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
article
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
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
Topics
Analytical researchHIVIntracellularIon-pairNucleoside reverse transcriptase inhibitorNucleotideabacavircarbovirstavudinetenofovirzidovudinecell lysatecontrolled studydrug cell levelhuman cellion pair chromatographyisolation procedureperformance measurement systemperipheral blood mononuclear cellprotein phosphorylationquadrupole mass spectrometryseparation technique
TNO Identifier
241169
ISSN
18628346
Source
Proteomics - Clinical Applications, 2(10-11), pp. 1557-1562.
Pages
1557-1562
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