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 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.