Abstract
An HPLC method for the determination of 2-hydroxyfluorene (2-OHF), various hydroxyphenanthrene metabolites (1-, 2-, 3-, 4- and 9-hydroxyphenanthrene, OHPhs), 1-hydroxypyrene (1-OHPy) and 3-hydroxybenzo[a]pyrene (3-OHB[a]Py) in human urine, has been developed using fast scanning fluorimetric detection and gradient elution mode. All reagents were of analytical grade. Standard solutions were prepared separately, by exact weighing or dilution with ultrapure acetonitrile, and were stored at 4 ºC in darkness. The standard addition method was used for the analysis of urine samples. In the optimized conditions, 2- and 3-hydroxyphenanthrene, and 1- and 9-hydroxyphenanthrene metabolites eluted at the same retention time; however, all other hydroxy-polycyclic aromatic hydrocarbons were well resolved. Multi-emission detection allows us to monitor each metabolite at its most sensitivity emission wavelength. Detection limits ranged between 0.9 and 4.26 ng ml-1. Fortified urine samples of nonexposure and nonsmoker volunteers, previous precipitation step with acetonitrile, were used to test the proposed method. The obtained results confirm the goodness of the method.
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