Urinary Polycyclic Aromatic Hydrocarbons: simultaneous determination of metabolites in human biomonitoring studies

Abstract

Introduction: exposure assessment of residents in industrial areas is a crucial issue in environmental epidemiology . We propose a novel analytical method for simultaneous quantification of urinary polycyclic aromatic hydrocarbons metabolites (OH-PHAs), to assess exposure to urban pollution, diesel exhaust and biomass burning and to verify the contribution of smoking to the selected biomarkers . Method: 1-hydroxypyrene, 1-and 2-hydroxynapthalene, 3-hydroxybenzo[a]pyrene, 6-hydroxynitropyrene were measured in the urine samples of 614 healthy residents in an industrial area of Central Italy, aged 35-69 years, non-occupationally exposed to PAHs. Samples were subjected to enzymatic hydrolysis, to SPE, and further analyzed on ESI-HPLC-MS/MS in the negative ion MRM mode, using isotope labelled internal standards. Smoking status was assessed by measuring urinary nicotine metabolite (cotinine) levels. We studied the association between each OH-PAH log-transformed value and cotinine levels (<100 µg/l, ≥100 µg/l) by using regression analysis (Geometric Mean Ratios (GMR) and p-value). Results: method accuracy was never less than 90%, variability never higher than 19%, and limits of detection were low enough to detect the five proposed biomarkers in the general population. 6-OHNP was present in the 65% of the subjects and 3-OHBaP in the 70% and the concentrations of these metabolites were not affected by smoking: we found no association between their log-transformed values and cotinine levels (GMR=1.33 (p=0.137) for 6-OHNP; GMR=1.05 (p=0.825) for 3-OHBaP). Conclusion: The novel HPLC-MS/MS method made it possible to simultaneously quantitate a metabolite of nitropyrene, a diesel exhaust pollution marker, and a metabolite of benzo[a]pyrene, a IARC class 1 carcinogenic substance, other than more known and usually determined metabolites; the fact that 6-OHNP and 3-OHBaP are not affected by smoking makes these biomarkers suitable for studying exposure to environmental pollution.