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

A high-performance liquid chromatography (HPLC) method utilizing triple qua drupole mass spectrometry (MS) detection was developed and validated for th e 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 mononucle ar cells (PBMCs) which are the sites of HIV replication and drug action. Io n-exchange solid phase extraction (SPE) followed by enzymatic digestion wit h alkaline phosphatase was utilized to yield the measurable nucleoside form s of the nucleotides. Reversed phase C-18 SPE with addition of a nucleoside internal standard, 3'-azido-2',3'-dideoxyuridine (AzdU) allowed for the in direct measurement of the original 5'-triphosphate concentration by HPLC/MS /MS. Quantitation was performed from calibration curves generated from auth entic 5'-triphosphate standards spiked in PBMCs from healthy volunteers. An alytical range for the three 5'-triphosphates was equivalent to 50-45,000 p g. 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%, respec tively. Recovery of the extraction method was 79.2%, 83.1%, and 98.3% for 3 TC-TP, d4T-TP, and ZDV-TP, respectively. This method can be utilized to mea sure the intracellular 5'-triphosphate levels in HIV infected patients rece iving antiretroviral therapy containing the nucleoside reverse transcriptas e inhibitors 3TC, d4T, or ZDV. (J Am Soc Mass Spectrom 2000, 11, 1134-1143) (C) 2000 American Society for Mass Spectrometry.