Reversed-phase high-performance liquid chromatographic determination of tricyclic nucleoside and tricyclic nucleoside 5'-phosphate in biological specimens.

An isocratic, sensitive, high-performance liquid chromatographic assay was developed for the determination of the tricyclic nucleoside 1,4,5,6,8-pentaazaacenaphthylene -3-amino-1,5-dihydro-5-methyl-1-beta-D-ribofuranosyl 5'-monophosphate (TCN-P; NSC 280594) and its dephosphorylated metabolite TCN (NSC 154020). Separation was obtained using a C18 Sep-Pak precolumn, a reversed-phase column, and a mobile phase of phosphate buffer--methanol (87.5:12.5, v/v) containing 0.0025 M tetrabutylammonium hydroxide. The absorbance of both TCN and TCN-P was monitored at 280 nm with a sensitivity limit of 10 ng/ml. The recovery was 54 +/- 6% and 51 +/- 8% (mean +/- S.D.) from plasma for TCN and TCN-P, respectively. Rapid enzymatic dephosphorylation of TCN-P in plasma and tissue samples was prevented by adding a large excess of adenosine 5'-monophosphate. The assay was used to determine plasma and tissue concentrations of TCN-P and TCN after administration of TCN-P to cancer patients in a Phase I clinical study.