Fluorometric determination of 2 '-beta-fluoro-2 ',3 '-dideoxyadenosine 5 '-triphosphate, the active metabolite of a new anti-human immunodeficiency virus drug, in human lymphocytes

A sensitive precolumn derivatization method has been developed to measure t he 5'-triphosphate of 2'-beta -fluoro-2',3'-dideoxyadenosine (F-ddA, lodeno sine), a new anti-HIV drug, in human lymphocytes by HPLC using fluorescence detection. Reaction of chloroacetaldehyde with F-ddA triphosphate in extra cts from human lymphocytes produces a highly fluorescent etheno adduct. Thi s derivative is then separated and quantitated by reverse-phase paired-ion chromatography. Degradation of natural nucleic acid ribosides, such as ATP, using periodate oxidation simplifies the chromatogram and minimizes interf erence with detection of the target analyte. This method, modeled using cul tured MOLT-4 T-lymphocytes, achieves a linear detector response for peak ar ea measurements over the range 2.5 to 22.5 pmol (50-450 nM using 50 yl samp le). Analyte recovery is greater than 90%, and the method achieves a limit of detection and limit of quantitation of 1.4 and 2.5 pmol per HPLC injecti on (50 mul sample containing cellular extract from 2.5 x 10(6) cells), resp ectively. Application of this method to measure F-ddATP in peripheral blood mononuclear cells from HIV-infected patients treated with F-ddA at 3.2 mg/ kg twice daily for 22 days shows F-ddATP levels which range from 1.5 to 3.5 pmol/10(6) cells. (C) 2001 Academic Press.