Simultaneous determination of nucleosides, modified nucleosides and mononucleotides in gastric cancer cells by liquid chromatography coupled with tandem mass spectrometry.

Nucleosides and their modified forms serve as fundamental building blocks of nucleic acids and play critical roles in epigenetic regulation, RNA metabolism, and cellular signaling. In this study, we developed and validated a rapid, sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous quantification of 27 endogenous nucleosides-including 12 canonical nucleosides, 11 modified nucleosides, and 4 mononucleotides-in cellular samples. Under optimized chromatographic conditions, good separations and peak shapes for 27 target compounds were achieved within a 13.0-min gradient elution. The overall LOQs were between 0.2 and 25.0 ng/mL. The intra-day precision of the method was less than 8.2 %, and the inter-day precision was less than 14.2 %. The accuracy was in the range of 89.9-112.2 % for all analytes. The developed method was successfully applied to analyze the metabolic profiles of gastric cancer cells with varying differentiation degrees (NCI-N87, SGC-7901, MGC-803). The results revealed distinct nucleotide metabolism alterations linked to tumor progression and identified potential biomarkers for gastric cancer advancement.