A Coupled HPLC/Radioimmunoassay for Analysis of Zidovudine Metabolites in Mononuclear Cells

Abstract

Reverse-phase HPLC, with the ion-pairing agent tetrabutyl ammonium phosphate, was used to separate zidovudine (ZDV) and its 5′-phosphorylated metabolites in extracts from peripheral blood mononuclear cells incubated with 2 μM ZDV. Because intracellular concentrations were too small to be visualized using UV detection, 1 ml fractions were collected and assayed for ZDV and metabolites using a commercial radioimmunoassay (RIA). Resolution of components was satisfactory, with a total chromatography time of 32 minutes. Interassay variability of peak areas was less than 14%. Comparison of UV detected chromatograms to RIA detected chromatograms from a standard mixture of ZDV and metabolites showed no significant difference between corresponding relative peak areas. The ion-pairing agent elevated baseline concentrations as measured by RIA. Quantitation was therefore performed by concurrent measurement of total phosphorylated ZDV using an established procedure, followed by comparison of relative peak areas. Results indicate that ZDV 5′-triphosphate, the active metabolite, is a major component of total phosphorylated ZDV, with peak heights significantly above baseline in extracts from less than 10⁷ mononuclear cells. Therefore, it should be possible to reliably quantitate this metabolite in cells from HIV-infected patients using only 10 to 20 ml of blood.