Fractionation of diesel particle extracts by sephadex LH-20 and thin-layer chromatography

A method was developed to concentrate fractions high in bacterial mutagenicity from diesel exhaust particle (DEP) extracts. The two-step fractionation of organic extracts of DEP employed Sephadex LH-20 followed by thin-layer chromatography (TLC). In both steps, nitro-substituted polycyclic aromatic hydrocarbons (nitro-PAHs) were found to co-elute with the fractions of highest specific bacterial mutagenic activity. Although recoveries of mass and mutagenicity from the LH-20 fractionation were high, very low (20-30%) recovery of mutagenicity was found after TLC. Recovery studies using 14C-labeled 1-nitropyrene showed that 78% of the compound was recovered unaltered through the combined fractionation. The most bioactive TLC subfraction was chromatographed using gas chromatography with thermionic specific detection. A sizeable peak was found to co-elute with a nitropyrene standard, but no peak was found co-eluting with nitrofluoranthene. Triple quadrapole mass spectrometry was used to measure the concentrations of nitropyrene in each of the fractions. Nitropyrene was enriched 160-fold in the most bioactive TLC subfraction over the starting material, for a final concentration of over 3200 micrograms g-1. Masses corresponding to several two- and three-ring methylated nitro-PAHs were found in the same fraction, including nitro-(methylbiphenyls/methylacenapthenes) and nitro-(methylanthracenes/methylphenanthrenes).