Reversed‐phase high‐performance liquid chromatographic analysis of endogenous purine ribonucleotide pools in BHK monolayer cultures

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Abstract
A rapid, sensitive, and reproducible separation of purine bases, nucleosides and nucleotides by reversed‐phase high‐performance liquid chromatography is described. A multi‐step gradient of acetonitrile in 0.1 M potassium phosphate was used to detect picomole amounts of each compound within 22 min. The high sensitivity of the method made it suitable for the analysis of purine nucleotide pools extracted from 1‐‐2 × 106 hamster fibroblasts grown as monolayer (BHK line) and for the detection of purine bases and nucleosides leaked form the cells into the incubation media. The chromatographic procedure was applied to study the effects of hexavalent chromium (potassium dichromate) on purine metabolism. Several steps are affected by this treatment, causing a marked unbalance of both the guanylate and adenylate pool, which was seen as a strong decrease in the level of triphosphates and accumulation of their precursors.