A quantitative lipid extraction method for residue analysis of fish involving nonhalogenated solvents

Numerous intercalibration exercises have indicated that the in Sweden frequently used, so called Jensen extraction method for total lipids and lipophilic pollutants gave satisfactory yields when applied to fatty aquatic organisms. However, a comparison with the classical Bligh and Dyer method and the forerunner, the Folch methods, revealed that in the case of very lean fish (fat content below 1%, e.g., cod), the lipid yields were about 25% too low for the Jensen method; consequently, residue levels quoted on a lipid weight basis were correspondingly too high. To rectify the unacceptably low fat recovery from lean marine organisms, the Jensen extraction method has been modified to give recoveries not significantly different from the Folch and Bligh and Dyer methods. In the modified version, acetone is replaced by 2-propanol and part of the hexane is replaced by diethyl ether. Comparison between the modified Jensen method and the Folch method for cod muscle gave the same recovery of total lipids but slightly lower than that obtained with the Bligh-Dyer method. A possible explanation for this small difference is discussed. It is anticipated that the reported increased yield for cod is due to the superior solubility of phospholipids in 2-propanol as compared to acetone. The possible use of correction factors for previously reported contaminant residual levels of lean and medium fat fish calculated on lipid basis in the future is suggested.     

Dichloromethane as a solvent for lipid extraction and assessment of lipid classes and fatty acids from samples of different natures

The usefulness of the solvent mixture dichloromethane/methanol for lipid extraction and the determination of lipid classes and fatty acids in samples of different natures was conducted. Two different extraction methods were compared, one containing chloroform/methanol, another containing dichloromethane/methanol. Total lipid extraction showed some minor differences but no variation in the lipid classes. Regarding the fatty acid profile, in Echium virescens seeds, 17 major fatty acids could be identified and quantified, and all were equally extracted when either solvent system was employed. In Echium acanthocarpum hairy roots, 17 major fatty acids were quantified, showing some statistical differences for one cell line in favor of chloroform. The data obtained from the liquid nutrient medium were also comparable. The cod roe sample showed 31 major fatty acids, showing no statistical differences between the two solvent systems. Contrarily, the CH 2Cl 2 method was able to extract 31 main fatty acids found in European seabass dorsal muscle more efficiently than the CHCl 3 method. The results indicate that, for lipid extraction and fatty acid assessment, dichloromethane/methanol can readily replace the commonly employed chloroform/methanol, thus avoiding the major health, security, and regulatory problems associated with the use of chloroform.     

Pressurized fluid extraction for quantitative recovery of chloroacetanilide and nitrogen heterocyclic herbicides in soil

Pressurized fluid extraction (PFE) is a new sample extraction method operated at elevated temperatures and pressures with liquid solvents. The use of PFE was investigated for the extraction of four Hawaiian clayey soils fortified with the selected chloroacetanilide and nitrogen heterocyclic herbicides Alachlor, Bromacil, Hexazinone, Metribuzin, and Tebuthiuron. The effects of operation temperature, pressure, flush volume, and static cycles on PFE performance were studied. Water was the most effective modifier of PFE for quantitative recoveries of the five herbicides in soils. The simple extraction method required pretreatment of the soil with 37.6% water and subsequent two-static-cycle extraction with a total of 32 mL of acetone at 1500 psi and 100 degrees C. Average recoveries of Alachlor, Bromacil, Hexazinone, Metribuzin, and Tebuthiuron ranged from 93 to 103% by the water-assisted PFE, compared with only 68-83% recoveries of the corresponding chemicals when no water was used. The extraction time and total organic solvent consumption were reduced from 18 h and 300 mL by Soxhlet to 22 min or less and 80 mL or less of organic solvent by PFE.     

马铃薯茎叶挥发性成分的两种提取方法比较

应用水蒸气蒸馏-溶剂萃取法和微波辅助-溶剂萃取法提取了成熟期马铃薯茎叶挥发性成分。用乙醚为萃取溶剂的水蒸气蒸馏法提取时,以0.533%（m/m）得油率获得精油;以正己烷为溶剂的微波辅助萃取法的最适提取条件为：温度60℃,时间9 min,液料比10︰1（V/m）,在此条件下的得油率为0.528%（m/m）。用GC-MS分析检测出水蒸气蒸馏法获得的精油含有81个成分,解析鉴定出占精油相对含量79.386%的43种物质,醇类化合物（24.789%）为主要成分;微波法获得的精油检测出78个成分,解析鉴定出占总精油82.226%的36种成分,酯类化合物（44.482%）为主要成分。2种提取方法获得的精油成分有很大的差别。     

Antioxidant activity of extracts of black sesame seed (Sesamum indicum L.) by supercritical carbon dioxide extraction

Antioxidant activities of extracts derived from sesame seed by supercritical carbon dioxide (SC-CO(2)) extraction and by n-hexane were determined using alpha,alpha-diphenyl-beta-picylhydrazyl (DPPH) radical scavenging and linoleic acid system methods. The highest extracted yield was given at 35 degrees C, 40 MPa, and a CO(2) flow rate of 2.5 mL min(-1) by an orthogonal experiment. The yields of extracts increased with increasing pressure, and yields at 40 and 30 MPa were higher than that by solvent extraction at 46.50%. Results from the linoleic acid system showed that the antioxidant activity follows the order: extract at 35 degrees C, 20 MPa > BHT > extract at 55 degrees C, 40 MPa > extract at 55 degrees C, 30 MPa > Trolox > solvent extraction > alpha-tocopherol. The SC-CO(2) extracts exhibited significantly higher antioxidant activities comparable to that by n-hexane extraction. The extracts at 30 MPa presented the highest antioxidant activities assessed in the DPPH method. At 20 MPa, the EC(50) increased with temperature, which indicated that the antioxidant activity was decreased in a temperature-dependent manner. The significant differences of antioxidant activities were found between the extracts by SC-CO(2) extraction and n-hexane. However, no significant differences were exhibited among the extracts by SC-CO(2) extraction. The vitamin E concentrations were also significantly higher in SC-CO(2) extracts than in n-hexane extracts, and its concentrations in extracts corresponded with the antioxidant activity of extracts.     

Effects of released iron, lipid peroxides, and ascorbate in trout hemoglobin-mediated lipid oxidation of washed cod muscle

Approximately 7% of the iron associated with hemoglobin was released from the heme protein during 2 degrees C storage in washed cod muscle. EDTA (2.2 mM) neither accelerated nor inhibited hemoglobin-mediated lipid oxidation based on the formation of lipid peroxides and TBARS. This suggested that low molecular weight iron was a minor contributor to hemoglobin-mediated lipid oxidation in washed cod muscle. Ascorbate (2.2 mM) was a modest to highly effective inhibitor of hemoglobin-mediated lipid oxidation depending on which washed cod preparation was assessed. Experimental evidence suggested that the ability of residual ascorbate to breakdown accumulating lipid hydroperoxides to reactive lipid radicals can explain the shift of ascorbate from an antioxidant to a pro-oxidant. Increasing the lipid peroxide content in washed cod muscle accelerated hemoglobin-mediated lipid oxidation and decreased the ability of ascorbate to inhibit lipid oxidation. Preformed lipid peroxide content in cod muscle was highly variable from fish to fish.     

Development of a rapid method for the sequential extraction and subsequent quantification of fatty acids and sugars from avocado mesocarp tissue

Methods devised for oil extraction from avocado (Persea americana Mill.) mesocarp (e.g., Soxhlet) are usually lengthy and require operation at high temperature. Moreover, methods for extracting sugars from avocado tissue (e.g., 80% ethanol, v/v) do not allow for lipids to be easily measured from the same sample. This study describes a new simple method that enabled sequential extraction and subsequent quantification of both fatty acids and sugars from the same avocado mesocarp tissue sample. Freeze-dried mesocarp samples of avocado cv. Hass fruit of different ripening stages were extracted by homogenization with hexane and the oil extracts quantified for fatty acid composition by GC. The resulting filter residues were readily usable for sugar extraction with methanol (62.5%, v/v). For comparison, oil was also extracted using the standard Soxhlet technique and the resulting thimble residue extracted for sugars as before. An additional experiment was carried out whereby filter residues were also extracted using ethanol. Average oil yield using the Soxhlet technique was significantly (P < 0.05) higher than that obtained by homogenization with hexane, although the difference remained very slight, and fatty acid profiles of the oil extracts following both methods were very similar. Oil recovery improved with increasing ripeness of the fruit with minor differences observed in the fatty acid composition during postharvest ripening. After lipid removal, methanolic extraction was superior in recovering sucrose and perseitol as compared to 80% ethanol (v/v), whereas mannoheptulose recovery was not affected by solvent used. The method presented has the benefits of shorter extraction time, lower extraction temperature, and reduced amount of solvent and can be used for sequential extraction of fatty acids and sugars from the same sample.     

耦合浮珠-超声辅助溶剂萃取法用于微藻采收及油脂提取

为了优化微藻生物柴油生产工艺,开发高效低耗的微藻采收与油脂提取技术,该研究使用优化浮珠浮选工艺对小球藻进行采收,随后选取小球藻-表面层状聚合物浮珠聚集体进行破壁提油处理,并通过响应面优化破壁工艺,建立一种新型耦合浮珠-超声辅助溶剂萃取工艺。结果表明,在超声时间为13 min,正己烷：异丙醇体积比例为4,微藻质量浓度为13.6 g/L,超声功率为254 W时,油脂提取效率较高,为18.91%。相比传统气浮法与超声辅助溶剂萃取法,该法采收效率、细胞破壁效率和饱和脂肪酸含量都达到了较高水平,分别为98.36%、90.19%和37.03%。因此,耦合浮珠-超声辅助溶剂萃取工艺是一种有效提取小球藻细胞中油脂的工艺。研究结果为微藻生物柴油制备工艺的发展提供科学依据。     

Lipid fractions with aggregatory and antiaggregatory activity toward platelets in fresh and fried cod (Gadus morhua): correlation with platelet-activating factor and atherogenesis

Cod (Gadus morhua) is a popular part of the diet in many countries on both sides of the North Atlantic; in most cases it is consumed fried. In this study, total lipids of cod muscle were separated into neutral and polar lipids, which were further fractionated by HPLC. The lipid fractions were tested in vitro, against washed rabbit platelets, for the probable existence of lipid compounds that either exhibit an action similar to that of platelet-activating factor (PAF) or inhibit the action of PAF. The platelet bioassay was used to evaluate total lipids, total polar lipids, and total neutral lipids, before any further separation. Detection of these compounds in fresh and fried cod could be used to evaluate the nutritional value of this important fish. The in vitro biological study of lipids showed that in fresh cod lipid fractions, ranges of PAF-like and anti-PAF-like activities were present, whereas in fried cod lipid fractions, both neutral and polar, anti-PAF activities were mainly observed. Because it has already been reported that PAF is involved in atheromatosis generation, the existence of PAF inhibitors in cod may contribute to the possible protective role of fish, in this case cod, against atherosclerosis.     

Ultrasonic extraction of phenols from olive mill wastewater: comparison with conventional methods

Recovery of phenols from olive mill wastewater (OMWW) was studied, comparing five sample preparation methods: filtration, solid-phase (SPE), liquid-liquid (LLE) and ultrasonic (US)-assisted extraction of liquid and solid (freeze-dried) OMWW. Results showed that ultrasonication is a good alternative to conventional solvent extractions, providing higher recoveries at both levels of individual and total phenol yields. Sonication of liquid OMWW in organic solvent was more efficient vs its nonassisted counterpart (agitation), but did not provide a representative phenol chromatogram due to ethyl acetate use. By contrast, the US-assisted extraction of freeze-dried OMWW (3 × 20 min) in 100% methanol (1.5 g/25 mL, w/v) offered the highest qualitative-quantitative phenol yields without any US-induced alterations. Moreover, freeze-drying is an excellent preservation of initial liquid OMWW, holding a great potential for delayed analysis. This study is also the first report that Slovenian OMWW may be utilized as a valuable source of phenols, especially hydroxytyrosol and tyrosol.     

Lipid [corrected] classes, fatty acids, and sterols in seafood from Gilbert Bay, southern labrador

Seafood from Gilbert Bay, southern Labrador, was sampled for lipid classes, fatty acid, and sterol composition. Gilbert Bay is a proposed Marine Protected Area, and the composition of seafood from this region is interesting from both human health and ecological perspectives. Analyses included four species of bivalves and flesh and liver samples from four fish species. Lipids from a locally isolated population of northern cod (Gadus morhua) were also compared to lipids from other cod populations. Lipid classes were analyzed by Chromarod/Iatroscan TLC-FID, fatty acids by GC, and sterols by GC-MS. Three cod populations had similar levels of total lipid per wet weight (0.6%) with triacylglycerols (TAG), sterols, and phospholipids comprising on average 13, 11, and 51%, respectively, of their total lipids. Fatty fish such as capelin and herring contained on average 8.4% lipid with 86% present as TAG. Fish livers from cod and herring showed opposite trends, with cod having elevated lipid (27%) and TAG (63%) and herring containing only 3.8% lipid and 20% TAG. Shellfish averaged 0.6% lipid; however, significant lipid class differences existed among species. Fatty acid analysis showed few significant differences in cod populations with on average 57% polyunsaturated fatty acids (PUFA), 18% monounsaturated fatty acids (MUFA), and 24% saturated fatty acids (SFA). Cod livers had lower PUFA (34%) and elevated MUFA (44%) relative to flesh. Bivalves averaged 25% SFA, 18% MUFA, and 57% PUFA, whereas scallop adductor muscle had the highest PUFA levels (63%). Bivalves contained 20 different sterols with cholesterol present as the major sterol (19-39%). trans-22-Dehydrocholesterol, brassicasterol, 24-methylenecholesterol, and campesterol individually accounted for >10% in at least one species. High levels of PUFA and non-cholesterol sterols observed in Gilbert Bay seafood demonstrate their positive attributes for human nutrition.     

Hydroxytyrosol prevents oxidative deterioration in foodstuffs rich in fish lipids

Hydroxytyrosol, a natural phenolic compound obtained from olive oil byproduct, was characterized as an antioxidant in three different foodstuffs rich in fish lipids: (a) bulk cod liver oil (40% of omega-3 PUFAs), (b) cod liver oil-in-water emulsions (4% of omega-3 PUFAs), and (c) frozen minced horse mackerel ( Trachurus trachurus) muscle. Hydroxytyrosol was evaluated at different concentration levels (10, 50, and 100 ppm), and its antioxidant capacity was compared against that of a synthetic phenolic, propyl gallate. Results proved the efficiency of hydroxytyrosol to inhibit the formation of lipid oxidation products in all tested food systems, although two different optimal antioxidant concentrations were observed. In bulk oil and oil-in-water emulsions, a higher oxidative stability was achieved by increasing the concentration of hydroxytyrosol, whereas an intermediate concentration (50 ppm) showed more efficiency, delaying lipid oxidation in frozen minced fish muscle. The endogenous depletion of alpha-tocopherol and omega-3 polyunsaturated fatty acids (omega-3 PUFAs) was also inhibited by supplementing hydroxytyrosol in minced muscle; however, the consumption of the endogenous total glutathione was not efficiently reduced by either hydroxytyrosol or propyl gallate. A concentration of 50 ppm of hydroxytyrosol was best to maintain a longer initial level of alpha-tocopherol (approximately 300 microg/g of fat), whereas both 50 and 100 ppm of hydroxytyrosol were able to preserve completely omega-3 PUFAs. Hydroxytyrosol and propyl gallate showed comparable antioxidant activities in emulsions and frozen fish muscle, and propyl gallate exhibited better antioxidant efficiency in bulk fish oil.     

Added triacylglycerols do not hasten hemoglobin-mediated lipid oxidation in washed minced cod muscle

Hemoglobin-mediated lipid oxidation in washed, minced cod muscle was related to the triacylglycerol to membrane lipid ratio. The same rapid development of thiobarbituric acid reactive substances (TBARS) and painty odor occurred with and without the presence of up to 15% menhaden oil. Without hemoglobin, development of TBARS and painty odor was slow, despite a high amount of hydroperoxides in samples with oil added (1135 micromol/kg muscle). This suggested that hemoglobin reacted by cleaving preformed hydroperoxides into secondary oxidation products. Nearly doubling the hemoglobin concentration approximately doubled the extent of lipid oxidation with and without added oil. This indicated that hemoglobin was limiting for the oxidation reaction. The noneffect of added oil suggests that membrane lipids and/or preformed membrane lipid hydroperoxides provided sufficient substrate in hemoglobin-catalyzed oxidation of washed minced cod muscle. Fe(2+-)ADP did not induce any oxidation of washed minced cod with/without added oil. Results suggest that lipid oxidation in fatty fish may be more related to the quantity and type of the aqueous pro-oxidant and the membrane lipids than to variations in total fat contents.     

Fractionation of honey carbohydrates using pressurized liquid extraction with activated charcoal

This article describes the development of a new procedure that combines the use of activated charcoal and pressurized liquid extraction (PLE) to obtain enriched fractions of di- and trisaccharides from honey. Honey was adsorbed onto activated charcoal and packed into a PLE extraction cell. Optimum results were obtained at 10 MPa and 40 degrees C using two consecutive PLE cycles: first, 1:99 (v/v) ethanol/water for 5 min and second, 50:50 (v/v) ethanol/water for 10 min. Di- and trisaccharide fractions were enriched after PLE treatment, accounting for 73% and 8% of total carbohydrates, respectively. This procedure was also compared with other methodologies reported in the literature for the fractionation of honey carbohydrates (yeast treatment and extraction from activated charcoal). While the removal of monosaccharides was more efficient with yeast treatment, recovery of di- and trisaccharides was higher when either the PLE or the activated charcoal treatment was used. PLE was found to be the faster technique; it also required less solvent volume and minimized handling of the sample.     

Rapid determination of methyl mercury in fish and shellfish: method development

The AOAC official first action method for methyl mercury in fish and shellfish was modified to provide more rapid determination. Methyl mercury is isolated from homogenized, acetone-washed tissue by addition of HCl and extraction by toluene of the methyl mercuric chloride produced. The extract is analyzed by electron capture gas chromatography (GC) on 5% DEGS-PS treated with mercuric chloride solution. The quantitation limit of the method is 0.25 micrograms Hg/g. Swordfish, shark, tuna, shrimp, clams, oysters, and NBS Research Material-50 (tuna) were analyzed for methyl mercury by the AOAC official first action method. All products also were analyzed by the modified method and the AOAC official method for total Hg. In addition, selected extracts obtained with the modified method were analyzed by GC with Hg-selective, microwave-induced helium plasma detection. There was no significant difference between the results for the various methods. Essentially all the Hg present (determined as total Hg) was in the organic form. Coefficients of variation from analyses by the modified method ranged from 1 to 7% for fish and shellfish containing methyl mercury at levels of 0.50-2.30 micrograms Hg/g. The overall average recovery was 100.5%.     

溶剂萃取与生物降解法联合修复老化石油污染土壤

This study aimed to evaluate the efficacy, practicality and sustainability of a combined approach based on solvent extraction and biodegradation to remediate the soils contaminated with high levels of weathered petroleum hydrocarbons. The soils used in this study were obtained from the Shengli Oilfield in China, which had a long history of contamination with high concentrations of petroleum hydrocarbons. The contaminated soils were washed using a composite organic solvent consisting of hexane and pentane (4:1, v/v) and then bioremediated in microcosms which were bioaugmentated with Bacillus subtilis FQ06 strains and/or rhamnolipid. The optimal solvent extraction conditions were determined as extraction for 20 min at 25 °C with solvent-soil ratio of 6:1 (v/w). On this basis, total petroleum hydrocarbon was decreased from 140 000 to 14 000 mg kg-1, which was further reduced to < 4 000 mg kg-1 by subsequent bioremediation for 132 d. Sustainability assessment of this integrated technology showed its good performance for both short- and long-term effectiveness. Overall the results encouraged its application for remediating contaminated sites especially with high concentration weathered hydrocarbons.     

Comparison of peroxide value methods used for semihard cheeses

The objective was to evaluate alternatives to the peroxide value method of choice in the dairy industry, the method issued by the International Dairy Federation. Furthermore, the study evaluated the feasibility of alternative solvents for extracting lipids and subsequent peroxide value determinations. Packaged cheeses were stored under illuminated display at 4 degrees C to obtain samples with various peroxide contents but with uniform gross composition. The hydroperoxide contents were measured during 3 weeks of storage by applying two lipid extraction methods, Folch and Bureau of Dairy Industry (BDI) extractions, and three different hydroperoxide extraction solutions [chloroform/methanol (7:3, v/v), hexane/2-propanol/methanol (5:7:2, v/v/v), and methanol/decanol/hexane (3:2:1, v/v/v)], prior to standard colorimetric measurements. Extraction yields of fat from Havarti cheeses using the Folch and BDI extraction methods were approximately 109 and 61%, respectively, of the yields obtained by the International Dairy Federation gravimetric reference method. Although differences in fat extraction yields were compensated for, significantly higher peroxide values resulted from the Folch extraction method than from the BDI extraction method. The peroxide values obtained by the three methods were all in the same range, and pronounced linear correlations between peroxide contents determined using the three solutions were noted (r (2) in the range of 0.951-0.983). Peroxide value levels were not significantly different in samples stored in the dark or exposed to light.     

Supercritical fluid extraction of organochlorine pesticides in eggs

The efficacy of supercritical fluid extraction (SFE) for the recovery of 16 common organochlorine pesticides (OCPs) from liquid whole eggs was investigated by employing supercritical carbon dioxide (SC-CO(2)) without the use of a solvent modifier to minimize interfering coextractives. The OCPs tested included aldrin; alpha-, beta-, delta-, and gamma-BHCs; p,p'-DDD, -DDE, and -DDT; dieldrin; endosulfans I, II, and sulfate; endrin; endrin aldehyde; heptachlor; and heptachlor epoxide. The SFE conditions were as follows: 10000 psi (680 bar), 40 degrees C, SC-CO(2) flow rate of 3.0 L/min with an extraction time of 40 min for a total of 120 L of CO(2). The OCPs were trapped off-line in an SPE cartridge containing Florisil and then eluted by an acetone/hexane mixture and analyzed by gas chromatography-electron capture detection (GC-ECD). Recovery studies were carried out on homogenized eggs fortified at the 0.05, 0.10, and 0.20 ppm levels. At the lowest level, 0.05 ppm, recoveries ranged from 81.8 to 108.3%, with CVs < 9.8%. All recoveries were significantly higher than those obtained by an AOAC/FDA solvent extraction method. Eggs containing incurred endosulfan I were also effectively extracted by SFE. This study suggests that the application of SFE for the extraction of OCPs from eggs will result in significant savings in analysis time and lower solvent use and disposal costs compared to conventional solvent extraction procedures.     

Fat extraction from acid- and base-hydrolyzed food samples using accelerated solvent extraction

This paper describes a new in-cell method for pursuing accelerated solvent extraction (ASE) prior to lipid analysis from food samples. It is difficult to pursue direct ASE with acid- or base-hydrolyzed samples due to the corrosive nature of the reagents and material limitations. In this study ion exchange based materials were used to remove acid or base reagents in-cell without compromising the recovery of lipids. The performance data are presented here for the new methods for lipid extraction for a variety of food samples and compared to the Mojonnier method. NIST Standard Reference Materials (SRM-1546 and SRM-1849) were used to validate the ASE methods. Excellent fat recoveries were obtained for the ASE methods. The new methods presented here enhance the utility of ASE and eliminate labor intensive protocols.     

Ethyl acetate/ethyl alcohol mixtures as an alternative to folch reagent for extracting animal lipids

The lipids of fresh egg yolk, boiled yolk, yolk powder, and raw animal tissues including pork loin, belly pork, and pork fat were extracted with the mixed solvents composed of ethyl acetate (EtOAc) and ethyl alcohol (EtOH) at 2:1 and 1:1 volume ratios, and the results were compared with those obtained with Folch reagent, that is, a mixture of chloroform and methyl alcohol (2:1, v/v). Extraction yields, lipid profiles, and fatty acid compositions were determined by weighing, TLC-FID, and GC, respectively. Data of the extracts obtained with the mixtures of EtOAc and EtOH were not significantly different from those obtained with Folch reagent, implying that the mixed solvent composed of EtOAc and EtOH (1:1 to 2:1, v/v) may replace Folch reagent, which is considered to be toxic and mutagenetic due to its component of CHCl3, for lipid extraction.