Ultra-high-performance Liquid Chromatography-high-resolution Mass Spectrometry Research Articles

A systematic evaluation of quenching, extraction and analysis procedures for metabolomics study of the mechanism of QYSLD intervention in A549 cells.

The preparation of cellular metabolomics samples and how to achieve comprehensive coverage of different polar metabolites in cell samples in the analysis pose a challenge for cellular metabolomics. In this study, we optimized a metabolomics protocol based on ultra-high-performance liquid chromatography high-resolution mass spectrometry (UPLC/HRMS) for theextraction and detection of metabolites in A549 cells and exploration of the intervention effect of Qi-Yu-San-Long decoction (QYSLD) on A549 cells. The results indicate that the lowest level of ATP leakage was observed when A549 cells were quenched under liquid nitrogen. MeOH/chloroform/H2O (1:2:1) extraction yielded more chromatographic peaks and excellent reproducibility, and the relative extraction efficiency of most target metabolites was also high. And we optimized thechromatographic separation conditions in both HILIC and RPLC modes, enabling comprehensive detection and analysis of metabolites with varying polarities. Then, we applied the optimized method to UPLC-Q-TOF/MS-based metabolomics of A549 cells to study the mechanism of QYSLD intervention in non-small cell lung cancer (NSCLC). The CCK-8, EdU staining, and cell cycle assay showed that QYSLD inhibited the proliferation of A549 cells by interfering with the cell cycle and blocking them in theG1 phase. A total of 36 differential metabolites associated with the antitumor effects of QYSLD on NSCLC were identified, mainly involving nicotinate and nicotinamide metabolism, sphingolipid metabolism, and glycerophospholipid metabolism. And western blotting confirmed that thechange in 1-methylnicotinamide levels after QYSLD intervention was associated with theinhibition of nicotinamide N-methyltransferase expression in A549 cells.

Chemical Profiling of Polar Lipids and the Polyphenolic Fraction of Commercial Italian Phaseolus Seeds by UHPLC-HRMS and Biological Evaluation

The common bean (Phaseolus vulgaris L.) is one of the oldest food crops in the world. In this study, the ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-MS/MS) technique was used to characterize the polar lipid composition and polyphenolic fraction of five bean varieties commonly consumed in Italy: Cannellino (PVCA), Controne (PVCO), Borlotti (PVBO), Stregoni (PVST), and Vellutina (PVVE). Lipid content represents a minor fraction of the whole metabolome in dry beans, and little is known about their polar lipids, which could be potentially bioactive components. Thirty-three compounds were detected through UHPLC-MS/MS, including oxylipins, phospholipids, N-acyl glycerolipids, and several fatty acids. The dichloromethane extracts were subjected to principal component analysis (PCA), with the results showing greater differentiation for the Borlotti variety. Moreover, 27 components belonging to different polyphenol classes, such as phenolic acids, flavonoids, catechins, anthocyanins and their glycosides, and some saponins, were identified in the hydroalcoholic seed extracts. In addition, the mineral content of the beans was determined. Considering the high number of compounds in the five apolar seed extracts, all samples were examined to determine their in vitro inhibitory activity against the enzyme cyclooxygenase-2 (COX-2), which is inducible in inflammatory cells and mediates inflammatory responses. Only PVCO showed the best inhibition of the COX-2 enzyme with an IC50 = 31.15 ± 2.16 µg/mL. In light of these results, the potential anti-inflammatory properties of PVCO were evaluated in the LPS-stimulated murine macrophage cell line J774A.1. Herein, we demonstrate, for the first time, that PVCO at 30 µg/mL can significantly reduce the release of TNF-α, with a less significant anti-inflammatory effect being observed in terms of IL-6 release.

Determination of tetramethylammonium hydroxide in air using ion chromatography or ultra-performance liquid chromatography - high-resolution mass spectrometry

Tetramethylammonium hydroxide (TMAH) is extensively utilized in the semiconductor and optoelectronic industries, serving as either a developer or an etchant. TMAH possesses acute toxicity and alkaline corrosiveness, having caused several fatal and injurious accidents in Taiwan. However, there is currently a lack of methods available for assessing TMAH exposure. This study developed a method involving sampling with quartz fiber filters followed by ultrasonic agitation in 10 mL mobile phase, filtering, and analysis using either ion chromatography (IC) or ultra-performance liquid chromatography - high-resolution mass spectrometry (UPLC-HRMS). Utilizing the characteristics of HRMS, the compound analyzed was identified as tetramethylammonium ion (TMA+) through mass spectrometry analysis. The method exhibits excellent trapping capacity and recovery rate. The linear range of TMA+ for IC is 0.3 to 100 μg/mL, with a correlation coefficient of R = 0.9998. The linear range of TMA+ for UPLC-HRMS is 0.002 to 1 μg/mL, with a correlation coefficient of R = 0.996. The recovery rates for IC and HRMS were 116.6% and 90.6%, with corresponding coefficient of variation (CV) of 2.25% and 3.53%, respectively. Samples can be stored at room temperature for 28 days. Verification of this method was conducted at the TMAH factory, and the air concentrations measured in the TMAH filling area were 3.29 and 4.75 μg/m3 from IC and UPLC-HRMS, respectively. This method will be applied in the on-site analysis of occupational hazards for operators in chemical plants, technology factories, and recycling plants, aiming to evaluate risk values to protect the safety and health of workers and prevent occupational diseases.

Multisample lipidomic profiles of irritable bowel syndrome and irritable bowel syndrome-like symptoms in patients with inflammatory bowel disease: new insight into the recognition of the same symptoms in different diseases.

Overlapping clinical manifestations of irritable bowel syndrome (IBS) and IBS-like symptoms in patients with inflammatory bowel disease (IBD-IBS) present challenges in diagnosis and management. Both conditions are associated with alterations in metabolites, but few studies have described the lipid profiles. Our aim was to pinpoint specific lipids that contribute to the pathogenesis of IBS and IBD-IBS by analyzing multiple biologic samples. Diarrhea-predominant IBS (IBS-D) patients (n = 39), ulcerative colitis in remission with IBS-like symptoms patients (UCR-IBS) (n = 21), and healthy volunteers (n = 35) were recruited. IBS-D patients meet the Rome IV diagnostic criteria, and UCR-IBS patients matched mayo scores ≤ two points and Rome IV diagnostic criteria. Serum, feces, and mucosa were collected for further analysis. Lipid extraction was carried out by ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). Lipidomics of mucosa and serum samples significantly differed among the three groups. Feces showed the most altered lipid species, and the enrichment analysis of 347 differentially abundant metabolites via KEGG pathway analysis revealed that alpha-linolenic acid metabolism was significantly altered in the two groups (P < 0.01). The ratio of omega-6/omega-3 fatty acid were imbalance in serum samples. This study revealed a comprehensive lipid composition pattern between IBS-D patients and UCR-IBS patients. We found several distinctive lipids involved in alpha-linolenic acid metabolism, reflecting an imbalance in the omega-6/omega-3 fatty acid ratio. Compared to mucosa and serum samples, fecal samples might have more advantages in lipidomics studies due to the convenience of sample collection and effectiveness in reflecting metabolic information.

High-coverage characterization and discovery of molecular markers for quality control of natural fragrant plant extracts using UPLC-HRMS-based untargeted metabolomics.

The chemical components of natural fragrant plant extracts are of high complexity, and the strategies for quality control (QC) and further discovery of fragrance mechanisms still need to be further investigated. This study integrated the strategies and methods of untargeted metabolomics and chemometrics and statistical modeling to attain the goal. The techniques of reversed-phase and HILIC analysis of ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) were simultaneously used to collect data in both positive and negative ion modes. The pattern analysis of fingerprints and discovery of characteristic molecular markers for QC analysis were comprehensively employed to reach in-depth analysis of the quality variation and discovery of differential molecules among natural fragrant plant extracts. The former uses fingerprint technique to analyze their overall similarities and differences, and the latter comprehensively discovers molecular substances characterizing the chemical characteristics of fragrant extracts with the help of metabolomics and univariate and multivariate methods. The findings are expected to be used as the molecular markers in product manufacturing, sales, and consumption to achieve accurate quality control and recognition of targeted molecules for potential quality monitoring using spectroscopy techniques. In this work, 27 natural fragrant extracts were applied as examples, and their chemical components were comprehensively analyzed with discovery of markers for quality control. After data integration, 1178 molecules were annotated, and 267 differential metabolite molecules with the values of variable importance in the projection (VIP) larger than 1.0 were found. The results show that the method proposed in this work is of great significance for high-coverage analysis, QC marker discovery, and aroma mechanism elucidation, which has potential applications in the areas of food, cosmetics, pharmaceuticals, tobacco, and others.

Phytochemical Characterization and Bioactivity of Extracts from Different Fruit Parts of Opuntia leucotricha DC.: A Comparison between a Conventional Organic Solvent and Green Natural Deep Eutectic Solvents

The objective of this study was to analyze the chemical profile of extracts from different fruit parts of the perennial arborescent cactus Opuntia leucotricha by applying ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS) and to evaluate their biological properties (antioxidant and enzyme inhibitory capacities). Extracts were obtained from the fruit pulp (without or with seeds), seeds, and peel by ultrasound-assisted extraction (UAE) using the conventional solvent methanol 50% and two Natural Deep Eutectic Solvents (NADESs) (glycerol:urea, 1:1 and citric acid:sorbitol, 1:2). A total of 33 compounds were identified, including phenolics, fatty acids, and others. Phenolic acids were the most abundant class of phenolics identified in all fruit parts, with the highest concentration observed in the methanol extracts from peel (593.02 µg/gDW), followed by seed-containing pulp (69.03 µg/gDW), pulp (57.83 µg/gDW), and seeds (39.97 µg/gDW). The second most effective extractant was NADES 1 (glycerol:urea, 1:1), which was also successful in extracting compounds with antioxidant capacity. Overall, the extracts demonstrated considerable enzyme inhibitory activities, with the greatest effects observed against α-amylase and α-glucosidase. The results indicate that O. leucotricha fruits could be a promising source of bioactive compounds, and NADES a viable alternative to organic solvents for their industrial exploitation.

A strategy integrating improved mass defect filtering, diagnostic product ions, and molecular network for rapid screening and systematic characterization of steroidal alkaloids in Fritillaria ussuriensis

An ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) method was employed to efficiently and rapidly analyze the chemical composition of steroidal alkaloids in Fritillaria ussuriensis Maxim (FM). The integration of nitrogen rule and improved mass defect filtering eliminated 84.61% of interfering MS1 peaks and facilitated subtype classification. Subsequently, six diagnostic product ions (DPIs) for Fritillaria alkaloids were summarized, guiding structural identification. Additionally, the adoption of molecular networks to infer potential compounds forms a new four-step comprehensive strategy for systematic screening and identification of Fritillaria alkaloids. By screening with the MDF window constructed from the Fritillaria alkaloids MS database combined with the nitrogen rule, a significant number of interfering MS1 peaks were eliminated, and different subtypes were distinguished. In FM, the number of positive-mode MS1 peaks was reduced by 84.61% (from 8,728 to 1,343). By summarizing the fragmentation behaviors of 22 reference standards, the fragmentation pathways and DPIs of five types of steroidal alkaloids were confirmed, providing the first evidence for DPIs of cevanine-seven-membered type Fritillaria alkaloids. Structural confirmation of MS1 peaks was carried out using an in-house database and DPIs. Furthermore, a feature-based molecular network (FBMN) was employed to cluster similar compounds into network clusters for rapid classification and to infer potential steroidal alkaloids using known compounds. A total of 103 Fritillaria alkaloids (65 cevanine type, 7 cevanine-seven-membered type, 18 jervine type, 5 veratramine type, 5 secosolanidine type, and 3 solanidine type) were identified, including 4 potential new compounds and 20 compounds that have been identified for the first time. This innovative comprehensive strategy, which includes NRF, improved MDF, DPIs, and FBMN, can be applied to rapidly screen, classify components, and conduct comprehensive characterization of complex samples such as herbal medicines.

Plasma metabolomic and lipidomic profiles accurately classify mothers of children with congenital heart disease: an observational study

IntroductionCongenital heart disease (CHD) is the most common congenital anomaly, representing a significant global disease burden. Limitations exist in our understanding of aetiology, diagnostic methodology and screening, with metabolomics offering promise in addressing these.ObjectiveTo evaluate maternal metabolomics and lipidomics in prediction and risk factor identification for childhood CHD.MethodsWe performed an observational study in mothers of children with CHD following pregnancy, using untargeted plasma metabolomics and lipidomics by ultrahigh performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). 190 cases (157 mothers of children with structural CHD (sCHD); 33 mothers of children with genetic CHD (gCHD)) from the children OMACp cohort and 162 controls from the ALSPAC cohort were analysed. CHD diagnoses were stratified by severity and clinical classifications. Univariate, exploratory and supervised chemometric methods were used to identify metabolites and lipids distinguishing cases and controls, alongside predictive modelling.Results499 metabolites and lipids were annotated and used to build PLS-DA and SO-CovSel-LDA predictive models to accurately distinguish sCHD and control groups. The best performing model had an sCHD test set mean accuracy of 94.74% (sCHD test group sensitivity 93.33%; specificity 96.00%) utilising only 11 analytes. Similar test performances were seen for gCHD. Across best performing models, 37 analytes contributed to performance including amino acids, lipids, and nucleotides.ConclusionsHere, maternal metabolomic and lipidomic analysis has facilitated the development of sensitive risk prediction models classifying mothers of children with CHD. Metabolites and lipids identified offer promise for maternal risk factor profiling, and understanding of CHD pathogenesis in the future.

A classic prescription alleviates inflammation in CUMS model mice via modulating MYDGF/MAP4K4/NF-κB signaling pathway, verified through UPLC-HRMS and proteomics analysis

BackgroundXiaoyaosan (XYS), a renowned classical traditional Chinese medicinal formula utilized in addressing major depressive disorder (MDD), has garnered significant acclaim for its remarkable efficacy in clinical application. The onset of major depressive disorder (MDD) often correlates with chronic unpredictable mild stress (CUMS), a pivotal instigating factor in its development.Aim of the study: This study aims to clarify the potential anti-inflammatory mechanisms of XYS in treating CUMS model mice. Materials and methodsUtilizing cutting-edge ultra high-performance liquid chromatography - high-resolution mass spectrometry (UPLC-HRMS), the active constituents of XYS were discerned, while employing proteomics analysis to delve into the potential mechanisms of its efficacy. Molecular docking studies, alongside subsequent in vivo experiments utilizing CUMS model mice, were conducted to corroborate the findings derived from the proteomics analysis. ResultsIn vivo experiments demonstrated that XYS not only markedly ameliorated behavioral markers but also attenuated serum inflammatory markers and suppressed IL-6 and TNF-α expression within the brains of CUMS model mice. Proteomics analysis suggested that the pivotal anti-inflammatory mechanism of XYS against CUMS-induced damage might involve modulation of the MAPK signaling pathway. Utilizing UPLC-HRMS, the active constituents of XYS were successfully identified, while molecular docking investigations explored interactions between XYS and MYDGF, PKC, MAP4K4, P-p65, p65, P-IKBα, and IKBα. The findings revealed XYS's regulatory influence on the MYDGF/MAP4K4/NF-κB signaling cascade. ConclusionsThis study is the first to our knowledge to demonstrate that XYS can alleviate inflammation in CUMS model mice by modulating the MYDGF/MAP4K4/NF-κB signaling pathway.

Characterization of the Components and Metabolites of Achyranthes Bidentata in the Plasma and Brain Tissue of Rats Based on Ultrahigh Performance Liquid Chromatography-High-Resolution Mass Spectrometry (UHPLC-HR-MS).

Achyranthes bidentata (AR) is a traditional Chinese herb used for the treatment of hypertension and cerebral ischemia, but its pharmacological effects are not known. We aimed to detect and accurately identify the components and metabolites of AR in the plasma and brain tissue of Sprague Dawley rats. We employed ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HR-MS) to detect AR components in the plasma and brain tissue of rats. The absorption and metabolites in the plasma and brain tissue of normal control rats and rats that underwent middle cerebral artery occlusion (MCAO) were characterized and compared. A total of 281 compounds, including alkaloids, flavonoids, terpenoids, phenylpropanes, sugars and glycosides, steroids, triterpenes, amino acids, and peptides, was identified in samples of Achyranthes bidentata (TCM-AR). Four types of absorbable prototype components and 48 kinds of metabolites were identified in rats in the normal control plasma group which were given AR (AR plasma group), and five kinds of metabolites were identified in rats of the normal control brain tissue group which were given AR (AR brain group). Three absorbed prototype components and 13 metabolites were identified in the plasma of rats which underwent MCAO and were given AR (MCAO + AR plasma group). Six absorbed prototype components and two metabolites were identified in the brain tissue of rats who underwent MCAO and were administered AR (MCAO + AR brain group). These results showed that, after the oral administration of AR, the number of identified components in plasma was more than that in brain tissue. The number of prototype components in the AR plasma group was higher than that in the MCAO + AR plasma group, which may indicate that metabolite absorption in rats undergoing MCAO was worse. The number of prototype components in the MCAO + AR brain group was higher than that in the AR brain group, indicating that the blood-brain barrier was destroyed after MCAO, resulting in more compounds entering brain tissue. UHPLC-HR-MS was used to rapidly analyze the components and metabolites of AR in the blood and brain of rats under normal and pathologic conditions, and to comprehensively characterize the components of TCM-AR. We also analyzed and compared the absorbable components and metabolites of normal rats under cerebral ischemia-reperfusion injury to explore the potential mechanism of action. This method could be applied to various Chinese herbs and disease models, which could promote TCM modernization.

Metabolomics combined with network pharmacology reveals a role for astragaloside IV in inhibiting enterovirus 71 replication via PI3K-AKT signaling

BackgroundAstragaloside IV (AST-IV), as an effective active ingredient of Astragalus membranaceus (Fisch.) Bunge. It has been found that AST-IV inhibits the replication of dengue virus, hepatitis B virus, adenovirus, and coxsackievirus B3. Enterovirus 71 (EV71) serves as the main pathogen in severe hand-foot-mouth disease (HFMD), but there are no specific drugs available. In this study, we focus on investigating whether AST-IV can inhibit EV71 replication and explore the potential underlying mechanisms.MethodsThe GES-1 or RD cells were infected with EV71, treated with AST-IV, or co-treated with both EV71 and AST-IV. The EV71 structural protein VP1 levels, the viral titers in the supernatant were measured using western blot and 50% tissue culture infective dose (TCID50), respectively. Network pharmacology was used to predict possible pathways and targets for AST-IV to inhibit EV71 replication. Additionally, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was used to investigate the potential targeted metabolites of AST-IV. Associations between metabolites and apparent indicators were performed via Spearman’s algorithm.ResultsThis study illustrated that AST-IV effectively inhibited EV71 replication. Network pharmacology suggested that AST-IV inhibits EV71 replication by targeting PI3K-AKT. Metabolomics results showed that AST-IV achieved these effects by elevating the levels of hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-hydroxy-1 H-indole-3- acetamide, oxypurinol, while reducing the levels of PC (14:0/15:0). Furthermore, AST-IV also mitigated EV71-induced oxidative stress by reducing the levels of MDA, ROS, while increasing the activity of T-AOC, CAT, GSH-Px. The inhibition of EV71 replication was also observed when using the ROS inhibitor N-Acetylcysteine (NAC). Additionally, AST-IV exhibited the ability to activate the PI3K-AKT signaling pathway and suppress EV71-induced apoptosis.ConclusionThis study suggests that AST-IV may activate the cAMP and the antioxidant stress response by targeting eight key metabolites, including hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-Hydroxy-1 H-indole-3-acetamide, oxypurinol and PC (14:0/15:0). This activation can further stimulate the PI3K-AKT signaling to inhibit EV71-induced apoptosis and EV71 replication.Graphical

Characterization of spironolactone and metabolites derivatized using Girard's reagent P using mass spectrometry and ion mobility spectrometry.

Spironolactone is a steroidal drug prescribed for a variety of medical conditions and is extensively metabolized quickly after administration. Measurement of spironolactone and its metabolites remains challenging using mass spectrometry (MS) due to in-source fragmentation and relatively poor ionization using electrospray ionization. Therefore, improved methods of measurements are needed, particularly in the case of small sample volumes. Girard's reagent P (GP) derivatization of spironolactone was employed to improve response and provide an MS-based solution to the measurement of spironolactone and its metabolites. We performed ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) and ion mobility spectrometry (IMS)-high-resolution mass spectrometry (HRMS) to fully characterize the GP derivatization products. Analytes were studied in positive ionization mode, and MS/MS was performed using nonresonance and resonance excitation collision-induced dissociation. We observed the successful GP derivatization of spironolactone and its metabolites using authentic chemical standards. A signal enhancement of 1-2 orders of magnitude was observed for GP-derivatized versions of spironolactone and its metabolites. Further, GP derivatization eliminated in-source fragmentation. Finally, we performed GP derivatization and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) in a small volume of murine serum (20 μL) from spironolactone-treated and control animals and observed multiple spironolactone metabolites only in the spironolactone-treated group. GP derivatization was proven to have advantageous mass spectral performance (e.g., limiting in-source fragmentation, enhancing signals, and eliminating isobaric analytes) for spironolactone and its metabolites. This work and the detailed characterization using ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) and IMS serve as the foundation for future developments in reaction optimization and/or quantitative assay development.

A multi-technique based analytical platform for characterization and identification of a newly emerged steroid 6β-chlorotestosterone and its in-vivo epimeric metabolites

Doping athletes tend to use new and non-approved drugs in an attempt to circumvent the dope test. Recently, a package of unknown drug was obtained from black market claiming to contain steroid-like compound, which could not be readily identified due to the lack of commercially available reference standards. In order to characterize and identify the chemical structure of the unknown compound, ultra performance liquid chromatography high resolution mass spectrometry (UPLC-HRMS), high performance liquid chromatography tandem quadrupole mass spectrometry (HPLC-MS/MS), gas chromatography tandem quadrupole mass spectrometry (GC–MS/MS) and nuclear magnetic resonance (NMR) instruments were applied in this work. Based on the combined data, the unknown compound was identified as 6β-chlorotestosterone. The aim of this study was to find urinary metabolic biomarkers of 6β-chlorotestosterone for clinical research and doping control analysis. Ten in-vivo metabolites including four phase I metabolites and six phase II metabolites were characterized and potentially identified. The detailed structure of the phase I metabolite was successfully obtained by GC–MS/MS analysis of trimethylsilylation (TMS) released upon dissolution, overcoming the absence of useful fragment ions to demonstrate the structure of sulfate metabolites. Two strategies including metabolic mechanisms and retention time variation of steroids were introduced to determine and distinguish 3α/β- and 5α/β- epimeric metabolites. A comprehensive evaluation was conducted on time of appearance and detection of each metabolite. The sulfate metabolite 6β-chloro-5β-androstan-17-one-3β-O-sulfate (S1) could be detected up to 12 days after oral administration, which was considered to be the long term biomarker for 6β-chlorotestosterone abuse in clinical research and doping control analysis.

Exploring the antimicrobial activity of Origanum majorana L. against the highly virulent multidrug-resistant Acinetobacter baumannii AB5075: UPLC-HRMS profiling with in vitro and in silico studies

BackgroundThe infamous multidrug-resistant (MDR) bacterium Acinetobacter baumannii is becoming a nightmare in intensive care units across the globe. Since there are now very few effective antimicrobial agents, it is necessary to explore unconventional resources for novel antimicrobials. This study investigated the potential antimicrobial activity of Origanum majorana L. against A. baumannii employing multiple approaches including antimicrobial susceptibility, fractionation, ultra-performance liquid chromatography–high-resolution mass spectrometry (UPLC-HRMS) dereplication, and in silico analysis for target/ligand identification.ResultsOn the extremely pathogenic MDR strain A. baumannii AB5075, O. majorana L. has shown a significant growth inhibitory effect (MIC = 0.675 mg/mL). The polar 50% methanol fraction was the most active (MIC = 0.5 mg/mL). The UPLC-HRMS dereplication of the bioactive fraction detected 29 metabolites belonging to different chemical classes. Justicidin B, one of the identified metabolites, was projected by preliminary in silico analysis to be the most highly scoring metabolite for binding with molecular targets in A. baumannii with a Fit score = 8.56 for enoyl-ACP reductase (FabI) (PDB ID: 6AHE), suggesting it to be its potential target. Additionally, docking, molecular dynamics simulation, and bioinformatics analysis suggested that this interaction is similar to a well-known FabI inhibitor. The amino acids involved in the interaction are conserved among different MDR A. baumannii strains and the effectiveness could extend to Gram-negative pathogens within the ESKAPE group.ConclusionsOriganum majorana L. extract exhibits antimicrobial activity against A. baumannii using one or more metabolites in its 50% methanol fraction. The characterized active metabolite is hypothesized to be justicidin B which inhibits the growth of A. baumannii AB5075 via targeting its fatty acid synthesis.

Differences in Metabolite Profiles of Dihydroberberine and Micellar Berberine in Caco-2 Cells and Humans-A Pilot Study.

We investigated the pharmacokinetic pathway of berberine and its metabolites in vitro, in Caco-2 cells, and in human participants following the administration of dihydroberberine (DHB) and micellar berberine (LipoMicel®, LMB) formulations. A pilot trial involving nine healthy volunteers was conducted over a 24 h period; blood samples were collected and subjected to Ultra High-Performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS) analyses to quantify the concentrations of berberine and its metabolites. Pharmacokinetic correlations indicated that berberrubine and thalifendine follow distinct metabolic pathways. Additionally, jatrorrhizine sulfate appeared to undergo metabolism differently compared to the other sulfated metabolites. Moreover, berberrubine glucuronide likely has a unique metabolic pathway distinct from other glucuronides. The human trial revealed significantly higher blood concentrations of berberine metabolites in participants of the DHB treatment group compared to the LMB treatment group-except for berberrubine glucuronide, which was only detected in the LMB treatment group. Similarly, results from in vitro investigations showed significant differences in berberine metabolite profiles between DHB and LMB. Dihydroberberine, dihydroxy-berberrubine/thalifendine and jatrorrhizine sulfate were detected in LMB-treated cells, but not in DHB-treated cells; thalifendine and jatrorrhizine-glucuronide were detected in DHB-treated cells only. While DHB treatment provided higher blood concentrations of berberine and most berberine metabolites, both in vitro (Caco-2 cells) and in vivo human studies showed that treatment with LMB resulted in a higher proportion of unmetabolized berberine compared to DHB. These findings suggest potential clinical implications that merit further investigation in future large-scale trials.

Deciphering the chemical constituents of Phlomis monocephala extracts using UHPLC-HRMS and their antioxidant, neuroprotective, antidiabetic and toxic potentials

In the current study, five different extracts (n-hexane, ethyl acetate, dichloromethane, ethanol, and water) of Phlomis monocephala were analyzed for the first time by ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) to identify their phenolic compounds. The extracts were also evaluated for their non-enzyme antioxidant activities using a variety of methods, including DPPH and, ABTS•+ scavenging activities, reduction of ferric (Fe3+), and cupric ions (Cu2+), metal chelating (MCA) activities, and phosphomolybdenum (PBD). Additionally, the extracts were assessed for their in vitro enzyme inhibition potential (acetylcholinesterase (AChE)/butyrylcholinesterase (BChE), α-amylase, α-glucosidase, and tyrosinase). Furthermore, cell viability was evaluated on HepG2 (human hepatocellular carcinoma), S17 cells (murine bone marrow stromal) and RAW (murine macrophages). UHPLC-HRMS allowed for the identification of 115 compounds from different chemical groups including flavonoids, iridoid glycosides, phenylethanoid glycosides and others. Most of the extracts had strong antioxidant potential and were rich in phenolic compounds. Ethanol and water extracts appear as the most promising antioxidant extracts providing the highest values followed by ethyl acetate by all the methodologies employed in this study. Furthermore, all the extracts, except the aqueous extract, inhibited all the enzymes significantly. Surprisingly, the aqueous extract did not show a prominent inhibition (low or no inhibition) against these enzymes. Hexane, ethyl acetate, and dichloromethane exhibited extremely significant cytotoxic effects against all cell lines at a higher concentration. The HepG2 cells demonstrated lower sensitivity to the extracts than RAW and S17 cells. In conclusion, P. monocephala can be considered as a valuable ingredient for the production of functional applications including nutraceuticals.

Relative quantitation of chiral thiol compounds labeled based on isotope novel mass spectrometry probes: Monitoring of the dynamic changes of chiral thiol compounds in human urine during normal, exercise, and rest recovery states

Monitoring changes in the content of chiral thiol compounds in the human body is crucial for the early diagnosis of oxidative stress-related diseases and the exploration of their pathogenesis. To address this, we synthesized a novel isotope mass spectrometry (MS) probe, denoted as (R)-(5-(3-isothiocyanato (13C) pyrrolidin-1-yl)-5-oxopentyl) triphenylphosphonium (N13CS-OTPP), with triphenylphosphine as its parent structure. In this study, we established a new ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLCHRMS) relative quantitative method to monitor chiral thiol compounds in human urine under varying oxidative stress conditions. This method relies on the ratio of 12C/13C isotope-labeled peak areas. To assess the chiral separation efficiency of N13CS-OTPP, we employed three types of thiol compounds (D/L-GSH, D/L-Cys, and D/L-Hcy) and observed separation degrees (Rs) ranging from 1.82 to 1.89. We further validated the accuracy and feasibility of our relative quantitative methods using D/L-Cys-as a model compound. N12C/13CS-OTPP-Cys-exhibited excellent linearity (R2 = 0.9993–0.9994) across different molar ratios (D/L-Cys = 10:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:10) and achieved a low limit of detection (LOD) of 2.5 fmol. Additionally, we monitored the dynamic changes in urine D/L-Cys-and D/L-Hcy ratios in 12 healthy volunteers (six males and six females) under various oxidative stress states. We generated fitting curves and investigated the trends in chiral thiol compounds in vivo. This study introduces a novel method for the relative quantitative monitoring of chiral thiol compounds in different oxidative stress states within the human body. It also presents a new strategy for understanding the pathogenesis of related diseases resulting from the abnormal metabolism of thiol compounds.

'Sorrento' and 'Tulare' Walnut Cultivars: Morphological Traits and Phytochemical Enhancement of Their Shell Waste.

Walnut processing generates considerable quantities of by-products that could be reprocessed into value-added products that have food and non-food applications. In this context, the aim of this study is to characterize the 'Sorrento' and 'Tulare' walnut cultivars using the UPOV guidelines and analyze the chemical composition and antioxidant activity of their shells. Insight into the chemical composition of the different granulometric fractions of walnut shell, obtained by sieving, was obtained following ultrasound-assisted extraction by Ultra-High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry (UHPLC-HRMS). The total phenolic, flavonoid, and tannin content and antiradical capacity, obtained by DPPH and ABTS assays, and the Fe(III) reducing power of the extracts were also evaluated. The UHPLC-HRMS analysis indicated the presence of thirty-two compounds ascribable to four major classes of specialized metabolites. Furthermore, the extraction efficiency of gallic acid, ellagic acid derivatives, as well as glansreginin A, increased with the decrease in shell matrix particle size in contrast to chlorogenic acids and flavonoid glycosides. This is the first study to highlight new knowledge on the chemical composition of walnut shells. The results obtained demonstrate the feasibility of recovering valuable bioactive components from agro-waste that may be further valorized.

Risk analysis of serum chemical residues for metabolic associated fatty liver disease based on exposome-lipidome wide association study

Metabolic associated fatty liver disease (MAFLD) is a common liver disease with a prevalence of up to 25%; it not only adversely affects human health but also aggravates the economic burden of society. An increasing number of studies have suggested that the occurrence of chronic noncommunicable diseases is affected by both environmental exposures and genetic factors. Research has also shown that environmental pollution may increase the risk of MAFLD and promote its occurrence and development. However, the relationship between these concepts, as well as the underlying exposure effects and mechanism, remains incompletely understood. Lipidomics, a branch of metabolomics that studies lipid disorders, can help researchers investigate abnormal lipid metabolites in various disease states. Lipidome-exposome wide association studies are a promising paradigm for investigating the health effects of cumulative environmental exposures on biological responses, and could provide new ideas for determining the associations between metabolic and lipid changes and disease risk caused by chemical-pollutant exposure. Hence, in this study, targeted exposomics and nontargeted lipidomics studies based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) were used to characterize exogenous chemical pollutants and endogenous lipid metabolites in the sera of patients with MAFLD and healthy subjects. The results demonstrated that fipronil sulfone, malathion dicarboxylic acid, and monocyclohexyl phthalate may be positively associated with the disease risk of patients diagnosed as simple fatty liver disease (hereafter referred to as MAFLD(0)). Moreover, fipronil sulfone, acesulfame potassium, perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluoroundecanoic acid (PFUnDA), 4-hydroxybenzophenone, and 3,5-di-tert-butyl-4-hydroxybenzoic acid (DBPOB) may be positively associated with the disease risk of patients diagnosed as fatty liver complicated by single or multiple metabolic disorders. Association analysis was carried out to explore the lipid metabolites induced by chemical residues. Triglyceride (TG) and diglyceride (DG) were significantly increased in MAFLD and MAFLD(0). The numbers of carbons of significantly changed DGs and TGs were mainly in the ranges of 32-40 and 35-60, respectively, and both were mainly characterized by changes in polyunsaturated lipids. Most of the lipid-effect markers were positively correlated with chemical residues and associated with increased disease risk. Our research provides a scientific basis for studies on the association and mechanism between serum chemical-pollutant residues and disease outcomes.

Emerging investigator series: in-depth chemical profiling of tire and artificial turf crumb rubber: aging, transformation products, and transport pathways.

Crumb rubber generated from end-of-life tires (ELTs) poses a threat to environmental and human health based on its widespread use. Of particular concern is the use of ELT crumb rubber as infill for artificial turf fields, as people are unknowingly exposed to complex mixtures of chemicals when playing on these fields. Additionally, there is concern regarding transport of rubber-related chemicals from artificial turf into the environment. However, existing knowledge does not fully elucidate the chemical profile, transformation products, and transport pathways of artificial turf crumb rubber across different ages. To address these knowledge gaps, we utilized a multi-faceted approach that consisted of targeted quantitation, chemical profiling, and suspect screening via ultra-high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). We collected and processed 3 tire and 11 artificial turf crumb rubber samples via solvent extraction, leaching, and a bioaccessibility-based extraction. Nineteen rubber-derived chemicals were quantified using parallel reaction monitoring and isotope dilution techniques. In solvent extracts, the most abundant analytes were 1,3-diphenylguanidine (0.18-1200 μg g-1), N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD, 0.16-720 μg g-1), 2-mercaptobenzothiazole (0.47-140 μg g-1), and benzothiazole (0.84-150 μg g-1). Chemical profiling assessed changes in sample diversity, abundance, polarity, and molecular mass. Suspect screening identified 81 compounds with different confidence levels (16 at level 1, 53 with level 2, 7 at level 3, and 5 at level 4). The formation rate of transformation products and clustering analysis results identified time-based trends in artificial turf field samples. We found that the first two years of aging may be critical for the potential environmental impact of artificial turf fields. Our analysis provides insight into the chemical complexity of artificial turf crumb rubber samples ranging from 0-14 years in age.