Reverse Phase HPLC Determination of 5,6-Dihydro-5-Azacytidine in Biological Fluids

Philipp N. Huguenin, Hiremagalur N. Jayaram, James A. Kelley

Research output: Contribution to journalArticle

8 Scopus citations


A sensitive and specific reverse phase HPLC method which allows measurement of the new antitumor agent 5,6-dihydro-5-azacytidine (DHAC) in oiological fluids at concentrations as low as 50 ng/ml (2 x 10–7 m) has been developed. After addition of 5’-chloro-5’-deoxy-5,6-dihydro-5-azacytidine as an internal stan-dard, 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 dimethyl ami nomethylene derivative with enhanced UV detectability (Xmax= 264 nm, log e = 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.

Original languageEnglish (US)
Pages (from-to)1433-1453
Number of pages21
JournalJournal of Liquid Chromatography
Issue number7
StatePublished - Jun 1 1984

ASJC Scopus subject areas

  • Molecular Medicine

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