Amino acids are a very interesting class of biologically active compounds, being the principal component of proteins and precursors of hormones, neurotransmitters and other relevant bio-molecules. Amino acid analysis finds many applications in biochemical, pharmaceutical and clinical research. The analysis of physiological samples is hampered by the presence of a substantial number of amino acids which occur in widely different concentrations at the same time. Thus, the chromatographic system should exhibit high specificity, sensitivity, linearity and reproducibility. The direct determination of amino acids poses problems due to their lack of significant chromophore and fluorophore groups. Between the many analytical methods, RP-LC with precolumn derivatization is usually preferred, because it is less time-consuming than other procedures and the used instrumentation is simple. Recently, phanquinone (4,7-phenanthroline-5,6-dione) was found to react selectively with primary amino function to give stable and highly fluorescent iminoquinols (1). The purpose of the present study was to apply the precolumn derivatization method, which involves phanquinone, to the selective and sensitive determination of amino acids with primary amino functional group in biological samples (plasma and urine). The derivatization reaction was carried out both at pH 8 (suitable condition for acidic and neutral amino acids) and in absence of buffer (generally, necessary condition for basic amino acids). The RP-HPLC separations were performed at 33±2°C on a Phenomenex Gemini 5m ODS (250 x 3.0 mm i.d.) with gradient elution using a mobile phase consisting of a mixture A/B, where A is TEA phosphate buffer (pH 4.0; 0.05 M) and B is methanol/water (80/20, v/v), at a flow-rate of 0.4 mL min-1. Fluorescence detection was used. Our studies suggest that the selectivity of the derivatization reaction, carried out at pH 8 or without catalysis, allows to modulate the application of the proposed method to the determination of target amino acids for diagnostic purposes in metabolic diseases. (1) R. Gatti, M.G. Gioia, A.M. Di Pietra, Anal. Chim. Acta 474 (2002) 11-20.
R.Gatti, M.G.Gioia (2007). LC determination with fluorescence detection of amino acids in plasma and urine by precolumn derivatization. CALENZANO (FIRENZE) : Grafiche Gelli.
LC determination with fluorescence detection of amino acids in plasma and urine by precolumn derivatization
GATTI, RITA;GIOIA, MARIA GRAZIA
2007
Abstract
Amino acids are a very interesting class of biologically active compounds, being the principal component of proteins and precursors of hormones, neurotransmitters and other relevant bio-molecules. Amino acid analysis finds many applications in biochemical, pharmaceutical and clinical research. The analysis of physiological samples is hampered by the presence of a substantial number of amino acids which occur in widely different concentrations at the same time. Thus, the chromatographic system should exhibit high specificity, sensitivity, linearity and reproducibility. The direct determination of amino acids poses problems due to their lack of significant chromophore and fluorophore groups. Between the many analytical methods, RP-LC with precolumn derivatization is usually preferred, because it is less time-consuming than other procedures and the used instrumentation is simple. Recently, phanquinone (4,7-phenanthroline-5,6-dione) was found to react selectively with primary amino function to give stable and highly fluorescent iminoquinols (1). The purpose of the present study was to apply the precolumn derivatization method, which involves phanquinone, to the selective and sensitive determination of amino acids with primary amino functional group in biological samples (plasma and urine). The derivatization reaction was carried out both at pH 8 (suitable condition for acidic and neutral amino acids) and in absence of buffer (generally, necessary condition for basic amino acids). The RP-HPLC separations were performed at 33±2°C on a Phenomenex Gemini 5m ODS (250 x 3.0 mm i.d.) with gradient elution using a mobile phase consisting of a mixture A/B, where A is TEA phosphate buffer (pH 4.0; 0.05 M) and B is methanol/water (80/20, v/v), at a flow-rate of 0.4 mL min-1. Fluorescence detection was used. Our studies suggest that the selectivity of the derivatization reaction, carried out at pH 8 or without catalysis, allows to modulate the application of the proposed method to the determination of target amino acids for diagnostic purposes in metabolic diseases. (1) R. Gatti, M.G. Gioia, A.M. Di Pietra, Anal. Chim. Acta 474 (2002) 11-20.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
