Author's personal copy Journal of Chromatography B, 877 (2009) 2142–2152 Contents lists available at ScienceDirect Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb Analysis of pyrene metabolites in marine snails by liquid chromatography using fluorescence and mass spectrometry detection Daniel G. Beach a,b , Michael A. Quilliam c , Jocelyne Hellou a,b,d,∗ a Department of Fisheries and Oceans, Bedford Institute of Oceanography, Dartmouth, NS, B2Y 4A2, Canada b Dalhousie University, Department of Chemistry, Halifax, NS, B3H 4R2, Canada c National Research Council, Institute for Marine Biosciences, 1411 Oxford St., Halifax, NS, B3H 3Z1, Canada d Dalhousie University, Department of Oceanography, Halifax, NS, B3H 4J1, Canada article info Article history: Received 7 April 2009 Accepted 5 June 2009 Available online 13 June 2009 Keywords: Polycyclic aromatic hydrocarbons Pyrene Biotransformation Mass spectrometry Tissue Metabolites abstract As part of a study of the metabolism of aromatic compounds in marine gastropods, a sensitive and selective method was developed to detect, identify and quantify pyrene (PY) and four of its metabolites in tissues: 1-hydroxypyrene (PYOH), pyrene sulfate (PYOS), pyrene glucuronide (PYOG) and pyrenediol disulfate (PYDS). Liquid chromatography (LC) with fluorescence detection was first used to detect the PY deriva- tives in the visceral mass of whelks exposed to PYOH. The identification of metabolites was accomplished through a combination of retention time and spectral matching with standards, enzymatic hydrolysis, solid phase extraction and LC coupled with electrospray ionization mass spectrometry. In addition to four known PY derivatives, two novel metabolites were identified as pyrenediol glucuronide sulfate and a sec- ond isomer of PYDS. The methanol extraction of metabolites from tissue gave excellent mean recoveries, ranging from 67 to 97%, for the available standards PY, PYOH, PYOS and PYOG spiked in both the mus- cle and visceral mass of Buccinum spp. The mean recoveries of a surrogate standard, 2-hydroxyfluorene, spiked in all tissue samples were 100% and 95% for visceral and muscle tissue samples, respectively. The method limits of detection for these compounds were all below 0.2ng/g of wet tissue, low enough to detect metabolites in reference animals. Results from the application of this method to the quantitative analysis of biotransformation products in the visceral mass of the whelk Neptunia lyrata exposed to PYOH contaminated food are also presented. This method will be useful to apply to the analysis of PY metabolites in soft tissues of other animals. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. 1. Introduction Polycyclic aromatic compounds are a widely studied class of contaminants, many of which are known to exhibit toxicity to humans and other organisms [1,2]. They are released into the environment both naturally and through human activities as com- bustion products and components of fossil fuels [3,4]. They are widely distributed throughout the environment and detected in highly variable concentrations in air [5], water [6], sediment and biota [7,8]. In the environment, polycyclic aromatic hydrocarbons (PAH) exhibit a diverse range of reactivities including photo- chemical oxidation [9], fungal and bacterial catabolism [10,11], biotransformation by organisms [12,13] and hydrolysis of conju- gated species [14]. Although smaller PAH with two or three rings ∗ Corresponding author at: Department of Fisheries and Oceans, Bedford Institute of Oceanography, Dartmouth, NS, B2Y 4A2, Canada. Tel.: +1 902 426 7451; fax: +1 902 426 6697. E-mail address: hellouj@dfo-mpo.gc.ca (J. Hellou). are known to degrade relatively easily, larger, more hydrophobic molecules tend to be more persistent. It is the magnitude of produc- tion and release into the environment of these chemicals that has led to characterizing them as persistent. Parent compounds and oxi- dation intermediates can accumulate to concentrations associated with a toxic risk [15,16]. It is because of this potential toxicity that PAH are referred to as priority pollutants and receive international attention. Pyrene (PY) is a tetracyclic PAH commonly studied as a model compound because it occurs as a major constituent in mixtures with many more harmful PAH. Because of the molecule’s high degree of symmetry, oxidation to 1-hydroxypyrene (PYOH) is the predom- inant initial pathway for PY reactivity in the environment. This makes the study of the compound’s fate simpler than that of other PAH which can form numerous oxidation products. In addition to being good model compounds, PY and PYOH are both environmen- tally relevant and detected in a wide variety of sources and sinks of contaminants including water, sediment and biota [8,17,18]. As is the case with many PAH, the toxicity of PY has been linked to an activated phase I oxidation product [19,20]. These types of inter- 1570-0232/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2009.06.006