Automated solid-phase extraction method for measuring urinary polycyclic aromatic hydrocarbon metabolites in human...

In order to perform comprehensive epidemiological studies where multiple metabolites of several PAHs are measured and compared in low-dose urine samples, fast and robust methods are needed to measure many analytes in the same sample. We have modified a previous method used for measuring polycyclic aromatic hydrocarbon (PAH) metabolites by automating the solid-phase extraction (SPE) and including an additional eight metabolites. We also added seven new carbon-13 labeled standards, which improves the use of isotope-dilution calibration. Our method included enzyme hydrolysis, automated SPE and derivatization with a silylating reagent followed by gas chromatography (GC), coupled with high-resolution mass spectrometry (HRMS). Using this method, we measured 23 metabolites, representing 9 parent PAHs, with detection limits in the low pg/mL range. All steps in the clean-up procedure were optimized individually, resulting in a method that gives good recoveries (69-93%), reproducibility (coefficient of variation for two quality control pools ranged between 4.6 and 17.1%, N>156), and the necessary specificity. We used the method to analyze nearly 3000 urine samples in the fifth National Health and Nutrition Examination Survey (NHANES 2001-2002).