Lipid phase extraction

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. 

Gustavson et al. (2000) developed a convenient and novel solid phase extraction (SPE) method for the removal of methyl oleate from SPMD dialysates containing PAHs. A small SPE column (1 g or 0.5 g) containing a dual-zone silica (normal phase)-based restricted-access sorbent (Diazem, Midland, MI, USA) is used for the separation. The capacity of this sorbent to remove methyl oleate is about 1.8% (lipid/sorbent wt wt ). The PAHs are eluted with 19 mL of hexane and methylene chloride (97 3 VV ) and recoveries of all PAHs are typically >72%. 

For the analysis, 140 pi of the apoA-I proteoliposomes is supplemented with 50pl of 8% BSA in analysis buffer, 10 pi of 0.1 M 2-mercaptoethanol, and 15 pi plasma. The mixture is incubated by shaking for 30 min at 37°C. The LCAT reaction is stopped by adding 4 ml of chloroform methanol (2 1, v/v). Lipids are extracted by incubation at room temperature for 2 h. Hydrophilic and lipophilic phases are separated by adding... 

Diethylstilbestrol is particularly difficult to quantitate below 1.0 ppb in bovine tissues, especially in liver, which is among the last tissues to contain diethystilbestrol after cattle are withdrawn from receiving tire drug (101, 102). Interferences from tissue matrix constitute a major problem that might be due to nonspecific interference of lipids and fatty compounds (103, 104). In addition, problems with false-positive results often appear in urine analysis unless a chromatographic step such as a solid-phase extraction cleanup (105, 106) is introduced. Simple sample preparation procedures such as those based on solvent extraction and liquid-liquid partitioning do not usually give satisfactory results (107, 108). 

Polyether antibiotics are hydrophobic compounds that are characterized chemically by their low polarities and their instability under acidic conditions. These antibiotics can be quantitatively extracted from the primary organic extract into carbon tetrachloride (393-395). When partitioning from a sodium chloride solution into an organic solvent, high yields have been achieved using dichloromethane (396, 397), carbon tetrachloride (391, 399), and chloroform (14, 398) as extraction solvents. In a different approach, water extracts containing lasalocid residues have been purified by partitioning into the mobile phase, which was a complex mixture of tetrahydrofuran, methanol, n-hexane, and ammonia (387, 389, 390, 392). To remove lipids, sample extracts have often been partitioned with n-hexane. 

The quality of FAME prepared by the methods described in this unit must be examined by GC analysis. Generally, impurities in the extracted lipid samples are not removed before methylation. If the GC results are not satisfactory due to sample contamination, additional steps may be necessary to clean the sample either before or after methylation. Commonly used techniques for purifying lipid samples are thin-layer chromatography (TLC), solid phase extraction (SPE), and column chromatography. 

Ultrasound-assisted extraction provides efficient extraction in a shorter processing time than is needed for conventional extraction. The aid of ultrasound will result in a higher extraction yield and reduce solvent consumption. The extract may exhibit a wide range of colors (pale yellow, brown, red). For anthocyanin extraction in an acidic environment, the extract will be deep red, pink, or purple. The extract may contain considerable amounts of lipophilic compounds (e.g., chlorophyll, carotenoids, lipids). Prior to solid-phase extraction, those compounds can be eliminated from the extracts using liquid-liquid extraction. 

Recently, however, prepacked solid-phase extraction (SPE) columns have been preferred. In this case, too, the phospholipid fraction is often recovered by elution with methanol. Thus, Nash and Frankel developed a method to recover the phospholipids from 1-g portions of crude oils using silica gel Sep-Pak cartridges (29). The oil was applied as a 10% solution in a 95/5 (v/v) mixture of petroleum ether and diethyl ether, and neutral lipids (about 94-97%) were eluted with 10 ml of additional solvent the so-called unsaponifiables (2-3%) were recovered using 20 ml of diethyl ether, and the phospholipid fraction was obtained using 10 ml of methanol. 

Actually, solid-phase extraction is used not only as a rough preliminary fractionation procedure. Prieto et al. described the complete fractionation of the total lipids from wheat into eight neutral lipid, two glycolipid, and four phospholipid classes in addition to PC and LPC, TV-acyl PE and A-acyl LPE were detected (37). However, two separate stationary phases (silica and aminopropyl) as well as seven different mobile phases were needed. Moreover, 14% crosscontamination of PC and LPC was observed, and the recovery of the phospholipids was limited to about 85%. Hence, SPE is a rapid and efficient technique for preliminary fractionation, but loses its advantages if more complex separations are tried. 

MN Vaghela, A Kilara. A rapid method for extraction of total lipids from whey protein concentrates and separation of lipid classes with solid phase extraction. J Am Oil Chem Soc 72 1117-1121, 1995. 

H Y Kim, N Salem Jr. Separation of hpid classes by solid phase extraction. J Lipid Res 31 2285-2289, 1990. 

Solid-phase extraction effectively separates vitamin D from its more polar 25-hydroxy metabolite. In the analysis of human milk (64), the dried lipid fraction of milk was dissolved in 35% dichloromethane in hexane and then applied to a preconditioned silica cartridge. The sample was fractionated using the following elution sequence 9 ml hexane (discard), 3 ml 7% ethyl acetate in hexane (discard), 15 ml 7% ethyl acetate in hexane (vitamins D2 and D3), 25 ml 15% ethyl acetate in hexane (discard), and 9.5 ml 3% 2-propanol in hexane (25-hydroxyvitamin D2 and 25-hydroxy vitamin D3). 

Vegetable juice add dihydrophylloquinone (internal standard) to sample. Extract with 2-PrOH/hexane (3 + 2) and water aided by sonication and vortexing. Silica solid-phase extraction in the sample cleanup mode. Milk as described for vegetable juice followed by liquid-phase reductive extraction for removal of lipids. 

Cleanup An additional separation of the mycotoxin from lipids and other components of the matrix is accomplished through the cleanup step. Most procedures include solid-phase extraction on stationary phases such as silica, C,8, florisil, and phenyl. Prepacked columns are largely used, with the variations between lots being recently ameliorated. Alternatively, the use of cleanup by immunoaffinity, based on the formation of mycotoxin-protein conjugate, is on the increase, since this is very rapid, selective, and usefully employed in various food matrices. One disadvantage is that the cost is still rather high, and cross-contamination phenomena (false-positive) can occur (30). 

There is substantial history regarding the application of conventional vibrational spectroscopy methods to study the intact surface of skin, the extracted stratum corneum and the ceramide-cholesterol-fatty acid mixtures that constitute the primary lipid components of the barrier. The complexity of the barrier and the multiple phases formed by the interactions of the barrier components have begun to reveal the role of each of these substances in barrier structure and stability. The use of bulk phase IR to monitor lipid phase behavior and protein secondary structures in the epidermis, as well as in stratum corneum models, is also well established 24-28 In addition, in vivo and ex vivo attenuated total reflectance (ATR) techniques have examined the outer layers of skin to probe hydration levels, drug delivery and percutaneous absorption at a macroscopic level.29-32 Both mid-IR and near-IR spectroscopy have been used to differentiate pathological skin samples.33,34 The above studies, and many others too numerous to mention, lend confidence to the fact that the extension to IR imaging will produce useful results. 

Lipids are extracted from 1 vol. of membrane suspension in a stoppered test-tube by shaking with 5 vol. chloroform/methanol reagent. The sample is centrifuged at 7000 xg for 5-10 min, and the upper phase removed by aspiration. A known volume of the lower chloroform phase (3 ml) is recovered and taken to dryness under nitrogen in a water bath at 45°C. While the lipid residue is still under the ni-... 

Determination of free 4-hydroxy-2,3-trans-alkenals by HPLC Esterbauer (1982) has developed a procedure for the qualitative detection and quantitative measurement of steady-state concentrations of free hydroxyalkenals (specifically HNE) in tissues, tissue extracts and lipid containing foodstuffs. Their method utilizes UV-detection of the free aldehyde at its 220 nm UV-absorption maximum and peak identification was confirmed by mass spectrometry. An effective purification and concentration step is employed using dichloromethane to extract hydroxyalkenals from samples trapped on Extrelut columns. The samples are subsequently purified by solid-phase extraction on octadecyl-bonded silica (ODS) disposable cartridges and then analysed by HPLC. 

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