Tographic separation techniques, have been successfully employed in plant lipidomics [80,81], foodomics

Tographic separation techniques, have been successfully employed in plant lipidomics [80,81], foodomics [82,83], health and disease [84], among others; due to the high resolution and sensitivity foodomics [82,83], health and disease [84], among others; due to the high resolution and sensitivity of of mass get SB 202190 spectrometers, analytical protocols are faster, less complex less require less sample mass spectrometers, analytical protocols are faster, less complex and requireand sample manipulation. manipulation. The typical lipidomics approach involves lipid extraction, in distinct of lipids in The typical lipidomics approach involves lipid extraction, separation of lipidsseparation lipid classes distinct lipid classes and lipid analysis by MS (Figure 6). and lipid analysis by MS (Figure 6).Figure 6. MS-based lipidomics to screen bioactive lipids from marine macrophytes. Figure 6. MSbased lipidomics to screen bioactive lipids from marine macrophytes.Mar. Drugs 2016, 14,11 of4.1. Methods of Lipid Extraction from Marine Macrophytes: Conventional vs. New Green Methods Lipid analysis requires a first step of lipid extraction from selected samples. There are several experimental protocols that can be used, but they must be fast and reproducible. Moreover, they must be chosen in order to obtain the best lipid recovery. The most common methods for lipid extraction that have been applied to marine macrophytes include liquid-liquid extraction (LLE), organic SB 202190MedChemExpress SB 202190 solvent precipitation or solid-phase extraction (SPE) [85]. Lipid extraction protocol should also be able to extract a wide range of analytes with different polarities, with the ultimate goal of extracting the most diversified lipid structures as possible. Currently, the methods to ascertain polar lipids from marine macrophytes are mainly supported by conventional solvent extraction using organic solvents (reviewed in Section 4.1.1). Nevertheless, an effort has been made in order to develop new and eco-friendly extraction processes to obtain valuable products from natural sources and more advantages to be used for human consumption as food additives, nutraceutics or pharmaceutics products. 4.1.1. Conventional Methods for Lipid Extraction The general procedures for lipid extraction, either from animal tissues, cells or plant tissues, use organic solvents, with the most used methods differing in the type of organic solvents and the number and proportion of different organic solvents being used. Traditionally, a chloroform–methanol–water mixture is the most commonly used approach. There are two main extraction protocols using this solvent mixture: one described by Folch et al. in 1957 [86] and the other described by Blight and Dyer in 1959 [87]. The difference between these two methods is the proportion between chloroform (CHCl3 ) and methanol (CH3 OH). The Folch method uses CHCl3 /CH3 OH (2:1), while the Blight and Dyer method uses CHCl3 /CH3 OH (1:2) with a subsequent addition of one volume of chloroform and one volume of water. The basic principle of these two methods is that a mixture of chloroform and methanol is initially added to the sample creating a mono-phase system that extracts the lipids from the sample matrix. Water is subsequently added to produce a biphasic system, the chloroform layer, the lower phase that contains lipids and methanol–water layer, the upper layer, with the non-lipid components. Chloroform dissolves fat, and methanol breaks down the lipid pro.Tographic separation techniques, have been successfully employed in plant lipidomics [80,81], foodomics [82,83], health and disease [84], among others; due to the high resolution and sensitivity foodomics [82,83], health and disease [84], among others; due to the high resolution and sensitivity of of mass spectrometers, analytical protocols are faster, less complex less require less sample mass spectrometers, analytical protocols are faster, less complex and requireand sample manipulation. manipulation. The typical lipidomics approach involves lipid extraction, in distinct of lipids in The typical lipidomics approach involves lipid extraction, separation of lipidsseparation lipid classes distinct lipid classes and lipid analysis by MS (Figure 6). and lipid analysis by MS (Figure 6).Figure 6. MS-based lipidomics to screen bioactive lipids from marine macrophytes. Figure 6. MSbased lipidomics to screen bioactive lipids from marine macrophytes.Mar. Drugs 2016, 14,11 of4.1. Methods of Lipid Extraction from Marine Macrophytes: Conventional vs. New Green Methods Lipid analysis requires a first step of lipid extraction from selected samples. There are several experimental protocols that can be used, but they must be fast and reproducible. Moreover, they must be chosen in order to obtain the best lipid recovery. The most common methods for lipid extraction that have been applied to marine macrophytes include liquid-liquid extraction (LLE), organic solvent precipitation or solid-phase extraction (SPE) [85]. Lipid extraction protocol should also be able to extract a wide range of analytes with different polarities, with the ultimate goal of extracting the most diversified lipid structures as possible. Currently, the methods to ascertain polar lipids from marine macrophytes are mainly supported by conventional solvent extraction using organic solvents (reviewed in Section 4.1.1). Nevertheless, an effort has been made in order to develop new and eco-friendly extraction processes to obtain valuable products from natural sources and more advantages to be used for human consumption as food additives, nutraceutics or pharmaceutics products. 4.1.1. Conventional Methods for Lipid Extraction The general procedures for lipid extraction, either from animal tissues, cells or plant tissues, use organic solvents, with the most used methods differing in the type of organic solvents and the number and proportion of different organic solvents being used. Traditionally, a chloroform–methanol–water mixture is the most commonly used approach. There are two main extraction protocols using this solvent mixture: one described by Folch et al. in 1957 [86] and the other described by Blight and Dyer in 1959 [87]. The difference between these two methods is the proportion between chloroform (CHCl3 ) and methanol (CH3 OH). The Folch method uses CHCl3 /CH3 OH (2:1), while the Blight and Dyer method uses CHCl3 /CH3 OH (1:2) with a subsequent addition of one volume of chloroform and one volume of water. The basic principle of these two methods is that a mixture of chloroform and methanol is initially added to the sample creating a mono-phase system that extracts the lipids from the sample matrix. Water is subsequently added to produce a biphasic system, the chloroform layer, the lower phase that contains lipids and methanol–water layer, the upper layer, with the non-lipid components. Chloroform dissolves fat, and methanol breaks down the lipid pro.