Journal of Chromatography B, 835 (2006) 47–54 Automated solid-phase extraction method for measuring urinary polycyclic aromatic hydrocarbon metabolites in human biomonitoring using isotope-dilution gas chromatography high-resolution mass spectrometry Lovisa C. Romanoff a,∗ , Zheng Li a , Kisha J. Young a , Nelson C. Blakely III a , Donald G. Patterson Jr. a , Courtney D. Sandau b a Centers for Disease Control and Prevention, National Center for Environmental Health, 4770 Buford Highway NE, Mailstop F17, Atlanta, GA 30341-3724, USA b TRIUM Environmental Solutions Inc., Calgary, Alta., Canada T3G 3T2 Received 3 September 2005; accepted 5 March 2006 Available online 24 March 2006 Abstract 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). © 2006 Elsevier B.V. All rights reserved. Keywords: PAH; Polycyclic aromatic hydrocarbon; Biomonitoring; Urinary metabolite; Hydroxylated PAH; Method development 1. Introduction Human exposure to polycyclic aromatic hydrocarbons (PAHs) has long been a concern because of the known health hazards associated with several of these compounds. A num- ber of PAHs have reported carcinogenic or genotoxic potential [1]. PAHs are formed during incomplete combustion of organic material such as coal, oil, gas, wood, or tobacco. Exposure is primarily through inhaling polluted air or tobacco smoke, and through ingesting contaminated and processed food and water [1]. Some studies also indicate that dermal exposure may be a ∗ Corresponding author. Tel.: +1 770 488 0192; fax: +1 770 488 4602. E-mail address: lromanoff@cdc.gov (L.C. Romanoff). major exposure pathway of some PAHs in occupational expo- sure settings [2,3]. Following absorption, PAHs are rapidly biotransformed (predominantly by cytochrome P450 mono-oxygenases) to hydroxylated metabolites, which are then further transformed to glucuronide or sulfate conjugates to increase their polarity and aid in urinary excretion [4,5]. Conjugated hydroxylated PAHs (HO-PAHs) are the most abundant and commonly measured metabolites in urine even though other metabolites such as dihydrodiols and DNA adducts also exist [5–8]. Over the years many reports have been published on separa- tion techniques used for measurement of PAHs. Jongeleen et al. pioneered measuring 1-hydroxypyrene by HPLC coupled with fluorescence spectrometry [9]. In their method, urine samples were pretreated with -glucuronidase/arylsulfatase to cleave off 1570-0232/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2006.03.004