Trap Quadrupole Orbitrap Mass Spectrometry Research Articles

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.

Comparative analysis of fatty acids, volatile and non-volatile components in red huajiao (Zanthoxylum bungeanum maxim.) and green huajiao (Zanthoxylum armatum DC.) using GC-MS, UPLC-LTQ-Orbitrap-MS/MS and HPLC-DAD

In China, huajiao, a traditional industrial crop, is categorized as red huajiao (RHJ; Zanthoxylum bungeanum maxim.) and green huajiao (GHJ; Zanthoxylum armatum DC.) based on the peel color. There were significant differences in sensory and biological activities between RHJ and GHJ in practical application. However, there is no sufficient and comprehensive metabolome-based explanation for these differences so far. The gas chromatography-mass spectrometry (GC-MS), ultra-performance liquid chromatography-linear trap quadrupole-orbitrap mass spectrometry (UPLC-LTQ-Orbitrap-MS/MS), and high-performance liquid chromatography-photodiode array detection (HPLC-DAD) were employed in this study to analyze the differences in fatty acids (FAs), volatile components (VCs) and non-volatile components (non-VCs) between 6 RHJ and 9 GHJ. The findings demonstrated that 12 FAs, 68 VCs, and 101 non-VCs were detected in huajiao, of which 5 FAs, 28 VCs, and 46 non-VCs were identified as differential metabolites (DMs) between RHJ and GHJ. And the DMs were enriched in multiple metabolic pathways by KEGG analysis. Moreover, kaempferol, kaempferide, berberrubine, scoulerine, saponaretin, naringenin, and 3-P-coumaroylquinic acid were identified as key bioactive DMs based on the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and analysis platform. In this study, the possible reasons for the differences in sensory and biological activities between RHJ and GHJ were comprehensively revealed at the metabolic level using non-targeted and targeted metabolomics, and the results provide a better theoretical understanding of the different application of them.

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.

Chemical constituents in different parts of Ixeris sonchifolia based on UPLC-LTQ-Orbitrap-MS~n

The chemical constituents in stem leaf, root, and flower of Ixeris sonchifolia were identified by the ultra performance li-quid chromatography coupled with linear ion trap quadrupole-orbitrap mass spectrometry(UPLC-LTQ-Orbitrap-MS~n). The separation was performed on an Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm) with a mobile phase of water(containing 0.1% formic acid, A)-acetonitrile(B) with gradient elution. With electrospray ionization source, the data of 70% methanol extract from stem leaf, root and flower of I. sonchifolia were collected by high-resolution full-scan Fourier transform spectroscopy, data dependent acquisition, precursor ion scan, and selected ion monitoring in the negative and positive ion modes. The compounds were identified based on accurate molecular weight, retention time, fragment ions, comparison with reference standard, Clog P and references. A total of 131 compounds were identified from the 70% methanol extract of I. sonchifolia, including nucleosides, flavonoids, organic acids, terpenoids, and phenylpropanoids, and 119, 110, and 126 compounds were identified from the stem leaf, root and flower of I. sonchifolia, respectively. In addition, isorhamnetin, isorhamnetin-7-O-sambubioside and caffeylshikimic acid were discovered from I. sonchifolia for the first time. This study comprehensively analyzed and compared the chemical constituents in different parts of I. sonchifolia, which facilitated the discovery of effective substances and the development and application of medicinal material resources of I. sonchifolia.

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.

Temporal lipid profiling in the progression from acute to chronic heart failure in mice and ischemic human hearts

Background and aimsMyocardial infarction (MI) is a leading cause of heart failure (HF). After MI, lipids undergo several phasic changes implicated in cardiac repair if inflammation resolves on time. However, if inflammation continues, that leads to end stage HF progression and development. Numerous studies have analyzed the traditional risk factors; however, temporal lipidomics data for human and animal models are limited. Thus, we aimed to obtain sequential lipid profiling from acute to chronic HF. MethodsHere, we report the comprehensive lipidome of the hearts from diseased and healthy subjects. To induce heart failure in mice, we used a non-reperfused model of coronary ligation, and MI was confirmed by echocardiography and histology, then temporal kinetics of lipids in different tissues (heart, spleen, kidney), and plasma was quantitated from heart failure mice and compared with naïve controls. For lipid analysis in mouse and human samples, untargeted liquid chromatography-linear trap quadrupole orbitrap mass spectrometry (LC-LTQ-Orbitrap MS) was performed. ResultsIn humans, multivariate analysis revealed distinct cardiac lipid profiles between healthy and ischemic subjects, with 16 lipid species significantly downregulated by 5-fold, mainly phosphatidylethanolamines (PE), in the ischemic heart. In contrast, PE levels were markedly increased in mouse tissues and plasma in chronic MI, indicating possible cardiac remodeling. Further, fold change analysis revealed site-specific lipid biomarkers for acute and chronic HF. A significant decrease in sulfatides (SHexCer (34:1; 2O)) and sphingomyelins (SM (d18:1/16:0)) was observed in mouse tissues and plasma in chronic HF. ConclusionsOverall, a significant decreased lipidome in human ischemic LV and differential lipid metabolites in the transition of acute to chronic HF with inter-organ communication could provide novel insights into targeting integrative pathways for the early diagnosis or development of novel therapeutics to delay/prevent HF.

Identification of the New Psychoactive Substance Eutylone.

To establish a method to identify unknown sample based on the combined use of Fourier transform infrared spectroscopy (FTIR), gas chromatography-quadrupole time-of-flight mass spectrometry (GC-QTOF-MS), ultra-high performance liquid chromatography-linear ion trap quadrupole-orbitrap mass spectrometry (UPLC-LTQ-Orbitrap MS) and 1H-nuclear magnetic resonance spectroscopy (1H-NMR) technique. The unknown sample was directly analyzed by FTIR. The unknown sample was dissolved in methanol solution containing internal standard SKF525A and the supernatant was detected by GC-QTOF-MS and UPLC-LTQ-Orbitrap MS. The unknown sample was dissolved in methanol-d4 solution for structural analysis of 1H-NMR. The characteristic absorption peaks of FTIR spectra obtained from unknown sample were 1 682 (C=O bond), 1 503, 1 488, 1 436, 1 363, 1 256, 1 092, 1 035, 935, 840 and 800 cm-1, the characteristic fragment ions (m/z) of GC-QTOF-MS were 86.096 4 (base peak), 58.065 1, 149.023 5, 121.028 6 and 65.038 6, the accurate mass [M+H]+ detected by UPLC-LTQ-Orbitrap MS was 236.127 7. The sample was identified as synthetic cathinone new psychoactive substance Eutylone by 1H-NMR. The method established in this study can be used for structural confirmation of Eutylone.

Evaluation of the chemical profile from four germplasms sources of Pruni Semen using UHPLC-LTQ-Orbitrap-MS and multivariate analyses

Pruni Semen, the seed of several unique Prunus plants, is a traditional purgative herbal material. To determine the authentic sources of Pruni Semen, 46 samples from four species were collected and analyzed. Ten compounds including multiflorin A (Mul A), a notable purative compound, were isolated and identified by chemical separation and nuclear magnetic resonance spectroscopy. Seventy-six communal components were identified by ultra-high performance liquid chromatography with linear ion trap-quadrupole Orbitrap mass spectrometry, and acetyl flavonoid glycosides were recognized as characteristic constituents. The flavonoids were distributed in the seed coat and cyanogenic glycosides in the kernel. Based on this, methods for identifying Pruni Semen from different sources were established using chemical fingerprinting, quantitative analysis of the eight principal compounds, hierarchical cluster analysis, principal component analysis, and orthogonal partial least squares discriminant analysis. The results showed that the samples were divided into two categories: one is the small seeds from Prunus humilis (Ph) and Prunus japonica (Pj), and the other is the big seeds from Prunus pedunculata (Pp) and Prunus triloba (Pt). The average content of Mul A was 3.02, 6.93, 0.40, and 0.29 mg/g, while the average content of amygdalin was 18.5, 17.7, 31.5, and 30.9 mg/g in Ph, Pj, Pp, and Pt, respectively. All the above information suggests that small seeds might be superior sources of Pruni Semen. This is the first comprehensive report on the identification of chemical components in Pruni Semen from different species.

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.

Plasma characteristic metabolites of pediatric community-acquired pneumonia in traditional Chinese medicine syndrome differentiation.

Community-acquired pneumonia (CAP) is the leading cause of lower respiratory tract infections in children. Heat syndrome (HS) and cold syndrome (CS) are two main syndrome types of pediatric CAP in traditional Chinese medicine (TCM). This study aimed to identify plasma metabolic profiles in pediatric CAP and to further select potential biomarkers to distinguish between HS and CS. An ultra-performance liquid chromatography coupled with linear ion trap quadrupole-orbitrap mass spectrometry method was applied to plasma samples of 296 patients and 55 healthy controls (HC). The samples were divided into the discovery group (n=213, HS=160, CS=23, HC=30) and the validation group (n=138, HS=93, CS=20, HC=25). The orthogonal partial least-squares discriminant analysis, the value of fold change, and Kruskal-Wallis test with false discovery rate correction (q-value <0.05) were applied to identify differential plasma metabolites. The area under the ROC curve (AUC) was used to evaluate the diagnostic performance of the screened metabolites. The results showed that the plasma levels of aspartic acid, phenylalanine, arginine, lysoPC20:1, lysoPE16:0, lysoPE18:0, and PE (16:0_22:6) were increased in CS compared with HC. The plasma levels of PC (18:1_18:1), PC (20:4_20:4), PE (16:0_18:2), lysoPE20:4, lysoPE18:2, and lysoPE22:6 were decreased, whereas, the plasma level of ceramide (d18:1_24:1) was increased in HS compared with HC. There were 13 differential metabolites in CS (AUC=0.995) and 15 differential metabolites in HS (AUC=0.954), compared with HC. A panel of seven biomarkers, including LysoPC20:1, lysoPE16:0, lysoPE18:2, lysoPE20:4, lysoPE22:6, PC (18:1_18:1), and PC (20:4_20:4) showed good discrimination between HS and CS with an AUC of 0.982. Altered plasma amino acids and lipids may provide an objective basis for TCM syndrome differentiation in pediatric CAP.

Identification of New Psychoactive Tryptamines 4-OH-MET and 4-AcO-DMT Using High Resolution Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy

Objective To detect the uncontrolled new psychoactive tryptamines involved in drug-related cases with high resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Methods White and brown powder obtained in actual cases were extracted and analyzed by gas chromatography-quadrupole time-of-flight mass spectrometry （GC-QTOF-MS）, ultra-high performance liquid chromatography-linear ion trap quadrupole-orbitrap mass spectrometry （UPLC-LTQ-Orbitrap MS） and 1H-nuclear magnetic resonance spectroscopy （1H-NMR）. Results After detection by GC-QTOF-MS, the components of white powder showed main characteristic fragment ion peaks at m/z 218.141 0 （molecular ion peak）, 72.080 6 （base peak）, etc. After detection by UPLC-LTQ-Orbitrap MS, its protonated molecular ion was m/z 219.149 4. The main ions in the secondary mass spectrum under the collision-induced dissociation （CID） mode were m/z 160.076 3 and 72.080 8. After detection by GC-QTOF-MS, the components of brown powder showed main characteristic fragment ion peaks at m/z 246.135 7 （molecular ion peak）, 58.065 1 （base peak）, etc. After detection by UPLC-LTQ-Orbitrap MS, its protonated molecular ion was m/z 247.145 0. The main ions in the secondary mass spectrum under CID mode were m/z 202.087 1, 160.076 3 and 134.060 5. NIST 17 library retrieval and 1H-NMR confirmed that the white powder and brown powder contained new psychoactive tryptamines 4-OH-MET and 4-AcO-DMT, respectively. Conclusion GC-QTOF-MS, UPLC-LTQ-Orbitrap MS and 1H-NMR can be used together to identify unknown new psychoactive substances.

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.

Lipidomic analysis of non-esterified furan fatty acids and fatty acid compositions in dietary shellfish and salmon by UHPLC/LTQ-Orbitrap-MS

Lipids such as furan fatty acids (F-acids) are the valuable minor bioactive components of food such as fatty fish and plants. They are reported to have positive health benefits, including antioxidant and anti-inflammatory activities. Despite their importance, limited studies are focusing on F-acid determination in dietary seafood. This study aimed to identify and profile non-esterified F-acids and free fatty acids in total lipid extract of seafood such as shellfish and salmon. The lipidomic analysis using liquid chromatography-linear trap quadrupole-orbitrap mass spectrometry led to identifying seven types of free F-acids in shellfish (n = 5) and salmon (n = 4). The identified F-acids were confirmed by their high-resolution masses and acquired mass spectra. The relative concentrations of F-acids in shellfish range from 0.01 to 10.93 mg/100 g of the fillet, and in salmon, 0.01 to 14.21 mg/100 g of the fillet. The results revealed the highest abundance of F-acids in Sakhalin surf clam, Japanese scallop, and a fatty salmon trout. Besides, relative levels of saturated, monounsaturated, and polyunsaturated fatty acids (PUFAs) in these seafoods were compared with each other, suggesting basket clams and salmon trout to have significantly higher levels of PUFAs. The dietary seafoods enriched with F-acids and PUFAs may have possible health benefits. Hence, the applied technique could be a promising tool for rapid detection and analysis of non-esterified fatty acids in food.

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.

Chemical Labeling Assisted Detection and Identification of Short Chain Fatty Acid Esters of Hydroxy Fatty Acid in Rat Colon and Cecum Contents.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are novel endogenous lipids with important physiological functions in mammals. We previously identified a new type of FAHFAs, named short-chain fatty acid esterified hydroxy fatty acids (SFAHFAs), with acetyl or propyl esters of hydroxy fatty acids of carbon chains, C ≥ 20. However, sensitive determination of SFAHFAs is still a challenge, due to their high structural similarity and low abundance in biological samples. This study employs one-step chemical derivatization following total lipid extraction using 2-dimethylaminoethylamine (DMED) for enhanced detection of SFAHFAs. The labeled extracts were subjected to ultrahigh performance liquid chromatography coupled to linear ion trap quadrupole-Orbitrap mass spectrometry (UHPLC/LTQ-Orbitrap MS). Our results demonstrated that the detection sensitivities of SFAHFAs increased after DMED labeling, and is highly helpful in discovering six additional novel SFAHFAs in the cecum and colon contents of WKAH/HKmSlc rats fed with normal and high-fat diet (HFD). The identified DMED labeled SFAHFAs were characterized by their detailed MS/MS analysis, and their plausible fragmentation patterns were proposed. The concentrations of SFAHFAs were significantly reduced in the cecum of HFD group compared to the control. Hence, the proposed method could be a promising tool to apply for the enhanced detection of SFAHFAs in various biological matrices, which in turn facilitate the understanding of their sources, and physiological functions of these novel lipids.

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.

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.