HPLC DETERMINATION OF TRITIATED POLYCYCLIC AROMATIC HYDROCARBON METABOLITES IN THE SUBFEMTOMOLE RANGE

In experiments to determine the toxicokinetics of polycyclic aromatic hydrocarbons (PAHs) in lungs at environmentally realistic levels, a te chnique to measure metabolites in tissue at subfemtomolar concentratio ns was needed. Postexposure blood concentrations of [H-3]benzo[a]pyren e and [H-3]pyrene metabolites were determined after intratracheal inst illation of similar to 10 ng in, three beagle dogs. (H2O)-H-3 was dist illed from samples in vacuo, and the residual radioactivity was fracti onated into water-soluble, bound (pellet-associated), and organic-extr actable fractions. Radioactivity in the pellet and aqueous fraction wa s determined by complete combustion and subsequent liquid scintillatio n counting. The organic soluble fractions, which contained the metabol ites of interest, also contained lipids that were inseparable from the highly Lipophilic PAHs. The lipids were saponified using CaO, extract ed with ethyl acetate, resuspended in. methanol, and fractionated usin g RP-HPLC. Metabolites were identified by coelution with authentic sta ndards. Saponification was a hey step in, the procedure because its om ission led to greatly inferior HPLC separation. The extraction efficie ncy for [H-3]BaP in organic extracts was 75%+/-similar to 5% (SD, N = 3) of total sample radioactivity. The smallest amounts of metabolites separated by HPLC in tissue samples were 200 amol/total sample on colu mn separated into fraction similar to 20 dpm above background. The met hod enables quantitation of PAHs and their metabolites in blood at con centrations orders of magnitudes lower than for those previously repor ted.