A Direct Method for Fatty Acid Methyl Ester (FAME) Synthesis

J. V. O'Fallon, J. R. Busboom, M. L. Nelson and C. T. Gaskinstissues, oils and feedstuffs

A direct method for fatty acid methyl ester (FAME) synthesis: Application to wet meat

published online February 12, 2007J ANIM SCI

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Direct Fame synthesis 1

Running head: Direct FAME synthesis 1

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A direct method for fatty acid methyl ester (FAME) synthesis: Application to wet 4

meat tissues, oils and feedstuffs 5

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J. V. O’Fallon, J. R. Busboom, M. L. Nelson, and C. T. Gaskins18

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

15 1 Correspondence: 135 Clark Hall (phone 509-335-6416; fax 509-335-4246; e-16

mail:[email protected]). 17

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Page 1 of 38 Journal of Animal Science

Published Online First on February 12, 2007 as doi:10.2527/jas.2006-491 by guest on May 4, 2012jas.fass.orgDownloaded from



ABSTRACT: A simplified protocol to obtain fatty acid methyl esters (FAME) directly 24

from fresh tissue, oils, or feedstuffs, without prior organic solvent extraction is presented. 25

With this protocol, FAME synthesis is conducted in the presence of up to 33% water. 26

Wet tissues, or other samples, are permeabilized and hydrolyzed for 1.5 hr at 55 °C in 1N 27

KOH in methanol containing C13:0 as internal standard. The KOH is neutralized and the 28

free fatty acids are methylated by H2SO4 catalysis for 1.5 hr at 55 °C. Hexane is then 29

added to the reaction tube, which is vortex-mixed and centrifuged. The hexane is pipetted 30

into a GC vial for subsequent gas chromatography. All reactions are conducted in a single 31

screw cap Pyrex tube for convenience. The method meets a number of criteria for fatty 32

acid analysis including not isomerizing conjugated linoleic acid (CLA) or introducing 33

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

addition to oils, waxes and feedstuffs. The method saves time, effort and is economical 35

when compared to other methods. Its unique performance, including easy sample 36

preparation, is achieved because water is included in the FAME reaction mixtures rather 37

than eliminated. 38

39

Key words: fatty acid analysis, FAME synthesis, longissimus muscle, conjugated linoleic 40

acid (CLA), fish oil, feedstuffs 41

42

Introduction 43

44

The analysis of fatty acids has become increasingly important because more people 45

have become aware of their nutritional and health implications. Because of this, a method 46

Page 2 of 38Journal of Animal Science




for analyzing fatty acids that provides both rapid and reliable results is of great value. 47

Many methods are currently used to analyze fatty acids (e.g., Morrison and Smith, 1964; 48

Sukhija and Palmquist, 1988; Kazala et al., 1999; Mir et al., 2002; Nuernberg et al., 2002; 49

Budge and Iverson, 2003; Cooper et al., 2004). These methods, however, are not 50

necessarily convenient, nor direct, and often must be optimized for reaction conditions, 51

including the catalyst and the temperature (Lewis et al., 2000; Park et al. 2002; Shahin et 52

al., 2003). On the other hand, the ideal method, as noted by Palmquist and Jenkins 53

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

determine the total fatty acid concentration in tissues, oils, and feed samples by 55

converting fatty acid salts, as well as the acyl components in all lipid classes such as 56

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

direct one-step esterification procedure. 58

In this paper we present a method that is established upon a surprising conception, i.e., 59

we add water to the fatty acid methyl ester (FAME) synthesis reagents. Until now, FAME 60

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

However by adding water, the dynamics of sample preparation and methyl ester 62

formation can be revisited and the ideal outcome of FAME synthesis discussed by 63

Palmquest and Jenkins (2003) mentioned above becomes possible. Although the method 64

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

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

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

it is obtained. Thus, for example, meat and milk samples can be analyzed directly without 68

any prior dehydration step. The presence of water in the reagents allows for tissue sample 69





dissolution and facilitates the total extraction of fatty acids not possible in the absence of 70

water. Importantly, water does not interfere with the methylation of any fatty acid. The 71

method uses familiar and economical chemicals, namely, methanol, potassium hydroxide, 72

and sulfuric acid, but uses these methylating reagents in the presence of water. As a result 73

of these characteristics, the direct FAME synthesis method is convenient, reliable and 74

efficient. 75

In short, the objective of this paper is to develop a method to directly methylate fatty 76

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

78

Materials and Methods 79

80

Methylation Agents and Sample Selection 81

82

To assess certain features of our method, we compared direct FAME synthesis to two 83

methylating agents routinely used for FAME synthesis, namely, the base catalyst sodium 84

methoxide and the acid catalyst boron trifluoride. Samples used with sodium methoxide 85

and boron trifluoride did not contain water, as this would compromise the performance of 86

these two methylating agents. The direct FAME synthesis method, however, always 87

contained water, even if the sample did not, because its reagents contained water, i.e., the 88

direct FAME synthesis reaction mixture contained 13.2% water due to the water in the 89

potassium hydroxide and sulfuric acid reagents. 90

The samples we used in this manuscript were chosen for distinct reasons. The Supelco 91

fatty acid standard mixture was chosen because it contained short and long chain 92





saturated, monounsaturated, and polyunsaturated fatty acids in defined amounts and thus 93

served as a primary test of the feasibility of our method. Fish oil was chosen because it is 94

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

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

acids. Conjugated linoleic acid (CLA), as the free acid, was chosen because it is of 97

medical importance as perhaps the only fatty acid that can directly inhibit cancer in 98

animal models (Belury, 2002) and because current FAME synthesis methods often cause 99

undesirable isomerizations of this fatty acid (Kramer et al., 1997). Beef longissimus 100

muscle was chosen because of our special interest in beef fatty acids and it serves as a 101

direct test of the ability of direct FAME synthesis to extract and methylate all of the fatty 102

acids present in meat tissue. To address the problem of refractory samples, we included 103

wax esters, cholesteryl lipid derivatives and alkyl methane sulfonates, as these are 104

difficult fatty acid derivatives to analyze (Palmquist and Jenkins, 2003). Finally, as a 105

concluding demonstration of the versatility of the direct FAME synthesis method, we 106

included a variety of oils, feedstuffs and foods. 107

108

Materials 109

110

Hexane (OmniSolv) was purchased from EM Science, Cherry Hill, NY. Absolute 111

methanol and potassium hydroxide were obtained from J.T. Baker Chemical Co., 112

Phillipsburg, NJ. Chloroform and sulfuric acid were purchased from Fisher Scientific, 113

Tustin, CA. Sodium methoxide and boron trifluoride-methanol were obtained from 114

Sigma-Aldrich, St. Louis, MO. The Supelco standard FAME mixture (47885-U) was 115





obtained from Supelco, Bellefonte, PA. Spring Valley fish oil capsules were distributed 116

by Leiner Health Products, Carson, CA. Tonalin 1000-CLA (conjugated linoleic acid) 117

capsules were obtained from Nature Bounty, Bohemia, NY. All other fatty acid standards 118

were purchased from Nu-Chek Prep. Inc., Elysian, MN. Beef longissimus muscle 119

samples (n=20) were obtained from department owned animals processed at an abattoir. 120

Nuts and sundry food items were purchased from local grocery stores. Coffee bean 121

grinders were purchased from Mr. Coffee, Inc., Cleveland, OH. Pyrex screw-cap culture 122

tubes (16 x 125 mm) were obtained from Corning Laboratory Science Company, 123

Corning, NY. The Tekmar VXR-10 multi-tube vortex was purchased from Jenke and 124

Kunkel, West Germany. 125

126

Folch Extraction of Fatty Acids from Longissimus Muscle 127

128

Longissimus muscle (1g) was extracted with chloroform/methanol (2:1, vol:vol), 129

containing C13:0 as internal standard, according to the method of Folch et al.(1956), 130

using a Brinkmann polytron at room temperature. The extraction mixture was then 131

filtered through a glass scintered filter and replicate aliquots were pipetted into a 16 x 125 132

mm screw-cap Pyrex culture tube and washed with 0.02% aqueous CaCl2. The organic 133

phase was dried with Na2SO4 and K2CO3 (10:1, wt:wt) and the solvent was subsequently 134

removed under nitrogen at 55 °C. 135

136

FAME Synthesis with Sodium Methoxide or Boron Trifluoride 137

138





Freeze-dried tissue samples were uniformly distributed by grinding for 10 -15 sec in a 139

room-temperature coffee bean grinder. Samples of freeze-dried tissue (0.25g), or oils (20 140

µl) were placed into a 16 x 125 mm screw- cap Pyrex culture tube to which 1.0 ml methyl 141

C13:0 internal standard (0.5mg methyl C13:0/ml methanol) was added. Two ml of 142

sodium methoxide (0.5 M) or 2 ml of boron trifluoride in methanol (14%, wt/vol) were 143

added to the Pyrex tubes containing the samples. The tubes were incubated in a 55 °C144

water bath for 1.5 h with vigorous 5 sec hand-shaking every 20 min. Two ml of a 145

saturated solution of NaHCO3 and 3 ml hexane were then added and the tubes were 146

vortex-mixed. After centrifugation, the hexane layer containing the FAME was placed 147

into a GC vial. The vial was capped and placed at –20 °C until GC analysis. 148

149

Direct FAME Synthesis 150

151

Samples were uniformly distributed by grinding for 10 -15 sec in a room-temperature 152

coffee bean grinder. Short grinding times minimize smearing fat on the grinder container 153

walls. Samples could be processed in the state obtained, e.g., wet, dry, freeze-dried, or 154

semi-frozen. Samples (0.5g wet, dry, or semi-frozen sample), (0.25g freeze-dried 155

sample), or oils (20 µl) were placed into a 16 x 125 mm screw-cap Pyrex culture tube to 156

which 1.0 ml C13:0 internal standard (0.5mg C13:0/ml methanol), 0.7 ml 10 N KOH in 157

water, and 5.3 ml methanol were added. The tube was incubated in a 55 °C water bath for 158

1.5 h with vigorous 5 sec hand-shaking every 20 min to properly permeate, dissolve and 159

hydrolyze the sample. After cooling below room temperature in a cold tap water bath, 160

0.58 ml of 24 N H2SO4 in water was added. The tube was mixed by inversion, and with 161





precipitated K2SO4 present, was incubated again in a 55 °C water bath for 1.5 h with 5 162

sec hand-shaking every 20 min. After FAME synthesis, the tube was cooled in a cold tap 163

water bath. Three ml of hexane was added and the tube was vortex-mixed for 5 min on a 164

multi-tube vortex. The tube was centrifuged for 5 min in a tabletop centrifuge and the 165

hexane layer, containing the FAME, was placed into a gas chromatography (GC) vial. 166

The vial was capped and placed at –20 °C until GC analysis. 167

168

Gas Chromatography 169

170

The fatty acid composition of the FAME was determined by capillary gas 171

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

(Supelco, Bellefonte, PA) installed on a Hewlett Packard 5890 gas chromatograph 173

equipped with a Hewlett Packard 3396 Series II integrator and 7673 controller, a flame 174

ionization detector and split injection. Initial oven temperature was 140 °C, held for 5 175

min, subsequently increased to 240 °C at a rate or 4 °C min-1, and then held for 20 min. 176

Helium was used as the carrier gas at a flow rate of 0.5 ml min-1 and column head 177

pressure was 280 kPa. Both the injector and detector were set at 260 °C. The split ratio 178

was 30:1. Fatty acids were identified by comparing their retention times with fatty acid 179

methyl standards described under Materials. 180

181

Statistical Analysis 182

183





Duplicate gas chromatograph results were averaged for animal and methylation 184

method. ANOVA of beef longissimus muscle FAME was calculated using Proc GLM of 185

SAS (SAS Institute, Cary, NC) using a model with methylation method as the treatments 186

and animal as a blocking factor in a randomized complete block design. When the F-ratio 187

for the methylation methods was significant, Student’s t-test was used to make pairwise 188

comparisons among the means. 189

190

Results 191

192

Sodium Methoxide, Boron Trifluoride, and Direct FAME Synthesis Methods Applied to a 193

Supelco Standard FAME Mixture 194

195

For an initial FAME synthesis analysis, we used a Supelco standard FAME mixture. 196

This standard mixture contained FAME in a defined ratio, and consisted of both short and 197

long chain saturated, monounsaturated, and polyunsaturated fatty acids. In order to 198

broadly assess certain features of our method, we compared the results of direct FAME 199

synthesis to those of the base catalyst sodium methoxide and the acid catalyst boron 200

trifluoride. 201

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

procedure. In the first step, sample fatty acid esters are hydrolyzed to free fatty acids and 203

in the second step the free fatty acids are converted to FAME. When the first step of 204

direct FAME synthesis was applied to the Supelco standard FAME mixture, the esters 205

were hydrolyzed to free fatty acids that were not volatile enough to enter the GC column. 206





These results, i.e., the absence of fatty acid peaks, provided formal evidence that the first 207

step in direct FAME synthesis completely hydrolyzed the Supelco standard FAME to free 208

fatty acids which was the desired general prerequisite for the subsequent methylation step 209

of direct FAME synthesis (data not shown). 210

When the second step of direct FAME synthesis, the methylation step, was applied to 211

the Supelco free fatty acids produced by the first step, the results shown in Table 1 were 212

obtained. All of the GC peaks present in the original Supelco standard mixture were 213

again observed, as can be seen by comparing the fatty acids of direct FAME synthesis to 214

those of the Supelco mix. When presented with a FAME sample, as in this experiment, 215

both sodium methoxide and boron trifluoride likewise gave the same FAME values 216

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

Fatty acid artifacts, e.g., the conversion of CLA to other isomers, are a concern in fatty 218

acid analysis (Kramer et al. 1997; Park et al., 2002; Shahin et al., 2003). Since direct 219

FAME synthesis, sodium methoxide, or boron trifluoride did not generate new fatty acid 220

peaks, no fatty acid artifacts were created in the Supelco standard FAME mixture. 221

222

The Methods of Sodium Methoxide, Boron Trifluoride, and Direct FAME Synthesis 223

Applied to Fish Oil 224

225

A comparison was made between the base catalyst sodium methoxide, the acid 226

catalyst boron trifluoride, and direct synthesis, on FAME production from fish oil 227

commercially obtained as a human nutritional supplement. The results are shown in 228

Table 2. 229





Twenty fatty acids were identified in the fish oil sample. Direct FAME synthesis 230

recovered more total amount of fatty acid than did either of the other two methods as 231

would be expected if direct FAME synthesis generated methyl esters of all the fatty acids 232

in the sample. In comparison, base catalysis with sodium methoxide methylated esterified 233

fatty acids, but not free fatty acids (Kramer et al., 1997), whereas, acid catalysis by boron 234

trifluoride should have methylated all fatty acids, including esterified, unesterified, and 235

those in salt form (Carrapiso and Garcia, 2000). 236

In analyzing the total fatty acids methylated, direct FAME synthesis converted 22% 237

more fatty acids to FAME than did sodium methoxide and 14% more than did boron 238

trifluoride indicating that there must be groups of fatty acids present that the latter two 239

methods did not recognize. Such limitations with these two reagents have been 240

previously noted (Kramer et al., 1997; Christie, 2003). The direct FAME synthesis 241

method apparently methylates all of the fatty acids present, as confirmed by the LECO 242

Fat Extractor (see Independent Assessment of Direct FAME Synthesis Efficiency), which 243

explains why the direct FAME synthesis recoveries were higher than the other two 244

methods. 245

When the peak areas were expressed as % of total fatty acid (% FA, wt/wt) present by 246

each method, the % FAs were similar for all three methods even though total recovery 247

among the three methods was somewhat different. This indicates that the fatty acids not 248

methylated by sodium methoxide or boron trifluoride were present in similar ratios for all 249

of the fatty acids present. The results with sodium methoxide, which does not methylate 250

free fatty acids, indicates that because it methylated only 82% of the total fatty acids 251

present the other 18% of the fatty acids present may have been free fatty acids. 252





253

Conjugated Linoleic Acid Analysis Using Sodium Methoxide, Boron Trifluoride or Direct 254

Synthesis 255

256

Table 3 presents the results obtained from an analysis of commercial CLA capsules 257

using sodium methoxide, boron trifluoride and direct synthesis. The CLA capsules 258

contained the two important CLA isomers C18:2c9,t11 and C18:2t10,c12, and also 259

palmitic (C 16:0), stearic (C18:0), oleic (C18:1n9), and linoleic (C18:2n6) fatty acids. 260

Most notable, was the difference in the results obtained with sodium methoxide 261

compared to that obtained with boron trifluoride and direct FAME synthesis. Only 0.4% 262

of the fatty acids present were methylated by sodium methoxide, indicating that they 263

were virtually all free fatty acids, and not esterified. As noted previously, sodium 264

methoxide methylates esterified fatty acids, but not free fatty acids (Kramer et al., 1997). 265

This example serves as a caution that a researcher who uses sodium methoxide, or 266

alkaline catalysts in general, is at great risk of missing fatty acids, whereas, direct FAME 267

synthesis methylates all of the fatty acids present. 268

Boron trifluoride and direct FAME synthesis gave essentially the same results for the 269

fatty acids present in the CLA capsules. Once again, the direct FAME synthesis method 270

did not generate fatty acid artifacts, including CLA artifacts, as all of the peaks were 271

essentially identical to those of the boron trifluoride method. The absence of CLA 272

artifacts confirmed the work of Park et al. (2002), who used similar H2SO4 conditions on 273

CLA samples as was used with direct FAME synthesis, but Park et al. (2002) did not 274

have water present. 275





276

The Analysis of Beef Longissimus Muscle Using Sodium Methoxide, Boron Trifluoride or 277

Direct FAME Synthesis 278

279

The analysis of freeze-dried beef longissimus muscle fatty acids using sodium 280

methoxide, boron trifluoride and direct FAME synthesis is presented in Table 4. Once 281

again, there were striking differences among the three methods. Direct FAME synthesis 282

recovered more (P< 0.01) fatty acids than did sodium methoxide and much more than did 283

boron trifluoride. Since most of the fatty acids were esterified in longissimus muscle, as 284

opposed to unesterified in the CLA capsule (Table 3), it was not surprising that sodium 285

methoxide performed much better in FAME synthesis of this sample, although it 286

methylated only 78% of the fatty acids present compared to direct FAME synthesis. This 287

sample also shows that boron trifluoride performed much better with the free fatty acids 288

in the CLA sample (Table 3) than with the esterified fatty acids in muscle tissue. This 289

latter result was surprising because boron trifluoride can methylate all families of fatty 290

acids (Carrapiso and Garcia, 2000). Boron trifluoride methylated all of the different fatty 291

acids present, because the same peaks were present with BF3 as with direct FAME 292

synthesis, but it did not do so quantitatively. It is unclear at this time why boron 293

trifluoride gave such poor results. It can be mentioned that Bolte et al. (2002) reported 294

satisfactory FAME synthesis results using boron trifluoride on freeze-dried lamb muscle 295

tissue fatty acids by using much higher temperature and more concentrated effort, i.e., by 296

incubating at 80°C and vortex-mixing two to three times/min. However, our results do 297

not seem to be due to the fact that boron trifluoride cannot permeate the meat sample, 298





because similar results were observed when using a chloroform:methanol extract 299

according to the method of Folch et al. (1956), where extraction of fatty acids by boron 300

trifluoride would no longer be an issue (data not shown). Apparently, and unexpectedly, 301

there are fatty acid structures in beef longissimus muscle that can be easily methylated by 302

direct FAME synthesis but not by boron trifluoride. 303

When expressed as % FA, sodium methoxide and direct FAME synthesis were quite 304

similar in their results, but boron trifluoride was different (P<0.01), and in this latter case 305

(BF3) the % FA values were higher when the concentration of fatty acid was lower. This 306

difference could be explained by the fact that boron trifluoride methylated only 46% of 307

the fatty acids present in longissimus muscle than did direct FAME synthesis, and did so 308

preferentially. With boron trifluoride, long chain unsaturated fatty acids appeared to be 309

methylated more efficiently than short chain or saturated fatty acids, whereas, direct 310

FAME synthesis methylated fatty acids without bias to chain length or structure. As a 311

result, direct FAME synthesis consistently methylated more fatty acid, averaging 1.3 312

times that of sodium methoxide and 2.2 times that of boron trifluoride. 313

314

Independent Assessment of Direct FAME Synthesis Efficiency 315

316

To determine if the direct FAME synthesis could extract all of the fatty acids present 317

in beef longissimus muscle, we independently compared it to the LECO TFE2000 Fat 318

Extractor (LECO Corporation, St. Joseph,MI). These results are shown in Table 5. For 319

the analysis of this experiment, wet tissue and freeze dried tissue was corrected to a dry 320

matter basis. Direct FAME synthesis, whether applied to dry or wet muscle tissue, 321





extracted all of the fatty acids present when compared to the LECO TFE200 Fat 322

Extractor, assuming that the LECO values should be 6-9 % higher since the LECO values 323

also contain glycerol and cholesterol. In this respect, direct synthesis gives a truer value 324

of fat content then does the LECO extractor, which does not differentiate fatty acids from 325

total lipid. 326

327

Direct FAME Synthesis Applied to Wax Esters, Cholesteryl Derivatives, and Fatty Acid 328

Salts 329

330

The ideal FAME synthesis method would be able to analyze fatty acids from samples 331

of wax esters, cholesteryl lipid derivatives, and alkyl methane sulfonates (Palmquist and 332

Jenkins, 2003). We applied the direct FAME synthesis method to such families of fatty 333

acids and the results are shown in Table 6. Direct FAME synthesis was able to identify 334

the fatty acids in wax esters as represented by palmityl stearate (saturated series) and 335

stearyl linoleate (unsaturated series), cholestery lipid derivatives as represented by 336

cholesteryl oleate, and fatty acid salts, as represented by oleyl methane sulfonate. As the 337

second component in each of these compounds was a fatty alcohol, and not a fatty acid, it 338

was not converted to the FAME. This is as it should be, since we were analyzing strictly 339

for fatty acid components. Depending on the concentration of these very hydrophobic 340

families of fatty acids, full quantification might require more shaking and a longer 341

incubation time during the first step of direct FAME synthesis. 342

343





Effect of Water Content on FAME Production by Direct FAME Synthesis 344

345

It is of great interest to know what the limiting concentration of water might be for the 346

direct FAME synthesis method. There has to be such a limit, for no other reason than the 347

fact there has to be a certain concentration of methylating reagents. In Figure 1, we show 348

the effect of water concentration on the direct FAME synthesis method. As the 349

percentage of water was increased, the total amount of fatty acids methylated decreased 350

(data not shown), but this was easily corrected for by the internal standard. Most 351

importantly, the percentage of each fatty acid remained constant up to 33% water. Only 352

above 33% water do the FAME production results become problematic. In comparison, 353

our reagents, without any sample present, constitute only 13% water in a final reaction 354

volume of 7.58 ml. Thus, from a practical standpoint, one can replace 1.5 ml of MeOH 355

with a 1.5 ml aqueous sample for a final concentration of 33% water. For example, using 356

the protocol as given, 1.5 ml of milk can be analyzed directly by our method. 357

358

The Versatility of Direct FAME Synthesis 359

360

The ideal method for the analysis of fatty acids would be applicable to any sample 361

whose fatty acid content was desired. With this in mind, direct FAME synthesis was 362

applied to various products and the results are shown in Figures 2 and 3. We evaluated a 363

number of oil sources including fish, Canola, and virgin olive; a number of nuts including 364

almond, cashew, peanut, sunflower, and walnut; a couple of feedstuffs, including pelleted 365

sheep concentrate and alfalfa; and foodstuffs including beef longissimus muscle, butter, 366





cheese, Crisco, margarine, Miracle Whip, and Slim Fast. The direct FAME synthesis 367

method readily generated a FAME profile for all of these different samples. 368

When % FA was plotted against retention time and presented as in Figures 2 and 3, it 369

was instantly seen that each sample has its own ‘fatty acid print’. Most samples were 370

dominated by 1 to 3 fatty acids, i.e., 1 to 3 fatty acids accounted for 85%, or more, of the 371

total fatty acid composition of the sample. When the graphs were arrayed as they were in 372

these two figures, it was easy to visually compare one sample to another, e.g., the 373

vegetable oil product Crisco contained 25% oleic and 25% linoleic fatty acids, while 374

olive oil contained 70% oleic acid; beef longissimus muscle contained 40% oleic and 375

25% palmitic acids, while Slim Fast contained 70% oleic acid of the total fatty acids 376

present. The dairy products, butter and Kraft American processed cheese, had very 377

similar fatty acid profiles. The fatty acid composition of the samples presented in Figures 378

2 and 3, determined by direct FAME synthesis, are in very good agreement with the fatty 379

acid tabulations in handbooks (Watt, 1975; McCance, 2002). 380

381

Discussion 382

383

An evaluation of numerous aspects in the isolation, separation, identification and 384

structural analysis of lipids was made by Christie (2003). Among his comments on the 385

various methods of FAME synthesis, he pointed out that care should be taken in the 386

evaporation of solvents as appreciable amounts of esters up to C14 can be lost if this step 387

is performed carelessly. He stated that the vigorous use of nitrogen to evaporate solvents 388

must be avoided. In direct synthesis we eliminate solvent evaporation completely because 389





we do not use any prior organic solvent extraction. Christie (2003) also reminded 390

researchers that boron trifluoride in methanol has a limited shelf life, even when 391

refrigerated, and the use of old or too concentrated solutions often resulted in the 392

production of artifacts and the loss of appreciable amounts of polyunsaturated fatty acids 393

by addition of methanol across the double bonds. On the other hand, Christie (2003) 394

recommended sulfuric acid in methanol, the principle of which we have incorporated into 395

direct synthesis. Others, using sulfuric acid in methanol, have shown that it does not 396

produce CLA artifacts (Park et al., 2002). Additionally, we have used C13:0 as internal 397

standard, rather than some other odd chain fatty acid such as C19:0 because it is readily 398

soluble in methanol, a major component of our initial reagents. In principle, any fatty 399

acid may be used as internal standard as long as all of the fatty acids in the sample are 400

methylated. 401

In our method of direct FAME synthesis we introduced water into fatty acid analysis. 402

In fact, water defines our method, and stands in definite contrast to all other FAME 403

synthesis methods. Other researchers, including those who created direct methods for 404

fatty acid analysis, took special care to avoid water. Even exposure to ambient air was 405

minimized in order to avoid moisture uptake by dry muscle tissue (Murrieta et al., 2003). 406

When water cannot be used, sample preparation was no longer convenient and hydrolysis 407

of fatty acids, cholesteryl esters, and waxes, the first step of our method, was not 408

possible. 409

Again, the principle behind the direct FAME synthesis method was to dissolve, or 410

thoroughly permeate a sample, for example, beef longissimus muscle, and in the process 411

hydrolyze fatty acid structures so they could be directly methylated without any prior 412





organic solvent extraction. Hydrolysis, of course, requires water as does solubilization 413

and permeation. KOH in methanol alone did not solubilize tissue properly, and H2SO4 in 414

methanol precipitated tissue (data not shown). In a typical assay, even without wet tissue, 415

the first step of direct FAME synthesis contained 10% water, while the second, and final 416

step, contained 13% water. 417

Even though certain fatty acids have limited solubility in water/methanol, hydrolysis 418

of fatty acid esters still occurs at the water/methanol:lipid interface and can, in fact, be 419

accounted for by the internal standard. For concentrated fat solutions, and waxes, the 420

hydrolysis step might take longer than 1.5 h at 55 º C to complete. All of our results show 421

the efficacy of the direct FAME synthesis method, which allows up to 33% water content 422

(e.g., see Figure 1). Again, KOH in MeOH alone could not solubilize and permeate 423

tissues as well as when water was added, and H2SO4 in MeOH precipitated tissues, rather 424

than solubilized them. 425

Of further interest were the results obtained by direct FAME synthesis when 426

compared, in various situations, to the sodium methoxide and boron trifluoride methods. 427

Most striking was the case of CLA analysis (Table 3) where sodium methoxide did not 428

methylate any CLA in a capsule full of it. Similarly, but not so pronounced, was the fact 429

that boron trifluoride methylated only 46% of the fatty acids present in beef longissimus 430

muscle (Table 4). In the latter instance, expressing the results as % FA could mask the 431

inadequacies of the method, but at closer examination this too would fail. If a method 432

results in a differential extraction and synthesis of FAME, then at some point the % FA 433

will be wrong. Such an example can be found with the boron trifluoride results in Table 434

4. The concentration of C20:4n6 in beef longissimus muscle was 1.6 % with the boron 435





trifluoride method, but only 0.9% with the direct FAME synthesis method. This two fold 436

discrepancy was accounted for by the fact that boron trifluoride differentially methylated 437

a higher percentage of C20:4n6 than it did of other fatty acids present. Direct FAME 438

synthesis provided the most accurate values because it methylated all of the fatty acids 439

present in beef longissimus muscle (Table 4) as verified by an independent analysis with 440

the LECO TFE2000 Fat Extractor (Table 5). 441

Finally, direct FAME synthesis is convenient. Since water is part of the method, and 442

not antagonistic to it, sample preparation is rapid; one only weighs-out or pipets the 443

sample into a Pyrex tube and then conducts the direct FAME synthesis. Gone is the 444

preparation time it takes to lyophilize a sample (usually days), or the prior organic 445

solvent extractions and nitrogen evaporations (usually hours) that are required to 446

eliminate water in the other fatty acid methods. 447

In summary, a simplified protocol was developed to obtain fatty acid methyl esters 448

(FAME) from any sample. The method consists of two steps, conducted in a single 449

reaction tube. The protocol relies on the presence of water, which heretofore, had been 450

rigorously and tediously eliminated in FAME synthesis methods. 451

452

Literature Cited 453

454

Belury, M. A. 2002. Inhibition of carcinogenesis by conjugated linoleic acid: potential 455

mechanisms of action. J. Nutr. 132:2995-2998. 456

Bolte, M. R., R. W. Hess, W. J. Means, G. E. Moss, and D. C. Rule. 2002. Feeding lambs 457

high-oleate or high-linoleate safflower seeds differentially influences carcass fatty 458





acid composition. J. Anim. Sci. 80:609-616. 459

Budge, S. M., and S. J. Iverson. 2003. Quantitative analysis of fatty acid precursors in 460

marine samples: direct conversion of wax ester alcohols and dimethylacetals to 461

FAMEs. J. Lipid Res. 44:1802-1807. 462

Carrapiso, A. I., and C. Garcia. 2000. Development in lipid analysis: some new extraction 463

techniques and in situ trans esterification. Lipids 35:1167-1177. 464

Christie, W. W. 2003. Lipid Analysis: Isolation, Separation, Identification and Structural 465

Analysis of Lipids, Third edition. The Oily Press, Bridgwater, England. 466

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2004. Manipulation of the n-3 polysaturated fatty acid content of muscle and adipose 468

tissue in lambs. J. Anim. Sci. 82:1461-1470. 469

Folch, J., M. Lees, and G. H. Sloane-Stanley. 1956. A simple method for the isolation 470

and purification of total lipids from animal tissue. J. Biol. Chem. 226:497-509. 471

Kazala, E. C., F. J. Lozeman, P. S. Mir, A. L. Laroche, D. R. C. Bailey, and R. J. 472

Weselake. 1999. Relationship of fatty acid composition to intramuscular fat content in 473

beef from crossbred Wagyu cattle. J. Anim. Sci. 77:1717-1725. 474

Kramer, J. K. G., V. Fellner, M. E. R. Dugan, F. D. Sauer, M. M. Mossoba, and M. P. 475

Yurawecz. 1997. Evaluating acid and base catalysts in the methylation of milk and 476

rumen fatty acids with special emphasis on conjugated dienes and total trans fatty 477

acids. Lipids 32:1219-1228. 478

Lewis, T., P. D. Nichols, and T. A. McMeekin. 2000. Evaluation of extraction methods 479

for recovery of fatty acids from lipid-producing microheterotrophs. J. Microb. 480

Methods 43:107-116. 481





McCance, R. A. 2002. McCance and Widdowson’s: The composition of foods. Royal 482

Society of Chemistry, Cambridge, Great Britain. 483

Mir, P. S., Z. Mir, P. S. Kuber, C. T. Gaskins, E. L. Martin, M. V. Dodson, J. A. Elias 484

Calles, K. A. Johnson, J. R. Busboom, A. J. Wood, G. J. Pittenger, and J. J. Reeves. 485

2002. Growth, carcass characteristics, muscle conjugated linoleic acid (CLA) content, 486

and response to intravenous glucose challenge in high percentage Wagyu, Wagyu X 487

Limousin, and Limousin steers fed sunflower oil-containing diets. J. Anim. Sci. 488

80:2996-3004. 489

Morrison, W. R., and L. M. Smith. 1964. Preparation of fatty acid methyl esters and 490

dimethylacetals from lipids with boron fluoride-methanol. J. Lipid Res. 5:600-608. 491

Murrieta, C. M., B. W. Hess, and D. C. Rule. 2003. Comparison of acidic and alkaline 492

catalysts for preparation of fatty acid methyl esters from ovine muscle with emphasis 493

on conjugated linoleic acid. Meat Sci. 65:523-529. 494

Nuernberg, K., G. Nuernberg, K. Ender, S. Lorenz, K. Winkler, R. Rickert, and H. 495

Steinhart. 2002. N-3 fatty acids and conjugated linoleic acids of longissimus muscle in 496

Beef cattle. Eur. J. Lipid Sci. Technol. 104:463-471. 497

Palmquist, D. L., and T. C. Jenkins. 2003. Challenges with fats and fatty acid methods. J. 498

Anim. Sci. 81:3250-3254. 499

Park, S. J., C. W. Park, S. J. Kim, J. K. Kim, Y. R. Kim, K. A. Park, J. O. Kim, and Y. L. 500

Ha. 2002. Methylation methods for the quantitative analysis of conjugated linoleic 501

acid (CLA) isomers in various lipid samples. J. Agric. Food Chem. 50:989-996. 502

Shahin, A. M., M. K. McGuire, K. L. Ritzenthaler, and T. D. Shultz. 2003. Determination 503

of c9,t11-CLA in major human plasma lipid classes using a combination of 504





methylating methodologies. Lipids 38:793-800. 505

Sukhija, P. S., and D. L.Palmquist. 1988. Rapid method for determination of total fatty 506

acid content and composition of feedstuffs and feces. J. Agric. Food Chem. 36:1202- 507

1206.508

Watt, B. K. 1975. Handbook of the nutritional contents of foods for the United States 509

Department of Agriculture, Dover Publications, New York.510





Table 1. Effect of sodium methoxide, boron trifluoride or direct FAME synthesis on a

standard Supelco FAME mixture

Supelco Mix Na Methoxide BF3 Direct synthesisFatty 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


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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).



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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

Palmitic C16:0 nd b 14.82 1.35 13.37 1.22

Stearic C18:0 nd b 24.78 1.49 24.25 2.45

Oleic C18:1n9 nd b 111.97 5.46 108.78 10.24

Linoleic C18:2n6 nd b 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

Lauric C12:0 0.21 ±0.02d 0.09d 0.17 ±0.01d 0.12e 0.24 ±0.02e 0.08d

Myristic C14:0 8.34 ±0.68e 3.46e 5.57 ±0.25d 3.90f 10.29 ±0.83f 3.32d

Myristoleic C14:1n5 2.01 ±0.14e 0.83e 1.38 ±0.08d 0.97f 2.29 ±0.21e 0.74d

Pentadecanoic C15:0 1.59 ±0.14d 0.66d 1.24 ±0.08d 0.87f 2.23 ±0.18e 0.72e

Palmitic C16:0 62.82 ±4.10e 26.07d 38.00 ±0.86d 26.61e 80.85 ±4.84f 26.08d

Palmitoleic C16:1n7 8.10 ±0.65e 3.36d 5.74 ±0.19d 4.02e 10.91 ±0.66f 3.52d

Heptadecanoic C17:0 3.89 ±0.35e 1.61e 2.03 ±0.08d 1.42d 5.05 ±0.37f 1.63e

Stearic C18:0 30.25 ±1.89e 12.56e 15.55 ±0.28d 10.89d 39.25 ±2.31f 12.66e

t-Vaccenic C18:1n11t 19.20 ±2.36e 7.97f 9.35 ±0.96d 6.55d 22.21 ±3.28e 7.16e

Oleic C18:1n9 87.10 ±5.28e 36.15e 47.60 ±1.03d 33.34d 113.91 ±6.43f 36.75e

Linoleic C18:2n6 10.70 ±0.62d 4.44d 10.50 ±0.46d 7.36e 13.98 ±0.82e 4.51d

Arachidic C20:0 0.60 ±0.06f 0.25f 0.00 ±0.00d 0.00d 0.40 ±0.05e 0.13e

Gamma- Linolenic C18:3n6 0.32 ±0.03e 0.13e 0.17 ±0.02d 0.12e 0.28 ±0.02e 0.09d

Eicosenoic C20:1n9 0.20 ±0.01e 0.08d 0.10 ±0.00d 0.07d 0.64 ±0.05f 0.21e



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Direct FAME synthesis 30

Linolenic C18:3n3 0.56 ±0.04e 0.23e 0.21 ±0.01d 0.15d 0.91 ±0.05f 0.29f

c-9,t-11 CLA C18:2c9,t11 0.70 ±0.04e 0.29d 0.59 ±0.02d 0.41e 0.85 ±0.09f 0.27d

Eicosadienoic C20:2 0.25 ±0.02e 0.10e 0.17 ±0.01d 0.12e 0.20 ±0.02e 0.06d

Behenic C22:0 0.40 ±0.04f 0.17e 0.25 ±0.02e 0.18e 0.18 ±0.01d 0.06d

Eicosatrienoic C20:3n6 0.41 ±0.06e 0.17d 0.31 ±0.06d 0.22e 0.85 ±0.03f 0.28f

Arachidonic C20:4n6 2.05 ±0.07d 0.85d 2.24 ±0.20d 1.57e 2.74 ±0.09e 0.89d

Eicosapentaenoic C20:5n3 0.40 ±0.03d 0.17d 0.52 ±0.03e 0.37e 0.54 ±0.03e 0.17d

Docosatetraenoic C22:4n6 0.18 ±0.01d 0.07d 0.24 ±0.01e 0.17e 0.26 ±0.01e 0.09d

Docosapentaenoic C22:5n3 0.68 ±0.03d 0.28d 0.84 ±0.06e 0.59e 0.93 ±0.03f 0.30d

Total 240.95 ±16.67e 100.00d 142.78 ±4.72d 100.00d 310.00 ±20.40f 100.00d

a mg/g of freeze dried tissue. b SE = Standard error (n=20). c % 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.



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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

Freeze dried tissueb 310.25 ± 18.23

Wet tissueb 312.37 ± 27.51

LECOc 336.59 ± 23.36 aSE = Standard error (n=12)

bSamples were analyzed by direct FAME synthesis. cLipid 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.





0

5

10

15

20

25

30

10 15 20 25 30 35 40 45 50 55Percent Water

Perc

entF

attyA

cid

C16:0C20:5n3C18:1n9C22:6n3C18:0C13:0C20:2n6C22:5n3C18:3n6





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

0.01.02.03.04.05.06.07.0

Ret ent ion t ime ( min)

Fish Oil

02468

1012141618

10 50Retention time (min)

%Fa

ttyac

id

Crisco

0

5

10

15

20

25

30

R et ent io n t ime ( min)

Pelleted Sheep Concentrate

05

1015

2025303540


Beef Longissimus Muscle

010

2030

4050


Canola

010203040506070


Alfalfa

05

1015

202530


Slim Fast

01020304050607080


Virgin Olive Oil

01020304050607080






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

010203040506070


Peanut

010203040506070


Blue Bonnet Margarine

05

1015

20253035404550


Walnut

010203040506070

R et ent ion t ime ( min)

Almond

01020304050607080


Butter

05

1015

202530


Cashew

010203040506070


Miracle Whip

0102030405060


Kraft Cheese

05

1015

202530





Citations

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A Direct Method for Fatty Acid Methyl Ester (FAME) Synthesis