GC-MS analysis of amino acid enantiomers as their N(O,S)-perfluoroacyl perfluoroalkyl esters: application to space analysis.
ABSTRACT 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.
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ABSTRACT: Metabolic fingerprinting in combination with gas chromatography and multivariate analysis is being extensively employed for the improved understanding of biological changes induced by endogenous or exogenous factors. Chemical derivatization increases the sensitivity and specificity of gas chromatography-mass spectrometry (GC-MS) for polar or thermally labile biological compounds, which bear derivatizable groups. Thus, there is a constant demand for simple methods of derivatization and separation that satisfy the need for metabolite analysis, identifying as many chemical classes of compounds as possible. In this study, an optimized protocol of extraction and derivatization is established as a generally applicable method for the analysis of a wide range of classes of metabolites in urine, whole blood and saliva. Compounds of biological relevance bearing hydroxyl- carboxyl- and amino-groups are derivatized using single-step fluoroacetylation/fluoroethylesterification after proper optimization of the protocol. Subsequently, the developed derivatization procedure is engaged in finding blood metabolic biomarkers, induced by lymphohyperplastic disease, through the metabolomic fingerprinting approach, the multivariate modeling (hierarchical cluster analysis) and GC-MS. Our preliminary, GC-MS-based metabolomic fingerprinting study underlines the contribution of certain metabolites to the discrimination of patients with lymphohyperplastic diseases.Journal of Chromatography A 06/2013; · 4.61 Impact Factor
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ABSTRACT: Enantioselective HPLC, SFC and GC methods were evaluated for separation and quantitative determination of chiral purity of (2R,4R)-1-(1-tert-butoxyvinyl)-4-methoxypyrrolidine-2-carboxylic acid [(2R,4R)-TBMPCA], a common building block in organic synthesis. All three separation methods can provide baseline resolution of (2R,4R)-TBMPCA and its enantiomer (2S,4S)-TBMPCA; however, both enantioselective HPLC and SFC are unsuitable for quantitation of low levels of the undesired enantiomer in (2R,4R)-TBMPCA. Comparatively, the enantioselective GC method not only separates the derivatized enantioselective pair with resolution as high as 4, but also was shown to be sufficiently linear, precise, and accurate to enable quantitation of derivatized (2S,4S)-TBMPCA down to 2.4 microg/ml (0.04% of nominal concentration). The sample derivatization procedure is simple, and no sample clean-up is needed before injecting samples for enantiomeric GC analysis. Compared to the enantioselective HPLC and SFC methods, the enantioselective GC method is advantageous because of its high efficiency and high sensitivity.Journal of pharmaceutical and biomedical analysis 12/2010; 53(4):878-83. · 2.45 Impact Factor
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ABSTRACT: Traces of prebiotic amino acids, i.e., the building blocks of proteins, are excellent biomarkers that could provide evidence of extinct or extant life in extra-terrestrial environments. In particular, characterization of the enantiomeric excess of amino acids gives relevant information about the biotic or abiotic origin of molecules, because it is generally assumed that life elsewhere could be based on either L or D amino acids, but not both. The analytical procedures used in in-situ space missions for chiral discrimination of amino acids must meet severe requirements imposed by flight conditions: short analysis time, low energy consumption, robustness, storage for long periods under extreme conditions, high efficiency and sensitivity, automation, and remote-control operation. Such methods are based on gas chromatography, high-pressure liquid chromatography, and capillary electrophoresis, usually coupled with mass spectrometry; of these, gas chromatography-mass spectrometry (GC-MS) is the only such combination yet used in space missions. Preliminary in-situ sample derivatization is required before GC-MS analysis to convert amino acids into volatile and thermally stable compounds. The silylation reagent most commonly used, N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide, is unsuitable for detection of homochirality, and alternative derivatization techniques have been developed that preserve the stereochemical configuration of the original compounds and are compatible with spaceflight conditions. These include the reagent N,N-dimethylformamide dimethylacetal, which has already been used in the Rosetta mission, a mixture of alkyl chloroformate, ethanol, and pyridine, a mixture of perfluorinated anhydrides and perfluoro alcohols, and hexafluoroacetone, the first gaseous derivatizing agent. In all the space instruments, solvent extraction of organic matter and chemical derivatization have been combined in a single automatic and remote-controlled procedure in a chemical reactor. Liquid-based separation systems have been used in space missions. In particular, microchip capillary electrophoresis, based on microfluidic lab-on-a-chip systems, enables high-performance chemical analysis of amino acids with low mass and volume equipment and low power and reagent consumption. Coupling with laser-induced fluorescence detectors results in ultra-low limits of detection. This critical review describes applications of the on-board instruments used in the Rosetta mission to comets and in the more recent Mars exploration program, i.e., the Mars Science Laboratory and ExoMars missions.Analytical and Bioanalytical Chemistry 03/2013; · 3.66 Impact Factor