Ion trap-Orbitrap Mass Spectrometer Research Articles

A comprehensive strategy based on ultra-high performance liquid chromatography with diode array detector fingerprinting and multi-component ultra-performance liquid chromatography with tandem mass spectrometry technology for quality control of Jiawei Huoxiang Zhengqi Pill.

Jiawei Huoxiang Zhengqi Pill (JHZP) is a commonly used Chinese patent medicine for the clinical treatment of headache, dizziness, chest tightness as well as abdominal distension, and pain caused by wind-cold flu. In this study, a comprehensive strategy combining ultra-high performance liquid chromatography with diode array detector (UHPLC-DAD) fingerprinting and multi-component quantitative analysis was established and validated for quality evaluation of JHZP. A total of 49 characteristic common peaks were selected in a chromatographic fingerprinting study to assess the similarity of 15 batches of JHZP. Furthermore, 109 compounds were identified or preliminarily identified from JHZP by coupling with an advanced hybrid linear ion trap-Orbitrap mass spectrometer. For quantification, the optimized ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was employed for the simultaneous determination of 13 target compounds within 12min. The sensitivity, precision, reproducibility, and accuracy of the method were satisfactory. This validated UPLC-MS/MS method was successfully applied to analyzing 15 batches of JHZP. The proposed comprehensive strategy combining UHPLC-DAD fingerprinting and multi-component UPLC-MS/MS analysis proved to be highly efficient, accurate, and reliable for the quality evaluation of JHZP, which can be considered as a reference for the overall quality evaluation of other Chinese herbal formulations.

Transformation of fluopyram during enzymatic hydrolysis of apple and its effect on polygalacturonase and apple juice yield

China is one of the largest apple-growing areas in the world. Fluopyram (FLP) is a novel pesticide that has been widely used in agriculture. This work investigated the behavior of pesticides during enzymatic hydrolysis of apple juice and its effect on polygalacturonase (PG), apple juice yield, and flavor. The study findings revealed that 27.5% to 34.2% FLP was degraded during the enzymatic hydrolysis of apple. The three degradation products (P1, P2, and P3) were identified by a hybrid ion trap-orbitrap mass spectrometer. Based on toxicity assessment, it was found that carcinogenicity was higher for P2 and P3 than for FLP. Furthermore, FLP affected the yield and flavor of apple juice. FLP reduced yield by 4.8%, because FLP inhibited the activity of PG. Through molecular docking, it was found that there was an interaction between the active center of PG and FLP, resulting in a reduction in catalytic ability.

A comprehensive profiling and identification of liquiritin metabolites in rats using ultra-high-performance liquid chromatography coupled with linear ion trap–orbitrap mass spectrometer

Liquiritin (LQ), a main component of liquorice, exerts various biological activities. However, insufficient attentions have been paid to the metabolism study on this natural compound until now. Our present study was conducted to investigate the LQ metabolites in rats urine, faeces and plasma using UHPLC-LTQ-Orbitrap mass spectrometer in both positive and negative ion modes. Meanwhile, post-acquisition data-mining methods including high-resolution extracted ion chromatogram (HREIC), multiple mass defect filters (MMDFs), neutral loss fragments (NLFs) and diagnostic product ions (DPIs) were utilised to screen and identify LQ metabolites from HR-ESI-MS to ESI-MS n stage. As a result, a total of 49 metabolites were detected and characterised unambiguously or tentatively. These metabolites were presumed to generate through glucuronidation, sulfation, deglucosylation, dehydrogenation, methylation, hydrogenation, hydroxylation, ring cleavage and their composite reactions. Our results not only provided novel and useful data to better understand the biological activities of LQ, but also indicated that the proposed strategy was reliable for a rapid discovery and identification drug-related constituents in vivo.

Degradation of fluopyram in water under ozone enhanced microbubbles: Kinetics, degradation products, reaction mechanism, and toxicity evaluation

The degradation of fluopyram (FLP) was investigated under ozone-microbubble treatment (OMBT). Kinetic models were established to study the influence of three treatments: ozonated water, microbubbles (MCB), and OMBT. FLP degraded completely in OMBT, and a clearance rate of 89.8–100% was achievable. Three direct transformation products [product 1 (F1), product 2 (F2), and product 3(F3)] were isolated and identified using a hybrid ion trap-orbitrap mass spectrometer. Moreover, a transformation theory of FLP degradation was developed according to targeted fragmentation, accurate mass measurements, and degradation profiles. These analyses showed that the products originated from a series of chemical reactions involving dechlorination, hydroxyl substitution, cleavage and oxidation, and were further confirmed based on molecular electrostatic potential and molecular orbital theory. In addition, the stability and toxicity of FLP and its transformation products were tested using the Toxicity Estimation Software Tool (T.E.S.T.) and the Ecological Structure Activity Relationships (ECOSAR) program. Products F1, F2 and F3 were found to be toxic substances, but their toxicity to aquatic organisms was lower than that of FLP. However, they were more toxic to rats than FLP, and their physicochemical properties were more stable. Overall, OMBT is a highly effective method for FLP removal during wastewater treatment.

Impurity identification and quantification for arginine vasopressin by liquid chromatography/high-resolution mass spectrometry.

For pharmaceutical quality control, impurities may have unexpected pharmacological or toxicological effects on quality, safety, and efficacy of drugs. Arginine vasopressin (AVP) is an important cyclic peptide drug that is mainly used for the treatment of diabetes insipidus and esophageal varices bleeding. With the advancement made in analytical techniques, liquid chromatography/high-resolution mass spectrometry (LC/HRMS) has emerged as a critical technique for the identification and quantification of structurally related peptide impurities in AVP. An LC/HRMS/MS-based method using a quadrupole ion trap-Orbitrap mass spectrometer operated in the positive ion electrospray ionization mode was developed for the determination and quantification of structurally related peptide impurities in AVP. Under optimized experimental conditions, three deamidation products, ([Glu4 ]AVP, [Asp5 ]AVP, and AVP acid), two amino acid deletion impurities (des-Pro7 -AVP and des-Gly9 -AVP), one amino acid insertion impurity (endo-Gly10a -AVP), one end chain reaction product (N-acetyl-AVP), and one AVP isomer were detected. Subsequent quantification using an external standard method estimated the total mass fraction of all structurally related peptide impurities in the AVP study material to be 30.3 mg/g with an expanded uncertainty of 3.0 mg/g (k = 2). This study complements the AVP impurity profile and improves the separation and discovery of other potential impurities in vasopressin analogues.

Profiling and identification of in vivo metabolism of rosmarinic acid in rats

Rosmarinic acid,a hydrosoluble polyphenolic hydroxyl compound,is the active ingredient in such traditional Chinese medicines as Menthae Haplocalycis Herba,Salviae Miltiorrhizae Radix et Rhizoma,Rosemary,Perillae Folium. Because of its good anti-inflammatory,anti-oxidant and anti-tumor effects,it is widely used in food,medicine and other fields. However,the metabolic process and metabolites of rosmarinic acid in vivo have not been completely defined. In this study,an efficient method of ultra-high performance liquid chromatography combined with linear ion trap-Orbitrap(UHPLC-LTQ-Orbitrap) mass spectrometer was used to analyze the metabolites in vivo of rosmarinic acid in rats. Plasma,urine and feces samples were collected after oral administration of rosmarinic acid. After biological samples were processed by solid phase extraction,Acquity UPLC  BEH C18 column(2. 1 mm × 100 mm,1. 7 μm) was used with 0. 1% formic acid(A)-acetonitrile(B) solution as the mobile phase at the speed of 0. 30 m L·min-1 and temperature of 35 ℃ under gradient conditions. The plasma,urine,feces and the blank samples were then analyzed by ESI-LTQ-Orbitrap under both negative and positive ion modes. Based on the accurate mass measurement(<5),MS/MS fragmentation patterns,standards and literatures,a total of 36 metabolites were screened out and identified in the biological samples collected from rats after intragastric administration. Three were identified 3 from rat plasma,31 from urine,and 7 from feces. The main metabolic pathways of rosmarinic acid in rats can be divided into five parts. Rosmarinic acid were first decomposed into small molecules,such as trans-caffeic acid,coumaric acid,m-hydroxybenzoic acid and Danshensu,which were followed by sulfation,methylation,glucuronic acid conjugation and glucose conjugation. The results showed that UHPLC-LTQ-Orbitrap mass spectrometer could be used to analyze the metabolism of rosmarinic acid in rats,and provide reference for further studies on toxicology,pharmacodynamics and secondary development of Chinese medicine.

Metabolomic Analysis Identifies Alterations of Amino Acid Metabolome Signatures in the Postmortem Brain of Alzheimer's Disease.

Despite significant advances in neuroscience research over the past several decades, the exact cause of AD has not yet fully understood. The metabolic hypothesis as well as the amyloid and tau hypotheses have been proposed to be associated with AD pathogenesis. In order to identify metabolome signatures from the postmortem brains of sporadic AD patients and control subjects, we performed ultra performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer (UPLC-LTQ–Orbitrap-MS). Not only our study identified new metabolome signatures but also verified previously known metabolome profiles in the brain. Statistical modeling of the analytical data and validation of the structural assignments discovered metabolic biomarkers associated with the AD pathogenesis. Interestingly, hypotaurin, myo-inositol and oxo-proline levels were markedly elevated in AD while lutamate and N-acetyl-aspartate were decreased in the postmortem brain tissue of AD patients. In addition, neurosteroid level such as cortisol was significantly increased in AD. Together, our data indicate that impaired amino acid metabolism is associated with AD pathogenesis and the altered amino acid signatures can be useful diagnostic biomarkers of AD. Thus, modulation of amino acid metabolism may be a possible therapeutic approach to treat AD.

Preliminary Automated Determination of Edibility of Alternative Foods: Non-Targeted Screening for Toxins in Red Maple Leaf Concentrate.

Alternative food supplies could maintain humanity despite sun-blocking global catastrophic risks (GCRs) that eliminate conventional agriculture. A promising alternative food is making leaf concentrate. However, the edibility of tree leaves is largely uncertain. To overcome this challenge, this study provides the methods for obtaining rapid toxics screening of common leaf concentrates. The investigation begins with a non-targeted approach using an ultra-high-resolution hybrid ion trap orbitrap mass spectrometer with electrospray ionization (ESI) coupled to an ultra-high pressure two-dimensional liquid chromatograph system on the most common North American leaf: the red maple. Identified chemicals from this non-targeted approach are then cross-referenced with the OpenFoodTox database to identify toxic chemicals. Identified toxins are then screened for formula validation and evaluated for risk as a food. The results after screening show that red maple leaf concentrate contains at least eight toxic chemicals, which upon analysis do not present substantial risks unless consumed in abundance. This indicates that red maple leaf is still a potential alternative food. The results are discussed in the context of expanding the analysis with open science and using leaf extract from other plants that are not traditionally used as foods to offset current global hunger challenges, and move to a more sustainable food system while also preparing for GCRs.

Rapidly identify compounds from danshen by using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer and predict its mechanisms of intervening thrombotic diseases

Danshen, having good efficacies of dilating the coronary artery, improving microcirculation and preventing thrombosis, is widely used in clinic to treat various thrombotic diseases in China. This study aimed to identifying and analyzing the chemical constituents of Danshen from different planting areas based on UHPLC-LTQ-Orbitrap MS and comprehensively predicting the potential pharmacological targets and pathways of the main chemical constituents that involve in the treatment of thrombotic diseases by using network pharmacology. As a result, we identified 870 single peaks from total ion current and accurately identified 19 compounds from Danshen in this study. Further analysis showed that these compounds had three types of accumulation patterns and contents of most compounds had significant differences in samples from different planting areas. Network pharmacological analysis showed that Tanshinone IIA, Tanshinone IIB, Dehydromiltirone, Cryptotanshinone, Danshenxinkun A, Danshenxinkun B and Neocryptotanshinone might intervene thrombotic diseases by adjusting the targets mainly involved in pathways of endocrine system, signal transduction, signaling molecules and interaction, cell motility, environmental adaptation and nervous system. This study provides a scientific basis for exploring the mechanism of Danshen in treating thrombotic diseases and controlling the quality of Danshen-related preparations.

Application of characteristic fragment filtering with ultra high performance liquid chromatography coupled with high-resolution mass spectrometry for comprehensive identification of components in Schisandrae chinensis Fructus.

An integrated strategy of characteristic fragment filtering combined with target database screening based on ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry was proposed for comprehensive profiling of components in Schisandrae chinensis Fructus. The strategy consisted of following five steps: (1) Representative standards were analyzed by ultra high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer for characteristic fragments and fragmentation rules of each structure type. (2) The raw data of 70% methanol extract was collected by ultra high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry. (3) The chemical components database that consisted of names, chemical formulas and structures of potential components in Schisandrae chinensis Fructus was established by summarizing previous literature to screen the collected liquid chromatography with mass spectrometry data and obtain matched compounds. (4) Characteristic fragments, literature, and reference standards were used to verify the matches. (5) Characteristic fragment filtering combined with online database querying was used to deduce potential new compounds. As a result, a total of 94 compounds were identified or characterized and 16 of them were potential new compounds. The study provided a reference for comprehensive characterization of ingredients in herbal medicine and formed the foundation for pharmacodynamic study of Schisandrae chinensis Fructus.

Abstract PD2-09: High-throughput phosphoproteomics (HTPS) in neoadjuvant (NEO) breast cancer (BC) reveals clusters of extreme sensitivity to paclitaxel (T)

Abstract Background: NGS has elucidated the mutational landscape in BC. However, the correlation between mutational landscapes and complex phenotypic traits such as drug response is yet unclear. The genomic and transcriptomic aberrations coalesce into a diverse number of phosphorylation-driven patterns of activation of the proteome. These patterns are and associated to clinical outcomes of interest. The specific nature of the phosphopeptides in a profile versus another can be driven by a small number of activated kinases. In the past we relied on mass-spectrometry-based HTPS to build a kinase-based classification of triple-negative breast cancer (TNBC, Nat. Comms, in press), which is more parsimonious than gene-centered classifications and uncovers new actionable targets. We implemented this methodology to find predictors of response to T in early BC. Methods: Training set: fresh baseline and day+15 tumor biopsies form a trial in NEO HER2-negative BC (N=139) that randomized patients to T (80 mg/m2 weekly x12) plus placebo or nintedanib (150 mg b.i.d) were processed with a hybrid ion trap-orbitrap mass spectrometer after TiO2 phospho-enrichment. Phosphoprofiles were compared pair-wise according to the following factors: day 0 versus day + 15, standard versus experimental arm, pathologic response Symmans/Pusztai (Symmans) 0/1 vs 2/3. Kinases driving each phosphoprofile were solved by an in silico algorithm termed kinase-set enrichment analysis (KSEAS). Validation set: an in-house designed and previously validated mass-spectrometry-to-immunohistochemistry translation algorithm was used to validate the kinases enriched in the profiles from the baseline samples of patients achieving Symmans 0/1 (pCR) or Symmans=3 in the training set. Probes against those kinases were validated in an independent dataset of 160 HER2-negative patients receiving NEO T followed by AC. H-score of each activated kinase was divided in quartiles (Q1 to Q4), and upper-quartiles (Q1) of each kinase were tested in a multivariate logistic regression model to predict pCR adjusted by T, N, age, G, ER/PR and Ki67. Results: &amp;gt;2.5 millions of spectra were captured, identifying &amp;gt;35000 unique phosphopeptides mapping to &amp;gt;2500 unique proteins per sample. Training set: KSEAS revealed that a high activity of CDK4 and pP70S6K drove the baseline phosphoprofiles of the patients obtaining pCR in the T arm, whereas pSTAT3, pSrc and BARD drove that of the patients achieving Symmans= 3. In the validation set, H-score cut-offs for Q1 were 1.21, 0.69, 0.79, 1.64 and 1.54 for pP70S6K, CDK4, pSTAT3, pSrc and BARD. TNBC patients with Q1 pP70S6K or Q1 CDK4 achieved pCR in 100% of the cases (versus 45% in Q2-4). In the HR+ cases, the pCR rate for Q1 patients was 50% and 61% respectively. In the multivariate model (all patients), having Q1 pP70S6K or CDK4 multiplied by 2.3- and 3.6-fold, respectively, the probability of achieving pCR (P&amp;lt;0.05). Regarding Symmans=3, BARD and pSTAT3 lost significance in the validation but not pSrc (2.5-fold less probability of pCR, P&amp;lt;0.005). Conclusions: HTPS is a useful tool to find associations with complex traits. TNBC and HR+ patients with high pP70S6K or CDK4 receiving NEO T-based chemotherapy achieve very high rates of pCR. Citation Format: Quintela-Fandino M, Lluch A, Manso L, Calvo I, Cortes J, Garcia-Saenz JA, Zagorac I, Tapial-Martinez P, Gomez-Lopez G, Fustero C, Muñoz J, Gonzalez-Cortijo L, Mouron S. High-throughput phosphoproteomics (HTPS) in neoadjuvant (NEO) breast cancer (BC) reveals clusters of extreme sensitivity to paclitaxel (T) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-09.

High-throughput proteomic analysis of candidate biomarker changes in gingival crevicular fluid after treatment of chronic periodontitis.

Untargeted, high-throughput proteomics methodologies have great potential to aid in identifying biomarkers for the diagnosis of periodontal disease. The application of such methods to the discovery of candidate biomarkers for the resolution of periodontal inflammation after periodontal therapy has been investigated. Gingival crevicular fluid samples were collected from 10 patients diagnosed with chronic periodontitis at baseline and 1, 5, 9 and 13weeks after completion of mechanical periodontal treatment. Clinical indices of periodontal disease, including probing depth, recession, clinical attachment level and bleeding on probing, were recorded at baseline and 13weeks. Samples were analyzed using an online liquid chromatography-nanoelectrospray-hybrid ion trap-Orbitrap mass spectrometer. Spectra were processed with the PILOT_PROTEIN proteomics software suite. Clinical parameters were significantly improved 13weeks after treatment (Wilcoxon signed ranks test, P<.05). From the substantial number of identified proteins, a small subset was extracted by filter methods that included temporal pattern matching, logistic function fitting and mixed-integer linear optimization. This subset includes azurocidin, lysozyme C and myosin-9 as candidate biomarkers prominent at baseline and alpha-smooth muscle actin as prominent 13weeks after treatment. Cross-validation studies yielded average predictive accuracy and area under the curve of 0.900 and 0.930, respectively. High-throughput proteomic analysis can contribute to identifying endpoints of periodontal therapy. These candidate biomarkers should be evaluated for clinical efficacy.

Metabolites of tanshinone Ⅰ and tanshinone ⅡA in vivo in rats

An efficient method of ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap (UHPLC-LTQ-Orbitrap) mass spectrometer was established to elucidate the in vivo metabolites of tanshinone Ⅰ and tanshinone ⅡA in rats. Urine and plasma samples were collected after oral gavage. After processing biological sample by solid phase extraction, Waters ACQUITY HPLC BEH C₁₈ column (2.1 mm×100 mm, 1.7 μm) was used with 0.1% formic acid (A) - acetonitrile (B) solution as the mobile phase for gradient elution. The plasma, urine and the blank samples were then analyzed by ESI-LTQ-Orbitrap equipped with an ESI ion source under positive ion mode. On the basis of the accurate mass measurements, multiple mass spectra and comparison of data with published literature, a total of 26 metabolites were tentatively identified and characterized in the rat samples. Among them, 7 metabolites were derived from tanshinone Ⅰ through metabolic pathways of glucuronide conjugation, hydroxylation, reduction reaction, demethylation reaction, methylation, sulfate conjugation and their composite reactions. Nineteen metabolites were derived from tanshinone ⅡA through metabolic pathways of hydroxylation, reduction reaction, methylation, sulfate conjugation, glucuronidation, glucosylation and their complicated reactions. The results showed that the metabolism of tanshinone Ⅰ and tanshinone ⅡA in rats could be comprehensively clarified by using UHPLC-LTQ-Orbitrap mass spectrometer, providing material basis for the further research in terms of pharmacodynamics, toxicology, and secondary development of Chinese medicine.

Flaxseed oil rich in omega-3 protects aorta against inflammation and endoplasmic reticulum stress partially mediated by GPR120 receptor in obese, diabetic and dyslipidemic mice models

The “first hit” to atherogenesis is driven by toll-like receptor 4, endoplasmic reticulum stress and ultimately metabolic dysfunction. In this study, we hypothesized that a flaxseed oil-enriched diet (FS) abolishes these inflammatory signaling pathway and restore metabolic homeostasis by activating the fatty acid receptor GPR120 in aorta of obese mice. Glucose homeostasis was assessed by GTT and ITT; lipidomics was performed using a Hybrid Ion Trap-Orbitrap Mass Spectrometer; serum lipids were measured using colorimetric assays; GPR120 and infiltrating macrophages were analyzed by immunofluorescence; protein immunoprecipitation and gene expression were evaluated by Western blot and RT-PCR, respectively. There were no differences in body weight and food intake between the groups from both strains (Swiss and LDLr-KO mice). GTT and cholesterol levels were improved by FS in both mice models. Lipidomics showed an increase in ω3 (C18:3) content, meanwhile stearic acid (C18:0) was not detected in endothelial tissue in response to FS. Moreover, FS markedly decreased pro-inflammatory (IL-1β, TNF-α, pIκBα, pIKKβ) and unfolded protein response markers (ATF6 and GRP78) in aorta. In Swiss mice, GPR120 was partially involved in the ω3-mediated anti-inflammatory actions, disrupting TLR4 pathway, but not in LDLr-KO mice. Partial replacement of dietary saturated by unsaturated ω3 fatty acids contributes to inhibition of cardiovascular risk markers, pro-inflammatory cytokines and ER stress sensors and effectors in the aorta. However, downregulation of inflammation is not mediated by arterial GPR120 activation.

Profiling and identification of chlorogenic acid metabolites in rats by ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer

1. Chlorogenic acids (CGAs), one kind of major bioactive constituents isolated from Flos Lonicera Japonica, possess many biological activities, such as antibacterial, antioxidant and antiviral activities. In this study, we established an efficient strategy using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) to profile the in vivo metabolic fate of CGAs in rat urine and plasma.2. The extract from Flos Lonicera Japonica was orally administrated to Sprague-Dawley (SD) rats at a dose of 1000 mg/kg body weight. Then, a combination of various post-acquisition data mining methods, including high-resolution extracted ion chromatogram (HREIC) and multiple mass defect filters (MMDFs) and diagnostic product ions (DPIs), were adopted to characterize the known and unknown CGA metabolites in SD rats.3. As a result, a total of 68 CGA metabolites were unambiguously or tentatively screened and characterized. These metabolites, including 18 prototype compounds and 50 metabolites, were deduced to be yielded via methylation, hydrogenation, demethylation, dehydration, sulfate conjugation, glucuronide conjugation, glycosylation conjugation and their composite reactions, which mainly occurred to caffeoylquinic acids, dicaffeoylquinic acids, p-coumaroylquinic acids and feruloylquinic acids.4. In conclusion, this study profiled CGA metabolites, which are useful in understanding the in vivo metabolic fate, effective forms, and pharmacological and toxic actions of CGAs.

Profiling and identification of (-)-epicatechin metabolites in rats using ultra-high performance liquid chromatography coupled with linear trap-Orbitrap mass spectrometer.

(-)-Epicatechin (EC), an optical antipode of (+)-catechin (C), possesses many potential significant health benefits. However, the in vivo metabolic pathway of EC has not been clarified yet. In this study, an efficient strategy based on ultra-high performance liquid chromatography coupled with a linear ion trap-Orbitrap mass spectrometer was developed to profile and characterize EC metabolites in rat urine, faeces, plasma, and various tissues. Meanwhile, post-acquisition data-mining methods including high-resolution extracted ion chromatogram (HREIC), multiple mass defect filters (MMDFs), and diagnostic product ions (DPIs) were utilized to screen and identify EC metabolites from HR-ESI-MS1 to ESI-MSn stage. Finally, a total of 67 metabolites (including parent drug) were tentatively identified based on standard substances, chromatographic retention times, accurate mass measurement, and relevant drug biotransformation knowledge. The results demonstrated that EC underwent multiple in vivo metabolic reactions including methylation, dehydration, hydrogenation, glucosylation, sulfonation, glucuronidation, ring-cleavage, and their composite reactions. Among them, methylation, dehydration, glucosylation, and their composite reactions were observed only occurring on EC when compared with C. Meanwhile, the distribution of these detected metabolites in various tissues including heart, liver, spleen, lung, kidney, and brain were respectively studied. The results demonstrated that liver and kidney were the most important organs for EC and its metabolites elimination. In conclusion, the newly discovered EC metabolites significantly expanded the understanding on its pharmacological effects and built the foundation for further toxicity and safety studies. Copyright © 2017 John Wiley & Sons, Ltd.

Simultaneous Qualitation and Quantitation of Chlorogenic Acids in Kuding Tea Using Ultra-High-Performance Liquid Chromatography-Diode Array Detection Coupled with Linear Ion Trap-Orbitrap Mass Spectrometer.

Kuding tea, the leaves of Ilex Kudingcha C.J. Tseng, has been applied for treating obesity, hypertension, cardiovascular disease, hyperlipidemia, and so on. The chlorogenic acids (CGAs) in Kuding tea have shown excellent antioxidative, antiobesity, anti-atherosclerotic and anticancer activities. Nevertheless, the chemical profiles of CGAs in Kuding tea have not been comprehensively studied yet, which hinders further quality control. In the present study, a sensitive ultra-high-performance liquid chromatography-diode array detection coupled with a linear ion trap-Orbitrap (UHPLC-DAD-LTQ-Orbitrap) method was established to screen and identify CGAs in Kuding tea. Six CGA standards were first analyzed in negative ion mode with a CID-MS/MS experiment and then the diagnostic product ions (DPIs) were summarized. According to the retention behavior in the RP-ODS column, accurate mass measurement, DPIs and relevant bibliography data, a total of 68 CGA candidates attributed to 12 categories were unambiguously or preliminarily screened and characterized within 18 min of chromatographic time. This was the first systematic report on the distribution of CGAs in Kuding tea. Meanwhile, the contents of 6 major CGAs in Kuding tea were also determined by the UHPLC-DAD method. All the results indicated that the established analytical method could be employed as an effective technique for the comprehensive and systematic characterization of CGAs and quality control of the botanic extracts or Chinese medicinal formulas that contain various CGAs.

Multi-Omics Evidence for Inheritance of Energy Pathways in Red Blood Cells

IntroductionEach year over 90 million units of blood are transfused worldwide. Our dependence on this blood supply requires optimized blood collection and storage. During storage, red blood cells (RBCs) undergo degenerative processes resulting in altered metabolic characteristics. In the past decade numerous studies have implicated longer storage of RBCs in adverse patient outcomes. The post-storage ATP level in blood is the single best predictor of transfused RBC in vivo recovery. Although the rate of ATP decline is highly variable between individuals, post-storage ATP levels are primarily determined by inheritance. Understanding the effect of storage on energy metabolism pathways is thus of vital importance to maintaining a safe and effective blood supply.MethodsWe performed comprehensive metabolomics and proteomics studies of mono- and di-zygotic twin pairs to measure heritability of molecules and identify correlations with ATP and other markers in energy metabolism. Metabolite levels were measured at six time points from 0-56 days to elucidate changes that occur during storage. An obstacle for RBC proteomics is the massive quantity of hemoglobin, constituting 97% of protein material. This was avoided by preparing RBC membrane fractions, which mitigated the need for hemoglobin depletion. All proteomics data was collected on an Orbitrap Elite hybrid ion trap-orbitrap mass spectrometer (Thermo Fisher Scientific). Metabolomics data was collected by Metabolon Inc. and was collated with proteomics results to give a complete view of RBC metabolism.Preliminary DataOur optimized method for collecting proteomics data in RBCs has yielded the greatest depth of coverage observed without the use of commercial hemoglobin depletion. Purified RBCs were lysed and centrifuged to collect membrane fractions allowing us to identify 1280 proteins and 330 metabolites from mono and di-zygotic twins. Of these, 146 proteins and 148 metabolites were found to be over 30% heritable. We observe a high degree of heritability in metabolites involved in energy metabolism, especially glycolysis. This is supported by the heritability in key regulatory enzymes including phosphofructokinase (PFK) (57%) and bisphosphoglycerate mutase (BPGM) (50%). Additionally we observe high correlations between both glycolytic proteins and metabolites suggesting that this crucial energy metabolism pathway is inherited en blocat various levels.A number of the correlations we observed can be combined to produce a model to predict post-storage ATP levels. Five key parameters in this model include PFK, carbonic anhydrase 1 (CA1), band 3, BPGM, and pH. Strikingly, concentrations of all protein components of this model were at least 45% heritable. Band 3, BPGM, and CA1 correlate negatively with post storage ATP levels and together shuttle flux away from glycolysis and ATP production. We also observe a positive correlation between pH and post-storage ATP.A negative correlation observed between CA1, which is 84% heritable, and post-storage ATP, is especially significant in that it provides a hypothetical model for the heritability of ATP decline during storage. Our model proposes that RBC units, which are stored in gas permeable bags allowing CO2 to diffuse into the bag, are subject to genetically determined, CA1-mediated production of carbonic acid, resulting in inhibition of PFK. This model is further supported by negative correlations between CA1 and pH during storage. We propose that heritable concentrations of CA1 negatively influence pH, which allosterically inhibits PFK and impedes energy metabolism and subsequently ATP production.We conclude that individuals inherit a phenotype composed of higher or lower concentrations of key energy metabolism proteins that regulate flux through glycolysis during RBC storage. Heritability of energy metabolism can result in markedly different RBC storage profiles and knowledge of heritable RBC energy metabolism can be used to improve and individualize RBC storage methods. DisclosuresHess:ASH: Patents & Royalties: 4 US patents related to RBC storage solution AS-7.

Proteotyping of biogas plant microbiomes separates biogas plants according to process temperature and reactor type.

BackgroundMethane yield and biogas productivity of biogas plants (BGPs) depend on microbial community structure and function, substrate supply, and general biogas process parameters. So far, however, relatively little is known about correlations between microbial community function and process parameters. To close this knowledge gap, microbial communities of 40 samples from 35 different industrial biogas plants were evaluated by a metaproteomics approach in this study.ResultsLiquid chromatography coupled to tandem mass spectrometry (Orbitrap Elite™ Hybrid Ion Trap-Orbitrap Mass Spectrometer) of all 40 samples as triplicate enabled the identification of 3138 different metaproteins belonging to 162 biological processes and 75 different taxonomic orders. The respective database searches were performed against UniProtKB/Swiss-Prot and seven metagenome databases. Subsequent clustering and principal component analysis of these data allowed for the identification of four main clusters associated with mesophile and thermophile process conditions, the use of upflow anaerobic sludge blanket reactors and BGP feeding with sewage sludge. Observations confirm a previous phylogenetic study of the same BGP samples that was based on 16S rRNA gene sequencing by De Vrieze et al. (Water Res 75:312–323, 2015). In particular, we identified similar microbial key players of biogas processes, namely Bacillales, Enterobacteriales, Bacteriodales, Clostridiales, Rhizobiales and Thermoanaerobacteriales as well as Methanobacteriales, Methanosarcinales and Methanococcales. For the elucidation of the main biomass degradation pathways, the most abundant 1 % of metaproteins was assigned to the KEGG map 1200 representing the central carbon metabolism. Additionally, the effect of the process parameters (i) temperature, (ii) organic loading rate (OLR), (iii) total ammonia nitrogen (TAN), and (iv) sludge retention time (SRT) on these pathways was investigated. For example, high TAN correlated with hydrogenotrophic methanogens and bacterial one-carbon metabolism, indicating syntrophic acetate oxidation.ConclusionsThis is the first large-scale metaproteome study of BGPs. Proteotyping of BGPs reveals general correlations between the microbial community structure and its function with process parameters. The monitoring of changes on the level of microbial key functions or even of the microbial community represents a well-directed tool for the identification of process problems and disturbances.Graphical abstractCorrelation between the different orders and process parameter, as well as principle component analysis of all investigated biogas plants based on the identified metaproteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0572-4) contains supplementary material, which is available to authorized users.

Profiling and Identification of the Metabolites of Evodiamine in Rats using Ultra–Performance Liquid Chromatography with Linear Ion Trap Orbitrap Mass Spectrometer

Purpose: To develop a highly sensitive and specific ultra-performance liquid chromatography with linear ion trap Orbitrap mass spectrometer (UPLC-LTQ-Orbitrap) method to profile and identify the metabolites of evodiamine in rats. Methods: First, blood samples were collected after oral administration of evodiamine to rats (50 mg/kg). Next, the plasma samples were pretreated using a solid-phase extraction (SPE) method. Finally, all the samples were analyzed by ultra-performance liquid chromatography LTQ-Orbitrap mass spectrometry (UPLC-LTQ-Orbitrap) coupled with electrospray ionization source (ESI) in negative mode. Results: A total of 7 metabolites (2 phase I and 4 phase II metabolites, including 4 new metabolites, viz, 10-hydroxyevodiamine sulfate, 10-hydroxyevodiamine sulfate, 10-hydroxyevodiamine glucuronide and 3-hydroxyevodiamine glucuronide) as well as the parent drug itself , were detected and identified based on accurate mass measurements, fragmentation patterns, and chromatographic retention times. The in vivo metabolic reactions of evodiamine in rats were hydroxylation, hydroxylation + sulfate conjugation, and hydroxylation + glucuronidation. Conclusion: These results provide better understanding of the metabolism of evodiamine as well as strong indications of the effective forms of the drug in vivo.