Analysis of polycyclic aromatic hydrocarbons in fish: Optimisation and validation of microwave-assisted extraction Maria João Ramalhosa , Paula Paíga, Simone Morais, Ana M.M. Sousa, Maria P. Gonçalves, Cristina Delerue-Matos, Maria Beatriz Prior Pinto Oliveira ABSTRACT An accurate and sensitive method for determination of 18 polycyclic aromatic hydrocarbons (PAHs) (16 PAHs considered by USEPA as priority pollutants, dibenzo[a,l]pyrene and benzo[j]fluoranthene) in fish samples was validated. Analysis was performed by microwave-assisted extraction and liquid chromatog- raphy with photodiode array and fluorescence detection. Response surface methodology was used to find the optimal extraction parameters. Validation of the overall methodology was performed by spiking assays at four levels and using SRM 2977. Quantification limits ranging from 0.15–27.16 ng/g wet weight were obtained. The established method was applied in edible tissues of three commonly consumed and commercially valuable fish species (sardine, chub mackerel and horse mackerel) originated from Atlantic Ocean. Variable levels of naphthalene (1.03–2.95 ng/g wet weight), fluorene (0.34–1.09 ng/g wet weight) and phenanthrene (0.34–3.54 ng/g wet weight) were detected in the analysed samples. None of the sam-ples contained detectable amounts of benzo[a]pyrene, the marker used for evaluating the occurrence and carcinogenic effects of PAHs in food. 1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants with the majority coming from anthropogenic activ- ities (WHO, 1998). The health concerns of PAHs are due to their po- tential cytotoxicity, mutagenicity and carcinogenicity in humans (WHO, 1998). U.S. Environmental Protection Agency (USEPA) listed 16 priority PAHs (USEPA, 1986). Excluding smokers and occupa- tionally exposed populations, most individuals are exposed to PAHs predominantly from dietary sources. Consequently, since the past decade, PAHs are under evaluation by the International Programme on Chemical Safety (IPCS), the Scientific Committee on Food (SCF) and by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Benzo[a]pyrene is regarded as a marker for the occurrence and genotoxic and carcinogenic effects of PAHs in food. Maximum levels had been set for diverse categories of food products, including fish and seafood (Commission Regulation, 2006). However, more carcinogenic PAHs, such as dibenzo[a,l]pyr- ene or dibenz[a,h]anthracene were found. Dibenzo[a,l]pyrene, as well as benzo[j]fluoranthene, are not included in the USEPA list but are considered in EC Recommendation 1881/2006 (2006). Studies that include the determination of dibenzo[a,l]pyrene, and of other EU priority PAHs that are not in USEPA list, in fish matrices are limited (Duedahl-Olesen & Ghorbani, 2008; Ramalhosa, Paíga, Morais, Delerue- Matos, & Oliveira, 2009; Serpe, Esposito, Gallo, & Serpe, 2010). Rising incomes, the ensuing diversification of diets and fish nutritional benefits related to its proteins of high biological quality as well as desirable lipid composition (rich in essential x-3 polyun- saturated fatty acids) are leading to a shift towards significantly higher fish consumption in developing countries. World apparent per capita finfish and seafood consumption has steadily increasing from an average of 9.9 kg in the 1960s reaching 16.4 kg in 2005 (FAO, 2009). Portuguese annual consumption of fish is 55.6 kg per capita year -1 , being Portugal the largest consumer among all of the EU countries (European Commission, 2010) and one of the biggest in the world. Sardine (Sardina pilchardus), chub mackerel (Scomber japonicus) and horse mackerel (Trachurus trachurus) rep- resent some of the most important pelagic fish groups captured; chub mackerel was the sixth species that contributed most to glo- bal catches in 2006 (FAO, 2009). In Portugal, they represent the three most consumed fish species (sardine > chub mackerel > horse mackerel) (European Commission, 2010). A large number of studies have been published on extraction of PAHs from fish, being a limiting step in the quantitative analysis.