Phospholipids derivatization

The correct ratio of lipid constituents is important to form stable liposomes. For instance, a reliable liposomal composition for encapsulating aqueous substances may contain molar ratios of lecithin cholesterol negatively charged phospholipid (e.g., phosphatidyl glycerol (PG)) of 0.9 1 0.1. A composition that is typical when an activated phosphatidylethanolamine (PE) derivative is included may contain molar ratios of phosphatidylcholine (PC) cholesterol PG derivatized PE of 8 10 1 1. Another typical composition using a maleimide derivative of PE without PG is PC male-imide-PE cholesterol of 85 15 50 (Friede et al., 1993). In general, to maintain membrane stability, the PE derivative should not exceed a concentration ratio of about l-10mol PE per lOOmol of total lipid. 

Historically, the target analytes in clinical mass spectrometric applications were small, volatile compounds that could be analyzed by GC-MS (see Chapter 4). With time, new chemical preparation techniques and derivatization schemes broadened the scope of these metabolites to include fatty acids, amino acids, intermediates of glucose oxidation, phospholipids, steroids, neurogenic amines, nucleic acids, etc. The molecular weights (molar masses) after derivatization were less than 1000 Da, a mass range easily within the limits of most conventional mass spectrometers. 

Gabizon AA, Barenholz Y, Bialer M. Prolongation of the circulation time of doxorubicin encapsulated in liposomes containing polyethylene glycol-derivatized phospholipid pharmacokinetic studies in rodents and dogs. Pharm Res 1993 10(5) 703. 

Uster P, et al. Insertion of poly(ethylene glycol) derivatized phospholipid into pre-formed liposomes results in prolonged in vivo circulation time. FEBS Lett 1996 386 243. 

Figure 1 Schematic structures of micelle and liposome, their formation and loading with a contrast agent, (a) A micelle is formed spontaneously in aqueous media from an amphiphilic compound (1) that consists of distinct hydrophilic (2) and hydrophobic (3) moieties. Hydrophobic moieties form the micelle core (4). Contrast agent (asterisk gamma- or MR-active metal-loaded chelating group, or heavy element, such as iodine or bromine) can be directly coupled to the hydrophobic moiety within the micelle core (5), or incorporated into the micelle as an individual monomeric (6) or polymeric (7) amphiphilic unit, (b) A liposome can be prepared from individual phospholipid molecules (1) that consists of a bilayered membrane (2) and internal aqueous compartment (3). Contrast agent (asterisk) can be entrapped in the inner water space of the liposome as a soluble entity (4) or incorporated into the liposome membrane as a part of monomeric (5) or polymeric (6) amphiphilic unit (similar to that in case of micelle). Additionally, liposomes can be sterically protected by amphiphilic derivatization with PEG or PEG-like polymer (7) [1].

The hydroperoxides obtained on thermal oxidation of cholesteryl acetate (191e) can be selectively separated by SPE and elution with a polar solvent. After reduction to the corresponding alcohols by NaBH4 and further derivatization to the trimethylsilyl ether, the products can be subjected to GLC with ion-trap MS detection. It can be thus demonstrated with the aid of standards that under the oxidation conditions (160 °C for 90 min) only the 7-position is attacked, leading to the la- and 7/3-hydroperoxy derivatives, while the plausible 4-position remains unscathed . Treatment of erythrocite ghosts with t-BuOOH causes a manyfold content increase of 5-hydroxyeicosatetraenoic acid (5-HETE), 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and 5-oxoeicosatetraenoic acid (5-oxo-ETE) residues of phospholipids. These acids can be separated by HPLC, identified and quantitized by tandem MS . ... 

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... 

The phosphoric acid esters of diacyl glycerides, phospholipids, are important constituents of cellular membranes. Lecithins (phosphatidyl cholines) from egg white or soybeans are often added to foods as emulsifying agents or to modify flow characteristics and viscosity. Phospholipids have very low vapor pressures and decompose at elevated temperatures. The strategy for analysis involves preliminary isolation of the class, for example by TLC, followed by enzymatic hydrolysis, derivatization of the hydrolysis products, and then GC of the volatile derivatives. A number of phospholipases are known which are highly specific for particular positions on phospholipids. Phospholipase A2, usually isolated from snake venom, selectively hydrolyzes the 2-acyl ester linkage. The positions of attack for phospholipases A, C, and D are summarized on Figure 9.7 (24). Appropriate use of phospholipases followed by GC can thus be used to determine the composition of phospholipids. 

Derivatization is never used in the analysis of the different phospholipid classes present, but is very widely used in the analysis of molecular species. The main reason for (mostly precolumn) derivatization in this case is to reduce significantly the detection limits. To this end, both UV-ab-sorbing and fluorescent derivatives are frequently used, as described in more detail in Secs. IV.B and V.3. 

Phospholipid-derived fatty acids are often used to identify bacteria by capillary GC analysis after liquid solvent extraction, concentration steps, and chemical derivatization to their methyl esters. Our initial investigations attempted to extract the intact phospholipids, but no significant recoveries were achieved using pure C02. Even if SFE conditions were developed that could extract intact phospholipids, an additional derivatization step would be required before GC analysis of the fatty acid components. For these reasons, chemical derivatization/SFE was investigated in an effort to eliminate the lengthy conventional liquid solvent extractions as well as to combine (and shorten) the extraction and derivatization steps. The derivatization/SFE procedure was performed on samples of whole bacteria using 0.5 mL of 1.5% TMPA in methanol. The static derivatization step was performed for 10 minutes at 80°C and 400 atm C02, followed by dynamic SFE for 15 minutes at a flow rate of ca. 0.5 mL/min of the pressurized C02. Extracts were collected in ca. 3 mL of methanol and immediately analyzed by capillary GC without any further sample preparation. 

SFE/derivatization of several chlorinated acid pesticides (those listed in EPA method 515.1) have been performed using conditions similar to those used for the bacterial phospholipids. The derivatized products from the SFE procedure for several representative organics are shown in Figure 6. As would be expected using the TMPA/methanol reagent, the carboxylic acids form the methyl esters (2,4-D and dicamba) while the phenols form the methyl ethers (pentachlorophenol). Esters of the carboxylic acids (e.g., the di-isopropyl amine ester of 2,4-D) also form the methyl esters. For ethers, two derivatized products resulted since the ether linkage could be cleaved on either side of the oxygen and methylated as shown by acifluorfen. 

Figure 5. GC/FTD analysis of the phospholipid-derived fatty acid methyl esters from derivatization/SFE extraction of Bacillus suhtilis. The front chromatogram shows the second extraction of the same sample (I.S.=internal standard).

---