A LC-MS/MS method for the analysis of intracellular nucleoside triphosphate levels

Abstract

Purpose: To simultaneously quantify intracellular nucleoside triphosphate (NTP) and deoxynucleoside triphosphate (dNTP) pools and to assess their changes produced by interfering with ribonucleotide reductase (RNR) expression in leukemia cells.

Methods: A HPLC-MS/MS system was used to quantify intracellular NTP and dNTP pools.

Results: The assay was linear between 50 nM, the lower limit of quantification (LLOQ), and 10 muM in cell lysate. The within-day coefficients of variation (CVs, n = 5) were found to be 12.0-18.0% at the LLOQ and 3.0-9.0% between 500 and 5,000 nM for dNTPs and 8.0-15.0% and 2.0-6.0% for NTPs. The between-day CVs (n = 5) were 9.0-13.0% and 3.0-11.0% for dNTPs and 9.0-13.0% and 3.0-6.0% for NTPs. The within-day accuracy values were 93.0-119.0% for both NTPs and dNTPs. ATP overlapped with dGTP and they were analyzed as a composite. This method was applied to measure basal intracellular dNTPs/NTPs in five leukemia cell lines exposed to the RNR antisense GTI-2040. Following drug treatment, dCTP and dATP levels were found to decrease significantly in MV4-11 and K562 cells. Additionally, perturbation of dNTP/NTP levels in bone marrow sample of a patient treated with GTI-2040 was detected.

Conclusions: This method provides a practical tool to measure intracellular dNTP/NTP levels in cells and clinical samples.

Fig. 1

A Total ion chromatogram of a standard mixture of ATP, GTP, CTP, UTP, dATP, dGTP, dCTP, dTTP and ClATP. B Full scan of a standard mixture 1 μM of ATP, GTP, CTP, UTP, dATP, dGTP, dCTP and dTTP with a direct infusion at 10 μL/min.

Fig. 2

Product ion mass spectra of the deprotonated molecular ions of dNTPs and NTPs.

Fig. 3

A The extract ion chromatograms (XIC) of dNTPs and NTPs, 50 nM each, spiked into acid phosphatase-treated blank K562 cell extracts. B The XICs of dNTPs and NTPs in blank acid phosphatase-treated K562 cell extracts. No significant interference peaks were observed.

Fig. 4

Standard curves of dNTPs and NTPs in K562 cell matrices.

Fig. 5

Stability study of dNTPs and NTPs in K562 cell matrices.

Fig. 6

A Alteration of dNTP and NTP levels in MV4-11 cells (blue column) or K562 cells (red column) following GTI-2040 treatment at various concentrations for 24 h (n=3). Asterisks indicate significant difference from untreated control at p<0.05.