N- (6-Hydroxy-Z-fluorenyl) acetamide, a Urinary Metabolite after Intraperitoneal Injection of N-Z-Fluorenylacetamide into Rats

In the solvent system used in this laboratory (1) for the resolution of the urinary metabolites of the carcinogen N-2-fluorenylacetamide,’ the mobility of synthetic N-(6-hydroxy-2-fluorenyl)acetamide (2) on paper chromatograms was similar (RF 3.4 to 14.5)2 to that of N-(7-hydroxy-2-fluorenyl)acetamide (RF 3.4 to 15.4), a known and quantitatively important metabolite of 2FAA (cf. (3)). Preliminary experiments by the inverse carrier isotope dilution technique (4,5) indicated that 6-hydroxy-2-FAA might also be a metabolite of 2-FAA, since a sample retained radioactivity even after repeated crystallizations and conversion to derivatives. However, in view of the close similarity of the various hydroxylated metabolites of 2-FAA, it was possible that this finding constituted an artifact owing to cocrystallization. An unambiguous demonstration that 6-hydroxy-a-FAA was actually produced by rats injected with 2-FAA was therefore desirable. This was achieved by modifying the usual technique (1) for separating the metabolites of 2-FAA by the use of a long column (approximately 2 meters) of silicic acid. Systematic examination by ultraviolet spectroscopy of the fractions obtained in this fashion revealed one with the characteristic features of the spectrum of 6-hydroxy-2-FAA. Carrier crystallization subsequently proved definitely that this sample contained 6-hydrosy-a-FAA, thereby establishing that this compound was a metabolite of 2-FAA in rats.