Hybrid Quadrupole-orbitrap High-resolution Mass Research Articles

Quality assessment and Q-markers discovery in Citri Sarcodactylis Fructus by integrating serum pharmacochemistry and network pharmacology.

Citri Sarcodactylis Fructus (CSF), a common fruit and traditional Chinese medicine (TCM), has been hindered in its further development and research owing to the lack of comprehensive and specific quality evaluation standards. This study aimed to establish clear TCM quality standards related to the therapeutic mechanisms of CSF and to provide a basis for subsequent research and development. Ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry (UPLC-Q-orbitrap HRMS) technology was used to comprehensively identify CSF components and explore their absorbance levels in rat serum. Network pharmacology research methods were employed to investigate the potential mechanisms of action of the identified components in the treatment of major clinical diseases. Subsequently, a combination of HPLC chromatographic fingerprinting for qualitative analysis and multi-index content determination was used to evaluate the detectability of the identified quality markers (Q-markers). Twenty-six prototype components were tentatively characterized in rat serum. Network pharmacology analysis showed six effective components, namely 7-hydroxycoumarin, isoscopoletin, diosmin, hesperidin, 5,7-dimethoxycoumarin, and bergapten, which played important roles in the treatment of chronic gastritis, functional dyspepsia, peptic ulcer, and depression and were preliminarily identified as Q-markers. The results of content determination in 15 batches of CSF indicated significant differences in the content of medicinal materials from different origins. However, compared with the preliminarily determined Q-markers, all six components could be measured and were determined as Q-markers of CSF. The chemical Q-markers obtained in this study could be used for effective quality control of CSF.

Biotransformation of 5-methoxy-N-isopropyl-N-methyltryptamine by zebrafish and human liver microsome with high-resolution mass spectrometry

To explore the metabolites of 5-Methoxy-N-isopropyl-N-methyltryptamine (5-MeO-MiPT) and unveil its toxicological effects, we examined its metabolic profiles using zebrafish and human liver microsome models. Employing ultra-high-performance liquid chromatography Q Exactive hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UPLC-QE-HRMS), we analyzed samples from intoxicated zebrafish and human liver microsomes. In the zebrafish model, we identified a total of six metabolites. Primary phase I metabolic pathways involved N-Demethylation and Indole-hydroxylation reactions, while phase II metabolism included Glucoside conjugation directly, Glucoside conjugation after Indole-hydroxylation, and Sulfonation following Indole-hydroxylation. In the human liver microsome model, nine metabolites were generated. Major phase I metabolic pathways encompassed N-Demethylation, 5-O-Demethylation, and N-Depropylation, N-Oxidation, Indole-hydroxylation, N-Demethylation combined with Indole-hydroxylation, and 5-O‐Methylation-carboxylation. Phase II metabolism involved Glucoside conjugation after Indole-hydroxylation, as well as Glucoside conjugation after 5-O-Demethylation. Proposed phase I metabolites, such as 5-MeO-MiPT-N-Demethylation (5-MeO-NiPT) and 5-MeO-MiPT-Indole-hydroxylation, alongside the phase II metabolite OH&Glucoside conjugation-5-MeO-MiPT, were identified as effective markers for screening 5-MeO-MiPT intake. This study systematically delineates the phase I and II metabolites of 5-MeO-MiPT, confirming their pathways through in vivo and in vitro extrapolation. Additionally, inclusion of the parent drug itself and OH&Glucoside conjugation-5-MeO-MiPT could serve as valuable confirmation tools.

In vivo comprehensive metabolite profiling of esculetin and esculin derived from chicory in hyperuricemia rats using ultra-high-performance liquid chromatography coupled with quadrupole-orbitrap high-resolution mass spectrometry.

Chicory, renowned for its multifaceted benefits, houses two vital coumarins, esculetin and esculin, both instrumental in reducing uric acid. This study emphasizes the metabolic pathways of esculetin and esculin under both standard and hyperuricemia conditions. Hyperuricemia was induced in Sprague-Dawley rats using oxonic acid potassium salt (300mg·kg-1 ) and a 10% fructose water regimen over 21 days. Leveraging the ultra-high-performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high resolution mass spectrometry, we analyzed the fragmentation behaviors of esculetin and esculin in rat bio-samples. Post oral-intake of esculetin or esculin, a notable dip in serum uric acid levels was observed in hyperuricemia rats. The investigation unveiled 24 esculetin metabolites and 14 for esculin. The metabolic pathways of both compounds were hydrolysis, hydroxylation, hydrogenation, dehydroxylation, glucuronidation, sulfation, and methylation. Interestingly, certain metabolites presented variations between standard and hyperuricemia rats, indicating that elevated levels of uric acid may affect enzyme activity linked to these metabolic reactions. This is the first systematic study on comparison of metabolic profiles of esculetin and esculin in both normal and hyperuricemia states, which was helpful to enrich our understanding of the complicated structure-activity relationships between esculin and esculetin and shed light to their action mechanism.

Shotgun Proteomic-Based Approach with a Q-Exactive Hybrid Quadrupole-Orbitrap High-Resolution Mass Spectrometer for Protein Adductomics on a 3D Human Brain Tumor Neurospheroid Culture Model: The Identification of Adduct Formation in Calmodulin-Dependent Protein Kinase-2 and Annexin-A1 Induced by Pesticide Mixture.

Pesticides are increasingly used in combinations in crop protection, resulting in enhanced toxicities for various organisms. Although protein adductomics is challenging, it remains a powerful bioanalytical tool to check environmental exposure and characterize xenobiotic adducts as putative toxicity biomarkers with high accuracy, facilitated by recent advances in proteomic methodologies and a mass spectrometry high-throughput technique. The present study aims to predict the potential neurotoxicity effect of imidacloprid and λ-cyhalothrin insecticides on human neural cells. Our protocol consisted first of 3D in vitro developing neurospheroids derived from human brain tumors and then treatment by pesticide mixture. Furthermore, we adopted a bottom-up proteomic-based approach using nanoflow ultraperformance liquid chromatography coupled with a high-resolution mass spectrometer for protein-adduct analysis with prediction of altered sites. Two proteins were selected, namely, calcium-calmodulin-dependent protein kinase-II (CaMK2) and annexin-A1 (ANXA1), as key targets endowed with primordial roles. De novo sequencing revealed several adduct formations in the active site of 82-ANXA1 and 228-CaMK2 as a result of neurotoxicity, predicted by the added mass shifts for the structure of electrophilic precursors. To the best of our knowledge, our study is the first to adopt a proteomic-based approach to investigate in depth pesticide molecular interactions and their potential to adduct proteins which play a crucial role in the neurotoxicity mechanism.

Chemical profiling and mechanistic studies of Zhi-Shang-Feng granules against influenza virus by high-performance liquid chromatography coupled with Q exactive focus hybrid quadrupole orbitrap high-resolution mass spectrometry in combination with network pharmacology analysis.

Zhi-Shang-Feng Granules are used in the clinical treatment of influenza to relieve headaches, chills and fever, bronchitis, nasal congestion, neuralgia and other symptoms. To decipher the components responsible for therapeutic effects of Zhi-Shang-Feng g ranules against influenza virus, an analytical method based on high-performance liquid chromatography coupled with Q exactive focus hybrid quadrupole orbitrap high resolution mass spectrometry was developed and the chemical profile of Zhi-Shang-Feng granules was characterized. Then, the identified components were used to conduct network pharmacological analysis and determine the potential mechanism of Zhi-Shang-Feng Granules. As a result, 177 compounds were putatively identified through comprehensive analysis by liquid chromatography coupled with high-resolution mass spectrometry, of which 23 compounds were unambiguously confirmed with reference standards. Components in Zhi-Shang-Feng Granules were found to specifically act on different enzymes, G-protein-coupled receptors, ion channels and transporters in the immune, endocrine, nervous, and circulatory systems. The potential mechanism was related to several biological processes, including cell growth and death, pattern recognition receptor signalling, signalling by interleukins, and lipid metabolism. The combination of chemical profile characterization and network construction provided useful insight into the overall chemical composition of Zhi-Shang-Feng granules and revealed their potential anti-infection, anti-inflammatory and immunoregulatory mechanisms against influenza virus infected disease.

Bio-based material-edible rosemary induced biodegradation of aflatoxin B1 via altering endogenous protective enzymes signatures in animal-derived foods

Aflatoxin B1 (AFB1) is the most hazardous mycotoxin, posing risks to public health. Utilization of bio-based materials to biodegrade AFB1 is a green strategy to overcome this issue. The investigation aimed to screen for endogenous protective enzymes in bio-based material-edible rosemary based on ultra-high performance liquid chromatography coupled to hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS)-proteomics and ascertain their impacts on the biodegradation and biotransformation of AFB1, and the trade-offs of multilevel metabolism of the animal-derived foods through untargeted metabolomics. The proteomics results verified that bio-based material-edible rosemary (0.20%, w/w) significantly up-regulated glutathione S-transferase and stimulated the down-regulation of cytochrome P450 1A2 levels via activating AhR nuclear translocation in rosemary-pickled AFB1-contaminated goat meat. Metabolomics results demonstrated that edible rosemary substantially increased histidine and glutathione implicated in the antioxidant status of goat meat. More importantly, edible rosemary with high endogenous protective enzyme content could efficiently biodegrade AFB1 in goat meat. We first unveiled that rosemary could not only efficiently biodegrade AFB1 up to 90.20% (20.00–1.96 μg kg−1) but also elevate the bio-ingestion quality of goat meat. These findings suggest that the bio-based material-rosemary is an efficient and environmentally friendly approach for biodegrading AFB1 and elevating the bio-ingestion composition of goat meat.

Rapid separation and characterization of the in vitro metabolites of moscatilin by ultra-high performance liquid chromatography coupled to hybrid quadrupole orbitrap tandem mass spectrometry.

Moscatilin, a bioactive ingredient isolated from Dendrobium moscatum, has been demonstrated to have excellent anti-cancer activity. The goals of the present study were to investigate the metabolic profiles of moscatilin and to identify and characterize its metabolites. In vitro studies were performed by incubating moscatilin (10 μM) with rat, dog, monkey, and human liver microsomes (0.5 mg protein/ml) to generate the metabolites. An analytical method of liquid chromatography combined with hybrid quadrupole orbitrap high-resolution mass spectrometry in full mass/data-dependent tandem mass spectrometry scan was utilized to separate and identify the metabolites in accordance with their accurate masses, formulas, and tandem mass spectrometry fragment ions determination. A total of six phase I metabolites were detected and structurally characterized. The phase I metabolic pathways of moscatilin were hydroxylation, demethylation, and dehydrogenation. In glutathione-supplemented liver microsomes, nine glutathione conjugates were detected and identified. Our results demonstrated that moscatilin was susceptible to bioactivation with the result of ortho quinone and quinone-methide intermediates. The present study provided an overview of the in vitro metabolic profiles of moscatilin, which will aid in the understanding of the efficacy and safety of this active compound.

Fructus Zanthoxyli extract improves glycolipid metabolism disorder of type 2 diabetes mellitus via activation of AMPK/PI3K/Akt pathway: Network pharmacology and experimental validation

ObjectiveThis study investigated the potential mechanisms behind the beneficial effects of Fructus Zanthoxyli (FZ) against type 2 diabetes mellitus (T2DM) based on network pharmacology and experimental validation. MethodsUltra-high-performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry, and gas chromatography-mass spectrometry were used to identify the constituents of FZ. Next, the differentially expressed genes linked to the treatment of diabetes with FZ were screened using online databases (including Gene Expression Omnibus database and Swiss Target Prediction online database), and the overlapping genes and their enrichment were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG). Finally, the pathway was verified by in vitro experiments, and cell staining with oil red and Nile red showed that the extract of FZ had a therapeutic effect on T2DM. ResultsA total of 43 components were identified from FZ, and 39 differentially expressed overlapping genes were screened as the possible targets of FZ in T2DM. The dug component-target network indicated that PPARA, PPARG, PIK3R3, JAK2 and GPR88 might be the core genes targeted by FZ in the treatment of T2DM. Interestingly, the enrichment analysis of KEGG showed that effects of FZ against T2DM were closely correlated with the adenosine monophosphate-activated protein kinase (AMPK) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signaling pathways. In vitro experiments further confirmed that FZ significantly inhibited palmitic acid-induced lipid formation in HepG2 cells. Moreover, FZ treatment was able to promote the AMPK and PI3K/Akt expressions in HepG2 cells. ConclusionNetwork pharmacology combined with experimental validation revealed that FZ extract can improve the glycolipid metabolism disorder of T2DM via activation of the AMPK/PI3K/Akt pathway.

Identifying quality markers of Mailuoshutong pill against thromboangiitis obliterans based on chinmedomics strategy

BackgroundMailuoshutong pill (MLSTP) is a traditional Chinese medicine (TCM) for the treatment of Thromboangiitis obliterans (TAO, Buerger's disease) which is a segmental non-atherosclerotic inflammatory occlusive disorder. However, the mechanism and quality standards of MLSTP have not been sufficiently studied. PurposeThis work aims to investigate the potential mechanisms and quality markers (Q-markers) of MLSTP treating TAO based on the chinmedomics strategy. MethodsThe therapeutical effect of MLSTP on TAO rats was evaluated by changes in body weight and clinical score, regional blood flow velocity and perfused blood vessel distribution, hematoxylin-eosin (H&E) staining, serum metabolic profile. Moreover, both endogenous metabolites and exogenous components were simultaneously detected in serum based on ultra-high performance liquid chromatography coupled with a Q Exactive hybrid quadrupole-orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS), and multivariate analysis was applied to identify the biomarkers, as well as the dynamic changes of metabolites were observed to explore the mechanism of action of MLSTP. In addition, the pharmacodynamic material basis were identified by correlation analysis between biomarkers and absorbed constituents. Finally, the Q-markers of MLSTP were determined according to the screening principles of Q-marker and validated the measurability. ResultsMLSTP treatment alleviated disease severity of TAO, reduced inflammatory infiltration, and ameliorated vascular function. 26 potential biomarkers associated with glutamate metabolism, linoleic acid metabolism, arachidonic acid metabolism and so on were identified. Besides, 27 prototypical components were identified in serum, 16 of which were highly correlated with efficacy and could serve as the pharmacodynamic material basis of MLSTP against TAO. In addition, 7 compounds, namely, sweroside, chlorogenic acid, calycosin-7-glucoside, formononetin, paeoniflorin, liquiritigenin and 3-butylidenephthalide, were considered as potential Q-markers of MLSTP. Ultimately, the measurability of the seven Q-markers was validated by rapid identifcation and quantifcation. ConclusionThis study successfully clarified the therapeutic effect and Q-markers of MLSTP by chinmedomics strategy, which is of great significance for the establishment of quality standards. Furthermore, it provides a certain reference for the screening of Q-markers in TCM prescriptions.

A strategy integrating parent ions list-modified mass defect filtering-diagnostic product ions for rapid screening and systematic characterization of flavonoids in Scutellaria barbata using hybrid quadrupole-orbitrap high-resolution mass spectrometry

In this study, full scan (FS)-parent ions list (PIL)-higher energy collision induced dissociation (HCD)-MS/MS (FS-PIL-HCD-MS/MS) was used to acquire the chemical profile of flavonoids in Scutellaria barbata. Mass defect filtering (MDF) induced subtype classification and diagnostic product ions (DPIs) dominated structural confirmation were integrated into an effective strategy for the systematic screening and identification of the flavonoids. An in-house flavonoid MS database based on molecular design was established to construct a modified triangle MDF algorithm for progressive screening and subtype classification. The obtained results demonstrated that the modified MDF was capable of simplifying the workload in formula editing and subsequent screening process, and distinguishing different subtypes. The fragmentation behaviors of eleven reference standards were evaluated to obtain the MS2 fragmentation pathway and DPIs which can provide a criterion to eliminate false-positive results and judge the target flavonoids with the exact number and position of substituents for the first time. Structure confirmation was characterized by comparing with the reference substance, searching the database, and analyzing DPIs. To distinguish some isomers, ClogP (the calculated lipophilicity parameter) was adopted. As a result, 127 target flavonoids, including 30 flavone/flavonol aglycones, 10 flavanone/flavanonol aglycones, 49 flavone/flavonol monoglycosides, 16 flavanone/flavanonol monoglycosides, 21 flavone/flavonol diglycosides and 1 flavanone/flavanonol diglycoside, were ultimately identified or tentatively characterized based on the MS fragmentation pathway and DPIs analysis. This study provides a novel MDF method with improved subtype classification and develops a novel strategy for the progressive screening, subtype classification and systematic characterization of complex components in herbal medicines.

Chemical constituents, anti-hyperuricemic and anti-gouty arthritis activities of extract of Herpetospermum caudigerum

In this paper, we studied the anti-hyperuricemia and anti-gouty arthritis effects of the ethyl acetate extract of Herpetospermum caudigerum (EH), and applied ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) technology to analyze and identify the chemical components of EH. EH can dramatically lower serum uric acid (UA) levels in the hyperuricemia mice model induced by potassium oxazine (PO). In addition, in the gouty arthritis rats model established by sodium urate, the ankle swelling degree, serum uric acid (UA), IL-1β, and TNF-α related inflammatory cytokines all decreased by treating with EH. Consequently, EH appears to have anti-hyperuricemia and anti-gouty arthritis activities, making it a viable option for anti-gout drugs without hepatic and renal side effects, and its effective substances of anti-gout can be further illustrated by UPLC-Q-Orbitrap HRMS analysis of 48 compounds.

Bidesmosidic Triterpenoid Saponins from Wild and Cultivated Gypsophila Paniculata L. (Caryophyllaceae)

Glucuronide oleanane-type triterpenoid carboxylic acid 3, 28-bidesmosides (GOTCAB) of wild and in vivo cultivated Gypsophila paniculata L. roots were assayed by ultra high-performance liquid chromatography coupled with hybrid quadrupole-Orbitrap high resolution mass spectrometry (UHPLCHRMS). More than 20 gypsogenin- and quillaic acid-based GOTCAB saponins were tentatively annotated including isobaric isomers. They are substituted at C-3 with a common 3-O-β-d-galactopyranosyl-(1→2)-[pentosyl-(1→3)]-βd-glucuronopyranoside. C-28 ester-bonded chain is mono- and diacetylated tetra- and pentasaccharide or possesses a methoxycinnamoyl residue. Nine core GOTACBs have not been previously reported in native G. paniculata. The structure of the major saponin of the wild G. paniculata roots was established by 1D and 2D NMR experiments as 3-O-β-d-galactopyranosyl-(1→2)-[β-d-xylopyranosyl-(1→3)]-β-d-glucuronopyranosyl quillaic acid 28-O − α-larabinopyranosyl-(1→4)-α-l-arabinopyranosyl-(1→3)-β-d-xylopyranosyl- (1→4)-α-l-rhamnopyranosyl-(1→2)-β-d-fucopyranosyl ester. For the first time the saponin was isolated from the G. paniculata roots.

Rapid and comprehensive identification of chemical constituents in Mai-Luo-Shu-Tong pill by UHPLC-Q-Orbitrap HRMS combined with a data mining strategy.

Mai-Luo-Shu-Tong pill is an effective traditional Chinese medicine formula for the treatment of superficial thrombophlebitis, but it was insufficiently chemically scrutinized. In this study, the mass spectral data of Mai-Luo-Shu-Tong pill were acquired by ultra-high performance liquid chromatography coupled with Q Exactive hybrid Quadrupole-Orbitrap high resolution mass spectrometry. Then, a data mining strategy combining multiple data processing methods was used to identify chemical constituents in Mai-Luo-Shu-Tong pill by constructing a database of precursor ions and summarizing the mass spectral fragmentation behaviors. As a result, a total of 211 compounds including 70 flavonoids, 56 terpenoids, 37 phenolic acids and 48 others were identified in positive and negative ion modes. Among them, 66 compounds have passed comparison verification with reference standards, 145 compounds were identified based on the data mining strategy combining the characteristic cleavage behaviour of homologous compounds and fragment ions and 4 compounds were potentially new compounds. This study provides a database for quality evaluation and further study of Mai-Luo-Shu-Tong pill in vivo. Moreover, it provides a reference for the characterization of the chemical constituents of other traditional Chinese medicine formulae.

Identification of the Metabolites of Scutebarbatine A in Rat Plasma, Bile, Urine, and feces by Using Ultra-high-performance Liquid Chromatography Coupled with Q Exactive Hybrid Quadrupole-orbitrap High-resolution Mass Spectrometry.

Scutebarbatine A is a new neo-Clerodane Diterpenoid Alkaloids from Scutellaria barbata, which has many pharmacological effects, such as anti-tumor, antibacterial, and anti-inflammatory. However, there are no studies on the metabolism of Scutebarbatine A. The objective of this study is to explore the metabolism of Scutebarbatine A in the body, the bile, plasma, urine, and feces samples of rats after administration were investigated. The biological samples were investigated using ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole-orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap-HRMS). A total of 20 metabolites were identified: 16 phase I metabolites and 4 phase II metabolites. The main metabolic pathways were hydrolysis, oxidation, hydrogenation, dehydration, and combination with sulfate. This study further elucidates the metabolism of Scutebarbatine A in rats and provides a reference for the study of its pharmacodynamic material basis and pharmacological mechanism.

Metabolic study of cafestol using in silico approach, zebrafish water tank experiments and liquid chromatography high-resolution mass spectrometry analyses

Coffee is one of the most consumed beverages worldwide. Cafestol is an endogenous coffee diterpene present in raw coffee beans and also found in hot beverages, with several biological activities. However, there is still little information on this molecule after ingestion of coffee infusion.Zebrafish (Danio rerio) is a promising in vivo model for metabolic studies due to the annotation of mammalian orthologs to encode enzymes related to drug metabolism. Experiments using Zebrafish Water Tank (ZWT) model produce more significant number of metabolites for molecular investigation in a cleaner matrix than other classical models, such as purified hepatocytes.This work aimed to investigate the biotransformation of cafestol by the ZWT model using ultra-performance liquid chromatography coupled to hybrid quadrupole-orbitrap high-resolution mass spectrometry equipped with electrospray ionization (UPLC-HRMS) supported by in silico approach using SMARTCyp, Way2Drug and XenoSite Softwares.Twenty-five metabolites of cafestol were proposed by in silico analysis, in which 5 phase I metabolites were confirmed in the ZWT by UPLC and MS/HRMS investigation: 6-hydroxy-cafestol, 6,12-dihydroxy-cafestol, 2-oxo-cafestol, 6-oxo-cafestol and one isomer whose position in the carboxyl group was not determined. These metabolites were observed during 9 h of the experiment, whose contents were associated with the behavioral responses of the fish.

Protein Biomarkers of Bovine Defective Meats at a Glance: Gel-Free Hybrid Quadrupole-Orbitrap Analysis for Rapid Screening.

An understanding of biological mechanisms that could be involved in the stress response of animal cattle prior to slaughter is critical to create effective strategies aiming at the production of high-quality meat. The sarcoplasmic proteome of directly extracted samples from normal and high ultimate pH (pHu) meat groups was studied through a straightforward gel-free strategy supported by liquid chromatography hybrid quadrupole-Orbitrap high-resolution mass spectrometry (LC-HRMS) analysis. A stepped proteomic pipeline combining rapid biomarker hunting supported by qualitative protein Mascot scores followed by targeted label-free peptide quantification revealed 26 descriptors that characterized meat groups assayed. The functional study of the proposed biomarkers suggested their relevant role in metabolic, chaperone/stress-related, muscle contractility/fiber organization, and transport activities. The efficiency, flexibility, rapidity, and easiness of the methodology proposed can positively contribute to the creation of innovative proteomic alternatives addressing meat quality assessment.

Analysis of the characteristic compounds of Citri Sarcodactylis Fructus from different geographical origins.

Citri Sarcodactylis Fructus (CSF) is widely used as a food ingredient and a traditional Chinese medicine. In China, CSF is cultivated in many places, including Sichuan, Guangdong, Zhejiang, and Fujian provinces. The types and chemical contents of CSF from different origins may vary greatly due to the difference in climate and environmental conditions. Therefore, comparing the chemical composition of CSF from various places is vital. To rapidly select potential characteristic compounds for differentiating CSF from different origins. Thirty-one batches of CSF samples from different regions were analysed using ultra-performance liquid chromatography with hybrid quadrupole-orbitrap high-resolution mass spectrometry. Thereafter, chemometric methods, including principal component analysis (PCA) and orthogonal partial least squares discrimination analysis (OPLS-DA), were employed to find differential metabolites among the CSF samples from various origins. PCA revealed 77.9% of the total variance and divided all CSF samples into three categories corresponding to their origins. OPLS-DA displayed better discrimination of CSF from different sources, with R2 X, R2 Y, and Q2 of 0.801, 0.985, and 0.849, respectively. Finally, 203 differential metabolites were obtained from CSF from different origins using the variable importance in projection of the OPLS-DA model, 30 of which were identified, and five coumarin compounds were selected as marker compounds discriminating CSF from different origins. This work provides a practical strategy for classifying CSF from different origins and offers a research foundation for the quality control of CSF.

First insight on in vitro metabolism of three newly identified aryl organophosphate esters via a suspect coupled with nontarget screening approach

The ubiquity of organophosphate esters (OPEs) in the environment has triggered research into metabolic pathways of OPEs. Using liquid chromatography coupled with a hybrid quadrupole Orbitrap high-resolution mass spectrometer, a suspect and characteristic fragment ion-based nontarget screening strategy for the identification of unknown OPE metabolites was developed and evaluated. Then, this integrated approach was successfully used for investigation of three newly identified organophosphate esters (NOPEs), namely 2-biphenylol diphenyl phosphate (BPDPP), tris(2-biphenyl) phosphate (TBPHP), and naphthalen-2-yl diphenyl phosphate (NDPHP), in human liver microsomes (HLMs). The results demonstrated that BPDPP, TBPHP, and NDPHP were effectively metabolized by HLMs, with zero-order kinetics (R2 = 0.48−0.94) within the time frame of the assay. The suspected approach identified a considerable number of dearylated phosphate (DP), and hydroxylated metabolites for each of NOPEs after incubation with HLMs for 2 h. In addition, the nontarget approach further identified 9 novel metabolites including 2 epoxide intermediates and 7 oxidative ring-opening compounds, which were first reported in the Phase I metabolism of OPEs. Collectively, this study provided a novel suspect coupled with nontarget screening approach and was successfully used to screen metabolites of three NOPEs. For the first time, we observed direct evidence that oxidative ring-opening might serve as another primary metabolic pathway regarding the metabolism of aryl OPEs.

An Entire Process Optimization Strategy for Comprehensive In Vivo Metabolite Profiling of Prucalopride in Rats Based on Ultra-Performance Liquid Chromatography With Q-Exactive Hybrid Quadrupole-Orbitrap High-Resolution Mass Spectrometry.

Prucalopride was widely used for chronic constipation, which is difficult to be adequately relieved by laxatives in adult patients in clinic. Due to the difficulty in metabolite identification, metabolic process of prucalopride had not been investigated in vivo. In this study, an efficient strategy was proposed for comprehensive metabolite profiling of prucalopride after oral administration in rat plasma, urine, and feces samples. This strategy was composed of five steps. First, the samples at multiple time points after oral administration were collected to increase the representativeness of the samples. Second, different sample preparation methods were investigated to obtain superior extraction efficiency. Third, the raw data of test sample and blank sample were acquired using ultra-performance liquid chromatography with Q-Exactive hybrid quadrupole–orbitrap high-resolution accurate mass spectrometry under the positive and negative full-scan/dd MS2 mode. Fourth, combined mass defect filter with background subtraction model in soft of compound discovery, all peaks were constructed to filter potential metabolites after retention time alignment and ion filtration, which could remove large amounts of interference ions. Besides, it can predict potential biotransformation, promoting to understand how to metabolize the drug. This provides multiple possibilities and prevents us conjecturing the potential metabolites blindly. Finally, the verification procedure was implemented through exporting the structure and MS2 spectrum to the analytical tool of Mass Frontier. The proposed strategy significantly improved the targeted detection and identification for metabolites in vivo. A total of 47 metabolites were tentatively characterized in the plasma, urine, and feces samples after oral administration of prucalopride. This study could provide a valuable reference for systematic metabolite profile of drug in vivo.

Establishment of a Target, Suspect, and Functional Group-Dependent Screening Strategy for Organophosphate Esters (OPEs): "Into the Unknown" of OPEs in the Sediment of Taihu Lake, China.

Current environmental monitoring studies are generally confined to several target organophosphate esters (OPEs), and there is a lack of strategies for comprehensively screening all potential OPEs in environmental samples. Here, an effective and accurate strategy was developed for the target, suspect, and functional group-dependent screening of OPEs by the use of ultrahigh-performance liquid chromatography-Q Exactive hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS), and this strategy was applied for the analysis of n = 74 sediment samples (including 23 surface sediment samples and 51 sediment core samples) collected from Taihu Lake (eastern China) in 2019. In these analyzed samples, we successfully identified n = 35 OPEs, and 23 of them were reported in this region for the first time. In addition, this strategy also presented other interesting findings, i.e., (1) OPE concentrations decreased with increasing distance from the coast of the lake; (2) the newly identified 3-hydroxyphenyl diphenyl phosphate (meta-OH-TPHP) was not statistically significantly correlated with triphenyl phosphate (TPHP; r = 0.02494, p = 0.9101) but with resorcinol bis(diphenyl phosphate) (RDP) (r = 0.9271, p < 0.0001) and three other OPEs; and (3) the summed concentrations of aryl OPEs (∑arylOPEs) in sediment core samples exhibited significantly increasing trends as the depth decreased. Collectively, this study provided an effective strategy that was successfully applied for comprehensive screening of OPEs in the sediments of Taihu Lake, and this strategy could have promising potential to be extended to other environmental matrices or samples.