temporaria. The problem of sequence inside C-terminal cycle formed by two Cys and differentiation of isomeric Leu and Ile residues was done in top-down mode without any derivatization steps. Besides general biomarkers of Rana temporaria species, Central Slovenian population of Rana temporaria demonstrates six novel temporins and one brevinin 1, which may be treated as biomarkers of that population. [ABSTRACT FROM AUTHOR]

Chemical hazardous substances in teethers may migrate into infant's body through oral exposure, resulting in a potential health risk. In recent years, researchers have performed a series of studies for detecting target chemicals in teethers and other toys, but the presence of unknown chemicals has not been systematically investigated yet. This paper reports the non-targeted identification of unknown chemical hazards that may have migrated from teethers to infants based on gas chromatography-Orbitrap high resolution mass spectrometry. In view of the difficulties that may be encountered in the qualitative analysis of substances, several typical cases and the corresponding reliable solutions are given from the perspective of comprehensive score and retention index, isotope-aided qualitative analysis, chemical ionization identification formula, and fragment ion detail comparison for distinguishing isomers. Finally, 28 substances are identified in 10 teether samples. Among them, phenol, N-methylaniline, 1,6-dioxacyclododecane-7,12-dione and cyclohexanone have higher detection rates. This study not only has valuable reference for the identification of unknown substances, but also has positive guiding role in monitoring potential chemical hazards in toys and promoting the safety of products.

Cultivated soybean (Glycine max (L.)), the world’s most important legume crop, has high-to-moderate salt sensitivity. Being the frontier for sensing and controlling solute transport, membrane proteins could be involved in cell signaling, osmoregulation, and stress-sensing mechanisms, but their roles in abiotic stresses are still largely unknown. By analyzing salt-induced membrane proteomic changes in the roots and leaves of salt-sensitive soybean cultivar (C08) seedlings germinated under NaCl, we detected 972 membrane proteins, with those present in both leaves and roots annotated as receptor kinases, calcium-sensing proteins, abscisic acid receptors, cation and anion channel proteins, proton pumps, amide and peptide transporters, and vesicle transport-related proteins etc. Endocytosis, linoleic acid metabolism, and fatty acid biosynthesis pathway-related proteins were enriched in roots whereas phagosome, spliceosome and soluble NSF attachment protein receptor (SNARE) interaction-related proteins were enriched in leaves. Using label-free quantitation, 129 differentially expressed membrane proteins were found in both tissues upon NaCl treatment. Additionally, the 140 NaCl-induced proteins identified in roots and 57 in leaves are vesicle-, mitochondrial-, and chloroplast-associated membrane proteins and those with functions related to ion transport, protein transport, ATP hydrolysis, protein folding, and receptor kinases, etc. Our proteomic results were verified against corresponding gene expression patterns from published C08 RNA-seq data, demonstrating the importance of solute transport and sensing in salt stress responses.

β-agonists have been illegally used for growth promoting purposes in animal husbandry, leading to residue concentrations capable of inducing acute toxic reactions among consumers of animal-based food. There is not only a need for detecting β-agonist residues at low concentrations, but also to increase the number of compounds to be monitored. It was therefore the aim of this paper to develop a unified method capable of detecting a wide range of different β-agonists (20 analytes including some metabolites) in a variety of matrices (muscle, liver, plasma, milk and urine). The developed procedure permits the quick processing of samples with limited labour input and consumption of consumables. The method has been validated according to the Commission Decision 98/536/EC. Detection is based on ultrahigh-performance chromatography coupled to high-resolution mass spectrometry. Validation was performed on two different instruments (Orbitrap and time of flight). The obtained limit of quantification (0.05 to 0.5 μg/kg and the average recovery of 78% in the most complex matrix (liver) satisfies current regulatory requirements. [ABSTRACT FROM AUTHOR]

Syrian golden hamsters (Mesocricetus auratus) infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests lung pathology. In this study, efforts were made to check the infectivity of a local SARS-CoV-2 isolate in a self-limiting and non-lethal hamster model and evaluate the differential expression of lung proteins during acute infection and convalescence. The findings of this study confirm the infectivity of this isolate in vivo. Analysis of clinical parameters and tissue samples show the pathophysiological manifestation of SARS-CoV-2 infection similar to that reported earlier in COVID-19 patients and hamsters infected with other isolates. However, diffuse alveolar damage (DAD), a common histopathological feature of human COVID-19 was only occasionally noticed. The lung-associated pathological changes were very prominent on the 4th day post-infection (dpi), mostly resolved by 14 dpi. Here, we carried out the quantitative proteomic analysis of the lung tissues from SARS-CoV-2-infected hamsters on day 4 and day 14 post-infection. This resulted in the identification of 1585 proteins of which 68 proteins were significantly altered between both the infected groups. Pathway analysis revealed complement and coagulation cascade, platelet activation, ferroptosis, and focal adhesion as the top enriched pathways. In addition, we also identified altered expression of two pulmonary surfactant-associated proteins (Sftpd and Sftpb), known for their protective role in lung function. Together, these findings will aid in understanding the mechanism(s) involved in SARS-CoV-2 pathogenesis and progression of the disease. [ABSTRACT FROM AUTHOR]

This paper reports a method for the differential analysis of volatile chemical components in five novel types of mosquito-repellent products based on chemometrics combined with headspace gas chromatography-Orbitrap high-resolution mass spectrometry (HS-GC-Orbitrap HRMS) nontargeted screening. A total of 358 unknown substances were detected in 30 samples under specific headspace conditions. Through principal component analysis and orthogonal partial least-squares discriminant analysis, 36 significantly different substances with variable importance in the projection values greater than 1 were further screened, and these substances were accurately identified by GC-Orbitrap HRMS. Most substances were found for the first time in mosquito-repellent products. The clustered heat map, Venn diagram and peak area histogram showed that the mosquito-repellent products had similar volatile composition, and the volatile species and content of different types of mosquito-repellent products significantly varied. Substances, such as eucalyptol, d -limonene, α -pinene, β -pinene, dl -menthol and methyl salicylate, may be the main sources of odour in mosquito-repellent products. This work explored the characteristic volatile components in mosquito-repellent products and comparatively analysed the chemical composition of different types of products. It can be generalised to consumer products as a case study and has positive implications for promoting product quality and safety and improving production processes. [Display omitted] • Volatile species and content varied significantly among mosquito-repellent products. • The 36 differential substances screened were identified by GC-Orbitrap HRMS. • Eucalyptol, d -limonene, α-pinene et al., may be the main sources of odour in products. • The method can be used to identify characteristic substances that cause differences in products. [ABSTRACT FROM AUTHOR]

In this work, we explore the processes involving food in contact with food contact materials (FCMs), and the subsequent changes in these materials. We delve into the historical context of "economic poisons", the development of food safety concerns, the regulation of food additives, and the rise of harmful chemicals in food. The focus is placed on mass spectrometry-based analyses of FCMs. We have examined substances, like bisphenols, and the chemical migration from various materials into food, highlighting the difficulty in identifying and managing these substances, especially unknown ones. This work further cements the inadequacy of current regulations in effectively controlling FCMs and mitigating associated risks. We conclude that despite the information provided by mass spectrometry, challenges persist in fully comprehending and regulating the interplay between FCMs and food under actual cooking conditions, emphasising the need for ongoing research and improved regulations to protect consumer health from these evolving chemical threats. • Historical context of food safety and "economic poisons". • Role of mass spectrometry in identifying chemicals in food contact materials. • Limitations of current regulations associated with food contact materials. • High-resolution mass spectrometry is a potent tool in food evaluations. • This technology has allowed to identify new substances of potential concern. [ABSTRACT FROM AUTHOR]

A method for the simultaneous screening of 258 pesticide residues in silage using modified QuEChERS combined with liquid chromatography (LC)- and gas chromatography (GC)- quadrupole-Orbitrap mass spectrometry (Q-Orbitrap/MS) has been developed. After hydration, the silage was homogenized with a 1% acetic acid–acetonitrile solution, and the extract was purified using C18, PSA, and anhydrous magnesium sulfate. Finally, the sample was detected using LC/GC-Q-Orbitrap/MS, and quantified using an external standard method. The results showed that 258 pesticides had an excellent linear relationship in the range of 0.1–50 μg L−1, and that the coefficients of determination (R2) were more than 0.99. The screening detection limit (SDL) of silage was in the range of 0.5–50 μg kg−1, and the limit of quantitation (LOQ) was in the range of 1–50 μg kg−1. The accuracy and precision of the method were verified at the spiked levels of 1-, 2- and 10-times LOQ, and the recovery of 258 pesticides was in the range of 66.5–119.8%, with relative standard deviations (RSDs) of less than 20% (n = 6). This method was simple, rapid, and reliable, and could be applied to screen and quantify multi-pesticide residues in silage.

We present a novel, scan-centric method for characterizing peaks from direct injection multi-scan Fourier transform mass spectra of complex samples that utilizes frequency values derived directly from the spacing of raw m/z points in spectral scans. Our peak characterization method utilizes intensity-independent noise removal and normalization of scan-level data to provide a much better fit of relative intensity to natural abundance probabilities for low abundance isotopologues that are not present in all of the acquired scans. Moreover, our method calculates both peak- and scan-specific statistics incorporated within a series of quality control steps that are designed to robustly derive peak centers, intensities, and intensity ratios with their scan-level variances. These cross-scan characterized peaks are suitable for use in our previously published peak assignment methodology, Small Molecule Isotope Resolved Formula Enumeration (SMIRFE).

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The evolution of the field of element speciation, from the targeted analysis for specific element species toward a global exploratory analysis for the entirety of metal- or metalloid-related compounds present in a biological system (metallomics), requires instrumental techniques with increasing selectivity and sensitivity. The selectivity of hyphenated techniques, combining chromatography, and capillary electrophoresis with element-specific detection (usually inductively coupled plasma mass spectrometry, ICP MS), is often insufficient to discriminate all the species of a given element in a sample. The necessary degree of specificity can be attained by ultrahigh-resolution (R >100,000 in the m/z < 1,000 range for a 1 s scan) mass spectrometry based on the Fourier transformation of an image current of the ions moving in an Orbitrap or an ion cyclotron resonance (ICR) cell. The latest developments, allowing the separate detection of two ions differing by a mass of one electron (0.5 mDa) and the measurement of their masses with a sub-ppm accuracy, make it possible to produce comprehensive lists of the element species present in a biological sample. Moreover, the increasing capacities of multistage fragmentation often allow their de novo identification. This perspective paper critically discusses the potential state-of-the-art of implementation, and challenges in front of FT (Orbitrap and ICR) MS for a large-scale speciation analysis using, as example, the case of the metabolism of selenium by yeast.

Escherichia coli normally cannot grow in the environment. One environmental stress that prevents E. coli growth may be the competition for growth substrates with co‐existing micro‐organisms. In this study, the growth substrates of E. coli were screened by high‐resolution mass spectrometry and compared with those of indigenous bacteria in river water. In an incubation experiment, E. coli multiplied in sterilized river water, but did not multiply when indigenous micro‐organisms were present in the water. By analysing dissolved organic matter in the river water before and after E. coli growth, 35 compounds were identified as putative growth substrates of E. coli. Among them, 33 compounds were also identified as putative growth substrates of indigenous bacteria. These results indicate that E. coli and indigenous bacteria compete for organic substrates in river water, which could suppress the growth of E. coli. [ABSTRACT FROM AUTHOR]

Food can contain unwanted compounds and need to be analyzed for compounds like carcinogenic polycyclic aromatic hydrocarbons to ensure consumer safety. The analytes need to be extracted from the sample matrix and cleaned‐up to enable detection. However, established methods for clean‐up are labor intensive and have a high expenditure on organic solvents. Here, we show a newly developed micro‐solid‐phase extraction cartridge method to automate the clean‐up for analysis of polycyclic aromatic hydrocarbons in sunflower oil using gas chromatography with quadrupole‐orbitrap mass spectrometry with a TriPlus autosampler. This automated micro‐solid‐phase extraction cartridge method needs only 4 μL of vegetable oil sample and requires only 360 μL acetonitrile for elution, and, therefore, it needs only small amounts of organic solvent. Two different micro‐solid‐phase extraction cartridge methods were developed, one using two commercially available cartridges with florisil and octadecylsilane/Z‐Sep/CarbonX, and the other method using one commercially available cartridge with florisil followed by one self‐made cartridge with octadecylsilane/Z‐Sep. The latter method showed successful lipid removal and was further validated for 22 of 24 polycyclic aromatic hydrocarbon compounds in sunflower oil at a spiked level of 1090 μg/kg with recoveries ranging from 53 to 118% and relative standard deviation below 22%. This method shows promising short‐time clean‐up with low expenditure of solvents. [ABSTRACT FROM AUTHOR]

Quadrupole time‐of‐flight (QTof) collision‐induced dissociation (CID) and Orbitrap higher‐energy collisional dissociation (HCD) are the most commonly used fragmentation techniques in mass spectrometry‐based proteomics workflows. The information content of the MS/MS spectra is first and foremost determined by the applied collision energy. How can we set up the two instrument types to achieve maximum transferability? To answer this question, we compared MS/MS spectra obtained on a Bruker QTof CID and a Thermo Q‐Exactive Focus Orbitrap HCD instrument as a function of collision energy using the similarity index. Results show that with a few eV lower collision energy setting on HCD (Orbitrap‐specific CID) than on QTof CID, nearly identical MS/MS spectra can be obtained for leucine enkephalin pentapeptide standard, for selected +2 and +3 enolase tryptic peptides and for a large number of peptides in a HeLa protein digest. The Bruker QTof was able to produce colder ions, which may be significant to study inherently labile compounds. Further, we examined energy dependence of peptide identification confidence, as characterized by Mascot scores, on the HeLa peptides. In line with earlier QTof results, this dependence shows one or two maxima (unimodal or bimodal behavior) on Orbitrap. The fraction of bimodal peptides is lower on Orbitrap. Optimal energies as a function of m/z show a similar linear trend on both instruments, which suggests that with appropriate collision energy adjustment, matching conditions for proteomics can be achieved. Data have been deposited in the MassIVE repository (MSV000086434). [ABSTRACT FROM AUTHOR]

Purpose: Validated biomarkers of food intake (BFIs) have recently been suggested as a useful tool to assess adherence to dietary guidelines or compliance in human dietary interventions. Although many new candidate biomarkers have emerged in the last decades for different foods from metabolic profiling studies, the number of comprehensively validated biomarkers of food intake is limited. Apples are among the most frequently consumed fruits and a rich source of polyphenols and fibers, an important mediator for their health-protective properties. Methods: Using an untargeted metabolomics approach, we aimed to identify biomarkers of long-term apple intake and explore how apples impact on the human plasma and urine metabolite profiles. Forty mildly hypercholesterolemic volunteers consumed two whole apples or a sugar and energy-matched control beverage, daily for 8 weeks in a randomized, controlled, crossover intervention study. The metabolome in plasma and urine samples was analyzed via untargeted metabolomics. Results: We found 61 urine and 9 plasma metabolites being statistically significant after the whole apple intake compared to the control beverage, including several polyphenol metabolites that could be used as BFIs. Furthermore, we identified several endogenous indole and phenylacetyl-glutamine microbial metabolites significantly increasing in urine after apple consumption. The multiomic dataset allowed exploration of the correlations between metabolites modulated significantly by the dietary intervention and fecal microbiota species at genus level, showing interesting interactions between Granulicatella genus and phenyl-acetic acid metabolites. Phloretin glucuronide and phloretin glucuronide sulfate appeared promising biomarkers of apple intake; however, robustness, reliability and stability data are needed for full BFI validation. Conclusion: The identified apple BFIs can be used in future studies to assess compliance and to explore their health effects after apple intake. Moreover, the identification of polyphenol microbial metabolites suggests that apple consumption mediates significant gut microbial metabolic activity which should be further explored. [ABSTRACT FROM AUTHOR]

To better understand cell-to-cell heterogeneity, advanced analytical tools are in a growing demand for elucidating chemical compositions of each cell within a population. However, the progress of single-cell chemical analysis has been restrained by the limitations of small cell volumes and minute cellular concentrations. Here, we present a rapid and sensitive method for investigating the lipid profiles of isolated single cells using infrared matrix-assisted laser desorption electrospray ionization mass spectrometry (IR-MALDESI-MS). In this work, HeLa cells were dispersed onto a glass slide, and the cellular contents were ionized by IR-MALDESI and measured using a Q-Exactive HF-X mass spectrometer. Importantly, this approach does not require extraction and/or enrichment of analytes prior to MS analysis. Using this approach, 45 distinct lipid species, predominantly phospholipids, were detected and putatively annotated from the single HeLa cells. The proof-of-concept study demonstrates the feasibility and efficacy of IR-MALDESI-MS for rapid lipidomic profiling of single cells, which provides an important basis for future work on differentiation between normal and diseased cells at various developmental states, which can offer new insights into cellular metabolic pathways and pathological processes. Although not yet accomplished, we believe this approach can be readily used as an assessment tool to compare the number of identified species during source evolution and method optimization (intra-laboratory), and also disclose the complementary nature of different direct analytical approaches for the coverage of different types of endogenous analytes (inter-laboratory). Graphical abstract [ABSTRACT FROM AUTHOR]

Hepatotoxic and genotoxic pyrrolizidine alkaloids have been involved in the acute poisoning of animals and humans. Crotalaria (Fabaceae) species contain these alkaloids. In this work, the diversity and distribution of pyrrolizidine alkaloids in roots, leaves, flowers, and seeds of Crotalaria pallida, Crotalaria maypurensis, Crotalaria retusa, Crotalaria spectabilis, Crotalaria incana, and Crotalaria nitens were studied. Matrix solid‐phase dispersion and ultra‐high‐performance liquid chromatography coupled with Orbitrap mass spectrometry were successfully employed in pyrrolizidine alkaloids extraction and analysis, respectively. Forty‐five pyrrolizidine alkaloids were detected and their identification was based on the mass spectrometry accurate mass measurement and fragmentation pattern analysis. The cyclic retronecine‐type diesters monocrotaline, crotaleschenine, integerrimine, usaramine, and their N‐oxides were predominantly present. Five novel alkaloids were identified for the first time in Crotalaria species, namely 14‐hydroxymonocrotaline, 12‐acetylcrotaleschenine, 12‐acetylmonocrotaline, 12‐acetylintegerrimine, and dihydrointegerrimine. Due to a lack of commercially available standards, the response factor of monocrotaline was used for quantification of pyrrolizidine alkaloids and their N‐oxides. Seeds and flowers possessed higher pyrrolizidine alkaloids amounts than roots and leaves. Due to their 1,2‐unsaturated pyrrolizidine alkaloids content, the ingestion of Crotalaria plant seeds or other parts through herbal products, infusions, or natural remedies is a serious health threat to humans and livestock. [ABSTRACT FROM AUTHOR]

Matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for highly multiplexed, untargeted detection of many hundreds of analytes from tissue. Recently, laser postionization (MALDI‐2) has been developed for increased ion yield and sensitivity for lipid IMS. However, the dependence of MALDI‐2 performance on the various lipid classes is largely unknown. To understand the effect of the applied matrix on MALDI‐2 analysis of lipids, samples including an equimolar lipid standard mixture, various tissue homogenates, and intact rat kidney tissue sections were analyzed using the following matrices: α‐cyano‐4‐hydroxycinnamic acid, 2′,5′‐dihydroxyacetophenone, 2′,5′‐dihydroxybenzoic acid (DHB), and norharmane (NOR). Lipid signal enhancement of protonated species using MALDI‐2 technology varied based on the matrix used. Although signal improvements were observed for all matrices, the most dramatic effects using MALDI‐2 were observed using NOR and DHB. For lipid standards analyzed by MALDI‐2, NOR provided the broadest coverage, enabling the detection of all 13 protonated standards, including nonpolar lipids, whereas DHB gave less coverage but gave the highest signal increase for those lipids recorded. With respect to tissue homogenates and rat kidney tissue, mass spectra were compared and showed that the number and intensity of neutral lipids tentatively identified with MALDI‐2 using NOR increased significantly (e.g., fivefold intensity increase for triacylglycerol). In the cases of DHB with MALDI‐2, the number of protonated lipids identified from tissue homogenates doubled with 152 on average compared with 76 with MALDI alone. High spatial resolution imaging (~20 μm) of rat kidney tissue showed similar results using DHB with 125 lipids tentatively identified from MALDI‐2 spectra versus just 72 using standard MALDI. From the four matrices tested, NOR provided the greatest increase in sensitivity for neutral lipids (triacylglycerol, diacylglycerol, monoacylglycerol, and cholesterol ester), and DHB provided the highest overall number of lipids detected using MALDI‐2 technology. [ABSTRACT FROM AUTHOR]

In an effort to characterize differently aged bourbons and to determine whether bourbons could be "fingerprinted" by their chemical compositions, we used Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to characterize 2 distinct sets of bourbon samples. The first set of bourbons were prepared using the same mash bill but were aged differently (unaged (0 years), 2 years, 4 years, and 6 years) in oak barrels. The results showed an increase in the number of chemical compounds present as the bourbon ages. Most of the large changes in chemical composition occur in the first 2 years of aging. We also analyzed single barrel bourbons, which were produced identically but maturated in different, adjacent barrels, and the results suggested that significant differences exist among these samples. These results suggest that "fingerprinting" of different bourbons for authentication purposes may be complicated and that careful analyses, coupled with more comprehensive identification of chemical compounds in bourbons, are needed. [ABSTRACT FROM AUTHOR]

Antarctic shelf systems play an important role in organic matter circulation on Earth; hence, identifying the characteristics of dissolved organic matter (DOM) can be a good indicator for understanding its origin, as well as climate change. In this study, to identify the characteristics of DOM in the ice shelf systems, surface water was collected from the open sea (OS) and Marian cove (fjord, FJ). Although there were no differences in DOM characteristics between sampling sites in the quantitative analyses, the DOM in the surface water of each region seemed to be more affected by terrestrial than marine biological sources in optical and molecular properties. This finding indicates that the terrestrial DOM related to mosses based on the results of molecular properties: high levels of lipid-like (35–39%) and unsaturated hydrocarbon-like (UH; 27–34%) in both the OS and the FJ regions and significantly higher tannin-like substance and condensed aromatic structures in the FJ than in the OS region. When comparing the FJ transect samples, those nearest to a glacier (FJ1; 0.93 km from the glacier) showed relatively low salinity, high dissolved organic carbon, and high chromophoric DOM, indicating that terrestrial DOM (possibly produced by moss) inflow occurred with the runoff from the freshly melting land ice and glacier. However, no significant differences in molecular composition were detected, suggesting that terrestrial DOM is introduced into the ice shelf systems by melting land ice, and glacier runoff could be a major source of DOM-rich seawater during austral fall when low marine biological activity occurs. This study has a great significance as background data for DOM characteristics in the ice shelf systems due to the enhanced biological activity during the austral summer.

Multi-attribute methods (MAM), based on proteolytic digestion followed by liquid chromatography-mass spectrometry analysis of proteolytic peptides, have gained substantial attention in the biopharmaceutical industry for quantifying a variety of quality attributes for therapeutic proteins. Most MAM developed so far have been based on high-resolution mass spectrometers, due to their superb resolving power to distinguish analyte signals from interferences. Lower-resolution instruments, if demonstrated suitable, may further promote the adoption of the technology due to their low cost, small footprint, and ease of use. In this work, we compared the performance of a high-resolution instrument with a few low-resolution quadrupole-type instruments in quantifying a diverse set of quality attributes in a monoclonal antibody product. Different modes of operation for the quadrupole instruments, including scan mode, selected-ion monitoring and multiple-reaction monitoring, were evaluated. The high-resolution instrument has superb performance, with a quantitation limit of 0.002%. Single-quadrupole instruments in scan mode, on the other hand, provide a quantitation limit of about 1%, which may be fit-for-purpose for many routine MAM applications.

The aim of this study was to develop and to validate a toxicological untargeted screening relying on LC-HRMS in meconium including the detection of the four main classes of drugs of abuse (DoA; amphetamines, cannabinoids, opioids and cocaine). The method was then applied to 29 real samples. Analyses were performed with a liquid chromatography system coupled to a benchtop Orbitrap operating in a data-dependent analysis. The sample amount was 300 mg of meconium extracted twice by solid phase extraction following two distinct procedures. Raw data were processed using the Compound Discoverer 3.2 software (Thermo). The method was evaluated and validated on 15 compounds (6-MAM, morphine, buprenorphine, norbuprenorphine, methadone, EDDP, amphetamine, MDA, MDMA, methamphetamine, cocaine, benzoylecgonine, THC, 11-OH-THC, THC-COOH). Limits of detection were between 0.5 and 5 pg/mg and limits of identification between 5 and 50 pg/mg. Mean matrix effect was between −79 and −19% (n = 6) and mean overall recovery between 18 and 73% (n = 6) at 100 pg/mg. The application allows the detection of 88 substances, including 47 pharmaceuticals and 15 pharmaceutical metabolites, cocaine and its metabolites, THC and its metabolites, and natural (morphine, codeine) and synthetic (methadone, buprenorphine, tramadol, norfentanyl) opioids. This method is now used routinely for toxicological screening in high-risk pregnancies

UHPLC/ESI/MS identification of organic compounds is the first step in the majority of screening techniques for the characterization of biologically active metabolites in natural sources. This paper describes a method for the fast identification and characterisation of secondary metabolites in Leptocarpha rivularis DC. (Palo negro) extracts by HPLC/UV (DAD)–Mass Spectrometry (HPLC/MS). The plant is used for the treatment of several diseases since pre-hispanic Mapuche times. Thirty-seven compounds were detected in the aqueous edible extract for the first time including 4 sesquiterpenes, 10 flavonoids, 9 oxylipins, 2 organic acids, and 11 phenolic acids. In addition, phenolic content antioxidant and cholinesterase inhibitory activities were measured for the first time using the edible infusion. The total polyphenol content of the infusion was 230.76 ± 2.5 mmol GAE/kg dry weight, while the antioxidant activity was 176.51 ± 28.84; 195.28 ± 4.83; and 223.92 ± 2.95 mmol TE/kg dry weight, for the DPPH, ABTS, and FRAP assays, respectively. The cholinesterase inhibitory activity was 7.38 ± 0.03 and 5.74 ± 0.06 mmol GALAE/kg, for the inhibition of acetylcholinesterase AChE and BChE, respectively, showing that this plant is a candidate for the isolation of compounds that can be useful for the treatment of neurodegenerative diseases. Furthermore, this plant could serve also as a raw material for the production of dietary supplements, due to its content of polyphenolic compounds.

The analysis of N-linked glycans using liquid chromatography and mass spectrometry (LC-MS) presents significant challenges, particularly owing to their hydrophilic nature. To address these difficulties, a variety of derivatization methods have been developed to facilitate improved ionization and detection sensitivity. One such method, the Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT)™ strategy for labeling glycans, has previously been utilized in the analysis of N- and O-linked glycans in biological samples. To assess the maximum sensitivity and separability of the INLIGHT™ preparation and analysis pipeline, several critical steps were investigated. First, recombinant and nonrecombinant sources of PNGase F were compared to assess variations in the released glycans. Second, modifications in the INLIGHT™ derivatization step were evaluated including temperature optimization, solvent composition changes, reaction condition length and tag concentration. Optimization of the modified method resulted in 20–100 times greater peak areas for the detected N-linked glycans in fetuin and horseradish peroxidase compared with the standard method. Furthermore, the identification of low-abundance glycans, such as (Fuc)1(Gal)2(GlcNAc)4(Man)3(NeuAc)1 and (Gal)3(GlcNAc)5(Man)3(NeuAc)3, was possible. Finally, the optimal LC setup for the INLIGHT™ derivatized N-linked glycan analyses was found to be a C18 reverse-phase (RP) column with mobile phases typical of RPLC. [ABSTRACT FROM AUTHOR]