Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography-tandem mass spectrometry. - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Analytical and Bioanalytical Chemistry Année : 2014

Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography-tandem mass spectrometry.

Résumé

An analytical method coupling online solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed to quantify 16 endogenous nucleoside mono- and triphosphates in cellular samples. Separation was achieved on a porous graphitic carbon (PGC) column without ion-pairing agent in the mobile phase. Low levels of the ion-pairing agent diethylamine (DEA) added to the reconstitution solution were necessary to prevent peak tailing of nucleoside triphosphates. The mass spectrometer, a triple quadrupole with an electrospray ionisation source, was operated in positive mode. Two multiple reaction monitoring (MRM) segments were programmed, each an internal standard. Extraction and separation of nucleoside mono- and triphosphates were obtained within 20 min. The total duration of a single run was 37 min. Calibration curves, performed with labelled nucleotides added to the sample matrix, ranged from 0.29 to 18.8 pmol injected for deoxyribonucleotides and from 3.9 to 3,156 pmol for ribonucleotides. Accuracy did not deviate more than −14.6 and 10.2 % from nominal values for all compounds at all levels. CV results were all lower than 17.0 % for the LLOQ level and 14.6 % for the other levels. Quality control (QC) samples were also in agreement with acceptance criteria, except for the lower QC of GMP. Ion suppression, matrix effect, extraction recoveries and stability were assessed. After validation, the method was applied to the evaluation of the effects of gemcitabine and hydroxyurea on nucleotide pools in Messa cells.

Domaines

Chimie organique
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Dates et versions

hal-01004663 , version 1 (11-06-2014)

Identifiants

Citer

Christelle Machon, Lars Petter Jordheim, Jean-Yves Puy, Isabelle Lefebvre, Charles Dumontet, et al.. Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography-tandem mass spectrometry.. Analytical and Bioanalytical Chemistry, 2014, 406, pp.2925-2941. ⟨10.1007/s00216-014-7711-1⟩. ⟨hal-01004663⟩
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