High-performance liquid chromatography and immunoassay techniques for monitoring urinary metabolites of polycyclic aromatic hydrocarbons
The purpose of this research was to investigate the feasibility of using High Performance Liquid Chromatography (HPLC) and Enzyme Linked Immunosorbent Assays (ELISA) as sensitive techniques for monitoring polycyclic aromatic hydrocarbon (PAH) metabolites in human urine. The method was tested using synthesized PAH conjugates as positive markers. Results showed that a PAH conjugate, S-(9,10-dihydro-9-hydroxy-10-phenanthryl)N-acetyl cysteine (PHONAC), present in HPLC effluent could be detected by ELISA at picomole levels, well below the sensitivity of the HPLC UV detector. Analyses of urine from mice dosed with phenanthrene demonstrated that a substance detected by HPLC which was not detected in ELISA tests was the principal phenanthrene metabolite. This substance was not hydrolyzed by Beta-glucuronidase. PHONAC was detected by ELISA in mouse urine extracts subjected to HPLC. Human urine samples were obtained from smoking and nonsmoking volunteers. Human urine was assayed directly in an inhibition (competition) ELISA system using monoclonal antibodies specific for PAH oxide and thiol metabolites. Median antibody inhibition was not significantly different in the two groups. Elimination of data from subjects smoking less than one pack of cigarettes per day did not alter the results. A few urine samples from smokers exhibited higher inhibitory activity than any of the control samples. HPLC conditions for separation of mixed isomers of synthetic PAH K-region oxide NAC conjugates were explored. Phenanthrene, pyrene (PY), and benzo(c)phenanthrene (BCP) NAC isomers were separated on all columns using isocratic elution with combinations of 10mM tris pH 7 and MeOH. Benzanthracene (BA) NAC isomers were separated on C-8 and C-18 columns, but not on a Dupont phenyl column using similar isocratic conditions. Benzo(a)pyrene (BP) NAC isomers were only separated using the phenyl column. A mixture of all of the NACs was separable except that BA and BCP NACs eluted together on all columns under all conditions.