An Ion-Pairing High-Performance Liquid Chromatographic Method for the Direct Simultaneous Determination of Nucleotides, Deoxynucleotides, Nicotinic Coenzymes, Oxypurines, Nucleosides, and Bases in Perchloric Acid Cell Extracts

Simultaneous Determination of Deoxyribonucleoside in the Presence of Ribonucleoside Triphosphates in Human Carcinoma Cells by High-Performance Liquid Chromatography

“…In our series of experiments, unneutralized control cell extracts stored at Ϫ80°C were found to be stable for at least 2 months, taking into account the experimental variability, in accordance with previously reported studies (4,23). However, after prolonged storage at Ϫ80°C (up to 8 months), additional peakspresumably from decomposition products formed on prolonged storage at Ϫ80°C-were eluted just before dATP.…”

“…The precipitation of proteins of cell extracts by 1.2 M HClO 4 (23) was not satisfactory, producing erratic HPLC profiles of supernatants even after careful neutralization. Diluted (3%) TCA was found to be suitable for adequately removing proteins from extracts which were centrifuged and neutralized with Na 2 CO 3 just prior to HPLC analysis.…”

“…To the best of our knowledge however, only a few studies reporting the simultaneous determination of NTP and dNTP by HPLC have been published so far (21)(22)(23). One of the reported methods had little success in separating dGTP from ADP (21), and another was not very sensitive and required a tedious three-step extraction with ethylic ether for removal of TCA (22).…”

“…One of the reported methods had little success in separating dGTP from ADP (21), and another was not very sensitive and required a tedious three-step extraction with ethylic ether for removal of TCA (22). A simplified method has been recently published for the simultaneous analysis of NTP and dNTP, along with other endogenous purine constituents from lymphocyte extracts (23). This was an important contribution, though the existing minute amount of dNTP could not be fully discriminated nor resolved from a relatively high baseline noise.…”

“…We report the adaptation, improvement, and optimization of the method of Di Pierro et al (23) leading to adequate sensitivity (down to the picomole range with a 40-l injection volume) and selectivity (separation from nearby interfering peaks) for the simultaneous separation and quantitation of dNTP and NTP.…”