Fenofibrate metabolism in the cynomolgus monkey using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry-based metabolomics

Fenofibrate, widely used for the treatment of dyslipidemia, activates the nuclear receptor, peroxisome proliferator-activated receptor alpha. However, liver toxicity, including liver cancer, occurs in rodents treated with fibrate drugs. Marked species differences occur in response to fibrate drugs, especially between rodents and humans, the latter of which are resistant to fibrate-induced cancer. Fenofibrate metabolism, which also shows species differences, has not been fully determined in humans and surrogate primates. In the present study, the metabolism of fenofibrate was investigated in cynomolgus monkeys by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS)-based metabolomics. Urine samples were collected before and after oral doses of fenofibrate. The samples were analyzed in both positive-ion and negative-ion modes by UPLC-QTOFMS, and after data deconvolution, the resulting data matrices were subjected to multivariate data analysis. Pattern recognition was performed on the retention time, mass/charge ratio, and other metabolite-related variables. Synthesized or purchased authentic compounds were used for metabolite identification and structure elucidation by liquid chromatographytandem mass spectrometry. Several metabolites were identified, including fenofibric acid, reduced fenofibric acid, fenofibric acid ester glucuronide, reduced fenofibric acid ester glucuronide, and compound X. Another two metabolites (compound B and compound AR), not previously reported in other species, were characterized in cynomolgus monkeys. More importantly, previously unknown metabolites, fenofibric acid taurine conjugate and reduced fenofibric acid taurine conjugate were identified, revealing a previously unrecognized conjugation pathway for fenofibrate.

Radiation metabolomics. 5. Identification of urinary biomarkers of ionizing radiation exposure in nonhuman primates by mass spectrometry-based metabolomics

Mass spectrometry-based metabolomics has previously demonstrated utility for identifying biomarkers of ionizing radiation exposure in cellular, mouse and rat in vivo radiation models. To provide a valuable link from small laboratory rodents to humans, γ-radiation-induced urinary biomarkers were investigated using a nonhuman primate total-body-irradiation model. Mass spectrometry-based metabolomics approaches were applied to determine whether biomarkers could be identified, as well as the previously discovered rodent biomarkers of γ radiation. Ultra-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry analysis was carried out on a time course of clean-catch urine samples collected from nonhuman primates (n = 6 per cohort) exposed to sham, 1.0, 3.5, 6.5 or 8.5 Gy doses of (60)Co γ ray (∼0.55 Gy/min) ionizing radiation. By multivariate data analysis, 13 biomarkers of radiation were discovered: N-acetyltaurine, isethionic acid, taurine, xanthine, hypoxanthine, uric acid, creatine, creatinine, tyrosol sulfate, 3-hydroxytyrosol sulfate, tyramine sulfate, N-acetylserotonin sulfate, and adipic acid. N-Acetyltaurine, isethionic acid, and taurine had previously been identified in rats, and taurine and xanthine in mice after ionizing radiation exposure. Mass spectrometry-based metabolomics has thus successfully revealed and verified urinary biomarkers of ionizing radiation exposure in the nonhuman primate for the first time, which indicates possible mechanisms for ionizing radiation injury.

A new Time-Of-Flight Mass Spectrometer (TOF-MS) for noble gas analysis

Noble gas analysis in early solar system materials, which can provide valuable information about early solar system processes and timescales, are very challenging because of extremely low noble gas concentrations (ppt). We therefore developed a new compact sized (33 cm length, 7.2cm diameter, 1.3 L internal volume) Time-of-Flight (TOF) noble gas mass spectrometer for high sensitivity. We call it as Edel Gas Time-of-flight (EGT) mass spectrometer. The instrument uses electron impact ionization coupled to an ion trap, which allows us to ionize and measure all noble gas isotopes. Using a reflectron set-up improves the mass resolution. In addition, the reflectron set-up also enables some extra focusing. The detection is via MCPs and the signals are processed either via ADC or TDC systems. The objective of this work is to understand the newly developed Time-Of-Flight (TOF) mass spectrometer for noble gas analysis in presolar grains of the meteorites. Chapter 1 briefly introduces the basic idea and importance of the instrument. The physics relevant to time-of-flight mass spectrometry technique is discussed in the Chapter 2 and Chapter 3 will present the oxidation technique of nanodiamonds of the presolar grains by using copper oxide. Chapter 4 will present the details about EGT data analysis software. Chapter 5 and Chapter 6 will explain the details about EGT design and operation. Finally, the performance results will be presented and discussed in the Chapter 7, and whole work is summarized in Chapter 8 and also outlook of the future work is given.

Performance evaluation of a miniature laser ablation time-of-flight mass spectrometer designed for in situ investigations in planetary space research

Key performance features of a miniature laser ablation time-of-flight mass spectrometer designed for in situ investigations of the chemical composition of planetary surfaces are presented. This mass spectrometer is well suited for elemental and isotopic analysis of raw solid materials with high sensitivity and high spatial resolution. In this study, ultraviolet laser radiation with irradiances suitable for ablation (< 1 GW/cm2) is used to achieve stable ion formation and low sample consumption. In comparison to our previous laser ablation studies at infrared wavelengths, several improvements to the experimental setup have been made, which allow accurate control over the experimental conditions and good reproducibility of measurements. Current performance evaluations indicate significant improvements to several instrumental figures of merit. Calibration of the mass scale is performed within a mass accuracy (Δm/m) in the range of 100 ppm, and a typical mass resolution (m/Δm) ~600 is achieved at the lead mass peaks. At lower laser irradiances, the mass resolution is better, about (m/Δm) ~900 for lead, and limited by the laser pulse duration of 3 ns. The effective dynamic range of the instrument was enhanced from about 6 decades determined in previous study up to more than 8 decades at present. Current studies show high sensitivity in detection of both metallic and non-metallic elements. Their abundance down to tens of ppb can be measured together with their isotopic patterns. Due to strict control of the experimental parameters, e.g. laser characteristics, ion-optical parameters and sample position, by computer control, measurements can be performed with high reproducibility. Copyright © 2012 John Wiley & Sons, Ltd.

Identification of animal Pasteurellaceae by MALDI-TOF mass spectrometry

Species of the family Pasteurellaceae play an important role as primary or opportunistic, predominantly respiratory, pathogens in domestic and wild animals. Some of them cause severe disease with high economic losses in commercial animal husbandry. Hence, rapid and accurate differentiation of Pasteurellaceae is important and signifies a particular challenge to diagnostic laboratories. Identification and differentiation of Pasteurellaceae is mostly done using phenotypic tests or genetic identification based on sequence similarity of housekeeping genes, such as the rrs gene encoding the 16S ribosomal RNA (16S rRNA). Both approaches are time consuming, laborious, and costly, therefore often delaying the final diagnosis of disease or epidemics. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry represents an alternative rapid and reliable method for the differentiation of most members of the family Pasteurellaceae. It is able to differentiate within a few minutes the currently known 18 genera and most of the over 60 species and subspecies of Pasteurellaceae including many members encountered in veterinary diagnostic laboratories. A few closely related species and subspecies that cannot be discriminated by MALDI-TOF are easily identified further by complementary simple tests, such as hemolysis done simultaneously or routinely during pathogen isolation.

Electrospray tandem mass spectrometry of mixed-sequence RNA/DNA oligonucleotides

The fragmentation of electrospray-generated multiply deprotonated RNA and mixed-sequence RNA/DNA pentanucleotides upon low-energy collision-induced dissociation (CID) in a hybrid quadrupole time-of-flight mass spectrometer was investigated. The goal of unambiguous sequence identification of mixed-sequence RNA/DNA oligonucleotides requires detailed understanding of the gas-phase dissociation of this class of compounds. The two major dissociation events, base loss and backbone fragmentation, are discussed and the unique fragmentation behavior of oligoribonucleotides is demonstrated. Backbone fragmentation of the all-RNA pentanucleotides is characterized by abundant c-ions and their complementary y-ions as the major sequence-defining fragment ion series. In contrast to the dissociation of oligodeoxyribonucleotides, where backbone fragmentation is initiated by the loss of a nucleobase which subsequently leads to the formation of the w- and [a-base]-ions, backbone dissociation of oligoribonucleotides is essentially decoupled from base loss. The different behavior of RNA and DNA oligonucleotides is related to the presence of the 2'-hydroxyl substituent, which is the only structural alteration between the DNA and RNA pentanucleotides studied. CID of mixed-sequence RNA/DNA pentanucleotides results in a combination of the nucleotide-typical backbone fragmentation products, with abundant w-fragment ions generated by cleavage of the phosphodiester backbone adjacent to the deoxy building blocks, whereas backbone cleavage adjacent to ribonucleotides induces the formation of c- and y-ions. (C) 2002 American Society for Mass Spectrometry.

The role of mass spectrometry-based metabolomics in medical countermeasures against radiation

Radiation metabolomics can be defined as the global profiling of biological fluids to uncover latent, endogenous small molecules whose concentrations change in a dose-response manner following exposure to ionizing radiation. In response to the potential threat of nuclear or radiological terrorism, the Center for High-Throughput Minimally Invasive Radiation Biodosimetry was established to develop field-deployable biodosimeters based, in part, on rapid analysis by mass spectrometry of readily and easily obtainable biofluids. In this review, we briefly summarize radiation biology and key events related to actual and potential nuclear disasters, discuss the important contributions the field of mass spectrometry has made to the field of radiation metabolomics, and summarize current discovery efforts to use mass spectrometry-based metabolomics to identify dose-responsive urinary constituents, and ultimately to build and deploy a noninvasive high-throughput biodosimeter.

Enantioselective determination of ondansetron and 8-hydroxyondansetron in human plasma from recovered surgery patients by liquid chromatography-tandem mass spectrometry

A liquid chromatographic-mass spectrometric assay with atmospheric pressure chemical ionization for quantification of ondansetron and its main metabolite 8-hydroxyondansetron in human plasma was presented. The enantiomeric separation was achieved on a Chiralcel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate). The validation data were within the required limits. The assay was successfully applied to authentic plasma samples. Quantitative results from postoperative patients receiving ondansetron demonstrated a great interindividual variability in postoperative plasma drug concentrations, the metabolites were not detected in their unconjugated form. A wide variation in the S-(+)-/R-(-)-ondansetron concentration ratio between 0.14 and 7.18 is indicative for a stereoselective disposition or metabolism. In further studies CYP2D6 and CYP3A4 genotype dependent metabolism of ondansetron enantiomers as well as of co-administered drugs and clinical efficacy of the medication should be tested.

Informatics for cross-sample analysis with comprehensive two-dimensional gas chromatography and high-resolution mass spectrometry (GCxGC-HRMS)

This paper describes informatics for cross-sample analysis with comprehensive two-dimensional gas chromatography (GCxGC) and high-resolution mass spectrometry (HRMS). GCxGC-HRMS analysis produces large data sets that are rich with information, but highly complex. The size of the data and volume of information requires automated processing for comprehensive cross-sample analysis, but the complexity poses a challenge for developing robust methods. The approach developed here analyzes GCxGC-HRMS data from multiple samples to extract a feature template that comprehensively captures the pattern of peaks detected in the retention-times plane. Then, for each sample chromatogram, the template is geometrically transformed to align with the detected peak pattern and generate a set of feature measurements for cross-sample analyses such as sample classification and biomarker discovery. The approach avoids the intractable problem of comprehensive peak matching by using a few reliable peaks for alignment and peak-based retention-plane windows to define comprehensive features that can be reliably matched for cross-sample analysis. The informatics are demonstrated with a set of 18 samples from breast-cancer tumors, each from different individuals, six each for Grades 1-3. The features allow classification that matches grading by a cancer pathologist with 78% success in leave-one-out cross-validation experiments. The HRMS signatures of the features of interest can be examined for determining elemental compositions and identifying compounds.

Analysis of llicit and illicit drugs in waste, surface and lake water samples using large volume direct injection high performance liquid chromatography – Electrospray tandem mass spectrometry (HPLC–MS/MS)

http://www.sciencedirect.com/science/article/pii/S0045653510008891

Tandem mass spectrometry identification and LC-MS quantification of intact cytokinin nucleotides in K-562 human leukemia cells

We describe here a new reversed-phase high-performance liquid chromatography with mass spectrometry detection method for quantifying intact cytokinin nucleotides in human K-562 leukemia cells. Tandem mass spectrometry was used to identify the intracellular metabolites (cytokinin monophosphorylated, diphosphorylated, and triphosphorylated nucleotides) in riboside-treated cells. For the protein precipitation and sample preparation, a trichloroacetic acid extraction method is used. Samples are then back-extracted with diethyl ether, lyophilized, reconstituted, and injected into the LC system. Analytes were quantified in negative selected ion monitoring mode using a single quadrupole mass spectrometer. The method was validated in terms of retention time stabilities, limits of detection, linearity, recovery, and analytical accuracy. The developed method was linear in the range of 1-1,000 pmol for all studied compounds. The limits of detection for the analytes vary from 0.2 to 0.6 pmol.