A method for a simultaneous separation of malondialdehyde (MDA), ascorbic acid and adenine nucleotide derivatives in biological samples by ion-pairing high-performance liquid chromatography is presented. The separation is obtained by an LC-18-T 15 cm x 4.6 mm 3 μm particle size column using tetrabutylammonium as the pairing ion. The starting buffer consists of 10 mM tetrabutylammonium hydroxide, 10 mM KH2PO4 plus 1% methanol, pH 7.00. A step gradient is formed using a second buffer consisting of 2.8 mM tetrabutylammonium hydroxide, 100 mM KH2PO4 plus 30% methanol, pH 5.5. Under these chromatographic conditions a highly resolved separation of MDA, ATP, ADP, AMP, adenosine, ascorbic acid, GTP, GDP, IMP, inosine, Hypoxanthine, Xanthine, uric acid, NAD, and NADP can be performed in about 36 min. In addition, the separation of NADH and NADPH can also be obtained; this renders the present method suitable for the detection of these reduced coenzymes in alkaline extracts from tissue samples. Data referring to PCA extracts from ischemic and reperfused isolated rat hearts and from human erythrocytes peroxidized in vitro by a challenge with 1 mM NaN3 and various concentrations of H2O2 are reported. The relevance of this chromatographic method lies in the possibility to determine directly MDA concentrations avoiding the unspecific thiobarbituric acid colorimetric test, any other manipulation of the sample out of the PCA extraction, and any possible coelution of other acid soluble compounds. The simultaneous determination of MDA, ascorbic acid, and of ATP and its degradation products gives the opportunity to correlate, by a single chromatographic run, peroxidative damages with the energy state of the cell which is of great importance in studies of ischemic and reperfused tissues.

Lazzarino, G., Di Pierro, D., Tavazzi, B., Cerroni, L., Giardina, B. (1991). Simultaneous separation of malondialdehyde, ascorbic acid, and adenine nucleotide derivatives from biological samples by ion-pairing high-performance liquid chromatography. ANALYTICAL BIOCHEMISTRY, 197(1), 191-196.

Simultaneous separation of malondialdehyde, ascorbic acid, and adenine nucleotide derivatives from biological samples by ion-pairing high-performance liquid chromatography

CERRONI, LOREDANA;
1991-01-01

Abstract

A method for a simultaneous separation of malondialdehyde (MDA), ascorbic acid and adenine nucleotide derivatives in biological samples by ion-pairing high-performance liquid chromatography is presented. The separation is obtained by an LC-18-T 15 cm x 4.6 mm 3 μm particle size column using tetrabutylammonium as the pairing ion. The starting buffer consists of 10 mM tetrabutylammonium hydroxide, 10 mM KH2PO4 plus 1% methanol, pH 7.00. A step gradient is formed using a second buffer consisting of 2.8 mM tetrabutylammonium hydroxide, 100 mM KH2PO4 plus 30% methanol, pH 5.5. Under these chromatographic conditions a highly resolved separation of MDA, ATP, ADP, AMP, adenosine, ascorbic acid, GTP, GDP, IMP, inosine, Hypoxanthine, Xanthine, uric acid, NAD, and NADP can be performed in about 36 min. In addition, the separation of NADH and NADPH can also be obtained; this renders the present method suitable for the detection of these reduced coenzymes in alkaline extracts from tissue samples. Data referring to PCA extracts from ischemic and reperfused isolated rat hearts and from human erythrocytes peroxidized in vitro by a challenge with 1 mM NaN3 and various concentrations of H2O2 are reported. The relevance of this chromatographic method lies in the possibility to determine directly MDA concentrations avoiding the unspecific thiobarbituric acid colorimetric test, any other manipulation of the sample out of the PCA extraction, and any possible coelution of other acid soluble compounds. The simultaneous determination of MDA, ascorbic acid, and of ATP and its degradation products gives the opportunity to correlate, by a single chromatographic run, peroxidative damages with the energy state of the cell which is of great importance in studies of ischemic and reperfused tissues.
1991
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore BIO/10 - BIOCHIMICA
English
Con Impact Factor ISI
adenine nucleotide; ascorbic acid; malonaldehyde; article; high performance liquid chromatography; human; human cell; ion pair chromatography; priority journal; Adenine Nucleotides; Animal; Ascorbic Acid; Chromatography, High Pressure Liquid; Comparative Study; Erythrocytes; Evaluation Studies; Human; Hydrogen Peroxide; In Vitro; Male; Malondialdehyde; Myocardial Reperfusion Injury; Rats; Rats, Inbred Strains; Support, Non-U.S. Gov't; Thiobarbiturates
Lazzarino, G., Di Pierro, D., Tavazzi, B., Cerroni, L., Giardina, B. (1991). Simultaneous separation of malondialdehyde, ascorbic acid, and adenine nucleotide derivatives from biological samples by ion-pairing high-performance liquid chromatography. ANALYTICAL BIOCHEMISTRY, 197(1), 191-196.
Lazzarino, G; Di Pierro, D; Tavazzi, B; Cerroni, L; Giardina, B
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/49636
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