Abstract For measurement of biomarkers from poly- cyclic aromatic hydrocarbon (PAH) exposure, an analyti- cal method is described quantifying hydroxylated PAH (OH-PAH) in urine samples. This method determined monohydroxy metabolites of naphthalene, fluorene, phen- anthrene, fluoranthene, pyrene, chrysene, benzo[c]phen- anthrene, and benz[a]anthracene. The sample preparation consisted of enzymatic hydrolysis, solid-phase extraction and derivatization with a silylating reagent. Five carbon-13 labeled standards were used for isotope dilution. Ana- lytes were separated by gas chromatography (GC) and quantified with high-resolution mass spectrometry (HRMS). This method produced good recoveries (41–70%), linear- ity, and specificity. Data were corrected for blank levels from the naphthalene, fluorene, and phenanthrene metab- olites. Method detection limits ranged from 2 ng L –1 for 1-hydroxypyrene to 43.5 ng L –1 for 1-hydroxynaphtha- lene. Using quality control charts from two urine pools, the method can be readily applied to biomonitoring PAH exposure. Keywords Polyaromatic hydrocarbons · Isotope dilution · High resolution mass spectrometry · Human urine · Solid-phase extraction Introduction Many classes of chemicals are thought to be carcinogenic or genotoxic. Among these, PAH are one of the most sig- nificant priority pollutants based upon the amounts pro- duced by combustion of biogenic and anthropogenic ma- terials [1]. Human exposure to PAH is generally caused by airborne particulates and by dietary consumption of food prepared by grilling, smoking, or storage in charred wooden casks. PAH exposure is also caused by cigarette and cigar smoking [2]. Biomarkers of human exposure to PAH are necessary for the accurate determination of ex- posure and body burden from the above-mentioned sources. By measuring metabolites of PAH (PAHm) in urine, short term (1 to 3 days) exposure may be evaluated. A widely adopted method was reported by Jongenee- len and coworkers for the quantification of 1-hydroxy- pyrene from urine [3]. Their protocol includes enzymatic hydrolysis of the conjugated metabolites, solid-phase ex- traction (SPE) with C-18 media, separation with high-per- formance liquid chromatography (HPLC), and detection by fluorescence emission. Recent exposure assessments using this method include workers at a gasworks plant, firefighters at a training facility, and children from an in- dustrial region [4, 5, 6]. Another HPLC method that de- termines hydroxypyrene, hydroxyphenanthrene, hydroxy- benz[a]anthracene, and hydroxybenzo[a]pyrene has been developed by Angerer and coworkers [7, 8]. They applied their method to monitor occupational exposure of workers in a fireproof stone-producing plant. Exposures to PAH were found with the conclusion that the workers’ expo- sure was too high when compared with other industrial plants and the exposure limits for benzo[a]pyrene. Several groups have worked on the analysis of PAHm using GC–MS techniques and incorporate varying sample preparation methods. With solid-phase microextraction, Gmeiner and coworkers developed a screening method for PAHm with GC–MS to detect 10 monohydroxy PAHm in urine samples [9]. Korfmacher and coworkers have used GC–MS with chemical ionization to analyze metab- olites of nitro-PAH from microsomes [10]. Incorporating liquid–liquid extraction and fractionation with a silica gel column, Wilson and coworkers have used GC–MS to monitor PAH exposures in children of low-income fami- lies [11]. A productive method has been applied by Grim- mer and coworkers who have used liquid extraction and GC–MS to determine metabolites of phenanthrene, fluo- ranthene, pyrene, chrysene, and benzo[a]pyrene in urine [12, 13, 14, 15]. They have demonstrated that the relative Christopher J. Smith · Wenlin Huang · Charisse J. Walcott · Wayman Turner · James Grainger · Donald G. Patterson Jr Quantification of monohydroxy-PAH metabolites in urine by solid-phase extraction with isotope dilution-GC–MS Anal Bioanal Chem (2002) 372 : 216–220 DOI 10.1007/s00216-001-1123-8 Received: 8 June 2001 / Revised: 28 July 2001 / Accepted: 25 August 2001 / Published online: 12 December 2001 ORIGINAL PAPER C.J. Smith (✉) · W. Huang · C.J. Walcott · W. Turner · J. Grainger · D.G. Patterson Jr Centers for Disease Control and Prevention, 4770 Buford Highway, MS F-17, Atlanta, GA 30341, USA e-mail: cps6@cdc.gov © Springer-Verlag 2001