Simultaneous quantitation of the 5′-triphosphate metabolites of zidovudine, lamivudine, and stavudine in peripheral mononuclear blood cells of HIV infected patients by high-performance liquid chromatography tandem mass spectrometry

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Abstract
A high-performance liquid chromatography (HPLC) method utilizing triple quadrupole mass spectrometry (MS) detection was developed and validated for the simultaneous measurement of the intracellular nucleoside 5′-triphosphate anabolites of zidovudine (ZDV-TP), lamivudine (3TC-TP), and stavudine (d4T-TP). These compounds were extracted from patient peripheral blood mononuclear cells (PBMCs) which are the sites of HIV replication and drug action. Ion-exchange solid phase extraction (SPE) followed by enzymatic digestion with alkaline phosphatase was utilized to yield the measurable nucleoside forms of the nucleotides. Reversed phase C-18 SPE with addition of a nucleoside internal standard, 3′-azido-2′,3′-dideoxyuridine (AzdU) allowed for the indirect measurement of the original 5′-triphosphate concentration by HPLC/MS/MS. Quantitation was performed from calibration curves generated from authentic 5′-triphosphate standards spiked in PBMCs from healthy volunteers. Analytical range for the three 5′-triphosphates was equivalent to 50–45,000 pg. Mean interassay accuracies for 3TC-TP, d4T-TP, and ZDV-TP (n > 90) were 99.4%, 100.1%, and 108.0%, respectively. Mean interassay precisions (%C.V.) for 3TC-TP, d4T-TP, and ZDV-TP (n > 90) were 8.8%, 10.4%, and 8.2%, respectively. Recovery of the extraction method was 79.2%, 83.1%, and 98.3% for 3TC-TP, d4T-TP, and ZDV-TP, respectively. This method can be utilized to measure the intracellular 5′-triphosphate levels in HIV infected patients receiving antiretroviral therapy containing the nucleoside reverse transcriptase inhibitors 3TC, d4T, or ZDV.

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