Analytical method development for the determination of polycyclic aromatic hydrocarbons in biological samples

P.-L. Cloutier,1-3 F. Fortin,3 M. Fournier,2 P. Brousseau,2 P.-É. Groleau,3 M. Desrosiers1 1Centre d’expertise en analyse environnementale du Québec, ministère du Développement durable, de l’Environnement et de la Lutte contre les changements climatiques, Québec, QC; 2Institut National de la recherche scientifique Institut Armand-Frappier (INRSIAF), Laval, QC; 3Centre d’expertise en analyse environnementale du Québec, ministère du Développement durable, de l’Environnement et de la Lutte contre les changements climatiques, Laval, QC, Canada


Introduction
Polycyclic aromatic hydrocarbons (PAHs) are major group of contaminants for sediment monitoring and are included in most of the priority pollutants lists of environmental regulatory agencies.PAHs are naturally produced by incomplete combustion and pyrolysis of organic matter and are largely present in fossil fuel.Their analysis in biological tissues can be useful for monitoring of anthropogenic activities related with combustion and heavy industries, and for the characterization of fossil fuel such as petroleum hydrocarbons spill. 1 PAHs are highly hydrophobic compounds and are preferably monitored in biota matrix or sediment than water samples (US-EPA, 2 EU Directive 2008/105/EC 3 ) since they are readily bioavailable towards biota.
5][6][7][8] The interaction between pollution and climatic changes program (IPOC), proposed to use mussels as bioindicator species for freshwater and marine water quality.The species selected are dressenids (Dreissena bugensis and Dreissena polymorpha) and blue mussels (Mytilus edulis) for freshwaters and marine waters respectively.
Instrumental analysis by mass spectrometry allows low-level (pg/g) quantification of PAHs in different matrices such as tissues but lipids and other matrix interferences must be removed to assure quality of results.Acid digestion usually used to remove the lipids cause decomposition of PAHs and saponification may cause degradation of deuterated internal standards.Lipids can also be removed by silica or alumina columns requiring large amount of solvent and efficiency is variable.Traditional size-exclusion with biobeads is proposed by the US EPA 3640A for such purpose, but it's however time-consuming and also requires large amount of organic solvent. 9Nowadays, commercial gel permeation columns (GPC) are coupled with conventional HPLC systems offer good alternative to easily remove lipids, with lower solvent consumption and overnight automation.This paper presents a method to remove lipids and macromolecules efficiently from mussels and salmon samples by GPC for PAHs analysis.

Stability of the gel permeation columns system with a performance standard
The stability of the GPC system for chromatography separation and the injection of large volume (1 mL) were evaluated with the commercial GPC performance standard by several injections (n=6).The reference standard solution used is the same as proposed in the EPA method 3640A for GPC system.The standard was kept in the freezer (T=-18°C) until the instrumental analysis.One ml is injected in the GPC system with a flow of 5 mL/min of methylene chloride.

Determination of the collected fractions for the recuperation of polycyclic aromatic hydrocarbons
Injection of the 49 highly concentrated native PAHs targeted was done in the GPC system and monitored through UV absorbance at 254 nm.The estimated fraction collection was then evaluated with 100 ng/mL of native 49 PAHs spiked and the 13 C-labeled and deuterated internal standard.The eluate was then concentrated to 0.5 mL in isooctane and injected on column in a GC-HRMS instrument equipped with a 30 m DB35-MS column.

Removal efficiency of the lipids by the gel permeation columns system for collected fractions
The removal efficiency of lipids for fish and mussel samples was evaluated by injection of extracts in the GPC system and the recording of the UV absorbance at 254 nm.Collection of the fractions was performed to selectively separate lipids from PAHs.A gravimetric analysis was performed for both fractions by concentration of the solvent to dryness under nitrogen stream.

Stability of the gel permeation columns system for a reference standard
The stability of the GPC system was evaluated for chromatography separation and the injection of large volume (1 mL) with the reference standard solution for GPC performance.The results show that for all the compounds the repeatability was elevated with less than 0.2% of RSD for the retention time and less than 2% RSD for the calculated area that corresponds to the injected volume.The elevated % RSD for the calculated area is due to a little tailing of for the corn oil, the perylene and the sulphur present in the chromatogram.

Recovery and accuracy for polycyclic aromatic hydrocarbons in the targeted fraction
The injection of a 1.4 mL of a 1.25 µg/mL solution of PAHs in the GPC system and the reading at 254 nm shows an important peak corresponding to the PAHs centered at 17.329 min that starts at 15.5 min and finish at 20 min.
Four different fractions (16-20 min, 14-20 min, 14-21.5 min and 15.5-20 min) were collected and confirmed by GC-HRMS analysis to ensure an adequate recovery of all the PAHs targeted in the proposed method (Table 1).
The recovery of the deuterated and 13 Clabeled standards was acceptable except for the 14-21.5 min was accidently evaporated to dryness, causing the loss of the volatile compounds (SURR-Methylnaphtalene-D 10, SURR-Acenaphtene-D 10, SURR-Anthracene-13 C).
There is no significant difference between the recovery of the 16-20 min and 15.5-20 min for the 13 C labeled and deuterated internal standards (Table 1).
Although the 14-21.5 min fraction was accidentally evaporated to dryness that causes loss of the volatiles PAHs compounds, the surrogates correct the accuracy adequately for the less volatile compounds (Figure 1).Results are better with the 15.5-20 min than the 16-20 min for 2,3,5-Trimethylnaphtalene (108% and 44% respectively).The fraction collection should start at 15.5 min to ensure a correct recovery of this compound.The fraction collection of the 21.5 min test shows a better recovery for 7H-Dibenzo(c,g)carbazole and Coronene (104% and 106% respectively) than the test until 20 min (around 3% and 60% respectively).For these reasons, the targeted fraction collection that would ensure adequate recovery of all the PAHs should be by 15.5 to 21.5 min.

Removal efficiency of the lipids by the gel permeation columns sys-tem for the collected fractions
Gravimetric analysis of the residual lipids for the PAHs newly determinated collected zone allows an efficient confirmation of the removal almost complete of the lipids (91±1% for the mussels extracts and 97±1% for the salmon extracts).Less than 10 mg of lipids are still present for mussels (≈1.5% of lipid) after the GPC purification and less than 15 mg (7-8% of lipid).Theses residual lipids are small and near of the error of the measurements for lipids.For that reasons, this method allows almost complete removal of lipids in biological tissues such as fish and mussels.

Conclusions
Table 1.Recovery percentage of the 13 C-labeled and deuterated internal standards for the four collections zones targeted for the polycyclic aromatic hydrocarbons.

Figure 1 .
Figure 1.Accuracy percentage of the targeted polycyclic aromatic hydrocarbons (PAHs) for the four fractions.