Triacylglycerols , derivatization

Saponification (see Section 7.4) is carried out to extract more recalcitrant lipids, and the yields are higher than for conventional solvent extraction (Stern et al. 2000). 3 ml of 0.5 M methanolic NaOH is added to 0.1 g of the shard powder and heated at 70°C for 3 hours in a sealed glass vial. After cooling, the supernatant is acidified with HC1 and extracted with three aliquots of 3 ml //-hexane. The hexane will not mix with the methanolic solution (unlike the DCM MeOH used above), but will absorb the lipids and can be transferred into a new clean vial. The removal of excess hexane is carried out as above. Saponification will hydrolyze and methylate any ester functionalities, which removes the requirement to derivatize the samples (Section 7.4) unless other molecules are suspected of being present. However, any wax esters or triacylglycerols will also be hydrolyzed to their fatty acid methyl esters and alcohols therefore, if information on their composition is required, then conventional solvent extraction is recommended as a first step. For subsequent characterization of the lipid extract, see Chapter 7. 

Triacylglycerols (esp. triolein) Chloroform-methanol extraction, HPLC with detection at 208 nm GC of derivatized or free fatty acids. Bacterial lipase Veeraragavan (1990), Choi and Jeon (1993)... 

Lipid extraction, preceding FA analysis, is an important process and it should be performed correctly to achieve plausible results. Sometimes, incomplete lipid recovery may not be a problem if the material recovered is a representative sample however, it can be misleading if FA compositional data are needed. If hydrolytic methods are used during extraction, or if the presence of free FAs is in order, an adequate derivatization method must be chosen to guarantee the methylation of both triacylglycerols and free FAs. 

Figure 8.17. High-temperature GC of derivatized plasma lipids on a WCOT column (8 m x 0.3 mm) of fused silica, coated with SE-54, and temperature-programmed to a maximum of 340 C [647]. Abbreviations A, TMS ester derivatives of free acids B, TMS ethers of monoacylglycerols (derived from lysophosphatidylcholine) C, TMS ether of cholesterol D, tridecanoin (internal standard) E, TMS ether of 16 0-sphingosine (derived from sphingomyelin) F, TMS ethers of diacylglycerols (derived from phosphatidylcholine) and ceramides G, more TMS ethers of ceramides H, cholesterol esters J, triacylglycerols. (Reproduced by kind permission of the authors and of the Journal of Biochemical and Biophysical Methods, and redrawn from the original paper).

In the United States, the total fat is defined as the total fatty acids expressed as triacylglycerols. Therefore, procedures for releasing fatty acids for total fat determination may be applicable to analysis of fatty acids per se. In the Association of Official Analytical Chemists (AOAC) official method 996.06 (AOAC, 1995), the sample is digested either with hydrochloric acid, to hydrolyze lipids and also carbohydrate and protein with which hpid material may be associated with, or with ammonia for dairy products, in the presence of an internal standard (triundecanoin). The fatty acids are then extracted with a petroleum ether-diethyl ether solvent mixture. As pointed out by Ackman (1999), the potential drawback of using hydrochloric acid is that not all hpids may be hydrolyzed totally to FFA. Presumably, this does not matter if an appropriate fatty acid derivatization procedure for FFA and esterified fatty acids (EFA) is subsequendy employed (see Section 2.3.3.1), as long... 

Although research laboratories use generally sophisticated analytical methods such as GLC to analyse and quantify lipid samples, chemical derivatizations are often used in hospitals. For these methods, the lipid samples are derivatized to yield a product which can be measured simply and accurately - usually by colour. Thus, total triacylglycerol, cholesterol or phospholipid-phosphorus can be quantitated conveniently without bothering with the extra information of molecular species, etc. which might be determined by more thorough analyses. 

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