A High Pressure Liquid Chromatography Procedure for the Separation of Metabolites of 2-Acetylaminofluorene from Cells in Culture

A method is presented for the determination of pmole levels of the carcinogenic metabolite N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF) formed from N-2-acetylaminofluorene (AAF) by cells in culture. The extract from the cells and cell incubation medium was given a preliminary cleanup using thin layer chromatography. N-hydroxy-AAF was then isolated using high-pressure liquid chromatography (HPLC) with a Waters Bondapak C 18 /Corasil column and an acetonitrile-water elution system. Ring hydroxylation products were eluted with 23% acetonitrile, and AAF and the deacetylation product 2-aminofluorene (AF) were eluted with 33% acetonitrile. Resolution of N-hydroxy-AAF was accomplished using a linear gradient of from 33% to 99% acetonitrile. When cells in culture were incubated with 22 nmoles/ml of AAF for 6 h, 690 pmoles of N-hydroxy-AAF per 10 6 cells were formed by hamster hepatocytes compared with 13 and 8 pmoles/10 6 cells for human embryo and hamster embryo cells respectively. The rate of formation of N-hydroxy-AAF by hamster liver microsomes was 140 nmoles per h of incubation per mg of microsomal protein.