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 technique to measure metabolites in tissue at subfemtomolar concentrations was needed. Postexposure blood concentrations of [H-3]benzo[a]pyrene and [H-3]pyrene metabolites were determined after intratracheal instillation of similar to 10 ng in, three beagle dogs. (H2O)-H-3 was distilled from samples in vacuo, and the residual radioactivity was fractionated into water-soluble, bound (pellet-associated), and organic-extractable fractions. Radioactivity in the pellet and aqueous fraction was determined by complete combustion and subsequent liquid scintillation counting. The organic soluble fractions, which contained the metabolites of interest, also contained lipids that were inseparable from the highly Lipophilic PAHs. The lipids were saponified using CaO, extracted with ethyl acetate, resuspended in. methanol, and fractionated using RP-HPLC. Metabolites were identified by coelution with authentic standards. Saponification was a hey step in, the procedure because its omission led to greatly inferior HPLC separation. The extraction efficiency 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 column separated into fraction similar to 20 dpm above background. The method enables quantitation of PAHs and their metabolites in blood at concentrations orders of magnitudes lower than for those previously reported.