Analysis of the free nucleotide pools of mammalian tissues by high-pressure liquid chromatography

• Published in: Analytical biochemistry Vol. 123; no. 2; pp. 378 - 388
• Main Authors: Brown, Eric G., Newton, Russell P., Shaw, Nicholas M.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.07.1982
• Subjects: HPLC
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(82)90461-4

Perchloric acid extracts of tissues were neutralized with tri- N-octylamine and, after removal of ClO 4 −, subjected to preliminary purification on a Cu 2+-loaded column of Chelex 100. A high-pressure liquid chromatographic (HPLC) anion-exchange procedure was developed and gave good resolution of the naturally occurring free nucleotides on a single column. Where heterogeneous peaks eluted, an effective supplementary analysis was achieved by reverse-phase HPLC. An HPLC paired-ion technique was also evaluated for use in nucleotide analysis. Although anion-exchange was best for overall separation of nucleotides, both reverse-phase and paired-ion chromatography gave excellent separation of cyclic nucleotides. Reduced pyridine nucleotides were detected and measured in the form of their acid-decomposition products. The recovery of nucleotides was examined throughout the described analytical techniques and shown to be quantitative.