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,N4-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.
Eisenberg
E J
EJ
Gilead Sciences, Foster City, CA 94404, USA.
Cundy
K C
KC
D000098:Acetophenones / Q000737:chemistry*; D000818:Animals; D000998:Antiviral Agents / Q000032:analysis* / Q000737:chemistry; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid; D000077404:Cidofovir; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D003597:Cytosine Nucleotides / Q000032:analysis* / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008252:Macaca fascicularis; D063065:Organophosphonates; D009943:Organophosphorus Compounds / Q000032:analysis* / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013696:Temperature
D000098:Acetophenones; D000998:Antiviral Agents; D002250:Carbon Radioisotopes; D003597:Cytosine Nucleotides; D063065:Organophosphonates; D009943:Organophosphorus Compounds; D012997:Solvents; C013190:phenacyl bromide; D003596:Cytosine; D000077404:Cidofovir