A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using...

Area of Science:

• Metabolomics
• Analytical Chemistry
• Cancer Biology

Background:

• Endogenous nucleosides and nucleotides are key differential metabolites in cancer research.
• Accurate quantification is crucial for understanding their role in cancer cell lines.

Purpose of the Study:

• To develop a rapid, sensitive, and high-throughput quantitative method for profiling 20 endogenous nucleosides and nucleotides.
• To apply this method for discriminating between different cancer cell lines.

Main Methods:

• Ultra-high performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS).
• Utilized a porous graphitic carbon column and a basic mobile phase (pH 11.5) with diethylamine (DEA) and ammonium acetate (NH4OAC).
• Method validation included linearity, sensitivity, accuracy, precision, matrix effects, recovery, and stability.

Main Results:

• Optimized basic mobile phase (pH 11.5) prevented adsorption and improved peak shape/sensitivity for multi-phosphorylated species.
• Achieved limits of quantification (LOQ) between 0.6-6 nM (6-60 fmol on column).
• Successfully discriminated between breast, colon, and lung cancer cell lines based on their nucleoside and nucleotide profiles.

Conclusions:

• The validated UHPLC-MS/MS method provides reliable quantification of 20 nucleosides and nucleotides in cancer cell lines.
• This method meets the demands of expanded metabolomics studies requiring high selectivity and sensitivity.
• The distinct metabolic profiles aid in distinguishing between different cancer types.