Abstract
Purpose
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).
Methods
Solid phase extraction of cell lysates followed by dephosphorylation to molar equivalent nucleoside and LC-MS/MS quantification.
Results
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.
Conclusion
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.
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Abbreviations
- AMP:
-
Adenosine monophosphate
- ATP:
-
Adenosine triphosphate
- DBS:
-
Dried blood spots
- DP:
-
Diphosphate
- GMP:
-
Guanosine monophosphate
- GTP:
-
Guanosine triphosphate
- HPLC:
-
High performance liquid chromatography
- IMP:
-
Inosine monophosphate
- IS:
-
Stable labeled internal standard
- ITP:
-
Inosine triphosphate
- LC-MS/MS:
-
Liquid chromatography tandem mass spectrometry
- LLOQ:
-
Lower limit of quantification
- MP:
-
Monophosphate
- NA:
-
Nucleoside analog
- PBMCs:
-
Peripheral blood mononuclear cells
- QC:
-
Quality control
- QH:
-
High control
- QL:
-
Low control
- QM:
-
Medium control
- RBCs:
-
Red blood cells
- RBV:
-
Ribavirin
- STD:
-
Standard
- TP:
-
Triphosphate
- UP-water:
-
Ultrapure water
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ACKNOWLEDGMENTS AND DISCLOSURES
This work was supported by R03 DK096121 (JJK), the International Maternal Pediatric Adolescent AIDS Clinical Trials Network (UM1AI068632 to JJK), and the Colorado Clinical Translational Sciences Institute (1UL1 RR025780).
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Jimmerson, L.C., Bushman, L.R., Ray, M.L. et al. A LC-MS/MS Method for Quantifying Adenosine, Guanosine and Inosine Nucleotides in Human Cells. Pharm Res 34, 73–83 (2017). https://doi.org/10.1007/s11095-016-2040-z
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DOI: https://doi.org/10.1007/s11095-016-2040-z