Systems for the separation of metabolites of the carcinogen, N-2-fluorenylacetamide, by high-performance liquid chromatography

Abstract

Separation procedures for N-2-fluorenylacetamide (2-FAA) and its metabolites have been hampered by the unusually strong adsorption of N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA), the proximate carcinogenic metabolite, to chromatographic packings. Results of investigatious with C 2, C 3, C 8, and C 18 column packings are presented showing that N-OH-2-FAA could be eluted and the separation of 2-FAA and eight other metabolites achieved with acidic mobile phases on C 2 and C 8 columns after a period of column conditioning. Elution of C 8 columns with mobile phases containing acetohydroxamic acid was less destructive to the columns while permitting the separation of 2-FAA and its metabolites with recovery yields of N-OH-2-FAA of 91.5%. Quantitation of these compounds was by integration of peaks detected by the spectroscopic method. Areas were linear for peaks representing from 5 to at least 50 ng of N-OH-2-FAA and of the eight other metabolites.