Highly sensitive routine method for urinary 3-hydroxybenzo[a]pyrene quantitation using liquid chromatography-fluorescence detection and automated off-line solid phase extraction

Many workers and also the general population are exposed to polycyclic aromatic hydrocarbons (PAHs), and benzo[a]pyrene (BaP) was recently classified as carcinogenic for humans (group 1) by the International Agency for Research on Cancer. Biomonitoring of PAHs exposure is usually performed by urinary 1-hydroxypyrene (1-OHP) analysis. 1-OHP is a metabolite of pyrene, a non-carcinogenic PAH. In this work, we developed a very simple but highly sensitive analytical method of quantifying one urinary metabolite of BaP, 3-hydroxybenzo[a]pyrene (3-OHBaP), to evaluate carcinogenic PAHs exposure. After hydrolysis of 10 mL urine for two hours and concentration by automated off-line solid phase extraction, the sample was injected in a column-switching high-performance liquid chromatography fluorescence detection system. The limit of quantification was 0.2 pmol L-1 (0.05 ng L-1) and the limit of detection was estimated at 0.07 pmol L-1 (0.02 ng L-1). Linearity was established for 3-OHBaP concentrations ranging from 0.4 to 74.5 pmol L-1 (0.1 to 20 ng L-1). Relative within-day standard deviation was less than 3% and relative between-day standard deviation was less than 4%. In non-occupationally exposed subjects, median concentrations for smokers compared with non-smokers were 3.5 times higher for 1-OHP (p < 0.001) and 2 times higher for 3-OHBaP (p < 0.05). The two urinary biomarkers were correlated in smokers (rho = 0.636; p < 0.05; n = 10) but not in non-smokers (rho = 0.09; p > 0.05; n = 21).