Comparison of sample preparation methods for the high-performance liquid chromatographic analysis of cell culture extracts for triphosphate ribonucleosides and deoxyribonucleosides.
We compared four different procedures for the purification and concentration of nucleoside triphosphates in cell extracts prior to HPLC analysis. Two methods involved precipitation, with either acetonitrile or calcium fluoride. The acetonitrile procedure yielded reasonable recovery and sufficient purity for the subsequent HPLC analysis. The calcium fluoride coprecipitation procedure gave both good recovery and purity; but the recovery was shown to be dependent on the concentration of the nucleoside triphosphates. The other two methods involved small Sep-Pak cartridges. The silica cartridge procedure yielded unfavorable recoveries in periodate-treated cell extracts, apparently due to poor solubility of nucleoside triphosphates in the requisite solvents. The strong anion exchange cartridge procedure yielded both good recovery and purity. This procedure was found to be fast, efficient, and reliable for purifying and concentrating nucleotides in cell extracts.
D000097:Acetonitriles; D000818:Animals; D002124:Calcium Fluoride; D002478:Cells, Cultured; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003854:Deoxyribonucleotides / Q000032:analysis*; D006801:Humans; D012265:Ribonucleotides / Q000032:analysis*; D012822:Silicon Dioxide
D000097:Acetonitriles; D003854:Deoxyribonucleotides; D012265:Ribonucleotides; D012822:Silicon Dioxide; D002124:Calcium Fluoride; C032159:acetonitrile