title: A LC-MS/MS Method for Quantifying Adenosine, Guanosine and Inosine Nucleotides in Human Cells.
abstract: To develop and validate a method for the simultaneous measurement of adenosine, guanosine, and inosine derived from mono (MP) and triphosphate (TP) forms in peripheral blood mononuclear cells (PBMCs), red blood cells (RBCs) and dried blood spots (DBS).
  
  
  
  Solid phase extraction of cell lysates followed by dephosphorylation to molar equivalent nucleoside and LC-MS/MS quantification.
  
  
  
  The assay was linear for each of the three quantification ranges: 10-2000, 1.0-200 and 0.25-50 pmol/sample for adenosine, guanosine, and inosine, respectively. Intraassay (n = 6) and interassay (n = 18) precision (%CV) were within 1.7 to 16% while accuracy (%deviation) was within -11.5 to 14.7% for all three analytes. Nucleotide monophosphates were less concentrated than triphosphates (except for inosine) and levels in PBMCs were higher than RBCs for all three nucleotides (10, 55, and 5.6 fold for ATP, GTP and ITP, respectively). DBS samples had an average (SD) of -26% (22.6%) lower TP and 184% (173%) higher MP levels compared to paired RBC lysates, suggesting hydrolysis of the TP in DBS.
  
  
  
  This method was accurate and precise for physiologically relevant concentrations of adenosine, guanosine and inosine nucleotides in mono- and triphosphate forms, providing a bioanalytical tool for quantitation of nucleotides for clinical studies.
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, 12850 E. Montview Blvd, V20 C238, Aurora, Colorado, 80045, USA.
authors.forename: Leah C
authors.initials: LC
authors.lastname: Jimmerson
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, 12850 E. Montview Blvd, V20 C238, Aurora, Colorado, 80045, USA.
authors.forename: Lane R
authors.initials: LR
authors.lastname: Bushman
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, 12850 E. Montview Blvd, V20 C238, Aurora, Colorado, 80045, USA.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Ray
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, 12850 E. Montview Blvd, V20 C238, Aurora, Colorado, 80045, USA.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Anderson
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, 12850 E. Montview Blvd, V20 C238, Aurora, Colorado, 80045, USA. jennifer.kiser@ucdenver.edu.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Kiser
mesh_terms: D000241:Adenosine / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods; D059788:Dried Blood Spot Testing / Q000379:methods; D004912:Erythrocytes / Q000737:chemistry; D006151:Guanosine / Q000097:blood*; D006801:Humans; D007288:Inosine / Q000097:blood*; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D009711:Nucleotides / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D009711:Nucleotides; D006151:Guanosine; D007288:Inosine; D000241:Adenosine
keywords: LC-MS/MS; analytical method; dried blood spots; intracellular quantification; purine nucleotides