Analytical Methods Lipid content of frozen fish: Comparison of different extraction methods and variability during freezing storage Maria João Ramalhosa a,b , Paula Paíga a , Simone Morais a , M. Rui Alves b,c , Cristina Delerue-Matos a , Maria Beatriz Prior Pinto Oliveira b,⇑ a Requimte, Instituto Superior de Engenharia do Porto, Rua Dr. António Bernardino de Almeida 431, 4200-072 Porto, Portugal b Requimte, Serviço de Bromatologia, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha 164, 4099-030 Porto, Portugal c ESTG, Instituto Politécnico de Viana do Castelo, Av. Atlântico, 4900-348 Viana do Castelo, Portugal article info Article history: Received 26 October 2010 Received in revised form 29 May 2011 Accepted 31 July 2011 Available online 5 August 2011 Keywords: Fish Extraction methods Lipids Fat content abstract In this work, a microwave-assisted extraction (MAE) methodology was compared with several conven- tional extraction methods (Soxhlet, Bligh & Dyer, modified Bligh & Dyer, Folch, modified Folch, Hara & Radin, Roese-Gottlieb) for quantification of total lipid content of three fish species: horse mackerel (Tra- churus trachurus), chub mackerel (Scomber japonicus), and sardine (Sardina pilchardus). The influence of species, extraction method and frozen storage time (varying from fresh to 9 months of freezing) on total lipid content was analysed in detail. The efficiencies of methods MAE, Bligh & Dyer, Folch, modified Folch and Hara & Radin were the highest and although they were not statistically different, differences existed in terms of variability, with MAE showing the highest repeatability (CV = 0.034). Roese-Gottlieb, Soxhlet, and modified Bligh & Dyer meth- ods were very poor in terms of efficiency as well as repeatability (CV between 0.13 and 0.18). Ó 2011 Published by Elsevier Ltd. 1. Introduction Total lipid content of fish samples is an important parameter used in biochemical, physiological, and nutritional studies. Many efforts are being made in order to include fish in human diet due to its numerous beneficial effects on health which are related to its high protein and unsaturated fatty acid contents. However, the reduction of fish stocks, restrictions on harvesting, the associ- ated operation costs and the need for waste reduction have con- tributed to the growing interest in maximising the use of fishery resources producing fish derivatives such as fillet and minced fish. These products are without smell, without spines and ready to eat or heat and serve foods (FAO/WHO, 2005). The process of freezing has been widely employed to maintain fish properties (FAO/WHO, 2005). However, the efficiency of freez- ing conservation is not always achieved due to the occurrence of lipid hydrolysis and oxidation which are directly related to the pro- duction of off-flavours and odours and influence fish acceptance. Some studies have been published about the changes in different fish species during the frozen storage (Aubourg, Lehmann, & Gal- lardo, 2002; Aubourg, Piñeiro, & González, 2004; Aubourg & Ugli- ano, 2002; Le Bihan, Perrin, & Koueta, 2007; Serdarog ˘lu & Felekog ˘lu, 2005). However, none was focused on chub mackerel (Scomber japonicus; whole fish or minced fillets) which was the sixth species that contributed most to global catches in 2006 (FAO, 2009). The lipid group consists of many types of lipids with different chemical composition. Lipids are usually classified into two groups: the neutral or non-polar lipids (triglycerides, diglycerides, monoglycerides, sterols, etc.) and the more polar lipids (free fatty acids, phospholipids, sphingolipids, etc.) (Manirakiza, Covaci, & Schepens, 2001). Lipid content has been estimated using Soxhlet which is the offi- cial recommended method (AOAC, 2005). Several alternative meth- ods were developed for total lipid extraction in fish samples. Folch, Less, and Sloane (1957) optimised a method using a chloroform/ methanol/water phase system which, under various modifications, continues to be considered the classical and most reliable mean for quantitative lipid extraction (Iverson, Lang, & Cooper, 2001). As previously referred (Priego-Capote, Ruiz-Jiménez, & Luque de Cas- tro, 2007), conventional fat extraction methods (Bligh and Dyer (1959); modified Bligh & Dyer (Smedes, 1999); modified Folch method (Lin, Liu, Yang, & Lee, 2004); Roese-Gottlied methodology (Manirakiza et al., 2001) and Hara & Radin procedure (Hara & Radin, 1978)) have not been greatly improved, despite modifications in sol- vent mixtures and laboratory practice. Long preparation times with re-extraction steps to ensure complete lipid isolation are still re- quired. In the interest of economy and environment, new technolo- gies such as Supercritical Fluid Extraction (SFE), Pressurised Liquid 0308-8146/$ - see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.foodchem.2011.07.123 ⇑ Corresponding author. Tel.: +351 222078927; fax: +351 222003977. E-mail address: beatoliv@ff.up.pt (M.B.P.P. Oliveira). Food Chemistry 131 (2012) 328–336 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem