Acylglycerols analysis

The potential for the preservation of lipids is relatively high since by definition they are hydrophobic and not susceptible to hydrolysis by water, unlike most amino acids and DNA. A wide range of fatty acids, sterols, acylglycerols, and wax esters have been identified in visible surface debris on pottery fragments or as residues absorbed into the permeable ceramic matrix. Isolation of lipids from these matrices is achieved by solvent extraction of powdered samples and analysis is often by the powerful and sensitive technique of combined gas chromatography-mass spectrometry (GC-MS see Section 8.4). This approach has been successfully used for the identification of ancient lipid residues, contributing to the study of artifact... 

Other Acylglycerols. If some of the DGs in freshly drawn milk are involved in biosynthesis, it is possible that they are enantiomeric and are probably the sn-1,2 isomer. If so, the constituent fatty acids are long chain. Their configuration can be determined by stereospecific or other analyses, but it is difficult to accumulate enough material for analysis. Nevertheless, Lok (1979) isolated the DGs from freshly extracted cream as the trityl derivatives. Trityl chloride reacts selectively with primary hydroxyls. The stereochemical configuration of the DGs was identified as sn-1,2 therefore, these residual DGs were most likely intermediates of biosynthesis. If the DGs were products of lipol-ysis, they would be a mixture of 1,2/2,3 isomers in a ratio of about 1 2, since milk lipoprotein lipase preferentially attacks the sn-1 position of TGs (Jensen et al. 1983). 

CRABP-GST is immobilized onto a glutathione-containing resin at a concentration that saturates the resin. A complex lipid mixture was prepared by combining a concentrated lipid extract derived from mouse tissue (brain) [5] (DMSO stock) with exogenously added retinoic acid (RA, positive control) and 13C-oleic acid (negative control). Analysis of this complex lipid mixture by LC-MS prior to incubation with immobilized CRABP demonstrated that the mixture contained RA (added), phospholipids, acylglycerols, cholesterol esters, and cholesterol. A portion of this mixture (corresponding to 1 nmol of RA) was added to PBS (DMSO concentration should not exceed 5%) and incubated with the CRABP-GST bound beads for 1 h. 

At present, mass spectrometry allows the analysis of an acylglycerol by identifying its various component fatty acids and their positions in the glycerol without necessitating prior chromatographic separation. The quantity of sample that is used during a mass spectrometric analysis is about 1 picomole. [288]... 

This method can be applied directly to acylglycerols present in a mixture, as is illustrated by the mass spectrometric analysis of natural cocoa butter. [299] This analysis (Figure 8.65) allowed the determination of the complete structure of the predominant acylglycerols in the cocoa (Table 8.10). 

APPI is derived from APCI, but instead of the corona discharge needle, the ionization takes place after irradiation with a krypton lamp that emits photons of 10.0-10.6 electron volt. Different methods with and without dopant have been recommended. APPI, in combination with LC, has been used to analyze glycosphingohpids (31). In addition, APPI has been reported to be more sensitive and efficient than APCI and ESI for the analysis of fatty acid esters and acylglycerols (32, 33). Because the method has not found widespread use to date, it is too early to estimate its potential. 

Cai SS, Syage JA. Atmospheric pressure photoionization mass spectrometry for analysis of fatty acid and acylglycerol lipids. J. Chromatogr. A 2006 1110 15-26. 

Nawabi, P., Lykidis, A., Ji, D., and Haidar, K. (2003). Neutral-lipid analysis reveals elevation of acylglycerols and lack of cholesterol esters in Plasmodium falciparum-infected erythrocytes. Eukaryot. Cell 2,1128-1131. 

The separated lipid classes can then be collected as discrete fractions in solvent. This is particularly useful for either the direct detection and quantitation of isolated lipid fractions or for further derivatization and analysis of lipid components. For example, acylglycerols, wax esters, and phospholipid classes can be separated by column chromatography and each fraction reacted as in Section 9.3.2 to analyze the FA present in each lipid class. 

The general application of reverse-phase HPLC can be employed directly in the separation and quantitation of total lipid classes. However, the high degree of resolution possible from HPLC columns is more frequently employed for the separation and analysis of individual lipid components from complex mixtures or individual lipid classes previously separated by other chromatographic procedures (e.g., column chromatography). Examples include HPLC analysis of acylglycerols (Plattner, 1981 Kuksis, 1994) and phospholipids (Porter and Weenen, 1981). 

Serum Lipid Analysis. Blood samples were withdrawn from the marginal ear-vein after overnight food deprivation every 2 wk until the termination of the experimental periods. Total cholesterol, high density lipoprotein cholesterol (HDL-C), and tri-acylglycerol (TG) concentrations were determined using enzymatic methods. Low density lipoprotein cholesterol (LDL-C) was calculated according to Friedewald et al. (14). 

Prior to the analysis a triacylglycerol internal standard, 1,3-diheptadecanoyl-2,6(Z)-octadecenoyl-glycerol (17 0, 18 1, 17 0), is added to the sample. The sample is then separated into its triacylglycerol components by silver nitrate TLC. The band for /8-SOS (symmetrically disaturated 2-oleoyl-l,3-diacylglycerols) is isolated and the total jS-SOS acylglycerols are converted to methyl esters which are then analysed by GLC. 

Finally, the analysis of the triacylglycerols by GLC (Section 6.2.19) can give information on adulteration by the determination of the composition of the fatty acids at the 2-position of the acylglycerols to check whether oils (e.g. olive oil) or fats have been re-esterified to check tallow on admixing with lard to check, as already mentioned under Section 6.2.19, cacao butter equivalent fats against the internationally proposed requirements (see also Padley, 1980 Fincke, 1980). 

High-frequency NMR has been used in the analysis of enantiomeric acylglycerols with the use of signal shift reagents, tri-(3-heptafluorobutyryl-... 

Glycerophospholipids may be hydrolyzed to phos-phatidic acid with phospholipase D, or to di-acylglycerols with phospholipase C. The latter hydrolysis allows the analysis of phospholipids by TLC, GC, or LC, whereas the hydrolysis with phospholipase D produces molecules that are best analyzed by LC. 

Capillary GC-MS is useful for identifying the carbon and unsaturation number of acylglycerols. However, APCI LC-MS can be used to analyze less volatile acylglycerols that may be unsuitable for GC-MS. APCI LC-MS has become the method of choice for qualitative and quantitative analysis of acylglycerols and, when combined with MS/MS, is capable of distinguishing fatty acid chains in the sn-2 position from those in the sn-1/3 positions. 

Some simple rules can be derived for preliminary quantitative analysis of reflectivity data. For a two-slab model of, e.g., a monolayer of close packed fatty acid, an acylglycerol or a phospholipid, consisting of a thin head group slab of higher density in-between a tail slab and subphase of nearly equal density (such as in Figure 7), it can be shown [16] that the first few minima in the observed R(q )IRf q.) data occur at, -values where... 

Cai, S.S., Short, L.C., Syage, J.A., Potvin, M. and Curtis, J.M. (2007) Liquid chromatography-atmospheric pressure photoionization-mass spectrometry analysis of Iri-acylglycerol lipids-effects of mobile phases on sensitivity. J. Chromatogr. A1173,88-97. 

Alternatively, the stereo-specific analysis can be carried out chemically. The TGs are partially hydrolyzed in the presence of ethyl magnesium bromide. The resulting diacylglycerols are isolated and their OH groups converted to urethane with (S)-l-(l-naphthyl)ethylisocyanate. The sn-1,3- and the diastereomeric sn-1,2-and 2,3-di-acylglycerol urethane derivatives are separated in a subsequent HPLC step. The fatty acid analysis of the urethanes show the distribution of the acyl residues in positions 1, 2 and 3. 

The acyl residues of an acylglycerol are released as methyl esters (cf. 3.3.1.3) and are analyzed as such by gas chromatography. However, free fatty acid analysis is also possible by using specially selected stationary solid phases. Capillary-column gas chromatography should be used to... 

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