Analysis of methylthiohydantoins of amino acids by gas-liquid chromatography of their trimethylsilyl derivatives

A procedure is described for the analysis of methylthiohydantoins of amino acids from the Edman degradation of proteins and peptides by gas-liquid chromatography of their trimethylsilyl derivatives. The procedure is applicable to the methylthiohydantoins of all the amino acids commonly found in proteins with the exception of arginine, which did not yield a volatile derivative, and hydroxyproline and hydroxylysine, which were not investigated. Chromatographic separation is achieved in a single run with only one unresolved pair, which can be separated by a supplementary procedure requiring only 4.5 min.     

Quantitative assay of conjugated and free bile acids as heptafluorobutyrate derivatives by gas-liquid chromatography.

Quantitative analyses of individual bile acids in biological samples are limited by the lengthy multistep preparations necessary. Using heptafluorobutyric acid anhydride in pyridine as derivatizing agent, we reduced several steps to one. Bile acids and their glycine and taurine conjugates form stable heptafluorobutyrate derivatives, climinating the need for deconjugation and preparation of methyl esters. The derivatives have been characterized by mass spectrometry, and optimum reaction yields have been determined. Operating conditions for analyzing the bile acid heptafluorobutyrates by gas-liquid chromatography on various column packings were investigated, and 0.5% QF-1 or 3% OV-255 was found suitable. The bile acid derivatives were identical whether starting with the bile acid or the glycine or taurine conjugates. The procedure was applied to a quantitative analysis of artificial mixtures of bile acids and bile conjugates, and also of human bile. The results compared favorably to those obtained with a 3 alpha- and 7 alpha-hydroxysteroid dehydrogenase fluorimetric method.     

Analysis of thiohydantoins from amino acids by gas-liquid chromatography on short glass capillary columns

A method for separation of amino acid methyl or phenyl thiohydantoins by GLC on a short glass capillary column is described. Calculations of required parameters of the capillary column are presented. By the described methods, nineteen of twenty silylated methylthiohydantoins were separated in one run. The last one (histidine) can be identified on the same column by starting the analysis at a higher temperature. Cysteine and arginine were analysed as S-methylcysteine and ornithine, respectively.     

Capillary gas chromatographic determination of proteins and biological amino acids as N(O)-tert.-butyldimethylsilyl derivatives

Forty-seven biological amino acids containing all 22 protein amino acids were derivatized to N(O)-tert.-Butyldimethylsilyl (tBDMSi) derivatives by a single-step reaction with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide and successfully separated on an HP-1 capillary column. The relative standard deviations of the relative molar responses of most amino acids were <5%. Cystine seems to be partially converted into cysteine during derivatization. An increase in carrier gas flow-rate towards the end of the analysis by inlet pressure programming with electron pressure control avoided the peak broadening and adsorption of the derivatives with high boiling points on the column and especially increased sensitivity of cystine to 5 pmol. Glutamine was converted almost completely into pyroglutamic acid during prolonged storage of a standard solution prepared in 0.01 M HCl but not during derivatization. These results compared with those for the phenylthiocarbamyl derivatives analysed by HPLC and the analytical results reported in the literature on soybean hydrolysate showed good agreement except for cysteine. The results for the amino acid composition of bovine serum albumin also showed good agreement with results in the literature except for cysteine. In human urine, seventeen free amino acids were detected as tBDMSi derivatives.     

Analysis of glycated amino acids by high-performance liquid chromatography of phenylthiocarbamyl derivatives

A method has been developed for the analysis of hexitolamino acids formed by acid-catalyzed hydrolysis of nonenzymatically glycated proteins that have been treated with sodium borohydride. The hexitolamino acids are converted into phenylthiocarbamyl (PTC) derivatives which are analyzed by reverse-phase HPLC. The PTC derivatives of N alpha-hexitolamino acids behave like lactones, migrating on the column more slowly than the corresponding PTC-amino acids. The PTC derivatives of N epsilon-glucitol- and N epsilon-mannitol-lysine are probably free acids, since they migrate faster than PTC-lysine. The method, which can be used to determine the degree of glycation of N-terminal and lysyl residues, has been applied successfully to human hemoglobin, serum albumin, and ocular lens proteins.     

O-methyl oximes of sugars. Analysis as O-trimethylsilyl derivatives by gas-liquid chromatography and mass spectrometry

The mass spectra of aldoses, partially methylated aldoses, deoxyaldoses, and ketoses containing 3–7 carbons, were recorded on the ethers of the trimethylsilyl O-methyl oxime derivatives. Each compound gave a distinctive spectrum indicating the carbon-chain length and the location of substituents. The gas-liquid chromatographic properties of most compounds in this study were also examined.     

Quantitative gas-liquid chromatography and mass spectrometry of the N(O)-perfluorobutyryl-O-isoamyl derivatives of amino acids.

A modification of a gas-liquid chromatographic method for quantitative analysis of amino acids as the N(O)-perfluorobutyryl-O-isoamyl derivatives is described. The modifications include changes in time and temperature for esterification, improved preparation of the esterification reagents and conducting the derivatizations in vacuo to obtain reproducible values for amino acids such as methionine and arginine. Mass spectral data are presented for all the derivatized amino acids.     

Preparative separation of amino acid enantiomers as N-TFA,O-isopropyl derivatives by continuous countercurrent gas-liquid chromatography

Preparative separation of derivatives of amino acid enantiomers was carried out by a countercurrent gas–liquid chromatography (CCGLC) with chiral liquid phases, N-stearoyl-L -valine tert-butylamide and/or N-stearoyl-L -leucine tert-butylamide. In order to make effective use of these phases and also to lower the viscosity, Apiezon C was added as diluent. Through a repeated operation of a temperature gradient, purities more than 99% of leucine and α-amino butyric acid derivatives were proved to be obtained. © 1993 Wiley-Liss, Inc.     

Quantitative extraction of methylmalonic and succinic acids and their determination by gas-liquid chromatography.

A method for routine small-volume solvent extraction, derivitization and gas-liquid chromatographic analysis of methylmalonic and succinic acids is described. The procedure allows for quantitative determination of mass and radioactivity of these acids and can be applied to metabolic studies of the genetic disorder methylmalonylacidurea.     

Analysis of the amino acid 3-methylhistidine by gas-liquid chromatography

A method for the quantitative separation of 3-methylhistidine from other amino acids, by gas-liquid chromatography, has been developed. This method gives complete resolution of the N-heptafluorobutyryl isobutyl esters of 20 amino acids with the use of a single column packed with 3% SE-30 on $\text{100}\text{120}$ mesh Gas Chrom Q. Using this method the 3-methylhistidine content of urine and meat has been determined.     

Determination of the specific radioactivity of fatty acids separated as their methyl esters by gas-liquid chromatography

Free or combined (3)H-labeled fatty acids are converted to their methyl-(14)C esters or, if labeled with (14)C, to their methyl-(3)H esters. For a given specific radioactivity of the methyl group, the nuclide ratio in the esters separated by GLC is a direct measure of the specific radioactivity of the fatty acids, and quantitative collection is unnecessary. Methods of methylation with minimum quantities of labeled methanol, and of deriving nuclide ratios from channel ratios in a scintillation spectrometer, are given.     

Gas chromatography-mass spectrometry analysis of tert.-butyldimethylsilyl derivatives of 2-acetylaminofluorene and metabolites in isolated rat hepatocytes

A new technique for the conversion of 2-acetylaminofuorene and several ring-hydroxylated metabolites to mono- and di-tert.-butyldimethylsilyl derivatives was developed to permit their analysis by gas chromatography-mass spectrometry in order to quantify the metabolism of 2-acetylaminofluorene incubated in freshly isolated rat hepatocytes. This new gas chromatography-mass spectrometry method allowed the separation, identification and quantitation of seven known metabolites comprising five arylhydroxylated compounds, 2-aminofuorene and N-hydroxy-2-acetylaminofuorene.Abbreviations 2-AAF 2-acetylaminofluorene - 2-AF 2-aminofluorene - DMF dimethylformamide - El electron impact ionization - FBS fetal bovine serum - GC-MS gas chromatography-mass spectrometry - MtBSTFA N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide - MU methylene unit - N-OH-2-AAF N-hydroxy-2-acetylaminofluorene - 4,4-OH-BP 4,4-hydroxybiphenyl - tBDMS tert.-butyldimethylsilyl     

Analysis of bile acids in serum and bile by capillary gas-liquid chromatography

Various liquid phases for glass capillary columns have been evaluated for gas-liquid chromatographic analysis of methyl ester trimethylsilylether derivatives of bile acids from serum and bile. Bile acid analysis is rapid and exhibits high separation efficiency with a 20 X 0.3 mm glass capillary column whose internal surface is covered with a crystal layer of barium carbonate and coated with polyethyleneglycol 20000 as liquid phase according to Grob et al.     

Analysis of unsaturated fatty acids, and their hydroperoxy and hydroxy derivatives by high-performance liquid chromatography.

A simple procedure for the detection of endo-β-N-acetylglucosaminidase H activity is described. The method utilizes N-[¹⁴C]methylribonuclease B as substrate. This is prepared from ribonuclease B by reductive alkylation of free amine groups in the protein with [¹⁴C]formaldehyde. Because the carbohydrate moiety of ribonuclease B has α-mannosyl residues at nonreducing terminal positions, the radioactive molecule binds to Sepharose-concanavalin A. Endo-β-N-acetylglucosaminidase action releases this mannose-containing oligosaccharide by splitting the di-N-acetylchitobiosyl residue that links it with the peptide and thereby renders the radioactive portion of the molecule unreactive with Sepharose-concanavalin A. This forms the basis of a convenient assay for screening column fractions during the purification of the endoglycosidase. Although protease or α-mannosidase activity might also be detected by the procedure, no difficulties were presented by these enzymes when the assay was used for the preparation of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus.     

Analysis of polyamines as their dabsyl derivatives by reversed-phase high-performance liquid chromatography

The polyamines putrescine, cadaverine, spermidine, and spermine and the corresponding mono-N-acetylpolyamines can be separated as their dimethylaminoazobenzenesulfonyl derivatives in a single analysis in less than 22 min. The method employs reversed-phase high-performance liquid chromatography (Spherisorb S5 ODS2 column) with an acetonitrile/acetate buffer gradient elution system and detection in the visible (436 nm) region. The detection limit for a single dimethylaminoazobenzenesulfonylpolyamine is less than 2 pmol.