Intracellular nucleotides of (-)-2',3'-deoxy-3'-thiacytidine in peripheral blood mononuclear cells of a patient infected with human immunodeficiency virus.
An analytical methodology was developed to quantitate the intracellular nucleotides including mono-, di-, and triphosphates and the diphosphocholine derivative of (-)-2', 3'-deoxy-3'-thiacytidine (3TC) in human peripheral blood mononuclear cells (PBMCs). The procedure includes the resolution of 3TC nucleotides by solid-phase extraction (SPE) on an anion-exchange cartridge, with subsequent enzyme digestion of the resulting phosphates to the parent drug that is ultimately quantitated by high-performance liquid chromatography with UV detection (HPLC-UV). Validation was performed with PBMCs from healthy donors exposed to [3H]3TC, leading to the formation of intracellular nucleotides that were quantitated by anion-exchange HPLC with radioactive detection (HPLC-RA). These nucleotide levels served as reference values and were used for cross-validation with data obtained by HPLC-UV. An excellent correlation was established between the results obtained by HPLC-RA and those obtained by HPLC-UV, with a slope of the regression lines close to unity and intercepts near nullity as well as a correlation coefficient close to unity for all 3TC phosphates. The assay was characterized by a limit of quantitation below 1 ng (amount on column) with a precision (percentage of coefficient of variation of repeated measurement) ranging from 0.8 to 18.1% and an accuracy (deviation of the amount determined by HPLC-UV from the nominal reference value) varying from -14.8 to 19.4%. This methodology was successfully applied to determine the quantity of 3TC nucleotides in PBMCs of a patient infected with human immunodeficiency virus after oral administration of 3TC and stavudine.
Solas
C
C
Department of Clinical Pharmacology, The Liver Center, Center for AIDS Research, University of Alabama at Birmingham, Birmingham, Alabama 35294-0019, USA.
Li
Y F
YF
Xie
M Y
MY
Sommadossi
J P
JP
Zhou
X J
XJ
D019380:Anti-HIV Agents / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D015658:HIV Infections / Q000188:drug therapy* / Q000378:metabolism; D006801:Humans; D019259:Lamivudine / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*
D019380:Anti-HIV Agents; D009711:Nucleotides; D019259:Lamivudine