Amino acids are very important molecules for their presence in biological tissues and fluids. Several diseases are associated with defects in amino acids metabolism and they are utilized as dietary supplements. The direct determination of these compounds is difficult because most of them lack of intrinsic chromophores and fluorophores, thus chemical derivatization represents an effective technique to enhance their poor detectability. Recently, 4,7-phenanthroline-5,6-dione (phanquinone) was found to react selectively with primary amino function and was successfully applied as pre-column derivatization reagent for HPLC analysis of amino acids by UV and fluorescence detection [1]. The aim of the present communication is the application of phanquinone as a labelling reagent for amino acids in micellar electrokinetic chromatography (MEKC). The derivatization reaction of the most important amino acids using phanquinone was carried out at 60°C for 1 hr in phosphate buffer (pH 8; 0.1M); the obtained stable adducts were directly analysed by MEKC. Precisely, the method involved the use of sodium cholate (200mM) as surfactant, in a phosphate buffer (pH 7; 20mM); the separations were carried out in a fused-silica capillary (50 mm ID x 32 cm effective length) using UV detection at 240 nm. Under these conditions the separation of more than ten derivatized amino acids was obtained within 20 min showing a different selectivity (separation profile) in comparison to that obtained in HPLC. The proposed method was validated and applied to the determination of phosphoserine and glutamine in commercial pharmaceutical formulations. The quantitative results obtained by MEKC were compared with those of HPLC; the two techniques performed similarly with regards to several aspects of validation. [1] R. Gatti, M.G. Gioia, P. Andreatta, G. Pentassuglia, J. Pharm. Biomed. Anal., 35 (2004) 339.
PHANQUINONE: A SUITABLE DERIVATIZATION REAGENT FOR MICELLAR ELECTROKINETIC CHROMATOGRAPHY (MEKC) OF AMINO ACIDS
GOTTI, ROBERTO;GIOIA, MARIA GRAZIA;GATTI, RITA;CAVRINI, VANNI
2005
Abstract
Amino acids are very important molecules for their presence in biological tissues and fluids. Several diseases are associated with defects in amino acids metabolism and they are utilized as dietary supplements. The direct determination of these compounds is difficult because most of them lack of intrinsic chromophores and fluorophores, thus chemical derivatization represents an effective technique to enhance their poor detectability. Recently, 4,7-phenanthroline-5,6-dione (phanquinone) was found to react selectively with primary amino function and was successfully applied as pre-column derivatization reagent for HPLC analysis of amino acids by UV and fluorescence detection [1]. The aim of the present communication is the application of phanquinone as a labelling reagent for amino acids in micellar electrokinetic chromatography (MEKC). The derivatization reaction of the most important amino acids using phanquinone was carried out at 60°C for 1 hr in phosphate buffer (pH 8; 0.1M); the obtained stable adducts were directly analysed by MEKC. Precisely, the method involved the use of sodium cholate (200mM) as surfactant, in a phosphate buffer (pH 7; 20mM); the separations were carried out in a fused-silica capillary (50 mm ID x 32 cm effective length) using UV detection at 240 nm. Under these conditions the separation of more than ten derivatized amino acids was obtained within 20 min showing a different selectivity (separation profile) in comparison to that obtained in HPLC. The proposed method was validated and applied to the determination of phosphoserine and glutamine in commercial pharmaceutical formulations. The quantitative results obtained by MEKC were compared with those of HPLC; the two techniques performed similarly with regards to several aspects of validation. [1] R. Gatti, M.G. Gioia, P. Andreatta, G. Pentassuglia, J. Pharm. Biomed. Anal., 35 (2004) 339.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
