Development and validation of an LC-MS/MS quantitative method for endogenous deoxynucleoside triphosphates in...

Area of Science:

• Biochemistry
• Pharmacology
• Analytical Chemistry

Background:

• The endogenous deoxynucleoside triphosphate (dNTP) pool, comprising dATP, dCTP, dGTP, and TTP, is crucial for DNA synthesis and repair.
• This pool is regulated by complex enzymatic pathways and is a target for antimetabolite drugs.
• dNTPs also compete with nucleoside/nucleotide analog drugs, influencing their therapeutic mechanisms.

Purpose of the Study:

• To develop and validate a sensitive method for quantifying the intracellular dNTP pool in cellular lysates.
• To establish a reliable assay for measuring individual dNTP concentrations (dATP, dCTP, dGTP, TTP).

Main Methods:

• Optimized extraction of dNTPs from cellular lysates.
• Separation of nucleotide moieties using strong anion exchange chromatography.
• Dephosphorylation to nucleosides followed by quantification via liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Main Results:

• Validated analytical range of 50-2500 fmol/sample for each dNTP.
• Quantified dNTPs in peripheral blood mononuclear cells from 40 participants (279 samples).
• Established median dNTP concentrations: dATP (143 fmol/10^6 cells), dCTP (737 fmol/10^6 cells), dGTP (237 fmol/10^6 cells), TTP (315 fmol/10^6 cells).

Conclusions:

• A sensitive and validated LC-MS/MS method for intracellular dNTP quantification has been developed.
• This assay enables future studies on dNTP disposition in clinical settings.
• Facilitates research on patients receiving antimetabolites or nucleos(t)ide analogs.