PAPER IN FOREFRONT Optimization and comparison of miniaturized extraction techniques for PAHs from crude oil exposed Atlantic cod and haddock eggs Lisbet Sørensen 1,2 & Marta S. Silva 2 & Andy M. Booth 3 & Sonnich Meier 1 Received: 14 October 2015 /Accepted: 26 November 2015 /Published online: 16 December 2015 # Springer-Verlag Berlin Heidelberg 2015 Abstract Two miniaturized extraction methods for a wide range of 2-6 ring polycyclic aromatic hydrocarbons (PAHs) and their alkylated homologues in small lipid-rich biota sam- ples (≤100 mg) have been developed. Both methods utilize liquid extraction (LE) prior to a clean-up step using either normal phase solid phase extraction (SPE) or mixed-phase dispersive SPE (dSPE). Optimization of the methods was achieved by comparing the type and amount of sorbents, dry- ing agents, and solvents used. In order to improve the limits of detection (LOD) of target PAHs under high sensitivity gas chromatography–tandem mass spectrometry analysis, specific emphasis was given to minimizing lipid co-extraction. The optimized LE–SPE method comprised extraction with dichlo- romethane/n-hexane (1:1, v/v) and clean-up by silica SPE, whereas the optimized LE–dSPE method comprised extrac- tion with acetonitrile and clean-up with PSA and C18 sor- bents. The methods were validated and directly compared through the analysis of Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) eggs exposed to oil. The LE–SPE method resulted in lower levels of co-extracted lipids (14.1±1.7 ng/μL) than the LE–dSPE method (60± 14 ng/μL). Achieved PAH LODs for the LE–SPE method were typically an order of magnitude lower (<5 ng/g) than for the LE–dSPE method (<125 ng/g). The LE–SPE method offers the possibility for PAH analysis of small samples of fish eggs (~100 mg) exposed to small quantities of crude oil (~1– 10 μg/L total PAHs). Keywords Polycyclic aromatic hydrocarbons . Crude oil . Fish eggs . Purification . Solid phase extraction . Dispersive SPE Introduction Polycyclic aromatic hydrocarbons (PAHs) are considered the main compound group responsible for eliciting acute and sub- lethal toxic effects in marine organisms following oil spills or operational production discharges. Close correlations between the concentration of total dissolved PAHs and the toxicity of weathered oils to early life stages (eggs and larvae) of marine fish have been shown previously [1, 2]. More recently, con- cerns are being raised about the influence of micro-sized dis- persed oil droplets on toxicity [3], especially towards early life stages of Atlantic cod ( Gadus morhua) and haddock (Melanogrammus aeglefinus) [4]. To understand this ob- served toxicity, accurate and sensitive measurement of low concentrations of PAHs and other oil-derived compounds ac- cumulated in small samples of fish eggs is necessary. Currently, there are few published methods that have suc- cessfully analyzed body residues of PAHs at low concentra- tions in small (<0.1 g) samples [5]. A range of traditional high volume solvent extraction methods (e.g., microwave-assisted extraction, pressurized liquid extraction, and Soxhlet extrac- tion) are suggested for miniaturization, but the main issue with most of these approaches is low recovery of volatile Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-9225-x) contains supplementary material, which is available to authorized users. * Lisbet Sørensen lisbet.soerensen@imr.no 1 Institute of Marine Research, P.O. Box 1870 Nordnes, 5817 Bergen, Norway 2 Department of Chemistry, University of Bergen, P.O. Box 7800, 5020 Bergen, Norway 3 SINTEF Materials and Chemistry, P.O. Box 4760 Sluppen, 7465 Trondheim, Norway Anal Bioanal Chem (2016) 408:1023–1032 DOI 10.1007/s00216-015-9225-x