Simultaneous and sensitive analytical methods of guanine-containing nucleic acid-related compounds by means of new selective fluoresence reaction

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Pre-column and post-column fluorescence derivatization methods for the high-performance liquid chromatographic (HPLC) determination of guanine-containing compounds have been studied on the basis of the fluorescence reaction with phenylglyoxal (PGO). The detection limits for the compounds tested were 140-720 fmol and 3-10 pmol per 100-ul injection volume in the pre- and post-column derivatization methods, respectively. The PGO reagent was also applied to the measurement of the nuceobase-pair of guanine-cytosine in double-stranded DNAs.Alkoxyphenylglyoxals as the analogues of PGO were synthesized and found to be more sensitive reagents for the guanine-containing compounds. A novel chemiluminogenic method was developed on the basis of the fluorescence reaction of the guanine-containing compounds with PGO in a phosphate buffer (pH 6.0) at 37 ﾟC for 20 min, followed by a chemiluminescence reaction with N, N-dimethylformamide in a weakly alkaline medium. The chemiluminescence method was approximately 20-fold more sensitive than the fluorimetric one.