High-performance liquid chromatographic analysis of 2'-fluoro-2',3'-dideoxyadenosine and 2'-fluoro-2',3'-dideoxyinosine in dog plasma and the urine

A high performance liquid chromatographic assay was developed and validated for a simultaneous determination of 2'-fluoro-2',3'-dideoxyadenosine (FddA) and its metabolite, 2'-fluoro-2',3'-dideoxyinosine (FddI) in dog plasma and urine. In vitro, FddA and FddI exhibit activity against human immunodeficiency virus (HIV). A solid phase extraction was applied to extract FddA, FddI, and the internal standard (IS; 3',5'-anhydrothymidine) from the biomatrices. The processed samples were chromatographed using a C8 column coupled with a mobile phase consisting of monobasic phosphate, dibasic phosphate, ethylene glycol monomethyl ether, and water. Detection was performed at 257 nm. The nominal retention times were 9, 14, and 26 min for FddI, IS, and FddA, respectively. The lower limits of quantitation were 0.1 and 2.0 mu g/mL in plasma and urine, respectively, for both analytes. The: accuracy of the assay deviated less than or equal to 10% from the nominal concentrations, and the precision was less than or equal to 44% coefficient of variation, In either matrix, both analytes were stable for at least three freeze-thaw cycles and in the injection media for at least 54 h. The extraction recoveries of the analytes were greater than 80%. The application of this assay was demonstrated in a preliminary pharmacokinetic study of FddA and FddI in dogs, Two male dogs per dose level received a 100, 250, or 500 mg/kg oral dose of FddA once daily for 14 clays. The early appearance of FddI in plasma (0.25 h; the first sampling time) and greater plasma levels of FddI than FddA (>50-fold of C-max), suggested that the conversion of FddA to FddI was rapid and extensive. Renal excretion appeared to be the major route of elimination of FddI.