Fatty acid composition of neutral lipid: Classes of Citrus seed oil

The constituents of total lipids and neutral lipid classes, i.e., hydrocarbons, wax esters, sterol esters, triacylglycerols, free fatty acids, 1,3-diacylglycerols, 1,2-diacylglycerols, free sterols, alcohols, and monoacylglycerols of three species of Citrus namely Citrus sinensis, Citrus paradisi, Citrus aurantium were investigated by thin layer and gas chromatography. Palmitic, oleic and linoleic acids were the major components in all the lipids and lipid classes studied.     

Analysis of human plasma lipids and soybean lecithin by means of high‐performance thin‐layer chromatography and matrix‐assisted laser desorption/ionization mass spectrometry

An improved analytical strategy for the analysis of complex lipid mixtures using matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) in combination with high‐performance thin‐layer chromatography (HPTLC) is reported. Positive ion MALDI RTOF MS was applied as a rapid screening tool for the various neutral (e.g. triacylglycerols) and polar (e.g. glycerophospholipids and ‐sphingolipids) lipid classes derived from crude lipid extracts of e.g. human plasma as well as soybean lecithin. Finally, MALDI seamless post‐source decay (PSD) product ion analysis was performed in order to obtain further structural information (head‐ and acyl‐group identification) of selected lipid species and structure verification. A Coomassie Brilliant Blue R‐250 staining protocol for lipids on HPTLC plates was evaluated and was found to be fully compatible with subsequent MALDI‐MS. Lipids were analyzed after elution from the HPTLC phase material of the selected band (corresponding to certain lipid classes) by using the proper organic solvent mixture or in few cases directly from the HPTLC plates (a type of on‐line HPTLC/MALDI‐MS coupling). More than 70 distinct lipid species from seven different lipid classes in the range between m/z 500 and 1500 could be identified from the lipid extracts of human plasma and soybean lecithin, respectively. The general high sensitivity of MALDI‐MS detection allowed the analysis of even minor lipid classes from only very small volumes of human plasma (50 µL). The combination of HPTLC, Coomassie staining and positive ion MALDI curved field RTOF‐MS represents a straightforward strategy during lipidomics studies of food and clinically relevant human lipid samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Lipid analysis by thin-layer chromatography--a review of the current state

High-performance thin-layer chromatography (HPTLC) is a widely used, fast and relatively inexpensive method of separating complex mixtures. It is particularly useful for smaller, apolar compounds and offers some advantages over HPLC. This review gives an overview about the special features as well as the problems that have to be considered upon the HPTLC analysis of lipids. The term "lipids" is used here in a broad sense and comprises fatty acids and their derivatives as well as substances related biosynthetically or functionally to these compounds. After a short introduction regarding the stationary phases and the methods how lipids can be visualized on an HPTLC plate, the individual lipid classes will be discussed and the most suitable solvent systems for their separation indicated. The focus will be on lipids that are most abundant in biological systems, i.e. cholesterol and its derivates, glycerides, sphingo- and glycolipids as well as phospholipids. Finally, a nowadays very important topic, the combination between HPTLC and mass spectrometric (MS) detection methods will be discussed. It will be shown that this is a very powerful method to investigate the identities of the HPTLC spots in more detail than by the use of common staining methods. Future aspects of HPTLC in the lipid field will be also discussed.     

Separation of acidic and neutral lipids by aminopropyl-bonded silica gel column chromatography.

The separation of acidic and neutral lipids by aminopropyl-bonded silica gel column chromatography is presented. Total lipid extracts from Escherichia coli and human spermatozoa were loaded onto pre-packed aminopropyl-bonded silica gel columns and the lipids separated into four fractions. Non-polar lipids including cholesterol esters, triglycerides, diglycerides, monoglycerides and cholesterol, were eluted with 4 ml of isopropanol-chloroform (1:2, v/v) (fraction 1); free fatty acids were eluted with 4 ml of 2% acetic acid in diethyl ether (fraction 2); neutral polar lipids, including phosphophatidylethanolamine, phosphatidylcholine, sphingomyelin and neutral glycolipids, were eluted with 4 ml of methanol (fraction 3); and, finally, polar acidic lipids, including phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylserine, seminolipid lipid A and acidic glycosphingolipids, were eluted with 4 ml of chloroform-methanol-0.8 M sodium acetate (60:30:4.5, v/V/V) (fraction 4). The recoveries for the different lipids ranged between 89 and 98% and the intra-assay variation, expressed as the standard deviation, was less than 5%.     

Gas chromatographic profiles of plasma total lipids as indicators of dietary history. Correlation with fat intake based on 24-h dietary recall

Fasting plasma total lipid profiles were determined by high-temperature gas chromatography on a total of 1246 free living urban subjects, ages 20-59 years, from the Toronto-McMaster Lipid Research Clinic Population Study. Quantitative estimates of the major molecular species, lipid classes and lipid class ratios were correlated with a total of twelve dietary lipid components, including total saturated and unsaturated fats. oleic and linoleic acids, and cholesterol, to give appropriate Spearman coefficients (rS) and tests of significance (P) for groups of 775 males and 471 females. The intake of the various nutrients was derived from a 24-h dietary recall. The most significant correlations varying from rs +/- 0.1-0.4 and P less than 0.0001-0.0005 were between the intake of total fat, individual saturated and unsaturated fats, and the ratios of C50/C54 triacylglycerols and the C34/C36 phosphatidylcholines, which reflected the nature and quantity of the dietary fat consumed. Increases in dietary cholesterol and saturated fat produced small increases in plasma cholesterol and saturated triacylglycerols, while unsaturated dietary fat produced small decreases in saturated and increases in unsaturated plasma triacylglycerols. These changes in the plasma lipid parameters are consistent with those observed previously in much more limited dietary experiments with accurately known composition of ingested fats. It is, therefore, concluded that direct gas chromatographic profiling of plasma total lipids provides a simple and rapid method of verifying the overall correctness of the dietary recall.     

Non‐polar lipids characterization of Quinoa (Chenopodium quinoa) seed by comprehensive two‐dimensional gas chromatography with flame ionization/mass spectrometry detection and non‐aqueous reversed‐phase liquid chromatography with atmospheric pressure chemical ionization mass spectrometry detection

A chemical characterization of major lipid components, namely, triacylglycerols, fatty acids and the unsaponifiable fraction, in a Quinoa seed lipids sample is reported. To tackle such a task, non‐aqueous reversed‐phase high‐performance liquid chromatography with mass spectrometry detection was employed. The latter was interfaced with atmospheric pressure chemical ionization for the analysis of triacylglycerols. The main triacylglycerols (>10%) were represented by OLP, OOL and OLL (P = palmitoyl, O = oleoyl, L = linoleoyl); the latter was present in the oil sample at the highest percentage (18.1%). Furthermore, fatty acid methyl esters were evaluated by gas chromatography with flame ionization detection. 89% of the total fatty acids was represented by unsaturated fatty acid methyl esters with the greatest percentage represented by linoleic and oleic acids accounting for approximately 48 and 28%, respectively. An extensive characterization of the unsaponifiable fraction of Quinoa seed lipids was performed for the first time, by using comprehensive two‐dimensional gas chromatography with dual mass spectrometry/flame ionization detection. Overall, 66 compounds of the unsaponifiable fraction were tentatively identified, many constituents of which (particularly sterols) were confirmed by using gas chromatography with high‐resolution time‐of‐flight mass spectrometry.     

Lipid classes of in vitro cultivar of Pakistani citrus

The in vitro cultivar of citrus contained 18.0% lipids after 12 weeks of germination of seed. The lipid was analyzed by GC procedure for its fatty acid composition. The oil contained seven major fatty acid constituents which were later identified by GC. The oil was further analyzed for its classes by means of thin layer chromatography and gas chromatography. The major portion of the lipid classes comprised of neutral lipids (93.9%) and polar lipids (6.1%). The identified neutral lipids comprised of hydrocarbon (1.4%), wax esters (1.5%), sterol esters (5.2%), triglycerides (52.3%), free fatty acids (1.3%), 1,3-diglycerides (6.0%), 1,2-diglycerides (5.0%), glycol (15.2%), sterols (6.0%), 2-monoglycerides (6.4%), 1-monoglycerides (5.3%) and the identified polar lipids comprised of phosphatidyl ethanolamine (1.8%) phosphatidyl choline (0.9%) lysophosphatidyl ethanolamine (1.8%) and phosphatidyl inositol (1.1%).     

A quantitative densitometric method for the rapid separation and quantitation of the major tissue and lipoprotein lipids by high-performance thin-layer chromatography. I. Sample preparation, chromatography, and densitometry

A rapid method for the separation and quantitation of the major lipids of tissues and lipoproteins by automated high-performance thin-layer chromatography is presented. Solvent systems for one-dimensional separation of neutral lipids, of cholesteryl esters, and of phospholipids are described. Separated lipids are measured following treatment with methanolic sulphuric acid containing manganese chloride and scanned in fluorescence or absorption mode. Absolute quantitation is obtained by the use of an internal standard and by references to standards for each lipid run on the same plates as samples. The method described here is particularly suitable for the rapid quantitation of small amounts of lipid (0.01-0.02 nmol per sample), for example in tissue culture studies; 100 micrograms of fibroblast or macrophage protein are sufficient for complete lipid analysis. The coefficients of variation due to the sample preparation, application to the plates and densitometry are in the range 7.2-9.1%. The method was compared with enzymatic determinations for cholesterol and gave correlation coefficients of 0.95 for total cholesterol and 0.91 for unesterified cholesterol. Phospholipid estimation was compared with large-plate thin-layer chromatography and phosphorus analysis and gave correlation coefficients of 0.90 for phosphatidylcholine and 0.89 for sphingomyelin.     

Gas chromatographic profiles of plasma total lipids as indicators of dietary history. Correlation with carbohydrate and alcohol intake based on 24-h dietary recall.

Quantitative gas chromatographic estimates of the major lipid classes and molecular species in fasting plasma were correlated with total carbohydrate, starch, fibre, sucrose and alcohol intake based on 24-h dietary recall. Spearman coefficients (rs) and tests of significance (P) were obtained for groups of 775 males and 471 females aged 20-59 years from a Toronto-McMaster Lipid Research Clinics Population Study. The most significant correlations varying from rs 0.1 to 0.2 and P 0.001 to 0.0005 (n = 400-773) were between increased intake of alcohol and increased ratios of C50/C54 triacylglycerols, C34/C36 phosphatidylcholines and phosphatidylcholine/free cholesterol (PC/FC) of plasma. Increase in total dietary carbohydrate, starch and fibre correlated with decreasing C50/C54 triacylglycerol, C34/C36 phosphatidylcholine and PC/FC ratios (rs = -0.1-0.2; P less than 0.002-0.04; n = 400-773). In contrast, consumption of high levels of alcohol was associated with increasing C50/C54 triacylglycerol, C34/C36 phosphatidylcholine and PC/FC ratios. A high intake of alcohol (50-150 ml per day) distinguished itself from other simple carbohydrate-induced lipid profiles by its marked effect on increased C50/C52 triacylglycerol and PC/FC ratio.     

Hypertension does not stimulate the development of hypercholesterolemia or fatty liver induced by a high cholesterol/cholate diet in rats

A high cholesterol/cholate diet induced hypercholesterolemia and fatty liver in both spontaneously hypertensive rats (SHR) and normotensive control rats (WKY). However, in contrast to previous concepts, the levels of cholesterol ester, triacylglycerol and phosphatidylcholine in plasma as well as triacylglycerol in liver were higher in WKY than in SHR fed a normal diet. The high cholesterol/cholate diet elevated the levels of plasma cholesterol, plasma cholesterol ester and hepatic triacylglycerol, and the extent of elevation was significantly higher in WKY than in SHR. Increases both in monoene/saturated ratios, an indication of elevated delta 9-desaturase activity, and in linoleate/arachidonate ratios, a possible indication of impaired desaturation-elongation activity, were observed in hepatic and plasma lipids of both strains fed the high cholesterol/cholate diet. The increases in monoene/saturated ratios were similar in both strains, but the increases in the linoleate/arachidonate ratios were higher for the plasma cholesterol esters of WKY than of SHR. The n-6/n-3 ratios of plasma and hepatic lipids were higher in WKY than in SHR throughout the experiments. These diet-induced changes observed in hepatic and plasma lipids were not reflected in the aortic lipids. Thus, hypertension per se does not promote the development of hyperlipemia and fatty liver induced by a high cholesterol/cholate diet. Our results also suggest that the metabolism of polyenoic fatty acids is different between SHR and WKY.     

Quantification of major lipid classes in human triacylglycerol-rich lipoproteins by high-performance liquid chromatography with evaporative light-scattering detection

Triacylglycerol-rich lipoproteins (TRL), comprising chylomicrons (CM) and very-low-density lipoproteins (VLDL), have been associated with cardiovascular disease. The lipid class content in the remnant particles of these lipoproteins is a determinant for the accumulation of lipids in macrophages and their transformation into foam cells. We have optimized a method for the simultaneous determination of cholesteryl esters (CE), triacylglycerols (TG), free cholesterol (FC), monoacylglycerols (MG), and phospholipids (PL) by HPLC coupled to a light-scattering detector (ELSD). A diol column and a ternary gradient of hexane, 2-propanol, and methanol were applied to CM and VLDL of human origin (n = 10), with excellent precision in terms of repeatability of peak areas and retention times. All peaks were baseline resolved although the resolution of CE and TG was compromised for the sake of simplicity of the solvent gradient. The ELSD response was fitted to second-order equations, with correlation coefficients (r2) higher than 0.999 for a wide range of concentrations (0.25-10 microg of lipid injected). TG were the major lipid class detected in human TRL, accounting for 62% in CM obtained 2 h after the oil intake. In addition we recorded a depletion of TG and CE in CM obtained 2 h after the oil intake of about 60%. We conclude that the method reported here is suitable for a rapid and precise determination of lipid classes in human TRL and, therefore, may be a useful tool for investigations on the atherogenicity of these lipoproteins.     

Automated separation and quantitation of lipid fractions by high-performance liquid chromatography and mass detection

This report describes an improved separation and quantitation of lipid fractions in a total lipid extract by high-performance liquid chromatography using a modified solvent and gradient system delivered by dual pumps and incorporating a mass detector and autosampler. The detector responses for various lipid fractions (cholesteryl esters, triacylglycerols, free cholesterol, and seven major phospholipid classes) were fitted to a quadratic equation, y = ax2 + bx + c, and quantified after detector calibration by a computer. This new system has the advantage of automation and reproducible separation. The present method was applied to rat liver analysis.     

Lipidomic-based investigation into the therapeutic effects of polyene phosphatidylcholine and Babao Dan on rats with non-alcoholic fatty liver disease

In recent years, with the improvement of people’s living standards, non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in the world. In this paper, the metabolic disorders in Sprague Dawley (SD) rats were induced by a choline-deficient, l -amino acid–defined (CDAA) diet. The therapeutic effects of polyene phosphatidylcholine (PPC) and Babao Dan (BBD) on NAFLD were observed. Lipidomic analysis was performed using ultra-high-performance liquid chromatography-Orbitrap MS, and data analysis and lipid identification were performed using the software LipidSearch. Both PPC and BBD can reduce lipid accumulation in the liver and improve abnormal biochemical indicators in rats, including reduction of triglycerides, total cholesterol, alanine transaminase and aspartate transaminase in serum. In addition, lipids in rat serum were systematically analyzed by lipidomics. The lipidomic results showed that the most obvious lipids with abnormal metabolism in CDAA diet–induced rats were glycerides (triglycerides and diacylglycerols), phospholipids and cholesterol esters. Both BBD and PPC partly reversed the disturbance to lipids induced by the CDAA diet. PPC may be more effective than BBD in alleviating NAFLD because it has a better effect on inhibiting the abnormal accumulation of lipids and reducing the inflammatory reaction in the body.     

Compressibility study of quaternary phospholipid blend monolayers

The mechanical properties of liposome membranes are strongly dependent on type and ratio of lipid compounds, which can have important role in drug targeting and release processes when liposome is used as drug carrier. In this work we have used Brewster's angle microscopy to monitor the lateral compression process of lipid monolayers containing as helper lipids either distearoyl phosphatidylethanolamine (DSPE) or dioleoyl phophatidylethanolamine (DOPE) molecules on the Langmuir trough. The compressibility coefficient was determined for lipid blend monolayers containing the helper lipids above, cholesterol, distearoyl phosphatidylcholine (DSPC) and pegylated-DSPE at room temperature. Two variables, the cholesterol fraction and the ratio ρ between the helper lipid (either DSPE or DOPE) and the reference lipid DSPC, were studied by multivariate analysis to evaluate their impact on the compressibility coefficient of the monolayers. The cholesterol level was found to be the most significant variable for DSPE blends while the ratio ρ was the most significant one for DOPE blend monolayers. It was also found that these two variables can exhibit positive interaction and the same compressibility value can be obtained with different blend compositions.     

Fluorescence detection by intensity changes for high-performance thin-layer chromatography separation of lipids using automated multiple development

Changes in emission of berberine cation, induced by non-covalent interactions with lipids on silica gel plates, can be used for detecting and quantifying lipids using fluorescence scanning densitometry in HPTLC analysis. This procedure, referred to as fluorescence detection by intensity changes (FDIC) has been used here in combination with automated multiple development (HPTLC/AMD), a gradient-based separation HPTLC technique, for separating, detecting and quantifying lipids from different families. Three different HPTLC/AMD gradient schemes have been developed for separating: neutral lipid families and steryl glycosides; different sphingolipids; and sphingosine-sphinganine mixtures. Fluorescent molar responses of studied lipids, and differences in response among different lipid families have been rationalized in the light of a previously proposed model of FDIC response, which is based on ion-induced dipole interactions between the fluorophore and the analyte. Likewise, computational calculations using molecular mechanics have also been a complementary useful tool to explain high FDIC responses of cholesteryl and steryl-derivatives, and moderate responses of sphingolipids. An explanation for the high FDIC response of cholesterol, whose limit of detection (LOD) is 5 ng, has been proposed. Advantages and limitations of FDIC application have also been discussed.     

Thin Layer Chromatographic Determination of Neutral Lipids and Phospholipids in Biomphalaria Glabrata Snails Fed Lettuce Versus Hen's Egg Yolk as a Function of Time

Abstract

TLC on HP preadsorbent silica gel plates was used to determine, at various times over a 21-day period, neutral lipids and phospholipids in the plasma and digestive glandgonad complex (DGG) of Biomphalaria glabrata snails maintained on diets of leaf lettuce and hen's egg yolk. The DGG of yolk-fed snails showed considerable elevation of triacylglycerols by day 3, and free fatty acids and sterols by day 5. Hemolymph from two pools of yolk-fed snails was analyzed on day 7, and the respective results for free fatty acids, sterols, and triacylglycerols were 0.036, 0.0023, and 0.028 g/dL. Comparable values in lettuce-fed snails were 0.0061 for free fatty acids, 0.0016 for sterols, and triacylglycerols were not detected. Semiquantitative TLC showed that compared to lettuce-fed snails, the plasma of yolk-fed snails had elevations in sterols, free fatty acids, triacylglycerols, and phospholipids from days 5 to 21. The DGG of snails fed both diets showed approximately equal amounts of phosphatidylcholine and phosphatidylserine throughout the 21 days, but phosphatidylethanolamine was elevated in the DGG of yolk-fed snails by day 5. All phospholipid fractions were elevated in the plasma of yolk-fed snails during the 21-day period, with plasma from lettuce-fed snails showing only negligible amounts of phospholipids.     

Quantification of brain lipids by FTIR spectroscopy and partial least squares regression

Brain tissue is characterized by high lipid content. Its content decreases and the lipid composition changes during transformation from normal brain tissue to tumors. Therefore, the analysis of brain lipids might complement the existing diagnostic tools to determine the tumor type and tumor grade. Objective of this work is to extract lipids from gray matter and white matter of porcine brain tissue, record infrared (IR) spectra of these extracts and develop a quantification model for the main lipids based on partial least squares (PLS) regression. IR spectra of the pure lipids cholesterol, cholesterol ester, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, galactocerebroside and sulfatide were used as references. Two lipid mixtures were prepared for training and validation of the quantification model. The composition of lipid extracts that were predicted by the PLS regression of IR spectra was compared with lipid quantification by thin layer chromatography.     

Interactions of Triton X-100 with sphingomyelin and phosphatidylcholine monolayers: influence of the cholesterol content

The presence of microdomains, called lipid rafts, in biological membranes is usually explained by lateral segregation between specific lipids and proteins. These rafts present similarities with the membrane domains isolated by their non-ionic detergent-resistance at 4 degrees C. They are enriched in sphingomyelin and cholesterol as compared with the outer leaflet of eukaryotic cell membranes. To understand the role played by the lipids enriched in rafts in their resistance to solubilization by detergents, the interactions between these lipids and the non-ionic detergent Triton X-100 were studied by using different lipid monolayers at the air-water interface. The influence of Triton X-100 on the Langmuir isotherms (i.e. surface pressure/area isotherms) of monolayers containing sphingomyelin and cholesterol at different mole ratios was analyzed and the results were compared with the influence of Triton X-100 on monolayers containing a phosphatidylcholine bearing a saturated and an unsaturated fatty acid (i.e. palmitoyloleylphosphatidylcholine) and cholesterol. This phosphatidylcholine was chosen since the phosphatidylcholines present in rafts isolated from bovine kidney could contain about 50% of saturated fatty acids. Triton X-100 induces an increase in the condensing effect observed as compared with ideal mixture of phospholipid/cholesterol. Triton X-100-induced changes in the morphology of the monolayers were visualized by Brewster angle microscopy, which confirmed the differences of behavior observed by analyzing the isotherms.     

Differential effects of cholesterol, ergosterol and lanosterol on a dipalmitoyl phosphatidylcholine membrane: a molecular dynamics simulation study

Lipid raft/domain formation may arise as a result of the effects of specific sterols on the physical properties of membranes. Here, using molecular dynamics simulation, we examine the effects of three closely-related sterols, ergosterol, cholesterol, and lanosterol, at a biologically relevant concentration (40 mol %) on the structural properties of a model dipalmitoyl phosphatidylcholine (DPPC) membrane at 309 and 323 K. All three sterols are found to order the DPPC acyl tails and condense the membrane relative to the DPPC liquid-phase membrane, but each one does this to a significantly different degree. The smooth alpha-face of ergosterol, together with the presence of tail unsaturation in this sterol, leads to closer interaction of ergosterol with the lipids and closer packing of the lipids with each other, so ergosterol has a higher condensing effect on the membrane, as reflected by the area per lipid. Moreover, ergosterol induces a higher proportion of trans lipid conformers, a thicker membrane, and higher lipid order parameters and is aligned more closely with the membrane normal. Ergosterol also positions itself closer to the bilayer/water interface. In contrast, the rough alpha-face of lanosterol leads to a less close interaction of the steroid ring system with the phospholipid acyl chains, and so lanosterol orders, straightens, and packs the lipid acyl chains less well and is less closely aligned with the membrane normal. Furthermore, lanosterol lies closer to the relatively disordered membrane center than do the other sterols. The behavior of cholesterol in all the above respects is intermediate between that of lanosterol and ergosterol. The findings here may explain why ergosterol is the most efficient of the three sterols at promoting the liquid-ordered phase and lipid domain formation and may also furnish part of the explanation as to why cholesterol is evolutionarily preferred over lanosterol in higher-vertebrate plasma membranes.     

High Resolution Separation of Non-Polar Lipid Classes by HPLC-ELSD Using Alumina as Stationary Phase

This study describes the performance and capacity of alumina as stationary phase in an HPLC-ELSD (evaporative light-scattering detection) method optimized for the separation of the non-polar lipid classes hydrocarbons, wax esters, sterol esters, triacylglycerols, and sterols, including quantitative determination of these lipid classes in natural samples. By using gradient elution and constant equilibration times between injections, highly reproducible separations of triacontane, stearyl oleate, and cholesterol oleate were accomplished with a binary mobile phase system. Phase A contained 0.5% tetrahydrofuran in hexane and phase B 20% isopropanol and 20% tetrahydrofuran in hexane. The same system was also used to determine the non-polar lipid classes in a zooplankton sample, the major lipid class being wax esters, followed by triacylglycerols, sterol esters, sterols, and hydrocarbons. Substantial amounts of an unknown compound, possibly acylated glyceryl ethers, were also found. The equilibration time of alumina was relatively slow compared to a polyvinyl alcohol stationary phase used earlier by the authors and calibration curves for different lipid classes were more uniform and linear with alumina.