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/106 cells (approximately 0.08 to 80 picomoles on‐column). Linear regression analysis with 1/x2 weighting yielded a coefficient of determination (r2) 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/106 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. Copyright © 2000 John Wiley & Sons, Ltd.

A mixed-mode liquid chromatography-tandem mass spectrometric method for the determination of cytarabine in mouse plasma

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Short-Term Metabolome Dynamics and Carbon, Electron, and ATP Balances in Chemostat-Grown Saccharomyces cerevisiae CEN.PK 113-7D following a Glucose Pulse

“…The nucleotides were separated by an ion-pairing reversed-phase high-performance liquid chromatography (HPLC) method adapted from a previously described method (8), using an XTerra MS C 18 column (100 mm ϫ 1 mm) equipped with a guard column (10 mm ϫ 2.1 mm) (both from Waters, Milford, MA). The standard solution and sample injection volume was 10 l; all standard solutions and samples were mixed 24:1 with a 2 M solution of the ion-pairing reagent tetrabutylammoniumacetate (Sigma-Aldrich, Steinheim, Germany) to obtain a final tetrabutylammoniumacetate concentration of 80 mM.…”

Determination of adenosine nucleotides in cultured cells by ion-pairing liquid chromatography–electrospray ionization mass spectrometry

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