High-Performance Liquid Chromatographic Analysis of 2′-Fluoro-2′,3′-dideoxyadenosine and 2′-Fluoro-2′,3′-dideoxyinosine in Dog Plasma and Urine

Abstract

A high-performance liquid chromatographic assay was developed and validated for a simulataneous determination of 2'-fluoro-2' 3'-dideoxyadenosine (FddA) and its metabolite, 2'-fluoro-2',3'-dideoxyinosine (Fddl) in dog plasma and urine. In vitro, FddA and Fddl exhibit activity against human immunodeficiency virus (HIV). A solid phase extraction was applied to extract FddA, Fddl, 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 Fddl, IS, and FddA, respectively. The lower limits of quantitation were 0.1 and 2.0 micrograms/mL in plasma and urine, respectively, for both analytes. The accuracy of the assay deviated < or = 10% from the nominal concentrations, and the precision was < or = 14% 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 Fddl in dogs. Two male dogs per dose level received a 100, 250, or 500 mg/kg oral dose of FddA once daily for 14 days. The early appearance of Fddl in plasma (0.25 h; the first sampling time) and greater plasma levels of Fddl than FddA (> 50-fold of Cmax), suggested that the conversion of FddA to Fddl was rapid and extensive. Renal excretion appeared to be the major route of elimination of Fddl.

Single dose and multiple dose pharmacokinetics of 2′-fluoro-2′,3′-dideoxyadenosine and 2′-fluoro-2′,3′-dideoxyinosine, anti-HIV agents, in rats

“…The C max and AUC o _ oo values for FddI were generally 2-3-fold greater than the corresponding FddA values at each dose level showing that the major circulating moiety in rats was FddI following the administration of FddA. These results were similar to the findings previously noted in dogs (8). Similar to FddA, the Tmax values of FddI showed a tendency to increase as the administered dose increased following single (0.50-0.96 h) or multiple (0.42-1.33 h) doses of FddA.…”

“…When the doses of FddI increased in a ratio of 1:6. were generally comparable to those after a single dose administration. The increments of either C max or AUCo-oo values after escalating doses of FddI appeared to plateau as the administered dose increased, consistent to the observations noted following escalating oral doses of FddA to dogs (8).…”

“…These data suggested that more FddI was bioavailable from FddA rather than FddI administration and supported the extensive conversion of FddA to FddI previously observed in dogs (8). Examination of %UR values for FddI (FddA treatment: 34.22-59.13%; FddI treatment: 14.87-35.12%) further confirmed the increased bioavailability of FddI from the FddA administration to rats.…”

“…Both plasma and urine samples were analyzed for intact Fddl by a validated HPLC/UV method (8). Suitable quality control samples were prepared and analyzed along with the standards and study samples to monitor the accuracy and precision of the quantitation.…”

“…Previously, in a preliminary pharmacokinetic study in dogs, we have shown that the in vivo conversion of FddA to Fddl was rapid and extensive (8). The conversion was so rapid that the major circulating moiety was FddI after the administration of FddA to dogs (the exposure of dogs to FddI was at least 9O-fold compared to FddA).…”