Preparation Of Fatty Acid Methyl Esters Research Articles

Determination of free fatty acids in plasma by gas chromatography.

A method was developed for determination of free fatty acids (FFAs) in plasma by gas chromatography. Plasma was extracted with 3vol of methanol. Most cholesterol esters and triacylglycerols did not dissolve in the aqueous methanol. FFAs in the crude lipid solution were directly and selectively methylated with (trimethylsilyl)diazomethane at room temperature. Fatty acid methyl esters (FAMEs) formed were extracted with hexane, and nonreactive phospholipids were washed out with 95% methanol. The partially purified FAME preparation was analyzed by gas chromatography. The composition and amount of plasma FFAs closely approximated those obtained using two different methods.

A Simplified Gas Chromatographic Fatty-Acid Analysis by the Direct Saponification/Methylation Procedure and Its Application on Wild Tuna Larvae.

A method for the direct preparation of fatty-acid methyl esters (FAME) was simplified for fatty-acid analysis of a single fish larva using gas chromatography (GC). The method included the isolation of a larval trunk and drying in a glass vial, followed by saponification of all the contents without prior lipid extraction. Thereafter, the fatty acids released were methylated by trimethylsilyldiazomethane. This method has advantages over another method, direct acid-catalyzed transesterification, because both the saponification and methylation at room temperature can reduce loss of unsaturated fatty acids and formation of artifacts unavoidable in acidic reaction at high temperature. GC of the products showed that the simplified method can yield methyl esters without artifacts interfering analysis. More than 50 fatty acids were determined, which are twice as many as those previously analyzed using high-performance liquid chromatography. Observation of consistent small impurities in GC of blank tests allowed the accurate determination of fatty acids by correcting the peak areas. Dry matter weights (<3 mg) and the total fatty-acid contents displayed a linear relationship. Fatty-acid analysis of wild larvae of bluefin tuna, yellowfin tuna, and skipjack tuna collected from the waters around Japan (n = 100) revealed that the eicosapentaenoic acid (EPA) level in bluefin tuna collected from the Japan Sea was significantly higher than that in the three species collected from Nansei Islands. The simplified direct saponification/methylation method will be a powerful tool for investigating growth and survival of individual larval tuna and other fish species.

Comparison of methylation methods for fatty acid analysis of milk fat

Three acid- and alkaline-catalysed transesterification methods were compared with the aim to validate a simple yet reliable protocol for fatty acid (FA) profiling of milk fat. While both the acid- and alkaline-catalysed methods were able to convert completely triglycerides and phospholipids into fatty acid methyl esters (FAMEs), the acid catalyst caused significant degradation of conjugated linoleic acid C18:2c9t11 at high temperature. Although a milder temperature can mitigate this negative impact, a long reaction time (2 h) is required to achieve full methylation. By contrast, despite being unable to methylate free fatty acids (FFA), the alkaline-catalysed transesterification yielded comparable results for all major FA due to the very low level of FFA in milk. The alkaline-catalysed methylation is benign for C18:2c9t11. We recommend here a simple one-step protocol based on 0.2 M methanolic KOH, a short reaction time (20 min) and a mild reaction temperature (50 °C) for milk FAME preparation.

The issue of reducing or removing phospholipids from total lipids of a microalgae and an oleaginous fungus for preparing biodiesel

ABSTRACTThis study is divided into three parts and examines recommendations made in the literature to remove or reduce phospholipids, a class of polar lipids, from microalgal and fungal lipids, so that transesterification of the neutral lipids with methanol improves the yield of fatty acid methyl esters (FAME) (biodiesel). Literature on the role of phospholipids lowering the yield of FAME during transesterification of vegetable oils is reviewed in the first part of this study. The second part of this study scrutinizes two published papers on the preparation and yield of FAME by transesterification of total lipids present in microalgae of Chlorella pyrenoidosa (CP) and oleaginous fungus Mucor circinelloides (MC) with or without biomass in different solvent systems. Mole balance between acyl groups present in total lipids of CP and MC with methanol in part 3 makes it possible to use the results reviewed in part 1 of reduction in the yield of FAME in vegetable oils due to phospholipids and excess methanol. In conclusion, it is recommended to separate neutral lipids from polar lipids/phospholipids present in the total lipids of microalgae and fungi by supercritical carbon dioxide to prepare FAME in improved yields and keep phosphorus content below the polluting limit in the biodiesel prepared.

Rapid microwave assisted preparation of fatty acid methyl esters for the analysis of fatty acid profiles in foods

A microwave (MW) assisted rapid high through-put method for the preparation of fatty acid methyl esters (FAME) for the analysis of fatty acid profiles in a selection of foods was evaluated by comparing fatty acid profiles with those resulting from conventional FAME preparation. The microwave method gave fatty acid profiles in close agreement with those arising from conventional methods and the protocol gave acceptable recoveries (98–102%) and repeatability (RSDs for replicate analyses were 0.56–5.2%). In comparison to conventional methods the MW assisted method was simple, rapid and universally effective across foods ranging from dairy products to ready meals. EU regulation (No. 1169/2011) requiring declaration of the saturated, monounsaturated and polyunsaturated content of foods is placing pressure on processors and contract laboratories to analyse fatty acid profiles using relatively low through-put conventional methods. MW assisted preparation of FAMEs offers analysts a high-through-put, rapid and universal method to help overcome this potentially costly and labour intensive regulatory hurdle.

A high-throughput fatty acid profiling screen reveals novel variations in fatty acid biosynthesis in Chlamydomonas reinhardtii and related algae.

Analysis of fatty acid methyl esters (FAMEs) by gas chromatography (GC) is a common technique for the quantitative and qualitative analysis of acyl lipids. Methods for FAME preparation are typically time-consuming and labor-intensive and require multiple transfers of reagents and products between reaction tubes and autosampler vials. In order to increase throughput and lower the time and materials costs required for FAME preparation prior to GC analysis, we have developed a method in which 10-to-20-mg samples of microbial biomass are transferred to standard GC autosampler vials, transesterified using an emulsion of methanolic trimethylsulfonium hydroxide and hexane, and analyzed directly by GC without further sample handling. This method gives results that are essentially identical to those obtained by the more labor- and material-intensive FAME preparation methods, such as transmethylation with methanolic HCl. We applied this method to the screening of laboratory and environmental isolates of the green alga Chlamydomonas for variations in fatty acid composition. This screening method facilitated two novel discoveries. First, we identified a common laboratory strain of C. reinhardtii, CC-620, completely lacking all ω-3 fatty acids normally found in this organism and showed that this strain contains an inactivating mutation in the CrFAD7 gene, encoding the sole ω-3 desaturase activity in this organism. Second, we showed that some species of Chlamydomonas make Δ6-unsaturated polyunsaturated fatty acids (PUFA) rather than the Δ5 species normally made by the previously characterized laboratory strains of Chlamydomonas, suggesting that there is species-specific variation in the regiospecificity and substrate selectivity of front-end desaturases in this algal genus.

Fast and accurate preparation fatty acid methyl esters by microwave-assisted derivatization in the yeast Saccharomyces cerevisiae

We present a fast and accurate method for preparation of fatty acid methyl esters (FAMEs) using microwave-assisted derivatization of fatty acids present in yeast samples. The esterification of free/bound fatty acids to FAMEs was completed within 5 min, which is 24 times faster than with conventional heating methods. The developed method was validated in two ways: (1) through comparison with a conventional method (hot plate) and (2) through validation with the standard reference material (SRM) 3275-2 omega-3 and omega-6 fatty acids in fish oil (from the Nation Institute of Standards and Technology, USA). There were no significant differences (P>0.05) in yields of FAMEs with both validations. By performing a simple modification of closed-vessel microwave heating, it was possible to carry out the esterification in Pyrex glass tubes kept inside the closed vessel. Hereby, we are able to increase the number of sample preparations to several hundred samples per day as the time for preparation of reused vessels was eliminated. Pretreated cell disruption steps are not required, since the direct FAME preparation provides equally quantitative results. The new microwave-assisted derivatization method facilitates the preparation of FAMEs directly from yeast cells, but the method is likely to also be applicable for other biological samples.

Comparison of Transesterification Methods for Fatty Acid Analysis in Higher Fungi: Application to Mushrooms

This study evaluated the effectiveness of two commonly used transesterification methods for the preparation of fatty acid methyl esters (FAMEs) for gas chromatographic analysis. A combination of base followed by acid catalysis [potassium hydroxide/sulfuric acid/methanol (KOH–H2SO4–MeOH)] was compared to an acid-only catalyst [methanolic–boron trifluoride (BF3–MeOH)] for the quantification of the fatty acid content in mushrooms after preextraction of total lipids with chloroform/methanol (2:1 v/v). The mushrooms studied were commonly cultivated species, Agaricus bisporus (brown) and A. bisporus (white), and three wild medicinal mushrooms, turkey tail (Trametes versicolor), artist conk (Ganoderma applanatum), and tinder polypore (Fomes fomentarius). The major fatty acids identified were palmitic (C16:0), stearic (C18:0), oleic (C18:1), and linoleic acid (18:2n6) which constituted 80% to 90% of the total amount. Variation due to the FAME preparation methods was observed in both minor and major fatty acids. Another major observation made between the two methods was the total number of individual fatty acids (FAs) methylated. The base and acid transesterification procedure resulted in the identification of 42 FAMEs, whereas with the acid 38 FAMEs were observed. Therefore, the research recommends the use of a base–acid combination for preparation of FAMEs in mushroom lipids.

Efficacy of fatty acid profile as a tool for screening feedstocks for biodiesel production

Fuel properties are largely dependent on the fatty acid (FA) composition of the feedstock from which biodiesel is prepared. Consequently, FA profile was employed as a screening tool for selection of feedstocks high in monounsaturated FAs for further evaluation as biodiesel. Those feedstocks included ailanthus (Ailanthus altissima L.), anise (Pimpinella anisum L.), arugula (Eruca vesicaria L.), cress (Lepidium sativum L.), cumin (Cuminum cyminum L.), Indian cress (Tropaeolum majus L.), shepherd’s purse (Capsella bursa-pastoris L.) and upland cress (Barbarea verna (Mill.) Asch.). Other selection criteria included saturated FA content, iodine value (IV), content of FAs containing twenty or more carbons and content of trienoic FAs. Anise oil satisfied all selection criteria and was therefore selected for further investigation. Arugula, cumin and upland cress oils were selected as antagonists to the selection criteria. Preparation of FA methyl esters (FAMEs, ≥ 92 wt % yield) following conventional alkaline-catalyzed methanolysis preceded fuel property determination. Of particular interest were oxidative stability and cold flow properties. Also measured were kinematic viscosity (40 °C), IV, acid value, free and total glycerol content, sulfur and phosphorous content, cetane number, energy content and lubricity. FAMEs prepared from anise oil yielded properties compliant with biodiesel standards ASTM D6751 and EN 14214 whereas the antagonists failed at least one specification contained within the standards. As a result, FA profile was an efficient predictor of compliance with biodiesel standards and is therefore recommended as a screening tool for investigation of alternative feedstocks.

Lower Serum Paraoxonase-1 Activity Is Related to Linoleic and Docosahexanoic Fatty Acids in Type 2 Diabetic Patients

Serum paraoxonase-1(PON-1) activity is decreased in clinical conditions associated with low high-density lipoprotein cholesterol (HDL-C), increased lipid peroxidation and low-grade chronic inflammation, as in type 2 diabetes mellitus (T2DM). Until now there are no data about the association of any fatty acid (FA) with PON-1 activity in T2DM. Twenty patients with T2DM and 16 healthy controls were included in this cross-sectional study. Serum PON-1 activity, superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity as well as plasma glucose, HbA1c, lipids, high-sensitivity C-reactive protein (hs-CRP) and insulin resistance, homeostasis model assessment (HOMA-IR) were measured. The preparation of FA methyl esters and their gas chromatography (GC) analysis were also performed. HbA1c, plasma insulin, HOMA-IR and triglycerides were higher in patients with T2DM, whereas HDL-C was lower in those subjects. Levels of pro-oxidative enzyme malondialdehyde (MDA) and hs-CRP were significantly higher, and anti-oxidative enzymes SOD and PON-1 activity were decreased in T2DM patients. N-6 PUFAs were higher in T2DM patients, particularly linoleic acid (LA, 18:2 n-6) and arachidonic acid (AA, 20:4 n-6), whereas n-3 PUFA, docosahexaenoic acid (DHA, 22:6 n-3) was lower in T2DM patients. Using regression analysis, we have shown that only LA and DHA independently predicted PON-1 activity of all participants, particularly in patients with T2DM. Decreased serum PON-1 activity may, in part, be influenced by higher levels of LA and lower levels of DHA in patients with T2DM. Prospective, randomized studies are necessary to confirm these preliminary findings.

A microfluidic device for the automated derivatization of free fatty acids to fatty acidmethylesters

Free fatty acid (FFA) compositions are examined in feedstock for biodiesel production, as source-specific markers in soil, and because of their role in cellular signaling. However, sample preparation of FFAs for gas chromatography-mass spectrometry (GC-MS) analysis can be time and labor intensive. Therefore, to increase sample preparation throughput, a glass microfluidic device was developed to automate derivatization of FFAs to fatty acid methyl esters (FAMEs). FFAs were delivered to one input of the device and methanolic-HCl was delivered to a second input. FAME products were produced as the reagents traversed a 29 μL reaction channel held at 55 °C. A Design of Experiment protocol was used to determine the combination of derivatization time (T(der)) and ratio of methanolic-HCl:FFA (R(der)) that maximized the derivatization efficiencies of tridecanoic acid and stearic acid to their methyl ester forms. The combination of T(der) = 0.8 min and R(der) = 4.9 that produced optimal derivatization conditions for both FFAs within a 5 min total sample preparation time was determined. This combination of T(der) and R(der) was used to derivatize 12 FFAs with a range of derivatization efficiencies from 18% to 93% with efficiencies of 61% for tridecanoic acid and 84% for stearic acid. As compared to a conventional macroscale derivatization of FFA to FAME, the microfluidic device decreased the volume of methanolic-HCl and FFA by 20- and 1300-fold, respectively. The developed microfluidic device can be used for automated preparation of FAMEs to analyze the FFA compositions of volume-limited samples.

Biofuels from Algae

Fatty acid methyl esters (FAME) are used as alternative diesel fuel originating from renewable sources. The attention is focused on the materials that do not compete with food and feed production. Algae have a significant potential as an alternative biodiesel feedstock. In comparison to other crops, they have the advantage in very fast reproduction cycles, enhanced resistance to high UV radiation doses and higher effectiveness of energy conversion to biomass due to low demands on other metabolic functions. Moreover, they fix waste CO2 very efficiently. The key problem is to destroy the algae cell in order to obtain the oil. In this work, the results of FAME preparation from received algae oils are presented. This study confirms that Nannochloropsis and Chlorella microalgae provide valuable triacyglycerols for the biofuel production. Characteristics of prepared FAME meet the EN 14 214 standard requirements. However, FAME from algae oil may exhibit impaired oxidative stability due to higher level of unsaturation.

Free-fatty acid profile obtained by enzymatic solvent-free hydrolysis of sunflower and soybean lecithins

The free-fatty acid profile of sunflower and soybean lecithins generated by enzymatic hydrolysis was determined by capillary gas chromatography using the internal standard method. This procedure involves the previous lipid extraction, clean-up and fatty-acid methyl-esters preparation. Different conventional methods commonly employed to calculate the hydrolysis degree in both substrates were compared. The fatty-acid profile of sunflower and soybean phospholipids and its initial acidity composition were chromatographically determined. Results from recovery studies were satisfactory (98% and 108% for soybean and sunflower, respectively). The effect of temperature (50 and 60 °C) and pH conditions (pH 7 and pH 8) on lecithin-hydrolysis degree was evaluated, being temperature the most affecting parameter. The profile of the fatty acids liberated after hydrolysis depended on both the enzyme and the pH of reaction mixture. Finally, the chromatographic methodology employed in this paper may be useful to study the lecithin enzymatic modification in order to produce emulsifiers with specific characteristics.

Rapid microwave-assisted transesterification for the preparation of fatty acid methyl esters from the oil of yellow horn (Xanthoceras sorbifolia Bunge.)

An efficient microwave-assisted transesterification (MAT) technique was developed to prepare fatty acid methyl esters (FAMEs) from yellow horn (Xanthoceras sorbifolia Bunge.) oil. To evaluate the applicability of this novel MAT method, the conversion yields of FAMEs and compositions were compared with those from the standard method of transesterification (AOAC Method 996.06). The results showed that MAT had obvious advantages to standard techniques in terms of short duration, high efficiency and stable composition. The parameters influencing the MAT procedure including microwave irradiation power, temperature, reaction time, molar ratio of methanol to oil and catalyst concentration were optimized. The maximum yield of FAMEs reached 96% under optimal MAT conditions of transesterification irradiation power 500 W, temperature 60 °C, 6 min, 1% wt catalyst and a molar ratio of methanol/oil 6:1 (v/v). Validation of the MAT method was performed in terms of repeatability and reproducibility; relative standard deviations for conversion yields were lower than 4.37 and 6.83%, respectively. These results revealed that the MAT method developed in the present investigation is suitable for the sample preparation of FAMEs from plant oils.

Fatty acid methyl esters preparation in the presence of maltolate and n-butoxide Ti(IV) and Zr(IV) complexes

Abstract Two series of complexes with general formula M ( n -butoxide) 4− x (maltolate) x , where M = Ti or Zr and x = 0–4, were studied as catalyst for transesterification and esterification, in order to obtain methyl esters. The compounds containing different ratios of maltolate and n -butoxide ligands were synthesized from the reaction of maltol (3-hydroxy-2-methyl-4-pyrone) and n -butoxide metal precursors. All complexes, containing maltolate as ligand, were very efficient as catalyst in esterification, mainly those based in zirconium. In counter part, the catalysts here displayed very poor activity for transesterification.

Application of factorial design in optimization of anion exchange resin based methylation of vegetable oil and fats

Abstract A simple, rapid and fairly selective method for the preparation of fatty acid methyl esters (FAMEs) based on anion exchange resin Amberlite IRA-904 catalyzed transesterification of vegetable oil/fat with iodomethane has been described. The vegetable oil and animal fats used were sunflower oil, palm oil, vanaspati (hydrogenated vegetable oil), olive oil, tallow and butter. A Plackett–Burman factorial experimental design was used as a multivariate strategy for the evaluation of the effects of varying several variables at once. The effects of five different variables amount of resin, strength of sodium hydroxide, volume of iodomethane, heating time and temperature of thermostatic water bath, on the yield of fatty acid methyl esters (FAMEs) have been investigated. From these studies, certain variable showed up as significant, and they were optimized by a using 23 + star central composite design, which involved 16 experiments. The best conditions for transesterification reaction were as follows: amount of resin 2 g, strength of sodium hydroxide 0.25 N, volume of iodomethane 400 µl, heating time 2 min at 70 °C. A standard IUPAC method was used to prepare FAMEs from vegetable oil/fats for comparative purpose. Finally samples of oil/fat obtained from both methods were analysed by Gas liquid chromatography. Analytical results for the FAMEs by resin based proposed method, and conventional IUPAC method showed a good agreement, thus indicating the possibility of using Amberlite IRA-904 based transesterification instead of intensive treatments inherent with the conventional time-consuming methods. Industrial relevance Fatty acids are the main components of edible oil and fats, therefore determination of fatty acid composition is so far one of the important parameters for quality evaluation and nutritional value determination of edible oil and fats. The analysis of fatty acid is usually carried out by Gas liquid chromatography (GLC) after conversion of volatile fatty acid methyl esters (FAME) although other ester may be prepared for specific purpose. The endeavor of present work was to improve the FAMEs preparation method, proposing the development of anion exchange resin Amberlite IRA-904 based transesterification of edible oil/fat with iodomethane as alkylating reagent. The present method besides being rapid and reproducible avoids the use of classical saponification, washing of esters and solvent extraction step.

One-Step Lipid Extraction and Fatty Acid Methyl Esters Preparation from Fresh Plant Tissues

A procedure for the digestion of fresh tissue, transmethylation of lipids, and the extraction of fatty acid methyl esters (FAMES) in one step is described. Fresh tissues or isolated lipids are heated with a reagent containing methanol:heptane:benzene:2,2-dimethoxypropane:H 2SO 4 (37:36:20:5:2, by vol), methanol:heptane:toluene:2,2-dimethoxypropane:H 2SO 4 (39:34:20:5:2, by vol), or methanol:heptane:tetrahydrofuran:2,2-dimethoxypropane:H 2SO 4 (31:42:20:5:2; by vol). At 80°C the simultaneous digestion and lipid transmethylation takes place in a single phase. After cooling at room temperature two phases are formed; the upper one contains the FAMES ready for GLC analysis. The procedure allows the determination of the fatty acid composition of lipids from tissues containing high proportions of triacylglycerols (oil seeds), water (leaves), or both (oil fruits). Due to the complete extraction of the lipids from the tissue, their content can be determined by adding an internal standard to the sample.

Simple Analysis Method for Fatty Acids in Food Samples

A simple and rapid analytical method for the determination of fatty acids in food samples by gas liquid chromatography (GC) was developedA sample of 10mg (as lipids) was dissolved in 2ml of hexane containing triphenylethylene as an internal standard (IS). Then 1ml of 2N sodium hydroxide-methanol solution was added, and the mixture was heated at 60° in a water-bath for 1min. Methyl-esterification was carried out with 1ml of 2N hydrochloric acid-methanol solution. The hexane layer was rinsed with water, and the fatty acid composition of food samples was estimated by GC on a Unisole 3000 column. Total fatty acids were determined from palmitic acid (16:0) composition and the peak area ratio of 16:0 methyl ester and IS.The proposed method was applied to mixed lipids and lipids extracted from food, and the results were compared with those obtained by the sodium methoxide (NaOMe) method and BF3 methanol (BF3-MeOH) method. The proposed method was superior to the NaOMe method and BF3-MeOH method in terms of detection limit and reproducibility.This method allows the rapid preparation of fatty acid methylesters and the simple and accurate determination of fatty acids, and is suitable for the routine analysis of fatty acids in food samples.

Evaluation of a rapid method for preparation of fatty acid methyl esters for analysis by gas—liquid chromatography

The major limitation to fatty acid analysis by gas—liquid chromatography is associated with preparation of fatty acid methyl esters (FAME). In the present study, FAME preparations were made from plant oils (corn, olive, sunflower), sunflower oil margarine, lard and various animal tissue fats by a rapid transesterification involving tetramethylammonium hydroxide in methanol, and also by a longer conventional saponification—esterification method. Fats from animal (beef, mutton, pork) adipose tissues were extracted by a simpler modified procedure and also by the Folch method prior to the rapid and the conventional FAME preparations, respectively. FAME analysis on a gas—liquid chromatograph equipped with a Silar 10C glass capillary column indicated similar fatty acid composition of a given fat or oil, whether FAME was prepared by the rapid or the longer conventional method. The data obtained by both methods were very highly correlated for all the fats ( r = 0.9895 – 0.9999). However, the rapid method showed a tendency for enhanced recoveries of lower chain fatty acids ( e.g. 14:0), and also of unsaturated C 18 isomers. Possibly, losses of fatty acids that occurred during the lengthy fat extraction, fatty acid esterification or ether-evaporation FAME concentration steps (conventional method) were minimised by the single transesterification step (rapid method). This rapid transesterification method appears to be an attractive alternative to FAME preparation from a wide variety of different fats for gas—liquid chromatographic analysis.