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Analyse d'Acides Aminés non dérivés par Chromatographie en Phase Liquide avec le Détecteur Evaporatif à Diffusion de la Lumière et Couplage avec la Spectrométrie de Masse

Abstract : Amino acids represent one of the most important classes of natural compounds. They take in many biological processes the most important of which is the formation of peptides and proteins. Since most of them are lacking in a chromophore group, the majority of the chromatographic methods proposed for their determination in mixtures involves pre- or post-column derivatization allowing a spectrophotometric detection.
In order to avoid the derivatization step, Liquid Chromatography (LC) was coupled with an Evaporative Light Scattering Detector (ELSD) or coupled with Mass Spectrometry (MS). Both detection modes are practically universal but require the development of volatile mobile phases.
Among all the LC systems tried, porous graphitic carbon was chosen and evaluated as stationary phase for underivatized amino acid analysis. Separation of sulfur amino acids (taurine analogous) and partial separations of proteinic amino acids have been obtained using an aqueous volatile mobile phase containing ammonium acetate ajusted to pH=9.3. A complete separation of the 20 underivatized proteinic amino acids was achieved using a mobile phase containing a perfluorinated carboxylic acid (nonafluoropentanoic acid) as volatile ion pairing agent, in a gradient elution mode, at 10°C, in approximatively 40 minutes.
LC systems that have been developed with an ESLD are directly transposable with MS. With the higher specificity of MS or MS tandem detection, the analysis of the 20 underivatized proteinic amino acids is achieved in 20 minutes. While ELSD does not permit detection limits below the mg/l level, MS tandem allows the detection of only about ten µg/l. Thus, direct quantitation of sulfur amino acids in some sea invertebrates have been allowed using LC-MS tandem.
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Submitted on : Wednesday, June 6, 2007 - 10:48:48 AM
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  • HAL Id : tel-00152167, version 1

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Patrick Chaimbault. Analyse d'Acides Aminés non dérivés par Chromatographie en Phase Liquide avec le Détecteur Evaporatif à Diffusion de la Lumière et Couplage avec la Spectrométrie de Masse. Autre. Université d'Orléans, 2000. Français. ⟨tel-00152167⟩

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