Selective Extraction of Nucleotides with Fluorous Biphasic System Utilizing Perfluoroalkylamine as an Ion-Pair Reagent

A novel and selective extraction method for nucleotides (AMP, ADP, ATP, GMP, GDP, and GTP) has been developed. In this study, we employed liquid-liquid based fluorous biphasic extraction system in the presence of a 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecylamine (HFUA) as an ion-pair reagent. The ion-pairs between nucleotides and HFUA were selectively extracted to a mixture of tetradecafluorohexane and 1H, 1H, 2H, 2H-tridecafluoro-1-n-octanol (3: 1, v/v) as fluorous solvent via the affinity of perfluoroalkyl-containing compounds with each other, i. e. fluorous interaction. Then, the nucleotides in the fluorous solvent could be back-extracted with aqueous ammonia in acetonitrile as non-fluorous solvent, and the extracts were directly injected to LC system. A hydrophilic interaction chromatography was utilized for separation of the nucleotides by gradient elution with the mixture of acetonitrile, water, and ammonium carbonate buffer (pH 10.0) as mobile phase, and an ultraviolet detection at 254 nm was used. The extraction recoveries of nucleotides were in the range from 70.4 to 96.8%. This method could be applied to the determination of nucleotides in cell samples.