Basic profiles of organic acids in urine

Altogether 143 of the organic acids regularly occurring in urine of healthy individuals are identified as methyl esters by gas chromatography-mass spectrometry with respect to their complete chemical structures. They are classified as dicarboxylic acids, oxocarboxylic acids, hydroxycarboxylic acids, aromatic acids, furancarboxylic acids, nitrogen-containing acids and acid conjugates. By pre-fractionating the complex mixture of the total organic acids, peak overlap is minimized, and substances in low concentrations can also be detected and identified. The qualitative patterns of the urinary organic acids in the fractions are constant and reproducible, and in many cases a reliable identification of organic acids is possible by gas chromatography alone, using methylene units and separation on OV-1701 capillary columns.     

Analysis of methyl and ethyl esters of hydroxybenzoic and hydroxycinnamic acids in plant material

A method is described for the extraction and purification of methyl and ethyl esters of hydroxybenzoic and hydroxycinnamic acids from plant material. The esters were analyzed as trimethylsilyl derivatives by glass capillary gas chromatography (OV-1, OV-73, Dexsil 300) and gas chromatography-mass spectrometry. The method has been applied to analysis of methyl and ethyl esters of hydroxybenzoic and hydroxycinnamic acids in vegetables and potato peels.     

Gas chromatography of alpha-keto acids as their O-trimethyl silylquinoxalinol derivatives.

The O-trimethylsilyl (TMS) quinoxalinols are very useful derivatives for the gas chromatography of alpha-keto acids because of their high stability and the absence of stereoisomerism and because of the presence of specific, common and abundant fragments in electron impact mass spectra, which allows the low-level detection of whole groups of keto acids by single-ion detection. In this paper, the chromatographic properties of eleven O-TMS-quinoxalinols on OV-1, OV-17 and Dexsil 300 are reported in terms of methylene units. Also by use of methylene units, the chromatographic isotope effect is analyzed in detail for nine perdeutero-TMS derivatives. The effect is explained by the diminished interaction of the deuterated compounds with the unlabelled liquid phase.     

Improved method for the determination of the major neutral steroids and unconjugated bile acids in human faeces using capillary gas chromatography

An improved method has been developed for the determination of the major neutral steroids (cholesterol and 5 beta-cholestan-3 beta-ol) and unconjugated bile acids (deoxycholic acid and lithocholic acid) in human faeces, using capillary gas chromatography with flame ionization detection. The freeze-dried faecal sample was subjected to a two-stage Soxhlet extraction followed by an aqueous alkali-organic solvent partition step to separate neutral steroids from bile acids. The neutral steroids were analysed as their trimethylsilyl ether derivatives on an OV-1 capillary column. The bile acids were further purified on a Sep-Pak C18 cartridge and then fractionated on a Sep-Pak SIL cartridge. Unconjugated bile acids were analysed as their methyl ester-trimethylsilyl ether derivatives also on an OV-1 capillary column. Quantitation of neutral steroids and unconjugated bile acids was achieved by reference to appropriate internal standards, added to the faecal extract immediately after the Soxhlet extraction stage. The method is being used in a study of the effect of diet on the metabolic activity of human gut flora.     

Analysis of the amino acid and sugar composition of streptococcal cell walls by gas chromatography-mass spectrometry

A procedure for determining the amino acid and sugar composition of streptococcal peptidoglycan-polysaccharide complexes by capillary gas chromatography-mass spectrometry (GC-MS) was established. Amino acids are analysed as butyl heptafluorobutyl derivatives and sugars as alditol acetates. These two different groups of compounds are derivatized independently but chromatography in both cases utilizes the same OV-1701 fused-silica capillary column which simplifies GC-MS analysis. The butyl heptafluorobutyl procedure incorporates new pre- and post-derivatization clean-up steps. Additionally, selected-ion monitoring MS allows amino acids to be readily analysed without interference from background noise.     

Gas Chromatographic Determination of Trimethylsilyl Derivatives of Free Amino Acids from a Botanical Source

Abstract

The application of gas chromatography for the separation of TMS-amino acids from a botanical source was demonstrated. The trimethyl-silyl derivatives of the extracts from germ free tobacco tissue cultures were prepared by reacting amino acid extracts with bis(trimethylsilyl)-trifluoroacetamide (BSTFA) using acetonitrile as a reaction solvent following preliminary separation of the free acids by ion exchange chromatography. Gas chromatographic separation was accomplished with a 10% OV-11 glass column and temperature programming. The findings compare favorably with other chromatographic methods. Structures of the TMS-amino acids were confirmed by gas chromatography-mass spectrometry combination. Mass spectral data for each derivative is presented for the principal protein amino acids observed as well as γ-aminobutyric acid, asparagine, α-aminobutyric acid and β-alanine.     

Chemometric study of retention on binary stationary phases in gas chromatography

Using gas chromatography, data analysis is performed on a dataset consisting of 486 retention indices, 27 standards (ramified alkanes, aliphatic alcohols, and aromatic compounds), 6 pure and binary stationary phases, and three temperatures. The behavior of the pure stationary phases (OV-3, OV-225, OV-61-OH, and OV-1701-OH) and the binary stationary phases (OV-3/OV-225 and OV-61-OH/OV-1701-OH) at different temperatures (60°C-100°C) is investigated with factor and topological analysis. The influence of temperature and the nature of the mixed stationary phases on the retention indices is studied by correspondence factor analysis (CFA). The non-additivity of the retention properties of the pure phases used as mixed phases is clearly established by CFA. The topological analysis of the substituent's effect is investigated with a DARC/PELCO procedure and shows the particular influence of the stationary phase composition on the retention. The substituent effect is measured for the pure and binary stationary phases at various temperatures. The evolution of the substituent effect from the pure stationary phases to the binary phases is discussed.     

Rapid screening and confirmation of amphetamine, methamphetamine, methadone, and methadone metabolite in urine by gas/thin layer chromatography.

A method suitable for large scale screening and confirmation of urine speciments for amphetamine, methamphetamine, methadone, and its primary metabolite (2-ethylidene-1,5-dimethyl-3,3-diphenlypyrrolidine) is described. The drugs are extracted from alkaline urine into an organic solvent. The amphetamine drugs are then back-extracted into a small volume of acid and identified by gas chromatography both as free bases on a 10% Apiezon L-10% KOH column and as their trifluoracetamide derivatives on a 3% OV-17 column. The organic layer, which still contains methadone and its primary metabolite, is analyzed by split-sample thin-layer chromatography using two solvent systems: ethyl acetate: methylene cloride: concentrated ammonium hydroxide (90:10:0.7) and methanol: chloroform: concentrated ammonium hydroxide (74:25:0.8). These solvent systems separate methadone from its primary metabolite without interference from other drugs or urinary substances.     

Polycyclic aromatic hydrocarbons in C8 isomer aromatic feed: analysis by GC, GC/MS, and GC/FTIR techniques

Polycyclic aromatic hydrocarbons (PAHs), apart from their carcinogenic and mutagenic nature, create many problems in the petrochemical industry due to their tendency toward carbonization. Compounds in C8 aromatic isomer feed are analyzed by means of sample concentration, followed by separation of individual compounds by gas chromatography on a stainless steel OV-101 phase capillary column and identification by gas chromatography/mass spectrometry and gas chromatography/Fourier transform infrared spectroscopy. Various compounds belonging to different classes (mainly monocyclic, dicyclic, and tricyclic aromatics), oxygenated aromatics, and aliphatic saturates are quantified in the concentrated hydrocarbon residue of C8 isomer feed. Both unsubstituted and alkyl substituted ring type compounds are present. Concentrations obtained for PAH compounds in the C8 isomer feed range from 0.2 to 0.42 micrograms/mL.     

Identification of organic acids in bayer liquors by GC-MS: a comparison using butylation and methylation

Two well known derivatization methods, butylation and methylation, are compared by gas chromatography (GC)-mass spectrometry (MS) to identify organic acids in Bayer liquors. These two derivatization methods should be combined together for the determination of the carboxylic acids. Twenty-four organic acids are identified by GC-MS and 13 organic acids are firstly found in Bayer liquors. The retention times and the carbon number of these seven n-dicarboxylic acids (C(4)-C(10)) are fit into the linear relationship in Microsoft Excel.     

Capillary gas chromatographic analysis of volatile and non-volatile organic acids from biological samples as the t-butyldimethylsilyl derivatives

A quantitative procedure for the analysis of volatile organic acids and lactic acid in silage is described. The samples were extracted with diethyl ether, derivatized by t-butyldimethylsilylation, and then separated by capillary gas chromatography. The same procedure was useful for the identification by gas chromatography/mass spectrometry of organic acids in samples such as the metabolic fermentation products of anaerobic bacteria.     

Rapid analysis of sorbitol, galactitol, mannitol and myoinositol mixtures from biological sources

The three commonly found hexitols mannitol, sorbitol and galactitol are well separated from each other and from myoinositol by gas chromatography as their butylboronate derivative on Dexsil-400, on a 1:1 mixture of OV-1 and OV-17, or on a DB-17 fused-silica capillary column. The method allows all four substances to be measured by autosampling electron ionization gas chromatography-mass spectrometry (GC-MS) in small tissue samples at organ concentrations as small as 5 mumol/kg wet mass in less than 4 min. Comparisons were made to determine the relative sensitivity of GC-MS and other detection methods. The order of sensitivity was electron ionization GC-MS greater than chemical ionization GC-MS greater than flame photometric detection using a boron-selective filter greater than hydrogen flame ionization detection.     

Metabolic profiling of Lactococcus lactis under different culture conditions

Gas chromatography mass spectrometry (GC-MS) and headspace gas chromatography mass spectrometry (HS/GC-MS) were used to study metabolites produced by Lactococcus lactis subsp. cremoris MG1363 grown at a temperature of 30 °C with and without agitation at 150 rpm, and at 37 °C without agitation. It was observed that L. lactis produced more organic acids under agitation. Primary alcohols, aldehydes, ketones and polyols were identified as the corresponding trimethylsilyl (TMS) derivatives, whereas amino acids and organic acids, including fatty acids, were detected through methyl chloroformate derivatization. HS analysis indicated that branched-chain methyl aldehydes, including 2-methylbutanal, 3-methylbutanal, and 2-methylpropanal are degdradation products of isoleucine, leucine or valine. Multivariate analysis (MVA) using partial least squares discriminant analysis (PLS-DA) revealed the major differences between treatments were due to changes of amino acids and fermentation products.     

Glass-capillary chromatography in medical research

Summary This paper deals with our experiences with glass-capillary chromatography in medical research. We reported earlier about chromatography of fatty acid methyl esters on a 50 m glass capillary, coated with FFAP. Now we have experimented with a 50 m Silar 10 C column, which gives even better results. The analysis of bile acids on 20 m columns, coated with OV-17, SE-54 or Silar-10 C yields good chromatograms and resolves the main bile acids by base line separation. Further, we are able to report the chromatography of steroid hormones extracted from human urine on a 50 m glass capillary coated with OV-101. Preliminary assessment can be made of the chromatography of the 20 major amino acids after butylation and acetylation on a 50 m Silar-10 C column. As an example of the analysis of vitamins we have chosen the chromatography of nicotinic acid and its isomer. In summary, we are able to show the wide variety of applications of the glass capillary column in medical research.     

A new derivative for improved GLC separation of mannose,galactose and glucose

Summary The procedure involving oxime formation followed by silylation for the analysis of standard hexoses by GC resulted in unseparated peaks for D-glucose, D-mannose and D-galactose. This procedure was therefore modified to obtain clear separation of hexoses. STOX (hydroxylamine containing phenyl--D-glucopyranoside as the internal standard) was mixed with trimethylsilylimidazol or hexamethyldisilazane, and then added to a standard sugar mixture containing D-glucose, D-galactose and D-mannose. The mixture was analyzed by gas chromatography with OV-17/OV-22 as the stationary phase, resulting in three distinct peaks.     

Chromatographic techniques in inborn errors of metabolism

Chromatography has played a pivotal role in the advances made during the last 30 years in our knowledge of inborn errors of metabolism. This review discusses the application of some of these techniques to the analysis of organic acids and acylcarnitines. The separation of organic acids needed a comprehensive approach that would permit all of the many organic acids present in urine or other complex mixtures to be extracted, analysed and identified in a single run. This required analytical methods of great resolving power, wide linear range and universal detectors such as gas chromatography (GC), or GC coupled with mass spectrometry. Sample preparation was another problem that has been tackled by a variety of approaches. Organic solvents have been employed widely for the extraction of organic acids from physiological fluids. Unfortunately, recoveries of the different organic acids by this method are sometimes less than quantitative and variable depending on the compound. Other methods, such as the use of DEAE-Sephadex columns, have the advantage of resulting in close to 100% recoveries, but are more tedious. Liquid partition chromatography on short silicic acid columns has also been recommended as a useful clean-up step prior to GC, permitting both the identification and quantitation of organic acids in urine, plasma or amniotic fluid. Although many derivatization procedure have been used to prepare organic acids for gas chromatography, the most common is trimethylsilylation. Oxo acids are usually reacted with one of several commonly used reagents to form oximes. GC analysis of organic acids was initially done using packed columns with methylsilicone-based, non-polar stationary phases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deconvolution gas chromatography/mass spectrometry of urinary organic acids--potential for pattern recognition and automated identification of metabolic disorders

The National Institute of Standards and Technology (NIST) Automated Mass Spectral Deconvolution and Identification System (AMDIS) is applied to a selection of data files obtained from the gas chromatography/mass spectrometry (GC/MS) analysis of urinary organic acids. Mass spectra obtained after deconvolution are compared with a special user library containing both the mass spectra and retention indices of ethoxime-trimethylsilyl (EO-TMS) derivatives of a set of organic acids. Efficient identification of components is achieved and the potential of the procedure for automated diagnosis of inborn errors of metabolism and for related research is demonstrated.     

Direct trace determination of organic compounds in aqueous solutions by capillary gas chromatography

The possibility of the direct determination of methyl alkyl ketones with four to eight carbon atoms in the alkyl chain and heterocyclic compounds (2-acetylpyridine, 2,3-diethylpyrazine, and 2-acetylthiophene) in aqueous solutions at a level ofn × 10^-3 vol % by gas chromatography with the injection of samples into an open tubular column with carrier gas flow splitting is studied. A significant effect of the calibration plot relating the concentration and the relative area of a gas-chromatographic peak on the results of determination is demonstrated. Factors that make it possible to optimize the calibration process, increase the rapidness of analysis, and decrease the relative error in the determination of organic compounds in water to 3% are discussed. Model mixtures of compounds were analyzed on a open tubular column with the nonpolar OV-101/KF stationary phase, which is stable in work with aqueous solutions for three years     

大鼠肝过氧化物酶体中脂肪酸的分析

 用双 (2 乙基己基 )酚酞酸酯 (DEHP)诱导大鼠肝过氧化物酶体增殖 ,再用蔗糖密度梯度离心法分离大鼠肝细胞过氧化物酶体 ,并用十七烷酸作内标 ,以毛细管气相色谱法在非极性SPB 1石英毛细管柱上对其中的 11种脂肪酸进行分离测定。正常组和诱导组的大鼠肝过氧化物酶体中的不饱和脂肪酸和长链脂肪酸所占总脂肪酸的比例及总脂肪酸的统计结果是 :诱导组的不饱和脂肪酸的含量高于正常组的 (P <0 0 5 ) ,而两个组的脂肪酸总量及长链脂肪酸的含量无明显差别。结果提示 :诱导组的大鼠肝过氧化物酶体的脂肪酸成分发生了变化 ,其膜结构与正常组的不相同。     

Kovats and lee retention indices determined by gas chromatography/mass spectrometry for organic compounds of environmental interest

Retention indices of standard organic compounds of environmental interest were determined by gas chromatography/mass spectrometry, using a DB-5 fused-silica capillary column. Retention indices are useful references for tentative compound identification by gas chromatography, or confirmation by gas chromatography/mass spectrometry. They provide elution order for isomers that might be indistinguishable based on mass spectra. Modified Kovats and Lee retention indices are given for polycyclic aromatic hydrocarbons; sulfur heterocycles; nitrogen heterocycles; aromatic amines; oxygen heterocycles; phenols; alcohols; ketones; alkanes; nitriles; and methylesters of fatty, dicarboxylic, and aromatic acids for comparison and reference. Retention index values for heterocycles by gas chromatography/mass spectrometry are comparable with gas chromatography values previously reported.