Principles and Methods of Lipid Extraction

Lipid extraction is one of the key steps for the successful analyses of cellular lipidomes by ESI-MS, in general, and by approaches using direct infusion, in particular. Traditionally, lipid samples from biological sources are extracted using a mixture of chloroform and methanol based on the Folch method [17] or the modified method of Bligh and Dyer [18] or other solvent combinations [19, 20]. 

For any method of analysis of lipids, if external standard(s) are used for quantitation, extraction recovery of individual species is always a concern. Therefore, this parameter has to be determined at an early stage during the development of a method. Different solvent systems may have different extraction recoveries for different lipid classes and molecular species. In contrast to the methods utilizing external standards, any methods utilizing internal standards should have less concerns for the extraction recovery since an incomplete extraction recovery, if present, is only a secondary effect on the quantitative analysis of a lipid class caused by the differential extraction recovery from different molecular species of the class relative to that of the internal standard(s) of the class. 

As lipidomic analysis is classified into the LC-MS-based approach and shotgun lipidomics, the requirement for sample preparation is also different. The former is more tolerant with the presence of inorganic ions in lipid extracts than the latter since the pre-column or even the analytical column can get rid of these extraction contaminants. However, the presence of inorganic residues may affect substantially the ionization stability as well as ionization efficiencies of different lipid classes and individual species of a class in shotgun lipidomics. Therefore, minimizing the inorganic residues, particularly eliminating any aqueous phase contamination, becomes crucial. 

A type of salt is usually used during lipid extraction to enhance the phase separation and extraction efficiency. It is advisable that it is better to add the salt that matches with the adduct preferred for analysis of lipids in both positive- and negative-ion modes by shotgun lipidomics. For example, if proton or ammonium is the preferred adduct for lipid analysis in the positive-ion mode and acetate adduct is the choice in the negative-ion mode, ammonium acetate is the preferred salt used for lipid extraction. If an inorganic salt is not introduced during the extraction step, sodium chloride is always the most abundant salt in nature. Therefore, sodium and chlorine adducts of zwitterionic lipids are always predominant in mass spectra 

Detergents can cause severe ion suppression in shotgun lipidomics and may affect the column separation of lipids. Therefore, the existence of detergents can complicate the MS analysis of lipids. Thus, utilization of detergents should be avoided in sample preparation when possible. If present, great efforts should be made to remove them as much as possible. 

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The principles of sonochemistry can also be applied to disrupt different species of oil-bearing microalgae cells. Detailed experimental results are necessary to support the cost-effectiveness and industrial scale applicability of ultrasound for microbial lipid extraction, and subsequent biodiesel production in comparison to conventional methods (Mata et al., 2010). 

The first step of nearly all methods for steroids involves extraction of lipid-soluble materials from plasma or urine and evaporation of the organic solvent. In the case of urine the water-soluble conjugates are usually hydrolyzed or solvolyzed prior to this step. There are a thousand different ways of carrying this out, but their empirical details are of little interest. A few general principles and the most useful specific examples of novel techniques will be discussed here. 

The best method of separation is worthless if the material to be analysed has been spoilt by inexpert treatment. Consequently, some methods for isolation of lipids from plant and animal material and some principles for handling the extracts are given here in detail. 

Several clean-up methods have incorporated the use of additional liquid-liquid steps with solvents such as chloroform and ethyl acetate. Alternatively, some authors have turned to ultrafiltration for further purification.Additionally, hexane is extensively used in several methods to further remove non-polar matrix components such as fats and lipids. By far, the most popular clean-up technique reported in the literature is the use of solid phase extraction (SPE). Some common phases reported include CIS cartridges such as SepPak C18, Strata Chromobond C18, " and Mega Bond Elut C18. The use of silica, alumina, and Extralut " " has also been reported, as well as polymeric cartridges such as Oasis HLB. The latter phase has been reported as the basis of an on-line clean-up procedure. Because of their amphoteric nature, as both weak acids and weak bases, the sulfonamides also lend themselves to analysis by ion exchange. Cationic SPE has been reported by several authors.On the basis of the same principles, anionic exchange has also been used. " ... 

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