Comparison of silylation and esterification/acylation procedures in GC‐MS analysis of amino acids

Three derivatization agents used in GC analysis of amino acids were compared: N,O‐bis(trimethylsilyl)trifluoroacetamide, (BSTFA), N‐methyl‐N‐(tert‐butyldimethylsilyl)trifluoroacetamide (MTBSTFA), and isobutyl chloroformate (iBuCF). It was shown that the analytical characteristics achieved in the case of silylation with MTBSTFA are comparable to those obtained for esterification/acylation. However, since the former approach requires laborious sample preparation to isolate the compounds in question prior to derivatization, determination of amino acids as N(O,S)‐alkoxycarbonyl alkyl esters seems to be preferable in many cases. Application of the esterification/acylation procedure to analysis of lyophilized E. coli microbial culture was demonstrated.     

Determination of amino acids by gas chromatography using a wide bore capillary column

A procedure is described in which a wide bore capillary column is used as an alternative to the more traditional packed column for the quantitative analysis of amino acids as their N-heptafluorobutyryl isobutyl ester (HBB) derivatives. The column, installed in a gas chromatograph previously configured for use with a packed column, is shown to give good reproducibility by repeated determination of amino acid response factors (RSD values for all amino acids are below 3%). A number of problems, encountered during the use of this column, are discussed and suitable techniques to overcome them are reported.     

Simple automated gas chromatographic analysis of amino acids and its application to brain tissue and urine

The separation of 21 free amino acids as their isobutyl N(O,S)-pentafluorobenzoyl derivatives by capillary gas chromatography with electron-capture detection is described. This method has been successfully applied to routine analysis of most of these amino acids in brain tissue and urine. The amino acids are converted to their isobutyl N(O,S)-pentafluorobenzoyl derivatives by acid-catalysed esterification followed by aqueous extractive pentafluorobenzoylation. The 21 amino acids can be separated on a single capillary column in 21 min. The high sensitivity permits analysis in minute quantities of sample and the high stability permits the use of an automatic sample injector at ambient temperatures. Thus, over 50 samples can be readily analyzed in a single day.     

Resolution of non-protein amino acids via the microbial protease-catalyzed enantioselective hydrolysis of their N-unprotected esters

In the Aspergillus oryzae protease-catalyzed ester hydrolysis, substitution of N-unprotected amino acid esters for the corresponding N-protected amino acid esters resulted in a large enhancement of the hydrolysis rate, while the enantioselectivity was deteriorated strikingly when the substrates employed were the conventional methyl esters. This difficulty was overcome by employing esters bearing a longer alkyl chain such as the isobutyl ester. Utilizing this ester, amino acids carrying an aromatic side chain were resolved with excellent enantioselectivities (E=50 to >200). With amino acids bearing an aliphatic side chain also, good results in terms of the hydrolysis rate and enantioselectivity were obtained by employing such an ester as the isobutyl ester. Moreover, the enantioselectivity proved to be enhanced further by conducting the reaction at low temperature. This procedure was applicable to the case where the enantioselectivity was not high enough even by the use of the isobutyl ester.     

Potential of Flame Ionization Detection Coupled On‐Line with Microcolumn Liquid Chromatography Using Aqueous Eluents and an Eluent‐Jet Interface

The potential of the on‐line coupling of microcolumn liquid chromatography (μLC) using aqueous eluents with a flame ionization detector (FID) was evaluated. An eluent‐jet interface was modified to allow the efficient introduction of the eluent into the FID. The potential of the method is demonstrated by the μLC–FID determination of lower alcohols and bis(2‐hydroxyethylthio)alkanes on porous and non‐porous stationary phases, respectively. Flow injection analysis (FIA)–FID experiments with highly polar, thermolabile, semi‐volatile and non‐volatile compounds like amino acids, organic acids, alkylphosphonic acids, and carbohydrates showed the developed configuration to be a promising approach for the detection of a wide range of analytes. Compared with a nebulization interface, the eluent‐jet interface showed 4–10 times higher peaks for citric acid. Detection limits by FIA for all compounds were in the range of 0.2–5 ng injected. With ribose as test compound, plots of peak height vs. amount injected showed good linearity (r² > 0.999) in the range of 75–12,000 μg/mL. The repeatability showed relative standard deviations of less than 5%.     

Determination of the enantiomeric purity of synthetic peptides by gas chromatography-mass spectrometry.

Hydrolysis using 2H-labelled HCl and H2O, derivatization of free amino acids as N.O.S-trifluoroacetyl isobutyl esters, separation by gas chromatography on a chiral stationary phase and detection by mass spectrometry in selected-ion monitoring mode have been used in order to determine the enantiomeric purity of several synthetic peptides. Chromatographic separation has been optimized for proline, whose two enantiomers are difficult to resolve under standard conditions. Electron impact and methane chemical ionization mass spectra and chromatographic resolution of unnatural amino acids, such as 3-(1-naphthyl)-alanine and p-chlorophenylalanine, are reported. For both natural and unnatural amino acids selected-ion monitoring of the different fragmentation peaks has been carried out. The results are interpreted from the point of view of whether or not the fragments contain a hydrogen atom on the alpha-carbon, and a comparison between electron impact and methane chemical ionization has been carried out. The main advantage of the latter method is that a quasimolecular ion can be observed for all the amino acids studied.     

Can gas chromatography combustion isotope ratio mass spectrometry be used to quantify organic compound abundance?

Quantifying the concentrations of organics such as phospholipid fatty acids (PLFAs) and n‐alkanes and measuring their corresponding ¹³ C/¹² C isotope ratios often involves two separate analyses; (1) quantification by gas chromatography flame ionisation detection (GC‐FID) or gas chromatography/mass spectrometry (GC/MS), and (2) ¹³ C‐isotope abundance analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC‐C‐IRMS). This requirement for two separate analyses has obvious disadvantages in terms of cost and time. However, there is a history of using the data output of isotope ratio mass spectrometers to quantify various components; including the N and C concentrations of solid materials and CO₂ concentrations in gaseous samples. Here we explore the possibility of quantifying n‐alkanes extracted from sheeps' faeces and fatty acid methyl esters (FAMEs) derivatised from PLFAs extracted from grassland soil, using GC‐C‐IRMS. The results were compared with those from GC‐FID analysis of the same extracts. For GC‐C‐IRMS the combined area of the masses for all the ions (m/z 44, 45 and 46) was collected, referred to as 'area all', while for the GC‐FID analysis the peak area data were collected. Following normalisation to a common value for added internal standards, the GC‐C‐IRMS 'area all' values and the GC‐FID peak area data were directly compared. Strong linear relationships were found for both n‐alkanes and FAMEs. For the n‐alkanes the relationships were 1:1 while, for the FAMEs, GC‐C‐IRMS overestimated the areas relative to the GC‐FID results. However, with suitable reference material 1:1 relationships were established. The output of a GC‐C‐IRMS system can form the basis for the quantification of certain organics including FAMEs and n‐alkanes. Copyright © 2011 John Wiley & Sons, Ltd.     

The nitrosation of Amino Acids

With a view to clarifying analogies and differences between the mechanisms involved in the nitrosation of amino acids and secondary amines, we studied the kinetics of the nitrosation of five imino acids (azetidine-2-carboxylic acid, pyrrolidine-2-carboxylic acid, piperidine-2-carboxylic acid, piperidine-3-carboxylic acid, and piperidine-4-carboxylic acid) and of the ethyl esters of three of them. Reaction kinetics were determined by the initial rate method, by spectrophotometric monitoring of the concentration of nitroso amino acid formed. The presence of the ? COO^? group in the amino acids opens a new mechanistic route for the nitrosation of the secondary amino group: a nitrosyl carboxylate formed initially acts as an internal nitrosating agent, resulting in intramolecular migration of ? N ? O from the carboxylate group to the secondary amino group. The observed order of the α?, β?, and γ-amino acids as regards the ease of N-nitrosation by this route is explained in terms of the relative energies of (a) the equatorial and axial orientations of the C_ring? C_carboxyl bond, and (b) the chair and boat forms of the piperidine ring. © 1994 John Wiley & Sons, Inc.     

N-phosphoryl amino acids and peptides. Part II: Fast atom bombardment mass spectrometry of N-di-isopropyloxyphosphoryl and N-dibutyloxyphosphoryl amino acids

The positive ion fast atom bombardment mass spectra of N-di-isopropyloxyphosphoryl (Dipp) and N-dibutyloxyphosphoryl (Dbp) amino acids or amino acid methyl esters are presented; and according to the observation of the metastable ions and the high-resolution accurate mass measurement their fragmentation patterns are postulated. Both types of compounds were found to undergo similar fragmentations to produce (HO)₂P(O)N?CH? R + H⁺, in most cases as the most abundant fragment ion of structural significance. An intrinsical difference between the two types of compounds is that N-Dippamino acids appear to favour the successive losses of two molecules of propylene, while the loss of HCOOH seems to be preferred by the Dbp amino acids. For those compounds containing an extra functional group on the side chain of amino acids such as serine or glutamic acid some other type of fragmentation was observed besides the normal phenomenon.     

Determination of Free D‐Amino Acids in Mammalia by Chiral Gas Chromatography–Mass Spectrometry

Quantities of D‐amino acids were determined in body fluids (urine, blood plasma and blood serum, milk) of mammals (hamster, horse, bovine, sheep, pig, and dog). Amino acids were isolated using a cation exchanger and converted into their N(O)‐pentafluoropropionyl (or trifluoroacetyl) amino acid 2‐propyl esters. Enantiomers were separated and quantified on a Chirasil‐L‐Val capillary column with mass spectrometric detection using selected ion monitoring. D‐Enantiomers of most protein L‐amino acids were detected. Largest absolute and relative amounts in most cases were determined for D‐Ser and D‐Ala in urine. Stereoisomers of 2,6‐diaminopimelic acid were also measured in bovine, ovine, and porcine urine. Since D‐amino acids were detected in all representative classes of the major orders of Mammalia, namely Artiodactyla, Perissodactyla, Rodentia, and Carnivora, and taking reports in the literature into account, it is postulated that D‐amino acids occur in all mammals.     

Radical Stability Directs Electron Capture and Transfer Dissociation of β‐Amino Acids in Peptides

We report on the characteristics of the radical‐ion‐driven dissociation of a diverse array of β‐amino acids incorporated into α‐peptides, as probed by tandem electron‐capture and electron‐transfer dissociation (ECD/ETD) mass spectrometry. The reported results demonstrate a stronger ECD/ETD dependence on the nature of the amino acid side chain for β‐amino acids than for their α‐form counterparts. In particular, only aromatic (e.g., β‐Phe), and to a substantially lower extent, carbonyl‐containing (e.g., β‐Glu and β‐Gln) amino acid side chains, lead to N? C_β bond cleavage in the corresponding β‐amino acids. We conclude that radical stabilization must be provided by the side chain to enable the radical‐driven fragmentation from the nearby backbone carbonyl carbon to proceed. In contrast with the cleavage of backbones derived from α‐amino acids, ECD of peptides composed mainly of β‐amino acids reveals a shift in cleavage priority from the N? C_β to the C_α? C bond. The incorporation of CH₂ groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical‐driven β‐amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research.     

Poly(Crown Ether) Catalyzed Derivatization of Lower Fatty Acids for Gas Chromatography

Abstract

Poly (crown ether) was employed as a catalyst of derivatization of lower fatty acids to the p-bromophenacyl esters for gas chromatographic determination of them. The esterification reaction proceeds quantitatively under mild conditions, i.e. at room temperature and within 30 min. The poly (crown ether) did not interfere with the gas chromatogram of the esters unlike monomeric crown ethers. Normal fatty acid (C₁-C₆) and the isomers could be determined simultaneously near to the detection limit of FID.     

Direct UV detection of underivatized amino acids using capillary electrophoresis with online sweeping enrichment

This is an original report proposed a CE method for direct analysis of the underivatized amino acids using UV detection with relatively higher sensitivity, which was based on coordination interactions between amino acids and Cu (II) ions. In addition, an online sweeping preconcentration technique was easily combined to improve the detection sensitivity. Satisfying separations of the amino acids were obtained under optimized conditions: 50 mmol/L CuSO₄–0.05% HAc–H₂O (pH 4.5), and the separation voltage of 15 kV. The LODs for the analytes ranged from 0.1 to 0.5 μmol/L. The linearity of detection for all analytes was two orders of magnitude with the correlation coefficients greater than 0.99. The repeatability was displayed with an RSD less than 3% for migration time and peak height (n = 5). Moreover, some amino acids in real samples of human saliva and green tea were analyzed by this direct UV detection CE method with acceptable sensitivity.     

Characterization and quantitative amino acids analysis of analgesic peptides in cinobufacini injection by size exclusion chromatography,matrix‐assisted laser desorption/ionization time of flight mass spectrometry and gas chromatography mass spectrometry

Cinobufacini injection that comes from the water extract of Bufo bufo gargarizans Cantor skin is widely used for cancer treatment in China. Peptide is one of its major types of constituents, however the biological effects and content of this injection are little reported. In present study, the analgesic effect of peptides was determined and evaluated by in‐vivo models. To characterize and quantitatively analyze these peptides, a reliable and efficient method combining size exclusion chromatography and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry with amino acid analysis was developed. The peptides presented as a series of analogs with similar molecular weights mostly ranging from 2 to 8 kDa. The amino acid analysis by gas chromatography mass spectrometry (GC‐MS) was developed to determine both free and combined amino acids (FAA and CAA) in cinobufacini injection. This method achieved good linearity (R², 0.9909–0.9999) and low limit of detection and quantification. FAA and CAA samples were efficiently analyzed by modified Phenomenex EZ: faast procedure. For the sample analysis, the method showed good repeatability (relative standard deviation, RSD ≤ 10%). For most FAA and CAA the mean recoveries were >80% with RSD <10%. The GC‐MS based method is useful for quality assurance of both FAA and CAA in cinobufacini injection. Copyright © 2014 John Wiley & Sons, Ltd.     

HS-SPME determination of volatile carbonyl and carboxylic compounds in different matrices

Specific chromatographic methodologies are developed for the analysis of carboxylic acids (C(2)-C(6), benzoic) and aldehydes (C(2)-C(10)) of low molecular weight in diverse matrices, such as air, automotive exhaust gases, human breath, and aqueous matrices. For carboxylic acids, the method is based on their reaction with pentafluorobenzyl bromide in aqueous solution, followed by the separation and identification of the resultant pentafluorobenzyl esters by means of headspace (HS)-solid-phase microextraction (SPME) combined with gas chromatography (GC) and electron capture detection (ECD). Detection limits in the microg/m(3) range are reached, with relative standard deviation (RSD) less than 10% and linear response (R(2) > 0.99) over two orders of magnitude. The analytical methodology for aldehydes is based on SPME with simultaneous derivatization of the analytes on the fiber, by reaction with pentafluorophenylhydrazine. The derivatization reagent is previously deposited on the SPME fiber, which is then exposed to the gaseous matrix or the HS of the sample solution. The pentafluorophenyl hydrazones formed on the fiber are analyzed selectively by means of GC-ECD, with detection limits in the ng/m(3) range, RSD less than 10%, and linear response (R(2) > 0.99) over two orders of magnitude.     

GC resolution of various D,L-amino acid derivatives on a chirasil-pval capillary column

An enantiomeric mixture of amino acids is derivatized to give the respective N(O)-perfluoroacyl alkyl esters which can be resolved directly by gas chromatography on a Chirasil-Val capillary column. The retention times, resolution numbers, and elution order are reported.     

Snytheses and Reactions of O,N,N′-Trisubstituted Isoureas

The CuCl₂-catalysed addition of alcohols to aliphatic caribodiimides (particularly diisopropylcarbodiimide) to form O,N,N′-trialkylisoureas may, in the case of bifunctional hydroxy compounds (esters of α-hydroxycarboxylic acids, diols, halogeno alcohols, amino alcohols, and cyclic hydroxy ethers), continue to give 5-, 6-, and 7-membered 1,3-O,N or 1,3-N,N heterocycles. O,N,N′-Trialkylisoureas are selective alkylating agents. Thus alcohols with allyl structures can be etherified with diisopropylcarbodiimide; a new method of esterification via O,N,N′-trialkylisoureas has been applied to mesitylenecarboxylic acid.     

Rapid determination of amino acids in neonatal blood samples based on derivatization with isobutyl chloroformate followed by solid-phase microextraction and gas chromatography/mass spectrometry

The purpose of this study was to develop a simple, rapid and sensitive analytical method for determination of amino acids in neonatal blood samples. The developed method involves the employment of derivatization and a solid-phase microextraction (SPME) technique together with gas chromatography/mass spectrometry (GC/MS). Amino acids in blood samples were derivatized by a mixture of isobutyl chloroformate, methanol and pyridine, and the N(O,S)-alkoxycarbonyl alkyl esters thus formed were headspace extracted by a SPME fiber. Finally, the extracted analytes on the fiber were desorbed and detected by GC/MS in electron impact (EI) mode. L-Valine, L-leucine, L-isoleucine, L-phenylanaline and L-tyrosine in blood samples were quantitatively analyzed by measurement of the corresponding N(O,S)-alkoxycarbonyl alkyl esters using an external standard method. SPME conditions were optimized, and the method was validated. The method was applied to diagnosis of neonatal phenylkenuria (PKU) and maple syrup urine disease (MSUD) by the analyses of five amino acids in blood samples. The results showed that the proposed method is a potentially powerful tool for simultaneous screening for neonatal PKU and MSUD.     

Synthesis of Germanium Oligomer from Germanium Dioxide

Several crystalline germanium oligomers Ge_nO_2n+1· L₂·xH₂O (L = amino acid or amine) have been prepared and characterized by IR and NMR spectra, EA (elemental analysis), TGA (thermogravimetric analysis) and ICP-AES (inductively coupled plasma-atomic emission spectroscopy). Oligomers of this type are based on chains of germanium-oxygen single and double bonds and synthesized in aqueous solution from germanium dioxide and basic ligands having an amino group. Two basic amino acids and mono-, and bidentate amines chosen as ligands to react with germanium dioxide gave L-lysine germanate (1a, 1b, 1c), L-arginine germanate (2a, 2b, 2c), Cyclohexylammonium hexagermanate (3), N-methylcyclohexylammonium hexagermanate (4), N,N-dimethylcyclohexylammonium hexagermanate (5), 2-aminoethylammonium trigermanate (6), 2-N′-methylammo)-N-methylethylammonium pentagermanate (7), 2-(N′N′-dimethylamino)-N′N-dimethylethylammonium heptagermanate (8).