Lipids sample application

A method which uses supercritical fluid/solid phase extraction/supercritical fluid chromatography (SE/SPE/SEC) has been developed for the analysis of trace constituents in complex matrices (67). By using this technique, extraction and clean-up are accomplished in one step using unmodified SC CO2. This step is monitored by a photodiode-array detector which allows fractionation. Eigure 10.14 shows a schematic representation of the SE/SPE/SEC set-up. This system allowed selective retention of the sample matrices while eluting and depositing the analytes of interest in the cryogenic trap. Application to the analysis of pesticides from lipid sample matrices have been reported. In this case, the lipids were completely separated from the pesticides. 

The basic technology for the preparation of sample material is similar in all TLC preparations, irrespective of the origin of the hpid and specific preparation method for a variety of biological samples [43]. The most important factor is the solubihty of the sample. The lipid sample must be completely soluble in the dissolving solvent prior to the application and must be free from water. Either toluene or chloroform is commonly used as the solvent to dissolve hpid materials. The dissolving solvent should be nonpolar in namre and volatile at such a concentration that the hpid components in the sample are completely adsorbed throughout the entire thickness of the layer as quickly as possible. Although sample sizes as small as 1 to 10 pi can... 

Only a very small proportion of the fatty acids are present in the free, unester-ified form and the vast majority are components of other lipids. Nevertheless it is important to be able to measure and identify the free fatty acids present in either form and for this they must be first extracted into an organic solvent and then usually converted to their methyl ester. The simplest method of methyla-tion, which is applicable to both esterified and non-esterified fatty acids, is to heat the lipid sample for 2 h under a current of nitrogen at 80-90°C with 4% sulphuric acid in methanol. After cooling and the addition of water, the resulting methyl esters are extracted several times into hexane and the combined extracts are dried over sodium carbonate and anhydrous sodium sulphate. The solvent fraction is then reduced in volume by a stream of nitrogen. 

Zhou, P., Chandan, V., Liu, X., Chan, K., Altman, E., Li, J. Microwave-assisted sample preparation for rapid and sensitive analysis of Helicobacter pylori lipid A applicable to a single colony. J Lipid Res 50 (2009) 1936-1944. 

Prior to spotting of samples, the plate is scored lengthwise at 0.5- to 1.0-cm widths by use of a stainless steel needle. Excess silica gel is removed by gentle tapping of the plate, and the samples are ready for application. The outer lanes of the scored plate are reserved for standards (e.g., phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lysolecithin, and others as desired), which are readily available from several biochemical companies. Aliquots of the unknown lipid sample, which contain sufficient P, as detected by the qualitative spray above, are spotted on two to three lanes (usually in a volume ratio of 1 2 3) approximately 1.0 cm above the bottom of the plate. This is called the origin. 

The major application described in the James-Martin landmark paper on gas-liquid chromatography is the separation and quantitation of fatty acids (FA). Indeed, it has remained as one of the major applications to this date. It has been estimated [350] that some 25% of all papers published in the field of GC involve, in one way or another, FA or their derivatives. A vast range of samples have been analyzed for FA animal and plant oils, foodstuffs, bacterial products, glandular secretions of animals, and various physiological fluids and tissues, just to mention a few. As more and more information is being sought concerning the composition of various lipids, the applications are expected to increase even further in the future. 

Gas chromatographic analysis of tobacco alkaloids does not require derivation. The general procedures for GC analyses are as follows (1) use the smallest sample applicable for analysis (2) utilize preparations that clean up the sample without loss of alkaloids (3) pre-extract the sample prior to alkaloid extraction with hexane (removes pigments and lipids) (4) extract alkaloids from tobacco with an aqueous acid solution, filter, partition the aqueous acid extract with organic solvent (hexane, methylene chloride, chloroform, etc.) (5) increase pH of the aqueous extract to pH 10 (5N NaOH) and partition the basic solution with chloroform (6) dry the chloroform solution over Na2S04, and (7) concentrate the sample or analyze as is. 

Application of these methods to a small subset (n = 10) of nonfasting plasma lipid samples showed that c9,t 1-CLA is most enriched in TAG, followed by CE and PL. The NEFA fraction was found to contain very little c9,rl 1-CLA (Table 12.1). 

Solid-phase extraction (SPE) offers an alternative way for enrichment of chlorinated fatty acids. Akesson-Nilsson presented an aminopropyl-based SPE technique for enrichment of chlorinated FAMEs in a cell-culture medium and for previously silver nitrate and urea treated eel lipids. In this application, a 500-mg aminopropyl column was connected to a vacuum manifold and conditioned with 2 ml of hexane. After transesterified lipid samples in 0.2 ml of hexane were loaded, the column was first eluted with 6 ml of hexane and then with 4 ml of a solvent mixmre made of hexane-diethyl ether-dichloromethane (89 1 10). In this way, the majority of non-chlorinated FAMEs were removed in the first elution and chlorinated FAMEs enriched in the later elution. 

The universal TLC facilities are utilized plates, adsorbents, microcapillaries, or micropipettes for sample application, development tanks, detection spray reagents, devices for spraying, and densitometers for quantification. Plates are either commercially precoated or handmade. Silica gel G (G, for gypsum as a binding substance), silica gel H (no binding substance) and, rarely, alumina and kieselguhr, form the thin-layer stationary phases. Complete sets of devices necessary for the preparation of handmade plates are commercially available. After the silica gel slurry is spread on the plates, they are left to dry in the air for at least 24 hr and shortly in an oven at 110°C. The plates are then ready for either direct use or for modification of the layer. From the great variety of precoated plates, which are commercially available and preferred nowadays, silica gel plates and plates with layers modified with carbon chains from C2 to C18 are of interest in lipid analysis. Understandably, precoated plates for Ag-TLC are not commercially available. Preparative TLC is performed mostly on 20 X 20 cm plates with layer thickness of... 

Because of the experimental difficulty of the technique and because more user-friendly and to some extent more powerful alternatives have become available, FDI is not frequently applied anymore, except for some specific applications. In this respect, an important development is liquid injection field desorption ionization (LIFDI), which enables sample application to the emitter without breaking the vacuum (see Fig. 7.1) [7, 8]. The specific applications where FDI and LIFDI are still applied comprise the analysis of some oiganometallic compounds [9,10], ionic liquids [11], and compound classes, such as (cyclo)paraffins, aromatic hydrocarbons, and nonpolar sulfur compounds (thiophenes) [7, 12-14], not readily amenable to ESI or MALDI. For such nonpolar analytes, mainly molecular ions M+ are observed, whereas for some more polar compounds, [M+H]+ and/or sodiated molecules ([M-l-Na] ) may be observed, e g., for glycosides (Sect. 7.5.2), lipids (Sect. 7.5.4), and peptides (Sect. 7.5.5). A detailed overview on technology and applications of FDI-MS was provided by Schulten et al. [15, 16]. 

Derivatives are often used in lipid chemistry to prepare fatty acid methyl esters needed to determine the fatty acid composition of lipids. Fried et al. (1992) published a technique on the transesterification of 500-mg samples of snail tissue. Because the technique is applicable to other biological tissues and fluids, it is presented herein. Lipids were extracted from snail bodies with 10 ml of chloroform-methanol (2 1) the extracts were filtered through a plug of glass wool contained in a Pasteur pipet, and nonlipid contaminants were removed by extraction with 8 ml of Folch wash (0.88% aqueous KCl). The lipid-containing lower phase was separated and evaporated just to dryness under a stream of nitrogen at room temperature. The total lipid sample was dissolved in about 30 ml of methanol, and 0.5-1.0 ml of concentrated sulfuric acid was added. The mixture was refluxed for 1 h the formed fatty acid methyl esters were extracted with 30-40 ml of petroleum ether and the extract dried over anhydrous sodium sulfate. The fatty acid methyl esters were concentrated on a Rotoevaporator at 40°C and the volume reduced to 1 ml. The fatty acid methyl esters can be separated by TLC on silica gel impregnated with silver nitrate (Christie, 1982). 

Figure 12.3 Sample application procedure using guide strips for PLC. Up to 1 mg of lipid was streaked across the center 10 cm of a 20 x 20-cm analytical plate, which was developed in the dual solvent system of Skipski et al. (Chapter 15). Major bands from the organism contained sterol (II) and phospholipid (I). The band labeled "blank" was lipid negative. Lanes 1 and 4 of the edge strips (5 cm wide) were spotted with neutral lipid standards, lanes 2 and 3 with sample. The edge strips were cut and visualized with 10% phosphomo-lybdic acid in ethanol and were then matched with the center to determine the position of the bands.

As with urine, saliva (spumm) is easy to collect. The levels of protein and lipids in saliva or spumm are low (compared to blood samples). These matrices are viscous, which is why extraction efficiency of xenobioties amoimts to only 5 to 9%. By acidifying the samples, extraction efficiencies are improved as the samples are clarified, and proteinaceous material and cellular debris are precipitated and removed. Some xenobioties and their metabohtes are expressed in hair. Hair is an ideal matrix for extraction of analytes to nonpolar phases, especially when the parent xenobioties are extensively metabolized and often nondetectable in other tissues (parent molecules of xenobioties are usually less polar than metabolites). Hair is a popular target for forensic purposes and to monitor drug compliance and abuse. Human milk may be an indicator of exposure of a newborn to compounds to which the mother has been previously exposed. The main components of human milk are water (88%), proteins (3%), lipids (3%), and carbohydrates in the form of lactose (6%). At present, increasing attention is devoted to the determination of xenobioties in breath. This matrix, however, contains only volatile substances, whose analysis is not related to PLC applications. 

Passi, S., Rothschildboros, M.C., Fasella, P., Nazzaroporro, M. and Whitehouse, D. (1981) An application of high performance liquid chromatography to analysis of lipids in archaeological samples. Journal of Lipid Research 22, 778 784. 

This technique was used by Delmas et al. [404] to separate lipid extracts in seawater into various classes. Lipid classes that have been eluted away from the point of application may be burnt off the rod in a partial scan, allowing those lipids remaining near the origin to be developed into the place that has just been simultaneously scanned and reactivated. By analysis of complex mixtures of neutral lipids in this stepwise manner it is possible to be more selective about lipid class separations as well as to be more confident about assigning identities to peaks obtained from a seawater sample. In addition, this approach also reduces the possibility of peak contamination by impurities which would normally coelute with marine lipid classes (e.g., phthalate esters [403]). 

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