A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS

Endogenous nucleosides and nucleotides in biosamples are frequently highlighted as the most differential metabolites in recent metabolomics studies. We developed a rapid, sensitive, high-throughput and reliable quantitative method to simultaneously profile 20 endogenous nucleosides and nucleotides in cancer cell lines based on ultra-high performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC- MS/MS) by using a porous graphitic carbon column and basic mobile phase. The results indicated that high pH value of mobile phase containing 0.12% diethylamine (DEA) and 5 mM NH₄OAC (pH 11.5) was the critical factor to prevent the adsorption of multi-phosphorylated species, and significantly improved peak shape and sensitivity. The optimized method was successfully validated with satisfactory linearity, sensitivity, accuracy, precision, matrix effects, recovery and stability for all analytes. The limit of quantification (LOQ) was in the range of 0.6–6 nM (6–60 fmol on column). The validated method was applied to the extract of three epithelial cancer cell lines, and the significant difference in the profiling of the nucleosides and nucleotides among the cancer cell lines enables discrimination of breast cancer cell line from the colon cancer cell line and the lung cancer cell line. This quantified analytical method of 20 endogenous nucleosides and nucleotides in cancer cell lines meets the requirement of quantification in specific expanded metabolomics studies, with good selectivity and sensitivity.