Linear Ion trap-Orbitrap Mass Spectrometer Research Articles

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.

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.

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.

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.

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.

Detection and characterization of the metabolites of rutaecarpine in rats based on ultra-high-performance liquid chromatography with linear ion trap-Orbitrap mass spectrometer

Context: Rutaecarpine is an active indoloquinazoline alkaloid ingredient originating from Evodia rutaecarpa (Wu-zhu-yu in Chinese), which possesses a variety of effects. However, its metabolism has not been investigated thoroughly yet.Objective: This study develops a highly sensitive and effective method for detection and characterization of the metabolites of rutaecarpine in Sprague–Dawley (SD) rats.Materials and methods: In this study, an efficient method was developed using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer (UHPLC–LTQ-Orbitrap MS) to detect the metabolism profile of rutaecarpine in rat plasma. First, a blood sample (1 mL) was withdrawn 2 h after oral administration of rutaecarpine in SD rats (50 mg/kg). Second, the blood was centrifuged at 4000 rpm for 10 min and pretreated by solid-phase extraction method. Third, 2 μL of the plasma was injected into UHPLC–LTQ-Orbitrap MS for analysis. Finally, the metabolites of rutaecarpine were tentatively identified based on accurate mass measurements, fragmentation patterns and chromatographic retention times.Results: A total of 16 metabolites (four new metabolites, viz., dihydroxylation and sulphate conjugation products of rutaecarpine (M8–M11)) as well as parent drug itself, including three phase I and 12 phase II metabolites were detected and identified in rat plasma. Hydroxylation, sulphate conjugation and glucuronidation were confirmed as the primary metabolic pathways for rutaecarpine in rat plasma.Discussion and conclusion: These results provide an insight into the metabolism of rutaecarpine and also can give strong indications on the effective forms of rutaecarpine in vivo.

Neuroprotective Activity of Grape Seed and Skin Extract Against Lithium Exposure Using Proteomic Research.

Lithium (Li) has raised scientific concern because it represents a serious problem threatening human health. This study aimed firstly at analyzing and potentially quantifying the impact of Li and grape seed and skin extract (GSSE) separately and, secondly, describing the possible neuroprotective activity of GSSE against Li toxicity. To this end, rats were exposed for30 days to different Li concentrations (0, 2, and 100mg/kg bw), to GSSE (4000mg/kg bw), and to binary mixture of Li and GSSE. Liquid chromatography (HPLC-MS/MS) analysis used for GSSE showed that 15 phenolic compounds are present in the extract. Significant modifications of proteins were detected in the brain using proteomics research after treatment. Proteins were successfully identified by a linear ion trap-Orbitrap mass spectrometer. These proteins can be roughly related to oxidative stress, glycolysis, signaling pathway, and inflammation. Additionally, proteins involved in cell junction such as myosin, spectrin, tubulin, ERM-binding phosphoprotein, and dynein were also affected by Li exposure. Dose response was detected for most expressed proteins after Li treatment. In contrast, GSSE induced the expression and/or the stabilization of some proteins changed after Li treatment in the brain showing its neuroprotective activity. These data demonstrate that proteomic analysis is a powerful tool to provide valuable insights into mechanisms of toxicity of Li in the nervous system of Wistar rats. To our knowledge, this is the first evidence of using GSSE as neuroprotective model against Li toxicity. These findings provide impetus for future investigation on GSSE against other toxic chemicals.

Simultaneous analysis of tocopherols, tocotrienols, phospholipids, γ-oryzanols and β-carotene in rice by ultra-high performance liquid chromatography coupled to a linear ion trap-orbitrap mass spectrometer

An UHPLC-LTQ-Orbitrap MS method was developed for the simultaneous analysis of tocopherols, tocotrienols, phospholipids, γ-oryzanols and β-carotene in rice.

A strategy for comprehensive identification of sequential constituents using ultra-high-performance liquid chromatography coupled with linear ion trap–Orbitrap mass spectrometer, application study on chlorogenic acids in Flos Lonicerae Japonicae

The analytical methodologies for evaluation of multi-component system in traditional Chinese medicines (TCMs) have been inadequate or unacceptable. As a result, the unclarity of multi-component hinders the sufficient interpretation of their bioactivities. In this paper, an ultra-high-performance liquid chromatography coupled with linear ion trap–Orbitrap (UPLC–LTQ–Orbitrap)-based strategy focused on the comprehensive identification of TCM sequential constituents was developed. The strategy was characterized by molecular design, multiple ion monitoring (MIM), targeted database hits and mass spectral trees similarity filter (MTSF), and even more isomerism discrimination. It was successfully applied in the HRMS data-acquisition and processing of chlorogenic acids (CGAs) in Flos Lonicerae Japonicae (FLJ), and a total of 115 chromatographic peaks attributed to 18 categories were characterized, allowing a comprehensive revelation of CGAs in FLJ for the first time. This demonstrated that MIM based on molecular design could improve the efficiency to trigger MS/MS fragmentation reactions. Targeted database hits and MTSF searching greatly facilitated the processing of extremely large information data. Besides, the introduction of diagnostic product ions (DPIs) discrimination, ClogP analysis, and molecular simulation, raised the efficiency and accuracy to characterize sequential constituents especially position and geometric isomers. In conclusion, the results expanded our understanding on CGAs in FLJ, and the strategy could be exemplary for future research on the comprehensive identification of sequential constituents in TCMs. Meanwhile, it may propose a novel idea for analyzing sequential constituents, and is promising for quality control and evaluation of TCMs.

Comprehensive characterization of the in vitro and in vivo metabolites of geniposide in rats using ultra-high-performance liquid chromatography coupled with linear ion trap–Orbitrap mass spectrometer

1. Geniposide (genipin 1-O-glucose), one of the major bioactive constituents isolated from Fructus Gardeniae, possesses many biological activities. In this study, an efficient strategy was developed using ultra-high-performance liquid chromatography coupled with linear ion trap–Orbitrap mass spectrometer (UPLC–LTQ–Orbitrap) to profile the in vitro and in vivo metabolic patterns of geniposide in rat liver microsomes (RLMs), plasma, urine, and various tissues. And post-acquisition data-mining methods including extracted ion chromatogram (EIC), multiple mass defect filters (MMDF), fragment ion searching (FISh), and isotope pattern filtering (IPF) were adopted to characterize the known and unknown metabolites.2. A total of 33 metabolites were detected and interpreted according to accurate mass measurement, diagnostic fragment ions, relevant drug biotransformation knowledge, and bibliography data. Among them, 17 metabolites were detected in the plasma, 31 metabolites were identified in the urine, six metabolites could be found in rat heart, 12 in liver, three in spleen, six in lung, 12 in kidney, six in brain, and four in RLMs.3. A series of corresponding reactions such as hydrolysis, hydroxylation, taurine conjugation, hydrogenation, decarboxylation, demethylation, sulfate conjugation, cysteine S-conjugation, glucosylation, and their composite reactions were all discovered.4. The results could provide comprehensive insights and guidance for elucidation of side effect mechanism and safety monitoring as well as for rational formulation design in drug delivery system. The newly discovered geniposide metabolites could be targets for future metabolism studies on the important chemical constituents from herbal medicines.

Profiling and identification of the metabolites of baicalin and study on their tissue distribution in rats by ultra-high-performance liquid chromatography with linear ion trap-Orbitrap mass spectrometer.

Baicalin (baicalein 7-O-glucuronide), which is one of the major bioactive constituents isolated from Scutellariae Radix, possesses many biological activities, such as antiallergic, antioxidation, and anti-inflammatory activities. In the present study, an efficient strategy was established using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer (UPLC-LTQ-Orbitrap MS) to profile the in vivo metabolic fate of baicalin in rat plasma, urine, and various tissues. A combination of post-acquisition mining methods including extracted ion chromatogram (EIC) and multiple mass defect filters (MMDF) was adopted to identify the common and uncommon baicalin metabolites from the full mass scan data sets. Their structures were elucidated based on the accurate mass measurement, relevant drug biotransformation knowledge, the characteristic collision induced fragmentation pattern of baicalin metabolites, and bibliography data. Based on the proposed strategy, a total of 32 metabolites were observed and characterized. The corresponding reactions in vivo such as methylation, hydrolysis, hydroxylation, methoxylation, glucuronide conjugation, sulfate conjugation, and their composite reactions, were all discovered in the study. The results demonstrated that the rat liver and kidney are the most important organs for the baicalin metabolites presence. Six metabolites might play an important role in exerting pharmacological effects of baicalin in vivo. The newly discovered baicalin metabolites significantly expanded our understanding on its pharmacological effects, and could be targets for future studies on the important chemical constituents from herbal medicines.

Identification of metabolites of gardenin A in rats by combination of high-performance liquid chromatography with linear ion trap-Orbitrap mass spectrometer based on multiple data processing techniques.

Gardenin A is one of the less abundant hydroxylated polymethoxyflavonoids (OH-PMFs) in nature, and has many potential significant health benefits. In the present study, an efficient strategy was established using high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer to profile the in vivo metabolic fate of gardenin A in rat plasma and various tissues. First, an online LC-MS(n) data acquisition method was developed to trace all the probable metabolites. Second, a combination of offline data processing methods including extracted ion chromatography and multiple mass defect filters was employed to screen the common and uncommon metabolites from the background noise and endogenous components. Finally, structures of the metabolites were elucidated based on an accurate mass measurement, the diagnostic product ions of PMFs, and relevant drug biotransformation knowledge. Based on the proposed strategy, a total of 26 metabolites were observed and characterized. The results indicate that some biotransformations, such as methylation, demethoxylation, demethylation, glucuronide conjugation, sulfate conjugation and their composite reactions, have been discovered for OH-PMFs. Moreover, some diagnostic biotransformation pathways are summarized. Overall, this study gives us a first insight into the in vivo metabolism of gardenin A. The study also provides a practical strategy for rapidly screening and identifying metabolites, which can be widely applied for the other biotransformations.

Application of ultra-high pressure liquid chromatography linear ion-trap orbitrap to qualitative and quantitative assessment of pesticide residues

The analysis of pesticides residues using a last generation high resolution and high mass accuracy hybrid linear ion trap-Orbitrap mass spectrometer (LTQ-Orbitrap-MS) was explored. Pesticides were extracted from fruits, fish, bees and sediments by QuEChERS and from water by solid-phase with Oasis HLB cartridges. Ultra-high pressure liquid chromatography (UHPLC)–LTQ-Orbitrap mass spectrometer acquired full scan MS data for quantification, and data dependent (dd) MS2 and MS3 product ion spectra for identification and/or confirmation. The regression coefficients (r2) for the calibration curves (two order of magnitude up to the lowest calibration level) in the study were ≥0.99. The LODs for 54 validated compounds were ≤2ngmL−1 (analytical standards). The relative standard deviation (RSD), which was used to estimate precision, was always lower than 22%. The recovery of extraction and matrix effects ranged from 58 to 120% and from −92 to 52%, respectively. Mass accuracy was always ≤4ppm, corresponding to a maximum mass error of 1.6millimass units (mmu). This procedure was then successfully applied to pesticide residues in a set of the above-mentioned food and environmental samples. In addition to target analytes, this method enables the simultaneous detection/identification of non-target pesticides, pharmaceuticals, drugs of abuse, mycotoxins, and their metabolites.

Rapid screening of drugs of abuse in human urine by high-performance liquid chromatography coupled with high resolution and high mass accuracy hybrid linear ion trap-Orbitrap mass spectrometry

A novel analytical toxicology method has been developed for the analysis of drugs of abuse in human urine by using a high resolution and high mass accuracy hybrid linear ion trap-Orbitrap mass spectrometer (LTQ-Orbitrap-MS). This method allows for the detection of different drugs of abuse, including amphetamines, cocaine, opiate alkaloids, cannabinoids, hallucinogens and their metabolites. After solid-phase extraction with Oasis HLB cartridges, spiked urine samples were analysed by HPLC/LTQ-Orbitrap-MS using an electrospray interface in positive ionisation mode, with resolving power of 30,000 full width at half maximum (FWHM). Gradient elution off of a Hypersil Gold PFP column (50mm×2.1mm) allowed to resolve 65 target compounds and 3 internal standards in a total chromatographic run time of 20min. Validation of this method consisted of confirmation of identity, selectivity, linearity, limit of detection (LOD), lowest limits of quantification (LLOQ), accuracy, precision, extraction recovery and matrix effect. The regression coefficients (r2) for the calibration curves (LLOQ – 100ng/mL) in the study were ≥0.99. The LODs for 65 validated compounds were better than 5ng/ml except for 4 compounds. The relative standard deviation (RSD), which was used to estimate repeatability at three concentrations, was always less than 15%. The recovery of extraction and matrix effects were above 50 and 70%, respectively. Mass accuracy was always better than 2ppm, corresponding to a maximum mass error of 0.8 millimass units (mmu). The accurate masses of characteristic fragments were obtained by collisional experiments for a more reliable identification of the analytes. Automated data analysis and reporting were performed using ToxID software with an exact mass database. This procedure was then successfully applied to analyse drugs of abuse in a real urine sample from subject who was assumed to be drug addict.

Multiplex Mass Spectrometric Imaging with Polarity Switching for Concurrent Acquisition of Positive and Negative Ion Images

We have recently developed a multiplex mass spectrometry imaging (MSI) method which incorporates high mass resolution imaging and MS/MS and MS(3) imaging of several compounds in a single data acquisition utilizing a hybrid linear ion trap-Orbitrap mass spectrometer (Perdian and Lee, Anal. Chem. 82, 9393-9400, 2010). Here we extend this capability to obtain positive and negative ion MS and MS/MS spectra in a single MS imaging experiment through polarity switching within spiral steps of each raster step. This methodology was demonstrated for the analysis of various lipid class compounds in a section of mouse brain. This allows for simultaneous imaging of compounds that are readily ionized in positive mode (e.g., phosphatidylcholines and sphingomyelins) and those that are readily ionized in negative mode (e.g., sulfatides, phosphatidylinositols and phosphatidylserines). MS/MS imaging was also performed for a few compounds in both positive and negative ion mode within the same experimental set-up. Insufficient stabilization time for the Orbitrap high voltage leads to slight deviations in observed masses, but these deviations are systematic and were easily corrected with a two-point calibration to background ions.

Glycoproteomic Analysis of the Secretome of Human Endothelial Cells

Previous proteomics studies have partially unraveled the complexity of endothelial protein secretion but have not investigated glycosylation, a key modification of secreted and membrane proteins for cell communication. In this study, human umbilical vein endothelial cells were kept in serum-free medium before activation by phorbol-12-myristate-13 acetate, a commonly used secretagogue that induces exocytosis of endothelial vesicles. In addition to 123 secreted proteins, the secretome was particularly rich in membrane proteins. Glycopeptides were enriched by zwitterionic hydrophilic interaction liquid chromatography resins and were either treated with PNGase F and H218O or directly analyzed using a recently developed workflow combining higher-energy C-trap dissociation (HCD) with electron-transfer dissociation (ETD) for a hybrid linear ion trap–orbitrap mass spectrometer. After deglycosylation with PNGase F in the presence of H218O, 123 unique peptides displayed 18O-deamidation of asparagine, corresponding to 86 proteins with a total of 121 glycosylation sites. Direct glycopeptide analysis via HCD-ETD identified 131 glycopeptides from 59 proteins and 118 glycosylation sites, of which 41 were known, 51 were predicted, and 26 were novel. Two methods were compared: alternating HCD-ETD and HCD-product-dependent ETD. The former detected predominantly high-intensity, multiply charged glycopeptides, whereas the latter preferentially selected precursors with complex/hybrid glycans for fragmentation. Validation was performed by means of glycoprotein enrichment and analysis of the input, the flow-through, and the bound fraction. This study represents the most comprehensive characterization of endothelial protein secretion to date and demonstrates the potential of new HCD-ETD workflows for determining the glycosylation status of complex biological samples.