High-performance liquid chromatographic separation of ring-oxidized metabolites of nitro-polycyclic aromatic hydrocarbons.

Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are widespread genotoxic environmental pollutants, which require metabolic activation to exert their biological activities. Metabolism of nitro-PAHs generates ring-oxidized metabolites including epoxides, phenols, dihydrodiols and tetrahydrotetrols. Separation of the oxidized metabolites and related compounds of a series of isomeric nitro compounds derived from anthracene, benz[a]anthracene, benzo[a]pyrene and benzo[e]pyrene was studied by high-performance liquid chromatography (HPLC) of different types of columns (monomeric and polymeric; reversed-phase and normal-phase). In the reversed-phase HPLC system, the general elution order of these compounds is: parent nitro-PAHs greater than phenolic derivatives greater than epoxides greater than dihydrodiols greater than tetrahydrotetrols. Among the geometric isomers, trans-dihydrodiols with both hydroxyl groups at the quasiaxial positions were eluted earlier than those with the hydroxyl groups at the quasiequatorial positions. Orientation of the nitro substituent has also been found to be an important structural feature for determining the relative retention order. Among the geometric isomers of nitro-PAHs and trans-dihydrodiols, the isomers with their nitro groups perpendicular or nearly perpendicular to the aromatic rings were eluted faster than the analogues with their nitro groups parallel or nearly parallel to the aromatic rings. Normal-phase HPLC gave opposite retention order, but with different separability among some of the compounds. Therefore, combination of both reversed- and normal-phase HPLC provides efficient separation of the ring-oxidized derivatives of nitro-PAHs. Results are also presented to compare the separation efficiency among different types of columns used. The results suggest that the polarity of solutes is the principal factor for determining their HPLC retention time.