Journal of Chromatography B: Biomedical Sciences and Applications
Determination of 2′-β-fluoro-2′,3′-dideoxyadenosine, an experimental anti-AIDS drug, in human plasma by high-performance liquid chromatography
Introduction
2′-β-Fluoro-2′,3′-dideoxyadenosine (F-ddA, lodenosine Fig. 1) is an experimental anti-AIDS drug that has been rationally designed to have improved chemical stability relative to its parent compound, dideoxyadenosine (ddA) or the currently approved anti-AIDS drug, dideoxyinosine (ddI, didanosine, Videx) [1]. This synthetic dideoxynucleoside has a fluorine atom substituted for the 2′-β-hydrogen of the dideoxyribose sugar in ddA. This structural modification increases the hydrolytic and metabolic stability of the analog relative to its parent compound. In fact, F-ddA is completely stable in acid at pH 1 for more than 24 h and is degraded by adenosine deaminase (ADA) at a much slower rate than ddA 2, 3. It is hoped that these properties will lead to enhanced oral bioavailability, an important consideration for AIDS drugs because chronic dosing is likely to be required. In preclinical testing, F-ddA has shown better oral bioavailability than ddI in dogs [4]. However, the addition of fluorine has not affected the compound's in vitro activity or potency against HIV. In fact, F-ddA protects ATH8 cells against HIV over the range 5–100 μM which is similar to didanosine, one of the five reverse transcriptase inhibitors (AZT, ddC, ddI, d4T and 3TC) approved by the Food and Drug Administration for the treatment of AIDS 5, 6. F-ddA is currently being evaluated in both adult and pediatric Phase I clinical trials at the National Cancer Institute (NCI) [7].
Chromatographic methods for analysis of antiviral agents, including anti-AIDS drugs, were reviewed a few years ago [8]. More recently, other methods for the analysis of nucleoside analogs, especially didanosine, have appeared 9, 10, 11, 12, 13, 14, 15, 16, 17. We reported an HPLC method for the analysis of F-ddA and its major metabolite, 2-β-fluoro-2′,3′-dideoxyinosine (F-ddI), in biological fluids used for analysis of samples from preclinical studies of rodents and primates 14, 18. That methodology employed solid-phase extraction (SPE) followed by gradient HPLC analysis on a phenyl reversed-phase column to achieve a limit of quantitation of 50 ng/ml (0.2 μM) for both compounds. However, that method was inadequate for analysis of clinical samples from AIDS patients since the sample work-up was not compatible with the necessary HIV viral decontamination procedure. In addition, the endogenous interferences in human plasma were different from those in animal plasma.
In this report we describe a modified method appropriate for the analysis of both F-ddA and F-ddI in plasma from patients who have AIDS or are HIV positive. Although based on the original analytical strategy, major modifications were necessary in the procedure to permit analysis of human samples. For safer sample handling, we chose a nonionic detergent, Triton X-100, to inactivate the HIV without adversely affecting the compounds of interest. The use of this detergent necessitated a modification of the SPE procedure. In addition, the chromatography was adjusted to optimize the separation in human plasma and a new internal standard was chosen and validated. Sample storage conditions were also evaluated since the large volume of samples from a clinical study means samples often have to be stored before analysis. This modified method is being used to analyze samples from the Phase I F-ddA clinical trials conducted by the NCI.
Section snippets
Chemicals and reagents
F-ddA (NSC 613792), F-ddI (NSC 616290), dideoxycytidine (ddC, zalcitabine), dideoxyadenosine (ddA) and dideoxyinosine (ddI, didanosine) were supplied by the Pharmaceutical Resources Branch, Developmental Therapeutics Program (DTP), Division of Cancer Treatment, Diagnosis and Centers (DCTDC), NCI (Bethesda, MD, USA). The adenosine deaminase inhibitor, 2′-deoxycoformycin (2′-dCF, NSC 218321) [19]was obtained from the Drug Synthesis and Chemistry Branch, DTP, DCTDC, NCI. The experimental anti-AIDS
Chromatography
Representative chromatograms of blank and spiked human plasma, using the initial mobile phase buffer at pH 6.8, are shown in Fig. 2. The same reversed-phase phenyl column was utilized for the analysis of F-ddA and F-ddI in human plasma as was previously used for analysis in the biological fluids of rat and monkey [14]. However, it was necessary to change the gradient program and internal standard due to the presence of different endogenous peaks in the human samples. A 30-min linear program
Conclusions
A robust, semi-automated method has been developed that is suitable for the analysis of F-ddA and F-ddI in clinical samples. It is based on an earlier method that was utilized for preclinical animal studies, but several modifications have been made. Because Triton X-100 is used to inactivate HIV, the SPE step has been changed to prevent HPLC column contamination and degradation. In addition, a different gradient program and new internal standard are employed to improve the chromatographic
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