Determination of amino acid enantiomers in orange juices by chiral phase capillary gas chromatography

Summary The configurations of free amino acids (AAs) in orange juice beverages (commercial products of satisfactory and unsatisfactory quality), an orange juice concentrate (bulk product suspected of being adulterated), and in an orange juice that has been contaminated by addition ofLactobacillus plantarum as a model for microbial spoilage, were determined, after derivatization, by means of gas-liquid chromatography (GC) using fused-silica capillary columns coated with Chirasil-L-Val or Chirasil-D-Val as stationary phases. AAs were isolated from juices by treatment with Dowex WX8 ion-exchanger and were investigated, by GC, as theirN(O)-pentafluoropropionylorN(O)-trifluoroacetyl 1-propyl esters. It was found that the high quality orange juice beverage contained L-AAs exclusively whereas this juice, after fermentation withLactobacillus, contained free D-Ala (32.7%), D-Val (62.3%), D-Phe (20.0%), D-Glu (24.3%), D-Ser (2.6%), D-Asp (0.8%), and significant amounts of D-Pro [% D=100 D/(D+L)]. D-Ala (8.8%) and D-Ser (4.2%) were found in a sensory and analytically unsatisfactory orange juice beverage, whilst D-Ala (27.5%) and D-Ser (14.3%) were detected in the orange juice concentrate suspected of being adulterated.Although capillary GC on chiral stationary phases is regarded as being highly suitable for the determination of AA enantiomers in fruit juice beverages, detection of D-AAs is currently not considered as conclusive proof of fruit juice adulteration caused by addition of AA racemates since a non-microbial origin of D-AAs in the respective juice, or an original occurrence of D-AAs, in either the free, substituted, or peptide-bonded form in the fruits, cannot be excluded with certainty.Presented in part at the Deutscher Lebensmittelchemikertag, Sept. 18–21, 1990, Frankfurt and at the 14. Jahrestagung Deutscher Lebensmitteltechnologen, Nov. 15–17, 1990, Düsseldorf.     

High-performance ligand-exchange chromatography of amino acids on chiral stationary phases

Three chiral stationary phases, obtained by grafting silica gel with (-)-trans-1,2-cyclohexanediamine, were studied for the resolution of α-amino acids by ligand-exchange chromatography. The packings were prepared by bonding the chiral ligand to silica gel via different hydrocarbon spacers. Separation of the optical isomers was accomplished by eluents containing a constant concentration of copper(II) acetate (0.05mM). The elution sequence of amino acids was found to be dependent on the grafting reaction selected to prepare the chiral packings.     

Resolution of sulphur-containing amino acids by chiral phase gas chromatography

Summary Methyl esters of the pentafluoropropionyl-amino acid derivatives of the tetrafunctional, sulphur-bridged, stereoisomeric lanthionines, cystathionines and -methyl-lanthionines were resolved on glass capillaries coated with the chiral stationary phase N-propionyl-L-valine-N-tert-butylamide-polysiloxane (Chirasil-Val) within 35min. Interestingly, L-cystathionine elutes before its D-enantiomer in contrast to the usual order of emergence on an L-phase. The method was applied to the polypeptide antibiotic nisin, which contains mesolanthionine and 2S,3S,6R-3-methyl-lanthionine.N-Pentafluoropropionyl-S-alkylthiocysteine methyl esters (R=methyl, ethyl, n- and iso-propyl, n- and sec-butyl, n-octyl, neo-pentyl, cyclohexyl-, benzyl-, tolyl-) were separated on Chirasil-Val within 30min. The identity of all derivatives was shown by combined gas chromatography-mass spectrometry.     

Novel chiral selectors anchored on polydimethylsiloxane as stationary phases for separation of derivatized amino acid enantiomers by capillary gas chromatography

Six chiral selectors of S-(-)-t-Leu-cyclopropylamide, S-(-)-t-Leu-cyclopentylamide, S-(-)-t-Leu-cyclohexylamide, S-(-)-t-Leu-cycloheptylamide, S-(-)-t-Leu-cyclooctylamide, S-(-)-t-Leu-cyclododecylamide have been prepared and anchored individually through amide bonding to a polydimethylsiloxane functionalized with 2,2,2-trifluoroethyl ester groups by way of nucleophilic displacement reaction. The resulting chiral polysiloxanes have been provided as stationary phases for the separation of amino acid enantiomers by capillary GC. Amino acids were derivatized into N(O)-trifluoroacetyl isopropyl esters. Especially, polydimethylsiloxane anchored with S-(-)-t-Leu-cyclooctylamide was found to be efficient for the separation of aspartic acid (Asp) enantiomers. The method was applied to the estimation of ages from the extent of Asp racemization in human dentines.     

Cyclodextrin dimer derivatives used as stationary phase for capillary gas chromatography

Summary Four cyclodetrin dimer derivatives were synthesized by linking two single cyclodextrin derivatives with difunctional spacer at the primary side of cyclodetrin. The separation properties of these cylodextrin dimer derivatives as CGC stationary phases were investigated and compared with those of the unbridged native cyclodextrin derivative. The results show that two recognition sites and one link spacer of these cyclodextrin dimer derivatives cooperate in separation and affect the separation of disubstituted benzene positional isomers.     

Determination of amino acids in urine by gas chromatography

This report describes a convenient means of reducing the complex matrix which is responsible for interference during gas chromatographic determination of amino acids in urine. The pre-chromatographic clean-up employs the principle of solid phase extraction using bonded silica incorporating cation exchange groups. This approach avoids the detrimental effects on amino acid recoveries associated with resin-based cation exchangers. In spite of significant reduction in the complexity of chromatograms, only the high efficiency and resolving power offered by the analytical capillary column (e. g. fused silica open tubular, FSOT) is sufficient for quantitative and analysis of amino acids in urine. Reproducibility data from the complete procedure are determined, coefficients of variation (CV) for most amino acids being better than 5% with a mean recovery of 96%.     

Enantiomer separation of amino acids as their N-alkyloxycarbonyl alkylamide derivatives by chiral phase capillary GC

The enantiomers of amino acids were first converted into N-alkyloxycarbonyl 2,2,2-trifluoroethyl esters, and then into N-alkyloxycarbonyl alkylamides by nucleophilic substitution of the ester group with amines. The first reaction proceeds instantaneously, while the second substitution occurs smoothly with n-propylamine and isobutylamine. The final derivatives were produced for separation on a capillary column coated with Chirasil-Val by GC. Pro, which is difficult to separate completely as its N-perfluoroacyl alkyl ester derivative, showed complete separation of the enantiomeric pair. All amino acids examined in this study showed an increased separation factor.     

Chiral separation and determination of the optical purity of thiamphenicol-related chiral compound by capillary zone electrophoresis with cyclodextrins as chiral selectors

Summary One classical method for quantitation of amino acids in proteins is hydrolysis of the proteins and determination of the free amino acids. Although the drastic experimental conditions necessary for complete hydrolysis always cause degradation of some of the amino acids, if mild hydrolysis conditions are used, a mixture of amino acids and oligopeptides is obtained. If these conditions are adequately tuned, the oligopeptides are almost exclusively dipeptides. For this reason we have initiated a study to find a derivatizing agent suitable for the analysis of amino acids and dipeptides by an absolute method of quantitation already tested for amino acids. FMOC-Cl was found to be a suitable derivatizing agent for this purpose.     

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.     

Cyclodextrin carbamates as novel chiral stationary phases for capillary gas chromatography

The following carbamate derivatives of cyclodextrins (CDs) were prepared as novel chiral stationary phases for capillary gas chromatography: hexakis(2,6-di-O-pentyl)-α-cyclodextrin hexa(3-n-propyl, 3-isopropyl, and 3-phenylcarbamate), heptakis-(2,6-di-O-pentyl)-β-cyclodextrin hepta(3-n-propyl, 3-isopropyl, and 3-phenylcarbamate), and octakis(2,6-di-O-pentyl)-γ-cyclodextrin octa(3-n-propyl, 3-isopropyl, and 3-isopropyl, and 3-phenylcarbamate). Metal capillary columns coated with these stationary phases resolved many kinds of racemic mixture. In general, they were especially effective towards polar compounds such as free alcohols, amines, and epoxides. The types of sample which were effectively resolved depended on the cavity size of the CD: α-CD derivatives were specifically effective toward compounds having linear alkyl chains, and β-CD derivatives toward compounds with phenyl groups. The results indicate that chiral separation with the cyclodextrin carbamates depends on the formation of inclusion complexes and also on the hydrogen-bonding interactions between the samples and the CD carbamates.     

Determination of the configuration of histrelin,and LHRH analog,by chiral capillary gas chromatography

Summary Summary The assigned chirality at each center of the synthetic nonapeptide histrelin (L-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-N^im-benzyl-histidyl-L-leucyl-L-arginyl-L-proline-ethylamide) was verified using chiral gas chromatography. The procedure involved acid hydrolysis of histrelin to the constituent amino acids, derivatization as the N-pentafluoropropionyl/isopropyl esters and the analysis of the mixture using a commercially available 25m chiral capillary column (Chirasil-L-Val). There was no significant difference in the retention time of the amino acids obtained from the hydrolysate mixture when compared to the appropriate standards. Additionally, the hydrolysate was spiked with the D and L amino acids to prove the identity of closely eluting peaks. Luteinizing hormone-releasing hormone     

Chiral separation of newly synthesized arylpropionic acids by capillary electrophoresis using cyclodextrins or a glycopeptide antibiotic as chiral selectors

Summary The chiral separation of two newly synthesized arylpropionic acids of pharmaceutical interest, namely 2-[(5′-benzoil-2′-hydroxy)phenyl]-propionic acid (DF-1738y) and 2-[(4′-benzoiloxy-2′-hydroxy)phenyl]-propionic acid (DF-1770y), was performed by Capillary Zone Electrophoresis (CZE) using either cyclodextrins or antibiotics as chiral selectors in coated capillary. In order to optimize the separation, the effect on the migration time and resolution of type and concentration of the chiral selector, the buffer pH and the capillary temperature were studied. Several cyclodextrins, namely the charged 6^A-monomethylamino-β-cyclodextrin (MeNH-β-CD) and the neutral methyl-β-cyclodextrins (M-β-CD) and heptakis-2,3,6-tri-O-methyl-β-cyclodextrin (TM-β-CD), were tested for the enantiomeric separation of aryl propionic acids (APAs) compounds. Of these TM-β-CD provided the highest enantiomeric resolution at pH 5, however only DF-1738y optical isomers were baseline resolved. Using background electrolytes (BGEs) at higher pHs (pH=6–7) supported with the above listed CDs, an enantioresolution increase was recognized only for compound DF-1738y. In contrast DF-1770y exhibited the highest resolution at the lowest pH value studied (pH 4). The macrocyclic antibiotic vancomycin was therefore added to the BGE and tested as chiral selector using the partial filling counter current mode in order to obtain a sensitive analysis, high resolution and reduced antibiotic adsorption on the capillary wall. 5 mM vancomycin dissolved in the BGE at pH 5 and 25°C provided relatively high enantiomeric resolution (R _DF-1738y=3.4,R _DF-1770y=2.22) of both compounds.     

Recent advances in peptide chiral selectors for electrophoresis and liquid chromatography

The application of peptides in chiral separations using techniques such as capillary electrophoresis (CE), electrokinetic capillary chromatography (EKC) and liquid chromatography is the focus of this review. Methods for finding peptide selectors using combinatorial library approaches are discussed, as well as recent advances in the use of peptides as general chiral selectors for electrophoresis and liquid chromatography. One example shows the effectiveness of polymeric dipeptide surfactants as general chiral selectors for electrophoresis. Another example shows the versatility of oligoproline chiral stationary phases, exhibiting resolution for a number of racemic analytes comparable to other well-established chiral stationary phases.     

Profiling propolis flavonoids by means of micellar electrokinetic capillary chromatography, capillary gas chromatography and bactericidal action

Summary There is no proportional correlation between increasing organic solvent polarity from hexane to methanol and the extractability of propolis solids by the solvents or the bactericidal action of the propolis extracts obtained. Hexane (17.7%) and chloroform (62.1%) were the poorest and the best extractants, respectively, for propolis solids. The antibiotic activity of the extracts againstStaphylococcus strains decreased in the order hexane≥ethanol>methanol. Different capillary GC profiles were obtained for persilyl derivatives of propolis extracted with hexane and methanol and for propolis collected in different Brazilian provinces, suggesting the influence of flora variability on propolis composition. Wavelength-selected DAD detection revealed MEKC to be an innovative and sensitive method for monitoring the occurrence in propolis of the flavonoids and phenolcarboxylic acids thought to be responsible for the antimicrobial activity of propolis.     

手性配体交换毛细管电泳在氨基酸手性分析中的应用研究进展

氨基酸的手性分析在生命科学中具有重要的研究意义。手性配体交换毛细管电泳法作为常用的氨基酸的手性分析方式之一,具有高效、快速、样品迁移顺序可调等优点,引起了研究者们极大的兴趣。本文主要综述了近年来手性配体交换毛细管电泳法在氨基酸的手性分析中的应用研究进展。     

Study of the behaviour of chiral alcohols by gas chromatography on chiral phases

Summary In order to study the resolution of chiral alcohols by gas chromatography twelve chiral phases derived from (+) tartaric acid were synthetized. The different factors whichcould modify the resolution were studied: importance of trapped fractions of the peak, structures of the chiral phases and racemic compounds.     

New chiral derivatization reagent for the resolution of amino acids as diastereomers by TLC and HPLC

Summary The applicability of a new chiral reagent to the resolution of amino acid enantiomers has been investigated. The new reagent, S(-)-N-1-(2-naphthylsulphonyl)-2-pyrrolidinecarbonyl chloride (NSP-C1), was synthesized by the chlorination of S(-)-N-1-(2-naphthylsulphonyl)-2-pyrrolidinecarboxylic acid which was prepared by the reaction of 2-naphthalene sulphonyl chloride with L-proline. Derivatization of the amino acids proceeds rapidly at ambient temperature and no racemization takes place during the reaction. The resolution of the diastereomeric amides was performed by TLC and normal phase HPLC. Complete resolutions were observed for the enantiomers of all amino acids examined except cysteine, cystine and histidine. The favourable UV absorption of the derivatives enabled the optical antipode to be determined down to the 0.1% level.     

Capillary gas chromatography of amino acids as their tert-butyldimethylsilyl derivatives in the analysis of serum amino acids in metabolic diseases

Reaction of amino acids with N-methyl-N-(tert-butyldimethylsilyl)trifluoroaceamide (MTbSTFA) in acetonitrile affords good yields of amino acid derivatives with excellent gas chromatographic and mass spectrometric properties. The single-step derivatization procedure is highly reproducible. The TBDMS amino acids are stable at room temperature for at least three days. Only a single peak is observed for each amino acid. The procedure allows simultaneous analysis of asparagine and glutamine together with other serum amino acids. Separation is achieved on a borosilicate glass capillary coated with OV-1. The mass spectra of the TBDMS amino acids possess characteristic diagnostic ions. These properties were used in the sensitive detection by GC-MS and SIM-GC-MS of GABA and pipecolic acid in the serum of a newborn suspected of a Zellweger-type syndrome, which could not be detected by other methods.