A Method of Determining Intracellular Nucleotides and Nucleosides by Ion-pair Liquid Chromatography for Metabonomics of Cancer Cell Lines

Objective:To establish a reversed-phase ion-pair high-performance liquid chromatographic method for simultaneous determination of nucleotides and nucleosides in K562 and K562/A cell lines.Method:The sam- ples were treated with 1.2 mol· L~(-1) perchloric acid,and nucleotides and nucleosides were purified with solid phase extraction method.The separation was performed on a Hanbon C_(18)column (4.6 mm×250 nm,5μm) using linear gradient elution of A (containing 10 mmol·L~(-1) tetrabutylammonium hydroxide,10 mmol·L~(-1) KH_2PO_4 and 0.25% MeOH,pH7.0) and B (containing 5.6 mmol·L~(-1) tetrabutylammonium hydroxide,50 mmol·L~(-1) KH_2PO_4 and 30% MeOH,pH7.0).The HPLC columns were kept at 25℃ and detection wavelength was 260 nm. Results:The nucleotides and nucleosides were well separated,and mean recovery for the determination of intracellu- lar nucleotides and nucleosides in K562 and K562/A cell lines were 88.3%-109.2% and 84.5%-115.6%,re- spectively.Conclusion:The proposed method is simple,reproducible and it can be applied to the determination of nucleotides and nucleosides in K562 and K562/A cell lines.