J. Anim Sci. 2007. 85:1511-1521. doi:10.2527/jas.2006-491
© 2007 American Society of Animal Science
A direct method for fatty acid methyl ester synthesis: Application to wet meat tissues, oils, and feedstuffs
J. V. O’Fallon,
J. R. Busboom,
M. L. Nelson and
C. T. Gaskins1
Department of Animal Sciences, Washington State University, Pullman 99164
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Abstract
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A simplified protocol to obtain fatty acid methyl esters (FAME) directly from fresh tissue, oils, or feedstuffs, without prior organic solvent extraction, is presented. With this protocol, FAME synthesis is conducted in the presence of up to 33% water. Wet tissues, or other samples, are permeabilized and hydrolyzed for 1.5 h at 55°C in 1 N KOH in MeOH containing C13:0 as the internal standard. The KOH is neutralized, and the FFA are methylated by H2SO4 catalysis for 1.5 h at 55°C. Hexane is then added to the reaction tube, which is vortex-mixed and centrifuged. The hexane is pipetted into a gas chromatography vial for subsequent gas chromatography. All reactions are conducted in a single screw-cap Pyrex tube for convenience. The method meets many criteria for fatty acid analysis, including not isomerizing CLA or introducing fatty acid artifacts. It is applicable to fresh, frozen, or lyophilized tissue samples, in addition to oils, waxes, and feedstuffs. The method saves time and effort and is economical when compared with other methods. Its unique performance, including easy sample preparation, is achieved because water is included rather than eliminated in the FAME reaction mixtures.
Key Words: conjugated linoleic acid • fatty acid analysis • fatty acid methyl ester synthesis • feedstuff • fish oil • longissimus muscle
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INTRODUCTION
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The analysis of fatty acids has become increasingly important, because more people have become aware of their nutritional and health implications. Because of this, a method for analyzing fatty acids that provides both rapid and reliable results is of great value. Many methods are currently used to analyze fatty acids (Morrison and Smith, 1964
; Sukhija and Palmquist, 1988; Kazala et al., 1999; Mir et al., 2002; Nuernberg et al., 2002; Budge and Iverson, 2003; Cooper et al., 2004). These methods, however, are not necessarily convenient, nor direct, and often must be optimized for reaction conditions, including the catalyst and the temperature (Lewis et al., 2000; Park et al., 2002; Shahin et al., 2003).
On the other hand, the ideal method, as noted by Palmquist and Jenkins (2003) in discussing challenges encountered in developing fatty acid methods, would determine the total fatty acid concentration in tissues, oils, and feed samples by converting fatty acid salts, as well as the acyl components in all lipid classes, such as triacylglycerols, phospholipids, sphingolipids, and waxes, to methyl esters using a simple, direct, 1-step esterification procedure.
In this paper, we present a method that is based on a surprising concept [i.e., we add water to the fatty acid methyl ester (FAME) synthesis reagents]. Until now, FAME synthesis methods have rigorously avoided water as a matter of standard procedure. However, by adding water, the dynamics of sample preparation and methyl ester formation can be revisited, and the ideal outcome of FAME synthesis discussed by Palmquist and Jenkins (2003) becomes possible. Although the method described herein requires 2 steps, it does so in 1 reaction tube.
In short, the objective of this paper was to develop a method to directly methylate fatty acids from muscle tissue, oils, and feedstuffs in aqueous solution.
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MATERIALS AND METHODS
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Methylation Agents and Sample Selection
To assess certain features of our method, we compared direct FAME synthesis to 2 methylating agents routinely used for FAME synthesis, namely, the base catalyst NaOCH3 and the acid catalyst BF3. Samples used with NaOCH3 and BF3 did not contain water, because this would compromise the performance of these 2 methylating agents. The direct FAME synthesis method, however, always contained water, even if the sample did not, because its reagents contained water; (i.e., the direct FAME synthesis reaction mixture contained 13.2% water due to the water in the KOH and H2SO4 reagents).
The samples we used in this manuscript were chosen for distinct reasons. The Supelco fatty acid standard mixture was chosen, because it contained short- and long-chain SFA, MUFA, and PUFA in defined amounts and thus served as a primary test of the feasibility of our method. Fish oil was chosen, because it is an important source of the long-chain polyunsaturated n-3 esterified eicosapentaenoic, docosapentaenoic, and docosahexaenoic fatty acids. Conjugated linoleic acid, as the free acid, was chosen, because it is of medical importance as perhaps the only fatty acid that can directly inhibit cancer in animal models (Belury, 2002) and because current FAME synthesis methods often cause undesirable isomerizations of this fatty acid (Kramer et al., 1997). Beef LM was chosen because of our special interest in beef fatty acids and because it serves as a direct test of the ability of direct FAME synthesis to extract and methylate all of the fatty acids present in meat tissue. To address the problem of refractory samples, we included wax esters, cholesteryl lipid derivatives, and alkyl methane sulfonates, because these are difficult fatty acid derivatives to analyze (Palmquist and Jenkins, 2003). Finally, as a concluding demonstration of the versatility of the direct FAME synthesis method, we included a variety of oils, feedstuffs, and foods.
Materials
Hexane (OmniSolv) was purchased from EM Science, Cherry Hill, New Jersey. Absolute MeOH and KOH were obtained from J. T. Baker Chemical Co., Phillipsburg, New Jersey. Chloroform and H2SO4 were purchased from Fisher Scientific, Tustin, California. Sodium methoxide and BF3-MeOH were obtained from Sigma-Aldrich, St. Louis, Missouri. The Supelco standard FAME mixture (47885-U) was obtained from Supelco, Bellefonte, Pennsylvania. Spring Valley fish oil capsules were distributed by Leiner Health Products, Carson, California. Tonalin 1000 CLA capsules were obtained from Nature’s Bounty, Bohemia, New York. All other fatty acid standards were purchased from Nu-Chek Prep Inc., Elysian, Minnesota. Beef LM samples (n = 20) were obtained from department-owned animals (Animal Care and Use Committee protocol 3088) processed at an abattoir (Toppenish, WA). Nuts and sundry food items were purchased from local grocery stores. Coffee bean grinders were purchased from Mr. Coffee Inc., Cleveland, OH. Pyrex screw-cap culture tubes (16 x 125 mm) were obtained from Corning Laboratory Science Company, New York. The Tekmar VXR-10 multitube vortex was purchased from Jenke and Kunkel, Staufen, West Germany.
Folch Extraction of Fatty Acids from LM
Longissimus muscle (1 g) was extracted with CHCl3:MeOH (2:1, vol/vol) containing C13:0 as the internal standard, according to the method of Folch et al.(1956), using a Brinkmann polytron at room temperature. The extraction mixture was then filtered through a scintered glass filter, and replicate aliquots were pipetted into a 16 x 125 mm screw-cap Pyrex culture tube and washed with 0.02% aqueous CaCl2. The organic phase was dried with Na2SO4 and K2CO3 (10:1, wt/wt), and the solvent was subsequently removed under N at 55°C.
FAME Synthesis with NaOCH3 or BF3
Freeze-dried tissue samples were uniformly distributed by grinding for 10 to 15 s in a room-temperature coffee bean grinder. Samples of freeze-dried tissue (0.50 g) or oils (40 µL) were placed into a 16 x 125 mm screw-cap Pyrex culture tube to which 1.0 mL of methyl C13:0 internal standard (0.5 mg of methyl C13:0/mL of MeOH) was added. Two milliliters of NaOCH3 (0.5 M) or 2 mL of BF3 in MeOH (14%, wt/vol) was added to the Pyrex tubes containing the samples. The tubes were incubated in a 55°C water bath for 1.5 h with vigorous hand-shaking for 5 s every 20 min. Two milliliters of a saturated solution of NaHCO3 and 3 mL of hexane were then added, and the tubes were vortex-mixed. After centrifugation, the hexane layer containing the FAME was placed into a gas chromatography (GC) vial. The vial was capped and placed at –20°C until GC analysis.
Direct FAME Synthesis
Samples were uniformly distributed by grinding for 10 to 15 s in a room-temperature coffee bean grinder. Short grinding times minimized smearing of the fat on the walls of the grinder container. Alternatively, samples were cut into 1.5-mm rectangular strips with a razor blade or scalpel. Samples could be processed in the state obtained (e.g., wet, dry, freeze-dried, or semifrozen). Samples (1.0 g of wet, dry, or semifrozen sample), 0.50 g of freeze-dried sample, or oils (40 µL) were placed into a 16 x 125 mm screw-cap Pyrex culture tube to which 1.0 mL of the C13:0 internal standard (0.5 mg of C13:0/mL of MeOH), 0.7 mL of 10 N KOH in water, and 5.3 mL of MeOH were added. The tube was incubated in a 55°C water bath for 1.5 h with vigorous hand-shaking for 5 s every 20 min to properly permeate, dissolve, and hydrolyze the sample. After cooling below room temperature in a cold tap water bath, 0.58 mL of 24 N H2SO4 in water was added. The tube was mixed by inversion and with precipitated K2SO4 present was incubated again in a 55°C water bath for 1.5 h with hand-shaking for 5 s every 20 min. After FAME synthesis, the tube was cooled in a cold tap water bath. Three milliliters of hexane was added, and the tube was vortex-mixed for 5 min on a multitube vortex. The tube was centrifuged for 5 min in a tabletop centrifuge, and the hexane layer, containing the FAME, was placed into a GC vial. The vial was capped and placed at –20°C until GC analysis.
GC
The fatty acid composition of the FAME was determined by capillary GC on a SP-2560, 100 m x 0.25 mm x 0.20 µm capillary column (Supelco) installed on a Hewlett Packard 5890 gas chromatograph equipped with a Hewlett Packard 3396 Series II integrator and 7673 controller, a flame ionization detector, and split injection (Agilent Technologies Inc., Santa Clara, CA). The initial oven temperature was 140°C, held for 5 min, subsequently increased to 240°C at a rate of 4°C min–1, and then held for 20 min. Helium was used as the carrier gas at a flow rate of 0.5 mL·min–1, and the column head pressure was 280 kPa. Both the injector and the detector were set at 260°C. The split ratio was 30:1. Fatty acids were identified by comparing their retention times with the fatty acid methyl standards described previously.
Statistical Analysis
Duplicate GC results were averaged for animal and methylation method. An ANOVA of the beef LM FAME was calculated using PROC GLM (SAS Inst. Inc., Cary, NC) using a model with methylation method as the treatment and animal as a blocking factor in a randomized complete block design. When the F-value for the methylation methods was significant, a Student’s t-test was used to make pairwise comparisons among the means.
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RESULTS
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NaOCH3, BF3, and Direct FAME Synthesis Methods Applied to a Supelco Standard FAME Mixture
For an initial FAME synthesis analysis, we used a Supelco standard FAME mixture. This standard mixture contained FAME in a defined ratio and consisted of both short- and long-chain SFA, MUFA, and PUFA. To broadly assess certain features of our method, we compared the results of direct FAME synthesis to those of the base catalyst NaOCH3 and the acid catalyst BF3.
Direct FAME synthesis, as described in "Materials and Methods," is a 2-step procedure. In the first step, sample fatty acid esters are hydrolyzed to FFA, and in the second step, the FFA are converted to FAME. When the first step of direct FAME synthesis was applied to the Supelco standard FAME mixture, the esters were hydrolyzed to FFA that were not volatile enough to enter the GC column. These results (i.e., the absence of fatty acid peaks) provided formal evidence that the first step in direct FAME synthesis completely hydrolyzed the Supelco standard FAME to FFA, which was the desired general prerequisite for the subsequent methylation step of direct FAME synthesis (data not shown).
When the second step of direct FAME synthesis, the methylation step, was applied to the Supelco FFA produced by the first step, the results shown in Table 1 were obtained. All of the GC peaks present in the original Supelco standard mixture were again observed, as can be seen by comparing the fatty acids of direct FAME synthesis to those of the Supelco mix. When presented with a FAME sample, as in this experiment, both NaOCH3 and BF3 likewise gave the same FAME values present in the original Supelco mix (Table 1).
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Table 1. Effect of NaOCH3, BF3, or direct fatty acid methyl ester (FAME) synthesis on a standard Supelco FAME mixture1
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Fatty acid artifacts (e.g., the conversion of CLA to other isomers) are a concern in fatty acid analysis (Kramer et al., 1997; Park et al., 2002; Shahin et al., 2003). Because direct FAME synthesis, NaOCH3, or BF3 did not generate new fatty acid peaks, no fatty acid artifacts were created in the Supelco standard FAME mixture.
The Methods of NaOCH3, BF3, and Direct FAME Synthesis Applied to Fish Oil
A comparison was made among the base catalyst NaOCH3, the acid catalyst BF3, and direct synthesis on FAME production from fish oil commercially obtained as a human nutritional supplement. The results are shown in Table 2.
Twenty fatty acids were identified in the fish oil sample. Direct FAME synthesis recovered more total amounts of fatty acid than did either of the other 2 methods, as would be expected if direct FAME synthesis generated methyl esters of all the fatty acids in the sample. In comparison, base catalysis with NaOCH3 methylated esterified fatty acids but not FFA (Kramer et al., 1997), whereas acid catalysis by BF3 should have methylated all fatty acids, including esterified, unesterified, and those in salt form (Carrapiso and Garcia, 2000).
In analyzing the total fatty acids methylated, direct FAME synthesis converted 22% more fatty acids to FAME than did NaOCH3 and 14% more than did BF3, indicating that there must be groups of fatty acids present that the latter 2 methods did not recognize. Such limitations with these 2 reagents have been previously noted (Kramer et al., 1997; Christie, 2003). The direct FAME synthesis method apparently methylates all of the fatty acids present, as confirmed by the Leco fat extractor (see "Independent Assessment of Direct FAME Synthesis Efficiency"), which explains why the direct FAME synthesis recoveries were greater than the other 2 methods.
When the peak areas were expressed as a percentage of total fatty acids (%FA; wt/wt) present by each method, the %FA were similar for all 3 methods, even though total recovery among the 3 methods was somewhat different. This indicates that the fatty acids not methylated by NaOCH3 or BF3 were present in similar ratios for all of the fatty acids present. The results with NaOCH3, which does not methylate FFA, indicates that because it methylated only 82% of the total fatty acids present, the other 18% of the fatty acids present may have been FFA.
CLA Analysis Using NaOCH3, BF3, or Direct Synthesis
Table 3 presents the results obtained from an analysis of commercial CLA capsules using NaOCH3, BF3, and direct synthesis. The CLA capsules contained the 2 important CLA isomers C18:2c9,t11 and C18:2t10,c12 and also palmitic (C16:0), stearic (C18:0), oleic (C18:1n9), and linoleic (C18:2n6) fatty acids.
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Table 3. Conjugated linoleic acid fatty acid methyl ester (FAME) synthesis by NaOCH3, BF3, or direct FAME synthesis1
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Most notable was the difference in the results obtained with NaOCH3 compared with that obtained with BF3 and direct FAME synthesis. Only 0.4% of the fatty acids present were methylated by NaOCH3, indicating that they were virtually all FFA and not esterified. As noted previously, NaOCH3 methylates esterified fatty acids but not FFA (Kramer et al., 1997). This example serves as a caution that a researcher who uses NaOCH3, or alkaline catalysts in general, is at great risk of missing fatty acids, whereas direct FAME synthesis methylates all of the fatty acids present.
Boron trifluoride and direct FAME synthesis gave essentially the same results for the fatty acids present in the CLA capsules. Once again, the direct FAME synthesis method did not generate fatty acid artifacts, including CLA artifacts, because all of the peaks were essentially identical to those of the BF3 method. The absence of CLA artifacts confirmed the work of Park et al. (2002), who used similar H2SO4 conditions on CLA samples, as was used with direct FAME synthesis, but Park et al. (2002) did not have water present.
The Analysis of Beef LM Using NaOCH3, BF3, or Direct FAME Synthesis
The analysis of freeze-dried beef LM fatty acids using NaOCH3, BF3, and direct FAME synthesis is presented in Table 4. Once again, there were striking differences among the 3 methods. Direct FAME synthesis recovered more (P < 0.01) fatty acids than did NaOCH3 and much more than did BF3. Because most of the fatty acids were esterified in LM, as opposed to unesterified in the CLA capsule (Table 3
), it was not surprising that NaOCH3 performed much better in FAME synthesis of this sample, although it methylated only 78% of the fatty acids present compared with direct FAME synthesis. This sample also shows that BF3 performed much better with the FFA in the CLA sample (Table 3) than with the esterified fatty acids in muscle tissue. This latter result was surprising, because BF3 can methylate all families of fatty acids (Carrapiso and Garcia, 2000). Boron trifluoride methylated all of the different fatty acids present, because the same peaks were present with BF3 as with direct FAME synthesis, but it did not do so quantitatively. It is unclear at this time why BF3 gave such poor results. It can be mentioned that Bolte et al. (2002) reported satisfactory FAME synthesis results using BF3 on freeze-dried lamb muscle tissue fatty acids by using much greater temperature and more concentrated effort (i.e., by incubating at 80°C and vortex-mixing 2 to 3 times/min). However, our results do not seem to be because BF3 cannot permeate the meat sample, because similar results were observed when using a CHCl3:MeOH extract according to the method of Folch et al. (1956), in which extraction of fatty acids by BF3 would no longer be an issue (data not shown). Apparently, and unexpectedly, there are fatty acid structures in beef LM that can be easily methylated by direct FAME synthesis but not by BF3.
When expressed as %FA, NaOCH3 and direct FAME synthesis were similar in their results, but BF3 was different (P < 0.01). In this latter case (BF3), the %FA values were greater when the concentration of fatty acid was lower. This difference could be explained by the fact that BF3 methylated only 46% of the fatty acids present in LM than did direct FAME synthesis and did so preferentially. With BF3, long-chain unsaturated fatty acids appeared to be methylated more efficiently than short-chain or SFA, whereas direct FAME synthesis methylated fatty acids without bias to chain length or structure. As a result, direct FAME synthesis consistently methylated more fatty acid, averaging 1.3 times that of NaOCH3 and 2.2 times that of BF3.
Independent Assessment of Direct FAME Synthesis Efficiency
To determine if the direct FAME synthesis could extract all of the fatty acids present in beef LM, we independently compared it to the Leco TFE2000 fat extractor (Leco Corp., St. Joseph, MI). These results are shown in Table 5. For the analysis of this experiment, wet tissue and freeze-dried tissue were corrected to a DM basis. Direct FAME synthesis, whether applied to dry or wet muscle tissue, extracted all of the fatty acids present when compared with the Leco TFE200 fat extractor, assuming that the Leco values should be 6 to 9% greater, because the Leco values also contain glycerol and cholesterol. In this respect, direct synthesis gives a truer value of fat content than does the Leco extractor, which does not differentiate fatty acids from total lipid.
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Table 5. A comparison of fatty acid concentrations obtained from beef LM using direct fatty acid methyl ester (FAME) synthesis or the Leco fat extractor1
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Direct FAME Synthesis Applied to Wax Esters, Cholesteryl Derivatives, and Fatty Acid Salts
The ideal FAME synthesis method would be able to analyze fatty acids from samples of wax esters, cholesteryl lipid derivatives, and alkyl methane sulfonates (Palmquist and Jenkins, 2003). We applied the direct FAME synthesis method to such families of fatty acids, and the results are shown in Table 6. Direct FAME synthesis was able to identify the fatty acids in wax esters, as represented by palmitoyl stearate (saturated series) and stearyl linoleate (unsaturated series), cholesteryl lipid derivatives, as represented by cholesteryl oleate, and fatty acid salts, as represented by oleyl methane sulfonate. Because the second component in each of these compounds was a fatty alcohol, and not a fatty acid, it was not converted to the FAME. This is as it should be, because we were analyzing strictly for fatty acid components. Depending on the concentration of these very hydrophobic families of fatty acids, full quantification might require more shaking and a longer incubation time during the first step of direct FAME synthesis.
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Table 6. Direct fatty acid methyl ester synthesis of samples from wax esters, cholesterol lipid derivatives, and alkyl methane sulfates
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Effect of Water Content on FAME Production by Direct FAME Synthesis
It is of great interest to know what the limiting concentration of water might be for the direct FAME synthesis method. There has to be such a limit, for no other reason than the fact there has to be a certain concentration of methylating reagents. In Figure 1, we show the effect of water concentration on the direct FAME synthesis method. As the percentage of water was increased, the total amount of fatty acids methylated decreased (data not shown), but this was easily corrected for by the internal standard. Most importantly, the percentage of each fatty acid remained constant up to 33% water. Only above 33% water do the FAME production results become problematic. In comparison, our reagents, without any sample present, constituted only 13% water in a final reaction volume of 7.58 mL. Thus, from a practical standpoint, one can replace 1.5 mL of MeOH with a 1.5-mL aqueous sample for a final concentration of 33% water. For example, using the protocol as given, 1.5 mL of milk can be analyzed directly by our method.
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Figure 1. Effect of water concentration on fatty acid methyl ester (FAME) production of fish oil fatty acids and the internal standard (C13:0) by direct FAME synthesis.
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The Versatility of Direct FAME Synthesis
The ideal method for the analysis of fatty acids would be applicable to any sample whose fatty acid content was desired. With this in mind, direct FAME synthesis was applied to various products, and the results are shown in Figures 2 and 3. We evaluated many oil sources, including fish, canola, and virgin olive; many nuts, including almond, cashew, peanut, sunflower, and walnut; a couple of feedstuffs, including pelleted sheep concentrate and alfalfa; and foodstuffs including beef LM, butter, cheese, Crisco (J. M. Smucker Co., Orville, OH), margarine, Miracle Whip (Kraft Foods, Northfield, IL), and Slim Fast (Unilever NV, Rotterdam, the Netherlands). The direct FAME synthesis method readily generated a FAME profile for all of these different samples.
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Figure 2. Direct fatty acid methyl ester (FAME) synthesis from samples of Supelco FAME mixture (Supelco, Bellefonte, PA), fish oil, Crisco (J. M. Smucker Co., Orville, OH), pelleted sheep concentrate, beef LM, canola, alfalfa, Slim Fast (Unilever NV, Rotterdam, the Netherlands), and virgin olive oil. %FA = % of total fatty acids identified in the sample (wt/wt).
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Figure 3. Direct fatty acid methyl ester (FAME) synthesis from samples of sunflower seeds, peanuts, Blue Bonnet margarine (ConAgra Foods, Omaha, NE), walnuts, almonds, butter, cashews, Miracle Whip (Kraft Foods, Northfield, IL), and Kraft American processed cheese (Kraft Foods). %FA = % of total fatty acids identified in the sample (wt/wt).
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When %FA was plotted against retention time and presented as in Figures 2 and 3, it was instantly seen that each sample has its own fatty acid print. Most samples were dominated by 1 to 3 fatty acids (i.e., 1 to 3 fatty acids accounted for 85%, or more, of the total fatty acid composition of the sample). When the graphs were arrayed as they were in these 2 figures, it was easy to visually compare one sample to another (e.g., the vegetable oil product Crisco contained 25% oleic and 25% linoleic fatty acids, whereas olive oil contained 70% oleic acid; beef LM contained 40% oleic and 25% palmitic acids, whereas Slim Fast contained 70% oleic acid of the total fatty acids present). The dairy products, butter and Kraft American processed cheese, had very similar fatty acid profiles. The fatty acid composition of the samples presented in Figures 2 and 3, determined by direct FAME synthesis, are in very good agreement with the fatty acid tabulations in handbooks (Watt, 1975; McCance, 2002).
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DISCUSSION
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An evaluation of numerous aspects in the isolation, separation, identification, and structural analysis of lipids was made by Christie (2003). Among his comments on the various methods of FAME synthesis, he pointed out that care should be taken in the evaporation of solvents, because appreciable amounts of esters up to C14 can be lost if this step is performed carelessly. He stated that the vigorous use of N to evaporate solvents must be avoided. In direct synthesis, we eliminate solvent evaporation completely, because we do not use any prior organic solvent extraction. Christie (2003) also reminded researchers that BF3 in MeOH has a limited shelf life, even when refrigerated, and the use of old or too-concentrated solutions often resulted in the production of artifacts and the loss of appreciable amounts of PUFA by addition of MeOH across the double bonds. On the other hand, Christie (2003) recommended H2SO4 in MeOH, the principle of which we have incorporated into direct synthesis. Others, using H2SO4 in MeOH, have shown that it does not produce CLA artifacts (Park et al., 2002). Additionally, we have used C13:0 as an internal standard rather than some other odd chain fatty acid such as C19:0, because it is readily soluble in MeOH, a major component of our initial reagents. In principle, any fatty acid may be used as an internal standard as long as all of the fatty acids in the sample are methylated.
In our method of direct FAME synthesis, we introduced water into fatty acid analysis. In fact, water defines our method and stands in definite contrast to all other FAME synthesis methods. Other researchers, including those who created direct methods for fatty acid analysis, have taken special care to avoid water. Even exposure to ambient air has been minimized to avoid moisture uptake by dry muscle tissue (Murrieta et al., 2003). When water cannot be used, sample preparation was no longer convenient, and hydrolysis of fatty acids, cholesteryl esters, and waxes, the first step of our method, was not possible.
Again, the principle behind the direct FAME synthesis method was to dissolve or thoroughly permeate a sample, for example, beef LM, and in the process hydrolyze fatty acid structures so they could be directly methylated without any prior organic solvent extraction. Hydrolysis, of course, requires water, as does solubilization and permeation. The KOH in MeOH alone did not solubilize tissue properly, and H2SO4 in MeOH precipitated tissue (data not shown). In a typical assay, even without wet tissue, the first step of direct FAME synthesis contained 10% water, whereas the second, and final, step contained 13% water.
Even though certain fatty acids have limited solubility in water and MeOH, hydrolysis of fatty acid esters still occurs at the water-methanol:lipid interface and can, in fact, be accounted for by the internal standard. For concentrated fat solutions and waxes, the hydrolysis step might take longer than 1.5 h at 55°C to complete. All of our results show the efficacy of the direct FAME synthesis method, which allows up to 33% water content (Figure 1). Again, KOH in MeOH alone could not solubilize and permeate tissues as well as when water was added, and H2SO4 in MeOH precipitated tissues rather than solubilized them.
Of further interest were the results obtained by direct FAME synthesis when compared, in various situations, to the NaOCH3 and BF3 methods. Most striking was the case of CLA analysis (Table 3), in which NaOCH3 did not methylate any CLA in a capsule full of it. Similarly, but not so pronounced, was the fact that BF3 methylated only 46% of the fatty acids present in beef LM (Table 4). In the latter instance, expressing the results as %FA could mask the inadequacies of the method, but at closer examination, this too would fail. If a method results in a differential extraction and synthesis of FAME, then at some point the %FA will be wrong. Such an example can be found with the BF3 results in Table 4. The concentration of C20:4n6 in beef LM was 1.6% with the BF3 method but only 0.9% with the direct FAME synthesis method. This 2-fold discrepancy was accounted for by the fact that BF3 differentially methylated a greater percent-age of C20:4n6 than it did of other fatty acids present. Direct FAME synthesis provided the most accurate values, because it methylated all of the fatty acids present in beef LM (Table 4), as verified by an independent analysis with the Leco TFE2000 fat extractor (Table 5).
Finally, direct FAME synthesis is convenient. Because water is part of the method, and not antagonistic to it, sample preparation is rapid; one only weighs out or pipets the sample into a Pyrex tube and then conducts the direct FAME synthesis. Gone is the preparation time it takes to lyophilize a sample (usually days) or the prior organic solvent extractions and N evaporations (usually hours) that are required to eliminate water in the other fatty acid methods.
In summary, a simplified protocol was developed to obtain FAME from any sample. The method consists of 2 steps, conducted in a single reaction tube. The protocol relies on the presence of water, which heretofore had been rigorously and tediously eliminated in FAME synthesis methods.
1 Corresponding author: gaskins{at}wsu.edu
Received for publication July 21, 2006.
Accepted for publication January 30, 2007.
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Abstract
INTRODUCTION
