HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF GUANINE AND ITS NUCLEOSIDES AND NUCLEOTIDES BY PRECOLUMN FLUORESCENCE DERIVATIZATION WITH PHENYLGLYOXAL REAGENT

A pre-column fluorescence derivatization method for the high-performance liquid chromatographic determination of guanine and its nucleosides and nucleotides is described. The compounds are converted into fluorescent derivatives by reaction with phenylglyoxal in a phosphate buffer (pH 6.0) at 37-degrees-C for 15 min. The derivatives are separated on a reversed-phase column (TSKgel ODS-120T) by gradient elution of acetonitrile in mobile phase containing 5 mM phosphate buffer (pH 6.5), and then detected fluorimetrically. The derivatization results in the guanine-containing compounds eluting as single fluorescent peaks. The method is highly selective and sensitive; the limits of detection for the compounds tested are 140-720 fmol per 100-mul injection volume.