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 a pplicability of the method to various biogenic and antiviral compounds is demonstrated. Quantitative determination of cidofovir, a potent an tiviral drug currently undergoing evaluation in the clinic for treatme nt of cytomegalovirus retinitis is also reported. The limit of detecti on 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 f ive replicate samples at 20, 160 and 320 ng/ml, respectively. The with in-day precision was 15.9, 5.9 and 2.1%, respectively. The method desc ribed has broad applicability and may offer significant advantages ove r existing HPLC methods in antiviral drug development as well as in nu cleic acid research.