Amino acids are important compounds in a wide range of samples such as biological tissues and fluids, food and industrial products. In particular they have a considerable role in pharmaceutical field because several diseases are associated with defects in amino acids metabolism and they are utilized as dietary supplements. The analysis of these compounds is essential and often difficult owing to their poor detectability. High performance liquid chromatography (HPLC) in combination with a pre-column chemical derivatization constitutes an effective and convenient technique to overcome the problem. To develop additional useful reagents, our attention was directed to 2,7-dimethyl-3,8-dinitrodipyrazolo[1,5-a:1’,5’-d]pyrazine-4,9-dione (I), which showed an excellent reactivity toward nucleophylic reagents, such as aliphatic, benzylic and aromatic amines [1]. The aim of the present work was the use of the compound I as labelling reagent for the pre-chromatographic derivatization of amino acids. The derivatization and chromatographic conditions were optimised by a series of experiments. The compound was found to react with amino function of the amino acid in 30 min at 68°C in presence of triethylamine (TEA) (0.04 M) to give adducts (II), which can be chromatographed and detected by UV-DAD (l=280 nm). The synthetized and purified derivatives were characterized by UV, IR, 1HNMR and MS. Routine chromatographic analyses were performed at 33±2°C on a reversed phase column (Phenomenex Gemini 5u C18 110A, 250 x 3 mm i.d.) by gradient elution using a mobile phase consisting of a mixture A/B where A=phosphate buffer (0.05 M; pH 3) and B=methanol at a flow rate of 0.5 mL/min. The proposed method was applied successfully to the determination of several amino acids, present as nutrients in commercial dosage forms. [1] Baraldi, P.G; Simoni, D; Periotto, V. Manfredini, S; Guarnieri, M; Manservigi, R; Cassai, E. Bertolasi V. Pyrazolo [4,3-d]pyrimidine Nucleosides. Synthesis and Antiviral Activity of 1-b-D-Ribofuranosyl-3-methyl-6-substituted-7H-pyrazolo[4,3-d]pyrimidin-7-ones, J. Med. Chem. 1984; 27: 986-990
M.G. Gioia , B. Cacciari, A. Leoni, R. Gatti (2005). 2,7-Dimethyl-3,8-dinitrodipyrazolo[1,5-a:1’,5’-d]pyrazine-4,9-dione: a new labelling reagent for HPLC analysis of amino acids. MODENA : s.n.
2,7-Dimethyl-3,8-dinitrodipyrazolo[1,5-a:1’,5’-d]pyrazine-4,9-dione: a new labelling reagent for HPLC analysis of amino acids
GIOIA, MARIA GRAZIA;LEONI, ALBERTO;GATTI, RITA
2005
Abstract
Amino acids are important compounds in a wide range of samples such as biological tissues and fluids, food and industrial products. In particular they have a considerable role in pharmaceutical field because several diseases are associated with defects in amino acids metabolism and they are utilized as dietary supplements. The analysis of these compounds is essential and often difficult owing to their poor detectability. High performance liquid chromatography (HPLC) in combination with a pre-column chemical derivatization constitutes an effective and convenient technique to overcome the problem. To develop additional useful reagents, our attention was directed to 2,7-dimethyl-3,8-dinitrodipyrazolo[1,5-a:1’,5’-d]pyrazine-4,9-dione (I), which showed an excellent reactivity toward nucleophylic reagents, such as aliphatic, benzylic and aromatic amines [1]. The aim of the present work was the use of the compound I as labelling reagent for the pre-chromatographic derivatization of amino acids. The derivatization and chromatographic conditions were optimised by a series of experiments. The compound was found to react with amino function of the amino acid in 30 min at 68°C in presence of triethylamine (TEA) (0.04 M) to give adducts (II), which can be chromatographed and detected by UV-DAD (l=280 nm). The synthetized and purified derivatives were characterized by UV, IR, 1HNMR and MS. Routine chromatographic analyses were performed at 33±2°C on a reversed phase column (Phenomenex Gemini 5u C18 110A, 250 x 3 mm i.d.) by gradient elution using a mobile phase consisting of a mixture A/B where A=phosphate buffer (0.05 M; pH 3) and B=methanol at a flow rate of 0.5 mL/min. The proposed method was applied successfully to the determination of several amino acids, present as nutrients in commercial dosage forms. [1] Baraldi, P.G; Simoni, D; Periotto, V. Manfredini, S; Guarnieri, M; Manservigi, R; Cassai, E. Bertolasi V. Pyrazolo [4,3-d]pyrimidine Nucleosides. Synthesis and Antiviral Activity of 1-b-D-Ribofuranosyl-3-methyl-6-substituted-7H-pyrazolo[4,3-d]pyrimidin-7-ones, J. Med. Chem. 1984; 27: 986-990I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
