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 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.
Moore
J D
JD
Division of Clinical Pharmacology, Center for AIDS Research, University of Alabama at Birmingham, 35294, USA.
Valette
G
G
Darque
A
A
Zhou
X J
XJ
Sommadossi
J P
JP
D019380:Anti-HIV Agents / Q000097:blood*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives / Q000097:blood*; D054306:Dideoxynucleotides; D004359:Drug Therapy, Combination; D015658:HIV Infections / Q000097:blood*; D006801:Humans; D019259:Lamivudine / Q000031:analogs & derivatives / Q000097:blood*; D013058:Mass Spectrometry; D009000:Monocytes / Q000737:chemistry / Q000378:metabolism*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D018119:Stavudine / Q000097:blood / Q000493:pharmacokinetics; D013942:Thymine Nucleotides / Q000097:blood*; D015215:Zidovudine / Q000031:analogs & derivatives / Q000097:blood*
D019380:Anti-HIV Agents; D054306:Dideoxynucleotides; D013942:Thymine Nucleotides; C417268:lamivudine triphosphate; D019259:Lamivudine; D015215:Zidovudine; D003570:Cytidine Triphosphate; C052873:zidovudine triphosphate; D018119:Stavudine