The simultaneous separation and quantitation of human milk lipids

A protocol using a dry column method was modified for the extraction of total lipids and the simultaneous separation and quantitation of neutral and polar lipids in human milk. The triacylglycerol, cholesterol, phospholipid and vitamin E contents of the lipid extracts were determined and compared with lipids extracted using a modified Folch procedure. Good precision for the extraction of neutral, polar and total lipids, as well as the different lipid classes, was demonstrated. No significant differences were found between the two methods with respect to the amount of cholesterol, phospholipid, total lipid or vitamin E extracted, thus validating the method as an extraction technique. We discuss the relationship between vitamin E and the three major milk lipids as an indicator of the vitamin's place of origin in the mammary gland. Our findings do not support the idea that vitamin E in mature milk has its original location in the apical membrane. Scientific Contribution No. 1254, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, CT 06269.     

Analysis of lipid classes by solid-phase extraction and high-performance size-exclusion chromatography

An improved method to analyze lipid classes of edible oils and fats by solid-phase extraction (SPE) and high-performance size-exclusion chromatography (HPSEC) is presented. A mixture of lipid standards was fractionated by the solid-phase extraction procedure (NH₂ phase) into polar and nonpolar fractions; these were then submitted to analysis by HPSEC. The size-exclusion chromatographic columns were three styrene/divinylbenzene columns with pore sizes of 100 Å and 50 Å. Light-scattering was used for the detection system, and the parameters of the detector were optimized to minimize the difference between the responses of the compounds studied. With this procedure it was possible to separate the following lipid classes: triacylglycerols, diacylglycerols, monoacylglycerols and free fatty acids, sterols, sterol esters, tocopherols and carotenoids. Quantitative analysis was studied for a light-scattering detector with several lipid standards of different molecular weights and unsaturation levels.     

Faster and easier methods for quantitative lipid extraction and fractionation from miniature samples of animal tissues

A less time-consuming dry-column method for total lipid extraction from tissue samples was scaled down and modified to permit the evaluation of 1-g samples of liver and muscle tissues from rats and from obese (ob/ob) and lean mice. Lipid yields obtained by the new dry-column method compared well with those obtained by the widely accepted traditional chloroform/methanol method. For subsequent separation of neutral and polar lipid classes, a solid-phase fractionation method was developed. Its performance was verified by thin-layer chromatogrphy. Both column chromatographic methods were found to be especially convenient when running multiple samples simultaneously.     

Cyclic fatty acid monomer: Isolation and purification with solid phase extraction

The application of a combined solid phase extraction (SPE)/clean-up procedure to the isolation of a purified fraction containing all the monomeric cyclic fatty acid methyl esters is described. Extraction of the nonpolar lipid components from nonurea-adducting (NUA) filtrates is performed on a reverse phase octadecyl bonded silica minicolumn. Stepwise elution of the SPE-retained materials through silica gel using several solvents allowed the separation of a pure fraction containing the cyclic monomers that can be used for a more reliable quantitative estimation of these compounds in edible fats and oils.     

Quantitative extraction of hamster liver lipid and cholesterol with supercritical carbon dioxide

A quantitative method for liver lipid and cholesterol extraction with supercritical CO₂ and ethanol entrainer (SCE) is reported and compared with the Folch (chloroform/methanol) procedure. Mean values for lipid and cholesterol in hamster livers (n=48) were similar between the SCE and Folch methods. Correlation coefficients between the two methods were 0.9866 for total lipid and 0.9546 for cholesterol. Similar mean values and high correlations between the two methods validate the SCE procedure as a precise alternative method for quantitative liver lipid extractions. The SCE method also reduces the use of hazardous solvents.     

Improved Butanol–Methanol (BUME) Method by Replacing Acetic Acid for Lipid Extraction of Biological Samples

Extraction of lipids from biological samples is a critical step in lipidomics, especially for shotgun lipidomics where lipid extracts are directly infused into a mass spectrometer. The butanol–methanol (BUME) extraction method was originally developed to extract lipids from plasma samples with 1 % acetic acid. Considering some lipids are sensitive to acidic environments, we modified this protocol by replacing acetic acid with lithium chloride solution and extended the modified extraction to tissue samples. Although no significant reduction of plasmalogen levels in the acidic BUME extracts of rat heart samples was found, the modified method was established to extract various tissue samples, including rat liver, heart, and plasma. Essentially identical profiles of the majority of lipid classes were obtained from the extracts of the modified BUME and traditional Bligh–Dyer methods. However, it was found that neither the original, nor the modified BUME method was suitable for 4‐hydroxyalkenal species measurement in biological samples.     

Quantitative analysis of brain and spinach leaf lipids employing silicic acid column chromatography and acetone for elution of glycolipids

Quantitative elution of acidic and neutral glycolipids of brain and spinach leaves from silicic acid columns with acetone was demonstrated. Cerebrosides and sulfatides of brain and sulfolipid and glycosyl diglycerides of spinach leaves were eluted quantitatively with acetone while prospholipids remained on the column. The observations provide the basis for an analytical procedure employing column and quantitative thin-layer chromatography (TLC). Sephadex column chromatography is utilized for separation of lipids from nonlipids; silicic acid column chromatography for separation into neutral lipid, glycolipid and phospholipid fractions; and quantitative TLC for analysis of lipid classes of each column fraction.     

Supercritical fluid extraction of minor lipids from pretreated sunflower oil deodorizer distillates

The recovery of minor lipid compounds (tocopherols and phytosterols) from sunflower oil deodorizer distillates using countercurrent supercritical carbon dioxide extraction has been studied. Since the raw material employed contains large amounts of triacylglycerols and free fatty acids, chemical transformation of these compounds into their corresponding fatty acid ethyl esters was previously carried out, in order to favor the concentration of minor lipids in the raffinate product. Extractions of the original and pretreated raw material were carried out in a pilot‐scale plant at 65 °C, with pressures ranging from 15 to 23 MPa and solvent‐to‐feed ratios from 15 to 30. The influence of the feed composition in the extraction process was analyzed by comparison of the tocopherol and phytosterol yields and enrichment factors obtained in each case. The chemical transformation of the deodorizer distillate composition significantly enhances the concentration of minor lipids in the raffinate product. Additionally, the reaction step produced a solid phase, mainly consisting of sterols, which was isolated from the liquid product.     

Microscale recovery of total lipids from fish tissue by accelerated solvent extraction

A number of techniques are available for the extraction of lipids from a variety of tissues; however, conventional methods are characteristically labor intensive, typically involve large volumes of toxic solvents, and usually require at least 1 g of tissue. With the availability of accelerated solvent extraction (ASE) technology, the opportunity exists to modify classical lipid extraction techniques such that automated high-pressure, high-temperature extractions may be performed with the use of far smaller volumes of costly and harmsul solvents. Moreover, the high extraction efficiency attainable by ASE suggests that significantly less tissue would be required than is routinely used. This paper describes the adaptation of previously developed lipid extraction solvent systems for use with ASE toward the purpose of extracting total lipids from 100 mg of fish tissue. The efficacy of three solvent systems for lipid extraction from representative fish tissues, including a standard reference material, was explored using gravimetry and FA analysis by GC. A TG was used as a surrogate to monitor overall method performance. The findings herein demonstrate that microscale ASE represents an effective and efficient alternative to traditional lipid extraction techniques based on quantity and composition of extracted lipid, surrogate recovery, and precision.     

Improved Methods for the Fatty Acid Analysis of Blood Lipid Classes

Two improved methods have been developed for preparation of fatty acid methyl esters (FAME) from major O-ester lipid classes in blood, i.e., cholesterol ester, triacylglycerol, and glycerophospholipids. The methods involve simple operations, and use neither harmful solvents such as chloroform or benzene nor highly reactive volatile reagents such as acetyl chloride. The FAME synthesis reaction proceeds under mild temperature conditions. The methods include (1) extraction of lipids from 0.2 ml of blood with 0.2 ml of tert-butyl methyl ether and 0.1 ml of methanol, (2) separation of the total lipids into lipid classes using a solid-phase extraction column or thin-layer chromatography, and (3) methanolysis of each lipid class at room temperature or at 45 °C. In all the operations, solvent concentration is performed only once prior to gas–liquid chromatography (GC). No noticeable differences in composition determined by GC have been found between FAME prepared by the present methods and those prepared by a conventional method involving lipid extraction with chloroform/methanol. The mild reaction and simplified procedures of the present methods enabled safe and reproducible analysis of the fatty acid compositions of the major ester-lipid classes in blood.     

Optimization of an Analytical Procedure for Extraction of Lipids from Microalgae

An optimized procedure for extraction of total and non-polar lipids from microalgae is proposed. The effects of solvent, pretreatment (lyophilization, inactivation of lipases, and addition of antioxidants) and cell-disruption (liquid nitrogen, sonication, and bead beating) on total lipid content, lipid class, and fatty acid composition were examined. Chloroform–methanol 1:1 was shown to be the best solvent mixture for extraction of total lipids from microalgae. When performing this extraction, lyophilized algae can be used, no pretreatment with isopropanol to inactivate the lipases is needed and addition of antioxidants is not necessary. Furthermore, cell-disruption is not essential, although in that case two extractions must be performed in series to ensure that, irrespective of the microalgal species, all lipids are extracted. Determination of non-polar lipid content should be performed by separation of the total lipid extract on an SPE column. Extraction using petroleum ether is only appropriate when a bead beater is used for pretreatment.     

A solvent extractor system for the rapid extraction of lipids and trace bioactive micronutrients in oilseeds

A low-cost laboratory extractor has been designed and constructed that selectively extracts polar and nonpolar components from oilseeds and other matrices. The extractor uses available high-performance liquid chromatography laboratory equipment for pumping the solvent into the extractor. Pressure, temperature, and valving arrangements are automatically controlled by commercially available components. Advantages of this system include low initial investment, reduced solvent consumption, shorter extraction times, quantitative lipid recovery, use of multiple extraction solvents, and reduction in cost per sample. The method has broader applications that include extraction of trace components from a variety of matrices, for example, the extraction of pesticides from foods and polychlorobiphenyls from soil. Class separation of components from different matrices can be achieved easily by selection of solvents with the appropriate polarity characteristics. Very small samples can be extracted simply by changing cell size or by adding an inert material to the cell to fill the void volume. Analyte collection can be accomplished by collecting in a test tube with an appropriate solvent, or on a solid-phase material. Optimization of extraction times, number of extractions, matrices, and solvent used is described. Neutral lipids were extracted from peanut meal in 70 min by the rapid extraction method compared to 1440 min required to extract the comparable amount of neutral lipids from a similar sample by the Soxhlet extraction method.     

Quantitative extraction of pecan oil from small samples with supercritical carbon dioxide

A procedure to determine total oil content of pecan was developed for samples weighing 500 and 10 mg by supercritical fluid extraction (SFE) with carbon dioxide as the extraction solvent, and chilled hexane as the trapping solvent. Fatty acid methyl esters (FAMEs) were prepared from the total lipid fraction by using either an aliquot (500 mg starting weight) or the entire extract (10 mg starting weight). Total oil content obtained for either sample size with SFE was similar to that obtained with an organic solvent extraction technique. The fatty acid composition for the total lipid fraction of oils extracted with SFE was the same as for oils extracted with organic solvents, and oil composition did not change during SFE. Both oil yield and fatty acid composition were similar to those reported previously for pecan. Samples could be extracted and placed into FAME-derivatizing reagents in one day, and fatty acid composition of the total lipid fraction could be determined by gas-liquid chromatography the next day. The procedure, as demonstrated for pecan, should be suitable for other oilseeds, especially those containing low amounts of water.     

Pressurized liquid extraction of polar and nonpolar lipids in corn and oats with hexane,methylene chloride,isopropanol, and ethanol

Samples of freshly ground corn kernels and freshly ground rolled oats were extracted via pressurized liquid extraction (accelerated solvent extraction) using four different organic solvents [hexane, methylene chloride (also known as dichloromethane), isopropanol, and ethanol] at two temperatures (40 and 100°C). Lipid yields varied from 2.9 to 5.9 wt% for ground corn and from 5.5 to 6.7 wt% for ground oats. With ground corn, more lipid was extracted as solvent polarity was increased, and for each individual solvent, more lipid was extracted at 100°C than at 40°C. With ground oats, the same temperature effects was observed, but the solvent polarity effect was more complex. For both corn and oats, methylene chloride extracted the highest levels of each of the nonpolar lipid classes. In general, for both corn and oats, icnreasing solvent polarity resulted in increasing yields of polar lipids, and for each solvent, more of each lipid class was extracted at 100°C, than at 40°C. Among the lipids in corn extracts, the phytosterols may be the most valuable, and total phytosterols ranged from about 0.6 wt% in the hot ethanol extracts to about 2.1 wt% in the hot hexane and methylene chloride extracts. Total phytosterols in all oat extracts were about 0.1 wt%. Digalactosyldiacylglycerol was the most abundant polar lipid in the oat extracts; its levels ranged from 1.6 wt% in the cold hexane extracts to 4.3 wt% in the hot ethanol extracts.     

Development trend and prospect of solid phase extraction technology

Solid phase extraction is widely used in sample pretreatment, concentration and analysis processes due to high selectivity and suitability for low concentration sample system. In this review, we systematically summarized and discussed the development trends of solid phase extraction by bibliometrics method. By analyzing papers output scale, the research and development direction of solid phase extraction technology is prospected. We also give an overview on current strategies of novel solid phase extraction from the separation medium and separation technology. The paper aims to describe the global research profile and the development trends of solid phase extraction, to help researchers to accurately grasp the research trend and to provide support for scientific research institutions to formulate scientific policies and strategic plans. Furthermore, the prospect of the development and application of solid phase extraction is also discussed.     

Lipid extraction from the microalga Phaeodactylum tricornutum

Ethanol was used for the extraction and purification of lipids from the biomass of the microalga Phaeodactylum tricornutum. This microalga is an oil‐rich substrate with a high proportion of eicosapentaenoic acid (EPA). The process consisted of two steps. First, ethanol (96% vol/vol) was used to extract the lipids from the lyophilized biomass. Second, a biphasic system was formed by adding water and hexane to the extracted crude oil. In this way, most of the lipids were transferred to the hexanic phase while most impurities remained in the hydroalcoholic phase. The first step was carried out by two consecutive extractions at room temperature, each with 5 mL ethanol per gram of biomass, for 10 and 1.25 h, respectively. Under these conditions, over 90% of the saponifiable lipids in the biomass were extracted. In the second step, the percentage of water in the hydroalcoholic phase, the hexane/hydroalcoholic phase ratio and the number of extraction steps were optimized. A water content of 40% vol/vol in the hydroalcoholic phase provided the highest lipid recovery. A recovery yield of 80% was obtained by four consecutive extractions with a hexane/hydroalcoholic phase ratio of 0.2 (vol/vol). Equilibrium distribution data of the lipids between the hydroethanolic and the hexanic phases were also obtained in order to predict the lipid recovery yield of the extraction. This process is an alternative to the traditional methods of lipid extraction, which uses less toxic solvents and reduces the total amount of solvents used.     

In-line and off-line trapping of lipids extracted from chicken liver by supercritical carbon dioxide

This supercritical fluid extraction study determined the retentive properties of neutral alumina sorbent as an in-line trap for lipids in the dynamic state over a pressure range of 490–680 bar and temperatures of 40 and 80°C. Lipids were extracted from a chicken liver matrix using supercritical carbon dioxide over a 40-min period at a flow rate of 3 L/min (expanded gas), then were quantified by high-performance liquid chromatography using an evaporative light-scattering detector. Approximately 30 and 18%, respectively, of the total extracted lipids were trapped on the in-line alumina sorbent bed at 40°C as the operating pressure increased from 490 to 680 bar, while the remaining lipids were trapped off-line after CO₂ decompression. The major lipid classes trapped in-line were fatty acids and cholesterol, whereas only minor amounts of the less polar lipid classes such as sterol esters and triacylglycerols were retained. At 80°C and 680 bar, less than 1.5% of the extracted total lipids was trapped in-line, indicating the lack of adsorptive selectivity for lipids by alumina under these conditions.     

Effect of absorbent in solid‐phase extraction on quantification of phospholipids in palm‐pressed fiber

Palm‐pressed fiber (PPF) is a by‐product of palm oil milling and it has been found to contain a high percentage of phospholipids (PL). The aim of this work was to evaluate the best solid‐phase extraction (SPE) method to purify PL from PPF. The purified PL were analyzed using high‐performance liquid chromatography (HPLC) with an evaporative light‐scattering detector (ELSD). The oil was extracted from PPF using the Folch method and classes of PL were purified using SPE. Different solvent phases and normal‐phase SPE cartridges [silica (Si), aminopropyl‐bonded silica (NH₂) and diol‐bonded silica (2OH)] at the same ratio of oil to sorbent mass were used to study the separation of PL from the extracted oil. The recovery of each standard PL was performed in a model oil system with the same amount of standard PL being added, and the repeatability of the SPE method was investigated. The isolation of PL by SPE diol cartridge and subsequent analysis by HPLC/ELSD have shown to be an accurate and reproducible analytical method for the determination of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and lysophosphatidylcholine in PPF. This method provided a high yield and rapid separation of PL in PPF with less contamination from other lipid groups.     

Rapid separation of neutral lipids,free fatty acids and polar lipids using prepacked silica sep-Pak columns

A method is described for the separation of neutral lipid, free fatty acid and polar lipid classes using small (600 mg), prepacked silica Sep-Pak columns. Combinations of hexane and methyltertiarybutylether were used to progressively elute cholesteryl ester first then triglyceride from the column. After column acidification, fatty acids were eluted followed by cholesterol. Recoveries of these lipids were 96% or greater. Polar lipids were eluted from the column using combinations of methyltertiarybutylether, methanol and ammonium acetate. Phospholipid classes could not be separated completely from each other. Phosphatidylethanolamine and phosphatidylinositol eluted together, whereas the more polar phosphatidylcholine, sphingomyelin and lysophosphatidylcholine were eluted as a second fraction. Recoveries of each phospholipid was greater than 98%.     

Rapid characterization of lipids by MALDI MS. Part 1: Bacterial taxonomy and analysis of food oils

Several new methods have been developed recently that allow the direct detection of lipids without resorting to derivatization or chromatographic separation. The simplest of these is direct MALDI (matrix‐assisted laser desorption/ionization) mass spectrometry. This approach is most useful for mixtures that contain minimal amounts of ion‐suppressing interfering components. However, even when such components are present, their effects can often be minimized by using simple separation techniques beforehand, such as solid phase extraction or thin layer chromatography. For example, direct MALDI has been used for rapid screening of lipids and for taxonomic identification of the source organisms with no sample pretreatment. Fractions collected from solid phase extraction cartridges have also been used to avoid the most extreme effects of ion suppression from more complex lipid mixtures. More recently, direct MALDI has been applied to the analysis of TLC plates allowing the detection of TLC‐separated lipids from the complex lipidome. Herein, we briefly describe the application of rapid MALDI MS to some typical research problems involving the characterization of lipids. In Part 1 these include bacterial taxonomy by direct analysis of intact lipids in simple extracts rather than by conversion to fatty acid methyl esters. Food oils such as triacylglycerols can be characterized simply and easily by direct MALDI MS without resort to any sort of separation. Part 2 (in the next issue of Lipid Technology) will cover the spontaneous fragmentation of protonated lipids, ion suppression and the use of solid phase extraction and thin layer chromatography with MALDI MS to characterize complex biological samples.