Mass spectra of alpha-amino acid oxazolidinones

2-Bis-(chlorodifluoromethyl)-4-substituted-1,3-oxazolidin-5-ones, a new type of alpha-amino acid derivative for gas chromatographic separation, have been studied by low resolution mass spectrometry. These derivatives are obtained by reacting alpha-amino acids with dichlorotetrafluoroacetone. Their structure has been established or confirmed for most protein amino acids and several non-protein alpha-amino acids. The mechanisms responsible for the mass spectral pattern have been rationalized. An interesting feature of this derivatization procedure is that it distinguishes aspartic and glutamic acid from the respective amides. The structure of asparagine and glutamine derivatives has been established. A survey of oxazolidinone mass spectra has provided a list of diagnostically useful ions. Each amino acid can be identified by one or two of its most abundant fragments.     

New procedure for isolation of amino acids based on selective hydrolysis of trimethylsilyl derivatives

A rapid procedure for the isolation of amino acids from physiological fluids by class separation suitable for gas chromatographic and gas chromatographic—mass spectrometric analysis is described. A physiological fluid such as plasma is adjusted to pH 2 and extracted with diethyl ether to remove organic acids and neutrals. After precipitation of proteins with trichloroacetic acid, the aqueous plasma is dried and derivatized by trimethylsilylation. Organic compounds like sugars and amino acids are rendered soluble in petroleum ether leaving inorganic salts when the soluble layer is transferred. Separation of sugars from amino acids is achieved by taking advantage of the different rates of aqueous hydrolysis of the trimethylsilyl (TMS) derivatives. Mixing the petroleum ether extract with a small volume of water results in two phases. The petroleum ether layer contains TMS-sugar constituents of plasma and the aqueous layer contains free amino acids and amines. This procedure was used to isolate L-dopa, 3-O-methyldopa and tyrosine from human plasma in a quantitation assay using ¹⁵O-labelled amino acids and gas chromatography—mass spectrometry.     

A new double-labelling procedure for determination of amino acid composition: application to bacteriorhodopsin

A new double-labelling procedure for amino acid analysis which requires only routine chromatographic equipment is described. When 1-fluoro-2,4-dinitro[3H]benzene is reacted with a mixture of 14C-labelled amino acids followed by reaction with the same 14C-labelled amino acid mixture diluted with an unlabelled sample of amino acids, the 3H:14C ratio in the resulting 2,4-dinitrophenyl (DNP) amino acid derivatives of the diluted sample will be increased in proportion to the quantity of unlabelled amino acid in the diluted sample. This procedure gave reliable results when applied to the known proteins insulin and lysozyme. The procedure is most advantageous when applied to amino acids which are unstable during acid hydrolysis or present in low molar fractions. When applied to the analysis of the bacteriorhodopsin in Halobacterium cutirubrum, this procedure showed the presence of one histidine residue and four tryptophan residues per mole protein but no cystine or cysteine; in general, the analyses obtained were consistent with those originally reported by Oesterhelt, D. and Stoeckenius, W. (1971) (Nature (London) New Biol. 233, 149-152) for bacteriorhodopsin of H. halobium.     

Analysis of all protein amino acids as their tert.-butyldimethylsilyl derivatives by gas-liquid chromatography

A gas chromatographic method for the separation and quantitation of the 20 protein amino acids is described using N-methyl-N(tert.-butyldimethylsilyl)trifluoroacetamide, with 1% tert.-butyldimethylchlorosilane as catalyst, to prepare the tert.-butyldimethylsilyl amino acid derivatives. Alkylsilylation of amino acids proceeds at 140 degrees C in 20 min. The derivatives formed in the one-step reaction are used directly for gas-liquid chromatographic analysis, using a flame-ionization detector, without prior isolation or purification. Complete separation and quantitation of all protein amino acids are readily achieved using a 15-m DB-5 capillary column. Strict linearity extends from less than 15 to about 100 ng for all amino acids except Arg, which has a linear range from 50 to 300 ng. The limits of detection, however, range from one to several hundred nanograms. The method was used to analyze the free amino acid pool in carnation petals.     

The stability of the o-phthalaldehyde/2-mercaptoethanol derivatives of amino acids: an investigation using high-pressure liquid chromatography with a precolumn derivatization technique

The investigation of the stabilities of o-phthalaldehyde/2-mercaptoethanol derivatives of amino acids using a precolumn reaction technique and a high-pressure liquid chromatographic procedure is reported. The amino acid derivatives are shown to be stable on the high-pressure liquid chromatography column. Optimal conditions for the development of these derivatives for their separation using this technique are recommended.     

A micromethod for the analysis of free amino acids by gas chromatography and its application to biological systems.

A gas chromatographic procedure was developed for determination of minute amounts of free amino acids in natural waters and laboratory models simulating biological systems. Sample pretreatment included removal of interfering organic substances by chloroform extraction and isolation of amino acids by cation exchange. Amino acids were converted to their N-heptafluorobutyryl isobutyl ester derivatives in glass capillary tubes, permitting considerable concentration of the sample prior to gc injection. The derivatives of 19 amino acids were successfully separated on either a glass column packed with a mixture of OV-101 and OV-17 on Chromosorb W, a glass capillary column coated with OV-101, or a support-coated capillary column supported with SE-30. One to five nanograms of individual amino acids were detected using flame ionization detector. The detection limit was reduced more than 100-fold using the electron capture detector and more than 1000-fold by mass fragmentography. The procedure allowed determination of less than 1 ppb of individual amino acids in lake and river water samples and was used to estimate the exeretion of free amino acids from microbial populations.     

Fast mass spectrometry-based enantiomeric excess determination of proteinogenic amino acids

A rapid determination of the enantiomeric excess of proteinogenic amino acids is of great importance in various fields of chemical and biologic research and industries. Owing to their different biologic effects, enantiomers are interesting research subjects in drug development for the design of new and more efficient pharmaceuticals. Usually, the enantiomeric composition of amino acids is determined by conventional analytical methods such as liquid or gas chromatography or capillary electrophoresis. These analytical techniques do not fulfill the requirements of high-throughput screening due to their relative long analysis times. The method presented allows a fast analysis of chiral amino acids without previous time consuming chromatographic separation. The analytical measurements base on parallel kinetic resolution with pseudoenantiomeric mass tagged auxiliaries and were carried out by mass spectrometry with electrospray ionization. All 19 chiral proteinogenic amino acids were tested and Pro, Ser, Trp, His, and Glu were selected as model substrates for verification measurements. The enantiomeric excesses of amino acids with non-polar and aliphatic side chains as well as Trp and Phe (aromatic side chains) were determined with maximum deviations of the expected value less than or equal to 10ee%. Ser, Cys, His, Glu, and Asp were determined with deviations lower or equal to 14ee% and the enantiomeric excess of Tyr were calculated with 17ee% deviation. The total screening process is fully automated from the sample pretreatment to the data processing. The method presented enables fast measurement times about 1.38 min per sample and is applicable in the scope of high-throughput screenings.     

Amino acid analysis by high-performance liquid chromatography after derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide

Amino acids are quantitatively determined by precolumn derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide and reversed-phase high-performance liquid chromatography with photometric detection at 340 nm. Excellent chromatographic resolution of a mixture of the derivatives of 20 amino acids including proline and cystine is achieved within 110 min by linear gradient elution with acetonitrile in 13 mM trifluoroacetate plus 4% (by vol) tetrahydrofuran. The limit of detection is 50 pmol. Amino acid analyses of acid hydrolysates of the several proteins gave results equivalent to those obtained by conventional ion-exchange-based amino acid analysis. The simplicity of the procedure allows its use on any multipurpose high-performance liquid chromatographic system.     

4-Methyleneglutamic acid and 4-methyleneglutamine: isolation from extracts of peanut seedlings and determination by high-performance liquid chromatography

The non-protein amino acids, 4-methyleneglutamic acid and 4-methyleneglutamine, are isolated from aqueous extracts of peanut seedlings in good yield and high purity using a simple HCl-gradient elution from a column of cation-exchange resin followed, in some instances, by a gradient elution with acetic acid from a column of an anion-exchange resin. All of the 4-substituted glutamic acids commonly found in legume species are resolved by a combination of these two system. For analytical purposes, resolution of the acidic amino acids as their phenylthiocarbamoyl derivatives is achieved by HPLC but not by conventional ion-exchange amino acid analysis. Although 4-methyleneglutamine undergoes cyclic deamidation in acidic medium at a slower rate than glutamine, this reaction occurs to a significant extent at 22 degrees C but not a 4 degrees C during the cation-exchange chromatographic fractionation.     

Preparation of α-substituted α-amino acids of high enantiomeric purity

Racemic 5,5-dialkyl hydantoins derived from ketones are resolved by preparative liquid chromatography as the diastereomeric 1-carboxamide derivatives afforded by the reaction with optically pure configurationally known α-phenylethyl isocyanate. Hydrolysis of the resolved diastereomers affords α-substituted α-amino acids of high enantiomeric purity. The synthetic route is short, overall yields are high, and the absolute configuration of the amino acid enantiomers may be deduced from the chromatographic and NMR properties of the diastereomers. © 1992 Wiley-Liss, Inc.     

Purification and characterization of cystathionine γ-lyase from Lactobacillus fermentum DT41

A homo-tetrameric ca. 140-kDa cystathionine γ-lyase was purified to homogeneity from Lactobacillus fermentum DT41 by four chromatographic steps. This was the first enzyme responsible for amino acid catabolism purified from lactobacilli. The activity is pyridoxal-5'-phosphate dependent and the enzyme catalyzes the α,γ-elimination reaction of l -cystathionine producing l -cysteine, ammonia and α-ketobutyrate. The cystathionine γ-lyase produced a free thiol group, a keto acid component and ammonia from several amino acids, including l -cysteine and methionine, and amino acid derivatives. l -Cystine was the best substrate. The enzyme was stable in the conditions of cheese ripening and may contribute to the biosynthesis of sulfur-containing compounds.     

Development of a protocol for the semi-synthesis and purification of betaxanthins

A method for the analytical and semi-preparative chromatographic purification of betaxanthins is described together with an improved procedure for the semi-synthesis of these compounds from betalamic acid. Standard conditions for obtaining preparative amounts of betaxanthins free of the precursor amino acids are provided. Following this procedure, 14 pure betaxanthins were obtained with yields of up to 100%. A simple reversed-phase HPLC protocol for pigment identification and quantification is also provided. Calibration for betaxanthins is reported for the first time using the synthesised and purified pigments as standards. Structures were confirmed by UV-vis spectroscopy, HPLC retention times and electrospray ionization mass spectrometry. Betaxanthins can be obtained pure, and in sufficient amounts for further studies, which opens up new perspectives in the research and applications of these pigments.     

Analysis of muramic acid in holocene microbial environments by gas chromatography,electron impact,and fast atom bombardment mass spectrometry

Analytical procedures have been modified to determine the abundance of muramic acid in four different Holocene sediment samples. Muramic acid is specific to the peptidoglycan moiety of the cell walls of most eubacterial pro‐karyotic organisms. The following procedure seemed to be the most appropriate for the detection of muramic acid and amino acids, including diaminopimelic acid. Hydrolysis of the samples (in 6 N HCl, 4.5 h, at 100°C) was followed by separation and purification of amino sugars and amino acids using Amberlite XAD‐2 and then Bio‐Rad AG 50W‐X8 resins. The N,O‐heptafluorobutyryl‐n‐butyl ester derivatives were prepared by esterification in acidified (3 N HCl) n‐butanol for 3 h at 100°C, followed by acylation by refluxing with heptafluorobutyric anhydride in acetonitrile (2:1 v/v) for 12 min at 150°C. The derivatives were analyzed by gas chromatography (GC) and gas chromatography‐mass spectrometry. Fast atom bombardment (FAB) ionization was used for the muramic acid derivative to determine its molecular weight and structure, d‐and l‐amino acids were separated by GC and a capillary chiral column. By using this technique a stable N,O‐heptafluo‐robutyryl‐n‐butyl ester derivative of muramic acid was identified at picogram levels in Holocene sedimentary microbial communities. It has been reported previously that microorganisms in sediments rapidly degrade muramic acid from cell walls of dead prokaryotes. Kinetic experiments revealed that muramic acid was relatively stable in intact cell walls but decomposed rapidly in the free form. These investigations noted above showed that the concentration of muramic acid may be used as an indicator of the presence of the intact cell walls of cyanobacteria and most other bacteria in Holocene microbial communities, and of microbial contamination in samples older than the Holocene.     

Reverse-phase liquid chromatographic analysis of amino acids after reaction with o-phthalaldehyde

Precolumn formation of o-phthalaldehyde (OPA) derivatives of amino acids, followed by reverse-phase separation and fluorescent detection, provides rapid, sensitive amino acid analysis. Eighteen OPA-amino acid derivatives are resolved on a Micropack MCH 5 column and can be measured at picomole levels. Ease of derivative preparation and separation makes liquid chromatographic analysis of OPA-amino acids a convenient and improved technique for measuring or confirming the presence of low levels of amino acids in aqueous solutions. Use of the method was demonstrated by measuring low concentrations of amino acids released from zooplankters stressed by contaminants.     

GC-MS analysis of amino acid enantiomers as their N(O,S)-perfluoroacyl perfluoroalkyl esters: application to space analysis

The target of the in-situ research of optical activity in extraterrestrial samples stimulated an extended investigation of a GC-MS method based on the derivatization of amino acids by using a mixture of perfluorinated alcohols and perfluorinated anhydrides. Amino acids are converted to their N(O,S)-perfluoroacyl perfluoroalkyl esters in a single-step procedure, using different combinations of the derivatization reagents trifluoroacetic anhydride (TFAA)-2,2,2-trifluoro-1-ethanol (TFE), TFAA-2,2,3,3,4,4,4-heptafluoro-1-butanol (HFB), and heptafluorobutyric anhydride (HFBA)-HFB. The derivatives obtained are analyzed using two different chiral columns: Chirasil-L-Val and gamma-cyclodextrin (Rt-gamma-DEXsa) stationary phases which show different and complementary enantiomeric selectivity. The mass spectra of the derivatives are studied, and mass fragmentation patterns are proposed: significant fragment ions can be identified to detect amino acid derivatives. The obtained results are compared in terms of the enantiomeric separation achieved and mass spectrometric response. Linearity studies and the measurement of the limit of detection (LOD) show that the proposed method is suitable for a quantitative determination of enantiomers of several amino acids. The use of the programmed temperature vaporiser (PTV) technique for the injection of the untreated reaction mixture is a promising method for avoiding manual treatment of the sample and decreasing the LOD.     

Free amino acid quantification by LC-MS/MS using derivatization generated isotope-labelled standards

The further development of derivatizing reagents for plasma amino acid quantification by tandem mass spectrometry is described. The succinimide ester of 4-methylpiperazineacetic acid (MPAS), the iTRAQ reagent, was systematically modified to improve tandem mass spectrometer (MS/MS) product ion intensity. 4-Methylpiperazinebutyryl succinimide (MPBS) and dimethylaminobutyryl succinimide (DMABS) afforded one to two orders of magnitude greater MS/MS product ion signal intensity than the MPAS derivative for simple amino acids. CD(3) analogues of the modified derivatizing reagents were evaluated for preparation of amino acid isotope-labelled quantifying standards. Acceptable accuracy and precision was obtained with d(3)-DMABS as the amino acid standards derivatizing reagent. The product ion spectra of the DMABS amino acid derivatives are diagnostic for structural isomers including valine/norvaline, alanine/sarcosine and leucine/isoleucine. Improved analytical sensitivity and specificity afforded by these derivatives may help to establish liquid chromatography tandem mass spectrometry (LC-MS/MS) with derivatization generated isotope-labelled standards a viable alternative to amino acids analysers.     

High-performance liquid chromatographic analysis of physiological amino acids in human brain tumors by pre-column derivatization with phenylisothiocyanate

A reversed-phase high-performance liquid chromatographic technique for the determination of free amino acids in five biopsies of human brain tumors (two meningiomas, one glioblastoma and two oligodendrogliomas) is described. The frozen tissues were homogenized, deproteinized with perchloric acid and neutralized with potassium hydroxide. Aliquots of the supernatant containing the physiological amino acids are used for pre-column derivatization with phenylisothiocyanate. The derivatized PTC-amino acids (phenylthiocarbamyl derivatives) are stable for a five day period if stored as a powder at −20°C in an inert atmosphere and they can be analyzed on a reversed-phase column (PicoTag) using a gradient of two eluents with absorption detection at a wavelength of 254 nm. Good resolution of several amino acids (>30) is achieved within ca. 60 min. For most amino acids this method is suitable for an accurate measurement over a wide range of physiological concentrations (50–400 pmol) starting from a very small amount of sample.     

A new method for the determination of optical purity of synthetic peptides

A novel and very accurate method was established for the determination of the optical purity of a peptide by use of the following procedure: (1) hydrolysis of the peptide in deuterium chloride, (2) gas chromatographic separation of each amino acid enantiomer on a chiral phase, and (3) determination of the D /L ratio by mass fragmentography. In this manner, one can estimate the true chiral purity of each amino acid residue with an accuracy of ～0.2%. The recemization effected during hydrolysis could be eliminated in principle, since the artificially formed DL -amino acids are necessarily labeled at the α-position with deuterium and can thus be distinguished mass spectrometrically from the D - and L -isomer originally present in the peptide. The versatility of the method was proven by analysis of model peptides, as well as by a racemization test in fragment condensation.     

Amino acids and growth substances in barley root excretions (Hordeum distichon L.) and their biological effect

The biological effect of barley root excretions was studied during a 4 to 10 days cultivation period. The effect of root excretions changes according to the cultivation period of barley. It was ascertained by means of a bioassay that growth was either conclusively stimulated or the root excretions did not affect growth of the roots and of the upper part ofNasturtium. No significant inhibitory effect was observed. The effect of the single amino acids and of their mixtures found in the root excretions of barley was quite different. The following amino acids were determined by paper chromatography in root excretions: alanine, asparagine, phenylalanine, glycine, leucine, lysine, serine, tyrosine, glutamic acid and valine plus methionine. During cultivation their total quantity increased from 5·48. 10^?5 up to 1·13. 10^?3 mg per plant. Most of the 20 amino acids observed, displayed in theNasturtium test at a concentration from 0·1 to 1,000 mg/l an inhibitory effect onNasturtium growth. The effect of amino acid mixtures, corresponding qualitatively and quantitatively to the free amino acids in barley root excretions was dependent on their concentration. Growth regulators of the auxin type were found in culture solutions by chromatographic separation and with bioassay. As it may be seen from the results obtained, there are besides amino acids and indole derivatives other non-identified compounds involved in the effect of barley root excretions.     

A chromatographic approach to the determination of relative free energies of interaction between hydrophobic and amphiphilic amino acid side chains.

A liquid chromatographic stationary phase was prepared by covalently binding to the surface of microparticulate silica gel functionality (benzylsilane), which mimics the side chain of the amino acid phenylalanine. The chromatographic retentions of the N-acetyl C-(N'-methyl) amides of various hydrophobic and amphiphilic amino acids on this stationary phase were measured using an aqueous mobile phase. A retention order of Gly < Ala < Cys < Val < Met < Pro < Ile < Leu < Tyr < Phe < Trp is seen at room temperature. Chromatographic retentions were used to derive free energies of adsorption of the amino acid derivatives on the chromatographic support relative to that of the glycine derivative. The temperature dependencies of the retention of aromatic and aliphatic amino acid derivatives differ in curvature, indicating a qualitative difference in the absorption mechanism. An adsorption model for retention is proposed, and arguments are made as to the suitability of an adsorption model for describing the contacts between amino acid side chains during the initial steps of protein folding.