High-performance liquid chromatographic determination of cytosine-containing compounds by precolumn fluorescence derivatization with phenacyl bromide: Application to antiviral nucleosides and nucleotides

A novel precolumn derivatization method for the HPLC determination of cytosine-containing compounds by HPLC is described. Highly fluorescent 2-phenyl-3,N-4-ethenocytosine derivatives are produced by a reaction of non-fluorescent cytosine-containing compounds with phenacyl bromide in weakly acidic acetonitrile solution at elevated temperature. The applicability of the method to various biogenic and antiviral compounds is demonstrated. Quantitative determination of cidofovir, a potent antiviral drug currently undergoing evaluation in the clinic for treatment of cytomegalovirus retinitis is also reported. The limit of detection for cidofovir in cynomolgus monkey plasma was 5 ng/ml (ca. 100 fmol on column) with the between-day precision of 16.6, 6.4 and 2.4% for five replicate samples at 20, 160 and 320 ng/ml, respectively. The within-day precision was 15.9, 5.9 and 2.1%, respectively. The method described has broad applicability and may offer significant advantages over existing HPLC methods in antiviral drug development as well as in nucleic acid research.