INTRACELLULAR NUCLEOTIDES OF (-)-2',3'-DEOXY-3'-THIACYTIDINE IN PERIPHERAL-BLOOD MONONUCLEAR-CELLS OF A PATIENT INFECTED WITH HUMAN-IMMUNODEFICIENCY-VIRUS
C. Solas et al., INTRACELLULAR NUCLEOTIDES OF (-)-2',3'-DEOXY-3'-THIACYTIDINE IN PERIPHERAL-BLOOD MONONUCLEAR-CELLS OF A PATIENT INFECTED WITH HUMAN-IMMUNODEFICIENCY-VIRUS, Antimicrobial agents and chemotherapy, 42(11), 1998, pp. 2989-2995
An analytical methodology was developed to quantitate the intracellula
r nucleotides including mono-, di-, and triphosphates and the diphosph
ocholine 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 a
nion-exchange cartridge, with subsequent enzyme digestion of the resul
ting phosphates to the parent drug that is ultimately quantitated by h
igh-performance liquid chromatography with UV detection (HPLC-UV). Val
idation was performed with PBMCs from healthy donors exposed to [H-3]3
TC, leading to the formation of intracellular nucleotides that were qu
antitated 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 correlati
on was established between the results obtained by HPLC-RA and those o
btained by HPLC-UV, with a slope of the regression lines close to unit
y and intercepts near nullity as well as a correlation coefficient clo
se to unity for all 3TC phosphates. The assay was characterized by a l
imit of quantitation below 1 ng (amount on column) with a precision (p
ercentage 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 quanti
ty of 3TC nucleotides in PBMCs of a patient infected with human immuno
deficiency virus after oral administration of 3TC and stavudine.