GC-MS analysis of amino acid enantiomers as theirN(O,S)-perfluoroacyl perfluoroalkyl esters: Application to space analysis

ArticleinChirality 18(4):279-95 · May 2006with60 Reads
DOI: 10.1002/chir.20241 · Source: PubMed
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.
    • "Among the different reagents studied—methyl (MCF), ethyl (ECF), and isobutyl chloroformates (IBCF) in combination with alcohols with an alkyl chain identical with or different from that of the chloroformate—the combination of HFB (2,2,3,3,4,4,4- heptafluoro-1-butanol) with MCF provided the best separation, because five pairs of enantiomers of the 20 proteinogenic amino acids could be separated with a good resolution close to 2 (R S values for pairs of enantiomers reported in Table 2, second column). Another one-step derivatization procedure uses a mixture of perfluorinated anhydride and perfluoro alcohols to obtain the N(O,S)-perfluoroacyl perfluoroalkyl derivatives by simultaneous esterification and acylation [49, 50] . Different combinations of the derivatization reagents, i.e., trifluoroacetic (TFAA) and heptafluorobutyric (HFBA) anhydrides, 2,2,2- trifluoro-1-ethanol (TFE), and 2,2,3,3,4,4,4-heptafluoro-1-bu- tanol (HFB) alcohols, have been investigated. "
    [Show abstract] [Hide abstract] 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. Figure Enantioselective separation of amino acids in in situ space missions could provide evidence of extinct or extant life in extra-terrestrial environments.
    Full-text · Article · Mar 2013
    • "The advantage of using GC-MS compared with HPLC-UV is that the HPLC-UV method relies only on the retention time, whereas in the GC-MS method verification of the amino acids is possible using both the retention time and the mass spectra of the derivatized amino acids. The GC-MS method using a chiral column would also give stereoisomeric information on the amino acids (Zampolli et al. 2006). LC-MS/MS methods have been developed to analyze underivatized amino acids (Qu et al. 2002, Piraud et al. 2005) and the micro-LC-MS method for stereoisomers analysis of underivatized amino acids (Zeleke et al. 2006). "
    [Show abstract] [Hide abstract] ABSTRACT: We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable. Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method. One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound. The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC. The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively. Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria. Tämän väitöskirjan aiheena on sisäilmasta ja ruoka-aineista eristettyjen itiöitä muodostavien Basillusten tuottamien lämmönkestävien toksisten ei-proteiini yhdisteiden rakenne ja toksisuus nisäkäs soluille Basillusten erittämien toksisten ei-proteiini yhdisteiden esiintymisestä, vaikutuksista ja kulkeutumistavoista kosteusvaurioisten rakennusten sisäilmassa ei ole paljon tutkimustietoa. Basillukset muodostavat ilmassa liikkuvia itiöitä, jotka voivat toimia toksiinien kantajina, kuten sisäilmasta eristetyt toksisten sienien itiöt. Ruoka-aineissa Basillusten itiöt ovat ongelmana, koska ne kestävät ruuanvalmistuksessa käytettäviä lämpötiloja ja ovat valmiita lisääntymään ruoka-aineissa suotuisissa olosuhteissa. Basillusten toksiset yhdisteet löydettiin käyttäen toksisuus indikaattorina sian siittiöiden liikkuvuuden muutosta toksisen altistuksen johdosta. Yhdisteiden puhdistuksessa, detektoinnissa ja rakenteen analysoimisessa käytettiin kaasu- ja nestekromatografiaa, massaspektrometriaa ja ydinmagneettista resonanssispektroskopiaa. Yhdisteiden toksisuutta ja vaikutustapoja tutkittiin käyttäen sian siittiöitä, kissan keuhkosoluja, ihmisen hermosoluja ja rotan maksasta eristettyjä mitokondrioita sekä BLM:ää (Black lipid membrane) ioniforisten ominaisuuksien selvittämiseen. Tämän väitöskirjatyön tuloksena löydettiin uusi K+ > Na+ > Ca2+ ionikanavan muodostava toksiini joka sai nimen amylosiini. Amylosiinia tuottivat kosteusvaurioisen rakennuksen sisäilmasta eristetyt B. amyloliquefaciens kannat. Amylosiini puhdistettiin nestekromatografilla ja monoisotooppinen atomipaino 1197 Da selvitettiin sähkösumutusionisaatio ioniloukku massaspektrillä. Lisäksi sen todettiin olevan peptidi happohydrolyysin ja aminohappojen kaasukromatografisen massaspektrometri analyysin avulla. Sen rakenteen todettiin sisältävän kromoforisen polyeeni ryhmän käyttämällä ydinmagneettista resonanssispektroskopiaa. Amylosiinille kehitettiin nestekromatografinen kvantitointimenetelmä käyttäen makrolaktoni polyeenia amphoterisin B:tä (moolimassa 924 g/mol) referenssiaineena. Ruoka-aine myrkytyksestä eristetty B. licheniformis kanta tuotti solukalvoa rikkovaa lipopeptidiä, likenysiini A:ta, joka puhdistettiin nestekromatografilla. Likenysiini A identifioitiin matriisiavusteisella laserionisaatio lentoaika-massaspektrometrillä sen tuottamien protonoituneiden sekä kationioituneiden ammonium, natrium, kalium molekyylien avulla. Sen tuottamat protonoidut molekyylit olivat m/z 1007, 1021 ja 1035. Likenysiini A:n aminohapot analysoitiin sähkösumutus-ionisaatio kolmoiskvadrupolin massaspektrometrin tuottaman tandem-massaspektrin sekä lipopeptidin happohydrolyysin ja aminohappojen nestekromatografisen analyysin avulla. Sisäilmasta eristetyn B. amyloliquefaciens kannan havaittiin tuottavan amylosiinin lisäksi lipopeptideja: sienille toksista fengysiinia ja solukalvoa hajottavaa surfaktiinia. Ne identifioitiin sähkösumutusionisaatio ioniloukku massaspektrillä niiden tuottamien protonoituneiden ja kationisoituneiden ammonium, natrium, kalium molekyylien sekä analysoimalla niiden tuottamat tandem massaspektrit. Surfaktiinin tuottamat protonoidut molekyylit olivat m/z 1009, 1023 ja 1037 ja fengysiinin m/z 1450, 1463, 1493 ja 1506. Kereulidi (moolimassa 1152 g/mol) puhdistettiin nestekromatografisesti ruokamyrkytys tapaukseen liittyvästä B. cereus kannasta. Kereulidi identifioitiin sen sähkösumutusionisaatio kolmoiskvadrupoli massaspektrometrin tuottamien protonoidun molekyylin m/z 1154, ammonium m/z 1171, natrium m/z 1176 ja kalium m/z 1192 kationisoituneiden molekyylien avulla sekä tulkitsemalla kereulidin protonoidun molekyylin m/z 1154 tandem-massaspektrin tuote-ionit. Kereulidille kehitettiin nestekromatografinen kvantitointimenetelmä käyttäen sitä muistuttavaa valinomysiinia (moolimassa 1110 g/mol) referenssiaineena. Lisäksi osoitettiin empiirisesti, käyttämällä BLM:ää, että B. cereuksen tuottama emeettinen toksiini, kereulidi on tehokas ja spesifinen kalium ionifori, jonka toksinen vaikutus kohdistuu erityisesti mitokondrioiden toimintaan.
    Full-text · Article · · Journal of Chromatography A
  • [Show abstract] [Hide abstract] ABSTRACT: This work describes a GC-MS method for enantioselective separation of amino acids. The method is based on a derivatization reaction which employs a mixture of alkyl chloroformate-alcohol-pyridine, as reagents to obtain the N(O,S)-alkyl alkoxy carbonyl esters of amino acids. Various reaction parameters are investigated and optimized to achieve a reproducible derivatization procedure suitable for separation of amino acid enantiomers on Chirasil-L-Val chiral stationary phase. In particular, the following topics are investigated for 20 proteinogenic amino acids: (i) the proper reagent and reaction conditions to obtain the highest derivative yield; (ii) the amino acid reactivity and the MS properties of the obtained derivatives; (iii) the linearity and sensitivity of the analytical method; (iv) the retention behavior of the derivatives and their enantiomeric separation on the Chirasil-L-Val chiral stationary phase. By combining the resolution power of the Chirasil-L-Val column and the high selectivity of the SIM MS detection mode, the described procedure enables the enantiomeric separation and quantification of 16 enantiomeric pairs of amino acids. The procedure is simple and fast and reproducible. It displays a wide linearity range at ppb detection limits for quantitative determinations: these properties make this derivatization method a suitable candidate for amino acid GC-MS analysis on board of the spacecrafts in space exploration missions of solar system body environments.
    Article · Jun 2007
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