Reverse Phase HPLC Determination OF 5,6-Dihydro-5-azacytidine in Biological Fluids

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Abstract
A sensitive and specific reverse phase HPLC method which allows measurement of the new antitumor agent 5,6-dihydro-5-azacytidine (DHAC) in biological fluids at concentrations as low as 50 ng/ml (2 × 10−7 M) has been developed. After addition of 5′-chloro-5′-deoxy-5,6-dihydro-5-azacytidine as an internal standard, sequential ultrafiltration, boronate gel affinity chromatography and cation exchange chromatography are employed to isolate DHAC from plasma or urine. DHAC is then reacted with N,N-dimethylformamide diethylacetal to form a dimethylaminomethylene derivative with enhanced UV detectability (λmax = 264 nm, log ε = 4.3) and better retention on a reverse phase column. Isocratic separation is then accomplished on a fully loaded and end-capped ODS column with 0.050 M formic acid in 20% acetonitrile/water. This assay has been used to determine the plasma pharmacokinetics of DHAC in rats given a single i.v. bolus dose of 50 mg/kg. Analysis of the drug in human plasma indicates that this method is suitable for determining DHAC disposition and pharmacokinetics in human subjects.