Determination of guanine and its nucleosides and nucleotides in human erythrocytes by high-performance liquid chromatography with postcolumn fluorescence derivatization using phenylglyoxal reagent.
A high-performance liquid chromatographic method with on-line postcolumn fluorescence derivatization is described for the simple and sensitive determination of guanine and its nucleosides and nucleotides in human erythrocytes. After deproteinization of the biospecimen, guanine and its nucleosides and nucleotides were separated on a reversed-phase column (TSKgel ODS-120T) by gradient elution with methanol in the aqueous mobile phase consisting of tetra-n-propylammonium phosphate (pH 6.0) and phosphate buffer (pH 6.0). The compounds were then automatically converted into fluorescent derivatives by reaction with phenylglyoxal. This derivatization was selective for guanine-containing compounds. The present method permitted the reliable quantification of GDP and GTP in human erythrocytes. The detection limits (at a signal-to-noise ratio of 3) for guanine and its nucleosides and nucleotides were 3.2-10.0 pmol in a 20-microliters injection volume. The concentrations of GDP and GTP in human erythrocytes were 17.2 +/- 6.2 and 40.2 +/- 5.8 nmol/ml, respectively.