Reverse Phase HPLC Determination and Murine Pharmacokinetics of Arabinosyl-5-azacytosine

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Abstract
A sensitive and specific reverse phase HPLC assay has been developed to measure the new antitumor agent arabinosyl-5-aza-cytosine (ara-AC) in biological fluids at concentrations as low as 50 ng/ml (0.2 μM). This assay also detects arabinosyl-N-formyl-guanylurea (AGU-CHO), the initial hydrolytic metabolite of ara-AC. 2′-Deoxy-5-azacytidine, an analogue with similar chemical stability, is used as an internal standard. Chromatographically interfering plasma ribosides are removed by solid phase extraction on a phenyl boronic acid cartridge. Separation of ara-AC, AGU-CHO and internal standard is then accomplished isocratically (1% CH3CN in 10 mM pH 6.8 phosphate buffer) on fully carbon loaded and end-capped C8 and C18 columns connected in tandem. The compounds of interest are detected by UV absorption at 240 nm and total analysis time is 20 min. This assay has been used to determine bolus dose plasma kinetics in male BDF1 mice given 200 mg/kg ara-AC as a tail vein injection. Plasma elimination of the ara-AC is triphasic with a terminal phase half-life of 52 min and the elimination of the AGU-CHO metabolite parallels that of the parent drug. Analysis of ara-AC in human plasma indicates that this method is suitable for determining drug disposition and pharmacokinetics in human subjects.