Title
Simultaneous determination of endogenous deoxynucleotides and phosphorylated nucleoside reverse transcriptase inhibitors in peripheral blood mononuclear cells using ion-pair liquid chromatography coupled to mass spectrometry
Author
Coulier, L.
van Kampen, J.J.A.
de Groot, R.
Gerritsen, H.W.
Bas, R.C.
van Dongen, W.D.
Brüll, L.P.
Luider, T.M.
TNO Kwaliteit van Leven
KvL
Publication year
2008
Abstract
Nucleoside reverse transcriptase inhibitors (NRTIs) are activated intracellularly to their triphosphate (TP) form, which compete with endogenous deoxynucleotide-triphosphates (dNTP) as substrate for HIV reverse transcriptase. The activity of NRTIs is thus described by the NRTI-TP to-dNTP ratio in relevant cell types. Therefore, we developed an ion-pair (IP) LC-MS method for the simultaneous analysis of the mono-, di-, and TP forms of NRTIs and endogenous deoxynucleosides in peripheral blood mononuclear cells (PBMC). The IP-LC method was applied on an ITmass spectrometer using the MS-mode as well as on a triple quadrupole mass spectrometer using the MS/MS mode. The MS/MS approach on the triple quadrupole mass spectrometer demonstrated the best clinical applicability due to its higher sensitivity. The LOD (minimum amount on column) were 25 fmol for the TP forms of zidovudine, lamivudine, and stavudine, as well as for their endogenous dNTP counterparts. The linearity (R2) of the calibration curves were>0.99. The obtained LOD readily allow for clinical applications using just one million PBMC obtained from HIV-infected patients under therapy. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Chemicals / CAS: RNA directed DNA polymerase, 37213-50-6, 9068-38-6; abacavir, 136470-78-5, 188062-50-2; carbovir, 118353-05-2; lamivudine, 134678-17-4, 134680-32-3; stavudine, 3056-17-5; tenofovir, 147127-19-3, 147127-20-6; zidovudine, 30516-87-1
Subject
Biology
Analytical research
HIV
Intracellular
Ion-pair
Nucleoside reverse transcriptase inhibitor
Nucleotide
abacavir
carbovir
stavudine
tenofovir
zidovudine
cell lysate
controlled study
drug cell level
human cell
ion pair chromatography
isolation procedure
performance measurement system
peripheral blood mononuclear cell
protein phosphorylation
quadrupole mass spectrometry
separation technique
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DOI
https://doi.org/10.1002/prca.200800002
TNO identifier
241169
ISSN
1862-8346
Source
Proteomics - Clinical Applications, 2 (10-11), 1557-1562
Document type
article