Analysis of Fatty Acids

AOAC Official Methods

969.33 996.06 991.39 994.15 985.21 etc Fatty acids in oils and fats Preparation methyl esters

Fat (total, saturated and unsaturated) GC-FID in foods Encapsulated fish oils cis- and trans-Octadecenoic isomers and general fatty acid composition trans-Fatty acid in margarines GC-FID GC-IR GC-FID

Typical analysis

Fat and fatty acids are extracted from food by hydrolytic method. Fat is extracted into ether, then methylated to fatty acid methyl esters (FAMEs). FAMEs are quantitatively measured by gas chromatography.

Typical analysis

Typical analysis

Fat Fat

Hydrolysis Hydrolysis

Methylated Methylated to to FAMEs FAMEs

GC-FID GC-FID (quantitation) (quantitation)

Typical analysis
Hydrolysis Weigh sample and pyrogallic acid into the Mojonnier flask

Typical analysis
Hydrolysis Add internal standard, ethanol and 8.3 M HCl.

Typical analysis
Hydrolysis Hydrolyse the sample at 70 80 oC

Typical analysis
Hydrolysis Remove flasks from water bath. Cool to room temperature.

Typical analysis
Extraction Add ethanol, Ether and shake.

10

Typical analysis
Extraction Centrifuge sample. (allow contents to sep. at least 1 h if centrifuge is not available)

11

Typical analysis
Extraction Decant the ether layer into a tube.

12

Typical analysis
Extraction Evaporate ether on water bath using nitrogen stream to aid in evaporation.

13

Typical analysis
Extraction Dissolve the residue in chloroform.

14

Typical analysis
Methylation Transfer mixture to a glass vial. Evaporate to dryness.

15

Typical analysis
Methylation Add 7% BF3 reagent and toluene.

16

Typical analysis
Methylation Seal vials with screwcap and heat them at 100 oC.

17

Typical analysis
Methylation Allow vials to cool to room temperature.

18

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

19

Typical analysis
Methylation Allow layers to separate and transfer top layer to another vial containing Na2SO4 (1 g).

20

Typical analysis
Methylation Top layer should contain FAMEs and internal standard.

21

Typical analysis
GC Determination Transfer to autosampler vial for GC analysis.

22

Fatty acids (saturated)

4:0 Butyric 6:0 Caproic 8:0 Caprylic 10:0 Capric 11:0 Undecanoic 12:0 Lauric 14:0 Myristic

23

Fatty acids (saturated)

15:0 Pentadecanoic 16:0 Palmitic 17:0 Margaric 18:0 Stearic 20:0 Arachidic 22:0 Behenic 24:0 Lignoceric

24

Definition

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

25

trans-Fatty acids
14:1 trans-Myristelaidic 16:1 trans-Palmitelaidic 18:1 trans 6-Petroselenic 18:1 trans-Elaidic 18:1 trans 11-Vaccenic 18:2 trans-Linolelaidic 18:2 trans 9-Linolelaidic 18:2 trans 12-Linolelaidic
26

trans-Fatty acids
18:2 cis-9, trans-11-Octadecadienic 18:2 Linoleic conjugated* 20:1 Eicosenic trans 11 22:1 13-trans Docosenic

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

27

trans-Fatty acids (18:3 trans)

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

28

pA

20

30

40

50

60

70

FAME (saturated and trans)

10 FID1 A, (090715\Q6F.D) 15 20 25 30 35 40 45 50 55 1A8.18 9 - C8:0 re a: 12 2. 22 2 1A2.61 0 - C4:0 re a: 84 . 1A4.6763 2 4 - C6:0 re 1 a: 94 .9 64 2 2A2.97 3 - C10 :0 re a: 14 8. 27 7 2 5.60 A r 1 - C11 :0 ea :5 24 .4 83 2A8.20 3 - C12 :0 re a: 14 6. 83 8 3A3.26 8 - C14 :0 re a: 16 3A4.63 3 - C14 :1T (9-tra ns ) 2. re 95.67 a: 3 A3r2 0 - C15 :0 58 ea .7 :1 78 83 3 . 3A7.96 8 - C16 :04 51 re a: 3A9.06 3 - C16 :1T (9-tra ns ) 15 re 4 a: 4A0.17 3 .-2 1 C17 :0 48 7 r ea .7 97 :1 2 52 .8 9 - C18 :0 4A2.28 re 3 3 a: 44 C18 :1T (6-tra ns 3.20 C18 :1T (9-tra ns A3.12 4A3.31 39 -4 -C18 :1T (11 -tran s ))) 16 re re a 4. a: : 5 29 42 0 3 4A4.67 C18 :2TT (9 , 12 -tran s ) 5 . re .9 53 5 a: 8 63 49 1 4A6.27 0 - C20 :0 .8 re 51 a: 4A7.11 1 -2 C20 :1T (11 -tran s ) re 1 a:4A8.03 7 - C18 :2T (9-c is , 11 -tran s ) 6 3. 4A8.33 46 5 re 3 - C18 :2T (10 -tran s , 1 2-c is ) re 0 6 a: a : .1 47 3. 58 68 4A9.95 2 - C22 :0 .5 8 4 9 re 97 6 5A0.74 0 - C22 :1T (13 -tran s ) a: 7 re 16 a: 4. 50 43 .2 15 4 5A3.54 1 - C24 :0 re a: 17 1. 15 9

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

29

min

pA

15 FID1 A, (090715\Q6F.D) 43 43.1

FAME (18:1 trans)

20

25

30 3. 12 9 - C18 : 1T (6-t ra ns ) A4 re a: 50 .3 56 3

35

40

45

ns a r t

43.2

4 3. 20 4 - C18 : 1T (9-t ra ns )

t s r an

43.3 3. 31 3 - C18 : 1T (11 -t ran s ) A4 re a: 42 .9 58 1

ns a r t

43.4

30

min

pA

15 FID1 A, (090715\Q6F.D) 46.75

20

25

30

35

40

45

50

ns a r t

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

47 47.25 47.5 47.75 48 48.25 48.5 min

7 . 1 1 1 - C 2 0 : 1 T (1 1 -t ra n s ) A4 re a: 50 .1 47 8

cis ns tra
8 . 0 3 7 - C 1 8 : 2 T (9 -c is , 1 1 -t ra n s ) A4 re a: 63 .8 49 6

ns a r t

8 . 3 3 3 - C 1 8 : 2 T (1 0 -t ra n s , 1 2 -c is ) A4 re a: 58 .5 97 7

cis

31

pA

15

20

25

30

35

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

FAME (18:2 trans)

25 25.568 Ar - C11:0 ea :4 97 .3 21 30 35 40 45 min - C18:2TT (9, 12-trans ) A44.633 re a - C18:2T (9-c is , 12-trans ) A45.079 - :C18:2T (9-trans , 12-c is ) 15 re A45.250 r ea : . 15 a: 6 09 6..3 2 4 97 09 96

32

pA

14 FID1 A, (090813\CHK-2F.D) 44.4 44.6 - C18:2TT (9, 12-trans ) A44.633 re a: 15 .1 50 9 44.8 45 - C18:2T (9-c is , 12 -tran s ) A45.079 re a: 6. 32 79 45.2 - C18:2T (9-trans , 12-c is ) A45.250 re a: 6. 43 12 9

FAME (18:2 trans)

16

18

20

22

24

26

28

33

min

pA

20 10 FID2 B, (GC-FTIR\080919\366.D) 15 20 25 30 35 40 45 50 55 min 7.367 - C12:0 A2 re a: 3 .7 04 47 2.423 - C14:0 A3 re a: 48 .1 6 4.82 2 - C15 :0 9 A3 re a: 8 .4 79 94 3 8 . 2 4 2 - C 1 6 : 1 T (9 -t ra n s ) 39.331 - C17:0 2.163 - C10:0 A2 re a: 1 .7 66 48 2.30 0 - C 12 8.:4 17 T2 ra ) T (1 2 5 (6 (9 1 -t-t ra nn ss )) A4 - -tC 1n 8s: 1 ra A4 re re a: a : 3 T (9 , 1 2 -t ra n s ) 4 .2 3.79 A4 21 - C 1 8 : 2 T 5. re 30 38 a: 04 9 0 . 5 24 1 8 C 2 0 : 0 A4 re 37 6 a: 7 28 9 4 1 5 - C 1 8 : 2 T (9 -c is , 1 1 -t ra n s ) A r 7 . .1 e a 09 3 :7 5 . 9 .093 337- C 2 2 : 0 A4 re 18 a: 0 .9 13 72 2.59 7 - C24 :0 A5 re a: 0 .3 72 42 3 7.424 - C8:0 A1 re a: 0 .5 68 31 5 24.7 A r4 9 - C 1 1 : 0 ea :1 95 .5 37 37.1 A r6 2 - C 1 6 : 0 ea :3 85 .0 24 4 1 . 4A 85 - C18:0 re a: 27 2 .0 4

Hard margarine

30

40

50

60

34

pA

22

24

26

28

30

32

34

36

38

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

Hard margarine

25 30 35 40 45 min 3 . 7 9 2 - C 1 8 : 2 TT (9 , 1 2 -t ra n s ) A4 re 4 4 . 1 9 1 - C 1 8 : 2 T (9 -c is , 1 2 -t ra n s ) aA: r0 4 4 . 4 3 9 - C 1 8 : 2 T (9 -t ra n s , 1 2 -c is ) eA .a 2r:e 33 a 7.:6 3 7.9 79 2 24 23 2

24.7 A r4 9 - C1 1 : 0 ea :1 95 .5 37

35

pA

22 42 FID2 B, (GC-FTIR\080919\366.D) 42.5 43 43.5 44 44.5 45 45.5 5. 41 8 - C20 : 0 A4 re a: 2. 80 93 5 3. 79 2 - C18 : 2TT (9 , 12 -t ran s ) A4 re a: 0. 23 76 79 2. 30 0 - C18 : 1T (6-t ra ns ) A4 re a: 4. 2. 42 5 - C18 : 1T (9-t ra ns ) 21 A4 38 4re 2. a:47 2 - C18 : 1T (11 -t ran s ) 9 35 .3 00 4

Hard margarine

24

26

28

30

32

34

36

38

ns a r t ns tra

ns a r t

36

min

pA

22

24

26

28

30

32

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

Hard margarine

42 42.5 43 43.5 44 44.5 45 min 3. 79 2 - C18 : 2TT (9 , 12 -t ran s ) A4 re a: 0. 23 76 79 4. 18 7 - C18 : 2T (9-c is , 12 -t ran s ) A4 re a: 3. 62 98 8 4 4. 43 4 - C18 : 2T (9-t ra ns , 12 -c is ) Ar ea :3 .5 14 42

37

Corn oil and 18:3 trans

38

Olive oil and 18:3 trans

39

Points to note (1)

Definition of 0 Saturated fatty acids 0.5/100 g Trans 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

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Points to note (2)

For prepackaged product with Free of sat fatclaim: 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.

41

Points to note (3)

Availability of FAME standards for calibration is crucial to this test method. Insufficient FAME standards would underestimate the level of saturated fat and trans fat.

42

Availability of FAME standards

Single and/or mixed FAMEs are available from different suppliers, including: 1. Supelco 2. Fluka 3. Nu-check Prep 4. Chem Service 5. Sigma
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Proficiency test
FAPAS AOAC LGC

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Thank You

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