Cost effective,robust, and reliable coupled separation techniques for the identification and quantification of phospholipids in complex biological matrices: Application to insects

The quantification of phospholipid classes and the determination of their molecular structures are crucial in physiological and medical studies. This paper's target analytes are cell membrane phospholipids, which play an important role in the seasonal acclimation processes of poikilothermic organisms. We introduce a set of simple and cost‐effective analytical methods that enable efficient characterization and quantification of particular phospholipid classes and the identification and relative distribution of the individual phospholipid species. The analytical approach involves solid‐phase extraction and high‐performance thin‐layer chromatography, which facilitate the separation of particular lipid classes. The obtained fractions are further transesterified to fatty acid methyl esters and subjected to gas chromatography coupled to flame ionization detection, which enables the determination of the position of double bonds. Phospholipid species separation is achieved by high‐performance liquid chromatography with mass spectrometry, which gives information about the headgroup moiety and attached fatty acids. The total content of each phospholipids class is assessed by phosphorus determination by UV spectrophotometry. The simultaneous analysis of phosphorus, fatty acid residues, and phospholipid species provides detailed information about phospholipid composition. Evaluation of these coupled methods was achieved by application to an insect model, Pyrrhocoris apterus. High correlation was observed between fatty acid compositions as determined by gas chromatography and high‐performance liquid chromatography analysis.     

红细胞膜上各种磷脂中脂肪酸的测定

应用自制硅胶板的薄层色谱法分离了红细胞膜上四种主要磷脂。将分离后的每种磷脂斑点硅胶涂层刮下，不经萃取，直接在无水甲醇－苯－乙酰氯溶液中进行转移甲基化，然后用毛细管相色谱分离测定其脂肪酸组成和含量。上述方法已成功地用于先天愚型病人和正常人红细胞膜上各种磷脂中脂肪酸轮廓分析，获得有意义的结果。     

Quantification of fatty acids as methyl esters and phospholipids in cheese samples after separation of triacylglycerides and phospholipids

Determination of the individual fatty acid composition of neutral- and phospholipids as well as the phospholipid content of dairy food and other foodstuffs are important tasks in life sciences. For these purposes, a method was developed for the separation of lipids (standards of triolein and diacylphosphatidylcholines as well as three cheese samples) by solid-phase extraction using a self-packed column filled with partly deactivated silica. Non-halogenated solvents were used for the elution of the lipid classes. Cyclohexane/ethyl acetate (1:1, v/v) served for the elution of neutral lipids, while polar lipids were eluted with three solvents (ethyl acetate/methanol, methanol, and methanol/water) into one fraction. The separated lipid fractions were transesterified and the individual fatty acids were quantified by using gas chromatography coupled to electron ionization mass spectrometry (GC/EI-MS) in the selected ion monitoring (SIM) mode. The recovery rate for standard phosphatidylcholines was ∼90% and cross-contamination from neutral lipids was negligible. The method was applied to cheese samples. Quantitative amounts of individual fatty acids in the phospholipid fraction were <0.002-0.29% of total lipids from camembert, <0.002-0.12% of total lipids from mozzarella, and <0.002-0.18% of total lipids in a goat cream cheese. Differences in the fatty acid pattern of neutral and polar lipids were detected. The quantity of the fatty acids determined in the phospholipid fraction was divided by the factor 0.7 in order to convert the fatty acid content into the phospholipid content of the cheese samples. This factor is based on the contribution of 16:0 to dipalmitoylphosphatidylcholine (DPPC). The resulting DPPC equivalents (DPPC_eq) were found to be representative for the average contribution of fatty acids to all classes of phospholipids in dairy products. Using this approach, the phospholipid content of lipids from mozzarella, camembert, and goat cream cheese was 0.60%, 1.42% and 0.79%, respectively.     

Fatty acid composition of human erythrocyte membranes by capillary gas chromatography-mass spectrometry

Capillary gas chromatographic and gas chromatographic--mass spectrometric methods were employed for profiling total fatty acid content of human erythrocyte membranes. The protocol was designed to efficiently separate, identify, and accurately quantify the fatty acid composition in human erythrocyte membranes. Washed erythrocyte "ghosts" were saponified in aqueous methanolic sodium hydroxide solution and methylated with boron trichloride and acid catalysis. Extracted total fatty acid methyl esters (FAMEs) were analyzed using a highly polar cyanopropylsiloxane SP 2560 fused-silica capillary column. Total run time was 55 min, and 45 FAMEs were tentatively identified by relative retention times compared to those of known FAMEs. Confirmation of identities by mass spectral structure elucidation revealed saturated, mono- and polyunsaturated, and branched-chain FAMEs. The presence of four fatty aldehydes was also confirmed as dimethyl acetal derivatives. Identification of cis/trans isomers was based on relative retention times and characteristic profile of the cis/trans FAME standard. Quantification of FAMEs for normal subjects showed some variation in relative amounts, consistent with expectations based on literature reports on total or phospholipid FAMEs from human erythrocytes. Separation of individual components of fatty acid families (n-3), (n-6), and (n-9) is demonstrated. Losses in relative amounts of polyunsaturated fatty acids upon storing samples were also detectable by this rapid method.     

兔骨骼肌肌质网磷脂组分及脂肪酸的分析

分析了兔骨骼肌肌质网磷脂的组成及摩尔分数,并用气相色谱法对两种主要磷脂的脂肪酸组成及摩尔分数进行了测定。     

兔骨骼肌肌质网磷脂组分及脂肪酸的分析

 分析了兔骨骼肌肌质网磷脂的组成及摩尔分数,并用气相色谱法对两种主要磷脂的脂肪酸组成及摩尔分数进行了测定。     

Lipid composition of membranes of Escherichia coli by liquid chromatography/tandem mass spectrometry using negative electrospray ionization

A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method using reversed-phase chromatography was developed for the analysis of phospholipids from bacterial extracts of a wild-type strain of Escherichia coli. Product ion mass spectra from [M--H](-) precursor ions allowed an identification of individual phospholipid species that includes both fatty acid composition and fatty acyl location on the glycerol backbone using diagnostic product ions. Thus, complete assignment, including sn-1/sn-2 fatty acyl position, was achieved for this strain of E. coli. In addition, the phospholipids were quantified relative to one another using an internal standard method.     

Separation and quantitation of fatty acids by high-performance liquid chromatography

To facilitate the determination of the fatty acid composition of tissues and the investigation of fatty acid metabolism, we developed a method for the rapid separation by high-performance liquid chromatography and quantitation (by ultraviolet light absorption) of p-bromophenyl esters of fatty acids which vary in chain length from 10 to 22 carbon atoms. The utility of the method was demonstrated by evaluating the fatty acid composition of human uterine decidua vera tissue and human endometrial stromal cells that are maintained in monolayer culture.     

Fatty acid profiling of blood cell membranes by gas chromatography with mass spectrometry

Fatty acids, which are well‐known for their influence on human metabolism and signal transduction, are also a substantial component of cellular membranes and regulate the basic properties and functions of membranes. Owing to their multiple functions, fatty acid profiles of cell membranes are of great interest to those who are studying the relationship between membrane biochemical compositions and functions. A HCl‐catalyzed derivation method and a gas chromatography with mass spectrometry analysis method were developed to accurately profile the fatty acids in cell membranes of erythrocytes, leukocytes, and platelets. The detection limits of all 35 fatty acids ranged from 0.58 to 22 ng/mL and the limits of quantitation were between 2.1 and 72 ng/mL. Finally, the established method was used to profile the membrane fatty acids of 44 healthy volunteers from the north and south of China. Results revealed significant differences in the fatty acid profiles from the two regions, particularly those of the erythrocytes. This technique may be applied to cell membrane studies to generate new biological hypotheses concerning fatty acid composition and membrane functions as well as to construct related disease profiles.     

Profiling and quantifying polar lipids in milk by hydrophilic interaction liquid chromatography coupled with evaporative light-scattering and mass spectrometry detection

In this work, a high-performance liquid chromatography with evaporative light scattering detection method has been developed and applied for quantification of the polar content of the lipid fraction in milk samples of different origin. From a chromatographic stand-point, a 4.6-mm I.D. hydrophilic interaction liquid chromatography column was employed to attain a baseline separation of major phospholipid classes contained in the various milk samples tested. Quantitative analysis was performed by the external calibration method using reference material solutions in the 5–100 mg/L concentration range. Analytical recoveries ranging from 57 to 100 %, and repeatability data lower than 8.04 % were obtained on a skimmed cow’s milk sample. The crude cow milk was the most abundant (0.04 %) in phospholipids and donkey milk was the poorest (0.004 %). Quantitative differences were determined in the phospholipid content of the milk samples tested. Finally, characterization of phospholipid profile and fatty acid composition of the different samples was carried out by an ion trap-time of flight mass spectrometer and gas chromatography coupled to flame ionization and mass spectrometry detection. A thorough screening of the polar lipid composition of milk samples of different origin is here outlined, for the first time.     

Determination of phospholipid fatty acid and triacylglycerol composition of rat caecal mucosa.

The lipid composition of rat caecal mucosa, including the fatty acid composition of phospholipids and triacylglycerols, has been examined by capillary gas chromatography. Thirty-seven peaks were resolved, ranging in chain length from 12 to 24 carbon atoms. Preliminary identification of fatty acids by comparison with authentic standards was confirmed by gas chromatography-mass spectrometry using electron-impact ionization. The neutral and polar components were examined. Fatty acid methyl esters were quantified in absolute amounts with respect to the percentage of total phospholipid and triacylglycerols. The results show significantly higher levels of 16:0, 18:0, 18:1(n-9), 18:1(n-7), 18:2(n-6) and 20:4(n-6) in phospholipids, and higher levels of 16:0, 18:1(n-9) and 18:2(n-6) in triacylglycerols. On the other hand, analysis of caecal triacylglycerols revealed sn-glycerol-palmitate-oleate-palmitate, sn-glycerol-palmitate-linoleate-palmitate and sn-glycerol-palmitate-linoleate-oleate as major components.     

Fatty acid composition of glycerophospholipids in seven tissues of cod (Gadus morhua), determined by combined high-performance liquid chromatography and gas chromatography

A method for the separation from fish tissues of the four main glycerophospholipid classes, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine, using adsorption high-performance liquid chromatography with ultraviolet detection and consecutive gas chromatographic analysis, based on one injection for their fatty acid compositions, is described. Fatty acid 19:0 was used as an internal standard for the calculation of the relative concentrations of the phospholipids. The patterns of fatty acid distribution within each of the phospholipids from seven cod organs had some general similarities. Phosphatidylcholine had the highest levels of 16:0 and 18:2 n - 6, and the ratio of 20:5 to 22:6 varied between 0.5 and 0.9. Phosphatidylethanolamine had the highest total polyunsaturated fatty acids, (n -3) polyunsaturated fatty acids, and 22:6 n -3, and the ratio of 20:5 to 22:6 varied between 0.2 and 0.5. Phosphatidylinositol showed the highest level of 18:0 and 20:4 n - 6 and had the lowest ratio of (n - 3) to (n - 6). Phosphatidylserine had the highest ratio of (n - 3) to (n - 6) and the lowest ratio of 20:5 to 22:6. A generally low level (less than 1.5%) of the long-chain monoene, 22:1, was found in the phospholipids in all tissues.     

Measurement of polyunsaturated fatty acids of myocardial lipids by high performance liquid chromatography

Summary This report describes a modified method for the separation and analysis of polyunsaturated fatty acids such as 20:4, 20:5, and 22:6, using HPLC. The results show that these fatty acids are well separated from the saturated acids. Since the unsaturated fatty acids elute earlier than saturated acids, and this method does not require the fractionation of free fatty acids using thin layer chromatography, a necessary step for the gas chromatographic analysis, the recoveries of polyunsaturated fatty acids were significantly higher as compared to those from gas chromatography. Furthermore, HPLC and gas chromatographic methods gave identical results for the acyl chain composition of phosphatidylserine. The advantages of using HPLC over gas chromatography in determining the acyl chain composition of free fatty acids and phospholipids are also discussed.     

Improvement of a solid phase extraction method for analysis of lipid fractions in muscle foods

The most used method for muscle lipid fractionation into major lipid classes was modified for improving its separation efficiency. Extracted lipids from a masseter muscle of one Iberian pig were separated into neutral lipids (NL), free fatty acids (FFA) and polar lipids (PL) using aminopropyl minicolumns, following the extensively used method of Kaluzny et al. [1] (old method-OM-) and a method based on that, developed by Pinkart et al. [2] with some (modifications modified method–MM). Obtained lipid classes were further analysed by TLC and lipid fractions were identified. TLC evidenced the presence of a certain amount of PL in the NL fraction obtained with the OM. On the other hand, using the MM only an almost undetectable presence of PL was evidenced in the NL fraction. Fatty acid composition of NL, PL and FFA obtained with each method was studied by gas chromatography. Fatty acid profile of NL was strongly influenced by the separation method used. Thus, NL obtained using the OM showed higher amounts of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) and lower of monounsaturated fatty acids (MUFA) than those obtained using the MM. Moreover, NL obtained using the OM showed the presence of fatty alcohols, constituents of phospholipids (PhL) absent or present only in trace amounts in acylglycerols. This profile reflects the coelution of PL in the NL fraction. Fatty acid profile of FFA and PL fractions was also influenced by the solid phase extraction (SPE) method used, but to a lesser extent.     

Study of fatty acid profiles in cancer cells grown in culture using gas chromatography--mass spectrometry

The determination of long-chain fatty acids in the phospholipid, triglyceride and free fatty acid fractions of HT29/219 colon cancer cells grown in a medium containing either foetal calf serum or horse serum, was carried out using gas chromatography - mass spectrometry. Several bonded-phase capillary columns were tested for the separation of the fatty acid methyl esters, and a 30-m poly(ethylene glycol) column was found to give optimum separation. The mass spectrometer was set to the multiple ion detection mode to increase the sensitivity of the recording of the characteristic ions, consisting of the molecular ion and the base peak. The phospholipid and triglyceride compositions of the cells were different when the cells were grown in media containing different sera. Differences were also found in the turnover of the acids in the different lipid fractions, the phospholipids being the most important, when the cells were grown in different media. The cellular metabolism and turnover of certain fatty acids differed from others in the same cell. These differences emphasise the importance of a precise knowledge of the lipid composition of the culture medium in in vitro studies of cancer cells.     

Two-dimensional analysis of phospholipids by capillary liquid chromatography/electrospray ionization mass spectrometry

A phospholipid mixture extracted from cultured cells was directly analyzed by capillary (Cap) liquid chromatography (LC)/electrospray ionization (ESI) mass spectrometry (MS). Using a quadrupole mass spectrometer, we analyzed positive molecular ions, negative molecular ions, positive fragment ions and negative fragment ions under four different functions. In the analysis of the elution patterns of the phospholipids, a two-dimensional map, in which the first dimension is elution time and the second dimension is mass, proved useful. Consequently, four different maps can be obtained by each of four different functions. Among them, from negative fragment ions at high cone voltage in the negative ion mode, ions that originated from acyl fatty acid and phosphorylcholine, phosphorylethanolamine and cyclic inositol phosphate can be detected at specific elution times. The map from positive fragment ions at high cone voltage in the positive ion mode indicated ions such as diradylglycerol and derivatives of 1-alkyl or 1-alkenyl cyclic phosphatidic acid from phosphatidylethanolamine (PE), and phosphorylcholine from choline-containing phospholipids. The map produced from positive molecular ions indicated choline-containing phospholipids such as phosphatidylcholine, sphingomyelin, lysophosphatidylcholine and PE. The map of negative molecular ions effectively indicated acidic phospholipids such as phosphatidylinositol. We were able to obtain more than 500 molecular species of phospholipids by this method within a few hours immediately after extraction from culture cells using a mixture of chloroform and methanol (2:1). In this context, we concluded that the combination of Cap-LC and ESIMS seems to be very effective in the analysis of phospholipid classes and their molecular species.     

A new high-performance liquid chromatographic method with evaporative light scattering detector for the analysis of phospholipids. Application to Iberian pig subcutaneous fat

A new method for the analysis of phospholipids by normal-phase HPLC is described using a silica column. Addition of ammonia and triethylamine to a gradient based on chloroform/methanol/water promoted a good and rapid separation of phospholipid classes (20 min run). The use of an evaporative light scattering detector permitted an accurate analysis of a mixture of phospholipids. Calibration curves were linear within different range for each phospholipid class. The LOD and LOQ obtained were below 0.03 and 0.05 mg kg⁻¹ for all cases, respectively. Besides, a new method for the separation of phospholipids from total lipids before HPLC analysis by a solid-phase extraction (SPE) with Si cartridges has been developed. This methodology gave a good recovery ranging from 97 to 117%. The method was validated with a standard mixture of phospholipids. This method has been applied to characterize the phospholipid fraction of subcutaneous fat from Iberian pig. Cardiolipin, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin have been described for first time in these samples. The fatty acid composition of the different phospholipid classes and their HPLC electrospray ionization mass spectrometry have been used for characterizing the molecular species present in each one.     

Characterization of medieval proteinaceous painting media using gas chromatography and gas chromatography — mass spectrometry

Proteinaceous organic materials used as ancient painting media were investigated by capillary gas chromatography (GC) and capillary gas chromatography — mass spectrometry (GC/MS). Medieval wall paintings made by the tempera technique were considered and their binding media were studied by the characterization of their main chemical components. The basic methodology is based on the determination of amino acids in samples of paint layers after hydrolysis and derivatization and on the comparison with reference proteinaceous materials. Multivariate chemometric techniques were used to facilitate the recognition of the protein source from chromatographic data. To characterize the binders further, a method was developed for the determination of fatty acids, present as minor components, by GC/MS. The use of fused-silica capillary columns coated with selected stationary phases allowed the separation of amino acid and fatty acid derivatives in a single analytical run.     

High-performance liquid chromatography with evaporative light-scattering detection for the determination of phospholipid classes in human milk,infant formulas and phospholipid sources of long-chain polyunsaturated fatty acids

We developed and validated a new high-performance liquid chromatographic method for the separation of phospholipid classes in human milk, infant formulas and phospholipidic sources of long-chain polyunsaturated fatty acids (LC-PUFAs) used in paediatric nutrition. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin were separated in less than 25 min using an Extrasil silica column (150 x 4.0 mm I.D., 3-microm particle size) by isocratic elution with a mixture of isopropanol-hexane-water. Phospholipids were determined by an evaporative light-scattering detector. Several chromatographic conditions were assayed to optimise the method, whose suitability is shown by the detection limits, linearity ranges and precision rates obtained. The main advantages of the proposed method are its speed and the direct determination of the main phospholipids present in human milk, infant formulas and the phospholipid sources of LC-PUFAs used in paediatric nutrition.     

Profiling of plasma cholesterol ester and triglyceride fatty acids as their methyl esters by capillary gas chromatography, preceded by a rapid aminopropyl-silica column chromatographic separation of lipid classes

A rapid procedure for the isolation of plasma cholesterol ester and triglyceride fractions with aminopropyl-silica columns, followed by analysis of their fatty acid compositions by capillary gas chromatography with flame ionization detection, is described. Within-series and long-term (six months) series-to-series precision were investigated. The isolation procedure caused minimal cross-over between the two lipid classes. Reference values for 57 apparently healthy Dutch adults were established and compared with data reported from other countries. Feeding of rats with four diets differing in their fatty acid compositions showed the relationship between the composition of the fatty acids in the diet and those esterified to cholesterol in plasma. The method is of potential usefulness to the establishment of the compliance of dietary interventions and basic dietary experiments.