High-performance liquid chromatographic method with fluorescence detection for the determination of 3-hydroxybenzo[a]pyrene and 3-hydroxybenz[a]anthracene in the urine of polycyclic aromatic hydrocarbon-exposed workers

Abstract

The described high-performance liquid chromatographic method with fluorescence detection (HPLC-FD) permits the simultaneous determination of 3-hydroxybenzo[a]pyrene and 3-hydroxybenz[a]anthracene as the most important metabolites of the carcinogenic polycyclic aromatic hydrocarbons (PAHs) benzo[a]pyrene and benz[a]anthracene in human urine. After enzymatic hydrolysis, to release the conjugated metabolites, the analytes are separated from the matrix by means of a liquid-solid extraction step which is followed by a coupled column HPLC procedure using an enriching precolumn consisting of silica modified with copper phthalocyanine. This special precolumn selectively adsorbs PAHs with at least three condensed rings and thus separates them from the urine matrix. The quantitative analysis was carried out using a switchable fluorescence detector. The detection limits were 6 ng/l urine (3-hydroxybenzo[a]pyrene) and 8 ng/l urine (3-hydroxybenz[a]anthracene). The relative standard deviations of the within-series imprecision ranged between 4.0% and 9.0%. The between-day imprecision was 7.7% (3-hydroxybenz[a]anthracene) and 12.9% (3-hydroxybenzo[a]pyrene). The recovery rates ranged between 102% and 124%. Using this analytical method we determined PAH metabolites in post shift urine samples from 19 workers engaged in the production of fire-proof materials. The urinary concentrations ranged from 3 to 198 ng 3-hydroxybenzo[a]pyrene per g creatinine and from 15 to 1871 ng 3-hydroxybenz[a]anthracene per g creatinine.