Optimization and comparison of miniaturized extraction techniques for PAHs from crude oil exposed Atlantic cod and haddock eggs

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 samples (≤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, drying 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 dichloromethane/n-hexane (1:1, v/v) and clean-up by silica SPE, whereas the optimized LE-dSPE method comprised extraction with acetonitrile and clean-up with PSA and C18 sorbents. 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).

Oil droplet fouling and differential toxicokinetics of polycyclic aromatic hydrocarbons in embryos of Atlantic haddock and cod

“…The samples were preserved by flash-freezing in liquid nitrogen and stored in poly ethylene cryovials at -80°C until further handling. Extraction was performed as described in Sørensen et al [32]. After transfer to glass vials and addition of surrogate standards (100 ng/g sample), the samples were homogenized in n -hexane-dichloromethane (1:1 v/v, 2 mL) using a microprocessor (Virtis Tempest IQ 2.0), followed by addition of sodium sulphate (150 mg), vortex extraction (30 s) and centrifugation (2000 rpm, 2 min).…”

“…N 2 was used as collision gas at a flow of 1.5 mL/min and helium was used as a quench gas at a flow of 4 mL/min. Target PAH analytes were identified by two unique multiple reaction monitoring (MRM) transitions and quantified by the most intense peak [32]. Alkyl PAH clusters were determined by MRM using transitions from the molecular ion, as described previously in Sørensen et al [31].…”

“…Calibration tandards were run daily to monitor system performance and a variation of no more than 25% was accepted. Method performance characteristics are reported in Sørensen et al [31, 32]. Alkanes were measured by GC-MS/MS using MRM transitions 99 + → 57 + and 127 + → 85 + .…”

“…This is due, in part, to the need for large samples masses (1–10 g wet weight) to achieve the instrumental detection limits [29, 30], particularly for samples that contain low PAH concentrations. Recently, methodologies were developed for the analysis of trace amounts of both parent and a wide range of alkylated PAHs in small (< 0.1 g) tissue samples [31, 32]. These methods enable the determination of total PAH body burden in fish egg samples from low dose exposure experiments using as little as 100 individuals or less per sample.…”

“…Both species have their main spawning grounds in the Lofoten-Vesterålen area [33]. The two species are biologically very similar, both in terms of egg lipid content (0.6–0.7%) [32], egg size (1.3–1.5 mm diameter) [34] and developmental biology [35–37]. Although individual studies of crude oil effects on ELS of Atlantic haddock embryos and larvae and cod larvae have previously been published [3, 17, 19, 20, 38], there has yet to be a direct comparison of the toxicological response in the two species.…”