The International Lecithin and Phospholipid

Up to now, however, HPLC has remained the method of choice. This is due mainly to the fact that this technique is much easier to automate as compared to TLC. Besides, a wide variety of stationary and mobile phases are available, so the technique is highly flexible. Besides, the investment cost is much lower as compared to P-NMR, whereas quantitation is more straightforward than in MEKC. Hence, the more recent official methods for the analysis of phospholipids, as proposed by the American Oil Chemists Society (AOCS), by the International Union for Pure and Applied Chemistry (IUPAC), and by the International Lecithin and Phospholipid Society (ILPS), all use HPLC. In this chapter, a review is presented of HPLC procedures that have been described during the past 10 years the older literature was discussed in a previous edition of this handbook (21). 

The International Lecithin and Phospholipid Society proposed a method including a gradient of two mixtures of H/2-P/W/acetic acid/triethylamine with a composition of 81.5/17/0/1.5/ 0.08 and 0/84.5/14/1.5/0.08, respectively (68). In it a 5-/zm LiChroCart 100 diol-bonded silica stationary phase was selected to give more stable retention times. Quantification is performed us-... 

Although phospholipids are natural components of nearly all food products, the analysis of the phospholipid composition is of importance mainly in the certification and quality control of lecithins. According to the European Analytical Subgroup of the International Lecithin and Phospholipid Society (ILPS), there is an urgent need for a standard method for the determination of the PL composition, for this would allow a better characterization of lecithin and PL products (15,16). Besides, the nonavailability of good calibration standards is a major problem when comparing analytical results between companies. In order to try to solve the latter problem, the ILPS proposes a calibration standard whose composition is certified by 31P-NMR as an absolute tech-... 

Many HPLC methods for phospholipids have been developed, but chromatographic resolution and dynamics of detection are not always satisfactory. For each source of phospholipids, special standards are needed due to the different distribution of fatty acids. These standards are expensive and in some cases are not available. Another problem is represented by the analysis of phospholipids in complex matrices. In many cases, separation is impossible or very difficult, not least due to the surface activity, which is desired in the application of phospholipids, but which complicates the analysis of these compounds. Therefore, a method is needed which is selective in the detection of phospholipids in order to avoid a separation from the matrix. The P NMR spectroscopy of phospholipids meets these requirements. The I.L.P.S. (Internationa Lecithin and Phospholipid Society) has chosen the P NMR method as the reference method [62],[63],[64]. It has been tested world-wide by round robin tests in comparison to various HPLC and TLC methods. With triphenylphosphate as internal standard, a pulse angle of 15°, 10-s relaxation delay, and 32-256 accumulations, the method has a precision of <0,5%. 

The internal hydrophobic core of lipospheres is composed of fats and biodegradable polymers, mainly triglycerides and lactide-based polymers, whereas the surface activity of liposphere particles is provided by the surrounding lecithin layer, composed of phospholipid molecules. 

Unlike surfactants, these agents do not appreciably lower surface and interfacial tension thus, they have little tendency to create foam or wet particles. Most deflocculants, however, are not generally considered safe for internal use, and as a result the only acceptable dispersant for internal products is lecithin or a lecithin derivative (naturally occurring mixture of phospha-tides and phospholipids). Because lecithins vary in water solubility and dispersibility characteristics, proper control of product specifications must be maintained to obtain reproducibility. 

Bi-directional flux of free cholesterol between cells and lipoproteins occurs, and rate constants characteristic of influx and efflux can be measured [17]. The direction of any net transfer of free cholesterol is determined by the relative free cholesterol/phospholipid molar ratios of the donor and acceptor particles. Cholesterol diffuses down its gradient of chemical potential generally partitioning to the phospholipid-rich particle. Such a surface transfer process can lead to delivery of cholesterol to cells. This mechanism operates independently of any lipoprotein internalization by the receptor-mediated endocytosis. The influence of enzymes such as lecithin-cholesterol acyltransferase and hepatic lipase on the direction of net transfer of free cholesterol between lipoproteins and cells can be understood in terms of their effects of the pool sizes and the rate constants for influx and efflux. 

One of the several shapes that micelles can take is laminar. Since the ends of such micelles have their lyophobic portions exposed to the surrounding solvent, they can curve upwards to form spherical structures called vesicles. Vesicles are spherical and have one or more surfactant bilayers surrounding an internal pocket of liquid. Multi-lamellar vesicles have concentric spheres of uni-lamellar vesicles, each separated from one another by a layer of solvent [193,876] (Figure 14.1). The bilayers are quite thin (-10 nm) and are stabilized by molecules such as phospholipids, cholesterol, or other surfactants (Figure 14.2). Vesicles made from phospholipid bi-layers are called liposomes. Liposomes can be made by dispersing phospholipids (such as lecithin) into water and then agitating with ultrasound. 

Aboofazeli, R., Lawrence, C. B., Wicks, S. R., and Lawrence, M. J. 1994. Investigations into the formation and characterization of phospholipid microemulsions. Part 3. Pseudo-ternary phase diagrams of systems containing water-lecithin-isopropyl myristate and either an alkanoic acid, amine, alkanediol, polyethylene glycol alkyl ether or alcohol as cosurfactant. International Journal of Pharmaceutics, 111, 63-72. 

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