A Direct Method For Fatty Acid Methyl Ester Synthe

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A direct method for fatty acid methyl ester synthesis: Application to wet meat
tissues, oils, and feedstuffs
Article in Journal of Animal Science · July 2007

DOI: 10.2527/jas.2006-491 · Source: PubMed
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A direct method for fatty acid methyl ester (FAME) synthesis: Application to wet meat
tissues, oils and feedstuffs
J. V. O'Fallon, J. R. Busboom, M. L. Nelson and C. T. Gaskins
J Anim Sci published online Feb 12, 2007;
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Page 1 of 38 Journal of Animal Science
Direct Fame synthesis 1
1 Running head: Direct FAME synthesis
4 A direct method for fatty acid methyl ester (FAME) synthesis: Application to wet
5 meat tissues, oils and feedstuffs
8 J. V. O’Fallon, J. R. Busboom, M. L. Nelson, and C. T. Gaskins1
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14 Department of Animal Sciences, Washington State University, Pullman 99164-6351
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16 Correspondence: 135 Clark Hall (phone 509-335-6416; fax 509-335-4246; e-
17 mail:gaskins@wsu.edu).
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Published Online First on February 12, 2007 as 15, 2011.
doi:10.2527/jas.2006-491
Journal of Animal Science Page 2 of 38
24 ABSTRACT: A simplified protocol to obtain fatty acid methyl esters (FAME) directly
25 from fresh tissue, oils, or feedstuffs, without prior organic solvent extraction is presented.
26 With this protocol, FAME synthesis is conducted in the presence of up to 33% water.
27 Wet tissues, or other samples, are permeabilized and hydrolyzed for 1.5 hr at 55 °C in 1N
28 KOH in methanol containing C13:0 as internal standard. The KOH is neutralized and the
29 free fatty acids are methylated by H2SO4 catalysis for 1.5 hr at 55 °C. Hexane is then
30 added to the reaction tube, which is vortex-mixed and centrifuged. The hexane is pipetted
31 into a GC vial for subsequent gas chromatography. All reactions are conducted in a single
32 screw cap Pyrex tube for convenience. The method meets a number of criteria for fatty
33 acid analysis including not isomerizing conjugated linoleic acid (CLA) or introducing
34 fatty acid artifacts. It is applicable to fresh, frozen, or lyopholyzed tissue samples, in
35 addition to oils, waxes and feedstuffs. The method saves time, effort and is economical
36 when compared to other methods. Its unique performance, including easy sample
37 preparation, is achieved because water is included in the FAME reaction mixtures rather
38 than eliminated.
39
40 Key words: fatty acid analysis, FAME synthesis, longissimus muscle, conjugated linoleic
41 acid (CLA), fish oil, feedstuffs
42
43 Introduction
44
45 The analysis of fatty acids has become increasingly important because more people
46 have become aware of their nutritional and health implications. Because of this, a method

47 for analyzing fatty acids that provides both rapid and reliable results is of great value.
48 Many methods are currently used to analyze fatty acids (e.g., Morrison and Smith, 1964;
49 Sukhija and Palmquist, 1988; Kazala et al., 1999; Mir et al., 2002; Nuernberg et al., 2002;
50 Budge and Iverson, 2003; Cooper et al., 2004). These methods, however, are not
51 necessarily convenient, nor direct, and often must be optimized for reaction conditions,
52 including the catalyst and the temperature (Lewis et al., 2000; Park et al. 2002; Shahin et
53 al., 2003). On the other hand, the ideal method, as noted by Palmquist and Jenkins
54 (2003) in discussing challenges encountered in developing fatty acid methods, would
55 determine the total fatty acid concentration in tissues, oils, and feed samples by
56 converting fatty acid salts, as well as the acyl components in all lipid classes such as
57 triacylglycerols, phospholipids, sphingolipids, and waxes, to methyl esters using a simple
58 direct one-step esterification procedure.
59 In this paper we present a method that is established upon a surprising conception, i.e.,
60 we add water to the fatty acid methyl ester (FAME) synthesis reagents. Until now, FAME
61 synthesis methods have rigorously avoided water as a matter of standard procedure.
62 However by adding water, the dynamics of sample preparation and methyl ester
63 formation can be revisited and the ideal outcome of FAME synthesis discussed by
64 Palmquest and Jenkins (2003) mentioned above becomes possible. Although the method
65 described herein requires two steps, it does so in one reaction tube.
66 By introducing water into FAME synthesis, the drudgery of special sample
67 preparation, e.g., freeze-drying, is no longer necessary. A sample is analyzed in the state
68 it is obtained. Thus, for example, meat and milk samples can be analyzed directly without
69 any prior dehydration step. The presence of water in the reagents allows for tissue sample

70 dissolution and facilitates the total extraction of fatty acids not possible in the absence of
71 water. Importantly, water does not interfere with the methylation of any fatty acid. The
72 method uses familiar and economical chemicals, namely, methanol, potassium hydroxide,
73 and sulfuric acid, but uses these methylating reagents in the presence of water. As a result
74 of these characteristics, the direct FAME synthesis method is convenient, reliable and
75 efficient.
76 In short, the objective of this paper is to develop a method to directly methylate fatty
77 acids from muscle tissue, oils, and feedstuffs in aqueous solution.
78
79 Materials and Methods
80
81 Methylation Agents and Sample Selection
82
83 To assess certain features of our method, we compared direct FAME synthesis to two
84 methylating agents routinely used for FAME synthesis, namely, the base catalyst sodium
85 methoxide and the acid catalyst boron trifluoride. Samples used with sodium methoxide
86 and boron trifluoride did not contain water, as this would compromise the performance of
87 these two methylating agents. The direct FAME synthesis method, however, always
88 contained water, even if the sample did not, because its reagents contained water, i.e., the
89 direct FAME synthesis reaction mixture contained 13.2% water due to the water in the
90 potassium hydroxide and sulfuric acid reagents.
91 The samples we used in this manuscript were chosen for distinct reasons. The Supelco
92 fatty acid standard mixture was chosen because it contained short and long chain

93 saturated, monounsaturated, and polyunsaturated fatty acids in defined amounts and thus
94 served as a primary test of the feasibility of our method. Fish oil was chosen because it is
95 an important source of the long chain polyunsaturated omega-3 esterified
96 eicosapentaenoic (EPA), docosapentaenoic (DPA), and docosahexaenoic (DHA) fatty
97 acids. Conjugated linoleic acid (CLA), as the free acid, was chosen because it is of
98 medical importance as perhaps the only fatty acid that can directly inhibit cancer in
99 animal models (Belury, 2002) and because current FAME synthesis methods often cause
100 undesirable isomerizations of this fatty acid (Kramer et al., 1997). Beef longissimus
101 muscle was chosen because of our special interest in beef fatty acids and it serves as a
102 direct test of the ability of direct FAME synthesis to extract and methylate all of the fatty
103 acids present in meat tissue. To address the problem of refractory samples, we included
104 wax esters, cholesteryl lipid derivatives and alkyl methane sulfonates, as these are
105 difficult fatty acid derivatives to analyze (Palmquist and Jenkins, 2003). Finally, as a
106 concluding demonstration of the versatility of the direct FAME synthesis method, we
107 included a variety of oils, feedstuffs and foods.
108
109 Materials
110
111 Hexane (OmniSolv) was purchased from EM Science, Cherry Hill, NY. Absolute
112 methanol and potassium hydroxide were obtained from J.T. Baker Chemical Co.,
113 Phillipsburg, NJ. Chloroform and sulfuric acid were purchased from Fisher Scientific,
114 Tustin, CA. Sodium methoxide and boron trifluoride-methanol were obtained from
115 Sigma-Aldrich, St. Louis, MO. The Supelco standard FAME mixture (47885-U) was

116 obtained from Supelco, Bellefonte, PA. Spring Valley fish oil capsules were distributed
117 by Leiner Health Products, Carson, CA. Tonalin 1000-CLA (conjugated linoleic acid)
118 capsules were obtained from Nature Bounty, Bohemia, NY. All other fatty acid standards
119 were purchased from Nu-Chek Prep. Inc., Elysian, MN. Beef longissimus muscle
120 samples (n=20) were obtained from department owned animals processed at an abattoir.
121 Nuts and sundry food items were purchased from local grocery stores. Coffee bean
122 grinders were purchased from Mr. Coffee, Inc., Cleveland, OH. Pyrex screw-cap culture
123 tubes (16 x 125 mm) were obtained from Corning Laboratory Science Company,
124 Corning, NY. The Tekmar VXR-10 multi-tube vortex was purchased from Jenke and
125 Kunkel, West Germany.
126
127 Folch Extraction of Fatty Acids from Longissimus Muscle
128
129 Longissimus muscle (1g) was extracted with chloroform/methanol (2:1, vol:vol),
130 containing C13:0 as internal standard, according to the method of Folch et al.(1956),
131 using a Brinkmann polytron at room temperature. The extraction mixture was then
132 filtered through a glass scintered filter and replicate aliquots were pipetted into a 16 x 125
133 mm screw-cap Pyrex culture tube and washed with 0.02% aqueous CaCl2. The organic
134 phase was dried with Na2SO4 and K2CO3 (10:1, wt:wt) and the solvent was subsequently
135 removed under nitrogen at 55 °C.
136
137 FAME Synthesis with Sodium Methoxide or Boron Trifluoride
138

139 Freeze-dried tissue samples were uniformly distributed by grinding for 10 -15 sec in a
140 room-temperature coffee bean grinder. Samples of freeze-dried tissue (0.25g), or oils (20
141 Il) were placed into a 16 x 125 mm screw- cap Pyrex culture tube to which 1.0 ml methyl
142 C13:0 internal standard (0.5mg methyl C13:0/ml methanol) was added. Two ml of
143 sodium methoxide (0.5 M) or 2 ml of boron trifluoride in methanol (14%, wt/vol) were
144 added to the Pyrex tubes containing the samples. The tubes were incubated in a 55 °C
145 water bath for 1.5 h with vigorous 5 sec hand-shaking every 20 min. Two ml of a
146 saturated solution of NaHCO3 and 3 ml hexane were then added and the tubes were
147 vortex-mixed. After centrifugation, the hexane layer containing the FAME was placed
148 into a GC vial. The vial was capped and placed at –20 °C until GC analysis.
149
150 Direct FAME Synthesis
151
152 Samples were uniformly distributed by grinding for 10 -15 sec in a room-temperature
153 coffee bean grinder. Short grinding times minimize smearing fat on the grinder container
154 walls. Samples could be processed in the state obtained, e.g., wet, dry, freeze-dried, or
155 semi-frozen. Samples (0.5g wet, dry, or semi-frozen sample), (0.25g freeze-dried
156 sample), or oils (20 Il) were placed into a 16 x 125 mm screw-cap Pyrex culture tube to
157 which 1.0 ml C13:0 internal standard (0.5mg C13:0/ml methanol), 0.7 ml 10 N KOH in
158 water, and 5.3 ml methanol were added. The tube was incubated in a 55 °C water bath for
159 1.5 h with vigorous 5 sec hand-shaking every 20 min to properly permeate, dissolve and
160 hydrolyze the sample. After cooling below room temperature in a cold tap water bath,
161 0.58 ml of 24 N H2SO4 in water was added. The tube was mixed by inversion, and with

162 precipitated K2SO4 present, was incubated again in a 55 °C water bath for 1.5 h with 5
163 sec hand-shaking every 20 min. After FAME synthesis, the tube was cooled in a cold tap
164 water bath. Three ml of hexane was added and the tube was vortex-mixed for 5 min on a
165 multi-tube vortex. The tube was centrifuged for 5 min in a tabletop centrifuge and the
166 hexane layer, containing the FAME, was placed into a gas chromatography (GC) vial.
167 The vial was capped and placed at –20 °C until GC analysis.
168
169 Gas Chromatography
170
171 The fatty acid composition of the FAME was determined by capillary gas
172 chromatography on a SPTM-2560, 100 m x 0.25 mm x 0.20 µm capillary column
173 (Supelco, Bellefonte, PA) installed on a Hewlett Packard 5890 gas chromatograph
174 equipped with a Hewlett Packard 3396 Series II integrator and 7673 controller, a flame
175 ionization detector and split injection. Initial oven temperature was 140 °C, held for 5
176 min, subsequently increased to 240 °C at a rate or 4 °C min-1, and then held for 20 min.
177 Helium was used as the carrier gas at a flow rate of 0.5 ml min-1 and column head
178 pressure was 280 kPa. Both the injector and detector were set at 260 °C. The split ratio
179 was 30:1. Fatty acids were identified by comparing their retention times with fatty acid
180 methyl standards described under Materials.
181
182 Statistical Analysis
183

184 Duplicate gas chromatograph results were averaged for animal and methylation
185 method. ANOVA of beef longissimus muscle FAME was calculated using Proc GLM of
186 SAS (SAS Institute, Cary, NC) using a model with methylation method as the treatments
187 and animal as a blocking factor in a randomized complete block design. When the F-ratio
188 for the methylation methods was significant, Student’s t-test was used to make pairwise
189 comparisons among the means.
190
191 Results
192
193 Sodium Methoxide, Boron Trifluoride, and Direct FAME Synthesis Methods Applied to a
194 Supelco Standard FAME Mixture
195
196 For an initial FAME synthesis analysis, we used a Supelco standard FAME mixture.
197 This standard mixture contained FAME in a defined ratio, and consisted of both short and
198 long chain saturated, monounsaturated, and polyunsaturated fatty acids. In order to
199 broadly assess certain features of our method, we compared the results of direct FAME
200 synthesis to those of the base catalyst sodium methoxide and the acid catalyst boron
201 trifluoride.
202 Direct FAME synthesis, as described in Materials and Methods, is a two-step
203 procedure. In the first step, sample fatty acid esters are hydrolyzed to free fatty acids and
204 in the second step the free fatty acids are converted to FAME. When the first step of
205 direct FAME synthesis was applied to the Supelco standard FAME mixture, the esters
206 were hydrolyzed to free fatty acids that were not volatile enough to enter the GC column.

207 These results, i.e., the absence of fatty acid peaks, provided formal evidence that the first
208 step in direct FAME synthesis completely hydrolyzed the Supelco standard FAME to free
209 fatty acids which was the desired general prerequisite for the subsequent methylation step
210 of direct FAME synthesis (data not shown).
211 When the second step of direct FAME synthesis, the methylation step, was applied to
212 the Supelco free fatty acids produced by the first step, the results shown in Table 1 were
213 obtained. All of the GC peaks present in the original Supelco standard mixture were
214 again observed, as can be seen by comparing the fatty acids of direct FAME synthesis to
215 those of the Supelco mix. When presented with a FAME sample, as in this experiment,
216 both sodium methoxide and boron trifluoride likewise gave the same FAME values
217 present in the original Supelco mix (Table 1).
218 Fatty acid artifacts, e.g., the conversion of CLA to other isomers, are a concern in fatty
219 acid analysis (Kramer et al. 1997; Park et al., 2002; Shahin et al., 2003). Since direct
220 FAME synthesis, sodium methoxide, or boron trifluoride did not generate new fatty acid
221 peaks, no fatty acid artifacts were created in the Supelco standard FAME mixture.
222
223 The Methods of Sodium Methoxide, Boron Trifluoride, and Direct FAME Synthesis
224 Applied to Fish Oil
225
226 A comparison was made between the base catalyst sodium methoxide, the acid
227 catalyst boron trifluoride, and direct synthesis, on FAME production from fish oil
228 commercially obtained as a human nutritional supplement. The results are shown in
229 Table 2.

230 Twenty fatty acids were identified in the fish oil sample. Direct FAME synthesis
231 recovered more total amount of fatty acid than did either of the other two methods as
232 would be expected if direct FAME synthesis generated methyl esters of all the fatty acids
233 in the sample. In comparison, base catalysis with sodium methoxide methylated esterified
234 fatty acids, but not free fatty acids (Kramer et al., 1997), whereas, acid catalysis by boron
235 trifluoride should have methylated all fatty acids, including esterified, unesterified, and
236 those in salt form (Carrapiso and Garcia, 2000).
237 In analyzing the total fatty acids methylated, direct FAME synthesis converted 22%
238 more fatty acids to FAME than did sodium methoxide and 14% more than did boron
239 trifluoride indicating that there must be groups of fatty acids present that the latter two
240 methods did not recognize. Such limitations with these two reagents have been
241 previously noted (Kramer et al., 1997; Christie, 2003). The direct FAME synthesis
242 method apparently methylates all of the fatty acids present, as confirmed by the LECO
243 Fat Extractor (see Independent Assessment of Direct FAME Synthesis Efficiency), which
244 explains why the direct FAME synthesis recoveries were higher than the other two
245 methods.
246 When the peak areas were expressed as % of total fatty acid (% FA, wt/wt) present by
247 each method, the % FAs were similar for all three methods even though total recovery
248 among the three methods was somewhat different. This indicates that the fatty acids not
249 methylated by sodium methoxide or boron trifluoride were present in similar ratios for all
250 of the fatty acids present. The results with sodium methoxide, which does not methylate
251 free fatty acids, indicates that because it methylated only 82% of the total fatty acids
252 present the other 18% of the fatty acids present may have been free fatty acids.

253
254 Conjugated Linoleic Acid Analysis Using Sodium Methoxide, Boron Trifluoride or Direct
255 Synthesis
256
257 Table 3 presents the results obtained from an analysis of commercial CLA capsules
258 using sodium methoxide, boron trifluoride and direct synthesis. The CLA capsules
259 contained the two important CLA isomers C18:2c9,t11 and C18:2t10,c12, and also
260 palmitic (C 16:0), stearic (C18:0), oleic (C18:1n9), and linoleic (C18:2n6) fatty acids.
261 Most notable, was the difference in the results obtained with sodium methoxide
262 compared to that obtained with boron trifluoride and direct FAME synthesis. Only 0.4%
263 of the fatty acids present were methylated by sodium methoxide, indicating that they
264 were virtually all free fatty acids, and not esterified. As noted previously, sodium
265 methoxide methylates esterified fatty acids, but not free fatty acids (Kramer et al., 1997).
266 This example serves as a caution that a researcher who uses sodium methoxide, or
267 alkaline catalysts in general, is at great risk of missing fatty acids, whereas, direct FAME
268 synthesis methylates all of the fatty acids present.
269 Boron trifluoride and direct FAME synthesis gave essentially the same results for the
270 fatty acids present in the CLA capsules. Once again, the direct FAME synthesis method
271 did not generate fatty acid artifacts, including CLA artifacts, as all of the peaks were
272 essentially identical to those of the boron trifluoride method. The absence of CLA
273 artifacts confirmed the work of Park et al. (2002), who used similar H2SO4 conditions on
274 CLA samples as was used with direct FAME synthesis, but Park et al. (2002) did not
275 have water present.

276
277 The Analysis of Beef Longissimus Muscle Using Sodium Methoxide, Boron Trifluoride or
278 Direct FAME Synthesis
279
280 The analysis of freeze-dried beef longissimus muscle fatty acids using sodium
281 methoxide, boron trifluoride and direct FAME synthesis is presented in Table 4. Once
282 again, there were striking differences among the three methods. Direct FAME synthesis
283 recovered more (P< 0.01) fatty acids than did sodium methoxide and much more than did
284 boron trifluoride. Since most of the fatty acids were esterified in longissimus muscle, as
285 opposed to unesterified in the CLA capsule (Table 3), it was not surprising that sodium
286 methoxide performed much better in FAME synthesis of this sample, although it
287 methylated only 78% of the fatty acids present compared to direct FAME synthesis. This
288 sample also shows that boron trifluoride performed much better with the free fatty acids
289 in the CLA sample (Table 3) than with the esterified fatty acids in muscle tissue. This
290 latter result was surprising because boron trifluoride can methylate all families of fatty
291 acids (Carrapiso and Garcia, 2000). Boron trifluoride methylated all of the different fatty
292 acids present, because the same peaks were present with BF3 as with direct FAME
293 synthesis, but it did not do so quantitatively. It is unclear at this time why boron
294 trifluoride gave such poor results. It can be mentioned that Bolte et al. (2002) reported
295 satisfactory FAME synthesis results using boron trifluoride on freeze-dried lamb muscle
296 tissue fatty acids by using much higher temperature and more concentrated effort, i.e., by
297 incubating at 80°C and vortex-mixing two to three times/min. However, our results do
298 not seem to be due to the fact that boron trifluoride cannot permeate the meat sample,

299 because similar results were observed when using a chloroform:methanol extract
300 according to the method of Folch et al. (1956), where extraction of fatty acids by boron
301 trifluoride would no longer be an issue (data not shown). Apparently, and unexpectedly,
302 there are fatty acid structures in beef longissimus muscle that can be easily methylated by
303 direct FAME synthesis but not by boron trifluoride.
304 When expressed as % FA, sodium methoxide and direct FAME synthesis were quite
305 similar in their results, but boron trifluoride was different (P<0.01), and in this latter case
306 (BF3) the % FA values were higher when the concentration of fatty acid was lower. This
307 difference could be explained by the fact that boron trifluoride methylated only 46% of
308 the fatty acids present in longissimus muscle than did direct FAME synthesis, and did so
309 preferentially. With boron trifluoride, long chain unsaturated fatty acids appeared to be
310 methylated more efficiently than short chain or saturated fatty acids, whereas, direct
311 FAME synthesis methylated fatty acids without bias to chain length or structure. As a
312 result, direct FAME synthesis consistently methylated more fatty acid, averaging 1.3
313 times that of sodium methoxide and 2.2 times that of boron trifluoride.
314
315 Independent Assessment of Direct FAME Synthesis Efficiency
316
317 To determine if the direct FAME synthesis could extract all of the fatty acids present
318 in beef longissimus muscle, we independently compared it to the LECO TFE2000 Fat
319 Extractor (LECO Corporation, St. Joseph,MI). These results are shown in Table 5. For
320 the analysis of this experiment, wet tissue and freeze dried tissue was corrected to a dry
321 matter basis. Direct FAME synthesis, whether applied to dry or wet muscle tissue,

322 extracted all of the fatty acids present when compared to the LECO TFE200 Fat
323 Extractor, assuming that the LECO values should be 6-9 % higher since the LECO values
324 also contain glycerol and cholesterol. In this respect, direct synthesis gives a truer value
325 of fat content then does the LECO extractor, which does not differentiate fatty acids from
326 total lipid.
327
328 Direct FAME Synthesis Applied to Wax Esters, Cholesteryl Derivatives, and Fatty Acid
329 Salts
330
331 The ideal FAME synthesis method would be able to analyze fatty acids from samples
332 of wax esters, cholesteryl lipid derivatives, and alkyl methane sulfonates (Palmquist and
333 Jenkins, 2003). We applied the direct FAME synthesis method to such families of fatty
334 acids and the results are shown in Table 6. Direct FAME synthesis was able to identify
335 the fatty acids in wax esters as represented by palmityl stearate (saturated series) and
336 stearyl linoleate (unsaturated series), cholestery lipid derivatives as represented by
337 cholesteryl oleate, and fatty acid salts, as represented by oleyl methane sulfonate. As the
338 second component in each of these compounds was a fatty alcohol, and not a fatty acid, it
339 was not converted to the FAME. This is as it should be, since we were analyzing strictly
340 for fatty acid components. Depending on the concentration of these very hydrophobic
341 families of fatty acids, full quantification might require more shaking and a longer
342 incubation time during the first step of direct FAME synthesis.
343

344 Effect of Water Content on FAME Production by Direct FAME Synthesis
345
346 It is of great interest to know what the limiting concentration of water might be for the
347 direct FAME synthesis method. There has to be such a limit, for no other reason than the
348 fact there has to be a certain concentration of methylating reagents. In Figure 1, we show
349 the effect of water concentration on the direct FAME synthesis method. As the
350 percentage of water was increased, the total amount of fatty acids methylated decreased
351 (data not shown), but this was easily corrected for by the internal standard. Most
352 importantly, the percentage of each fatty acid remained constant up to 33% water. Only
353 above 33% water do the FAME production results become problematic. In comparison,
354 our reagents, without any sample present, constitute only 13% water in a final reaction
355 volume of 7.58 ml. Thus, from a practical standpoint, one can replace 1.5 ml of MeOH
356 with a 1.5 ml aqueous sample for a final concentration of 33% water. For example, using
357 the protocol as given, 1.5 ml of milk can be analyzed directly by our method.
358
359 The Versatility of Direct FAME Synthesis
360
361 The ideal method for the analysis of fatty acids would be applicable to any sample
362 whose fatty acid content was desired. With this in mind, direct FAME synthesis was
363 applied to various products and the results are shown in Figures 2 and 3. We evaluated a
364 number of oil sources including fish, Canola, and virgin olive; a number of nuts including
365 almond, cashew, peanut, sunflower, and walnut; a couple of feedstuffs, including pelleted
366 sheep concentrate and alfalfa; and foodstuffs including beef longissimus muscle, butter,

367 cheese, Crisco, margarine, Miracle Whip, and Slim Fast. The direct FAME synthesis
368 method readily generated a FAME profile for all of these different samples.
369 When % FA was plotted against retention time and presented as in Figures 2 and 3, it
370 was instantly seen that each sample has its own ‘fatty acid print’. Most samples were
371 dominated by 1 to 3 fatty acids, i.e., 1 to 3 fatty acids accounted for 85%, or more, of the
372 total fatty acid composition of the sample. When the graphs were arrayed as they were in
373 these two figures, it was easy to visually compare one sample to another, e.g., the
374 vegetable oil product Crisco contained 25% oleic and 25% linoleic fatty acids, while
375 olive oil contained 70% oleic acid; beef longissimus muscle contained 40% oleic and
376 25% palmitic acids, while Slim Fast contained 70% oleic acid of the total fatty acids
377 present. The dairy products, butter and Kraft American processed cheese, had very
378 similar fatty acid profiles. The fatty acid composition of the samples presented in Figures
379 2 and 3, determined by direct FAME synthesis, are in very good agreement with the fatty
380 acid tabulations in handbooks (Watt, 1975; McCance, 2002).
381
382 Discussion
383
384 An evaluation of numerous aspects in the isolation, separation, identification and
385 structural analysis of lipids was made by Christie (2003). Among his comments on the
386 various methods of FAME synthesis, he pointed out that care should be taken in the
387 evaporation of solvents as appreciable amounts of esters up to C14 can be lost if this step
388 is performed carelessly. He stated that the vigorous use of nitrogen to evaporate solvents
389 must be avoided. In direct synthesis we eliminate solvent evaporation completely because

390 we do not use any prior organic solvent extraction. Christie (2003) also reminded
391 researchers that boron trifluoride in methanol has a limited shelf life, even when
392 refrigerated, and the use of old or too concentrated solutions often resulted in the
393 production of artifacts and the loss of appreciable amounts of polyunsaturated fatty acids
394 by addition of methanol across the double bonds. On the other hand, Christie (2003)
395 recommended sulfuric acid in methanol, the principle of which we have incorporated into
396 direct synthesis. Others, using sulfuric acid in methanol, have shown that it does not
397 produce CLA artifacts (Park et al., 2002). Additionally, we have used C13:0 as internal
398 standard, rather than some other odd chain fatty acid such as C19:0 because it is readily
399 soluble in methanol, a major component of our initial reagents. In principle, any fatty
400 acid may be used as internal standard as long as all of the fatty acids in the sample are
401 methylated.
402 In our method of direct FAME synthesis we introduced water into fatty acid analysis.
403 In fact, water defines our method, and stands in definite contrast to all other FAME
404 synthesis methods. Other researchers, including those who created direct methods for
405 fatty acid analysis, took special care to avoid water. Even exposure to ambient air was
406 minimized in order to avoid moisture uptake by dry muscle tissue (Murrieta et al., 2003).
407 When water cannot be used, sample preparation was no longer convenient and hydrolysis
408 of fatty acids, cholesteryl esters, and waxes, the first step of our method, was not
409 possible.
410 Again, the principle behind the direct FAME synthesis method was to dissolve, or
411 thoroughly permeate a sample, for example, beef longissimus muscle, and in the process
412 hydrolyze fatty acid structures so they could be directly methylated without any prior

413 organic solvent extraction. Hydrolysis, of course, requires water as does solubilization
414 and permeation. KOH in methanol alone did not solubilize tissue properly, and H2SO4 in
415 methanol precipitated tissue (data not shown). In a typical assay, even without wet tissue,
416 the first step of direct FAME synthesis contained 10% water, while the second, and final
417 step, contained 13% water.
418 Even though certain fatty acids have limited solubility in water/methanol, hydrolysis
419 of fatty acid esters still occurs at the water/methanol:lipid interface and can, in fact, be
420 accounted for by the internal standard. For concentrated fat solutions, and waxes, the
421 hydrolysis step might take longer than 1.5 h at 55 º C to complete. All of our results show
422 the efficacy of the direct FAME synthesis method, which allows up to 33% water content
423 (e.g., see Figure 1). Again, KOH in MeOH alone could not solubilize and permeate
424 tissues as well as when water was added, and H2SO4 in MeOH precipitated tissues, rather
425 than solubilized them.
426 Of further interest were the results obtained by direct FAME synthesis when
427 compared, in various situations, to the sodium methoxide and boron trifluoride methods.
428 Most striking was the case of CLA analysis (Table 3) where sodium methoxide did not
429 methylate any CLA in a capsule full of it. Similarly, but not so pronounced, was the fact
430 that boron trifluoride methylated only 46% of the fatty acids present in beef longissimus
431 muscle (Table 4). In the latter instance, expressing the results as % FA could mask the
432 inadequacies of the method, but at closer examination this too would fail. If a method
433 results in a differential extraction and synthesis of FAME, then at some point the % FA
434 will be wrong. Such an example can be found with the boron trifluoride results in Table
435 4. The concentration of C20:4n6 in beef longissimus muscle was 1.6 % with the boron

436 trifluoride method, but only 0.9% with the direct FAME synthesis method. This two fold
437 discrepancy was accounted for by the fact that boron trifluoride differentially methylated
438 a higher percentage of C20:4n6 than it did of other fatty acids present. Direct FAME
439 synthesis provided the most accurate values because it methylated all of the fatty acids
440 present in beef longissimus muscle (Table 4) as verified by an independent analysis with
441 the LECO TFE2000 Fat Extractor (Table 5).
442 Finally, direct FAME synthesis is convenient. Since water is part of the method, and
443 not antagonistic to it, sample preparation is rapid; one only weighs-out or pipets the
444 sample into a Pyrex tube and then conducts the direct FAME synthesis. Gone is the
445 preparation time it takes to lyophilize a sample (usually days), or the prior organic
446 solvent extractions and nitrogen evaporations (usually hours) that are required to
447 eliminate water in the other fatty acid methods.
448 In summary, a simplified protocol was developed to obtain fatty acid methyl esters
449 (FAME) from any sample. The method consists of two steps, conducted in a single
450 reaction tube. The protocol relies on the presence of water, which heretofore, had been
451 rigorously and tediously eliminated in FAME synthesis methods.
452
453 Literature Cited
454
455 Belury, M. A. 2002. Inhibition of carcinogenesis by conjugated linoleic acid: potential
456 mechanisms of action. J. Nutr. 132:2995-2998.
457 Bolte, M. R., R. W. Hess, W. J. Means, G. E. Moss, and D. C. Rule. 2002. Feeding lambs
458 high-oleate or high-linoleate safflower seeds differentially influences carcass fatty

459 acid composition. J. Anim. Sci. 80:609-616.
460 Budge, S. M., and S. J. Iverson. 2003. Quantitative analysis of fatty acid precursors in
461 marine samples: direct conversion of wax ester alcohols and dimethylacetals to
462 FAMEs. J. Lipid Res. 44:1802-1807.
463 Carrapiso, A. I., and C. Garcia. 2000. Development in lipid analysis: some new extraction
464 techniques and in situ trans esterification. Lipids 35:1167-1177.
465 Christie, W. W. 2003. Lipid Analysis: Isolation, Separation, Identification and Structural
466 Analysis of Lipids, Third edition. The Oily Press, Bridgwater, England.
467 Cooper, S. L., L. A. Sinclair, R, G. Wilkinson, K. G. Hallett, M. Enser, and J. D. Wood.
468 2004. Manipulation of the n-3 polysaturated fatty acid content of muscle and adipose
469 tissue in lambs. J. Anim. Sci. 82:1461-1470.
470 Folch, J., M. Lees, and G. H. Sloane-Stanley. 1956. A simple method for the isolation
471 and purification of total lipids from animal tissue. J. Biol. Chem. 226:497-509.
472 Kazala, E. C., F. J. Lozeman, P. S. Mir, A. L. Laroche, D. R. C. Bailey, and R. J.
473 Weselake. 1999. Relationship of fatty acid composition to intramuscular fat content in
474 beef from crossbred Wagyu cattle. J. Anim. Sci. 77:1717-1725.
475 Kramer, J. K. G., V. Fellner, M. E. R. Dugan, F. D. Sauer, M. M. Mossoba, and M. P.
476 Yurawecz. 1997. Evaluating acid and base catalysts in the methylation of milk and
477 rumen fatty acids with special emphasis on conjugated dienes and total trans fatty
478 acids. Lipids 32:1219-1228.
479 Lewis, T., P. D. Nichols, and T. A. McMeekin. 2000. Evaluation of extraction methods
480 for recovery of fatty acids from lipid-producing microheterotrophs. J. Microb.
481 Methods 43:107-116.

482 McCance, R. A. 2002. McCance and Widdowson’s: The composition of foods. Royal
483 Society of Chemistry, Cambridge, Great Britain.
484 Mir, P. S., Z. Mir, P. S. Kuber, C. T. Gaskins, E. L. Martin, M. V. Dodson, J. A. Elias
485 Calles, K. A. Johnson, J. R. Busboom, A. J. Wood, G. J. Pittenger, and J. J. Reeves.
486 2002. Growth, carcass characteristics, muscle conjugated linoleic acid (CLA) content,
487 and response to intravenous glucose challenge in high percentage Wagyu, Wagyu X
488 Limousin, and Limousin steers fed sunflower oil-containing diets. J. Anim. Sci.
489 80:2996-3004.
490 Morrison, W. R., and L. M. Smith. 1964. Preparation of fatty acid methyl esters and
491 dimethylacetals from lipids with boron fluoride-methanol. J. Lipid Res. 5:600-608.
492 Murrieta, C. M., B. W. Hess, and D. C. Rule. 2003. Comparison of acidic and alkaline
493 catalysts for preparation of fatty acid methyl esters from ovine muscle with emphasis
494 on conjugated linoleic acid. Meat Sci. 65:523-529.
495 Nuernberg, K., G. Nuernberg, K. Ender, S. Lorenz, K. Winkler, R. Rickert, and H.
496 Steinhart. 2002. N-3 fatty acids and conjugated linoleic acids of longissimus muscle in
497 Beef cattle. Eur. J. Lipid Sci. Technol. 104:463-471.
498 Palmquist, D. L., and T. C. Jenkins. 2003. Challenges with fats and fatty acid methods. J.
499 Anim. Sci. 81:3250-3254.
500 Park, S. J., C. W. Park, S. J. Kim, J. K. Kim, Y. R. Kim, K. A. Park, J. O. Kim, and Y. L.
501 Ha. 2002. Methylation methods for the quantitative analysis of conjugated linoleic
502 acid (CLA) isomers in various lipid samples. J. Agric. Food Chem. 50:989-996.
503 Shahin, A. M., M. K. McGuire, K. L. Ritzenthaler, and T. D. Shultz. 2003. Determination
504 of c9,t11-CLA in major human plasma lipid classes using a combination of

505 methylating methodologies. Lipids 38:793-800.
506 Sukhija, P. S., and D. L.Palmquist. 1988. Rapid method for determination of total fatty
507 acid content and composition of feedstuffs and feces. J. Agric. Food Chem. 36:1202-
508 1206.
509 Watt, B. K. 1975. Handbook of the nutritional contents of foods for the United States
510 Department of Agriculture, Dover Publications, New York.

Table 1. Effect of sodium methoxide, boron trifluoride or direct FAME synthesis on a
standard Supelco FAME mixture
Supelco Mix Na Methoxide BF3 Direct synthesis

Fatty acid Structure mg/g SDa mg/g SDa mg/g SDa
mg/g SDa
Capric C10:0 40.02 0.28 38.93 0.29 39.91 0.55 39.40 0.24
Undecanic C11:0 20.41 0.25 19.77 0.17 20.00 0.00 20.23 0.03
Lauric C12:0 39.57 0.36 38.66 0.14 39.06 0.28 39.01 0.39
Tridecanic C13:0 20.80 0.02 20.34 0.27 20.69 0.07 20.71 0.05
Myristic C14:0 41.12 0.16 39.43 0.35 40.84 0.34 40.91 0.41
Myristoleic C14:1n5 20.38 0.32 18.53 0.17 19.88 0.05 20.05 0.01
Pentadecanoic C15:0 21.54 0.05 20.62 0.39 21.09 0.25 21.16 0.22
Pentadecenoic C15:1n5 20.60 0.22 18.90 0.25 20.47 0.29 20.22 0.07
Palmitic C16:0 63.25 0.52 62.99 0.78 63.04 0.35 62.90 0.07
Palmitoleic C16:1n7 20.86 0.15 20.07 0.15 20.83 0.09 21.07 0.14
Heptadecanoic C17:0 18.66 0.55 21.42 0.21 18.62 0.08 21.23 0.36
Heptadecenoic C17:1n7 21.29 0.07 20.93 0.57 21.42 0.12 21.57 0.20
Stearic C18:0 42.68 0.25 43.72 0.64 42.66 0.52 42.97 0.35
Elaidic C18:1n9t 21.85 0.01 22.57 1.43 21.67 0.03 21.58 0.08
Oleic C18:1n9 43.06 0.27 43.78 1.37 43.58 0.26 42.70 0.08
Linolelaidic C18:2n6t 20.15 0.19 19.92 0.13 20.02 0.51 20.26 0.05
Linoleic C18:2n6 20.89 0.14 20.25 0.61 20.87 0.11 20.58 0.10
Arachidic C20:0 41.68 0.20 42.50 0.13 41.61 0.74 42.32 0.10
Gamma- Linolenic C18:3n6 19.54 0.07 18.36 0.02 19.12 0.05 19.50 0.18
Eicosenoic C20:1n9 21.06 0.11 21.08 0.01 20.62 0.06 20.87 0.15
Linolenic C18:3n3 19.84 0.25 18.69 0.15 20.33 0.25 19.92 0.19

Heneicosanoic C21:0 21.62 0.35 21.98 0.10 21.77 0.08 21.66 0.34
Eicosadienoic C20:2 20.44 0.07 20.30 0.16 20.65 0.08 19.85 0.67
Behenic C22:0 42.20 0.08 43.27 0.66 42.18 0.04 41.91 0.16
Eicosatrienoic C20:3n6 19.49 0.03 19.36 0.00 20.08 0.12 19.26 0.06
Erucic C22:1n9 19.86 0.58 20.88 0.13 19.40 0.36 19.05 0.22
Eicosatrienoic C20:3n3 18.50 0.03 18.82 0.21 20.47 0.48 19.51 0.13
Arachidonic C20:4n6 18.52 0.46 19.38 0.07 18.94 0.06 18.71 0.22
Tricosanoic C23:0 18.54 0.49 19.25 0.07 18.86 0.05 18.67 0.20
Docosadienoic C22:2n6 19.16 0.39 19.86 0.37 19.23 0.06 19.09 0.04
Lignoceric C24:0 34.34 1.45 37.72 0.31 31.84 0.70 32.00 0.22
Eicosapentaenoic C20:5n3 19.58 2.10 17.60 0.23 22.29 0.11 23.21 0.28
Nervonic C24:1n9 17.04 0.12 18.53 0.31 16.74 0.76 16.13 0.18
Docosahexaenoic C22:6n3 11.48 0.50 11.60 0.17 11.24 0.16 11.82 0.08
Total 880.02 10.56 880.01 10.55 880.02 7.51 880.03 6.00
a
SD = Standard deviation. Three Supelco FAME standard vials were pooled and then
divided into two separate samples for methylation as described in Materials and Methods.

Table 2. Fish oil FAME synthesis by sodium methoxide, boron trifluoride or direct FAME synthesis
Na methoxide BF3 Direct synthesis
Fatty acid Structure mg/g SDa % FAb mg/g SDa % FAb mg/g SDa % FAb
Lauric C12:0 1.01 0.05 0.2 1.14 0.11 0.2 1.01 0.11 0.1
Myristic C14:0 55.74 2.93 9.0 61.53 1.80 9.2 66.69 3.33 8.8
Myristoleic C14:1n5 3.86 0.20 0.6 4.25 0.13 0.6 4.70 0.29 0.6
Palmitic C16:0 126.54 6.66 20.4 134.06 3.15 20.1 155.95 8.34 20.5
Palmitoleic C16:1n7 59.02 3.11 9.5 63.66 1.81 9.5 70.95 3.50 9.3
Heptadecanoic C17:0 3.33 0.18 0.5 3.40 0.07 0.5 4.11 0.28 0.5
Heptadecenoic C17:1n7 1.13 0.06 0.2 1.22 0.06 0.2 1.35 0.07 0.2
Stearic C18:0 25.17 1.32 4.1 25.66 0.50 3.8 31.18 1.60 4.1
Oleic C18:1n9 81.31 4.28 13.1 85.95 2.55 12.9 98.36 4.90 12.9
Linoleic C18:2n6 20.54 1.08 3.3 19.79 0.67 3.0 21.35 0.85 2.8
Arachidic C20:0 1.83 0.10 0.3 1.71 0.01 0.3 2.43 0.16 0.3
Gamma- Linolenic C18:3n6 2.70 0.14 0.4 3.11 0.06 0.5 3.27 0.18 0.4
Eicosenoic C20:1n9 10.43 0.55 1.7 10.06 0.04 1.5 12.83 0.66 1.7
Linolenic C18:3n3 6.71 0.35 1.1 7.16 0.22 1.1 8.04 0.40 1.1
Eicosadienoic C20:2n6 21.67 1.14 3.5 23.62 0.77 3.5 25.43 1.11 3.3
Docosadienoic C22:2n6 5.57 0.29 0.9 5.85 0.19 0.9 6.71 0.33 0.9
Eicosapentaenoic C20:5n3 117.67 6.19 18.9 127.90 3.22 19.2 142.59 6.82 18.8
Nervonic C24:1n9 2.22 0.12 0.4 2.46 0.08 0.4 3.10 0.11 0.4
Docosapentaenoic C22:5n3 12.37 0.65 2.0 12.51 0.12 1.9 14.67 0.73 1.9
Docosahexaenoic C22:6n3 62.43 3.29 10.0 71.86 0.75 10.8 85.27 4.06 11.2
Total 621.23 32.70 100.0 666.89 16.13 100.0 760.00 37.63 100.0
a
SD = Standard deviation. Three fish oil capsules were pooled and then divided into two separate samples for methylation as
described in Materials and Methods.

b
% FA = % of total fatty acids identified in sample (wt/wt).
Table 3. Conjugated linoleic acid (CLA) FAME synthesis by sodium methoxide, boron
trifluoride or direct FAME synthesis
Fatty acid Structure mg/g SDa mg/g SDa mg/g SDa

b
Palmitic C16:0 nd 14.82 1.35 13.37 1.22
b
Stearic C18:0 nd 24.78 1.49 24.25 2.45
b
Oleic C18:1n9 nd 111.97 5.46 108.78 10.24
b
Linoleic C18:2n6 nd 3.32 0.15 3.25 0.34
c9,t11 CLA C18:2c9,t11 1.35 0.12 295.08 13.91 311.90 40.40
t10,c12 CLA C18:2t10,c12 1.45 0.21 289.44 11.92 308.45 37.25
Total 2.81 0.33 739.42 34.27 770.00 91.90

a
SD = Standard deviation. Three CLA capsules were pooled and then divided into two
samples for methylation as described in Materials and Methods.

b
nd = Not detected.

Table 4.Beef Longissimus muscle FAME synthesis by sodium methoxide, boron trifluoride or direct FAME synthesis
Fatty acid Structure mg/ga ± SEb % FAc mg/ga ± SEb % FAc mg/ga ± SEb % FAc
d d d e e d
Lauric C12:0 0.21 ±0.02 0.09 0.17 ±0.01 0.12 0.24 ±0.02 0.08
e e d f f d
Myristic C14:0 8.34 ±0.68 3.46 5.57 ±0.25 3.90 10.29 ±0.83 3.32
e e d f e d
Myristoleic C14:1n5 2.01 ±0.14 0.83 1.38 ±0.08 0.97 2.29 ±0.21 0.74
d d d f e e
Pentadecanoic C15:0 1.59 ±0.14 0.66 1.24 ±0.08 0.87 2.23 ±0.18 0.72
e d d e f d
Palmitic C16:0 62.82 ±4.10 26.07 38.00 ±0.86 26.61 80.85 ±4.84 26.08
e d d e f d
Palmitoleic C16:1n7 8.10 ±0.65 3.36 5.74 ±0.19 4.02 10.91 ±0.66 3.52
e e d d f e
Heptadecanoic C17:0 3.89 ±0.35 1.61 2.03 ±0.08 1.42 5.05 ±0.37 1.63
e e d d f e
Stearic C18:0 30.25 ±1.89 12.56 15.55 ±0.28 10.89 39.25 ±2.31 12.66
e f d d e e
t-Vaccenic C18:1n11t 19.20 ±2.36 7.97 9.35 ±0.96 6.55 22.21 ±3.28 7.16
e e d d f e
Oleic C18:1n9 87.10 ±5.28 36.15 47.60 ±1.03 33.34 113.91 ±6.43 36.75
d d d e e d
Linoleic C18:2n6 10.70 ±0.62 4.44 10.50 ±0.46 7.36 13.98 ±0.82 4.51
f f d d e e
Arachidic C20:0 0.60 ±0.06 0.25 0.00 ±0.00 0.00 0.40 ±0.05 0.13
e e d e e d
Gamma- Linolenic C18:3n6 0.32 ±0.03 0.13 0.17 ±0.02 0.12 0.28 ±0.02 0.09
e d d d f e
Eicosenoic C20:1n9 0.20 ±0.01 0.08 0.10 ±0.00 0.07 0.64 ±0.05 0.21
Direct FAME synthesis 30
e e d d f f
Linolenic C18:3n3 0.56 ±0.04 0.23 0.21 ±0.01 0.15 0.91 ±0.05 0.29
e d d e f d
c-9,t-11 CLA C18:2c9,t11 0.70 ±0.04 0.29 0.59 ±0.02 0.41 0.85 ±0.09 0.27
e e d e e d
Eicosadienoic C20:2 0.25 ±0.02 0.10 0.17 ±0.01 0.12 0.20 ±0.02 0.06
f e e e d d
Behenic C22:0 0.40 ±0.04 0.17 0.25 ±0.02 0.18 0.18 ±0.01 0.06
e d d e f f
Eicosatrienoic C20:3n6 0.41 ±0.06 0.17 0.31 ±0.06 0.22 0.85 ±0.03 0.28
d d d e e d
Arachidonic C20:4n6 2.05 ±0.07 0.85 2.24 ±0.20 1.57 2.74 ±0.09 0.89
d d e e e d
Eicosapentaenoic C20:5n3 0.40 ±0.03 0.17 0.52 ±0.03 0.37 0.54 ±0.03 0.17
d d e e e d
Docosatetraenoic C22:4n6 0.18 ±0.01 0.07 0.24 ±0.01 0.17 0.26 ±0.01 0.09
d d e e f d
Docosapentaenoic C22:5n3 0.68 ±0.03 0.28 0.84 ±0.06 0.59 0.93 ±0.03 0.30
e d d d f d
Total 240.95 ±16.67 100.00 142.78 ±4.72 100.00 310.00 ±20.40 100.00
a b c
mg/g of freeze dried tissue. SE = Standard error (n=20). % FA = % of total fatty acids identified in sample (wt/wt). d,e,f ANOVA
analysis was determined as described in Materials and Methods. Different superscripts indicate significant difference at P < .01 for
pairwise comparisons among the methylation methods. Columns containing mg/g were compared among themselves and columns
containing %FA were compared among themselves.

Table 5. A comparison of fatty acid concentrations obtained from beef longissimus
muscle using direct FAME synthesis or the LECO fat extractor
Condition Fatty acid (mg / g dry matter) ± SEa
FAME synthesis
b
Freeze dried tissue 310.25 ± 18.23
b
Wet tissue 312.37 ± 27.51
c
LECO 336.59 ± 23.36
a
SE = Standard error (n=12)
b
Samples were analyzed by direct FAME synthesis.
c
Lipid content was determined in freeze dried tissue by the LECO TFE2000 Fat
Extractor.

Table 6. Direct FAME synthesis of samples from wax esters, cholesterol lipid derivatives
and alkyl methane sulfates
Compound Acid component Ester observed
Palmityl stearate Stearic Methyl stearate
Stearyl linoleate Linoleic Methyl linoleate
Cholesteryl oleate Oleic Methyl oleate
Oleyl methane sulfonate Methane sulfonic Methyl methane sulfonate

Figure 1.Effect of water concentration on FAME production of fish oil fatty acids and
internal standard by direct FAME synthesis.

30
25
C16:0
C20:5n3
Percent Fatty Acid
20 C18:1n9
C22:6n3
15 C18:0
C13:0
10 C20:2n6
C22:5n3
C18:3n6
5
0
10 15 20 25 30 35 40 45 50 55
Percent Water

Figure 2. Direct FAME synthesis from samples of Supelco FAME mixture, fish oil,
Crisco, pelleted sheep concentrate, beef longissimus muscle, canola, alfalfa, Slim Fast,
and virgin olive oil. % Fatty acid = % of total fatty acid present in sample.

Supelco FAME Mixture Fish Oil Crisco
7.0 18 30
6.0 16
25
14
5.0
12 20
% Fatty acid
4.0
10
15
3.0 8
6 10
2.0
4
1.0 5
2
0.0 0 0
10 50
R et ent i o n t i me ( min) Retention time (min) R et ent io n t i me ( mi n)
Pelleted Sheep Beef Longissim us Canola

Concentrate Muscle
70
40 50 60
35 50
40
30
40
25 30
20 30
15 20
20
10
10 10
5
0 0 0
R et ent io n t i me ( mi n) R et ent i o n t ime ( mi n) R et ent i o n t ime ( mi n)
Alfalfa Slim Fast Virgin Olive Oil
30 80 80
70 70
25
60 60
20 50 50
15 40 40
30 30
10
20 20
5 10 10
0 0 0
R et ent io n t i me ( mi n) R et ent io n t i me ( mi n) R et ent i o n t i me ( min)

Figure 3. Direct FAME synthesis from samples of sunflower seeds, peanuts, Blue
Bonnet margarine, walnuts, almonds, butter, cashews, Miracle Whip, and Kraft American
processed cheese. % Fatty acid = % of total fatty acid present in sample.

Sunflower Peanut Blue Bonnet Margarine
70 70 50
45
60 60
40
50 50 35
30
40 40
25
30 30 20
20 20 15
10
10 10 5
0 0 0
R et ent io n t i me ( mi n) R et ent io n t i me ( mi n) R et ent io n t i me ( mi n)
Walnut Alm ond Butter
70 80 30
60 70 25
60
50 20
50
40
40 15
30
30 10
20 20
10 5
10
0 0 0
R et ent i o n t i me ( mi n) R et ent io n t i me ( mi n) R et ent i o n t i me ( min)
Cashew Miracle Whip Kraft Cheese
70 60 30
60 50 25
50 20
40
40
30 15
30
20 10
20
10 10 5
0 0 0
R et ent i o n t i me ( min) R et ent i o n t i me ( min) R et ent i o n t i me ( min)

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Article in Journal of Animal Science · July 2007

Jan Busboom Charles T Gaskins

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J. V. O'Fallon, J. R. Busboom, M. L. Nelson and C. T. Gaskins

J Anim Sci published online Feb 12, 2007;

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Direct Fame synthesis 1

1 Running head: Direct FAME synthesis

5 meat tissues, oils and feedstuffs

8 J. V. O’Fallon, J. R. Busboom, M. L. Nelson, and C. T. Gaskins1

14 Department of Animal Sciences, Washington State University, Pullman 99164-6351

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Direct Fame synthesis 2

34 fatty acid artifacts. It is applicable to fresh, frozen, or lyopholyzed tissue samples, in

41 acid (CLA), fish oil, feedstuffs

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Direct Fame synthesis 3

54 (2003) in discussing challenges encountered in developing fatty acid methods, would

57 triacylglycerols, phospholipids, sphingolipids, and waxes, to methyl esters using a simple

58 direct one-step esterification procedure.

61 synthesis methods have rigorously avoided water as a matter of standard procedure.

65 described herein requires two steps, it does so in one reaction tube.

66 By introducing water into FAME synthesis, the drudgery of special sample

67 preparation, e.g., freeze-drying, is no longer necessary. A sample is analyzed in the state

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Direct Fame synthesis 4

77 acids from muscle tissue, oils, and feedstuffs in aqueous solution.

79 Materials and Methods

81 Methylation Agents and Sample Selection

90 potassium hydroxide and sulfuric acid reagents.

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Direct Fame synthesis 5

95 an important source of the long chain polyunsaturated omega-3 esterified

96 eicosapentaenoic (EPA), docosapentaenoic (DPA), and docosahexaenoic (DHA) fatty

107 included a variety of oils, feedstuffs and foods.

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Direct Fame synthesis 6

125 Kunkel, West Germany.

127 Folch Extraction of Fatty Acids from Longissimus Muscle

135 removed under nitrogen at 55 °C.

137 FAME Synthesis with Sodium Methoxide or Boron Trifluoride

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Direct Fame synthesis 7

150 Direct FAME Synthesis

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Direct Fame synthesis 8

169 Gas Chromatography

172 chromatography on a SPTM-2560, 100 m x 0.25 mm x 0.20 µm capillary column

180 methyl standards described under Materials.

182 Statistical Analysis

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Direct Fame synthesis 9

189 comparisons among the means.

194 Supelco Standard FAME Mixture

202 Direct FAME synthesis, as described in Materials and Methods, is a two-step

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Direct Fame synthesis 10

210 of direct FAME synthesis (data not shown).

217 present in the original Supelco mix (Table 1).

224 Applied to Fish Oil