Ion-pair RP-HPLC determination of sugars, amino sugars, and uronic acids after derivatization with p-aminobenzoic acid

A new, selective, and sensitive ion-pair RP-HPLC method for the simultaneous determination of three classes of natural organic compounds, i.e., carbohydrates, amino sugars, and uronic acids, in environmental samples is presented. p-Aminobenzoic acid is used for precolumn derivatization of the analytes, enabling fluorescence (lambda(ex) 313 nm, lambda(em) 358 nm) or photometric detection (303 nm). The dependence of the derivatization yield on the reaction conditions is examined. Derivatives of lactose, galactose, glucose, mannose, xylose, arabinose, galacturonic acid, glucuronic acid, N-acetylglucosamine, and glycerinealdehyde were separated on a RP-C18 column with hydrophilic end capping within 35 min, applying TBAHSO4 as the ion-pair reagent. The concentration detection limits range between 20 and 30 microg L(-1) ((1-2) x 10(-7) M) for fluorescence detection and between 30 and 75 microg L(-1) for UV detection. A good linearity is achieved in the concentration range from 50 microg L(-1) to 100 mg L(-1) (r2 > 0.99). The described method has been applied for the determination of mono-/disaccharides, uronic acids, and amino sugars in soil solutions and in landfill leachates.     

Automatic generation of peak-shaped models

We describe how parametric spectral models for analytical applications can be generated by an automatic curve-fitting algorithm. The algorithm does not require initial choices of parameters or other human intervention, in contrast to established approaches that rely on deconvolution or derivative spectroscopy. This algorithm has been applied for quantitative analysis but can potentially be used in other applications that are based on parametric representations of peak-shaped models or could benefit from using such models, such as calibration transfer.     

Mass spectrometric method for determining the uronic Acid epimerization in heparan sulfate disaccharides generated using nitrous Acid

Heparan sulfate (HS) glycosaminoglycans (GAGs) regulate a host of biological functions. To better understand their biological roles, it is necessary to gain understanding about the structure of HS, which requires identification of the sulfation pattern as well as the uronic acid epimerization. In order to model HS structure, it is necessary to quantitatively profile depolymerization products. To date, liquid chromatography-mass spectrometry (LC-MS) methods for profiling heparin lyase decomposition products have been shown. These enzymes, however, destroy information about uronic acid epimerization. Deaminative cleavage using nitrous acid (HONO) is a classic method for GAG depolymerization that retains uronic acid epimerization. Several chromatographic methods have been used for analysis of deaminative cleavage products. The chromatographic methods have the disadvantage that there is no direct readout on the structures producing the observed peaks. This report demonstrates a porous graphitized carbon (PGC)-MS method for the quantification of HONO generated disaccharides to obtain information about the sulfation pattern and uronic acid epimerization. Here, we demonstrate the separation and identification of uronic acid epimers as well as geometric sulfation isomers. The results are comparable to those expected for benchmark HS and heparin samples. The data demonstrate the utility of PGC-MS for quantification of HS nitrous acid depolymerization products for structural analysis of HS and heparin.     

Complete monosaccharide analysis by high-performance anion-exchange chromatography with pulsed amperometric detection

Monosaccharide analysis is a critical way to profile the composition of complex carbohydrates. Methods to analyze neutral and amino sugars have been established for a long time, but methods for acidic sugars are rare. The acidic sugars, including uronic acids and sialic acids, are also important components in some complex carbohydrates. In this report, a high-performance anion-exchange chromatography method with pulsed amperometric detection was initially developed to analyze acidic sugars including different uronic acids and sialic acids. Subsequently, a method to profile complete monosaccharides, including most neutral, amino, and acidic sugars, was developed. This method has a limit of quantitation of ~12.5 × 10(-3) nmol for each sugar as well as good linearity over a wide range. This is a convenient procedure because it avoids additional derivatization of monosaccharides and has a broad application to a wide range of complex carbohydrates. The monosaccharide compositions of a variety of complex carbohydrates such as different glycosaminoglycans, alginate, fucoidan, and glycans were profiled by this comprehensive method. In addition, the hydrolysis patterns of these complex carbohydrates are discussed.     

Recovery of microcrack parameters in mortar using quantitative acoustic emission

A three-dimensional quantitative acoustic emission (AE) analysis of microcracking in unreinforced mortar beams was conducted. In order to facilitate the analysis of the large amounts of data generated by an AE test, a simplified method for the inversion of AE signals was developed. By applying the theoretical Green's function for an infinite space, the multichannel deconvolution normally required of AE data inversion reduces to a nonlinear curve-fitting problem. Using this procedure, microcracking in a mortar specimen was evaluated using a seismic moment tensor representation. Source-time functions for the microcracks were also recovered. The locations of the AE events were calculated, and damage localization was observed. The moment tensor analysis showed the dominant mode of microfracture to be mode II, with a limited number classified as mixed mode. A microstructural mechanism for this behavior is presented.     

傅立叶变换红外光谱法分析市售食品中反式脂肪酸

建立了一种快速测定食品中反式脂肪酸含量的方法,用索氏提取法或氯仿—甲醇提取法提取食品中脂肪,经甲醇-BF3快速甲酯化后,采用Avatar 370傅立叶变换红外光谱对反式脂肪酸进行定性定量分析,回收率为89.5%~103.3%,相对标准偏差为1.80%,重复性好,准确度高,适用于一般食品营养标签的测定。对我国市场上的涂抹奶油、派和蛋糕、饼干、巧克力、冰淇淋、薯片和薯条等6大类食品中20种样品中反式脂肪酸进行测定。结果表明,16种检出样品中反式脂肪酸含量在0.18%~10.34%之间,反式脂肪酸含量随食品种类不同存在显著差异,涂抹奶油、蛋黄派、威化饼干是反式脂肪酸含量较高的食品类别。     

Carbon isotope composition of dissolved humic and fulvic acids in the Tokachi River system

This study reports carbon isotopic ratios (Δ(14)C and δ(13)C) of dissolved humic and fulvic acids in the Tokachi River system, northern Japan. These acids have a refractory feature and they represent the largest fraction of dissolved organic matter in aquatic environments. The acids were isolated using the XAD extraction method from river water samples collected at three sites (on the upper and lower Tokachi River, and from one of its tributaries) in June 2004 and 2005. δ(13)C values were -27.8 to -26.9 ‰ for humic and fulvic acids. On the other hand, the Δ(14)C values ranged from -247 to +26 ‰ and the average values were -170 ± 79 ‰ for humic acid and -44 ± 73 ‰ for fulvic acid. The difference was attributed to the residence time of fulvic acid in the watershed being shorter than that of humic acid. The large variation suggested that humic substances have a different pathway in each watershed environment.     

Enhancement of the LC/MS analysis of fatty acids through derivatization and stable isotope coding

This paper focuses on the development of an enhanced LC/ESI-MS method for the identification and quantification of fatty acids through derivatization. Fatty acids were derivatized with 2-bromo-1-methylpyridinium iodide and 3-carbinol-1-methylpyridinium iodide, forming 3-acyloxymethyl-1-methylpyridinium iodide (AMMP). This process attaches a quaternary amine to analytes and enabled ESI-MS in the positive mode of ionization with common LC mobile phases. Moreover, detection sensitivity was generally 2500-fold higher than in the negative mode of ionization used with underivatized fatty acids. The limits of detection were roughly 1.0-4.0 nM (or 10 pg/injection) for standard fatty acids from C10 to C24 and spanned approximately 2 orders of magnitude in linearity. AMMP derivatives had unique tandem mass spectra characterized by common ions at m/z 107.0, 124.0, and 178.0. Individual fatty acids also had unique fingerprint regions that allowed identification of their carbon skeleton number, number of double bonds, and double bond position. The derivatization method also allowed coding of analytes as a means of recognizing derivatives and enhancing quantification. 2H-Coding was achieved through derivatization with deuterated 3-carbinol-1-methyl-d3-pyridinium iodide. The 2H-coded derivatization reagent, 3-acyloxymethyl-1-methyl-d3-pyridinium iodide, was used in two ways. One was to differentially label equal fractions of a sample such that after being recombined and analyzed by ESI-MS all fatty acids appeared as doublet clusters of ions separated by roughly 3 amu. This greatly facilitated identification of fatty acids in complex mixtures. Another use of stable isotope coding was in comparative quantification. Control and experimental samples were differentially labeled with nondeuterated and deuterated isotopomers of CPM, respectively. After mixing the two samples, they were analyzed by ESI-MS. The abundance of a fatty acid in an experimental sample relative to the control was established by the isotope ratio of the isotopomeric fatty acids. Absolute quantification was achieved by adding differentially labeled fatty acid standards to experimental samples containing unknown quantities of fatty acids. Utility of the method was examined in the analysis of human serum samples.     

中温沥青中甲苯可溶组分的热解特性及热转化产物的性质

以中温煤沥青的甲苯可溶组分为原料,进行元素分析和TG/DTG分析,并且利用偏光显微镜、X射线单晶衍射仪、拉曼光谱以及相应的分峰拟合等数学方法对热转化的产物进行研究。以实验获得的数据为基础,采用Flynn-Wall-Ozawa法和Kissinger-Akahira-Sunose法计算得到反应活化能,采用Satava-Sastak法求解热解反应动力学参数。实验结果表明,中温沥青中甲苯可溶组分的热解反应活化能为E=88.48kJ/mol,指前因子lgA=10.22,反应级数为2级,热解反应机理适合随机成核及其随后的增长模型。1 000℃焙烧后的炭化产物的光学显微组分含量为:镶嵌型23.74%,粗纤维型14.80%,细纤维17.88%,大片结构30.45%。由XRD研究结果可知,趋于规整结构的碳微晶含量为41.86%。Raman光谱分析研究结果表明,石墨结构的碳微晶含量为11.59%,缺陷石墨碳含量为79.31%,无定形碳含量为9.10%。热转化产物具有很好的可石墨化性。     

气相色谱法测定工蜂幼虫和蛹中的脂肪酸组成

建立了一种测定工蜂幼虫和蛹中脂肪酸的气相色谱分析方法。采用湿法研磨工蜂幼虫和蛹以破坏细胞和提取脂肪,提取液用氢氧化钾-甲醇进行甲脂化反应,上清液用于气相色谱分析。共检出了10种脂肪酸,其中幼虫和蛹的不饱和脂肪酸含量约占脂肪酸总量的46-47%。该方法简便、快速,且有较好的精密度和重复性、可靠性,适用于工蜂幼虫和蛹中脂肪酸的测定。     

Characterization of natural resin shellac by reactive pyrolysis-gas chromatography in the presence of organic alkali

A method to determine chemical composition of natural resin shellac was developed on the basis of reactive pyrolysis-gas chromatography (Py-GC) in the presence of an organic alkali, tetramethylammonium hydroxide ((CH(3))(4)NOH, TMAH). Py-GC using 25% TMAH aqueous solution enabled the highly sensitive determination of terpenic acids, aleuritic acid, several minor fatty acids, and the wax components of shellac as their methyl derivatives on the resulting pyrograms with less than 2.0% relative standard deviations without using any cumbersome pretreatment. The observed average distributions of each resin acid component determined by reactive Py-GC for shellac samples from India and Thailand showed that the average ratios among terpenic acids, aleuritic acid, and the other fatty acids were about 53:34:14 for Indian shellac and 51:35:14 for Thailand shellac, respectively, suggesting a slightly significant difference. However, clearer discrimination of the shellac samples from the two different growing places was attainable by applying principal component analysis for the mole percent distributions of all the acidic components determined by reactive Py-GC.     

Determination of stable carbon isotopic compositions of low molecular weight dicarboxylic acids and ketocarboxylic acids in atmospheric aerosol and snow samples

We report a new method developed for the determination of stable carbon isotopic composition of homologous alpha,omega-dicarboxylic acids and phthalic acid isolated from environmental samples such as atmospheric aerosols and snow. Dicarboxylic acids are derivatized with BF3/1-butanol to dibutyl esters, which are analyzed for the stable carbon isotopic composition using a capillary GC interfaced to on-line combustion isotope ratio mass spectrometer. The delta13C values for individual dicarboxylic acid are then calculated from delta13C of 1-butanol and butyl ester derivative using a mass balance equation. The accuracy of the delta13C measurement for C2-C10 diacids is within 0.8 per thousand. We report a few examples of the delta13C ratios of saturated C2-C9 alpha,omega-dicarboxylic acids, unsaturated (maleic, phthalic) diacids, and oxocarboxylic acids in the aerosol and snow samples.     

Validated quantitation of underivatized amino acids in human blood samples by volatile ion-pair reversed-phase liquid chromatography coupled to isotope dilution tandem mass spectrometry

Quantitation of amino acids in complex matrixes without derivatization is advantageous; however, difficulties exist in both the separation and the detection of those compounds. A validated method that is based on the use of volatile ion-pair liquid chromatography coupled to stable isotope dilution tandem mass spectrometry has been developed for the simple and accurate quantitation of underivatized amino acids in biological samples. Sufficient separation of 22 underivatized amino acids was achieved on a C18 column in 36 min using perfluoroheptanoic acid (PFHA) and trifluoroacetic acid (TFA) as mobile phase modifiers. The collisionally activated dissociation spectra of the amino acids were investigated and the transitions of [M + H]+ --> [M + H - 46]+, which are specific to alpha-amino acids, were used for the detection of most amino acids and their stable isotopes. The calibration curves were linear over the range of 0.10-100 microg/mL, and the detection limits were 0.03-20 pmol on column. The quantitative results by this method were compared with those by an established OPA-derivatization HPLC method in the assay of 8 human serum samples, and better recovery and precision data of this method were observed. The method was also applied to the neonatal screening for phenylketonuria (PKU) with dry blood spots, and the results were satisfactory. This is the first time that all proteinogenic amino acids have been quantified directly from biological extracts without any kind of derivaization. The technique shows potential for routine determination of amino acids and analogous compounds in complex matrixes.     

R410A热力性质的拟合计算

采用拟合关联式简便计算方法,对制冷剂R22主要替代工质之一的R410A的热力性质在饱和温度-40～60℃,过热度0～60℃范围内进行了拟合,并分析了误差。结果表明：采用该方法得到的拟合模型与参考数据源相比,在保证物性计算速度和稳定性的前提下,精度满足工程计算所需的要求,适用于系统的仿真和优化计算以及过程的实时控制。     

Determination of the Acidic Degradation Products, Acetic, Propionic, Butyric, and Phthalic Acid in Aqueous Pseudolatexes of Cellulosic Esters by High Performance Liquid Chromatography

An isocratic, reversed-phase HPLC method was developed to quantify the organic acids, acetic, propionic, butyric, and phthalic acid, formed as a result of ester hydrolysis, in pseudolatexes of cellulosic esters. Colloidal dispersions of cellulose acetate, cellulose acetate butyrate, and cellulose acetate propionate were prepared by a microfluidization-solvent evaporation method. Dispersions of cellulose acetate phthalate were prepared by redispersion of a spraydried commercial pseudolatex. The acids were detected at 210 nm, the mobile phase being 0.025 M phosphate buffer: methanol (80:20 v/v%, pH 3.0). The peak height response was linear over the studied concentration range of 2 - 10 mM/L for the aliphatic acids and 20-100 μM/L for phthalic acid. The minimum detectable quantities for acetic, propionic, butyric, and phthalic acid were 0.02 mM/L, 0.05 mM/L, 0.1 mM/L, and 0.0005 mM/L, corresponding to a % change in acetyl, propionyl, butyryl, and phthalyl content of 4.0 × 10⁴, 1.2 × 10³, 2.9 × 10³, and 2.8 × 10^-5 for a 30% w/v pseudolatex. The colloidal polymer particles were separated by ultracentrifugation, filtration, or flocculation with aluminum chloride solution before analysis of the aqueous phase. Similar acid concentrations were obtained for the three separation methods. The recovery from spiked samples was almost complete for acetic, approximately 90% for propionic acid, and less than 80% for butyric acid.     

Liquid chromatography-high resolution mass spectrometry analysis of fatty acid metabolism

We present a liquid chromatography/mass spectrometry (LC/MS) method for long-chain and very-long-chain fatty acid analysis and its application to (13)C-tracer studies of fatty acid metabolism. Fatty acids containing 14 to 36 carbon atoms are separated by C(8) reversed-phase chromatography using a water-methanol gradient with tributylamine as ion pairing agent, ionized by electrospray and analyzed by a stand-alone orbitrap mass spectrometer. The median limit of detection is 5 ng/mL with a linear dynamic range of 100-fold. Ratios of unlabeled to (13)C-labeled species are quantitated precisely and accurately (average relative standard deviation 3.2% and deviation from expectation 2.3%). In samples consisting of fatty acids saponified from cultured mammalian cells, 45 species are quantified, with average intraday relative standard deviations for independent biological replicates of 11%. The method enables quantitation of molecular ion peaks for all labeled forms of each fatty acid. Different degrees of (13)C-labeling from glucose and glutamine correspond to fatty acid uptake from media, de novo synthesis, and elongation. To exemplify the utility of the method, we examined isogenic cell lines with and without activated Ras oncogene expression. Ras increases the abundance and alters the labeling patterns of saturated and monounsaturated very-long-chain fatty acids, with the observed pattern consistent with Ras leading to enhanced activity of ELOVL4 or an enzyme with similar catalytic activity. This LC/MS method and associated isotope tracer techniques should be broadly applicable to investigating fatty acid metabolism.     

A strategy for identifying differences in large series of metabolomic samples analyzed by GC/MS

In metabolomics, the purpose is to identify and quantify all the metabolites in a biological system. Combined gas chromatography and mass spectrometry (GC/MS) is one of the most commonly used techniques in metabolomics together with 1H NMR, and it has been shown that more than 300 compounds can be distinguished with GC/MS after deconvolution of overlapping peaks. To avoid having to deconvolute all analyzed samples prior to multivariate analysis of the data, we have developed a strategy for rapid comparison of nonprocessed MS data files. The method includes baseline correction, alignment, time window determinations, alternating regression, PLS-DA, and identification of retention time windows in the chromatograms that explain the differences between the samples. Use of alternating regression also gives interpretable loadings, which retain the information provided by m/z values that vary between the samples in each retention time window. The method has been applied to plant extracts derived from leaves of different developmental stages and plants subjected to small changes in day length. The data show that the new method can detect differences between the samples and that it gives results comparable to those obtained when deconvolution is applied prior to the multivariate analysis. We suggest that this method can be used for rapid comparison of large sets of GC/MS data, thereby applying time-consuming deconvolution only to parts of the chromatograms that contribute to explain the differences between the samples.     

Spectral characterization of eucalyptus wood

The main difficulties in wood and pulp analyses arise principally from their numerous components with different chemical structures. Therefore, the basic problem in a specific analytical procedure may be the selective separation of the main carbohydrate-derived components from lignin due to their chemical association and structural coexistence. The processing of the wood determines some structural modification in its components depending on the type of wood and the applied procedure. Fourier transform infrared (FT-IR) spectrometry and X-ray diffraction have been applied to analyze Eucalyptus g. wood chips and unbleached and chlorite-bleached pulp. The differences between samples have been established by examination of the spectra of the fractions obtained by successive extraction (acetone extractives, acetone free extractive samples, hemicelluloses, and lignins) by evaluating the derivative spectra, band deconvolution, etc. The energy and the hydrogen bonding distance have been evaluated. The relationship between spectral characteristics and sample composition has been established, as well as the variation of the degree of crystallinity after pulping and bleaching. The integral absorption and lignin/carbohydrate ratios calculated from FT-IR spectra of the IR bands assigned to different bending or stretching in lignin groups are stronger in the spectrum of eucalyptus chips than those from brown stock (BS) pulp spectra because of the smaller total amount of lignin in the latter. FT-IR spectra clearly show that after chlorite bleaching the structure of the wood components is partially modified or removed. Along with FT-IR data, the X-ray results confirmed the low content of lignin in the pulp samples by increasing the calculated values of the crystalline parameters. It was concluded that FT-IR spectroscopy can be used as a quick method to differentiate Eucalyptus globulus samples.     

Oxidation and reduction of multiwalled carbon nanotubes — preparation and characterization

This work presents the use of a modified titration (Boehm's) process which is a simple and efficient method to quantify functional groups formed on the surface of oxidized multiwalled carbon nanotubes (MWCNTs). The MWCNTs were synthesized via chemical vapor deposition (CVD) and were purified through a hydrochloric acid treatment. Purified material was oxidized in a mixture of nitric and sulfuric acids. A part of oxidized sample was reduced with sodium borohydrate (NaBH₄). Boehm's titration is a complimentary method to Fourier Transform Infrared spectroscopy (FT-IR) with which to investigate the changes to the surface of oxidized MWCNTs after the reduction process. The reduction process led to threefold increase in the hydroxyl group content. In addition, the pristine, oxidized and reduced samples were investigated by thermogravimetry analysis (TGA) and Raman spectroscopy.     

LC-Biosensor System for the Determination of the Neurotoxin β-N-Oxalyl-l-α,β-diaminopropionic Acid

An analysis system is described for the determination of the neurotoxin β-N-oxalyl-l-α,β-diaminopropionic acid (β-ODAP). The system is based on liquid chromatographic separation of β-ODAP from interfering amino acids on an anion exchange column coupled with an amperometric enzyme electrode for the registration of β-ODAP. The electrode is based on a graphite rod modified with an Os(2+/3+) redox polymer cross-linked with l-glutamate oxidase and horseradish peroxidase. This LC-bienzyme electrode analytical system enabled monitoring of as low as 4 μM β-ODAP (injection volume 100 μL). The β-ODAP content in real grass pea samples was measured to range between 3.3 and 5.2 g kg(-)(1) in dry grass pea.