1

Analysis of Fatty Acids

2

AOAC Official Methods

GC-IRcis- and trans-Octadecenoic isomers and general fatty acid composition

994.15

GC-FIDtrans-Fatty acid in margarines985.21etc

GC-FIDEncapsulated fish oils991.39

GC-FIDFat (total, saturated and unsaturated) in foods

996.06

Preparation methyl esters

Fatty acids in oils and fats969.33

3

Typical analysis

Fat and fatty acids are extracted from food by hydrolytic method.

Fat is extracted into ether, then methylatedto fatty acid methyl esters (FAMEs).

FAMEs are quantitatively measured by gas chromatography.

4

Typical analysis

5

Typical analysis

FatFat HydrolysisHydrolysis

Methylatedto FAMEsMethylatedto FAMEs

GC-FID(quantitation)GC-FID

(quantitation)

6

Typical analysis

Hydrolysis

Weigh sampleand pyrogallicacid into the Mojonnier flask

7

Typical analysis

Hydrolysis

Add internalstandard, ethanoland 8.3 M HCl.

8

Typical analysis

Hydrolysis

Hydrolyse thesample at 70 –80 oC

9

Typical analysis

Hydrolysis

Remove flasksfrom water bath.Cool to roomtemperature.

10

Typical analysis

Extraction

Add ethanol, Ether and shake.

11

Typical analysis

Extraction

Centrifuge sample.(allow contents tosep. at least 1 h if centrifuge is notavailable)

12

Typical analysis

Extraction

Decant the etherlayer into a tube.

13

Typical analysis

Extraction

Evaporate etheron water bathusing nitrogenstream to aidin evaporation.

14

Typical analysis

Extraction

Dissolve the residue in chloroform.

15

Typical analysis

Methylation

Transfer mixture to a glass vial. Evaporate todryness.

16

Typical analysis

Methylation

Add 7% BF3

reagent and toluene.

17

Typical analysis

Methylation

Seal vials withscrewcap andheat them at 100 oC.

18

Typical analysis

Methylation

Allow vials to coolto roomtemperature.

19

Typical analysis

Methylation

Add water (5 mL),hexane (1 mL),Na2SO4 (1 g).Shake

20

Typical analysis

Methylation

Allow layers to separate and transfer top layer to anothervial containing Na2SO4 (1 g).

21

Typical analysis

Methylation

Top layer shouldcontain FAMEsand internal standard.

22

Typical analysis

GC Determination

Transfer to autosampler vialfor GC analysis.

23

Fatty acids (saturated)

14:0 Myristic12:0 Lauric

10:0 Capric11:0 Undecanoic

8:0 Caprylic6:0 Caproic4:0 Butyric

24

Fatty acids (saturated)

15:0 Pentadecanoic

24:0 Lignoceric22:0 Behenic20:0 Arachidic18:0 Stearic17:0 Margaric16:0 Palmitic

25

Definition

Trans fatty acids means the sum of all unsaturated fatty acids which contains at least one nonconjugated and trans double bond.

26

trans-Fatty acids

18:2 trans 9-Linolelaidic18:2 trans 12-Linolelaidic

18:2 trans-Linolelaidic18:1 trans 11-Vaccenic 18:1 trans-Elaidic18:1 trans 6-Petroselenic 16:1 trans-Palmitelaidic14:1 trans-Myristelaidic

27

trans-Fatty acids

*Three 18:2 linoleic – conjugated fatty acids were listed in method 996.06

22:1 13-trans Docosenic20:1 Eicosenic trans 11

18:2 cis-9, trans-11-Octadecadienic18:2 Linoleic – conjugated*

28

trans-Fatty acids (18:3 trans)

18:3 cis-9, trans-12, cis-15-octadecatrienoic18:3 trans-9, cis-12, cis-15-octadecatrienoic

18:3 cis-9, trans-12, trans-15-octadecatrienoic18:3 cis-9, cis-12, trans-15-octadecatrienoic

18:3 trans-9, cis-12, trans-15-octadecatrienoic

18:3 trans-9, trans-12, trans-15-octadecatrienoic18:3 trans-9, trans-12, cis-15-octadecatrienoic

29

FAME (saturated and trans)

min10 15 20 25 30 35 40 45 50 55

pA

20

30

40

50

60

70

FID1 A, (090715\Q6F.D)

Area: 8

4.6241

Area:

94.96

42 A

rea: 12

2.222 A

rea: 148.277

Area:

524.4

83 A

rea: 146.8

38

Area: 1

62.932

Area:

58.77

83

Area:

183.4

51 A

rea: 15

4.217

Area:

48.7972

Area: 1

52.833

Area:

164.2

95

Area:

50.3563

Area:

42.95

81

Area:

49.8512

Area:

163.4

66

Area:

50.14

78

Area:

63.84

96

Area: 5

8.5977

Area:

164.4

3

Area: 5

0.2154

Area:

171.1

59

12.

610

- C

4:0

14.

673

- C

6:0

18.

189

- C

8:0

22.

973

- C

10:0

25.

601

- C

11:0

28.

203

- C

12:0

33.

268

- C

14:0

34.

633

- C

14:1

T (9

-tra

ns)

35.

670

- C

15:0

37.

968

- C

16:0

39.

063

- C

16:1

T (9

-tran

s)

40.

173

- C

17:0

42.

289

- C

18:0

43.

129

- C

18:1

T (6

-tra

ns)

43.

204

- C

18:1

T (9

-tra

ns)

43.

313

- C

18:1

T (1

1-tr

ans)

44.

673

- C

18:2

TT (

9, 1

2-tra

ns)

46.

270

- C

20:0

47.

111

- C

20:1

T (1

1-tr

ans)

48.

037

- C

18:2

T (9

-cis

, 11

-tra

ns)

48.

333

- C

18:2

T (1

0-tr

ans,

12-

cis)

49.

952

- C

22:0

50.

740

- C

22:1

T (1

3-tr

ans) 5

3.54

1 -

C24

:0

SP-2560, 100m x 0.25mm ID, 0.2 µm, flow rate: 0.7 mL/min

30

FAME (18:1 trans)

min43 43.1 43.2 43.3 43.4

pA

15

20

25

30

35

40

45

FID1 A, (090715\Q6F.D)

Area: 5

0.3563

Area: 4

2.9581

43.

129

- C

18:1

T (6

-tra

ns)

43.

204

- C

18:1

T (9

-tra

ns)

43.

313

- C

18:1

T (1

1-tr

ans)

transtrans trans

31

FAME (20:1 trans and 18:2 trans)

min46.75 47 47.25 47.5 47.75 48 48.25 48.5

pA

15

20

25

30

35

40

45

50

FID1 A, (090715\Q6F.D)

Area: 5

0.1478

Area: 6

3.8496

Area: 5

8.5977

47.

111

- C

20:1

T (1

1-tr

ans)

48.

037

- C

18:2

T (9

-cis

, 11

-tra

ns)

48.

333

- C

18:2

T (1

0-tr

ans,

12-

cis)

transcis

trans

transcis

32

FAME (18:2 trans)

min25 30 35 40 45

pA

15

20

25

30

35

FID1 A, (090813\CHK-2F.D)

Area:

497.3

21

Area:

15.15

09

Area:

6.327

9

Area:

6.490

96

25.

568

- C

11:0

44.

633

- C

18:2

TT (9

, 12-

tran

s) 4

5.07

9 -

C18

:2T

(9-c

is,

12-t

rans

) 4

5.25

0 -

C18

:2T

(9-tr

ans,

12-

cis)

33

FAME (18:2 trans)

min44.4 44.6 44.8 45 45.2

pA

14

16

18

20

22

24

26

28

FID1 A, (090813\CHK-2F.D)

Area:

15.1509

Area:

6.3279

Area:

6.43129

44.

633

- C

18:2

TT (

9, 1

2-tr

ans)

45.

079

- C

18:2

T (9

-cis

, 12-

trans

)

45.

250

- C

18:2

T (9

-tra

ns, 1

2-ci

s)

34

Hard margarine

min10 15 20 25 30 35 40 45 50 55

pA

20

30

40

50

60

FID2 B, (GC-FTIR\080919\366.D)

Area: 0

.568315

Area: 1

.76648 A

rea: 195.537

Area: 3

.70447

Area: 4

8.169 A

rea: 8.47994

Area: 3

85.024

Area: 2

72.04

Area: 4

.21389

Area: 3

5.3004

Area: 0

.237679

Area: 2

.80935 A

rea: 7.33718

Area: 0

.91372 A

rea: 0.372423

17.

424

- C

8:0

22.

163

- C

10:0

24.

749

- C

11: 0

27.

367

- C

12:0

32.

423

- C

14:0

34.

822

- C

15:0

37.

162

- C

16: 0

38.

242

- C

16:1

T (9

-tra

ns)

39.

331

- C

17:0

41.

485

- C

18: 0

42.

300

- C

18:1

T (6

-tra

ns)

42.

425

- C

18:1

T (9

-tra

ns)

42.

472

- C

18:1

T (1

1-tr

ans)

43.

792

- C

18:2

TT (

9, 1

2-tr

ans)

45.

418

- C

20:0

47.

115

- C

18:2

T (9

-cis

, 11

-tra

ns)

49.

093

- C

22:0

52.

597

- C

24:0

35

Hard margarine

min25 30 35 40 45

pA

22

24

26

28

30

32

34

36

38

FID2 B, (GC-FTIR\080919\366.D)

Area: 1

95.537

Area: 0

.237679

Area: 3

.63294

Area: 3

.72232

24.

749

- C

11:0

43.

792

- C

18:2

TT (

9, 1

2-tr

ans)

44.

191

- C

18:2

T (9

-cis

, 12

-tra

ns)

44.

439

- C

18:2

T (9

-tra

ns,

12-c

is)

36

Hard margarine

min42 42.5 43 43.5 44 44.5 45 45.5

pA

22

24

26

28

30

32

34

36

38

FID2 B, (GC-FTIR\080919\366.D)

Area: 4

.21389 A

rea: 35.3004

Area: 0

.237679

Area: 2

.80935

42.

300

- C

18:1

T (6

-tran

s)

42.

425

- C

18:1

T (9

-tran

s) 4

2.47

2 -

C18

:1T

(11-

tran

s)

43.

792

- C

18:2

TT (

9, 1

2-tr

ans)

45.

418

- C

20:0

trans

trans

trans

37

Hard margarine

min42 42.5 43 43.5 44 44.5 45

pA

22

24

26

28

30

32

FID2 B, (GC-FTIR\080919\366.D)

Area: 0

.237679

Area: 3

.62988 A

rea: 3.51442

43.

792

- C

18:2

TT (

9, 1

2-tra

ns)

44.

187

- C

18:2

T (9

-cis

, 12-

trans

)

44.

434

- C

18:2

T (9

-tra

ns,

12-c

is)

38

Corn oil and 18:3 trans

39

Olive oil and 18:3 trans

40

Points to note (1)

Definition of “0”Saturated fatty acids ≤0.5/100 gTrans fatty acids ≤0.3/100 g

Limit of detection of saturated fatty acids and trans fatty acids should be better than 0.5 g/100 g and 0.3 g/100 g respectively

41

Points to note (2)

For prepackaged product with “Free of sat fat”claim:

Sum of sat and trans fat ≤0.1/100 g

Limit of detection of saturated fatty acids and trans fatty acids should be better than 0.05 g/100 g respectively.

42

Points to note (3)

Availability of FAME standards for calibration is crucial to this test method.

Insufficient FAME standards wouldunderestimate the level of saturated fat and trans fat.

43

Availability of FAME standards

Single and/or mixed FAMEs are available from different suppliers, including:

1. Supelco2. Fluka3. Nu-check Prep4. Chem Service5. Sigma

44

Proficiency test

FAPAS

AOAC

LGC

45

Thank You