Identification Of Individual Components Research Articles

Combined Analysis by GC(RI), GC-MS and 13C NMR of Leaf and Wood Essential Oils from Vietnamese Glyptostrobus pensilis (Staunton ex D. Don) K. Koch

Glyptostrobus pensilis (Staunton ex D. Don) K. Koch is a critically endangered species, native to southeastern China and also very locally found in Dak Lak Province, Vietnam. Essential oil isolated from leaves is a monoterpene-rich oil containing mainly limonene (33.3%), α-pinene (23.4%) and bornyl acetate (9.2%). The composition of G. pensilis wood oil is rather complex and the identification of individual components needed fractionation over column chromatography. The main components, identified by GC(RI), GC-MS and 13C NMR, were cedrol (29.3%), occidentalol (6.6%) and occidentalol isomer (5.9%).

Protective properties of the cultured stem cell proteome studied in an animal model of acetaminophen-induced acute liver failure

Chronic overuse of common pharmaceuticals, e.g. acetaminophen (paracetamol), often leads to the development of acute liver failure (ALF). This study aimed to elucidate the effect of cultured mesenchymal stem cells (MSCs) proteome on the onset of liver damage and regeneration dynamics in animals with ALF induced by acetaminophen, to test the liver protective efficacy of MSCs proteome depending on the oxygen tension in cell culture, and to blueprint protein components responsible for the effect. Protein compositions prepared from MSCs cultured in mild hypoxic (5% and 10% O2) and normal (21% O2) conditions were used to treat ALF induced in mice by injection of acetaminophen. To test the effect of reduced oxygen tension in cell culture on resulting MSCs proteome content we applied a combination of high performance liquid chromatography and mass-spectrometry (LC–MS/MS) for the identification of proteins in lysates of MSCs cultured at different O2 levels. The treatment of acetaminophen-administered animals with proteins released from cultured MSCs resulted in the inhibition of inflammatory reactions in damaged liver; the area of hepatocyte necrosis being reduced in the first 24 h. Compositions obtained from MSCs cultured at lower O2 level were shown to be more potent than a composition prepared from normoxic cells. A comparative characterization of protein pattern and identification of individual components done by a cytokine assay and proteomics analysis of protein compositions revealed that even moderate hypoxia produces discrete changes in the expression of various subsets of proteins responsible for intracellular respiration and cell signaling. The application of proteins prepared from MSCs grown in vitro at reduced oxygen tension significantly accelerates healing process in damaged liver tissue. The proteomics data obtained for different preparations offer new information about the potential candidates in the MSCs protein repertoire sensitive to oxygen tension in culture medium, which can be involved in the generalized mechanisms the cells use to respond to acute liver failure.

Non-targeted determination of 13C-labeling in the Methylobacterium extorquens AM1 metabolome using the two-dimensional mass cluster method and principal component analysis

A novel analytical workflow is presented for the analysis of time-dependent 13C-labeling of the metabolites in the methylotrophic bacterium Methylobacterium extorquens AM1 using gas chromatography time-of-flight mass spectrometry (GC-TOFMS). Using 13C-methanol as the substrate in a time course experiment, the method provides an accurate determination of the number of carbons converted to the stable isotope. The method also extracts a quantitative isotopic dilution time course profile for 13C uptake of each metabolite labeled that could in principle be used to obtain metabolic flux rates. The analytical challenges encountered require novel analytical platforms and chemometric techniques. GC-TOFMS offers advanced separation of mixtures, identification of individual components, and high data density for the application of advanced chemometrics. This workflow combines both novel and traditional chemometric techniques, including the recently reported two-dimensional mass cluster plot method (2D m/z cluster plot method) as well as principal component analysis (PCA). The 2D m/z cluster plot method effectively indexed all metabolites present in the sample and deconvoluted metabolites at ultra-low chromatographic resolution (RS≈0.04). Using the pure mass spectra extracted, two PCA models were created. Firstly, PCA was used on the first and last time points of the time course experiment to determine and quantify the extent of 13C uptake. Secondly, PCA modeled the full time course in order to quantitatively extract the time course profile for each metabolite. The 2D m/z cluster plot method found 152 analytes (metabolites and reagent peaks), with 54 pure analytes, and 98 were convoluted, with 65 of the 98 requiring mathematical deconvolution. Of the 152 analytes surveyed, 83 were metabolites determined by the PCA model to have incorporated 13C while 69 were determined to be either metabolites or reagent peaks that remained unlabeled.

Osmium tetroxide post-fixation and periodic acid-Schiff dual-staining technique to demonstrate intracellular lipid and glycogen in the mouse liver section – a novel method for co-visualization of intracellular contents in paraffin-embedded tissue

Intracellular accumulation of excessive glycogen or lipid in the liver occurs as a manifestation of metabolic dysfunction or drug-induced toxicity in humans or animals. The identification of individual components (lipids, glycogen, or water) involved is essential to the diagnosis of disease and pathogenesis. The aqueous fixatives and processing solvents used in paraffin embedding for routine hematoxylin and eosin stains remove both lipid and glycogen, leaving clear vacuoles of which the precise content is unknown in routine hematoxylin and eosin-stained sections. Traditional methods for detecting intracellular lipids and glycogen require two separate techniques, such as periodic acid-Schiff (PAS) reaction, with or without diastase digestion for glycogen, and osmium tetroxide (OsO4) post-fixation for lipid and utilization of different tissue sections. We describe here, for the first time, a novel OsO4/PAS dual-staining technique that permits a simple color-coded detection and direct visualization of both lipids and glycogen in the same paraffin-embedded tissue section. In this study, liver samples were collected from diet-induced obese (C57BL/6) or db/db mice and fixed in 10% neutral buffered formalin for 24 h. The formalin-fixed samples were post-fixed with OsO4, processed, sectioned, stained with PAS reaction, and evaluated microscopically for lipid and glycogen contents. Both lipid and glycogen were clearly identifiable with subjectively quantifiable severity and distribution in the same liver section. In conclusion, OsO4/PAS dual-staining, in our opinion, could be used in routine practice and provides a resource-saving innovation in differentiating glycogen from lipids in the same section of paraffin-embedded tissue sections.

Resolution-enhanced 2D NMR of complex mixtures by non-uniform sampling.

NMR is a powerful tool for the analysis of complex mixtures and the identification of individual components. Two-dimensional (2D) NMR potentially offers a wealth of information, but resolution is often sacrificed in order to contain experimental times. We explore the use of non-uniform sampling (NUS) to increase substantially the resolution of 2D NMR spectra of complex mixtures of small molecules, with no increase in experimental time. Two common pulse sequences for metabolomics applications are analysed, HSQC and TOCSY. Specific attention is paid to sensitivity in resolution-enhanced NUS spectra, using the signal-to-maximum-noise ratio as a metric. With a careful choice of sampling schedule and reconstruction algorithm, resolution in the (13) C dimension for HSQC is increased by a factor of at least 32, with no loss in sensitivity and no spurious peaks. For TOCSY, multiplets can be resolved in the indirect dimension in a reasonable experimental time. These properties should increase the usefulness of 2D NMR for metabolomics applications by, for example, increasing the chances of metabolite identification.

Identification of metabolites from 2D (1)H-(13)C HSQC NMR using peak correlation plots.

BackgroundIdentification of individual components in complex mixtures is an important and sometimes daunting task in several research areas like metabolomics and natural product studies. NMR spectroscopy is an excellent technique for analysis of mixtures of organic compounds and gives a detailed chemical fingerprint of most individual components above the detection limit. For the identification of individual metabolites in metabolomics, correlation or covariance between peaks in 1H NMR spectra has previously been successfully employed. Similar correlation of 2D 1H-13C Heteronuclear Single Quantum Correlation spectra was recently applied to investigate the structure of heparine. In this paper, we demonstrate how a similar approach can be used to identify metabolites in human biofluids (post-prostatic palpation urine).ResultsFrom 50 1H-13C Heteronuclear Single Quantum Correlation spectra, 23 correlation plots resembling pure metabolites were constructed. The identities of these metabolites were confirmed by comparing the correlation plots with reported NMR data, mostly from the Human Metabolome Database.ConclusionsCorrelation plots prepared by statistically correlating 1H-13C Heteronuclear Single Quantum Correlation spectra from human biofluids provide unambiguous identification of metabolites. The correlation plots highlight cross-peaks belonging to each individual compound, not limited by long-range magnetization transfer as conventional NMR experiments.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-014-0413-z) contains supplementary material, which is available to authorized users.

Formation of the urine proteome of healthy humans

The review deals with modern ideas on the processes that determine the urine protein composition of healthy people. In the past decade, the development of highly sensitive mass-spectrometric methods of protein detection stimulated studies of the protein composition of various human body fluids, including urine. Nowadays, the methods of separating complex protein mixtures and identification of individual components of these mixtures provide an opportunity to detect a significant amount of proteins and peptides of different origins in human urine. Physiological variation of the urine protein composition determined by the methods of proteomics remains a poorly studied but very important problem. Under physiological conditions, there are many factors that influence the filtering of plasma proteins in the glomeruli and reabsorption in the proximal tubules of the nephron. These are hypoxia, oxidative stress, changes in the acid-base balance and blood pressure, the effects of the parathyroid hormone, angiotensin-II, and other substances that control water and electrolyte metabolism. It is demonstrated that, because of the close structural and functional relationships between reabsorption processes in the proximal tubules of the nephron, reabsorption and modulation of sodium, water, chloride, phosphate, and bicarbonate depend on changes in various parts of the process of protein reabsorption.

Direct Identification of Two Major Components of an Essential Oil Using “Extraction NMR”

Identification of individual components of essential oils by comparative techniques (GC-MS and/or13C NMR) can be carried out exclusively for compounds whose spectroscopic data are compiled in computerized libraries. Otherwise, the compound should be isolated and submitted to a full set of spectroscopic analyses. In this paper we report on the direct identification of the two major components of Chamaemelum fuscatum (Brot.) Vasconcellos essential oil using “Extraction NMR”. Indeed, 2-methylallyl isobutyrate and ( E)-2-methyl-2-butenyl methacrylate have been identified, without previous isolation, by combination of the data from one and two-dimensional NMR spectra of the essential oil.

Criminalistic identification of PGM-containing products of mining and metallurgical companies

In early 1990s some organized criminal groups started to develop a new field of illegal business, which involved thefts of intermediary products from mining and metal-producing plants in Russia and in the south of Africa. Since local sulfide copper/nickel ores contain certain concentrations of precious and platinum group metals (PGMs), the intermediary products recovered at different stages of metallurgical transformation of these ores are materials of high commercial value. Illicit transportation and refining of these materials in Western Europe and North America has evolved into a large-scale business, where a lot of unlawful revenues are being laundered. The most important tasks in combating this organized crime are as follows: to establish the facts when some PGM-containing semi-products had been received at certain refineries; to carry out the identification of these semi-products; and to prove that these semi-products had been produced by a certain company. As a rule, it is not difficult to establish the identity of a “clean product”. However, when a material is a mix of several semi-products or a mix of some semi-product with masking substances, the identification of individual components becomes an extremely complicated task. The purpose of developing the “complex procedure for establishing the nature and source of origin of precious metal-bearing products of mining and metallurgical operations” was to make possible the identification of complex mixes comprised of various metallurgical semi-products. In the complex procedure that we have developed to characterize dispersed materials, distribution of particles by their elemental composition (the so-called “pseudophase” composition) was used instead of mineralogical composition. To determine the “pseudophase” composition by the method of scanning electron microscopy with X-ray spectral microanalysis (SEM-EDX), a representative sample of material containing not less than 1000 particles was analyzed. All microparticles can be divided into several types. Each type is characterized by an association of chemical elements contained. The first stage includes the study of elemental composition by inductively coupled plasma optical emission spectrometry and inductively coupled plasma mass-spectrometry; and the study of phase composition by X-ray diffractometry. The results of each study are compared with data in the Data Base. In case of coincidence with one of the products with respect to all diagnostic features, the type of product and its source of origin are defined accordingly. If the features of the sample under analysis detected by the aforementioned methods do not coincide with any of the product types represented in Data Base, it is necessary to examine elemental composition and morphology of separate particle contained in the substance using SEM-EDX. If some particles characterized by features coinciding with features of particles belonging to any product or products from Data Base are found, this product or a mixture of products is assumed to be present in the composition of the substance under analysis. The assumption that the substance is a mixture can be verified by juxtaposing all previously determined features of the analyzed sample with the features of the pattern mixture (superposition) of the appropriate types of products represented in Data Base. Depending on the results of this verification the corresponding conclusion can be made.

The Splicing Factor PSF is Part of a Large Complex That Assembles in the Absence of pre-mRNA and Contains All 5 snRNPs

PSF (PTB-associated splicing factor) is a large nuclear protein that has been implicated in numerous processes including transcription and RNA splicing. It has been shown to directly associate with U5 snRNA and has also been found within numerous purified splicing complexes. Here, we show that when HeLa nuclear extracts are adjusted to splicing conditions, PSF is found as part of a large complex that contains all five snRNPs and most known splicing factors. Formation of the complex does not require addition of exogenous pre-mRNA substrate and occurs at 40C but is salt sensitive. Sedimentation experiments and identification of individual components by mass spectrometry revealed association with multiple nuclear factors, most of which overlap with spliceosome components.

Evaluation of High Molecular Weight Species Produced during Thermal Degradation of Poly(Vinyl Alcohol)

Thermal decomposition during pyrolysis of poly(vinyl alcohol) (PVA) was investigated using off-line pyrolysis gas chromatography mass spectrometry (Py-GCMS). Observation of a yellow-brown smoke produced when PVA was heated at elevated temperatures indicated that the liquid products may contain highly conjugated chromophores not previously reported. Collection of the resulting pyrolysis mixtures and subsequent analysis by GC-MS confirmed the presence of conjugated, high-molecular-weight compounds in the product mixture. Identification of individual components indicates that the mechanism of degradation involves dehydration followed by random chain scission and eyclization to produce conjugated molecules of moderate to high molecular weight.

Morphology Characterization in Multicomponent Polymer Systems using Scanning Probe Microscopy

ABSTRACTA newly available scanning probe microscopy, phase imaging, has been used to image polyethylene (PE) blends and composites. By providing not only a topographical map of the surface but also information on material properties, this technique has allowed ready identification of individual components within the mixtures investigated.

Artificial neural network and classical least-squares methods for neurotransmitter mixture analysis

Identification of individual components in biological mixtures can be a difficult problem regardiess of the analytical method employed. In this work, Raman spectroscopy was chosen as a prototype analytical method due to its inherent versatility and applicability to aqueous media, making it useful for the study of biological samples. Artificial neural networks (ANNs) and the classical least-squares (CLS) method were used to identify and quantify the Raman spectra of the small-molecule neurotransmitters and mixtures of such molecules. The transfer functions used by a network, as well as the architecture of a network, played an important role in the ability of the network to identify the Raman spectra of individual neurotransmitters and the Raman spectra of neurotransmitter mixtures. Specifically, networks using sigmoid and hyperbolic tangent transfer functions generalized better from the mixtures in the training data set to those in the testing data sets than networks using sine functions. Networks with connections that permit the local processing of inputs generally performed better than other networks on all the testing data sets, and better than the CLS method of curve fitting, on novel spectra of some neurotransmitters. The CLS method was found to perform well on noisy, shifted, and difference spectra.

Determination of Composition of Reaction Mixtures from Alkylation of Toluene on Phosphorus-Modified H-ZSM-5 Zeolite

Determination of analytical composition of reaction mixtures formed by alkylation of toluene with ethylene on a phosphorus-modified H-ZSM-5 zeolite was made by a capillary high resolution gas chromatography. Identification of individual components of these reaction mixtures was performed by GS-MS method, using the samples obtained at 320 and 400 °C on H-ZSM-5 catalyst (modul 43.6) modified with 3.4 wt.% phosphorus at toluene to ethylene molar ratio 4.5 : 1 and the catalyst loading expressed as weight hour space velocity WHSV (for toluene) = 6.9 h-1. The reaction mixtures contained a total of 86 hydrocarbons. It was confirmed that in the addition to the main alkylation reaction, there proceeds also ethylene oligomerization with subsequent cyclization and aromatization, disproportionation, alkylation and cyclization reactions of alkylaromatic hydrocarbons to give alkylnaphthalenes and alkylindanes.

The identification of individual components in flavourings and flavoured foods

The identification of individual components in flavourings and flavoured foods

High performance liquid chromatography and nuclear magnetic resonance analysis of longipinene derivatives fromStevia subpubescens var.intermedia

The methodology for the separation of complex mixtures of longipin-2-ene-7β, 9α-diol-1-one diesters using high performance liquid chromatography followed by the identification of individual components by pulse Fourier transform 1H nuclear magnetic resonance spectroscopy is described. The identification methodology takes advantage of the spectral comparison between the isolated natural product and a wide series of previously prepared longipinene derivatives, and is applied to the study of the extracts of Stevia subpubescens var. intermedia which contain 11 previously isolated longipinene diesters and the new natural product longipin-2-ene-7β, 9α-diol-1-one 7-seneciate-9-angelate.

Midline and temporal lobe MLRs in the guinea pig originate from different generator systems: a conceptual framework for new and existing data

In the guinea pig and gerbil, individual components within the MLR time frame differ in optimal recording location. Specifically, MLR components obtained from the midline differ from those obtained over the temporal lobe. In the present paper midline and temporal lobe components were shown to differ not only in scalp topography but also in response to the following experimental manipulations: intracortical injection of neural inactivating agents (lidocaine and kainic acid), temporal lobe ablation, electrolytic lesions, systemic anesthesia, stimulation rate and course of development. Since midline and temporal lobe components respond differently to experimental manipulations, it can be concluded that the midline and temporal lobe responses are mediated by different generator sources. The particular orientation of the generators responsible for the MLR in the guinea pig and gerbil facilitates the identification of individual components. Results from simultaneous recordings of these components during experimental manipulations support the hypothesis of multiple MLR generators in laboratory animals and provide insight into the generators and developmental aspects of the MLR in humans.

Extractable metal salts of carboxylic acids in Green River oil shale

A study of solvent-extractable metal salts of carboxylic acids from Colorado Green River oil shale is presented. Sodium was found to be the most abundant cation present in these acid metal salts. Identification of individual components is based on combined gas Chromatographic and mass spectrometric results obtained for their respective volatile methyl esters. Normal acid salts (C 11 to C 34), isoprenoid acid salts (C 15 to C 17, C 19 to C 22), nonisoprenoid branched acid salts (C 14 to C 19), terpenoid acid salts (C 30 to C 32), and α,ω-dicarboxylic acid salts (C 11 to C 29) were identified. Small concentrations of cyclohexyl acid salts and aromatic acid salts were also found. The high even-odd predominance for the distribution of normal acid salts indicates a mild thermal history for Green River oil shale. Some acid salts (45.6 wt%) were not identified because of nonvolatility of their methyl esters.

Separation and characterization of the aldehydic products of lipid peroxidation stimulated by ADP-Fe2+ in rat liver microsomes.

1. Methods using t.l.c. and high-pressure liquid chromatography (h.p.l.c.) have been used to separate the complex variety of substances possessing a carbonyl function that are produced during lipid peroxidation. 2. The major type of lipid peroxidation studied was the ADP-Fe2+-stimulated peroxidation of rat liver microsomal phospholipids. Preliminary separation of the polar and non-polar products was achieved by t.l.c.: further separation and identification of individual components was performed by h.p.l.c. Estimations were performed on microsomal pellets and the supernatant mixture after incubation of microsomes for 30 min at 37 degrees C. 3. The polar fraction was larger than the non-polar fraction when expressed as nmol of carbonyl groups/g of liver. In the non-polar supernatant fraction the major contributors were n-alkanals (31% of the total), alpha-dicarbonyl compounds (22%) and 4-hydroxyalkenals (37%) with the extraction method used. 4. Major individual contributors to the non-polar fraction were found to be propanal, 4-hydroxynonenal, hexanal and oct-2-enal. Other components identified include butanal, pent-2-enal, hex-2-enal, hept-2-enal, 4-hydroxyoctenal and 4-hydroxyundecenal. The polar carbonyl fraction was less complex than the non-polar fraction, although the identities of the individual components have not yet been established. 5. Since these carbonyl compounds do not react significantly in the thiobarbituric acid reaction, which largely demonstrates the presence of malonaldehyde, it is concluded that considerable amounts of biologically reactive carbonyl derivatives are released in lipid peroxidation and yet may not be picked up by the thiobarbituric acid reaction.

System for identification of individual components in mixtures on the basis of shpol'skii spectra

System for identification of individual components in mixtures on the basis of shpol'skii spectra