N-trifluoroacetyl N-butyl Esters Research Articles

Determination of sulfur amino acids, glutathione, and related aminothiols in biological samples by gas chromatography with flame photometric detection.

The determination of sulfur amino acids, glutathione (GSH), and related aminothiols has been carried out by isotachophoresis (), amino acid analyzer (AAA) (,), gas chromatography (GC) (, , , , ), high-performance liquid chromatography (HPLC) (, , , , , , , , , , , , , , , , , , , , , , , ), GC-mass spectrometry (GC-MS) (,), liquid chromatography-mass spectrometry (LC-MS) (), and capillary zone electrophoresis (CZE) (, , ). However, isotachophoresis and AAA methods are nonselective for sulfur amino acids and lack sensitivity. GC methods based on the conversion into trimethylsilyl (,), neopentylidine (), and N-trifluoroacetyl n-butyl ester () derivatives lack sensitivity, gives a tailing peak and requires anhydrous derivatization conditions. Although GC method based on the preparation of N-heptafluorobutyryl isobutyl or ethyl esters () is selective and sensitive by flame photometric detection (FPD), this method is not applied to the analysis of biological samples. As the HPLC methods, the ultraviolet (UV) (, , , ), and the postcolumn UV derivatization with 4,4′-dithiopyridine () and 5,5-dithiobis (2-nitrobenzoic acid) (), the precolumn fluorescence derivatization with 4-(aminosulfonyl)- or ammonium-7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (,), methyl 4-(6-methoxynaphthalene-2-yl)-4-oxo-2-butenoate (), N-(1-pyrenyl)maleimide (), monobromobimane (, , , , ), o-phthaldialdehyde (,), 9-fluorenylmethyloxycarbonyl chloride (), and 2-chloro-1-methylpyridinium (), and electrochemical detection (, , , , ) have been reported. Many of these HPLC methods were highly sensitive, but some of these methods lack specificity and require cleanup of the sample to remove the excess reagent and coexisting substances. GC-MS methods based on the conversion into tert-butyldimethylsilyl () and N(O,S)-propoxycarbonyl propyl ester () derivatives, and LC-MS method were highly sensitive and specific, but these methods require expensive equipment. Furthermore, CZE methods with electrochemical detection are capable of achieving higher separation efficiency, use less organic solvents, and require small amounts of samples in comparison with HPLC, but these methods are not applied enough to the analysis of biological samples. Current methods for the determination of sulfur amino acids, GSH, and related aminothiols have also been described in detail in refs. , , , , .

Variations in the amino acid composition of cyanophycin in the cyanobacterium Synechocystis sp. PCC 6308 as a function of growth conditions.

Gas chromatography-mass spectrometry studies of the nitrogen isotopic composition of the N-trifluoroacetyl n-butyl ester derivatives of the amino acids from isolated hydrolyzed cyanophycin from 15N-enriched cells led to two major findings: (1) the amino acid composition of this granular polypeptide, isolated using procedures optimized for extracting and purifying cyanophycin from cells in the stationary growth phase, varied with the culture growth condition; (2) the rate of incorporation of exogenous nitrate differed for each nitrogen atom of the amino acid constituents of cyanophycin or cyanophycin-like polypeptide. Arginine and aspartic acid were the principle components of cyanophycin isolated from exponentially growing cells and from light-limited stationary phase cells, with glutamic acid as an additional minor component. The cyanophycin-like polypeptide from nitrogen-limited cells contained only aspartic and glutamic acids, but no arginine. The glutamic acid content decreased and arginine content increased as nitrate was provided to nitrogen-limited cells. These cells rapidly incorporated nitrate at different rates at each cyanophycin nitrogen site: guanidino nitrogens of arginine > aspartic acid > alpha-amino nitrogen of arginine > glutamic acid. Little media-derived nitrogen was incorporated into cyanophycin of exponentially growing cells during one cellular doubling time.

Analysis of Amino Acids by Gas Chromatography as the N-Trifluoroacetyl η-Butyl Esters

This presentation describes amino acid analysis with the gas chromatographic method and experimental conditions using the N-trifluoroacetyl n-butyl ester derivatives; the study we describe here was undertaken to compare gas chromatographic (GC) and ion-exchange chromatographic (IEC) analyses of amino acids in hydrolysates of 9 diverse sample types to gain insight into effects of these 2 chromatographic methods of analysis on variation in amino acid results. Our study showed that values for samples prepared by 2 separate laboratories using the same procedure were generally in good agreement when all of the hydrolysates were analyzed by a single laboratory using a single method of analysis. To compare results from gas chromatography with those from ion-exchange chromatography analyses were performed by 2 different laboratories on the same hydrolysates and on different hydrolysates prepared by the same method by both laboratories. The data demonstrate that GC and IEC can be expected to yield essentially identical results when applied to the same hydrolysate. Agreement is so close that interlaboratory differences in hydrolysate preparation of the same sample contribute as much to variation in amino acid results as does the method of analysis, a fact which should be noted in planning collaborative studies.

Gas—liquid chromatography of free amino acids in the cytosol of mammalian atrium and ventricle of the heart

N-Trifluoroacetyl n-butyl ester derivatives of amino acids were studied. The investigations, carried out on random slaughter-house material, were based on the results of histological and biochemical differences between the atrium and ventricle of the mammalian heart muscle. The data indicate a high level of glutamic acid, and a predominance of tyrosine in the atrium and phenylalanine in the ventricle. The results provide evidence for metabolic differences between the atrium and the ventricle of the heart. This finding may have some significance in heart muscle defects.

Gas-liquid chromatography of free amino acids in the hyaloplasm of rat liver and kidney cells

Gas-liquid chromatography of free protein amino acids has a high and verified reproducibility of quantitative and qualitative estimation: Cytoplasmic matrix obtained from the 105,000 g supernatant fraction of rat liver and kidney cells was investigated. The ultracentrifuged product was passed through ion-exchange resin, thus isolating free amino acids from the eluate. After lyophilization the product was derivatized to N-trifluoroacetyl n-butyl esters and separated on the OV 17 + 210 and ethylene glycol adipate phases. Flame ionization detection was used. Comparison of liver and kidney tissues showed significant differences in the concentrations of the amino acids studied.

Gas chromatography-mass spectrometry of lysinoalanine and the related crosslinked amino acids in alkali-treated food proteins.

Mass spectra of N-trifluoroacetyl n-butyl ester (BTFA) of ornithinoalanine (OAL) and lanthionine (LAN) were compared with those of the BTFA derivatives of lysinoalanine (LAL) and the related amino acids. A difference of m/z 14, corresponding to one methylene group, was found in each pair of characteristic fragments between BTFA-OAL and BTFA-LAL. A temperature-programmed gas-liquid chromatography and gas chromatography-mass spectrometry were developed to analyze BTFA-LAN, BTFA-OAL and BTFA-LAL. More LAL and LAN were formed in α-lactalbumin than lysozyme by high-temperature treatment in water. OAL was detected in lysozyme treated at 100° and 120°C in alkali solution, but not in α-lactalbumin.

Gas chromatography and mass spectrometry of N-trifluoroacetyl n-butyl esters of alkylated tyrosines and lysines

The gas chromatographic behavior of three O- n-alkyltyrosines, four N t - n-alkyllysines and three N t , N t -di- n-alkyllysines was studied as their N-trifluoroacetyl n-bytyl ester (BTFA) derivatives on columns of OV-17 and Dexsil 300 GC. The retention indices and some other retention parameters are discussed in relation to the chemical structures of the amino acids. Mass fragmentation patterns of the BTFA derivativs of these amino obtained by gas chromatography-mass spectrometry with electron impact ionizaton are also described.

A method for the measurement of sulfur-34 abundance in bound cysteine and methionine

A technique is described for determining the abundance of sulfur-34 in bound cysteine and methionine. Samples to be assayed are first boiled with water to remove any nonbound cysteine and methionine, reduced with mercaptoethanol, and alkylated with methyl iodide in order to convert the cystine and cysteine residues into S-methylcysteine. Following hydrolysis with 6 m hydrochloric acid, the released amino acids are desalted in a Dowex 50W-8X column and converted into their N-trifluoroacetyl n-butyl esters for gas chromatographic-mass spectrometric analyses. Analysis of the sulfur isotope abundances in ions from the S-methylcysteine and methionine derivatives allows the determination of the atom% abundance of sulfur-34 in these molecules with a standard deviation of less than 1 atom%. Data are presented on the incorporation of 34S from sulfate by Escherichia coli under a variety of growth conditions.

The enantiomer as internal standard for the quantitation of the alkylated amino acid S-methyl-L-cysteine in haemoglobin by gas chromatography—chemical ionisation mass spectrometry with single ion detection

A method has been developed using gas chromatography—chemical ionisation mass spectrometry for he determination of S-methyl-L-cysteine in rat and human haemoglobin. The D-enantiomer of S-methylcysteine is added as an internal standard prior to the protein hydrolysis and a partial purification of the protein hydrolysate made by ion-exchange column chromatography. The enantiomers of S-methylcysteine are separated as their N-trifluoroacetyl n-butyl ester derivatives on a capillary column coated with the chiral stationary phase Chirasil-Val. The use of single ion detection in the assay is three times more sensitive than the use of multiple ion detection with a deuterium labelled internal standard. The method has been applied to haemoglobin samples from rats and human exposed to methylating agents.

Studies on the gas chromatographic analysis of lysinoalanine in alkali-treated food proteins.

In order to analyze the cross-linked amino acid formed in the food protein by alkali-treatment, an isothermal gas Chromatographic procedure was developed. After acid-hydrolysis of protein, amino acids were converted to N-trifluoroacetyl n-butyl esters and then analyzed on a mixed column of OV–17/OV-210 at 230°C. Identification of the substances was performed by gas chromatography-mass spectrometry. The presence and the formation of lysinoalanine in several kinds of food proteins were investigated. The amount of lysinoalanine formed depended on alkali concentration, reaction temperature and exposure time. 2-Mercaptoethanol inhibited lysinoalanine formation.

Protein amino acid analysis by an isotope ratio gas chromatography mass spectrometry computer technique.

A method for quantitative analysis of protein amino acids by a gas chromatography mass spectrometry computer system is described. Amino acids were analysed as their N-trifluoroacetyl n-butyl ester derivatives. Isotope ratio determination was used as the quantitating technique via multiple internal standards. The exact composition of a deuterated amino acid mixture was determined against a standard amino acid calibration mixture and in turn the protein amino acid composition was determined against the deuterated amino acid mixture. The amount of protein taken for analysis was 100 micrograms and the procedure, excluding hydrolysis, could be performed with 2 1/2 hours. The introduction of the internal standards prior to protein hydrolysis provides a method with good precision (mean coefficient of variation less than 5%). The method, tested on insulin, gave results which agreed well with the known composition of the protein and with simultaneous analysis on ion exchangers.

Gas-Liquid Chromatography of Histidine, Arginine, and Cystine. Interactions with the Liquid and Solid Support Phases

The interactions of the N-trifluoroacetyl n-butyl ester derivatives (TAB) of histidine, arginine, and cystine with the liquid phase, ethylene glycol adipate (EGA), and/or solid support were investigated. Glutamic acid was used as internal standard to determine the relative weight response (RWR) values of the three amino acids. Changes in the RWR values served as an index of substrate or solid support interaction. Essentially nonpolar Gas-Chrom Q and more reactive Chromosorb W AW were coated with a nonpolar OV-101 polymer and tested to determine whether the solid support contributed to the breakdown of the TAB derivatives of these three amino acids. Then a bilayer column was prepared by coating OV- 101/Gas-Chrom Q with polar EGA to determine the interaction of the EGA polymer with the TAB derivatives of histidine, arginine, and cystine. It was found that the polar substrate, EGA, was primarily responsible for the breakdown of histidine. The decomposition of arginine and cystine was caused mainly by the solid support (Chromosorb W AW), and the more active support significantly increased the degree of breakdown. As the loading of the EGA polymer on the Gas-Chrom Q of the polymer bilayer was decreased, an increased histidine response was observed while the arginine and cystine response remained unaffected. Also, the histidine response was substrate contact time and temperature dependent and increased significantly on a bilayer column with decreased residence times; whereas, the arginine and cystine response decreased, again indicating complex temperature dependent interactions with the EGA-polymer and/or solid support phase. Nickel and glass tubing filled with the EGA bilayer packing were compared in the chromatography of the TAB derivatives. The nickel tubing completely destroyed the derivatives of histidine, arginine, and cystine. For the quantitation of all 20 protein amino acids by gas-liquid chromatography (GLC) the two-column system introduced by Gehrke and Kuo, et al. (33) still remains a valuable method.

Gas-liquid chromatographic separation of common amino acids in pine needle extracts

An improved gas—liquid chromatographic method based on the separation of N-trifluoroacetyl n-butyl esters of amino acids on a “two-column” setup  Tabsorb and Tabsorb HAC  was developed for the identification and estimation of amino acids in pine needles ( Pinus banksiana Lamb.). A comparative study was made of various available gas—liquid chromatographic methods for separation and estimation of amino acids from pine needle extracts.

On the derivatives for the ultramicrodetermination of amino acids by mass fragmentography

The fragmentation of eight volatile derivatives of alanine, valine, leucine, proline, and glutamic acid upon electron impact is studied for the concurrent ultramicrodetermination or the selective identification of amino acids by mass fragmentography. The relationship between the sensitivity of mass fragmentography and Σ 10% values (percentage of the total ionization over m e ) of N-trifluoroacetyl- l-prolyl, N-benzoyl, and N-pentafluorobenzoyl derivatives of alanine, valine, leucine, and proline is also investigated. The results obtained in this study suggest that N-trifluoroacetyl- l-prolyl n-butyl ester, N-benzoyl n-butyl ester, N-pentafluorobenzoyl n-butyl ester, and N-trifluoroacetyl l-menthyl ester derivatives can be used for the concurrent ultramicrodetermination or selective identification of amino acids by mass fragmentography because these four derivatives produce common intense fragment ions at m e 166, 105, 195, and 83 or diagnostic ions, respectively. Although N-trifluoroacetyl- l-4-thiazolidinecarbonyl n-butyl ester, N-trifluoroacetyl n-butyl ester, N-trimethylsilyl n-butyl ester, N-trimethylsilyl l-menthyl ester, and trimethylsilylated derivatives do not produce the common intense fragment ions, these derivatives are useful for the selective identification of ultra amounts of amino acids using mass fragmentography monitored the diagnostic ions, respectively. The general common rules between sensitivity and Σ 10% values were not adhered among the derivatives studied.

GAS-LIQUID CHROMATOGRAPHIC ANALYSIS OF AMINO ACIDS FOR SYNIHETIC POLY (AMINO ACID)

Copoly (Ne-carbobenzyloxy-L-lysine γ-methyl-L-glutamate) prepared from N-carboxy anhydrides (NCA) of Ne-carbobenzyloxy-L-lysine and γ-methyl L-glutamate was hydrolyzed in a mixture of trifluoroacetic acid and 12N HCl at 100°C. The amino acids in the hydrolyzate were converted to their N-trifluoroacetyl n-butyl esters and quantitatively analyzed by gas-liquid chromatography. Accuracy of the analytical data was discussed.

The structure of mycosubtilin, an antibiotic isolated from Bacillus subtilis (author's transl)

Mycosubtilin, an antifungal agent isolated from Bacillus subtilis is a mixture of homologous lipopeptides essentially C54H83N14O16 and C55H85N15O16. The differences in their structures was found in the lipid moiety which contains several beta-amino acids; the structure of these beta-amino acids was demonstrated by combined gas chromatography-mass spectrometry of the N-trifluoroacetyl n-butyl esters; a strong peak at m/e = 240 indicates a beta-amino group. The comparison of the derivatives of natural amino acids with synthetic 3-aminohexadecanoic and 3-aminoheptadecanoic acids indicates that natural beta-amino acids are a mixture of 3-amino-14-methylpentadecanoic acid (35%), 3-amino-14-methylhexadecanoic acid (59%), 3-aminohexadecanoic acid (6%) and a trace of C18 beta-amino acid. The peptide moiety contains 8 moles of amino acids, two of D-aspargine, two of L-asparagine, and one of L-glutamine, L-proline, D-serine and D-tyrosine. The peptide sequence was determinated by partial acid hydrolysis of mycosubtilin and isolation and structural determination of the peptides from the hydrolysates. Four liposoluble peptides and four hydrosoluble peptides were studied. The results gave the cyclic structure shown on Formula 1 for mycosubtilin.

Quantitative gas-liquid chromatography of non-protein amino acids

The quantitative gas-liquid chromatographic analysis of non-protein amino acids, in the presence of protein amino acids, is described. The amino acids were determined as their N-trifluoroacetyl n-butyl esters on an ethylene glycol adipate column. The relative molar responses of 38 amino acids are reported.

Determination of Lanthionine and Lysinoalanine as N-Trifluoroacetyl n-Butyl Esters by Gas-Liquid Chromatography

A gas-liquid chromatographic method was explored for the analysis of lanthionine and lysinoalanine as well as lysine, cystine, and S-carboxyethylcysteine in chemically modified wool samples. The amino acids in the wool hydrolyzate were converted to n-butyl esters of N-trifluoroacetyl derivatives according to the method of Gehrke et al. The retention indices of the five amino acids mentioned above as well as other protein amino acids such as proline, phenytalanine, histidine (as mono and diacyl derivatives), tyrosine, arginine, and tryptophane were determined on OV-17 and Dexsil 300 GC. It was found that Dexsil 300 GC was suitable for the intended analysis with n-butyl stearate as an internal standard. The molar responses of these amino acids were determined.

Gas—liquid chromatographic behaviour of n-trifluoroacetyl n-butyl esters of various s-substituted cysteines

The gas—liquid chromatographic behaviour of four S- n-alkyl-, three S-ω-carboxyalkyl- and S- ′ p -aminoethylcysteines, lanthionine, cystine. S,S′-methylene- and S,S′-ethylenebiscysteines, and some homocysteine analogues, as their N-trifluoroacetyl n-butyl esters, was studied on columns of OV-17 and Dexsil 300 GC and compared with the behaviour of similar non-sulphur-containing amino acids. The retention indices and some other parameters are discussed in relation to the structure of the amino acids. Relative molar responses were determined and an empirical additive method for calculating relative molar responses from the structural feature is described.

Quantitative gas chromatographic analysis of amino acids in the form of N-trifluoroacetyl N-butyl esters

Quantitative gas chromatographic analysis of amino acids in the form of N-trifluoroacetyl N-butyl esters