Automated solid-phase extraction method for measuring urinary polycyclic aromatic hydrocarbon metabolites in human biomonitoring using isotope-dilution gas chromatography high-resolution mass spectrometry

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).