Fluorometric determination of adenine nucleotides and adenosine by ion-paired, reverse-phase, high-performance liquid chromatography

✍ Scribed by Alfonso Ramos-Salazar; Andrew D. Baines

• Publisher: Elsevier Science
• Year: 1985
• Tongue: English
• Weight: 389 KB
• Volume: 145
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(85)90319-7

✦ Synopsis

A sensitive and specific assay for measurement of adenine nucleotides and adenosine by paired-ion high-performance liquid chromatography is described. The I,N6-ethenoderivatives of ATP (c-ATP), ADP (+ADP), AMP (c-AMP). and adenosine (c-Ado), formed by reaction with chloroacetaldehyde at 37°C. were separated under isocratic conditions in 20 min. These compounds are strongly fluorescent at an emission wavelength of 280 nm, rendering a lowest detection limit of 2-5 pmol per injection. The detector responded linearly over the measured ranges (5-100 pmol for C-Ado and 5-4000 pmol for nucleotides). Specificity was confirmed enzymatically. a$-Methyleneadenosine 5'-diphosphate could be used as an internal standard for measurement of the nucleotides. Significant amounts of NADH appeared as a separate peak in hypoxic tissue. Recoveries from snap-frozen kidney were 88. 92, 76, and 63% for AMP, ADP, ATP, and adenosine, with SD for recovery of 1 .O, 10.5, 8.3, and 5.6%, respectively. This method was successfully used to measure adenine nucleotides and adenosine in oxygenated and hypoxic perfused rat kidneys. G 1985 Academic PES. inc.