Automated capillary liquid chromatography for simultaneous determination of neuroactive amines and amino acids

A method for the separation and quantitative determination of neuroactive amino acids (aspartate, glutamate, citrulline, arginine, glycine, taurine, gamma-aminobutyric acid) and neuroactive amines (noradrenaline, dopamine and serotonin) in a single chromatographic analysis is presented. The method is based on pre-column derivatization with o-phthalaldehyde and tert.-butyl thiol, on-column preconcentration and separation using 50 microns I.D. packed capillary columns, and detection by amperometry. Mass limits of detection are 80-900 amol for all neurotransmitters with RSDs of 0.71 and 4.6% or better for retention time and peak area, respectively. The method was demonstrated by application to the determination of neurotransmitters in microdialysis samples collected from striatum of live rats and tissue samples extracted from butterfly brains.     

Determination of 4-aminobutyric acid, aspartate, glutamate and glutamine and their 13C stable-isotopic enrichment in brain tissue by gas chromatography-mass spectrometry

A selected-ion monitoring method was developed for measuring 4-aminobutyric acid, aspartate, glutamate, and glutamine in brain tissue. Natural isotopes of these amino acids and their stable-isotopic enrichment following intravenous infusion of a precursor, [13C]glucose, were quantitated. Frozen mouse brain tissue was homogenized in cold 80% ethanol, and the supernatant, equivalent to 1 mg of wet weight brain tissue, was extracted using solid-phase bonded silica ion-exchange columns. Aspartate and glutamate (dicarboxylic acids) were isolated from strong anion-exchange columns, whereas 4-aminobutyric acid and glutamine (neutral amino acids) were isolated from strong-cation exchange columns. n-Butyl ester pentafluoropropionyl amide derivatives of these amino acids were analyzed by gas chromatography-mass spectrometry using a methane positive chemical ionization mode after gas chromatographic separation on a wide-bore, fused-silica capillary column. The method is applicable to determination of brain concentrations of these amino acids as well as their fluxes following administration of a stable-isotopic tracer.     

Applications of gas-liquid chromatography in protein chemistry. Determination of C-terminal sequences on nanomolar amounts of proteins.

A method for the determination of C-terminal amino acids and C-terminal amino acid sequences in nanomolar amounts of proteins is described, based on carboxypeptidase A digestion of the protein, followed by removal of the partially digested protein and quantitative gas-liquid chromatographic determination of the amino acids released after known time intervals. Sequences deduced from the kinetics of release of specific amino acids are compared with the known C-terminal sequences of well-characterized proteins.     

Application de la chromatographie sur échangeur d'ions a l'étude de la composition des aliments en acides amines

A detailed procedure is presented for the determination of the amino acid composition of foods by chromatography of the hydrolysates on columns of Dowex-50 by the method of Moore and Stein. Nearly all of the common amino acids can be determined. The measurement of methionine, however, is complicated by partial oxidation of the amino acid to the sulfoxide during acid hydrolysis. A satisfactory chromatographic method for tryptophan has not been obtained as yet. Cross-reference is given to the auxiliary development of a Chromatographic method for the determination of cystine (in the presence of carbohydrate) as cysteic acid and to the utilization of these combined techniques in the analysis of several foods of importance in human and animal nutrition.     

Capillary electrophoresis and column chromatography in biomedical chiral amino acid analysis

Free amino acids are typically quantified as the sum of their enantiomers, because in terrestrial organisms they mainly exist in the left-handed form. However, with increasing understanding of the biological significance of right-handed amino acids interest in enantioselective quantification of amino acids has steadily increased. Initially, electrophoretic and chromatographic methods using chiral (pseudo)-stationary phases or chiral eluents were applied to the separation of amino acid enantiomers. Later, derivatization of amino acids prior to chromatography with chiral reagents gained in popularity, because the diastereomers formed can be resolved on conventional reversed-phase columns. Novel multi-interaction chiral columns turned attention back to direct chiral chromatographic methods. Hyphenation to mass spectrometry has increasingly replaced optical detection because of superior selectivity, although this has not obviated the need for baseline resolution of amino acid enantiomers. Despite the progress made, enantioselective separation and quantification of amino acids remains an analytical challenge owing to frequently incomplete resolution of all naturally occurring enantiomers and insufficient sensitivity for the determination of the trace amounts of d-amino acids typically found in biological fluids and tissues. Chiral GC-MS analysis of heptafluorobutanol/pentafluoropropionanhydride amino acid derivatives on an Rt-gDEXsa column     

Gas chromatography of amino acids: Use of a fused-silica capillary column in the determination of plasma amino acids

A capillary chromatographic procedure using a fused silica column is described which can be used to quantitatively determine amino acids in plasma following the pre-chromatographic “clean-up” described in a recent paper [1]. In substituting this procedure for that involving a packed column, advantage has been taken of the greater resolving power to separate amino acids from background component peaks. In order to extend this advantage and provide a sound basis for quantitative analysis, the technique of cold on-column injection was employed. As a result, good precision of standard analysis was obtained with relative standard deviation (RSD) values for all amino acids of less than 4%. Application of the entire procedure to plasma samples yields RSD values of better than 10% for all amino acids with recoveries ranging from 72% to 104%. Simultaneous determination of plasma amino acid levels by gas chromatography (GC) using capillary columns and by classical ion exchange (CIE) showed reasonable agreement. Statistical evaluation showed no significant difference between twelve amino acids. Values for the remaining two, namely, phenylalanine and histidine are significantly different (p < 0.005). Comparison of the values obtained from GC capillary and packed columns reveals no significant difference between fourteen amino acids. Significant differences exist between results for phenylalanine and tyrosine (p < 0.001). It is concluded that there is good agreement between data obtained by GC capillary and CIE techniques and that differences between results for phenylalanine and histidine are method related.     

Capillary gas chromatographic analysis of amino acids in blood and protein hydrolysates as tert.-butyldimethylsilyl derivatives

A method is given for a one-step derivatization and gas chromatography of amino acids in blood and protein hydrolysates. Blood samples are partially purified by solvent extraction. Protein hydrolysates are neutralized with a triethylamine solution. Then tert.-butyldimethylsilyl derivatives of the amino acids are prepared in a one-step procedure and separated on a 30-m fused-silica SE-30 capillary column. Except for tryptophan and cystine, amino acids are eluted within 30 min. Amino acids are derivatized more rapidly than their corresponding trimethylsilyl derivatives and do not degrade on the long fused-silica columns.     

Enantioseparation of chiral amino acids as the N(O,S)-ethoxycarbonylated diastereomeric esters by achiral dual-capillary column gas chromatography

The enantioseparation of 30 racemic amino acids in a single analysis is described for the determination of their absolute configurations. Two-phase extractive ethoxycarbonyl (EOC) reaction with ethyl chloroformate present in the dichloromethane phase was performed to recover amino acids from alkaline aqueous solutions. The resulting N(O,S)-EOC amino acids extracted into an organic solvent after acidification were reacted with a chiral alcohol such as (S)-(+)-3-methylbutan-2-ol, (S)-(+)-butan-2-ol and (S)-(+)-octan-2-ol for gas chromatographic analysis on achiral dual-capillary DB-5 and DB-17 columns of different polarities. Among the chiral reagents examined, (S)-(+)-3-methylbutan-2-ol provided the best diastereomeric structures in resolving all the racemic amino acids into their enantiomeric pairs with high resolution factors (1.2-8.0). Moreover, the temperature-programmed retention index (I) values measured on the two columns were characteristic of each enantiomer. Hence simple I matching with the reference values was useful in cross-checking for chemical identification and also chiral discrimination. When the present method was applied to a fermented dairy product (Yakult), D-alanine, D-aspartic acid, D-glutamic acid and D-proline were positively detected along with their respective L-forms in addition to glycine.     

Gas chromatography-mass spectrometry of monohydroxyeicosatetraenoic acids as their methyl esters trimethylsilyl, allyldimethylsilyl and tert.-butyldimethylsilyl ethers

The gas chromatographic and mass spectrometric properties of the monohydroxy acids 5-hydroxyeicosatetraenoic acid (5-HETE), 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) as their methyl ester trimethylsilyl, methyl ester allyldimethylsilyl and methyl ester tert.-butyldimethylsilyl ethers were investigated. The gas chromatographic properties of the trimethylsilyl and tert.-butyldimethylsilyl derivatives were found to be excellent while the allyldimethylsilyl derivative required a well deactivated column. The mass spectra of these silyl derivatives with the exception for 12-HETE did not exhibit particularly intense ions in the upper mass region. A quantitative analysis by selected-ion monitoring of the most intense ion in the upper mass region of respective mass spectrum demonstrated that a detection limit in the low picogram range could only be obtained for 12-HETE. Since the mass spectra indicated that the double bonds exerted a strong influence on the fragmentation pattern, the trimethylsilyl, allyldimethylsilyl and tert.-butyldimethylsilyl ethers of the methyl esters of the reduced analogues of the monohydroxy acids were prepared. The saturation of the double bonds completely altered the fragmentation patterns and very intense ions carrying a high percentage of the total ion abundance were found in all of the mass spectra. The developed technique was utilized for measurements of 5-HETE in lung tissue samples from patients with lung cancer.     

Analysis of tissue free fatty acids isolated by aminopropyl bonded-phase columns

Gas chromatographic analysis revealed that polyunsaturated fatty acids such as arachidonic acid and total tissue free fatty acids isolated from an aminopropyl bonded-phase column yield a two- to three-fold higher recovery of arachidonic acid as compared to those isolated from thin-layer chromatographic plates. This method was further improved by packing the aminopropyl bonded phase in glass columns, since the glass column significantly eliminated the other contaminants (from polypropylene columns) coeluting with fatty acids in both a neutral lipid thin-layer chromatographic system and on a 5% DEGS-PS column of gas chromatographic analysis. In aminopropyl bonded-phase columns, the standard triglycerides and phospholipids were completely separated from free fatty acids as judged by gas chromatographic analysis. These results warrant the use of an aminopropyl bonded-phase column for the isolation of free fatty acids to obtain better recovery of polyunsaturated fatty acids.     

Chromatography of amino acids and short peptides. New advances

The newest results in the application of various chromatographic methods (gas-liquid chromatography, liquid chromatographic techniques, electrically driven systems) for the separation and quantitative determination of amino acids and short peptides in pure state and in complicated matrices are compiled. The results are concisely described and critically evaluated. The future trends of the chromatographic analysis of amino acids and short peptides are briefly discussed.     

C18柱串联冠醚手性柱高效分离3种芳香族氨基酸对映体

将C18柱与手性冠醚柱串联,建立了一种反相高效液相色谱法用于3种芳香族氨基酸对映体同时拆分的方法.考察了反相色谱流动相的组成、pH值、柱温、流速对对映体拆分的影响.实验结果表明,当流动相为HClO4-乙睛溶液(86:14,V/V,pH 2.0)、柱温20℃、流速0.4 mL/min时,3种氨基酸对映体可获得基线分离.进一步对比了C18柱、冠醚手性柱和串联顺序不同的4种分离模式,结果表明,C18柱不能拆分氨基酸对映体,仅能分离不同种类氨基酸;冠醚手性柱可分离氨基酸映体,但不同种类氨基酸色谱峰出现重叠;串联模式能实现3种氨基酸对映体的基线分离,实现双柱优势互补,而串联顺序对分离影响不大,仅影响色谱峰的峰形.     

Non-aqueous capillary electrophoretic enantioseparation of N-derivatized amino acids using cinchona alkaloids and derivatives as chiral counter-ions

A non-aqueous capillary electrophoretic method developed with quinine and tert.-butyl carbamoylated quinine as chiral selectors for the enantioseparation of N-protected amino acids was applied to the investigation of other quinine derivatives as chiral additives. The optimum composition of the background electrolyte was found to be 12.5 mM ammonia, 100 mM octanoic acid and 10 mM chiral selector in an ethanol-methanol (60:40, v/v) mixture. Under these conditions, a series of chiral acids, as various benzoyl, 3,5-dinitrobenzoyl and 3,5-dinitrobenzyloxycarbonyl amino acid derivatives were investigated with regards to selectand-selector relationships and enantioselectivity employing quinine, quinidine, cinchonine, cinchonidine, tert.-butyl carbamoylated quinine, tert.-butyl carbamoylated quinidine, dinitrophenyl carbamoylated quinine and cyclohexyl carbamoylated quinine as chiral selector.     

Normal-phase high-performance liquid chromatography of tocopherols and tocotrienols. Comparison of different chromatographic columns

Natural vitamin E is composed of eight different vitamers (alpha-, beta-, gamma- and delta-tocopherols and alpha-, beta-, gamma- and delta-tocotrienols). As these eight vitamers have different antioxidant and biological activities, it is necessary to have quantitative data on each substance separately. The aim of this study was to find universal HPLC columns for the separation of all eight components and to test if a few columns of the same material (different batches) will give reproducible results. Normal-phase HPLC separations of vitamin E compounds in a prepared mixture (containing oat extracts, palm oil and tocopherol standards) were tried on six silica, three amino and one diol columns. As shown by calculations of retention factors (k), separation factors (alpha), numbers of theoretical plates (N) and resolutions (Rs), the best separations were obtained on three silica columns and two amino columns using 4 or 5% dioxane in hexane as the mobile phase as well as on a diol column using 4% tert.-butyl methyl ether in hexane as the mobile phase.     

A study of the chromatographic determination of amino acids in the presence of large amounts of carbohydrate

Synthetic mixtures of 15 of the 18 most common amino acids (cystine, methionine and tryptophan excepted) were boiled with 6N HCl in the presence and absence of carbohydrate. The decomposition products from starch and glucose do not interfere with the chromatographic determination of the amino acids on ion exchange columns. In no instance was the observed recovery of an amino acid lowered by as much as 3 per cent by the addition of carbohydrate (2 g) to 25–50 mg of amino acids per 200 ml 6N HCl. The dilute conditions of hydrolysis correspond to those adopted for the determination of the amino acid composition of foods of high carbohydrate content.     

The use of a moving wire detector system for the study of liquid chromatographic columns

A detector system for liquid columns which employs a moving wire system and a gas-liquid chromatographic detector, is described. The performance of silicic acid columns was studied by investigating the effect of particle size, sample size and flow rate of the mobile phase on the HETP of squalane for coiled and straight columns. The effect of sample size on the resolution of methyl oleate and olive oil on coiled and straight columns was also studied.     

Direct High‐Performance Liquid Chromatographic Enantioseparation of β‐Methyl‐Substituted Unusual Amino Acids on a Quinine‐Derived Chiral Anion‐Exchange Stationary Phase

A quinine‐derived chiral anion‐exchange stationary phase was used for the direct high‐performance liquid chromatographic separation of the enantiomers of the N‐protected unusual β‐substituted α‐amino acids, β‐methylphenylalanine, β‐methyltyrosine, β‐methyltryptophan, and β‐methyl‐1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid. The readily prepared 2,4‐dinitrophenyl and tert‐butyloxycarbonyl derivatives were well separated, and in most cases the separation of all four stereoisomers of these β‐methyl‐α‐amino acids could be obtained in one chromatographic run. The elution sequences of the enantiomers of the different derivatives were determined and revealed a dependence on the type of the N‐protecting group. In this context, the effects of different protecting groups (acetyl, tert‐butyloxycarbonyl, benzoyl, 3,5‐dinitrobenzoyl, benzyloxycarbonyl, 3,5‐dinitrobenzyloxycarbonyl, 2,4‐dinitrophenyl, and 9‐fluorenylmethoxycarbonyl) on the chromatographic behavior were investigated.     

Quantitative chromatographic method for the determination of low levels of nitrilotriacetic and ethylenediaminetetraacetic acids in diethylenetriaminepentaacetic acid

A quantitative method for determination of low levels (0.05%, w/w) of nitrilotriacetic and ethylenediaminetetraacetic acids in diethylenetriaminepentaacetic acid is described. Palmitic acid is added to the chelator as an internal standard before esterification with methanol containing 2%(v/v)H2SO4. The methyl esters of palmitic, nitrilotriacetic, and ethylenediaminetetraacetic acids are first separated from diethylenetriaminepentaacetate by silicic acid column chromatography and are subsequently quantitated by gas-liquid chromatography. The method is both accurate and reproducible with less than 10% relative error. Thin-layer chromatographic separations of the methyl esters, and quantitation at the 1% level, are also described.     

Characterization of medieval proteinaceous painting media using gas chromatography and gas chromatography — mass spectrometry

Proteinaceous organic materials used as ancient painting media were investigated by capillary gas chromatography (GC) and capillary gas chromatography — mass spectrometry (GC/MS). Medieval wall paintings made by the tempera technique were considered and their binding media were studied by the characterization of their main chemical components. The basic methodology is based on the determination of amino acids in samples of paint layers after hydrolysis and derivatization and on the comparison with reference proteinaceous materials. Multivariate chemometric techniques were used to facilitate the recognition of the protein source from chromatographic data. To characterize the binders further, a method was developed for the determination of fatty acids, present as minor components, by GC/MS. The use of fused-silica capillary columns coated with selected stationary phases allowed the separation of amino acid and fatty acid derivatives in a single analytical run.