N-heptafluorobutyryl Isobutyl Esters Research Articles

Determination of isotopic ratios of L-leucine and L-phenylalanine and their stable isotope labeled analogues in biological samples by gas chromatography/triple-stage quadrupole mass spectrometry.

A gas chromatographic/triple-stage quadrupole mass spectrometric (GC/MS/MS) method for measuring very low levels of enrichment of [5,5,5-2H3]-L-leucine and [ring-13C6]-L-phenylalanine in plasma and lipoprotein hydrolysates is described. The amino acids were derivatized to their N-heptafluorobutyryl isobutyl ester derivatives and the isotope ratio was determined by GC/MS/MS in the negative-ion chemical ionization mode. Parent ions were the [M-HF]- ions and fragment ions used for quantification were [P-2HF-C3H7]- (leucine) and [P-HF-OC4H9]- (phenylalanine), respectively. The limit of quantification was about 10 pg of the labeled compound co-eluting with 20 ng of the endogenous compound. The calibration curves were linear in the investigated range from 0.1% to 100% of the labeled compound. In biological samples, the higher selectivity of GC/MS/MS compared with GC/MS was demonstrated.

Determination of amino acids in the hepatic and brain tissue of the rat by gas chromatography

A procedure is described for the quantitative determination of amino acids in hepatic and brain tissue samples from the rat. Because the presence of certain matrix components in the tissue material led to interference with chromatographic analysis they were removed by a prechromatographic “clean-up” step. Quantitative analysis of amino acids, as their N-heptafluorobutyryl iso-butyl ester derivatives, was achieved by high resolution gas chromatography on an apolar fused silica open tubular column. Reproducibility data from the complete procedure are presented; coefficients of variation for arginine and histidine in hepatic tissue varied between 7.1 and 10.1% whereas those for most other amino acids were better than 5%, with a mean recovery of 90%.

Determination of environmental levels of peptidoglycan and lipopolysaccharide using gas chromatography with negative-ion chemical-ionization mass spectrometry utilizing bacterial amino acids and hydroxy fatty acids as biomarkers

d-Alanine and diaminopimelic acid originating from bacterial peptidoglycans and hydroxy fatty acids from lipopolysaccharides (endotoxins) were analysed by gas chromatography using a chiral column (Chirasil-Val as stationary phase) and selected-ion monitoring detection with negative-ion chemical-ionization mass spectrometry. The amino acids were analysed as N-heptafluorobutyryl isobutyl esters after rapid hydrolysis of peptidoglycan followed by isolation of the amino acids with disposable ion-exchange columns. Racemization of amino acid enantiomers was controlled by using deuterium chloride in the hydrolysis. The hydroxy acids were analysed as O-pentafluorobenzoyl methyl esters. Most of the bacteria present in airborne dust from a poultry confinement building were found to be Gram-positive according to the analytical chemical method whereas the Limulus amoebocyte lysate test suggested the presence of appreciable amounts of lipopolysaccharides of Gram-negative bacteria. Further studies are required to compare the utility of these two methods for determining endotoxins in complex environments.

Identification of a new, naturally occurring, non proteic amino acid in xylem sap of pisum sativum

The xylem sap of nitrogen-fixing Pisum Sativum cv Homesteader has been examined by capillary gas chromatography and by gas chromatography mass spectrometry in both electron-impact and chemical ionization modes following the formation of N-heptafluorobutyryl isobutyl ester derivatives. One of the compounds thus detected had a mass of 389 and contained chlorine, one carboxylic acid group and a nitrogen atom. High-resolution mass spectrometry indicated that there were no other hetero atoms present in the molecule. The composition was determined to be C4H8NO2Cl. This composition can be explained only in terms of a chloroamino butyric acid.

Amino acid analysis by gas—liquid chromatography using a nitrogen-selective detector

A nitrongen-specific gas-chromatographic detector has been used to analyse amino acids in the low picomole range as their N-heptafluorobutyryl isobutyl esters. The amino acid derivatives were studied over the range 3–60 pmol and a coefficient of variation of <4% was obtained for the relative molar reponses of most of the monobasic amino acids. A detection limit of ca. 250 fmol was established for the monobasic amino acids. No simple relation could be established between detector response and the atomic composition of the amino acids. The specificity of the detector allowed the nitrogenous components of complex physiological samples to be analyses without prior fractionation of the samples.

Analysis of (1-13C)leucine and (13C)KIC in plasma by capillary gas chromatography/mass spectrometry in protein turnover studies

The methods used for the determination of the concentration and isotope enrichment of (1-13C)leucine and its metabolite (13C) alpha-ketoisocaproic acid (KIC) in plasma for the study of whole-body protein turnover are described. Leucine was analysed as its N-heptafluorobutyryl isobutyl ester and KIC as its quinoxalinol-TMS derivative, both by chemical ionization selected ion monitoring gas chromatography/mass spectrometry (GC/MS). The sensitivity of the leucine assay was improved 30 times by monitoring the negative ions under the conditions described. The coefficient of variation for enrichment and concentration measurements were 0.5% and 2%, respectively, with a minimum detectable enrichment of 0.1 at% excess for both assays.

Free amino-acid composition of wax-stimulated whole saliva in human subjects with healthy periodontium, severe chronic periodontitis and post-juvenile periodontitis

The free amino-acid composition of wax-stimulated whole saliva of 3 subjects with different periodontal status was determined. The amino acids were analysed by gas chromatographic separation on a packed column and determined as their N-heptafluorobutyrylisobutyl esters. In 2 patients with periodontal diseases, the salivary concentrations of proline and glycine were significantly elevated, suggesting increased collagen and protein degradation and their increased release from the inflamed gingiva. δ-Amino- n-valeric acid is related to bacterial metabolism so that the changes found in its concentrations could suggest an altered plaque metabolism and different bacterial contents in gingival pockets of patients with chronic and post-juvenile periodontitis.

Gas Chromatography-Mass Spectrometry of N- Heptafluorobutyryl Isobutyl Esters of Amino Acids in the Analysis of the Kinetics of [15N]H4+ Assimilation in Lemna minor L

Rapid, sensitive, and selective methods for the determination of the (15)N abundance of amino acids in isotopic tracer experiments with plant tissues are described and discussed. Methodology has been directly tested in an analysis of the kinetics of [(15)N]H(4) (+) assimilation in Lemna minor L. The techniques utilize gas chromatography-mass spectrometry selected ion monitoring of major fragments containing the N moiety of N-heptafluorobutyryl isobutyl esters of amino acids. The ratio of selected ion pairs at the characteristic retention time of each amino acid derivative can be used to calculate (15)N abundance with an accuracy of +/-1 atom% excess (15)N using samples containing as little as 30 picomoles of individual amino acids. Up to 11 individual amino acid derivatives can be selectively monitored in a single chromatogram of 30 minutes. It is suggested that these techniques will be useful in situations where the small quantities of N available for analysis have hitherto hindered the use of (15)N-labeled precursors.

Glass capillary gas-chromatographic analysis of free amino acids in biological microenvironments using electron capture or selected ion-monitoring detection

Studies on analysis of free animo acids using a support-coated, open-tube capillary column, and electron-capture detection or selective ion monitoring have been performed on samples from biological microenvironments. For most amino acids the detection limit was found to be less than 1 pg. The preparation of the support-coated open-tube capillary column is described as well as the gas-chromatographic conditions for direct injection and temperature-programmed separation of the N-heptafluorobutyryl iso butyl ester derivatives. Electron-capture detection and selected ion monitoring are compared with respect to linearity and sensitivity and the bases for the greater sensitivity of electron-capture detection compared with flame-ionization detection using halogenated derivatives is discussed. Applications of the gas-chromatographic method for analysis of free amino acids in environments deliberately chosen very small are demonstrated.

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 100 120 mesh Gas Chrom Q. Using this method the 3-methylhistidine content of urine and meat has been determined.

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.

Amino acid analysis by gas—liquid chromatography of N-heptafluorobutyryl isobutyl esters : Complete resolution using a support-coated open-tubular capillary column

Amino acids have been separated by gas—liquid chromatography as their N-heptafluorobutyryl isobutyl esters. Complete resolution of derivatives of all the common amino acids has been achieved using a high-performance support-coated open-tubular capillary column. The analysis time was 30 min. Modifications to the derivatization procedure of MacKenzie and Tenashuk have been introduced. Acylation by heating at 150° was shown to be destructive; 110° has been selected for routine preparation. To obtain a volatile histidine derivative it has been found necessary to add an antioxidant and to heat samples with ethoxyformic anhydride prior to injection. Amino acid analysis of β-lactoglobulin after 6 N HCl digestion yielded results in good agreement with those obtained by the conventional ion-exchange method. The method has also been successfully applied to estimation of the different caseins in whole casein and in purified fractions by amino acid analysis of residues liberated by carboxypeptidase digestion.

Gas-liquid chromatography of the N-heptafluorobutyryl isobutyl esters of fifty biologically interesting amino acids

Fifty amino acids are analyzed as their N-heptafluorobutyryl isobutyl esters by gas chromatography on a column of 3% OV-101 on Gas-Chrom Q. The identification and quantification of unusual amino acids, specifically those occurring in the marine environment, in the presence of common protein amino acids is reported. Regular relationships between elution temperatures and amino acid structures are found, thus allowing the estimation of elution temperatures of other amino acids.

Gas-liquid chromatography of N-heptafluorobutyryl isobutyl esters of amino acids

The N-heptafluorobutyryl isobutyl esters of the protein amino acids have been separated by gas-liquid chromatography using a single column of 3% SE-30 on Gas-Chrom Q. The separation is superior to that of previous single-column methods and is particularly suitable for the analysis of plant seed proteins. The method is also applicable to glycoproteins.