Phospholipids analysis, separation

Supercritical fluid chromatography (SFC) has also been used in phospholipid analysis. According to Lafosse et al., phospholipid classes can be separated by SFC using a simple isocratic solvent consisting of 78.4/21.6 (w/w) mixture of carbon dioxide and a mixture of methanol, water, and triethylamine (95/4.95/0.05) in combination with a Zorbax Sil stationary phase detection was performed by evaporative light-scattering (20). 

Fig. 11.4 shows separately curve 1 from Fig. 11.3 which is the dependence of W on the DPPC concentrations in the AF. The W(C) curves allow to determine the threshold concentration C i.e. the minimum phospholipid concentration at which there is a 100% probability of observation of black films (see Eq. (3.130)). At concentrations lower than C, NBFs are no more observed, since W sharply decreases to zero (films rupture). At concentrations higher than C, (W = 1), NBFs always form. Special studies with phospholipid analysis of amniotic fluid indicate that of all phospholipids in the AF, it is the DPPC that stabilises the foam bilayers. This analysis gives grounds to conclude that the concentration of each phospholipid (except DPPC) in the native AF is of an order lower than the corresponding... 

Since increasing temperature leads to increased fluidity and thus to a faster probe diffusion, pyrene Hpids have been frequently used to study phase transition in membranes [161,162]. Phospholipid phase separation increases the local concentration of dye labeled Hpids and can, therefore, be investigated via the characterization of exdmer formation. The binding of proteins or ions, however, may induce phase separation as well as decreasing lateral lipid diffusion. Since these two effects are opposing in terms of excimer formation, the binding of such proteins or ions cannot be studied by the (Ex/Mo)-ratio. The time-resolved analysis of the monomer fluorescence of the labeled lipid, however, allows for the separation of... 

In many instances, oxidized products are considerably more stable when formed in complex lipids, like phospholipids or cholesteryl esters, and alkaline hydrolysis is required to release the oxidized lipid for analysis. Separation and quantitation of mono-hydroxyicosanoids by HPLC has been regularly used for the last 30 years. The procedures have become routine and there are now published procedures for automating the analysis [60]. A recent review by Yin et al. [36] describes in detail how to quantify both mono-hydroxyeicosatetraenoates and F2-isoprostanes. Similar procedures can be used to separate and identify products derived from (5Z,8Z,llZ,14Z,17Z)-eicosa-5,8,11,14,17-pentenoic acid, (7Z,10Z,13Z,16Z,19Z)-docosa-7,10,13,16,19-pentaenoic acid, and (4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoic acid. 

Detailed analysis of phospholipids, as well as neutral lipids and nonesterified fatty acids, has become progressively more important as these molecules have come to represent intercellular messenger molecules that are involved in a number of disease states. The analysis of lipid patterns of blood and lipoproteins may therefore constitute a useful diagnostic tool. As noted in the previous edition, suitable methods for phospholipid analysis are often time consuming and tedious, since most of them include several separation and derivatization steps. 

Two papers have dealt with model membrane systems. Shintani and co-workers reported an NOE and MD simulation study of phospholipid aggregates in the form of micelles and vesicles. In the case of these systems, the usual approximation in the NOE analysis - separability of the information on distances and dynamics - are not applicable and the MD data provide an important support for the analysis of experimental data. Uchino et described characterization of rigid and elastic vesicles, loaded with the drug keterolac. Among other techniques, they applied T2 (really line-width) studies for suitable isolated proton spins in the drug. 

For quantitation of cholesterol and its derivatives in muscle and liver tissues, the extracts of the tissue homogenates are evaporated, dissolved in a mobile phase such as hexane-isopropanol, and injected onto a normal-phase column. For analysis of soybean oil by reversed-phase HPLC, after extraction with chloroform-methanol (9 1), the neutral lipids, chlorophyls, and the phospholipids are separated by TLC. The lipids recovered from the TLC are analyzed by HPLC. 

Aliquots of the total lipids, total phospholipids, and separated phospholipid classes were subjected to methano-lysis at 80 C for 3 hours in 5% methanolic HCl. Resultant fatty acid methyl esters were extracted with hexane and analyzed by gas chromatography using a bonded fused silica column (cyanopropylmethylsilicone, 0.25 mm i.d. X 50 m, 0.25 jam film, Tokyo Kasei Kogyo). The fatty acid methyl esters were identified by comparing their retention times with those of standards. The double-bond positions and the cis and trans configurations of fatty acid methyl esters were determined by the pyrrolidide method (6) and IR analysis (7), respectively. 

The ACE analysis of interactions between drugs and phospholipid bilayers of liposomes present as a pseudostationary phase was performed by Zhang et al. (32). The capillaries were treated to eliminate electroendosmosis. Freshly prepared and essentially neutral small unilamellar liposomes composed of egg phosphatidylcholine were sucked into the capillary. These liposomes increased both the retention of four negatively charged drugs and the separation between the substances (Fig. 6). The chromatographic retentions of these drugs on immobilized phosphatidylcholine liposomes, ex-... 

IJ Cartwright. Separation and analysis of phospholipids by thin layer chromatography. In JM Graham, JA Higgins, eds. Methods in Molecular Biology. Vol. 19 Biomembrane Protocols I. Isolation and Analysis. Totowa, NJ Humana Press, 1993, pp 153-167. 

The marriage of HPLC to mass spectrometry (MS), now developed into a mature instrumentation, continues to greatly impact many of the separation sciences, especially in pharmaceutical analysis where it has been used in new drug discovery [23,24] and in drug metabolite identification [25-27]. HPLC-MS has also made an impact on lipid research, providing a convenient approach to the analysis of phospholipids and fatty acids [28,29]. It has also greatly benefited the field of proteomics [30-34], especially analysis of protein structure and function. 

Gas-liquid chromatography separates volatile components of a mixture according to their relative tendencies to dissolve in the inert material packed in the chromatography column and to volatilize and move through the column, carried by a current of an inert gas such as helium. Some lipids are naturally volatile, but most must first be derivatized to increase their volatility (that is, lower their boiling point). For an analysis of the fatty acids in a sample of phospholipids, the lipids are first... 

Bernhard et al., on the other hand, selected 5-fim spherical Nucleosil-NH2 as the stationary phase (43). In order to improve the resolution of the early-eluting components and to reduce the overall time needed per analysis, they set up an isocratic HPLC system, including two separate columns. By means of a switch valve, the second 175-mm column could be excluded after the elution of PC, SPH, LPC, /V-methyl PE, PG, and PE. This enabled the rapid elution of the acidic phospholipids PI and PS from the 50-mm column and hence significantly decreased the total run time (Fig. 3). The mobile phase contained 1460 ml A, 500 ml M, 30 ml W, and 600 jj, 1... 

K Verleysen, P Sandra. Analysis of phospholipids in lecithins separation according to hydrophobic-ity by lowering the temperature. J High Resol Chromatogr 20 337-339, 1997. 

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