Linear Trap Quadrupole Orbitrap Mass Research Articles

Sanhan Huashi Formula and Its Bioactive Compounds Exert Antiviral and Anti-Inflammatory Effects on COVID-19

Sanhan Huashi formula (SHHS), a traditional Chinese medicine (TCM), has shown significant therapeutic effects on coronavirus disease 2019 (COVID-19) in clinical settings. However, its specific mechanism and components still require further clarification. In vitro experiments with Vero-E6 cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demonstrated that SHHS effectively inhibited viral invasion and proliferation. Complementary in vivo experiments using K18-human angiotensin converting enzyme 2 (hACE2) mice exposed to virus-like particles (VLPs) further confirmed that SHHS impeded SARS-CoV-2 entry. Although SHHS did not demonstrate direct antiviral effects in K18-hACE2 mice challenged with SARS-CoV-2, it significantly alleviated pathological damage and decreased the expression of chemokines such as C–C motif ligand (CCL)-2, CCL-3, C–X–C motif ligand (CXCL)-1, CXCL-6, CXCL-9, CXCL-10, and CXCL-11 in the lungs, suggesting that SHHS exerts immunomodulatory and anti-inflammatory effects via the CCL-2–CXCL axis. Additional research using a lipopolysaccharide (LPS)-induced acute lung injury (ALI) and RAW264.7 cell model validated the ability of SHHS to reduce the levels of inflammatory biomarkers, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α). Using advanced analytical techniques such as ultrahigh-performance liquid chromatography coupled with linear trap quadrupole Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap-MS) and surface plasmon resonance (SPR), nodakenin was identified as a potent antiviral component of SHHS that targets the 3C-like protease (3CLpro), a finding supported by the hydrogen–deuterium exchange mass spectrometry (HDX-MS) and molecular docking analyses. Furthermore, nodakenin demonstrated a significant antiviral effect, reducing the viral load by more than 66%. This investigation reveals that SHHS can combat COVID-19 by inhibiting viral invasion and promoting anti-inflammatory effects.

The elucidation of plasma lipidome profiles during severe influenza in a mouse model

Although influenza virus infection has been shown to affect lipid metabolism, details remain unknown. Therefore, we elucidated the kinetic lipid profiles of mice infected with different doses of influenza virus A/Puerto Rico/8/34 (H1N1) (PR8) by measuring multiple lipid molecular species using untargeted lipidomic analysis. C57BL/6 male mice were intranasally infected with PR8 virus at 50 or 500 plaque-forming units to cause sublethal or lethal influenza, respectively. Plasma and tissue samples were collected at 1, 3, and 6 days post-infection (dpi), and comprehensive lipidomic analysis was performed using high-performance liquid chromatography–linear trap quadrupole–Orbitrap mass spectrometry, as well as gene expression analyses. The most prominent feature of the lipid profile in lethally infected mice was the elevated plasma concentrations of phosphatidylethanolamines (PEs) containing polyunsaturated fatty acid (PUFA) at 3 dpi. Furthermore, the facilitation of PUFA-containing phospholipid production in the lungs, but not in the liver, was suggested by gene expression and lipidomic analysis of tissue samples. Given the increased plasma or serum levels of PUFA-containing PEs in patients with other viral infections, especially in severe cases, the elevation of these phospholipids in circulation could be a biomarker of infection and the severity of infectious diseases.

Authentication, chemical profiles analysis, and quality evaluation of corn silk via DNA barcoding and UPLC-LTQ/Orbitrap MS chemical profiling

Corn silk is commonly consumed in teas, food ingredients, and herbal medicines. Several varieties of corn silk are grown in different habitats in China. However, as information regarding their phytochemistry and genetic diversity is limited, their medicinal potential has not been utilized thoroughly. Thus, we aimed to use a combination of DNA barcoding based on specific primer ITSC sequences and ultra-performance liquid chromatography coupled with linear trap quadrupole-Orbitrap mass spectrometry (UPLC-LTQ/Orbitrap MS) approach for identifying and evaluating corn silk. ITSC barcoding helped us to identify that 52 samples could be classified into 7 groups of corn silk varieties, but the widely used nrITS and psbA-trnH barcodes failed to identify these varieties. UPLC-LTQ/Orbitrap MS was used to study the components in alcohol extracts derived from different corn silk varieties, and the detected chemical components were analyzed via bioinformatics techniques. We proposed 199 components using untargeted UPLC-LTQ/Orbitrap MS-based metabolomics analysis and identified 67 components. PCA and OPLS-DA analysis revealed two distinct chemotypes by selecting 27 components that could act as difference indicators. KEGG analysis showed that the 199 components were enriched in 12 metabolic pathways. The results showed that corn silk is rich in many types of chemicals and DNA barcoding is better than UPLC-LTQ/Orbitrap MS in distinguishing the differences between different varieties of corn silk. Our findings provide new insights into the chemical and molecular characteristics of different varieties of corn silk, which play a crucial role in the utilization of corn silk resources.

The profiles of durian (Durio zibethinus Murr.) shell phenolics and their antioxidant effects on H2O2-treated HepG2 cells as well as the metabolites and organ distribution in rats

Durian is a nutritious tropical fruit with potent antioxidant, anti-inflammatory, antibacterial and anti-cancer effects. However, the durian shell was mainly discarded as waste, while there were few studies on the characterization of its phenolic profiles, antioxidant activities, and in vivo metabolites. In the present study, a total of 17 compounds were identified in durian shell extract (DSE) by using an ultra-high-performance liquid chromatography coupled with linear ion trap quadrupole Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap-MS/MS), while 33 metabolites were found in rats' plasma, urine and organ. Moreover, DSE could effectively reduce H2O2-induced oxidative damage in HepG2 cells, reduce the expression of Reactive Oxygen Species (ROS), Malondialdehyde (MDA) and Lactate Dehydrogenase (LDH) and inhibit apoptosis by regulating the expression of Bcl-2-Associated X (BAX), B-Cell Lymphoma 2 (BCL-2), Caspase-3 and Caspase-9 genes and proteins related to mitochondrial pathway apoptosis. This is the first comprehensive report on Durian shell phenolics, their metabolic profiles and underlying mechanisms of the in vitro antioxidant activities.

Comparative analysis of absorbed ingredients and metabolites, and pharmacokinetic studies of Zhimu-Huangbai herb pair in the plasma of normal and type 2 diabetes mellitus rats by UHPLC-linear trap quadrupole-orbitrap MS and LC-MS/MS.

A new rapid ultra-high-performance liquid chromatography coupled with linear trap quadrupole orbitrap mass spectrometry method was established for the qualitative analysis of absorbed ingredients and metabolites of Zhimu-Huangbai herb pair, which is used to treat type 2 diabetes mellitus. A total of 16 absorbed ingredients and 11 metabolites were identified in normal and type 2 diabetes mellitus rats, respectively. Such findings indicated that the diabetic model had no effect on the type of components in plasma. Seven absorbed ingredients and 11 metabolites were first identified after the oral administration of Zhimu-Huangbai herb pair. Thereafter, ultra-high-performance liquid chromatography coupled with linear trap quadrupole orbitrap mass spectrometry and liquid chromatography-API4000+ triple quadrupole mass spectrometer methods were established and validated for pharmacokinetic comparative studies of seven major bioactive components in normal and type 2 diabetes mellitus rats. Partial pharmacokinetic parameters in the plasma of type 2 diabetes mellitus rats were significantly different from those in normal rats. To our knowledge, this is the first comparison of absorbed ingredients and metabolites of Zhimu-Huangbai herb pair, and its use in pharmacokinetic studies between normal and type 2 diabetes mellitus rats. Ultimately, our findings provide insights into the clinical usage of Zhimu-Huangbai herb pair.

Identifying the active compounds and mechanism of action of Banxia Xiexin decoction for treating ethanol-induced chronic gastritis using network pharmacology combined with UPLC–LTQ–Orbitrap MS

BackgroundBanxia Xiexin decoction (BXD), a traditionally prescribed Chinese medicine, has been used to treat chronic gastritis for many years. However, the underlying mechanism and targets for its effects remain unknown. In the present study, we predicted the targets and active compounds of BXD in the treatment of chronic gastritis through network pharmacology and ultra-performance liquid chromatography coupled with linear trap quadrupole–Orbitrap mass spectrometry (UPLC–LTQ–Orbitrap MS). MethodA chronic gastritis model was established in rats by oral administration of 56 % ethanol. BXD was orally administered for 7 days. Stomach tissues were collected for histopathological analysis, and tumour necrosis factor (TNF)-α, interleukin (IL)-2, IL-8, and lactate dehydrogenase (LDH) levels were measured by enzyme-linked immunosorbent assay. UPLC–LTQ–Orbitrap MS was established to analyse compounds in rat plasma following oral BXD administration. The absorbed ingredients were selected as candidate active compounds. The chronic gastritis-related targets were screened using multiple databases. The potential targets for the treatment of chronic gastritis were used to construct a protein–protein interaction (PPI) network and were also analysed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Finally, molecular docking was used to uncover the interaction between multi-components and putative targets, and the results were verified by surface plasmon resonance (SPR). ResultsIntragastric administration of BXD ameliorated stomach injury resulting from chronic gastritis in rats and decreased the levels of TNF-α, IL-2, IL-8, and LDH. A comprehensive systematic strategy was used to successfully identify 38 candidate targets and 14 active compounds in BXD. Based on the network of compounds–targets and PPI, three hub genes that were associated with BXD therapy for chronic gastritis were selected and included intercellular adhesion molecule-1, peroxisome proliferator-activated receptor gamma and mitogen-activated protein kinase 14. The results of molecular docking and SPR demonstrated that the active compounds in BXD demonstrate affinity for these targets. Additionally, an enrichment analysis revealed that treatment of chronic gastritis with BXD primarily involves cytokine activation, the inflammatory response and nuclear factor-kappa B, hypoxia-inducible factor-1, phosphatidylinositol-3-kinase–protein–serine–threonine kinase and Janus kinase–signal transducer and activator of transcription signalling pathways, which may mediate the effects of BXD in the treatment of chronic gastritis. ConclusionBXD exhibits a therapeutic effect in ethanol-induced gastritis through multi-compound, multi-target and multi-pathway mechanisms. A strategy of network pharmacology combined with SPR may provide a feasible approach to explore the targets of herbal medicine and uncover novel bioactive components.

Profile chemical compounds and antioxidant activity of Korean commercial vinegars produced by traditional fermentation

Ultra-high-performance liquid chromatography coupled with linear trap quadrupole-orbitrap mass spectrometry (UHPLC-LTQ–Orbitrap MS) with multivariate statistical analysis was applied for the metabolite profiling and assessing antioxidant activity of ten commercial vinegars produced by traditional fermentation techniques. Totally, 28 metabolites including three organic acids, three phenolic acids, 11 flavonoids, nine lipids and fatty acids, 6-methoxymellein, and tyrosyl-valine, were identified in the ten vinegars. Using principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA), vinegar samples were differentiated based on raw materials used in their production. Based on the PC1 (42.10%) and PC2 (9.86%), mulberry fruit vinegar (OV) and citrus vinegar (CV) were classified as rest vinegars. Fruit vinegars were found to be rich in metabolites and antioxidant properties, as compared to grain vinegars. OV had the highest content of the quercetin derivatives and phenolic acids, and showed strongest antioxidant activity. Of the 28 metabolites identified, quercetin-3-O-rhamnoside-7-O-glucoside and quercetin-3-O-glucosyl-rutinoside were determined to be the main contributors to antioxidant activity in vinegars. The current study suggests that metabolite profiling by UHPLC–MS can be used to illuminate compounds associated with antioxidant properties as well as selection for vinegar type with the high content of bioactive metabolite profiles.

The Pharmacokinetics, Tissue Distribution, Metabolism, and Excretion of Pinostrobin in Rats: Ultra-High-Performance Liquid Chromatography Coupled With Linear Trap Quadrupole Orbitrap Mass Spectrometry Studies.

Pinostrobin is a natural flavonoid found in various plants, well known for its wide range of pharmacological activities. However, there are few reports regarding the pharmacokinetics, tissue distribution, metabolism, and excretion of pinostrobin in rats after oral administration as a single compound. Therefore, we established a method using ultra-high-performance liquid chromatography coupled with linear trap quadrupole orbitrap mass spectrometry (UPLC-LTQ orbitrap-MS/MS) to determine pinostrobin and its metabolites in rat plasma, urine, feces, bile, and tissue homogenates. Pharmacokinetic parameters were measured. The large apparent volume of distribution implied that pinostrobin preferentially bound to tissues and preferably remained within the body. Based on previous pharmacological studies of its antiulcer, anti-HP, anti-inflammatory, and antioxidant activities, pinostrobin is mostly distributed in the gastrointestinal tract, indicating its potential as an effective component of traditional Chinese medicines for the treatment of peptic ulcers. Furthermore, 30 flavonoid metabolites were screened using UPLC-LTQ orbitrap-MS/MS. The metabolism pathways (mainly hydroxylation, demethylation, glucuronidation, and sulfation) of pinostrobin in rats have also been proposed. A small amount of pinostrobin in its parent form is excreted through the urine, feces, and bile, indicating that it is mainly metabolized in vivo. In this study, we systemically investigated the pharmacokinetics, tissue distribution, metabolism, and excretion of pinostrobin in rats. Our results provide a significant basis for the clinical development and application of pinostrobin as well as traditional Chinese medicines containing pinostrobin.

Using serum peptidomics to discovery the diagnostic marker for different stage of ulcerative colitis

The use of peptidomics to find diagnostic markers has attracted increasing clinical attention. Ulcerative colitis (UC) is a type of inflammatory bowel disease, and the traditional auxiliary diagnostic technique is colonoscopy. However, this invasive method is not effective in distinguishing between patients with endoscopic remission and healthy people, which carries the risk of delayed diagnosis of UC. In this study, we used peptidomics to find serum diagnostic markers for different stages of UC. A total of 78 serum samples were collected to form a training set (60 samples) and a testing set (18 samples). Among them, patients with active UC, remitting UC and healthy people accounted for one third each. The nano-liquid chromatography coupled with hybrid linear trap quadrupole orbitrap mass spectrometry was used for detection of low molecular weight peptides in serum. According to the protein database search and de novo sequencing algorithm, forty peptides were simultaneously identified in all samples. Six biomarker peptides were screened in the training set through orthogonal partial least-squares-discriminant analysis and receiver operating characteristic curve analysis. These six peptides were derived from proteins involved in coagulation and complement activation. We evaluated the diagnostic ability of the six peptides in the testing set through hierarchical cluster analysis, and showed that perturbation of these peptides could distinguish patients with active UC, patients with remitting UC and healthy people. This study validated the feasibility of serum peptidomics for the discovery of diagnostic markers, and provided a potential method for diagnosing different stages of UC.

Identification of short-chain fatty acid esters of hydroxy fatty acids (SFAHFAs) in a murine model by nontargeted analysis using ultra-high-performance liquid chromatography/linear ion trap quadrupole-Orbitrap mass spectrometry.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are recently discovered endogenous lipids with outstanding health benefits. FAHFAs are known to exhibit antioxidant, antidiabetic and anti-inflammatory properties. The number of known long-chain FAHFAs in mammalian tissues and dietary resources increased recently because of the latest developments in high-resolution tandem mass spectrometry techniques. However, there are no reports on the identification of short-chain fatty acid esterified hydroxy fatty acids (SFAHFAs). Intestinal contents, tissues, and plasma of rats fed with high-fat diet (HFD) and normal diet (ND) were analyzed for fatty acids, hydroxy fatty acids, and FAHFAs using ultra-high-performance liquid chromatography (UHPLC) and linear trap quadrupole-Orbitrap mass spectrometry (LTQ Orbitrap MS) with negative heated electrospray ionization. Untargeted analysis of total lipid extracts from murine samples (male 13-week-old WKAH/HKmSlc rats) led to the identification of several new SFAHFAs of acetic acid or propanoic acid esterified long-chain (>C20)-hydroxy fatty acids. Furthermore, MS3 analysis revealed the position of the hydroxyl group in the long-chain fatty acid as C-2. The relative amounts of SFAHFAs were quantified in intestinal contents and their tissues (Cecum, small intestine, and large intestine), liver, and plasma of rats fed with HFD and ND. The large intestine showed the highest abundance of SFAHFAs with a concentration range from 0.84 to 57 pmol/mg followed by the cecum with a range of 0.66 to 28.6 pmol/mg. The SFAHFAs were significantly altered between the HFD and ND groups, with a strong decreasing tendency under HFD conditions. Identification of these novel SFAHFAs can contribute to a better understanding of the chemical and biological properties of individual SFAHFAs and their possible sources in the gut, which in turn helps us tackle the role of these lipids in various metabolic diseases.

Establishing the chromatographic fingerprints of flavan-3-ols and proanthocyanidins from rose hip (Rosa sp.) species.

The profile of flavan-3-ols and proanthocyanidins in five differentRosa species(R. canina, R. glutinosa, R. rubiginosa, R. multiflora,andR. spinosissima) was estimated on high performance thin layer chromatography cellulose plates. Differences in flavanol and proanthocyanidin profiles of the extracts were evident, among whichRosaspinosissimastood out with catechin as the only detected flavanol and red zones as indication of anthocyanins. Furthermore, the elution solvent for thin layer chromatography with mass spectrometry analyses of glycosylated flavan-3-ols and proanthocyanidins was optimized, enabling identification of catechin, (epi)catechin hexoside, proanthocyanidin dimer, and proanthocyanidin dimers and trimers hexosides. A total of 15 flavanols and their derivatives were identified using ultra-high-performance liquid chromatography with linear trap quadrupole-Orbitrap mass analyzer and epicatechin, gallocatechin, and proanthocyanidin trimer were identified only using this technique. However, proanthocyanidin trimer trihexoside was identified only by thin-layer chromatography with mass spectrometry. To establish the relationships between the flavanols and proanthocyanidins composition of rose hip and their origin, principal component analysis was performed on the entire set of liquid chromatography/mass spectrometry data. Both principal components' scores plots showed that Rosa spinosissima could be considered as an outlier. Our study demonstrated that flavanol and proanthocyanidin profiles of different rose hips depend on the geographical origin rather than on the cultivar and genotype.

Characterization and Quantification of Phenolic Constituents in Peach Blossom by UPLC-LTQ-Orbitrap-MS and UPLC-DAD

Peach blossom comes from the flower of Prunus persica (L.) Batsch, which is used as herbal tea and medicine in China and Korea. It could promote defecation and alleviate the abdominal pain. In this paper, the methods, ultra-performance liquid chromatography (UPLC) method coupled with electrospray ionization hybrid linear trap quadrupole orbitrap mass spectrometry (LTQ OrbitrapMS) and UPLC system coupled with a diode array detector (DAD), were developed for the qualitative and quantitative analysis of the flavonoids and phenolic acids in peach blossoms. Eight standards were divided into 3 types according to their basic skeletons: phenolic acids, quercetin-type flavonoids, and kaempferol-type flavonoids. The MSn fragmentation behaviors and diagnostic ions of these 3 types of compounds were proposed to aid the structural identification of components in peach blossom extract. By extracting the diagnostic ions from the mass spectrum in negative mode, a total of 25 compounds, including 8 phenolic acids and 17 flavonoids, were screened out. Among these compounds, 5 compounds (chlorogenic acid, ferulic acid, rutin, hyperoside, and isoquercetrin) were quantitated by UPLC-DAD. The linearity, precision, accuracy, limit of detection, and limit of quantitation were validated for the quantification method. The validated method was applied to assay 9 batches of peach blossoms from different regions. This study was the first report on the systematic qualitative analysis of compounds in peach blossom, providing insights into the quality control of peach blossom.

Characterization and Identification of Prenylated Flavonoids from Artocarpus heterophyllus Lam. Roots by Quadrupole Time-Of-Flight and Linear Trap Quadrupole Orbitrap Mass Spectrometry

In this study, a combination of quadrupole time-of-flight mass spectrometry (Q-TOF-MS) and linear trap quadrupole orbitrap mass spectrometry (LTQ-Orbitrap-MS) was performed to investigate the fragmentation behaviors of prenylated flavonoids (PFs) from Artocarpus plants. Fifteen PFs were selected as the model molecules and divided into five types (groups A–E) according to their structural characteristics in terms of the position and existing form of prenyl substitution in the flavone skeleton. The LTQ-Orbitrap-MSn spectra of the [M − H]− ions for these compounds provided a wealth of structural information on the five different types of compounds. The main fragmentation pathways of group A were the ortho effect and retro Diels–Alder (RDA), and common losses of C4H10, CO, and CO2. The compounds in group B easily lose C6H12, forming a stable structure of a 1,4-dienyl group, unlike those in group A. The fragmentation pathway for group C is characterized by obvious 1,4A−, 1,4B− cracking of the C ring. The diagnostic fragmentation for group D is obvious RDA cracking of the C ring and the successive loss of CH3 and H2O in the LTQ-Orbitrap-MSn spectra. Fragmentation with successive loss of CO or CO2, ·CH3, and CH4 in the LTQ-Orbitrap-MSn spectra formed the characteristics of group E. The summarized fragmentation rules were successfully exploited to identify PFs from Artocarpus heterophyllus, a well-known Artocarpus plant, which led to the identification of a total of 47 PFs in this plant.

Paracetamol Degradation Performance and Mechanisms Using Microwave-Assisted Heat-Activated Persulfate in Solutions

A microwave (MW) heat-activated the persulfate (PS) process was employed to treat paracetamol (PAM) in wastewater, and the powder-activated carbon (PAC) be used is used as a catalyst to accelerate this reaction process. The PAM added (100 mg) to the solution was nearly completely removed within 70 min, and the PH, temperature, PAC, and PS dosage have great influence on the degradation process; the total organic carbon (TOC) removal rate reached 98%. The PAC1 still had a good catalytic effect after being reused six times. The radical mechanism was investigated to determine the type of dominant active species involved in PAM degradation. Sulfate radicals ( $$ {\mathrm{SO}}_4^{-\bullet } $$) were the dominant oxidizing agent for PAM degradation under acidic conditions. The degradation mechanism was proposed based on the PAM degradation intermediates, which were identified using ultra-high-performance liquid chromatography coupled with linear trap quadrupole orbitrap mass spectrometry. Three types of possible reaction pathways for PAM were identified as follows: including hydroxylation of the benzene ring, amine group oxidation at the benzene ring, and amine (HN–C=O) functional group N–C bond cleavage.

Phenolic Profile of Grape Canes: Novel Compounds Identified by LC-ESI-LTQ-Orbitrap-MS.

Grape canes (Vitis vinifera L.) are a viticulture industry by-product with an important content of secondary metabolites, mainly polyphenols with a broad spectrum of demonstrated health benefits. Grape canes, therefore, have considerable economic potential as a source of high-value phytochemicals. In this work, liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole-Orbitrap mass spectrometry (LC–LTQ-Orbitrap) was used for the comprehensive identification of polyphenolic compounds in grape canes. Identification of polyphenols was performed by comparing their retention times, accurate mass measured, and mass fragmentation patterns with those of reference substances or available data in the literature. A total of 75 compounds were identified, including phenolic acids, flavanols, flavonols, flavanonols, flavanones, and stilbenoids. The most abundant polyphenols were proanthocyanidins and stilbenoids and their oligomers. Moreover, the high-resolution mass spectrometry analysis revealed the occurrence of 17 polyphenols never described before in grape canes, thereby providing a more complete polyphenolic profile of this potentially valuable by-product.

Phytochemistry and Antioxidant Activity of Aerial Parts of Phagnalon sordidum L.

One new natural monoterpene, 5-O-β-d-glucopyranosyl-2-hydroxy-p-cymene (1: ), and 11 known compounds were isolated through a biologically oriented approach from the aerial parts of Phagnalon sordidum L. The most active extract and fractions were selected using 3 complementary antioxidant activity assays. Results and the different methods were compared by relative antioxidant capacity index. In addition, the most active extract of P. sordidum was subjected to liquid chromatography coupled with electrospray ionization hybrid linear ion trap quadrupole Orbitrap mass spectrometry to quantify secondary metabolites. Antioxidant activities of ethyl acetate extract, and purified 3,4-dihydroxyacetophenone (3: ) and nebrodenside A (7: ) were demonstrated by in vitro cell free model assays, and their protective effect against H2O2-induced oxidative stress in a HepG2 (human hepatocellular carcinoma) cell line was established.

Identification of In Vivo Metabolites of Dictamnine in Mice Using HPLC-LTQ-Orbitrap Mass Spectrometry.

Dictamnine (4-methoxyfuro[2,3-b]quinolone, DIC), a common furoquinoline alkaloid in the family of Rutaceae, showed diverse biological activities. To investigate the in vivo metabolic pathways of DIC, metabolism of DIC in mice was studied using a high-performance liquid chromatography coupled to electrospray ionization of hybrid linear trap quadrupole orbitrap (HPLC-LTQ-Orbitrap) mass spectrometer. Nine metabolites were identified in the DIC-treated mouse urine, plasma, and fecal samples, of which two were identified as new metabolites. The major metabolic pathways of DIC in animal and human liver microsomes were confirmed in the present study, including o-demethylation, monohydroxylation, N-oxidation, and 2,3-olefinic epoxidation pathways. For the first time, a mono-acetylcysteine conjugate of DIC (M9) was detected from DIC-treated mouse urine and plasma samples, and 4-methoxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (M10) and 2-(2,8-dihydroxy-4-methoxyquinolin-3-yl)acetaldehyde (M11) were identified as new metabolites of DIC; furthermore, using an in vitro human fecal incubation model, furo[2,3-b]quinolin-4-ol (M1) was verified to be a microbial demethylated metabolite of DIC. Collectively, the present study provided new information on the in vivo metabolic fate of DIC.

Application of High-Performance Liquid Chromatography Coupled with Linear Ion Trap Quadrupole Orbitrap Mass Spectrometry for Qualitative and Quantitative Assessment of Shejin-Liyan Granule Supplements.

A method for high-performance liquid chromatography coupled with linear ion trap quadrupole Orbitrap high-resolution mass spectrometry (HPLC-LTQ-Orbitrap MS) was developed and validated for the qualitative and quantitative assessment of Shejin-liyan Granule. According to the fragmentation mechanism and high-resolution MS data, 54 compounds, including fourteen isoflavones, eleven ligands, eight flavonoids, six physalins, six organic acids, four triterpenoid saponins, two xanthones, two alkaloids, and one licorice coumarin, were identified or tentatively characterized. In addition, ten of the representative compounds (matrine, galuteolin, tectoridin, iridin, arctiin, tectorigenin, glycyrrhizic acid, irigenin, arctigenin, and irisflorentin) were quantified using the validated HPLC-LTQ-Orbitrap MS method. The method validation showed a good linearity with coefficients of determination (r2) above 0.9914 for all analytes. The accuracy of the intra- and inter-day variation of the investigated compounds was 95.0–105.0%, and the precision values were less than 4.89%. The mean recoveries and reproducibilities of each analyte were 95.1–104.8%, with relative standard deviations below 4.91%. The method successfully quantified the ten compounds in Shejin-liyan Granule, and the results show that the method is accurate, sensitive, and reliable.

Two-dimensional liquid chromatography separation and high resolution mass spectrometry analysis for proteome of rice leaves based on different extraction methods

A two-dimensional liquid chromatography (2D LC) system was developed to separate proteins from rice leaves, which was extracted by phenol method, followed by the analysis with linear trap quadrupole orbitrap mass spectrometry (LTQ/Orbitrap MS). After proteins were extracted with phenol method, the enzymolytic peptides were separated by offline two-dimensional RP-RP system and detected by LTQ/Orbitrap MS, yielding 2712 proteins. Liquid chromatography separation system (1D LC and 2D LC) and protein extraction methods (phenol method, sodium dodecyl sulfate method (SDS method) and trichloroacetic acid/acetone method (TCA/acetone method)) were compared. Proteins identified by 2D LC were 2712, 2415 and 1914 with the above three extraction methods, respectively. The proteins were 2.7-fold, 2.5-fold and 1.9-fold the number of proteins identified by 1D LC respectively. And in terms of 2D LC, the proteins identified by phenol method were 297 and 798 more than SDS method and TCA/acetone method, respectively. Some proteins with extreme properties, such as very acidic or basic protein and high relative molecular mass proteins, were only identified in phenol method. Furthermore, proteins, which were extracted by different extraction methods and separated by 2D LC, were classified according to biological functions. It was found that protein functions by the three extraction methods were complementary. However, phenol method had the most variety of functions. The method provides technological support for rice proteomics and reference for research techniques of other crop proteomics.

Pharmacokinetics and Tissue Distribution Study of Pinosylvin in Rats by Ultra-High-Performance Liquid Chromatography Coupled with Linear Trap Quadrupole Orbitrap Mass Spectrometry.

Pinosylvin is a potential anti-inflammatory and antioxidant compound and the major effective medicinal ingredient in the root of Lindera reflexa Hemsl. However, few investigations have been conducted regarding the pharmacokinetics, excretion, characteristics of tissue distribution, and major metabolites of pinosylvin in rats after oral administration. To better understand the behavior and mechanisms of action underlying the activity of pinosylvin in vivo, we established a simple, sensitive, and reliable ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for quantifying pinosylvin in rat plasma, urine, feces, and various tissues (including heart, liver, spleen, lung, kidneys, large intestine, small intestine, and stomach). Noncompartmental pharmacokinetic parameters indicated that pinosylvin is rapidly distributed and taken up by tissues. The time to peak (maximum) concentration (Tmax) was 0.137 h, and the apparent elimination half-life (t1/2) was 1.347±0.01 h. The results of the tissue distribution study suggest that pinosylvin is widely distributed to various tissues; the highest concentration was observed after 10 min in the stomach, followed by the heart, lung, spleen, and kidneys. Results of the excretion study suggest that a small amount of pinosylvin is excreted from the urine and feces in the parent form; the 73 h accumulative excretion ratios of urine and feces were 0.82% and 0.11%, respectively. It is likely that pinosylvin is mostly metabolized in vivo. Nine metabolites were found, and the main metabolic pathways of pinosylvin in rats included glucuronidation, hydroxylation, and methylation. Four metabolites had higher concentrations in the stomach, suggesting that the stomach is a potential target organ of pinosylvin. In conclusion, the present study may provide a material basis for studying the pharmacological action of pinosylvin and provides meaningful information for the clinical treatment of chronic gastritis and gastric ulcers using Radix Linderae Reflexae.