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Rationale: The oxygen stable isotope ratio (δ 18 O) of the sugar-rich fraction of fruit juice is important as a tracer of the geographical origin of raw material. This study sought to minimize the inter-day variation of δ 18 O attributable to the influence of water to accurately monitor geographical origin labeling., Methods: Two drying devices (freeze dryer and vacuum oven) were compared. Then, two humidity levels (normal and low humidity) at which the samples were placed after drying were compared. The low-humidity environment was constructed using a glove bag and pure argon gas. δ 18 O was measured using thermal conversion elemental analyzer/isotope ratio mass spectrometry. Improvements were made to the measurement method based on aforementioned analyses results, and the performance of the initial and improved methods was compared., Results: δ 18 O of juice dried in a vacuum oven was 3.30‰ lower than that of juice dried in a freeze dryer. Moreover, δ 18 O of juice samples exposed to normal humidity was 3.74‰ lower than that of samples exposed to low humidity. The combined inter-day and intra-day standard deviation was reduced from 1.20‰ in the initial method to 0.42‰ in the improved method., Conclusions: This study describes a pretreatment method for δ 18 O measurement in the sugar-rich fraction of fruit juice with less inter-day variation, and it will be useful for monitoring geographical origin labeling., (© 2024 John Wiley & Sons Ltd.)

Native proteomics measures endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid-phase separations. Native proteomics should provide the most accurate bird's-eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well-purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate). Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra-high mass range (UHMR) Orbitrap mass spectrometer. The nCZE-MS technique enabled the measurement of a 115-kDa standard protein complex while consuming only about 0.1 ng of protein material. nCZE-MS analysis of an E.coli cell lysate detected 72 proteoforms or protein complexes in a mass range of 30-400 kDa in a single run while consuming only 50-ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough in native proteomics for measuring complex proteomes., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Unknown impurities, metabolites and harmful pollutants present in pharmaceutical products, biological and environmental samples, respectively are of high concern in terms of their detection and quantification. The targeted analysis aims to quantify known chemical entities, but it lacks the ability to identify unknown components present in a sample. Non-targeted analysis is an analytical approach that can be made applicable to various disciplines of science to effectively search for unknown chemical, biological, or environmental entities that can answer various baffling mysteries of research. It employs various high-end analytical techniques that can specifically screen out multiple unknown compounds from complex mixtures. Non-targeted analysis is also applicable for complex studies such as metabolomics to search unidentified metabolites of new chemical entities. This review critically discusses the current advancements in non-targeted analysis related to the analysis of pharmaceutical, biological, and environmental samples. Various steps like sample collection, handling, preparation, extraction, its analysis using advanced techniques like high-resolution mass spectrometry, liquid chromatography mass spectrometry, and lastly interpretation of the huge amounts of complex data obtained upon analysis of complex matrices have been discussed broadly in this article. Besides the advantages of non-targeted analysis over targeted analysis, limitations, bioinformatics tools, sources of error, and research gaps have been critically analyzed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Terminalia bellirica (Gaertn.) Roxb. is an ethnomedicinal plant that has been utilized in Tibetan and traditional Chinese medicine (TCM). Nevertheless, its quality standard officially listed in the Chinese Pharmacopoeia does not include any content determination of the indicator components of Terminaliae Belliricae Fructus, which constrains the effective quality evaluation of medicinal material and related products. In this paper, a three-dimensional "content-pharmacokinetics-pharmacology" network strategy was developed to identify the quality markers (Q-markers) of Terminaliae Belliricae Fructus in terms of "measurability", "traceability" and "effectiveness". Chromatographic fingerprint analysis was performed to outline its chemical contour, and identify the differential components of 17 batches of Terminaliae Belliricae Fructus combined with multivariate statistics analysis and UPLC-QTOF-MS analysis. Serum pharmacochemistry analysis was implemented on rats, and 25 prototype components absorbed into the blood were identified. By network pharmacology analysis, a component-disease-target-pathway network was constructed, thus validating the effectiveness of the chemical components of Terminaliae Belliricae Fructus. Afterwards, the above screened candidate components were put into construction of three-dimensional "radar chart". According to the calculated regression area (RA) and coefficient of variation (CV) values, the potential Q-markers was determined, followed by "specificity" evaluation. Ultimately, ellagic acid (EA), chebulagic acid (CHA), gallic acid (GA), chebulinic acid (CA), corilagin (CO) and chebulanin (CH) were specified as the Q-markers of Terminaliae Belliricae Fructus. Owing to high content, good pharmacokinetic property, high pharmacological activities and specificity. The screened Q-markers could offer a scientific foundation for the quality control of Terminaliae Belliricae Fructus, and the proposed strategy is demonstrated to be reliable and feasible for deciphering Q-markers of TCM., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Juan Chen reports financial support was provided by Ministry of Science and Technology of the People's Republic of China. Juan Chen reports financial support was provided by National Medical Products Administration. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Heparin is the most widely used anticoagulant in clinical practice, with enoxaparin being one of the most important low molecular weight heparins (LMWHs). In this study, an antithrombin III (ATIII) affinity column was used. Enoxaparin and its oligosaccharides of varying sizes, prepared using preparative size exclusion chromatography (SEC), were fractionated through the ATIII affinity column. The different affinity fractions from each oligosaccharide size were profiled using strong anion exchange (SAX) chromatography. Each peak was automatically transferred to an SEC column for desalting prior to mass spectrometry (MS) analysis, which enabled structural identification using a multiple heart-cut (MHC) 2D LC-MS system (SAX-SEC-MS). The high-affinity fraction from enoxaparin was further analyzed using the MHC 2D LC system (SEC-SAX). SAX profiles of the high-affinity oligosaccharides, prepared by both size and affinity fractionation, were consistent with those obtained by direct SEC-SAX analysis. The possible sequences of several high-affinity hexasaccharides and the domain compositions of high-affinity octa- and decasaccharides in enoxaparin were further elucidated by disaccharide analysis after manual collection of the oligosaccharides. This work advances the understanding of enoxaparin's structural features and offers a potential approach to improve the quality of enoxaparin, as well as to identify key structural motifs in heparin/LMWHs that contribute to protein binding., Competing Interests: Declaration of competing interest There is no conflict of interest for authors of this manuscript., (Copyright © 2024. Published by Elsevier B.V.)

As a unique type of glycosylation, O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) on Ser/Thr residues of proteins was discovered 40 y ago. O-GlcNAcylation is catalyzed by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove O-GlcNAc, respectively. O-GlcNAcylation is an essential glycosylation that regulates the functions of many proteins in virtually all cellular processes. However, deep and site-specific characterization of O-GlcNAcylated proteins remains a challenge. We developed an ultradeep O-GlcNAc proteomics workflow by integrating digestion with multiple proteases, two mass spectrometric approaches (i.e., electron-transfer/higher-energy collision dissociation [EThcD] and HCD product-dependent electron-transfer/higher-energy collision dissociation [HCD-pd-EThcD]), and two data analysis tools (i.e., MaxQuant and Proteome Discoverer). The performance of this strategy was benchmarked by the analysis of whole lysates from PANC-1 (a pancreatic cancer cell line). In total, 2,831 O-GlcNAc sites were unambiguously identified, representing the largest O-GlcNAc dataset of an individual study reported so far. Unexpectedly, in addition to confirming known sites and identifying many other sites of Ser/Thr modification, O-GlcNAcylation was found on 121 tyrosine (Tyr) residues of 93 proteins. In vitro enzymatic assays showed that OGT catalyzes the transfer of O-GlcNAc onto Tyr residues of peptides and OGA catalyzes its removal. Taken together, our work reveals widespread O-GlcNAcylation on Tyr residues of proteins and that Tyr O-GlcNAcylation is mediated by OGT and OGA. As another form of glycosylation, Tyr O-GlcNAcylation is likely to have important regulatory roles., Competing Interests: Competing interests statement:The authors declare no competing interest.

Antibody-drug conjugates (ADCs) represent a novel class of immunoconjugates with growing therapeutic relevance, since they combine the efficacy of cytotoxic drugs with the specificity of antibodies. However, by design, ADCs introduce structural features into the monoclonal antibody scaffold that complicate their analysis. Payload attachment to cysteine or lysine residues can often result in product heterogeneity, regarding both the number of attached drug molecules and their conjugation site, necessitating the use of state-of-the-art MS instrumentation to elucidate their complexity. In middle-down mass spectrometry (MD MS), the gas-phase sequencing of ∼25 kDa ADC subunits with different ion activation techniques generally produces rich fragmentation mass spectra; however, spectral congestion can cause some fragment ions to go undetected, including those that can pinpoint the exact location of payload conjugation sites. Proton transfer charge reduction (PTCR) can substantially simplify fragment ion spectra, thereby unveiling the presence of product ions whose signals were previously suppressed. Herein, we present an MD MS strategy relying on the use of PTCR to investigate a cysteine-based ADC mimic with a variable drug-to-antibody ratio, targeting the unambiguous localization of payload conjugation sites. Unlike traditional tandem MS experiments (MS 2 ), which could not provide a complete map of conjugation sites, a single PTCR-based experiment (MS 3 ) proved to be sufficient to achieve this goal across all variably modified ADC subunits, including isomeric ones. Combining the results obtained from orthogonal ion activation techniques followed by PTCR further strengthened the confidence in the assignments.

High-resolution mass spectrometry (HRMS) has become an indispensable tool in the characterization of organic aerosols (OA) providing information on air quality, health assessment, climate trends, reactions, and source apportionment. Spectra-derived lists of formulas and their relative abundances are used to compare ambient OA from different sources or to monitor secondary OA formation under controlled laboratory conditions in smog chamber experiments. Various techniques are implemented to visualize common and unique features, series of precursors, and products. The disadvantage of this conventional approach is in associating elemental compositions to specific compounds, while due to several analytical limitations, the structural information remains hidden. We argue that some of the conclusions derived from this data analysis can be misleading. In this study, we applied in-ESI source H/D exchange (HDX), which facilitated enumeration of labile protons in molecules behind elemental compositions of OA. We applied this technique to compare OA from three different locations representing urban, forest, and marine environments and to examine tentative chemical information derived from Kendrick mass defect (KMD) series analysis. Significant discrepancies were found between numbers of labile protons in protogenic functional groups and the stoichiometry of chemical reactions, which are associated with KMD series. Only a portion of chemical pairs matched target stoichiometries, which highlights the existing limitations in environmental applications of conventional formula-based HRMS data interpretation strategies.

There is a potential synergistic effect between nonsteroidal anti-inflammatory drugs and hydrogen sulfide (H 2 S), but direct evidence for the study is lacking. With a single fluorescence detection method, it is difficult to accurately confirm the effectiveness of the synergistic effect. In this study, the fluorescent probe and the nonsteroidal anti-inflammatory drug naproxen were combined via different self-immolative spacer groups to obtain a diagnostic and therapeutic integrated fluorescent probe Nap-NP-NSB , which can release H 2 S. The quantitative release of H 2 S by Nap-NP-NSB was evaluated in vitro and in cells, and the synergistic effect of H 2 S and naproxen was confirmed by monitoring the treatment process of cellular inflammation and oxidative damage of gastric mucosa cells. Finally, in vivo fluorescence imaging and mass spectrometry imaging of the liver and stomach tissues and their sections were performed in the mouse model of acute hepatitis. The dual-modal detection method not only confirmed that Nap-NP-NSB had better anti-inflammatory activity and less gastric mucosal damage, but also enabled a more accurate visualization of the drug synergistic effect of naproxen and H 2 S. This work provides a dual visualization imaging method combining fluorescence and mass spectrometry imaging and develops a new idea for studying drug synergies based on self-immolative structures.

Colorectal cancer (CRC) lymph node metastasis (LNM) is a crucial factor affecting the prognosis and treatment outcomes of CRC patients. It has been confirmed that altered glycosylation is a key event during CRC lymphatic metastases. Sialylation is one of the most significant glycosylation alterations in tumors. However, the predictive role of sialylation and sialylated protein in CRC remains elusive, especially in CRC-LNM. In this study, we explored and identified 1102 sialylated glycoproteins in CRC-LNM using metabolic labeling strategy and proteomics analysis. Combined with comprehensive analysis with bioinformatics and machine learning algorithms, we screened 25 prognostic sialylation-related genes (SRGs) to construct a new molecular phenotype (LRSRGs-Phenotype) and a prognostic SRG signature (LRSRGs-related Gene Signature) in CRC. Then, we further confirmed that patients in different phenotypes had different prognosis, molecular biological characteristics, immune cell infiltration and could be closely linked to three previously reported immune phenotypes: immune-excluded (Phenotype A), immune-desert (Phenotype B), and immune-inflamed (Phenotype C). Besides, we evaluated and validated the LRSRGs-related gene (ACADM, EHD4, FLOT1, GPC1, GSR, LRRC8A, NGFR, SDHB, and SEC61G) signature and found the risk score was an independent risk factor for CRC prognosis. CRC patients in different risk groups had different somatic mutation, tumor microenvironment and immunotherapy response. Finally, we also identified the potential therapeutic agents for CRC patients in different risk groups. In conclusion, we explored the key sialylated glycoproteins, which may play a key role in tumor LNM and clinical outcomes. And constructed the LRSRGs-phenotype and signature with prognostic and therapeutic predictive value in CRC, hoping to provide reliable scientific basis for future treatments in CRC patients., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Skin health relies heavily on a well-maintained cutaneous barrier. While the detrimental effects of UV radiation on the epidermis are established, the impact of blue light, a significant component of sunlight and artificial sources, is less clear. This study aims to explore blue light's influence on the reconstructed human epidermis (RHE) using two complementary analytical approaches: Raman microspectroscopy and normal phase liquid chromatography coupled with high-resolution mass spectrometry (NPLC/HR-MS n ). RHE samples were exposed to blue light (415 nm and 455 nm) during different stages of their maturation. Raman spectra were acquired for both irradiated and non-irradiated (control) samples. Raman descriptors were analyzed to assess potential alterations in the structural organization of proteins and lipids' conformational changes. Additionally, lipids from RHE samples were extracted and analyzed using NPLC/HR-MS n . Blue light exposure led to changes in the structural organization of RHE lipids and proteins, as well as changes in the lipid composition. These changes varied depending on the wavelength and exposure dose. Exposure to blue light could disrupt the integrity of the skin's protective barrier, leading to increased sensitivity to environmental stressors and potential skin damage.

Cancer is one of the most serious health problems worldwide. Because cancer has no specific symptoms in its early stages, it is often not diagnosed until it is in advanced stages, reducing the likelihood of successful treatment. Therefore, early diagnosis of cancer is a formidable challenge. Mass spectrometry-based proteomics offers a robust technical foundation for cancer diagnosis. However, mass spectrometry data are characterized by high dimensionality, large data volume, and noise interference, which can lead to diagnostic errors in clinical applications. To address this challenge, an improved algorithm combining principal component analysis (PCA) with a convolutional neural network (CNN) algorithm (denoted as PCA-1DSE-ResCNN) was proposed to assist in analyzing high-dimensional mass spectral data. The algorithm initially reduced the dimensionality of the data through the PCA technique. Subsequently, the convolutional neural network algorithm (1DSE-ResCNN) integrating residual blocks and squeeze-and-excitation blocks was used as a classifier. This approach can not only alleviate the issues of overfitting and gradient vanishing caused by deep network layers but also reduce redundant information, enabling the algorithm to effectively learn high-dimensional data features and deal with nonlinear relationships. To validate the effectiveness of the algorithm, the high-dimensional ovarian cancer mass spectrometry dataset was selected as an example to examine its application performance in early diagnosis of ovarian cancer. The experimental results demonstrated that the PCA-1DSE-ResCNN algorithm outperforms other methods in terms of accuracy, specificity, and sensitivity on three high-dimensional ovarian cancer datasets. This study will contribute to the rapid diagnosis and early detection of cancer.

AMP-activated protein kinase (AMPK) is a key regulator of metabolism and a recognised target for the treatment of metabolic diseases such as Type 2 diabetes (T2D). Here, we review how mass spectrometry (MS) can be used to study short-term control by AMPK via protein phosphorylation and long-term control due to changes in protein expression. We discuss how MS can quantify AMPK subunit levels in tissues from different species. We propose hydrogen-deuterium exchange (HDX)-MS to investigate molecular mechanisms of AMPK activation and thermoproteomic profiling (TPP) to assess off-target effects of pharmacological AMPK activators/inhibitors. Lastly, because large MS data sets are generated, we consider different approaches that can be used for their interpretation., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Iron is the most abundant trace metal in the human brain and consistently shown elevated in prevalent neurological disorders. Because of its paramagnetism, brain iron can be assessed in vivo by quantitative MRI techniques such as R 2 * mapping and Quantitative Susceptibility Mapping (QSM). While Inductively Coupled Plasma Mass Spectrometry (ICP-MS) has demonstrated good correlations of the total iron content to MRI parameters in gray matter, the relationship to ferritin levels as assessed by Electron Paramagnetic Resonance (EPR) has not been systematically analyzed. Therefore, we included 15 postmortem subjects (age: 26-91 years) which underwent quantitative in-situ MRI at 7 Tesla within a post-mortem interval of 24 h after death. ICP-MS and EPR were used to measure the total iron and ferritin content in 8 selected gray matter (GM) structures and the correlations to R 2 * and QSM were calculated. We found that R 2 * and QSM in the iron rich basal ganglia and the red nucleus were highly correlated with iron (R² > 0.7) and ferritin (R² > 0.6), whereas those correlations were lost in cortical regions and the hippocampus. The neuromelanin-rich substantia nigra showed a different behavior with a correlation with total iron only (R² > 0.5) but not with ferritin. Although qualitative results were similar for both qMRI techniques the observed correlation was always stronger for QSM than R 2 *. This study demonstrated the quantitative correlations between R 2 *, QSM, total iron and ferritin levels in an in-situ MRI setup and therefore aids to understand how molecular forms of iron are responsible for MRI contrast generation., Competing Interests: Declaration of competing interest None, (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Single cell mass spectrometry (scMS) has shown great promise for label free proteomics analysis recently. To present single cell samples for proteomics analysis by MS is not a trivial task. Existing methods rely on robotic liquid handlers to scale up sample preparation throughput. The cost associated with specialized equipment hinders the broad adoption of these workflows, and the sequential sample processing nature limits the ultimate throughput., Results: In this work, we report a parallel sample processing workflow that can simultaneously process 10 single cells without the need of robotic liquid handlers for scMS. This method utilized 3D printed microfluidic devices to form reagent arrays on a glass slide, and a magnetic beads-based streamlined sample processing workflow to present peptides for LC-MS detection. We optimized key operational parameters of the method and demonstrated the quantification consistency among 10 parallel processed samples. Finally, the utility of the method in differentiating cell lines and studying the proteome change induced by drug treatment were demonstrated., Significance: The present method allows parallel sample processing for single cells without the need of expensive liquid handlers, which has great potential to further improve throughput and decrease the barrier for single cell proteomics., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Peng Li reports financial support was provided by the National Institutes of Health and the National Science Foundation., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Rationale: Sulfur isotopes are increasingly used as mobility indicators in humans and animals in biology, archaeology, and forensics. However, there has been a lack of modern sulfur isotope baseline "isoscape" studies using modern plants and animals, largely due to the possibility of contamination of the S isotope values by modern pollution., Methods: We collected plants from across a 900-km east-west transect of British Columbia Canada and measured their sulfur isotope values. We then used a random forest model to determine which variables best explained the isotope data patterning and produced a sulfur isoscape for the southern region of British Columbia., Results: We see clear patterning in the plant sulfur isotope values that relate to geographical location and rainfall. Our model also shows that for this study area, it is unlikely that there is a significant influence of anthropogenic pollution on plant δ 34 S values. We also discuss the use of plants as a substrate for sulfur isoscapes and possible explanations for the often-observed difference between plant and animal δ 34 S values from the same region, related to differing sources of sulfur in plants compared to amino acids in human and animal tissues., Conclusions: We found that for areas of the world where sulfur pollution is likely less widespread, it is possible to produce a modern plant S isoscape that should be an accurate baseline for mobility studies. Using random forest modelling, we have produced a baseline sulfur isoscape map of southern British Columbia that can be used for ecology, forensic and archaeological studies., (© 2024 The Author(s). Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

The increasing complexity and ubiquity of food processing and the emergence of fraudulent practices have made effective and reliable methods to authenticate food products of utmost importance. Carbohydrates, with various nutritional functions, are abundant in foods and can serve as potential markers for food authentication. However, the complex and diverse structures and properties of carbohydrates, especially polysaccharides, pose challenges. Nonetheless, significant progress has been made in this area. This paper provides an overview of the utilization of carbohydrates in food authentication since 2000, focusing on strategies involving carbohydrate-based markers, carbohydrate profiles, and carbohydrate-protein interaction-based assays. The analytical techniques, applications, challenges and limitations of these strategies are reviewed and discussed. The findings demonstrate that these strategies offer origin verification, quality assessment, adulteration detection, process control, and food species identification. Notably, oligosaccharide analysis has proven effective in food authentication and remains a promising marker, especially for analyzing intricate matrices. The advances in chromatography separation and mass spectrometry identification of isomers and trace amounts of these compounds have facilitated the discovery of such markers. In conclusion, carbohydrate analysis can play a crucial role in food authentication. Future research and development will make the authentication of carbohydrate-rich foods ever more accurate and efficient., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2024. Published by Elsevier Ltd.)

Rationale: Shiduqing Capsules, a well-known Chinese patent medicine, are widely used clinically for the treatment of pruritus. However, to date, there is a lack of research on its pharmacological substances and mechanisms of action., Methods: In the current study, the chemical components of Shiduqing Capsules were identified using UHPLC-QE-Orbitrap-MS technology. Molecular network analysis was employed to identify structurally similar compounds to the known chemical components. The potential molecular targets of the active ingredients were predicted using the SwissTargetPrediction website. The identified targets were further analyzed using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis through the DAVID database. Molecular docking was used to validate the network pharmacology results., Results: Ultimately, A total of 51 chemical components of Shiduqing Capsules were identified. Molecular network analysis identified 21 flavonoids and 13 terpenoids. The core targets of these ingredients include TP53, AKT1, and STAT3. GO and KEGG enrichment analysis revealed 1,371 different biological functions and 177 signaling pathways. Molecular docking confirmed the high affinity between multiple core active ingredients of Shiduqing Capsules and pruritus targets., Conclusion: In conclusion, the effective ingredients of Shiduqing Capsules exert a multifaceted therapeutic effect on pruritus through multiple targets and pathways., (© 2024 John Wiley & Sons Ltd.)

Several food regulatory bodies regard olive oil as highly susceptible to food fraud, largely due to its substantial economic worth. Precise analytical tools are being developed to uncover these types of fraud. This study examines an innovative approach to extract strontium (Sr) from the olive oil matrix (via EDTA complexation and ion-exchange chromatography) and to determine its isotope composition by MC-ICP-MS. This technique was compared to a commonly used technique (i.e. acid extraction and extraction chromatography), and then validated. Three olive oils that are sold in France were prepared and analyzed by two methods: 1) acid extraction prior to Sr purification by Sr-spec resin and 2) complexation by EDTA prior to Sr purification by AG50W-X8. These methods were applied for the determination of the 87 Sr/ 86 Sr isotope ratio of 23 olive oils from various countries. We also demonstrated the feasibility of the method for the detection of olive oil mixtures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Traditional Chinese medicine (TCM) and its preparations have become increasingly popular in recent years. Nonetheless, due to the high complexity of the compounds in Traditional Chinese Patent Medicine (TCPM), the quality differences between different dosage forms and products from various manufacturers pose numerous challenges and difficulties in quality evaluation. The Qiangli Tianma Duzhong (QLTMDZ) prescription, comprising twelve TCM, is widely used in China. Despite its prevalence, current research on QLTMDZ is limited and lacks in-depth and systematic analysis of the chemical composition of the prescription. In this study, a comprehensive strategy was proposed for characterizing the chemical profile of QLTMDZ based on UHPLC-Q-TOF-MS. A total of 122 compounds were identified in QLTMDZ under both positive and negative ion modes. Subsequently, multivariate statistical methods such as principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were conducted in the MS-DIAL software to further elucidate quality differences among 55 batches of QLTMDZ samples from seven manufacturers. Lastly, multiple reaction monitoring (MRM) mode was utilized in conjunction with UHPLC-QQQ-MS, for the precise quantification of the identified 24 compounds within the QLTMDZ preparation and providing supplementary information in quality evaluation. The established analytical method in this study is sensitive and efficient, enabling qualitative and quantitative analysis of the chemical constituents within QLTMDZ. The application of multivariate statistical analyses effectively discriminates samples based on different dosage forms and manufacturers, thereby providing new research directions and scientific support for further studies on the quality control of the prescription., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Development of monoclonal and bispecific antibody-based protein therapeutics requires detailed characterization of native disulfide linkages, which is commonly achieved through peptide mapping under non-reducing conditions followed by liquid chromatography-mass spectrometry (LC-MS) analysis. One major challenge of this method is incomplete protein digestion due to insufficient denaturation of antibodies under non-reducing conditions. For a long time, researchers have explored various strategies with the aim of efficiently digesting antibody drugs when the disulfide bonds remain intact, but few could achieve this by using a simple and generic approach with well controlled disulfide scrambling artifacts. Here, we report a simple method for fast and efficient mapping of native disulfides of monoclonal and bispecific antibody-based protein therapeutics. The method was optimized to achieve optimal digestion efficiency by denaturing proteins with 8 M urea plus 0-1.25 M guanidine-HCl at elevated temperature (50 °C), followed by two-step digestion with trypsin/Lys-C mix using a one-pot reaction. The only parameter that needs to be optimized for different proteins is the concentration of guanidine-HCl present. This simplified sample preparation eliminated buffer exchange and can be completed within three hours. By using this new method, all native disulfide bonds were confirmed for these monoclonal and bispecific antibodies with high confidence. When compared with a commercial kit utilizing low-pH digestion condition, the new method demonstrated higher digestion efficiency and shorter sample preparation time. These results suggest this new one-pot-two-step digestion method is suitable for the characterization of antibody disulfide bonds, particularly for those antibodies with digestion-resistant domains under typical digestion conditions., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Liqing Gu reports financial support was provided by Incyte Corporation. Tiger X. Hu reports financial support was provided by Incyte Corporation. Liqing Gu reports a relationship with Incyte Corporation that includes: employment and equity or stocks. Tiger X. Hu reports a relationship with Incyte Corporation that includes: employment and equity or stocks., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Dynamic conformational and structural changes in proteins and protein complexes play a central and ubiquitous role in the regulation of protein function, yet it is very challenging to study these changes, especially for large protein complexes, under physiological conditions. Here, we introduce a novel isobaric crosslinker, Qlinker, for studying conformational and structural changes in proteins and protein complexes using quantitative crosslinking mass spectrometry. Qlinkers are small and simple, amine-reactive molecules with an optimal extended distance of ~10 Å, which use MS2 reporter ions for relative quantification of Qlinker-modified peptides derived from different samples. We synthesized the 2-plex Q2linker and showed that the Q2linker can provide quantitative crosslinking data that pinpoints key conformational and structural changes in biosensors, binary and ternary complexes composed of the general transcription factors TBP, TFIIA, and TFIIB, and RNA polymerase II complexes., Competing Interests: JL, JR No competing interests declared, (© 2024, Luo and Ranish.)

The identification of individual protein-protein interactions (PPIs) began more than 40 years ago, using protein affinity chromatography and antibody co-immunoprecipitation. As new technologies emerged, analysis of PPIs increased to a genome-wide scale with the introduction of intracellular tagging methods, affinity purification (AP) followed by mass spectrometry (MS), and co-fractionation MS (CF-MS). Now, combining the resulting catalogs of interactions with complementary methods, including crosslinking MS (XL-MS) and cryogenic electron microscopy (cryo-EM), helps distinguish direct interactions from indirect ones within the same or between different protein complexes. These powerful approaches and the promise of artificial intelligence applications like AlphaFold herald a future where PPIs and protein complexes, including energy-driven protein machines, will be understood in exquisite detail, unlocking new insights in the contexts of both basic biology and disease., Competing Interests: Declaration of interests The Krogan Laboratory has received research support from Vir Biotechnology, F. Hoffmann-La Roche, Maze Therapeutics, and Rezo Therapeutics. N.J.K. is on the Board of Directors of Rezo Therapeutics, and he is a shareholder in Tenaya Therapeutics, Maze Therapeutics, Rezo Therapeutics, GEn1E Lifesciences, and Interline Therapeutics., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The measurement of trace breath gases is of growing interest for its potential to provide non-invasive physiological information in health and disease. While instrumental techniques such as selected-ion flow-tube mass spectrometry (SIFT-MS) can achieve this, these are less suitable for clinical application. Sensitive sensor-based systems for breath ammonia could be more widely deployed, but have proven challenging to develop. This work demonstrates the sequential analytical validation of an electrochemical impedance-based sensor system for the measurement of ammonia in breath using SIFT-MS. Qualitative and relative responses between the two methods were comparable, although there were consistent differences in absolute concentration. When tested in artificial breath ammonia, sensors had a relative impedance sensitivity of 3.43 × 10 -5 ppbv -1 for each breath in the range of 249-1653 ppbv ( r 2 = 0.87, p < 0.05). When correlated with SIFT-MS using human breath ( n = 14), ammonia was detected in the range of 100-700 ppbv ( r = 0.78, p < 0.001), demonstrating acceptable sensitivity, reproducibility and dynamic range for clinical application., (© 2024 IOP Publishing Ltd. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

This study aimed to establish an effective affinity-ultrafiltration coupled with mass spectrometry (AUF-MS) method to rapidly screen the active components of antithrombin in Xuefu Zhuyu decoction (XFZYD). The HPLC fingerprint of different batches of XFZYD was established to verify the stability of the compound ingredients. AUF-MS was established for the rapid screening of the active ingredients in XFZYD. Eight active ingredients were screened using AUF-MS: oxypaeoniflorin, chlorogenic acid, amygdalin, isoliquiritin, neohesperidin, hesperidin, glycyrrhizic acid and chikusetsu saponin IVa. Simultaneously, the anticoagulant effect of the active ingredients was verified by performing in vivo and in vitro experiments. The results of this study indicate that AUF-MS can be used for high-throughput screening of active components of traditional Chinese medicine compounds, thus providing a basis for the discovery of thrombin inhibitors and promoting the research progress of antithrombotic drugs. The compounds screened from XFZYD showed an inhibitory effect against thrombin and a certain antithrombotic effect.

Coordination compounds of lanthanides are indispensable in biomedical applications as MRI contrast agents and radiotherapeutics. However, since the introduction of the chelator DOTA four decades ago, there has been only limited progress on improving their thermodynamic stability and kinetic inertness, which are essential for safe in vivo use. Here, we present ClickZip, an innovative synthetic strategy employing a coordination-templated formation of a 1,5-triazole bridge that improves kinetic inertness up to a million-fold relative to DOTA, expanding utility of lanthanide chelates beyond traditional uses. Acting as unique mass tags, the ClickZip chelates can be released from (biological) samples by acidic hydrolysis, chromatographically distinguished from interfering lanthanide species, and sensitively detected by mass spectrometry. Lanthanides enclosed in ClickZip chelates are chemically almost indistinguishable, providing a more versatile alternative to chemically identical isotopic labels for multiplexed analysis. The bioanalytical potential is demonstrated on tagged cell-penetrating peptides in vitro, and anti-obesity prolactin-releasing peptides in vivo., Competing Interests: Competing interests M.P., T.D., M.S. and A.J. are co-inventors on a patent application no. PCT/CZ2022/050087 filed in the name of applicant Ustav Organicke Chemie a Biochemie AV CR V.V.I. The application covers some of the compounds and their use discussed in this work. M.P., T.D., M.S. and A.J. declare no other competing interests. The other authors declare no competing interests., (© 2024. The Author(s).)

TEPCO planned the release of Advanced Liquid Processing System (ALPS)-treated water, which is decontaminated stagnated water by ALPS, to the Pacific Ocean in 2023 after diluting it more than a hundred times in accordance with the policy of the Japan government. Since the low level of I-129 can remain in ALPS-treated water, the background I-129 concentration in seaweed samples around 1F NPS before the release of ALPS-treated water was recorded in this study. The iodine in seaweed samples was extracted via TMAH alkali-dissolution, and the I-129 concentration was measured by 8900 Triple Quadrupole ICP-MS. The resulting I-129 concentration was <5.4 × 10-2 Bq/kg-wet (sargassum) and <2.7 × 10-2 Bq/kg-wet (laminaria), respectively., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Introduction: Tempeh is an antioxidant-rich soybean fermentation product from Java, Indonesia. Cooking methods have an impact on the nutritional value and bioactivity of food., Objective: This study aims to investigate how the cooking process affects the metabolites and antioxidant activity in tempeh using ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS)., Methods: A nontargeted UHPLC-HRMS metabolomics and chemometric analysis were used to evaluate metabolite profiles and antioxidant activity changes because of food processing in tempeh., Results: The score plots of tempeh produced by boiling and frying methods displayed a distinct separation from raw tempeh, revealing that the cooking process altered the metabolite composition of tempeh. Due to processing, L-glutamic acid, L-pyroglutamic acid, DL-glutamine, and D-( +)-proline became the most affected metabolites on tempeh. There were 70 metabolites that showed antioxidant activity using the DPPH assay; 23 metabolites significantly differ from DPPH and control for antioxidant activity for all processing tempeh. Metabolites with significantly different antioxidant activity in raw and processed tempeh were dominated by flavonoids, vitamin E, and bioactive lipids., Conclusion: The DPPH antioxidant assay using UHPLC-HRMS is promising as a fast antioxidant assay by simplifying the conventional DPPH antioxidant assay. Further, it can be used to identify the name of metabolites responsible for its antioxidant activity., Competing Interests: Declarations Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Hydrogen-deuterium exchange (HDX) has become a pivotal method for investigating the structural and dynamic properties of proteins. The versatility and sensitivity of mass spectrometry (MS) made the technique the ideal companion for HDX, and today HDX-MS is addressing a growing number of applications in both academic research and industrial settings. The prolific generation of experimental data has spurred the concurrent development of numerous computational tools, designed to automate parts of the workflow while employing different strategies to achieve common objectives. Various computational methods are available to perform automated peptide searches and identification; different statistical tests have been implemented to quantify differences in the exchange pattern between two or more experimental conditions; alternative strategies have been developed to deconvolve and analyze peptides showing multimodal behavior; and different algorithms have been proposed to computationally increase the resolution of HDX-MS data, with the ultimate aim to provide information at the level of the single residue. This review delves into a comprehensive examination of the merits and drawbacks associated with the diverse strategies implemented by software tools for the analysis of HDX-MS data.

Although radioactive experiments are necessary in radiopharmaceutical drug discovery and theranostic cancer research, they are expensive, require special facilities, and face certain restrictions. Thus, finding techniques not involving radioactivity is highly beneficial for minimizing these disadvantages in such research. In this regard, methods using inductively coupled plasma-mass spectrometry (ICP-MS) have emerged as viable alternatives to traditional radioactive approaches. Despite its potential, practical applications of ICP-MS in radiopharmaceutical cancer research have only emerged in recent years. This Perspective focuses on the development and implementation of nonradioactive ICP-MS-based assays in radiopharmaceutical research and aims to inspire future research efforts in this area.

Covering: up to 2024The prompt identification of (bio)active natural products (NPs) from complex mixtures poses a significant challenge due to the presence of numerous compounds with diverse structures and (bio)activities. Thus, this review provides an overview of current and emerging tools and strategies for the identification of (bio)active NPs in complex mixtures. Traditional approaches of bioassay-guided fractionation (BGF), followed by nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis for compound structure elucidation, continue to play an important role in the identification of active NPs. However, recent advances (2018-2024) have led to the development of novel techniques such as (bio)chemometric analysis, dereplication and combined approaches, which allow efficient prioritization for the elucidation of (bio)active compounds. For researchers involved in the search for bioactive NPs and who want to speed up their discoveries while maintaining accurate identifications, this review highlights the strengths and limitations of each technique and provides up-to-date insights into their combined use to achieve the highest level of confidence in the identification of (bio)active natural products from complex matrices.

Bees produce honey through the collection and transformation of nectar, whose botanical origin impacts the taste, nutritional value, and, therefore, the market price of the resulting honey. This phenomenon has led some to mislabel their honey so that it can be sold at a higher price. Metabolomics has been gaining popularity in food authentication, but rapid data mining algorithms are needed to facilitate the discovery of new authenticity markers. A nontargeted high-resolution liquid chromatography-mass spectrometry (HR/LC-MS) analysis of 262 monofloral honey samples, of which 50 were blueberry honey, was performed. Data mining methods were demonstrated for the discovery of binary single-markers (compound was only detected in blueberry honey), threshold single-markers (compound had the highest concentration in blueberry honey), and interval ratio-markers (the ratio of two compounds was within a unique interval in blueberry honey). A novel convolutional algorithm was developed for the discovery of interval ratio-markers, which trained 14× faster and achieved a 0.2 Matthews correlation coefficient (MCC) units higher classification score than existing open-source implementations. The convolutional algorithm also had classification performance similar to that of a brute-force search but trained 1521× faster. A pipeline for shortlisting candidate authenticity markers from the LC-MS spectra that may be suitable for chemical structure identification was also demonstrated and led to the identification of niacin as a blueberry honey threshold single-marker. This work demonstrates an end-to-end approach to mine the honey metabolome for novel authenticity markers and can readily be applied to other types of food and analytical chemistry instruments.

Bispecific antibodies (bsAbs) and multispecific antibodies (msAbs) represent a promising frontier in therapeutic antibody development, offering unique capabilities not achievable with traditional monoclonal antibodies. Despite their potential, significant challenges remain due to their increased molecular complexity. One prominent challenge is the correct assembly of light and heavy chains, as improper pairing leads to mispaired or incompletely assembled species that lack therapeutic efficacy and possess undesired properties, impairing the developability, manufacturability, and safety. There is a critical need for rapid, sensitive analytical tools to monitor and control these undesired species and ensure the quality assessment of bsAbs and msAbs. To address this need, we present a novel high-throughput, format-agnostic intact mass workflow that significantly enhances the efficiency of detecting and quantifying biotherapeutic products and related impurities. This workflow integrates automated sample preparation, novel high-resolution rapid mass detection powered by SampleStream-MS, and an advanced data analysis pipeline. It offers increased throughput and data quality while substantially reducing analysis turnover time and labor. This was demonstrated in a pilot program where ∼800 multispecific antibodies were processed in 10 working days. The article details the evaluation and validation of our method, demonstrating its repeatability and intermediate precision in terms of measurement accuracy and relative quantification of various product-related species. We underscore the transformative potential of this end-to-end high-throughput workflow in expediting bispecific and multispecific antibody discovery, optimizing production processes, and ensuring high-quality development and manufacturing for therapeutic antibodies.

Bacterial infections are a major threat to human health worldwide. A better understanding of the properties and physiology of bacterial pathogens in human tissues is required to develop urgently needed novel control strategies. Mass spectrometry-based proteomics could yield such data, but identifying and quantifying scarce bacterial proteins against a preponderance of human proteins is challenging. Here, we explored the recently introduced SureQuant method for highly sensitive targeted mass spectrometry. Using a major human pathogen, the Gram-positive bacteria Staphylococcus aureus , as an example, we evaluated several parameters, including the number of targets and intensity thresholds, for optimal qualitative and quantitative protein analysis. By comparison, we found that SureQuant achieved the same quantitative performance as standard parallel reaction monitoring while allowing accurate and precise quantification of up to 400 targets. SureQuant also surpassed the sensitivity and quantification capabilities of global data-independent acquisition methods. Finally, to facilitate method development, we provide optimized MS parameters for the sensitive quantification of different peptide panel sizes. This study provides a foundation for the broader application of SureQuant in the analysis of clinical specimens containing trace amounts of bacterial proteins as well as other studies requiring ultrasensitive detection of low-abundant proteins.

Microcystins (MCs) can be detected in various matrices in two forms: a freely extractable fraction and a total (free and covalently protein-bound) fraction. Although the majority of MCs analyses are limited to the free fraction, they do not allow the analysis of all MCs variants or protein-bound forms. Other methods, known as total MCs analysis methods, enable simultaneous analysis of all MCs variants, as well as bound forms, which may be a major form of toxin accumulation in organisms. Among these techniques, the chemical oxidation method (e.g. Lemieux) allows the detection of total forms of MC (and nodularins) by oxidizing the common part to all MC and nodularins, and analyzing the resultant MMPB product (2-methyl-3-methoxy-4-phenylbutyric acid). However, the execution of this method in the context of health monitoring is challenging due to the variability of the protocols, the recoveries obtained with these protocols, and the important matrix effects associated with the method. The objectives of this study were i) to optimize an existing protocol of chemical oxidation "Lemieux1" on fresh fish fillet matrices, ii) to compare two existing protocols ("Lemieux1" and "Lemieux2"), and iii) apply Lemieux oxidation to fish fillets and livers naturally contaminated with MCs-producing cyanobacteria and to freshwater mussels contaminated with MCs in laboratories. Optimization of the "Lemieux1" protocol, in particular in the oxidation and SPE (solid phase extraction) steps improved the method's yields on the fresh fish fillet matrix (from <5 % to around 40 %). Moreover, several quantification methods have been compared through various calibration techniques (solvent calibration curve, matrix-matched calibration curve, oxidized MC-LR calibration curve and also by testing the addition of d 3 -MMPB as an internal standard). Comparison with the "Lemieux2" protocol showed the best results on the same matrix, with yields of around 65 %. MMPB was analyzed using this "Lemieux 2" protocol, in livers of carps sampled during an episode of cyanobacteria proliferation, at concentrations ranging from 17.9 to 27.5 μg/kg MMPB and at concentrations ranging from 50 to 2890 μg/kg MMPB in freshwater mussels laboratory contaminated to MCs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Salvia miltiorrhiza, a widely used traditional Chinese medicine, contains a complex matrix of hydrophobic diterpenoids and hydrophilic phenolic acids, presenting significant challenges in comprehensive analysis. In this study, an online polarity-partitioned two-dimensional liquid chromatography coupled with mass spectrometry (2D-LC-MS) method was developed for comprehensive analysis of both lipophilic and hydrophilic active components in Salvia miltiorrhiza. The method integrated hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC), facilitating the efficient separation of compounds across a wide range of polarities. An online dilution strategy was implemented, minimizing sample loss and enhancing the method's utility for quality control and chemical characterization of complex herbal matrices. Compared with other LC methods, this approach significantly improved analyte coverage, resolution, and analysis efficiency. Under optimal conditions, 150 active components were successfully identified, including 33 compounds newly discovered in Salvia miltiorrhiza. Additionally, the validated online method was applied to the quantitative determination of 16 quality markers of Salvia miltiorrhiza from different sources. The results demonstrated the online method's potential as a superior alternative to existing techniques, offering broader applicability in traditional Chinese medicine research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The characterization of plant extracts is usually accomplished by reverse-phase liquid chromatography, but the development of new complementary approaches, such as HILIC, offers an orthogonal method. In this study, five HILIC stationary phases were evaluated to assess their ability to retain polyphenols. They were selected to cover the main different HILIC mechanisms: bare silica; silica with ethylene bridge; neutral amide; amino; zwitterionic. A total of 31 polyphenol standards were used for the screening, including 9 stilbenes, 8 flavonoids, 6 anthocyanins, and 8 phenolic acids. Three different detections were tested: diode array detector, charged aerosol detector and mass spectrometry. Results indicated that silica supports were not suitable for retaining polyphenols, with no or low retention observed except for anthocyanins. The effectiveness of stationary phases in retention of phenolics following the order related to increased retention: zwitterionic, amide, and amino. The choice of mobile phase also influenced retention. Mobile phases containing TFA as pH modifier limited retention, while formic acid was found to be more effective for polyphenol retention. Ammonium buffers also improved retention but often compromised peak shape. pH changes mainly impacted ionizable compounds, such as phenolic acids, by increasing their retention when they were ionized. DAD was wellsuited for detecting polyphenols that possess aromatic rings, though peak wavelengths depend on the structures of the polyphenols. CAD, while less sensitive than DAD and MS, provided an almost similar response for structurally related compounds, even with gradient elution. MS was the preferred detector for quantification when resolution between compounds was challenging, as it is often the case with natural extracts. The study successfully demonstrated that best HILIC conditions were obtained using an amino stationary phase composed of a polyethylenimine and formic acid-based mobile phase. These conditions were successfully applied to the analysis of stilbenoid-rich extracts from different parts of the vine. The elution order of stilbenoids followed the degree of polymerization. With CAD, the chromatographic profile was more representative of sample composition. It was demonstrated for the first time the interest of a combination of HILIC and CAD for analyzing stilbenes, offering a complementary approach to the classic RP analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

It is important to develop specific adsorbents for malachite green and other fish drug residues. Herein, a simple strategy for synthesizing a novel magnetic covalent organic frameworks (rFe 3 O 4 @Py-COF) has been studied, and the materials were used as a magnetic absorbent for solid phase extraction (MSPE) of malachite green (MG) and its metabolite as leucomalachite green (LMG) in fishes. In this study, the mild reduction program of formic acid replacing traditional sodium borohydride as a reducing agent has been adopted to increase the stability of the framework, which can maintain the original high crystallinity and surface area of the reduced COF. The secondary amine bond is expected to be used as the reaction center for further functionalization of COF pore wall. Subsequently, rFe 3 O 4 @Py-COF (rmCOF) obtained after reduction was used as MSPE materials to detect MG and LMG by a portable mass spectrometer. After optimizing the conditions, the linearity is good within the range of 1.25∼100 μg/kg (R 2 ≥0.9954), the limits of detection (LODs) are 0.31∼0.44 μg/kg with satisfactory recovery (85.0 %∼106.0 %). These results indicate that the assay is suitable for monitoring MG and LMG in complex aquatic foods, providing protection for food safety., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Nitrosamine drug substance related impurities (NDSRIs) are often analyzed using high performance liquid chromatography (HPLC) with mass spectrometry (MS) detection. Due to high sensitivity requirements, high resolution MS or MS/MS is commonly used. However, it is difficult to implement this type of method for routine analysis at a supply site. Herein, we report a systematic approach to develop and validate a practical, robust, and user-friendly method for the analysis of NDSRIs using an inexpensive single quadrupole MS instrument such as QDa. We used 7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro- [1,2,4] triazolo [4,3-a] pyrazine (NTTP) as an example to demonstrate the method development process. By optimizing the HPLC and MS parameters, we were able to develop a simple HPLC-MS method that provides the desired specificity and sensitivity for the analysis of NTTP and can be easily implemented in an analytical lab. The limit of quantitation is 0.5 ng/mL, corresponding to 0.1 ppm with respect to 5 mg/mL sitagliptin. The method has been successfully validated per ICH guidelines., Competing Interests: Declaration of competing interest I have The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Qing-Kai-Ling oral liquid is commonly used clinically for the treatment of fever and upper respiratory tract infection. Moreover, studies have shown that Qing-Kai-Ling oral liquid has an anti-pneumonia effect. However, owing to its complex pharmacodynamic material basis, its pharmacological research and clinical application are limited. To address this problem, the chemical constituents of Qing-Kai-Ling oral liquid were identified by ultra-high performance liquid chromatography quadrupole-Exactive Orbitrap mass (UHPLC-Q-Exactive Orbitrap MS) and network pharmacology methods, which were used to predict its potential anti-pneumonia target and signalling pathway. A total of 150 compounds were identified and tentatively characterized, including 35 amino acids and their derivatives, 36 organic acids, 20 terpenoids, 20 alkaloids, 12 glycosides, 7 flavonoids, and 20 others. Among them, 14 compounds were accurately identified by comparing their retention time and mass spectrum data with those of reference substances. Additionally, we performed molecular simulation calculations via Density Function Theory to determine the plausibility of the compound cleavage reactions and further confirm compound structures. Furthermore, 90 key targets were screened through network pharmacology, with the particular focus on the PI3K-AKT, MAPK and TNF signalling pathways. This method achieved the first comprehensive identification of the chemical composition of Qing-Kai-Ling oral liquid and elucidated its potential mechanism of anti-pneumonia. The results provide valuable reference and data support for pharmacodynamic substance research and quality control., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study presents a method utilizing solid-phase microextraction Arrow (SPME Arrow) combined with ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) for the selective detection of three veterinary drugs-thiabendazole, sulfamethazine, and clenbuterol-in milk and pork. Two-dimensional metal-organic framework nanosheets (2D-MOFs) were employed as coating materials for the SPME Arrow. Three types of 2D-MOFs (Ni, Mn, and Co based) were synthesized and characterized using Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, and a physical adsorption analyzer. The 2D-MOF coatings were fabricated using the electrospinning technique, with polyacrylonitrile (PAN) serving as the binder. Comparative analysis of the three 2D-MOF coatings revealed that 2D-Ni-MOF was the optimal coating material for the SPME Arrow. Optimization of the coating preparation conditions and SPME procedures included determining the optimal mass ratio of 2D-Ni-MOF to PAN, electrospinning time, and extraction and desorption parameters. Equilibrium extraction was achieved within 60 min, and desorption was completed within 30 min. Subsequently, the 2D-Ni-MOF-SPME Arrow-UPLC-Q-TOF-MS method was established and validated under optimal conditions, demonstrating high precision with inter-day precision ranging from 3.8 % to 9.5 % and intra-day precision ranging from 5.1 % to 11.5 %. The reusability study indicated that the extraction performance of the new SPME Arrow remained consistent after 90 adsorption-desorption cycles. The method exhibited linearity in milk and pork over the ranges of 0.002-5 μg L -1 and 0.01-5 μg L -1 , respectively. The detection limits in milk and pork were 0.001-0.004 μg L -1 and 0.003-0.007 μg L -1 , respectively. This method demonstrated excellent applicability for determining residues of the three veterinary drugs in milk and pork., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hangzhen Lan reports financial support was provided by Ningbo University. Hangzhen Lan reports financial support was provided by One Health Interdisciplinary Research Project of Ningbo University. Hangzhen Lan reports financial support was provided by J National Natural Science Foundation of China. Hangzhen Lan reports financial support was provided by Key R&D Program of Zhejiang. Hangzhen Lan reports financial support was provided by Natural Science Foundation of Zhejiang Province. Hangzhen Lan reports financial support was provided by Science and Technology Programs of Ningbo., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Polyfunctional thiols are key contributors to wine aroma due to their extremely low odor thresholds, and their quantitative analysis remains challenging as a result of their ultratrace concentrations and high reactivity. This work presents the first method based on ultra-high-performance liquid chromatography (UHPLC) coupled to quadrupole Orbitrap high-resolution mass spectrometry (HRMS) in parallel reaction monitoring (PRM) mode for quantifying thiols at nanograms per liter (ng/L) levels in wine. Thiols in wine were derivatized using 4,4'-dithiodipyridine and isolated by liquid-liquid extraction. This protocol allowed rapid sample preparation with minimum labor input and low consumable expenses. Instrumental analysis was conducted using UHPLC-quadrupole Orbitrap HRMS in PRM mode. Twenty thiol analytes, including literature-known, recently identified, and novel thiols were selected and validated by the optimized method in three wine matrices. The overall analytical performances demonstrated by this method were equivalent, and in most cases, greater than many previously developed GC-MS or LC-MS methods. The validated method was applied to analyze a selection of wines in which 12 thiols were quantified., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This manuscript discusses the development of a reversed-phase ultra high-performance liquid chromatography (RP UHPLC) method based on phenyl-bonded stationary phases without ion-pairs for the separation and identification of oligonucleotides. The elimination of ion-pair reagents makes the proposed protocol as more compliant to the principles of green chemistry, compared to the traditional ion-pair reversed-phase liquid chromatography methods (IP RP LC). In detail, three phenyl-based stationary phases were tested, namely a C18/AR (a C18 stationary phase with the addition of aromatic groups), a Phenyl-hexyl, and a Diphenyl. Generally, the retention of oligonucleotides increases with the increase of salt concentration and the decrease of the pH, thus confirming the significant impact of van der Waals interactions, salting-out effect, and π-electrons interactions in the retention mechanism. The highest retention and best peak symmetry were observed for the C18/AR stationary phase, while the lowest retention for the Phenyl-hexyl, with retention influenced by the type of salt in the mobile phase. The obtained methods using C18/AR stationary phases allow for the effective separations of positional isomers and for identifying impurities and degradation products using RP UHPLC Q-TOF-MS in a comparatively short time. The application of RP UHPLC Q-TOF-MS provides reasonable selectivity for the resolution of 33 impurities and two degradation products. Both groups of compounds are mainly 3'N and 5'N-shortmers, but in the case of impurities, modifications of cyclic phosphate and phosphate groups were also identified. Nevertheless, Diphenyl and Phenyl-Hexyl may be applied to separate modified oligonucleotides with higher salt concentrations. The proposed separation methods without ion-pair reagents contribute to a more sustainable approach in oligonucleotide analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A nanospray emitter coupled to a supercritical fluid chromatograph (SFC-nSI-MS) for mass spectrometric (MS) analysis of fatty acids (FA) positional isomers is introduced. The experimental setup uses conventional bore columns before the SF back-pressure regulator (pre-BPR). The flow is then split and nanosprayed using a short emitter post-BPR. A C18 column was used to resolve positional isomers of unsaturated FA with a 5 min gradient. Chromatographic resolution of the nSFC was compared to a LC-MS system with superior resolving power demonstrated in the nSFC MS system. This system has proven quantitative performance for analyzing pharmaceutical effects on FA composition in a complex biological matrix like E coli lysate., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Recent advances in fluoropolymer polymerization have focused on replacing perfluorinated polymerization aids (PAs) with hydrocarbon-based alternatives. Hydrocarbon PAs are vulnerable to fluorinated radicals during polymerization, leading to the creation of hundreds of process-specific polyfluorinated residuals. These residuals, which include low molecular weight extractable or leachable impurities, are challenging to detect at trace levels. This study investigates a polytetrafluoroethylene (PTFE) dispersion prepared with a hydrocarbon-based surfactant (DOSS) to measure these process-specific fluorinated residues. Liquid chromatography high resolution mass spectrometry is one of the few analytical methods that offers the sensitivity and selectivity required to detect these residuals in complex matrices at concentrations as low as parts per billion. The results indicate that using a hydrocarbon PA during emulsion polymerization produces numerous polyfluorinated residuals. These must be identified and monitored to develop effective abatement strategies, ensuring responsible fluoropolymer manufacturing., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: John C. Sworen, Peter A. Morken, Adam P. Smith, Jill E. Boyle, Maria D. Cervantes Garcia, Jordyn Kramer, Michael P. Wadsley, and Michael C. Davis reports a relationship with The Chemours Company that includes employment., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The characterization of cysteine-linked antibody‒drug conjugates (ADCs) can be more challenging than that of monoclonal antibodies (mAbs) and lysine-linked ADCs because the interchain disulfide bonds are reduced for payload conjugation, and the chains are noncovalently bonded to each other. Furthermore, payload conjugation and disulfide bond reduction/scrambling may introduce additional charge heterogeneity to biomolecules. This study illustrates an innovative workflow employing multiple separation techniques and tandem high-resolution mass spectrometry for comprehensive and in-depth characterization of disitamab vedotin, a recent-generation cysteine-linked ADC, including reversed-phase liquid chromatography (RPLC), ion exchange chromatography (IEX) and image capillary isoelectric focusing (icIEF). RPLC was employed for reduced chains analysis, subunit analysis and peptide mapping. IEX and icIEF were used for charge heterogeneity analysis. The innovation of the integrated methodology emphasizes the importance of cutting-edge icIEF-MS online coupling under near-native conditions to reveal the heterogeneity of disitamab vedotin., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Immortalised cell lines that mimic their primary cell counterparts are fundamental to research, particularly when large cell numbers are required. Here, we report that immortalisation of bone marrow-derived macrophages (iBMDMs) using the J2 virus resulted in the loss of a protein of interest, MSR1, in WT cells by an unknown mechanism. This led us to perform an in-depth mass spectrometry-based proteomic characterisation of common murine macrophage cell lines (J774A.1, RAW264.7, and BMA3.1A7), in comparison with the iBMDMs, as well as primary BMDMs from both C57BL/6 and BALB/c mice. This analysis revealed striking differences in protein profiles associated with macrophage polarisation, phagocytosis, pathogen recognition, and interferon signalling. Among the cell lines, J774A.1 cells were the most similar to the gold standard primary BMDM model, whereas BMA3.1A7 cells were the least similar because of the reduction in abundance of several key proteins related closely to macrophage function. This comprehensive proteomic dataset offers valuable insights into the use and suitability of macrophage cell lines for cell signalling and inflammation research., (© 2024 Gudgeon et al.)

Chemical hair dye components can have allergenic, reproductive, and carcinogenic risks. Detecting restricted and prohibited ingredients in these products is challenging due to product diversity, isomer separation, instability, and wide polarity range. A method was developed using HPLC-high-resolution mass spectrometry for the qualitative and quantitative analysis of 54 hair dye components in various products. Samples were extracted with a 70% methanol solution, ultrasonicated in an ice bath, centrifuged, filtered, diluted with 25% methanol solution and 25% methanol solution containing 0.05% D-isoascorbic acid. Separation was achieved using an ACE Excel 3 C18 column (2.1 mm × 150 mm, 3 μm) with analysis conducted via quadrupole Orbitrap mass spectrometry. It showed good linear correlations, with detection limits of 0.1-23.5 ng mL -1 , and quantitation limits of 0.2-78.1 ng mL -1 . Average recovery ranged from 60.0% to 118.4%, with repeatability from 4.0% to 14.9%. Stability was confirmed within 48 hours. When applied to 20 batches of commercially available hair dyes, 24 hair dye components were found within permissible levels. The method is crucial for quality control of hair dyes, covering 10 common prohibited and 44 permissible hair dye components outlined in the Safety and Technical Standards for Cosmetics (2015 Edition). Compared to the standard methods, it can separate isomers in a single mobile phase system within 15 minutes in positive ion mode while maintaining sensitivity for phenol, hydroquinone, and other components in negative ion mode. Moreover, the pre-treatment strategy significantly improved stability and accuracy, enabling precise analysis of the 54 hair dye components.

This work was designed for the in-depth characterization and holistic comparison of up to 12 ginseng varieties, which can benefit the development of functional foods and ensure their authenticity in the food industry. An online comprehensive two-dimensional liquid chromatography/quadrupole time-of-flight mass spectrometry (2D-LC/QTOF-MS) approach was established by configurating the XCharge C18 and HSS Cyano columns. Under the optimal conditions, we characterized a total of 1146 ginsenosides (including 876 potentially new compounds) from 12 ginseng varieties by reference to an in-house library of 573 known ginsenosides and 70 reference compounds. The online 2D-LC/QTOF-MS-based untargeted metabolomics workflows were developed, by which 126 potential ginsenoside markers were unveiled and utilized to establish the key identification points for each ginseng species. Compared with the conventional liquid chromatography/mass spectrometry metabolomics, our multidimensional chromatography approach performed better in discriminating multiple ginseng varieties. This work demonstrates a potent and practical methodology to identify easily confused functional plants.