Benzo[a]pyrene-specific online high-performance liquid chromatography fractionation of air particulate extracts – A tool for evaluating biological interactions

Benzo[a]pyrene (B[a]P) is a known human carcinogen and is commonly used as a surrogate for assessing the carcinogenic risk posed by complex mixtures of polycyclic aromatic hydrocarbons (PAHs) present in air particulate matter (PM). However, studies have shown that using B[a]P as a surrogate may underestimate the carcinogenic potential of PAH mixtures, as the risk assessment approach does not consider interaction effects. Thus, toxicological studies using B[a]P to assess its carcinogenic potential in environmentally derived complex mixtures, as opposed to single compound experiments, could improve risk assessment. The intention of the present study was to develop an online HPLC fractionation system for the selective removal of B[a]P from air PM extracts. Two serial pyrenylethyl (PYE) columns enabled selective separation of B[a]P from its isomers and other PAHs as well as a short fractionation cycle of 30 min. One run consisted of three collection steps: the first fraction contained PAHs eluting earlier than B[a]P, the second contained B[a]P and the last contained later-eluting PAHs. The selectivity and recovery of the system was investigated using extracts of Stockholm air PM samples. The overall recovery for all PAHs was approximately 80%, and the system proved to be selective, as it removed 94% of B[a]P and less than 3% of benzo[b]fluoranthene from the complex PAH mixture. Exposing human cells to blanks generated by the fractionation system did not induce cytotoxicity or DNA damage signalling. In conclusion, the online HPLC system was selective for B[a]P fractionation whilst minimising run-to-run variation and allowing repeated fractionations for larger samples due to its relatively short run time.