Quantitative analysis of lipid classes by liquid chromatography via a flame ionization detector

A method is described for the direct quantitative analysis of the lipid classes of mammalian tissue lipids using high performance liquid chromatography (HPLC) with a flame ionization detector (FID). The lipid is extracted from the tissue with chloroform/methanol after deactivation of hydrolytic enzymes and removal of nonlipid substances by extraction with hot dilute acetic acid (0.05N). Separation of the lipid classes is performed with a column (45 cm × 0.2 cm id) of 8 μm silica (Spherisorb, Phase Sep, Hauppague, NY) treated with concentrated ammonium hydroxide at a solvent flow rate of 0.5 ml/min, which requires a pressure of ca. 900 psi. Cholesteryl esters (CE) and triglycerides (TG) are eluted first with Skellysolve B/methylene chloride (1∶1, v/v); cholesterol (CH) is eluted with chloroform/methylene chloride (1∶2, v/v) and the phospholipids with methanol containing 6% ammonium hydroxide added to the latter solvent in a linear gradient. The neutral lipids are eluted in ca. 12 min and the phospholipids in an additional 30 min. The relative amount of each lipid class was determined from standard curves of the peak areas obtained according to response factors using erucyl alcohol as an internal standard. The method was applied to samples of kidney, liver and serum of rats. Duplicate analyses were generally within ca. 1.0% and good agreement was obtained in the analysis of the lipid classes of Azolectin and liver mitochondria lipid compared to thin layer chromatography (TLC) via photodensitometry of charred spots or phosphorus analysis of recovered phospholipids.     

A simplified procedure for the quantitative extraction of lipids from brain tissue

A method is described for the quantitative extraction of lipid from brain tissue with chloroform/methanol (C/M) that eliminates secondary purification of the lipid extract by dextran-gel chromatography or aqueous washing of the organic extract. Nonlipid substances that generally contaminate C/M lipid extracts are separated by pre-extraction of the tissue with dilute (0.25%) aqueous acetic acid. The residual tissue is extracted twice with 40 volumes of C/M (1∶1, v/v). Approximately 97% of the lipid is recovered in these extractions. A third extraction which yields ca. 1% more lipid is performed if the process is discontinued at this stage in a shortened version of the method. The remainder of the lipid is recovered after treatment of the tissue with 1 N HCl by two additional extractions, the first with 40 volumes of C/M (1∶2, v/v) and the second with 40 volumes of methanol. The method, which was demonstrated with pig brain, gave a complete extraction of the lipid, including gangliosides, free of nonlipid substances.     

Lipid hydroperoxides determination in milk‐based infant formulae by gas chromatography

A simple gas chromatograph using a flame ionization detector and a polar capillary column method is proposed for determining hydroperoxides in the lipid fraction of milkbased infant formulas. Tricosanoic acid is used as internal standard. The method includes the following steps: a) the extraction of fat with chloroform/methanol (2:1, v/v), b) the reduction of hydroperoxide acids to hydroxide acids with hydrogen gas and palladium as a catalyst, c) the transmethylation to obtain the methyl esters with acetyl chloride and methanol/ hexane (4:1, v/v), and d) an acetylation with acetic anhydride/ pyridine (4:1, v/v) to obtain an acetoxy derivate of the hydroxide present. Two chromatography runs, before and after the acetylation step, are carried out. The decrease in the retention time observed after the acetylation process is the criterion used to detect the hydroperoxide. A gas chromatography‐mass spectrometry analysis is applied to confirm the conversion of hydroperoxide acids to the acetylated forms and to identify the specific hydroperoxide structures. The estimated precision expressed as relative standard deviation (12.1%) and the accuracy, estimated by recovery assays (105 ± 28.3%) confirm the analytical quality of the method. In the milk‐based infant formula analyzed after storage of 11 months at 22 °C, the total hydroxide found corresponded to C₁₈. The gas chromatography‐mass spectra obtained after the acetylation step confirmed the presence of methyl 9‐ and 13‐acetoxyoctadecanoate/s, respectively, derived from the corresponding peroxides.     

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.     

Effects of variety and maturity on lipid class composition of peanut oil

Oils extracted from three varieties of mature peanuts with chloroform/methanol (2:1, v/v) or petroleum ether and oils extracted with petroleum ether from one variety at eight distinct physiological maturity stages were fractionated by TLC. The three varieties of mature peanuts were quantitatively similar in relative percentages of lipid classes regardless of extraction solvent; however, oil composition of the one variety changed with maturity. Generally, triacyl-glycerol percentage increased and percentage of all other fractions decreased with maturity. The calculated weight per seed of most fractions increased from the lowest maturity stage tested up through stage 10 or 11, which corresponds closely with apparent physiological maturity of the seed.     

Lipid composition of oats (Avena sativa L.)

Compositions of lipids extracted from a sample of Hinoat oat by seven solvent systems and that extracted with chloroform/methanol (2:1 v/v) from six selected cultivars representing high and low lipid contents are reported. Lipid components (steryl esters, triglycerides, partial glycerides, free fatty acids, glycolipids and phospholipids) were separated by silicic acid column chromatography and thin layer chromatography and quantitated by GLC analysis of fatty acids and phosphorus determinations. Twelve oat cultivars were examined for the fatty acid composition of lipid extracted with n-hexane. Lipids extracted from Hinoat by different solvent systems ranged from 5.6 to 8.8%. Quantitative distribution of lipid components extracted with chloroform/methanol from six cultivars containing 4.6 to 11.6% lipid showed a significant correlation (γ=0.99) between the total lipid and the neutral lipid content. Phospholipid content was similar in all cultivars, but glycolipids showed a two-fold increase in high lipid oats. Triglycerides contained less palmitic and more oleic acid than the glycolipids or phospholipids. Nine glycolipids and 11 phospholipids have been identified, and the polar lipid composition of Hinoat oat is presented.     

Extraction of Algal Lipids and Their Analysis by HPLC and Mass Spectrometry

Algae are a promising source of biofuel but claims about their lipid content can be ambiguous because extraction methods vary and lipid quantitation often does not distinguish between particular lipid classes. Here we compared methods for the extraction of algal lipids and showed that 2-ethoxyethanol (2-EE) provides superior lipid recovery (>150–200 %) compared to other common extraction solvents such as chloroform:methanol or hexane. Extractions of wet and dry algal biomass showed that 2-EE was more effective at extracting lipids from wet rather than dried algal pellets. To analyze lipid content we used normal-phase HPLC with parallel quantitation by an evaporative light scattering detector and a mass spectrometer. Analysis of crude lipid extracts showed that all major lipid classes could be identified and quantified and revealed a surprisingly large amount of saturated hydrocarbons (HC). This HC fraction was isolated from extracts of bioreactor-grown algae and further analyzed by HPLC/MS, NMR, and GC/MS. The results showed that the sample consisted of a mixture of saturated, straight- and branched-chain HC of different chain lengths. These algal HC could represent an alternative biofuel to triacylglycerols (TAG) that could feed directly into the current petroleum infrastructure.     

Separation and determination of glycolipids from edible plant sources by high-performance liquid chromatography and evaporative light-scattering detection

Glycolipids from edible plant sources were accurately quantified by silica-based, normal-phase high-performance liquid chromatography using an evaporative light-scattering detector. Five major glycolipid classes (acylated steryl glucoside, steryl glucoside, ceramide monohexoside, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol) were separated and determined with a binary gradient system consisting of chloroform and methanol/water (95∶5, vol/vol) without any interference from other lipid classes and pigments. The described method was applied to 48 edible plants available in Japan including cereals, legumes, vegetables, and fruits. Examined plant species contained glycolipids in wide concentration ranges, such as 5–645 mg/100 g tissue.     

钝顶螺旋藻中γ-亚麻酸甲酯的提取研究

用１∶２∶０８（Ｖ／Ｖ）的氯仿 甲醇 水溶剂系统提取螺旋藻中总类脂,总类脂在硅胶柱上经氯仿、丙酮、甲醇依次洗脱,分离得到中性脂、总脂肪酸半乳糖脂、磷脂。总脂肪酸半乳糖脂与５∶９５（Ｖ／Ｖ）的氯乙酰 甲醇作用转变为总脂肪酸甲酯,再通过多次与尿素形成复合物,可从总脂肪酸甲酯中分离、纯化出 亚麻酸甲酯,其纯度达到了９６５％。     

The analysis of lipids via HPLC with a charged aerosol detector

Because most lipid extracts are a mixture of saturated and unsaturated molecules, the most successful strategies for the quantitative analysis of lipids have involved the use of so-called “mass” or universal detectors such as flame ionization detectors and evaporative light scattering detectors. Recently a new type of HPLC “mass” detector, a charge aerosol detector (CAD), was developed and is now commercially available. This detection method involves nebulizing the HPLC column effluent, evaporating the solvents charging the aerosol particles, and measuring the current from the charged aerosol flux. In the present study, the CAD was evaluated with several normal phase and reverse phase HPLC methods commonly used for the quantitative analysis of lipid classes and lipid molecular species. The CAD detected common lipids such as triacylglycerols, diacylglycerols, glycolipids, phospholipids, and sterols. Lower molecular weight lipids such as free FA had smaller peak areas (50–80% lower). FAME were not detected by the CAD, probably because they were completely evaporated and did not form aerosol particles. The minimum limits of detection of the CAD with lipids varied with different mobile phase solvents. Using solvent systems that were predominantly hexane, the minimum limits of detection of triacylglycerols, cholesterol esters, and free sterols were about 1 ng per injection and the mass-to-peak area ratio was nearly linear from the range of about 1 ng to about 20 mg per injection. Three other solvents commonly used for HPLC lipid analysis (methanol, isopropanol, and acetonitrile) caused higher levels of background noise and higher minimum limits of detection. These experiments indicate that the CAD has the potential to become a valuable tool for the quantitative HPLC analysis of lipids. Long-term studies are needed to evaluate full instrument performance.     

The isolation and characterization of lipid compounds from black soldier fly larvae

With the global demand for lipid compounds on the rise, concerns are growing about the environmental and economic impact of traditional lipid sources. This concern is exacerbated by the ever-increasing demand for plant-based lipids, which is contributing to unsustainable production practices and competition for land and food. Considering this challenge, this work aimed at exploring the potential of black soldier fly larvae (BSFL) as a low-cost and ecofriendly source of lipid classes. A fractionation scheme consisting of a mixture of polar and nonpolar solvents at different ratios was employed to isolate the lipid classes from BSFL oil using silica gel column chromatography, which is a conventional method of chromatography. The fraction's separation efficiency was validated using thin-layer chromatography and characterized with Fourier-transformed infrared spectroscopy and gas chromatography flame ionization (GC-FID). Triglycerides (30%) were found to be the most abundant component while cholesterols (0.6%) were the least abundant lipid fractions in the lipid mixture. GC-FID in the various lipid fractions analyzed, lauric acid exhibited the highest percentage among the triglycerides (16.64%), diglycerides (19.10%), monoglycerides (22.70%), and free fatty acids (27.65%) fractions. The fractionation scheme proposed achieves high efficiency in separating and recovering different lipid classes extracted from the BSFL.     

Lipids and fatty acids in roasted chickens

Total lipids from meat portions of breast, thigh, wing, side and back with and without skin from 10 roasted chickens were extracted with chloroform and methanol and gravimetrically determined, and their fatty acids were analysed as methyl esters by gaseous chromatography, using a flame ionization detector and capillary column. The main fatty acids found were: C16:0, C18:1 omega 9, and C18:2 omega 6. The average ratio observed between PUFA/SFA was of 0.98, mainly due to the great concentration of the C18:2 omega 6 fatty acid, with an average of 26.75%. Regarding to the lipids content, the skinless breast showed the lowest content, 0.78 g/100 g, while the back with skin was the one with the highest content, 12.13 g/100 g except for the pure skin, with 26.54 grams of lipids by 100 grams.     

Isolation and purification of deoxynivalenol and a new trichothecene by high pressure liquid chromatography

Deoxynivalenol (3,7,15-trihydroxy-12,l3-epoxytrichothec-9-ene-8-one) was extracted from corn with methanol/water (80:20, v/v) and purified by liquid:liquid partitioning and by preparative high pressure liquid chromatography (HPLC). This procedure was used to prepare mg quantities of toxin from field-inoculated corn for reference standards. Analysis of the isolated deoxynivalenol by analytical HPLC, gas liquid chromatography (GLC) and gas liquid chromatography/mass spectroscopy (GLC/MS) indicated the presence of a second compound similar to deoxynivalenol. This compound comigrates with deoxynivalenol on thin layer chromatography plates in chloroform/methanol (90:10, v/v), but can be separated by HPLC on a reverse-phase C₈ column with methanol/water (10:90, v/v). GC/MS of the compound and the trimethylsilyl ether derivative gave parent ions of m/e 280 and 424, respectively. These data and NMR data indicate that the compound is 3,15-dihydroxy-12,13-epoxytrichothec-9-ene-8-one, a previously unreported trichothecene.     

Dry column method for the quantitative extraction and simultaneous class separation of lipids from muscle tissue

A method for lipid isolation is presented that is alternative to the traditional chloroform/methanol extraction methods. This new method allows lipid isolation by solvent elution of a dry column composed of a tissue sample, anhydrous sodium sulfate, and Celite 545 diatomaceous earth groud together. To isolate total lipids, the dry column is eluted with a mixture of dichloromethane/methanol (90∶10, v/v). Alternatively, the lipids may be isolated and simultaneously separated into neutral and polar fractions by a sequential elution procedure; neutral lipids free of polar lipids are eluted first with dichloromethane, followed by elution of polar lipids with the dichloromethane/methanol (90∶10) mixture. The two dry column methods-isocratic or sequential elution-were compared with the traditional chloroform/methanol methods by gravimetric, thin layer chromatographic and phosphorus analyses. Presented in part at the AOCS 70th Annual Meeting, San Francisco, May 1979. Agricultural Research, Science and Education Administration, U.S. Department of Agriculture.     

Impact of extraction method on yield of lipid oxidation products from oxidized and unoxidized walnuts

The objective of this study was to measure and compare differences in oxidized products of oil extracted from unoxidized and oxidized walnuts using five different extraction methods: (i) mechanical pressing, or solvent extraction with (ii) hexane, (iii) methylene chloride, (iv) chloroform/methanol, or (v) supercritical carbon dioxide (SC−CO₂). Of the extraction methods evaluated, only chloroform/methanol and methylene chloride provided reasonable results for all parameters measured (total lipid yield, FA profile, PV, conjugated dienes, FFA content, and volatile content); however, chloroform/methanol extracted significantly greater levels of volatile compounds. The SC−CO₂ extraction with purified gas was simple and accurate for all data except collection of volatile compounds, as these materials are lost during the lipid extraction. Pressing was neither quantitative nor qualitative, and hexane extraction retrieved significantly lower levels of volatiles than the other methods, except for SC−CO₂.     

33.5%甲磺·异丙·莠悬浮剂含量的测定

采用高效液相色谱,以C18为固定相,以V(甲醇)∶V(水)=50∶50(用磷酸调pH=3)为流动相,检测波长220 nm及氢火焰离子化检测器气相色谱相结合的方法,对33.5%甲磺.异丙.莠悬浮剂进行定量分析。结果表明,甲基磺草酮、异丙草胺、莠去津线性相关系数分别为0.9993,0.999 1,0.999 6;变异系数分别为0.62%,0.34%,0.51%;平均回收率分别为99.4%,99.7%,99.7%。该方法简便、快捷,准确。     

Quantitative determination of isomeric glycerides,free fatty acids and triglycerides by thin layer chromatography-flame ionization detector system

Partial glyceride mixtures, which include 1-monoglyceride, 2-monoglyceride, free fatty acid, 1,2-diglyceride, 1,3-diglyceride and triglyceride, could be separated from each other on a 3% boric-acid-impregnated Chromarod S-II (silica gel sintered quartz rod) with either chloroform/acetone (96∶4, v/v) or chloroform/acetone/acetic acid (100∶1∶1, v/v) as the developing solvent mixtures. The components separated on the boric-acid-impregnated rod were automatically quantitated in a hydrogen flame ionization detector (Iatroscan). The relative responses of 1,2-diglyceride, 1,3-diglyceride, free fatty acid and triglyceride were slightly lower than theoretical responses based on weight percentage, whereas 1-monoglyceride and 2-monoglyceride showed slightly higher responses. These responses were converged within a maximal error of 5–10% (SD). Boric-acid-impregnated rods could be used repeatedly, ca. 5 times without any reconditioning procedure. Part of this investigation was reported at the 2nd JOCS-AOCS joint meeting, San Francisco, May 1979.     

Quantitation of 1,2-diacylglycerol in rat heart by latroscan TLC/FID

1,2-Diacylglycerol, which has been recognized as one of the intracellular second messengers, was measured quantitatively in the lipid extract from rat hearts using the thin layer chromatography and flame ionization detection (TLC/FID) method. Cholesterol acetate was added to the tissue as an internal standard, and the crude lipids from the tissue were purified with silicic acid column to eliminate phospholipids. Development of Chromarods was carried out using two solvent systems and a three-step development technique. The relationship of the peak area ratio detected by flame ionization detector to weight ratio was linear compared with cholesteryl acetate. The 1,2-diacylglycerol content in the rat heart in the unstimulated condition was 72.5±15.3 ng/mg wet wt (mean±SD).     

Accelerated Solvent Extraction Improves Efficiency of Lipid Removal from Dry Pet Food While Limiting Lipid Oxidation

Accelerated solvent extraction (ASE) was evaluated for extracting lipids from baked and extruded dry pet foods to determine factors controlling extraction efficiency and effects on lipid oxidation. Hydroperoxide decomposition and new lipid oxidation were minimal at 40 °C but increased at higher extraction temperatures without increasing yields. Maximum extraction required grinding samples to 250 µm particles, presence of polar solvents [chloroform, chloroform/methanol 2:1 (v/v) mixed, hexane/methanol 2:1 (v/v)], and a minimum of 20 min total static extraction time in repeat extraction cycles. Hexane and methanol injected into extraction cells simultaneously but separately was able to nearly duplicate extractions of chloroform/methanol, providing an option for replacing toxic chlorinated hydrocarbon solvents in ASE. However, lipid oxidation was higher in hexane. Yields were quantitative in baked biscuits but lower in extruded kibbles due to more dense, complex molecular structures. ASE extraction yields of 40 min or less were comparable to manual extraction yields of 24–48 h, with lower oxidation. Overall, one or two ASE extraction cycles with static times less than 20 min appeared to provide adequate lipid yields that accurately reflect lipid composition while inducing minimal modification when lipid oxidation products are the analytical endpoint.     

Two procedures to remove polar contaminants from a crude brain lipid extract by using prepacked reversed-phase columns

Two procedures were developed using prepacked, reversed-phase columns (Bond Elut) for the separation of lipids from water-soluble contaminants. A crude lipid extract from brain tissue, was diluted stepwise with a methanol/water (method 1) or a methanol/saline (method 2) mixture and, with each step, was passed through the column. As the polarity of the solvent was increased, all lipids became bound to the column, while the water-soluble compounds remained in the eluate. After three subsequent dilutions and column elutions, the eluate containing the more polar contaminants was discarded. The bound lipids were then eluted with a small volume of chloroform/methanol (1∶2, v/v). Alternatively two fractions were eluted, the first fraction eluted with methanol/water (12∶1, v/v), contained gangliosides, phosphatidyl-serine, phosphatidylinositol, phosphatidic acid and sulfatides. The second fraction, eluted with chloroform/methanol (1∶2, v/v), contained all remaining phospholipids, cerebrosides and cholesterol. For both methods a quantitative recovery of cholesterol and phospholipids was obtained. In method 2, when water was replaced by saline in the dilution solvent mixture, gangliosides were also bound and quantitatively recovered.