Positive ion electrospray ionization tandem mass spectrometry coupled to ion-pairing high-performance liquid chromatography with a phosphate buffer for the quantitative analysis of intracellular nucleotides.

Abstract

A novel analytical procedure has been developed for the analysis of intracellular nucleotide triphosphates. Positive ion electrospray ionization tandem mass spectrometry (MS/MS) was interfaced to ion-pairing high-performance liquid chromatography (HPLC) utilizing a mobile phase containing 10 mM ammonium phosphate, pH 6.4, with 2 mM tetrabutylammonium hydroxide and 15% acetonitrile. The methodology was developed to support the analysis of the 5'-triphosphate anabolite of the antiviral agent (-)-FTC ((2R, 5S)-5-fluoro-1-[2-(hydroxymethyl-1,3-oxathiolan-5-yl]cytosine) in human peripheral blood mononuclear cells (PBMCs). In this procedure, all nucleotides were extracted from PBMCs with aqueous methanol, isolated with high recovery using a novel ion-pairing solid phase extraction procedure, and then analyzed directly with LC/MS/MS with a 10-min analysis time. A calibration curve was generated representing (-)-FTC 5'-triphosphate ((-)-FTCTP) concentration over the range of 0.083 to 83 picomol/10(6) cells (approximately 0.08 to 80 picomoles on-column). Linear regression analysis with 1/x(2) weighting yielded a coefficient of determination (r(2)) of greater than 0.999. The back-calculated concentrations of all calibration standards had relative errors within the range of +5 to -3%. A preliminary assessment of intra-assay precision and accuracy, analyte stability, and LC/MS system stability indicated a robust method capable of being validated with a limit of quantitation estimated conservatively at 0.08 picomol/10(6) cells (approximately 0.08 picomoles on-column; signal-to-noise (S/N) = 5). The general method developed here should be adaptable to all purine- and pyrimidine-based nucleotide applications. This report provides a detailed discussion on the key HPLC, MS, and sample preparation procedures that hold the potential for even greater nucleotide sensitivity.