Optimization of cellular nucleotide extraction and sample preparation for nucleotide pool analyses using capillary electrophoresis.

Cell extraction and further sample preparation for nucleotide pool analysis using capillary electrophoresis was faster and simpler using volatile extraction solvents (e.g. organic solvents and de-ionized water) compared to the commonly applied acids dissolved in water (e.g. perchloric acid and trichloracetic acid). Temperature had to be controlled during the whole sample preparation process to prevent degradation, and extracts had to be cleaned from proteins and other large molecules prior to capillary electrophoretic analysis to improve reproducibility. Capillary electrophoresis using borate and cyclodextrins in the background electrolyte was used for determining 11 cellular nucleotides simultaneously. In order to optimize the assay, 0-100% acetonitrile, 0-100% ethanol, and 0-100% methanol in de-ionized water were applied to extract nucleotides from mouse lymphoma cells, and nucleotide yields, recovery, and reproducibility were compared. The assay met the commonly accepted validation limits for biological fluids, if 20-80% acetonitrile in water and 40-60% ethanol in water were used as extraction solvents.