Analysis of Triacylglycerols and Free Fatty Acids in Algae Using Ultra-Performance Liquid Chromatography Mass Spectrometry

To assess the suitability of microalgal strains for biodiesel production the lipid content and composition, especially individual triacylglycerols (TAG) and free fatty acids (FFA) must be determined. In this study, the compositions and concentrations of TAG and FFA were analysed in four halophytic algal species, Dunaliella salina, D. tertiolecta, D. bardawil, and D. granulata. These species were selected as part of a larger screen to identify species suitable for biofuel feedstocks. An accelerated solvent extraction instrument was used for lipids and fatty acid extraction using a dichloromethane–hexane solvent system. Ultra-performance liquid chromatography coupled with mass spectrometry (MS) detection was optimized and applied to the quantitative analysis of TAG and FFA in the different algal extracts. Individual TAG were characterized structurally using direct electrospray ionization (ESI) MS and MS/MS techniques. Cationic adducts (NH₄ ⁺) of TAG were detected and quantified in the positive ESI MS and MS/MS modes, while the negative ESI mode was used for FFA analysis. Over 20 TAG were identified and quantified in the four Dunaliella strains. Analysis of FFA compositions demonstrated that the most abundant FFA in these four algal species were palmitic, linolenic, linoleic, and oleic acids.     

不同亚临界溶剂从微拟球藻湿藻泥中提取油脂

以微拟球藻(Nannochlorsis sp.)湿藻泥为原料,研究了亚临界乙醇、亚临界乙醇-正己烷共溶剂及硫酸辅助亚临界乙醇-正己烷共溶剂3种萃取体系对微藻油脂提取的影响. 结果表明,亚临界乙醇-正己烷比亚临界乙醇对湿藻细胞有更高的油脂萃取率和低的溶剂用量,加入少量硫酸可进一步提高油脂的提取率、降低溶剂用量. 微拟球藻湿藻泥(含水约70%)优化提取条件为,正己烷/乙醇体积比3:1,液固比(溶剂/藻细胞干重)7 mL/g,加入藻细胞干重6%的硫酸,1.5 MPa下90℃萃取30 min,在此条件下油脂提取率可达90%以上. 3种萃取体系获得的微藻油脂均以甘油三酯为主,甘油三酯的脂肪酸主要为C16:0, C18:1和C16:1,其中硫酸辅助亚临界共溶剂萃取的微藻油脂中甘油三酯含量最高,约占总脂的86%以上.     

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.     

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.     

Identification and quantification of glycerolipids in cotton fibers: Reconciliation with metabolic pathway predictions from DNA databases

The lipid profiles of cotton fiber cells were determined from total lipid extracts of elongating and maturing cotton fiber cells to see whether the membrane lipid composition changed during the phases of rapid cell elongation or secondary cell wall thickening. Total FA content was highest or increased during elongation and was lower or decreased thereafter, likely reflecting the assembly of the expanding cell membranes during elongation and the shift to membrane maintenance (and increase in secondary cell wall content) in maturing fibers. Analysis of lipid extracts by electrospray ionization and tandem MS (ESI-MS/MS) revealed that in elongating fiber cells (7–10 d post-anthesis), the polar lipids—PC, PE, PI, PA, phosphatidylglycerol, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and phosphatidyl-glycerol—were most abundant. These same glycerolipids were found in similar proportions in maturing fiber cells (21 dpa). Detailed molecular species profiles were determined by ESI-MS/MS for all glycerolipid classes, and ESI-MS/MS results were consistent with lipid profiles determined by HPLC and ELSD. The predominant molecular species of PC, PE, PI, and PA was 34∶3 (16∶0, 18∶3), but 36∶6 (18∶3, 18∶3) also was prevalent. Total FA analysis of cotton lipids confirmed that indeed linolenic (18∶3) and palmitic (16∶0) acids were the most abundant FA in these cell types. Bioinformatics data were mined from cotton fiber expressed sequence tag databases in an attempt to reconcile expression of lipid metabolic enzymes with lipid metabolite data. Together, these data form a foundation for future studies of the functional contribution of lipid metabolism to the development of this unusual and economically important cell type.     

Subcritical co‐solvents extraction of lipid from wet microalgae pastes of Nannochloropsis sp.

In this paper subcritical co‐solvents extraction (SCE) of algal lipid from wet pastes of Nannochloropsis sp. is examined. The influences of five operating parameters including the ratio between ethanol to hexane, the ratio of mixed solvents to algal biomass (dry weight), extraction temperature, pressure, and time were investigated. The determined optimum extraction conditions were 3:1 (hexane to ethanol ratio), 10:1 ratio (co‐solvents to microalgae (dry weight) ratio), 90°C, 1.4 MPa, and 50 min, which could produce 88% recovery rate of the total lipids. In addition, electron micrographs of transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were conducted to show that the algal cell presented shrunken, collapsed with some wrinkles and microholes after SCE extraction. The main composition of total lipids extracted under the optimum conditions was TAG which represented more than 80%. And the fatty acid profile of triglycerides revealed that C16:0 (35.67 ± 0.2%), C18:1 (26.84 ± 0.044%) and C16:1 (25.96 ± 0.011%) were dominant. Practical applications: The reported method could save energy consumption significantly through avoiding deep dewatering (for example drying). The composition of the extracted lipid is suitable for the production of high quality biodiesel.     

Comparative evaluation of solvent extraction methods for the determination of neutral and polar lipids in beef

Samples of lean (< 5% fat), medium (13–15%) and high-fat (> 20%) ground beef were extracted for total lipid by 4 methods of wet extraction employing chloroform/methanol (CM), n-hexane/iso-propanol (HIP) and ethyl alcohol/ethyl ether (AE), and by 3 methods of soxhlet extraction of freeze-dried material by petroleum ether (PE) or eithyl ether (EE), CM and methylene chloride/ methanol (MM). The purified lipid was fractionated into neutral and polar lipid fractions by silicic acid chromatography and the frac-tions were analyzed for fatty acid distribution by gas liquid chroma-tography (GLC). The soxhlet procedure employing either PE or EE extracted less than 75% of total lipid, 89% of triglycérides and 15% of polar lipids from lean beef as compared to other methods, and as the fat content increased from 3 to 20%, extracted amounts of polar lipid which increased to 40% of that extracted by other methods. The fatty acid distribution of the fractionated triglycerides and polar lipids was generally within experimental error for each frac-tion, irrespective of the method of extraction. The percentages of 16:0 and 18:1 were significantly less in polar lipids than in trigly-cerides. In addition to significantly higher percentage of 18:2, the polar lipids contained up to 20% of long-chain fatty acids not detected in triglycerides. The soxhlet procedures with CM or MM were as effective as wet extraction procedures in extracting neutral and polar lipids. Presented at the 73rd AOCS annual meeting, Toronto, 1982. Contribution No. 512, Food Research Institute, Agriculture Canada.     

Extraction of pollen lipids by SFE‐CO2 and determination of free fatty acids by HPLC

Rape bee pollen lipids obtained by petrol ether extraction (PEE) or supercritical fluid (carbon dioxide) extraction (SFE) were compared with regard to their free fatty acid (FFA) components. Optimal SFE conditions were selected by carrying out the Taguchi method with an OA9 (3³) matrix design, and are as follows: extraction pressure at 35 MPa, temperature at 45 °C, and dynamic extraction time at 90 min. The lipid yield based on PEE was 7.42 wt‐% and the extracts of the desired analytes based on SFE varied in the range of 3.23–5.58 wt‐% under different conditions. With the optimized procedure, the lipid yield was 6.09 wt‐%. The FFA in the lipids were separated with a pre‐column derivation method and 1‐[2‐(p‐toluenesulfonate) ethyl]‐2‐phenylimidazole [4,5‐f]9,10‐phenanthrene as labeling regent, followed by high‐pressure liquid chromatography (HPLC) with fluorescence detection. HPLC analysis shows that the lipids contain abundant unsaturated fatty acids (UFA) in high to low concentrations as follows: linolenic acid (18:3), oleic acid (18:1), linoleic acid (18:2), nervonic acid (24:1), and lignoceric acid (20:4). The UFA contents in the SFE extracts were higher than those after PEE. The results indicated that SFE under suitable conditions is more selective than conventional PEE with regard to lipid extraction and preservation of their quality.     

Neutral lipids in non‐starch lipid and starch lipid extracts from Portuguese sourdough bread

This research effort was aimed at assessing the changes in extractable neutral lipids (NL) throughout the baking process of Broa, a Portuguese traditional sourdough bread. NL were accordingly isolated, purified and quantitated – starting from non‐starch lipid (NSL) and starch lipid (SL) extracts of maize and rye flours, as well as fermented dough and bread. NSL accounted for the major fraction of extracted lipids; furthermore, the NSL/SL ratio evolved throughout processing in agreement with the phenomena prevailing during dough preparation, fermentation, and baking. An analytical method based on resolution by normal‐phase HPLC coupled with detection by evaporative light scattering was accordingly developed for quantitation of the aforementioned NL classes. Distinct NL classes correlated well with the stage of bread making. The main NL in NSL were triacylglycerols (ca. 75% of the total), but relatively high concentrations of sterol esters and diacylglycerols were also found. Conversely, free fatty acids were the dominant component of SL, whereas monoacylglycerols and free sterols were comparable to those in NSL.     

从雨生红球藻藻泥中选择性提取虾青素

以雨生红球藻湿藻泥为原料,研究了不同有机溶剂对胞内油脂和虾青素选择性提取分离的影响,通过酸解破壁提高虾青素和油脂的提取效率。结果表明,连续乙醇提取可对胞内色素和油脂有效分级提取,先提取出极性组分(叶绿素和极性脂),再提取中性组分(类胡萝卜素和中性脂)。中等极性溶剂或溶剂体系对类胡萝卜素的选择性和提取率较好;乙醇/乙酸乙酯混合溶剂提取类胡萝卜素的总得率(干重)达25.31 mg/g,提取率为69.35%。对雨生红球藻湿细胞进行酸解破壁处理有助于提高虾青素和油脂的提取率。在最优酸解破壁条件(盐酸浓度1 mol/L,温度60℃,时间60 min)下,含水80%的雨生红球藻藻泥的油脂总得率(干重)达418 mg/g,总脂提取率达97%。     

Quantitative high-performance liquid chromatography analysis of plant phospholipids and glycolipids using light-scattering detection

Application of the evaporative light-scattering principle to quantitative high-performance liquid chromatography (HPLC) analyses of plant membrane lipids has received little study. Light-scattering detection response curves were generated for nine classes of plant membrane phospholipid and glycolipids. Quantitative results obtained by HPLC/light-scattering detection and conventional lipid analytical methods (thin-layer chromatography and lipid-P assay) were in close agreement, confirming the reliability of HPLC/evaporative light-scattering detection (ELSD) analyses. Only three of the nine plant lipid classes gave linear detector response functions above 10 μg injected lipid mass. This finding contradicts earlier precepts involving light-scattering detection of lipids. At a given mass, appreciable variation in ELSD signal intensity and detection limit was found to exist among the various plant membrane lipid classes. The variation in detector response among plant lipid classes is an important consideration in achieving accurate quantitative results in plant lipid analyses.     

Supercritical fluids extraction and anti-solvent purification of carotenoids from microalgae and associated bioactivity

This study investigated co-solvent modified supercritical carbon dioxide extraction of lipids and carotenoids from the microalgal species of Nannochloropsis oculata. Supercritical carbon dioxide (SCCO₂) anti-solvent precipitation of carotenoids from the extracts following purification of Zeaxanthin was also examined. Continuous modification by ethanol of supercritical carbon dioxide extractions showed that the addition ratio was important for extraction efficiency of lipids and carotenoids. SCCO₂ extraction at 350 bar, 323 K and 16.7 wt% of ethanol addition yielded 239.7 mg of triglycerides and 7.61 mg of carotenoids per gram extract with a total yield of 15.5%. SCCO₂ anti-solvent experiments showed that the content of Zeaxanthin in the precipitate was greater than that in the fraction of normal phase column chromatography. The purest Zeaxanthin (93.8%) was then successfully isolated from the purified fraction by using a reverse-phase HPLC column chromatography. Rat macrophages treated by ultra-sonicated water extracts of the SCCO₂ defatted algae showed a positive phagocytotic activity.     

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.     

Extraction of lipids from fermentation biomass using near-critical dimethylether

The extraction of lipids from both wet and dry biomass produced by fermentation has been carried out using near-critical dimethylether (DME) as the extraction solvent. Fermentations were carried out from a shake flask up to a 300 L scale using the microorganism Mortierella alpina, and up to a 20 L scale for Phaffia rhodozyma and Agrobacterium tumefaciens. The lipids extracted at a laboratory and pilot scale from the biomasses were enriched in arachidonic acid, astaxanthin, and co-enzyme Q10 respectively. Extractions were also performed on marine microalgae, produced by a proprietary fermentation process, to obtain lipids rich in EPA. Lipids were extracted from wet biomass using DME, which removes the need to dry the biomass. Water is also co-extracted, which has to be separated from the lipid. The biomass shrunk considerably during packed bed extraction of wet biomass, leading to channelling. Repacking and re-extraction of the packed bed enabled full lipid yields to be obtained. The extraction of lipids from biomass suspended in fermentation broth showed considerable promise, and lipid yields were improved due to the recovery of lipids that had been exuded into the broth from the microorganism. In contrast, the extraction of lipids from freeze-dried biomass using DME was routine, yields were substantially higher than using CO₂ or CO₂ + ethanol, but were lower than from wet biomass. DME also extracted polar lipids from both wet and dry biomass, leading to the higher total lipid yields compared to CO₂. Separate extraction of non-polar and polar lipids was possible by sequential extraction of dry biomass using initially CO₂ followed optionally with ethanol co-solvent; and then DME.     

Combined effect of ozone and ultrasound on disruption of microalgal cells

Among several disruption methods tested so far, ozonation and sonication were distinguished from others by their effective cell rupturing ability. In this study, these disruption methods were combined to enhance the retrieval of biofuel feedstock (carbohydrates and lipids) from microalgae. A mixed microalgal culture was subjected to first ozonation, followed by sonication. Compared to the extraction of 22% of total lipids and 47% of total carbohydrates by sole ozonation, the retrieval of algal metabolites reached 59% and 81% of total lipids and carbohydrates, respectively. The optimum process variables were found by means of a response surface modeling and quadratic programming.     

Lipid composition in tomato skin supercritical fluid extracts with high lycopene content

The use of supercritical fluid extraction is proposed for obtaining stable extracts with high added value from natural and economical sources. Lipid composition, namely, of FFA, TAG, and FAME, in tomato skin extracts with high lycopene content was determined. Separation of different classes of lipids was achieved from tomato extracts using TLC followed by transesterification and GC, and lycopene and other carotenoids were analyzed by HPLC with a photodiode array detector. In lycopene extracts obtained using supercritical fluids, no FFA were found and polyunsatureted TAG represented only 9.2% of the total TAG content.     

A Comprehensive GC–MS Sub-Microscale Assay for Fatty Acids and its Applications

Fatty acid analysis is essential to a broad range of applications including those associated with the nascent algal biofuel and algal bioproduct industries. Current fatty acid profiling methods require lengthy, sequential extraction and transesterification steps necessitating significant quantities of analyte. We report the development of a rapid, microscale, single-step, in situ protocol for GC–MS lipid analysis that requires only 250 μg dry mass per sample. We furthermore demonstrate the broad applications of this technique by profiling the fatty acids of several algal species, small aquatic organisms, insects and terrestrial plant material. When combined with fluorescent techniques utilizing the BODIPY dye family and flow cytometry, this micro-assay serves as a powerful tool for analyzing fatty acids in laboratory and field collected samples, for high-throughput screening, and for crop assessment. Additionally, the high sensitivity of the technique allows for population analyses across a wide variety of taxa.     

Fatty Acid and Lipid Profiles with Emphasis on n-3 Fatty Acids and Phospholipids from Ciona intestinalis

In order to establish Ciona intestinalis as a new bioresource for n‐3 fatty acids‐rich marine lipids, the animal was fractionated into tunic and inner body tissues prior to lipid extraction. The lipids obtained were further classified into neutral lipids (NL), glycolipids (GL) and phospholipids (PL) followed by qualitative and quantitative analysis using GC‐FID, GC–MS, ¹H NMR, 2D NMR, MALDI‐TOF‐MS and LC–ESI–MS methods. It was found that the tunic and inner body tissues contained 3.42–4.08 % and 15.9–23.4 % of lipids respectively. PL was the dominant lipid class (42–60 %) irrespective of the anatomic fractions. From all lipid fractions and classes, the major fatty acids were 16:0, 18:1n‐9, C20:1n‐9, C20:5n‐3 (EPA) and C22:6n‐3 (DHA). The highest amounts of long chain n‐3 fatty acids, mainly EPA and DHA, were located in PL from both body fractions. Cholestanol and cholesterol were the dominant sterols together with noticeable amounts of stellasterol, 22 (Z)‐dehydrocholesterol and lathosterol. Several other identified and two yet unidentified sterols were observed for the first time from C. intestinalis. Different molecular species of phosphatidylcholine (34 species), sphingomyelin (2 species), phosphatidylethanolamine (2 species), phosphatidylserine (10 species), phosphatidylglycerol (9 species), ceramide (38 species) and lysophospholipid (5 species) were identified, representing the most systematic PL profiling knowledge so far for the animal. It could be concluded that C. intestinalis lipids should be a good alternative for fish oil with high contents of n‐3 fatty acids. The lipids would be more bioavailable due to the presence of the fatty acids being mainly in the form of PL.     

Characterization of Lipid Components in Two Microalgae for Biofuel Application

A full characterization of lipid components is critical for selecting the most suitable microalgae and for downstream processing for biofuel production. This study demonstrates extraction, quantification, and diversity of lipid components from two microalgae of different types. For total lipid quantification, three extraction methods were compared and the method of pre-drying, dry ice-assisted grinding, and sequential solvent extraction gave the highest total lipid recovery. For lipid class composition determination, the photosynthetic Nannochloropsis contained 37.0% polar lipids, 41.1% triacylglycerols, and 12.5% unsaponifiable matter, whereas, the heterotrophic Schizochytrium had much less polar lipids and unsaponifiable matter. Further separation and quantification showed that Nannochloropsis contained more glycolipids (37.1% of total polar lipids) than that in Schizochytrium (14.3%), while Schizochytrium contained much more phospholipids (85.7%) than that in Nannochloropsis (44.7%). The major components in unsaponifiable matter of Nannochloropsis were hydrocarbon and cholesterol (55.8 and 37.7% of the total GC quantifiable matter), which only accounted for 29.3% of total unsaponifiable matter. For Schizochytrium, 15.4% squalene, 28.9% cholesterol, and 43.2% stigmasta-4,7,22-trien-3-ol were found in its GC quantifiable matter, and the total quantified hydrocarbon and sterols accounted for 50.5% of unsaponifiable matter. The lipid compositions of the two types of microalgae are very different, therefore, processing performance, such as lipid extraction and conversion to biodiesel may be different. Similar lipid characterization for other biofuel types of microalgae needs to be made to ensure optimal biofuel processing and fuel quality.     

Analysis of nonvolatile lipid oxidation products in vegetable oils by normal-phase high-performance liquid chromatography with mass spectrometric detection

Nonvolatile triacylglyceride (TAG) oxidation products play an important role in the oxidative degradation of lipids. They serve as a reservoir of oxygen-containing species and hence can act as off-flavor precursors or as initiators for further oxidation reactions. Possible nonvolatile lipid oxidation products are TAG with a hydroperoxy, hydroxy, epoxy, or oxo (ketone or aldehyde) group or combination of these groups. The breakdown of TAG hydroperoxides yields nonvolatile glyceride species with two intact fatty acid chains and one short chain mostly ending in an aldehyde or hydroxy group (2 1/2 glycerides). By means of normal-phase high-performance liquid chromatography (HPLC) with mass spectrometric (MS) detection, non-volatile lipid oxidation products can be separated according to polarity. This results in separation into classes of TAG oxidation products, such as epoxy-TAG, oxo-TAG, hydroperoxy-TAG, hydroxy-TAG and 2 1/2 glycerides, which can be identified using selected ion chromatograms. The retention times of TAG oxidation products on the normal-phase HPLC system and the signal intensity of the MS detector are stable enough to enable quantitative analysis based on external calibration. The normal-phase HPLC-MS method is very suitable for the characterization and quantitation of nonvolatile TAG oxidation products in oxidized TAG reference compounds as well as in real oils or oil phases isolated from emulsions, spreads, or other fat-based food products. This method can give detailed information for the study of lipid oxidation mechanisms. Presented at the 15th Montreux Symposium on Liquid Chromatography/Mass Spectrometry, Montreux, Switzerland, November 9–10, 1998.