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A LC-MS/MS Method for Quantifying Adenosine, Guanosine and Inosine Nucleotides in Human Cells

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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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Correspondence to Jennifer J. Kiser.

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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

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