Hybrid Quadrupole-orbitrap Mass Spectrometry Research Articles

Integrated UHPLC-Q-exactive-orbitrap MS/MS and ABTS analyses screen antioxidant and anti‑counterfeiting marker of Eucommiae Cortex (Duzhong) for Pharmacopoeias

Eucommiae Cortex (Duzhong) has been collected in several Pharmacopoeias. However, its Pharmacopoeia quality-marker is outdated and unreasonable. Therefore, the study used database-aided ultra-high-performance liquid chromatography coupled with hybrid quadrupole-orbitrap tandem mass spectrometry (UHPLC-Q-Exactive-Orbitrap MS/MS) analysis to putatively identify 33 compounds from Eucommiae Cortex. Especially, 14 “unexcavated” compounds were detected in Eucommiae Cortex for the first time; Meanwhile, all monocaffeoylquinic acid isomers (i.e., 1-O-, 3-O-, 4-O-, and 5-O- caffeoylquinic acids) have been strictly distinguished from each other, and three dicaffeoylquinic acid isomers (1,3-O-, 3,5-O-, and 4,5-O- dicaffeoylquinic acids) have been distinguished as well. All identified compounds were further subjected to a semi-quantification analysis, through which cholesteryl acetate (4.11 ± 0.46 %), sucrose (3.53 ± 0.063 %), and (−)-syringaresinol (2.81 ± 1.31 %) were found to be highly abundant. Furthermore, the semi-quantification results were integrated with ABTS+• antioxidant assay to individually calculate the antioxidant contribution of all compounds. (−)-Syringaresinol, neochlorogenic acid, vanillic acid, chlorogenic acid, and (−) pinoresinol were proven to be the top five antioxidant contributors, and their contribution values were 97.50, 46.31, 40.57, 35.05, and 30.90. Of the top contributors, chlorogenic acid was proposed as an additional pharmacopoeia Q-marker for its good testability and traceability. The additional Q-marker not only enhances the pharmacological relevance, but also effectively recognizes Eucommiae Cortex from its counterfeiting via liquid chromatography analysis. Thus, these findings will help Pharmacopoeias to update its Q-markers.

Comparative study on chemical constituents of different medicinal parts of Lonicera japonica Thunb. Based on LC-MS combined with multivariate statistical analysis

Lonicerae japonicae flos (LJF), Lonicerae japonicae caulis (LJC), Lonicerae folium (LF) and Lonicerae fructus (LFR) are derived from Lonicera japonica Thunb., which are formed due to different medicinal parts. The efficacy of the 4 medicinal materials has similarities and differences. However, little attention has been paid to illustrate the differences in efficacy from the perspective of phytochemistry. In this study, ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry (UPLC-Q-Exactive-Orbitrap-MS) was used to qualitatively analyze the ingredients in 4 herbs. A total of 86 compounds were plausibly or unambiguously identified, there were 54 common components among the 4 medicinal materials, and each kind of medicinal materials had its own unique components. On the basis of qualitative analysis, ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UPLC-QQQ-MS/MS) was used to quantitatively analyze 31 components contained in 4 medicinal materials, and principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and other multivariate statistical analysis were furtherly performed for comparing the component contents. The results showed that the samples from the same parts were clustered into one group, and the samples from different medicinal parts were significantly different. The analysis of variable importance projection (VIP) value of the OPLS-DA model showed that 10 components including chlorogenic acid, secologanic acid, isochlorogenic acid A, loganin, lonicerin, loganic acid, secoxyloganin, sweroside, luteolin and rhoifolin were the main difference components among the 4 medicinal materials. The study not only lays a solid foundation for the intrinsic quality control of 4 medicinal materials and the study of different effects of the 4 medicinal materials at the phytochemical level, but also provides a basis for more rational utilization of various parts of L. japonica and expansion of medicinal resources.

Exploring the gut microbiota mediated biotransformation of Senecio scandens Buch.-Ham.: Insights from metabolite spectrum with UHPLC-Q-Orbitrap HRMS and bioinformatics analysis of gut microbiota metabolites

Senecio scandens Buch.-Ham., a traditional Chinese medicine commonly used clinically, exhibits various pharmacological properties, including anti-inflammatory, anti-tumor, antiviral, and antibacterial activities. However, its water extracts' chemical components and metabolites are inadequately understood, limiting further research. In this study, the chemical components and metabolism processes of Senecio scandens, both in vivo (plasma, feces, urine, and bile) and in vitro (gut microbiota and liver microsomes), were characterized based on ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry. Additionally, metabolites detectable in fecal samples and intestinal microbiota incubated but absent in liver microsomes were identified as characteristic metabolites of intestinal microbiota. The targets of the characteristic metabolites of intestinal microbiota were collected, followed by exploration of potential pathways through KEGG enrichment analysis. As a result, a total of 133 chemical components were preliminarily identified, including 35 organic acids, 21 alkaloids, 19 flavonoids and their glycosides, 17 phenylpropanoids, 10 jacaranda ketones, and 31 other compounds. Notably, 12 of these were potentially novel compounds. In addition, 39 prototype components in rats and 109 metabolites were identified and characterized, including 102 in vivo and 52 metabolites in vitro (51 in rat gut microbiota and 24 in rat liver microsomes). The main metabolic pathways include oxidation, reduction, hydrolysis, methylation, glucuronidation, sulfonation, and acetylation reactions. Furthermore, KEGG enrichment analysis revealed that the characteristic metabolites of intestinal microbiota may be related to the ErbB, FoxO, mTOR, and MAPK signaling pathways, exhibiting anti-inflammatory and anti-tumor effects. In summary, the chemical components and metabolites of Senecio scandens were comprehensively identified using a rapid and accurate method, providing a scientific basis for the in-depth study of the material basis and its clinical application of Senecio scandens.

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.

Identification and Characterization of the Chemical Constituents of Qianlie Shule Capsules by UPLC-Q-Orbitrap-MS/MS.

The Qianlie Shule capsule is a classical Chinese medicine compound preparation frequently used in therapeutic settings to alleviate astringent pain in the urethra, prostatic hypertrophy, and chronic prostatitis or urinary frequency. However, a comprehensive analysis of the chemical composition of Qianlie Shule capsules has not been reported. To establish a quick and effective analytical method based on hybrid quadrupole-orbitrap mass spectrometry ultrahigh-performance liquid chromatography (UPLC-Q-Orbitrap-MS/MS) for the identification and characterization of chemical components in Qianlie Shule capsules. Using ultrahigh-performance liquid chromatography with hybrid quadrupole-orbitrap mass spectrometry and data post-processing, the samples of Qianlie Shule capsules were examined. First, the whole extract of the Qianlie Shule capsules was separated using a UPLC machine, and the fragmentation data were collected in both positive and negative ion mode. The target molecule is then quickly identified by comparing the fragmentation information of the neutral loss (NLs) and characteristic fragments (CFs) reported in the literature. A total of 145 chemical components were identified. It includes flavonoids, triterpenoids, phenylpropanoids, organic acids, alkaloids, phenylethanoids, iridoids, and anthraquinones. This study is a method for the rapid qualitative analysis of the chemical composition of Qianlie Shule capsules, which provides a method for the rapid, sensitive, and high-throughput identification of the prescription components of Chinese medicine. Systematic identification of the chemical composition of QLSL capsules provides a theoretical basis for studying the substance basis of QLSL capsules and the improvement of the quality control level.

Decoction regulating phytochemicals’ micromorphology changes and anti-inflammation activity enhancements originated from herb medicine supermolecules

BackgroundMahuang Fuzi decoction (MGF) is composed of three herb medicines that has been clinically used to treat inflammatory diseases for a long history. At present, more and more active phytochemicals’ aggregations have been found during the thermodynamic process of herb medicine decoction, and revealing the clinical efficacy of herb medicine through supramolecular strategies is the focus of current research. However, it is not clear whether decoction induced supermolecules’ morphological changes to modify activity.MethodsDynamic light scattering (DLS) and field emission scanning electron microscopy (FESEM) were used to analyze the micromorphology of MGF, MGF SA (MGF supermolecules), and MIX (physical mixture of MGF single decoction). The interaction and thermodynamic parameters of single herbs in a decoction were investigated by Isothermal titration calorimetry (ITC). The phytochemicals were systematically analyzed by ultra high performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry (UHPLC-Q-Orbitrap HRMS). Under the safe dose on RAW264.7 cells, NO, IL-6 and TNF-α were determined by Enzyme-Linked ImmunoSorbent Assay (ELISA) method. NF-κB p65 translocation from the cytoplasm into the nucleus was examined using the immunofluorescence assay and the western blot, respectively. Furthermore, Metabolomics was used to discover potential biomarkers and the associated metabolic pathways of MGF SA treatment.ResultsThere were nanoscale aggregations in MGF, and the micromorphology of the extracted MGF SA consisted of uniform particles; while the MIX micromorphology had no uniformity. ITC showed that the interaction MH-GC and FZ-GC were a spontaneous exothermic reaction, indicating that their phytochemicals had the property of self-assembly. Though the micromorphology between MGF, MGF SA, and MIX was obviously different, UHPLC-Q-Orbitrap HRMS results displayed that the main phytochemicals of MGF and MIX had nearly the same components. Interestingly, MGF and MGF SA could significantly inhibit the production of NO, and had better inhibition effect on the expression of nuclear protein NF-κB p65 than MIX, among which MGF SA had the best effect. Further investigation indicated that the perturbance of metabolic profiling in RAW264.7 inflammatory cells was obviously reversed by MGF SA.ConclusionsThe decoction enriched the key active phytochemicals and regulated the formation of homogeneous nanoparticles in MGF SA. The supermolecules in MGF SA significantly enhanced its anti-inflammatory activity, primarily affecting the NF-κB signaling pathway and the biosynthesis and metabolism of arginine in RAW264.7 inflammatory cells. Current study displayed that co-decocting herbal medicine were beneficial to the treatment of diseases than the mixture of the single herbs’ extraction.Graphical

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.

Comparative analysis of the metabolites and biotransformation pathways of fentanyl in the liver and brain of zebrafish.

The rise of fentanyl has introduced significant new challenges to public health. To improve the examination and identification of biological samples in cases of fentanyl misuse and fatalities, this study utilized a zebrafish animal model to conduct a comparative investigation of the metabolites and biotransformation pathways of fentanyl in the zebrafish's liver and brain. A total of 17 fentanyl metabolites were identified in the positive ion mode using ultra-high-pressure liquid chromatography Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometry (UHPLC-QE HF MS). Specifically, the zebrafish's liver revealed 16 fentanyl metabolites, including 6 phase I metabolites and 10 phase II metabolites. Conversely, the zebrafish's brain presented fewer metabolites, with only 8 detected, comprising 6 phase I metabolites and 2 phase II metabolites. Notably, M'4, a metabolite of dihydroxylation, was found exclusively in the brain, not in the liver. Through our research, we have identified two specific metabolites, M9-a (monohydroxylation followed by glucuronidation) and M3-c (monohydroxylation, precursor of M9-a), as potential markers of fentanyl toxicity within the liver. Furthermore, we propose that the metabolites M1 (normetabolite) and M3-b (monohydroxylation) may serve as indicators of fentanyl metabolism within the brain. These findings suggest potential strategies for extending the detection window and enhancing the efficiency of fentanyl detection, and provide valuable insights that can be referenced in metabolic studies of other new psychoactive substances.

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.

Tsantan Sumtang, a traditional Tibetan medicine, protects pulmonary vascular endothelial function of hypoxia-induced pulmonary hypertension rats through AKT/eNOS signaling pathway

Ethnopharmacological relevanceTsantan Sumtang (TS), originated from the Four Tantras, is an empirical Tibetan medicine prescription, which has been widely used for treating cardiovascular diseases in the clinic in Qinghai Province of China. Our previous studies found that TS alleviated hypoxia-induced pulmonary hypertension (HPH) in rats. However, the effect and bioactive fractions of TS on hypoxia-injured pulmonary vascular endothelium are unknown. Aim of the studyTo investigate the effect, bioactive fractions and pharmacological mechanism of TS on hypoxia-injured pulmonary vascular endothelium in vivo and in vitro. Materials and methodsIn vivo studies, HPH animal model was established, and TS was administrated for four weeks. Then, hemodynamic indexes, ex vivo pulmonary artery perfusion experiment, morphological characteristics, nitric oxide (NO) production, and the protein expression of protein kinase B (AKT)/endothelial nitric oxide synthase (eNOS) and AMP-activated protein kinase (AMPK)/eNOS signaling were determined. In vitro studies, 1% O2-induced pulmonary artery endothelial cells (PAECs) injury model was applied for screening bioactive fractions of TS by cell proliferation assay and NO production measurement. The associated proteins of AKT/eNOS signaling were further measured to elucidate underlying mechanism of bioactive fraction of TS via using phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002. Ultra-high performance liquid chromatography with hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS) was used to reveal the chemical profile of bioactive fraction of TS. ResultsTS showed protective effect on the integrity of distal pulmonary arterial endothelium in HPH rats. Tsantan Sumtang dilated pulmonary arterial rings in HPH rats. TS enhanced NO bioavailability in lung tissue via regulating AKT/eNOS signaling. Furthermore, in the cellular level, cell viability as well as NO content of hypoxia-injured PAECs were elevated by fraction 17 of water extract of TS (WTS), through activating the AKT/eNOS signaling. Ellagic acid could be one of compositions in fraction 17 of WTS to produce NO in hypoxia-injured PAECs. ConclusionTS restored pulmonary arterial endothelial function in HPH rats. The bioactive fraction 17 was screened, which protected hypoxia-injured PAECs via upregulating AKT/eNOS signaling.

Integrated Network Pharmacology and In-silico Approaches to Decipher the Pharmacological Mechanism of Dioscorea septemloba Thunb in Treating Gout and Its Complications

Background: Dioscorea septemloba Thunb. (DST) has demonstrated therapeutic potential in the treatment of gout and its associated complications. However, the underlying mechanisms of DST’s pharmacological activity remain unclear. This study aims to investigate the pharmacological substances and network regulatory mechanisms of DST in treating gout and its complications using network pharmacology. Methods: According to ultra-high performance liquid chromatography coupled with hybrid quadrupole-Orbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap-MS) data and Lipinski’s rule of five, 24 bioactive phytochemicals from DST were identified. The targets of gout were retrieved from Gene Expression Omnibus (GEO), GeneCards, and DisGeNET databases, followed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG pathway) enrichment analysis. The Cytoscape network analysis was used to identify the primary pathological pathways and key targets. Finally, LeDock was used for molecular docking to verify the active components of DST and their core target proteins. Results: DST contains several core active ingredients, such as tetrahydroimidazo[1,2-a]pyridine- 2,5-dione, diosgenin, beta-sitosterol, dioscorol B, montroumarin and 9,10-dihydro-5,7- dimethoxy-3,4-phenanthrenediol. Moreover, these active components were found to strongly bind to the key targets for treating gout and its complications, including HSP90AA1, STAT3, PTGS2, PPARG, MTOR, HIF1A, MMP9, ESR1, and TLR4. As a result, DST alleviates gout and its complications by inhibiting xanthine dehydrogenase (XDH) to reduce uric acid levels and regulating the HIF-1α, EZH2/STAT3, and COX-2/PPAR-γ pathways to reduce inflammation. Additionally, it also plays an analgesic role by regulating the neuroactive ligand-receptor interaction pathway and calcium ion signaling pathway. Conclusion: This study has provided insights into the underlying mechanisms of DST in the treatment of gout and its complications, which could serve as a scientific foundation for its clinical translation.

Comprehensive quality control of Qingjin Yiqi granule based on UHPLC-Q-Orbitrap-MS and UPLC-QQQ-MS.

Qingjin Yiqi granule (QYG) is a prescription medicine of traditional Chinese medicine which is widely used clinically for the recovery of coronavirus patients. However, there is currently limited research on the quality control of QYG. To evaluate the quality of QYG qualitatively and quantitatively by making full use of advanced chromatography-mass spectrometry techniques. Firstly, a multicomponent characterisation of QYG was performed by ultrahigh-performance liquid chromatography coupled with a Q Exactive™ hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) system using a rapid negative/positive switching mode. Secondly, the co-condition fingerprint analysis of constituted herbal medicines of QYG was performed to unveil active ingredients as the quality markers of QYG. Thirdly, the marker compounds in 10 batches of QYG were quantified by ultrahigh-performance liquid chromatography coupled with a Waters Xevo TQ-S triple quadrupole mass spectrometry (UPLC-QQQ-MS) system. A comprehensive method that combined the inclusion list and data-dependent acquisition (DDA) to achieve a systematic characterisation of QYG was established by UHPLC-Q-Orbitrap-MS. After analysis based on Compound Discoverer software and Global Natural Products Social (GNPS) platform, a total of 332 compounds were detected. Eleven Q-markers were determined for the quality evaluation of QYG by comparison with the fingerprint of nine constituted herbal medicines. An adjusted multiple reaction monitoring (MRM) quantification method was further established to simultaneously determine the 11 Q-markers for holistic quality evaluation of QYG. This is the first study to report comprehensive multicomponent characterisation, identification, and quality assessment of QYG, which could be used for effective guarantee of the quality of QYG.

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.

New UHPLC-Q-Orbitrap MS/MS-based Library-comparison method simultaneously distinguishes 22 phytophenol isomers from Desmodium styracifolium

Isomers distinction has become a bottleneck of the emerging ultra-high-performance liquid chromatography coupled with hybrid quadrupole-orbitrap tandem mass spectrometry (UHPLC-Q-orbitrap MS/MS) technology. The present study thus used Desmodium styracifolium as model plant to establish a new library-comparison method for isomers distinction. The method firstly established a data library comprising MS/MS characteristic fragment, MS/MS profile, and retention time (R.T.) value. Through subsequent comparisons, 22 phytophenol isomers in D. styracifolium have been simultaneously distinguished under MS spectra negative model. The results suggested that: (1) there were four main types of isomers, i.e., positional isomers, functional group isomers, skeleton isomers, and hybridized isomers. (2) The hybridized isomers (e.g., luteoloside and astragalin) could be easily distinguished, while positional isomers (e.g., schaftoside and isoschaftoside) were difficultly distinguished from each other. (3) The R.T. value comparison played a key role for distinction of highly similar isomers, especially positional isomers. On the basis of above results and computational chemistry, it is concluded that, the new UHPLC-Q-orbitrap MS/MS-based library-comparison method can be capable for isomers distinction and thus has developed the UHPLC-Q-orbitrap MS/MS technology. The development will benefit many fields, including food, herbal medicine, and analysis chemistry. Particularly, the data of 22 phytophenol isomers can be directly cited for their isomersdistinction in future by other researchers.

UHPLC-Quadrupole-Exactive-Orbitrap-MS/MS-based putative identification for Eucommiae Folium (Duzhongye) and its quality-marker candidate for pharmacopeia.

Eucommiae Folium (Duzhongye) is a traditional Chinese medicine with a long history of use in China. However, its quality-marker (Q-marker) in Chinese Pharmacopoeia is poorly defined nowadays. The study therefore conducted an ultra-high-performance liquid chromatography coupled with hybrid quadrupole-orbitrap tandem mass spectrometry (UHPLC-Quadrupole-Exactive-Orbitrap MS/MS) analysis to obtain accurate data. The obtained data were then compared with authentic standards library using Xcalibur 4.1 software package and TraceFinder General Quan. Through the comparison, the study has putatively identified 26 bioactive compounds, which include 17 flavonoid derivatives (catechin, quercetin 3-gentiobioside, quercetin 3-O-β-D-glucose-7-O-β-D-gentiobioside, taxifolin, myricetin 3-O-galactoside, myricitrin, hyperoside, rutin, isoquercitrin, quercetin 3-O-β-xylopyranoside, quercitrin, isorhamnetin 3-O-β-D-glucoside, quercetin, kaempferol, S-eriodictyol, S-naringenin, and phloridzin), four caffeoylquinic acids (neochlorogenic acid, chlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C), two alkaloids (vincamine and jervine), one lignan (pinoresinol), one xanthone (cowaxanthone B), and one steroid (cholesteryl acetate). Of these, flavonoid isoquercitrin is recommended as the new and additional pharmacopoeia Q-marker candidate, which can not only overcome the unreliability of old Q-marker but also recognize the possible counterfeit. This article is protected by copyright. All rights reserved.

Fermented Astragalus and its metabolites regulate inflammatory status and gut microbiota to repair intestinal barrier damage in dextran sulfate sodium-induced ulcerative colitis

Fermentation represents an efficient biotechnological approach to increase the nutritional and functional potential of traditional Chinese medicine. In this study, Lactobacillus plantarum was used to ferment traditional Chinese medicine Astragalus, the differential metabolites in the fermented Astragalus (FA) were identified by ultra-performance liquid chromatography-Q Exactive hybrid quadrupole-Orbitrap mass spectrometry (UPLC-Q-Exactive-MS), and the ameliorating effect of FA on dextran sulfate sodium (DSS)-induced colitis in mice were further explored. The results showed that 11 differential metabolites such as raffinose, progesterone and uridine were identified in FA, which may help improve the ability of FA to alleviate colitis. Prophylactic FA supplementation effectively improved DAI score, colon length and histopathological lesion in DSS-treated mice. The abnormal activation of the intestinal immune barrier in mice was controlled after FA supplementation, the contents of myeloperoxidase (MPO) and IgE were reduced and the contents of IgA were increased. The intestinal pro-inflammatory factors TNF-α, IL-1β, IL-6, and IL-17 were down-regulated and the anti-inflammatory factors IL-10 and TGF-β were up-regulated, suggesting that FA can intervene in inflammatory status by regulating the balance of Th1/Th2/Th17/Treg related cytokines. In addition, FA supplementation modified the structure of the intestinal microbiota and enriched the abundance of Akkermansia and Alistipes, which were positively associated with the production of short-chain fatty acids. These microbes and their metabolites induced by FA also be involved in maintaining the intestinal mucosal barrier integrity by affecting mucosal immunity. We observed that intestinal tight junction protein and mucous secreting protein ZO-1, occludin, and MUC2 genes expression were more pronounced in mice supplemented with FA compared to unfermented Astragalus, along with modulation of intestinal epithelial cells (IECs) apoptosis, verifying the intestinal mucosal barrier repaired by FA. This study is the first to suggest that FA as a potential modulator can more effectively regulate the inflammatory status and gut microbiota to repair the intestinal barrier damage caused by colitis.

Substance flow analysis on the leachate DOM molecules along five typical membrane advanced treatment processes

The processes combining biological treatment with membrane separation technologies have been widely adopted for leachate treatment. However, dissolved organic matter (DOM) of leachate membrane concentrates generated from various membrane separation technologies has not been systematically investigated in field scale. Therefore, substance flow analysis based on DOM molecular information of leachate membrane concentrates from primary membrane systems (i.e. nanofiltration (NF) and reverse osmosis (RO)) and secondary membrane systems (i.e. disk-tube reverse osmosis (DTRO) and humic substance filtration system (HSF)) in five engineering-scale leachate treatment facilities, obtained via ultra-performance liquid chromatography coupled with hybrid quadrupole Orbitrap mass spectrometry, was given and simultaneously compared. In NF concentrates (NFC), 45.1–98.5% of DOM originated from raw leachate (L-DOM) was concentrated, showing poor biodegradability. The L-DOM interception characteristics of NFC-fed HSF were mainly based on volume reduction but concentration effect. L-DOM in RO concentrates (ROC) showed a higher proportion of peak intensity reduced components, accounting for 50.3–96.8%, and organic composition changes were more dependent on water quality characteristics than membrane types. ROC-fed DTRO intercepted 49.3–72.6% of L-DOM, but DTRO may be less effective at intercepting DOM molecules in landfill leachate with higher oxidation levels. Considering risks from feasible treatment technologies, the difficulty for the treatment of leachate membrane concentrates followed the order of DTRO concentrates > ROC > NFC. This study suggests that ROC-fed DTRO need to be controlled to avoid amplifying the treatment difficulty. Besides, treatment technologies for RO and DTRO concentrates with low-concentrated but refractory DOM and high salts should be explored.

Identification of phytochemicals from Lentinus edodes and Auricularia auricula with UPLC-Q-Exactive Orbitrap MS

• Phytochemicals of Lentinus edodes was investigated by UPLC-Q-Exactive-Orbitrap MS. Twenty-three compounds were identified in L. edodes. • Phytochemicals of Auricularia auricula were investigated by UPLC-Q-Exactive-Orbitrap MS. Twenty-nine compounds were identified in A. auricula. • There are 9 common ingredients of L. edodes and A. auricula, including 4 amino acids and analogs, 2 flavonoids, 2 nucleosides and analogs and 1 phenol. Lentinus edodes and Auricularia auricula are the first and second largest edible fungi in China with high nutritional and medicinal values. Research about phytochemicals of L. edodes and A. auricula mainly focuses on polysaccharides, other phytochemicals are not fully studied. Phytochemicals of L. edodes and A. auricula were investigated by ultra-high performance liquid chromatography tandem hybrid quadrupole-orbitrap mass spectrometry (UPLC-Q-Exactive-Orbitrap MS). Twenty-three compounds were identified in L. edodes , mainly including amino acids and analogs, flavonoids, nucleosides and analogs, phenols and steroids. Twenty-nine compounds were identified in A. auricula , mainly including amino acids and analogs, nucleosides and analogs, flavonoids and phenols. There are 9 common ingredients of L. edodes and A. auricula , including 4 amino acids and analogs, 2 flavonoids, 2 nucleosides and analogs and 1 phenol.

Comprehensive plasma metabolomics and lipidomics of benign and malignant solitary pulmonary nodules.

Solitary pulmonary nodules (SPNs) are commonly found in imaging technologies, but are plagued by high false-positive rates. We aimed to identify metabolic alterations in SPN etiology and diagnosis using less invasive plasma metabolomics and lipidomics. In total, 1160 plasma samples were obtained from healthy volunteers (n = 280), benign SPNs (n = 157) and malignant SPNs (stage I, n = 723) patients enrolled from 5 independent centers. Gas chromatography-triple quadrupole mass spectrometry (GC‒MS) and liquid chromatography-Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometry (LC‒MS) were used to analyze the samples for untargeted metabolomics and lipidomics. GC‒MS-based metabolomics revealed 1336 metabolic features, while LC‒MS-based lipidomics revealed 6088 and 2542 lipid features in the positive and negative ion modes, respectively. The metabolic and lipidic characteristics of healthy vs. benign or malignant SPNs exhibited substantial pattern differences. Of note, benign and malignant SPNs had no significant variations in circulating metabolic and lipidic markers and were validated in four other centers. This study demonstrates evidence of early metabolic alterations that can possibly distinguish SPNs from healthy controls, but not between benign and malignant SPNs.

A high‐resolution mass spectrometric method for identification and characterization of the in vitro metabolites of senkyunolide H

Ligusticum chuanxiong Hort is a well-known herb medicine that has been widely prescribed to treat cardiovascular diseases in China for hundreds of years. Senkyunolide H (SNH) is one of the major bioactive ingredients extracted from L. chuanxiong, and it displayed neuroprotective effects. To fully understand its mechanism of action, the metabolism needs to be investigated. In vitro studies were conducted by incubating SNH with rat and human hepatocytes, and the metabolites were identified and characterized using liquid chromatography in combination with hybrid quadrupole Orbitrap mass spectrometry (LC-Orbitrap-MS). The structures of the metabolites were proposed by accurate mass analysis of respective precursor ions, indicative product ions, and elemental compositions. Under the current conditions, a total of 10 metabolites were identified, and among these metabolites, M3 and M4 were the most abundant metabolites both in rat and human hepatocytes. Our results demonstrated that hydroxylation, hydration, glucuronidation, and GSH conjugation were the primary metabolic pathways of SNH. The present study provides new information on the metabolism of SNH, which would help prospects of the disposition of SNH.