Zeitschrift für Chromatographie und Trenntechniken
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Separation and Analysis of Mono-Di- And Tri- Phosphate Nucleotides from Cell Extract Using Reversed Phase HPLC

Praveen Kusumanchi, Rachel Rogers, Ting Zhang, Suthat Liangpunsakul, Zhihong Yang, Hiremagalur N Jayaram

The focus of the present study was to develop a simple, reliable, and highly sensitive ion pair reversed phase high performance liquid chromatography for the separation and quantification of purine, pyrimidine and pyridine nucleotides. First, we optimized the column conditions to achieve baseline separation and quantification of 15 nucleotides. The least amount required for detecting all the ribonucleotides with reproducibility and high accuracy was 10 pmol. Calibration curves for all nucleotide standards were linear within the range of 10-200 pmol. This method can be successfully used to quantitate all nucleotides simultaneously in studies with nucleotide inhibitors such as Tiazofurin and Benzamide Riboside in cancer cell lines. This is the first report showing the separation of all 15 nucleotides in a single injection