Fluorous-assisted metal chelate affinity extraction for nucleotides followed by HILIC-MS/MS analysis

We herein developed a selective method for the determination of nucleotides by fluorous-assisted metal chelate affinity extraction followed by hydrophilic interaction liquid chromatography (HILIC) combined with tandem mass spectrometric (MS/MS) analysis. In this study, the nucleotides were selectively chelated by Fe(III)-immobilized perfluoroalkyliminodiacetic acid, and the resulting chelates were subsequently extracted into a fluorous solvent. The nucleotides present in the fluorous solvent were then back-extracted into a non-fluorous solution, such as a solution of ammonia in aqueous acetonitrile. The resulting non-fluorous solution containing the nucleotides was then directly injected into an amide-type HILIC column using a mixture of acetonitrile and aqueous ammonium bicarbonate as the mobile phase for gradient elution, and the nucleotides were detected using the negative electrospray ionization MS/MS mode. In this method, the extraction recoveries of the nucleotides ranged from 43.2 to 94.7% within a relative standard deviation of 17%. This method enabled the determination of intracellular concentrations of nucleotides.