Simultaneous detection of nucleotides, nucleosides and oxidative metabolites in myocardial biopsies

A new simple, simultaneous matrix HPLC methodology was developed to facilitate better peak separability and resolution for the determination of levels of myocardial tissue nucleotides, nucleosides and oxidative metabolites. The components of interests were ATP, AMP, ADP, IMP, hypoxanthine, xanthine,...

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Published inJournal of chromatography. B, Biomedical applications Vol. 678; no. 2; pp. 181 - 186
Main Authors Childs, Keith F., Ning, Xue-Han, Bolling, Steven F.
Format Journal Article
LanguageEnglish
Published Netherlands Elsevier B.V 12.04.1996
Subjects
Adenine Nucleotides - analysis
Biopsy
Chromatography, High Pressure Liquid - methods
Chromatography, High Pressure Liquid - statistics & numerical data
Energy Metabolism
Humans
Indicators and Reagents
Inosine Monophosphate - analysis
Myocardium - chemistry
Myocardium - metabolism
NAD - analysis
Nucleosides
Nucleosides - analysis
Nucleotides
Nucleotides - analysis
Oxidation-Reduction
Nucleosides
Nucleotides
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Summary:A new simple, simultaneous matrix HPLC methodology was developed to facilitate better peak separability and resolution for the determination of levels of myocardial tissue nucleotides, nucleosides and oxidative metabolites. The components of interests were ATP, AMP, ADP, IMP, hypoxanthine, xanthine, adenosine, inosine, NAD, and NADH, which are used to establish myocardial cellular energy status and effectiveness of cardioprotection. Their detection was achieved using a 4-μm spherical bead, 300 × 3.9 mm I.D. Nova-Pak C 18 column in a 12% methanol mobile phase solvent selection, ion-pairing reagents 1.47 m M TBAP (tetrabutylammonium phosphate) and 73.5 m M KH 2PO 4, at a pH of 4.0. The extraction method was modified for rapid determination to ensure diminished acid labile NADH effects. Comparisons of peak retention ( k), resolution ( R s) of solvents of varying concentrations and pH adjustments facilitated this method. This isocratic single run determination allows for simple, simultaneous rapid quantification and identification of alterations in high-energy phosphates, nucleoside degradation products and NAD/NADH levels associated with myocardial ischemia, with excellent reliability.
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ISSN:0378-4347
1572-6495
DOI:10.1016/0378-4347(95)00474-2