Methods for the fatty acid analysis and their confirmation

Methods for the fatty acid

analysis and their confirmation

University of the Basque Country

(UPV-EHU)

Specialist course on Lipids in Ruminants:

N. Aldai, J.K.G. Kramer, P. Delmonte

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Vitoria-Gasteiz

European Green Capital 2012

Research Group

www.ehu.es/lactiker

Quality and Safety of Foods

from Animal Origin

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• Analytical methods are tools we use

to obtain results.

• The results are only as good as the total sum of all

our mistakes.

• Only accurate analytical results provide the basis for

meaningful interpretations.

Lipid research - remember

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Outline

• Lipid extraction

• Derivatization (methylation)

• Fatty acid methyl ester (FAME) purification

• Normal GC/FID analysis & its limitations:

separations, quantification, peak ID, …

• Confirmatory methods:

Silver ion fractionation (Ag+-SPE)

GC-online reduction x GC (GC-OR x GC)

GC/MS

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Liquid

solvent extraction

direct methylation

methylation Freeze-dry

& pulverize

FAME

Several methods:

Homogenize

solvent extraction methylation

solvent extraction methylation

direct methylation Tissue

solvent extraction

direct methylation

methylation

Freeze-dry

& pulverize direct methylation

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Type of lipid structures common in ruminant samples and their

relative amounts (% of total lipids)

Samples

TG

CLA

CE

O-acyl

PL

O-alkenyl

PL* N-

acyl

FFA

Milk 98 0.7 0.2 0.5 - 0.2 <0.1

Meat 70 0.7 0.5 20 4 4 <0.1

Blood 35 0.7 5 50 3 3 1

Rumen

fluids/feces

10-20 0.2 - 1 - <0.1 80**

* Glycerol-O-CH=CH-R ** Salts and complex glycolipids

Nature of sample

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Extraction – nature of sample

Folch et al. (1957)

(JBC 226: 497)

Bligh & Dyer (1959) (Can.J.Biochem.Physiol. 37: 911)

Solvent systems that include chloroform:

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• Hexane or heptane/isopropanol (Hara & Radin, 1976)

• Dichloromethane/methanol (Marmer & Maxwell, 1981)

Note:

- Hexane will only extract neutral lipids, not phospholipids (PL).

- Suitable for sample types that consist mainly of TG (vegetable oils,

adipose tissue).

- Animal tissues that contain PL

require polar solvent extraction.

muscle

backfat

Extraction– other solvent mixtures

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Derivatization

1) Acid-catalyzed methylation

2) Base-catalyzed methylation

3) Combining acid- & base-methylations

4) Direct methylation

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Derivatization

Suitability of catalysts for the methylation of specific lipid classes

DAM: diazomethane. Y: catalyst suitable; No: catalyst not suitable; L, longer reaction times &

generally higher temperatures are required.

(Cruz-Hernandez et al., 2006)

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Derivatization

• Quantitative: methylation of all common lipid classes including:

FFA, O-acyl (esters, glycosides), N-acyl (sphingomyelin), alk-1-enyl

ethers (plasmalogenic lipids).

Example:

• 5% (w/v) solution of anhydrous methanolic HCl:

Methylation of all lipids is complete in 1h at 80ºC.

Bubbling dry HCl gas into anhydrous methanol (Stoffel et al., 1959).

Adding acetyl chloride into dry methanol (Lepage & Roy, 1986).

Commercial 0.5N or 3N methanolic HCl preparations can be purchased (Sigma-Aldrich).

1) Acid-catalyzed methylation:

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Derivatization

Major disadvantages:

• Isomerization of c,t-, t,c- and c,c- to t,t-CLA isomers (Kramer et al., 1997).

• Formation of methoxy artifacts depending on sample type (Yurawecz et

al., 1994; Kramer et al., 1997).

• Lowering the methylation temperature to 60ºC (Park et al., 2001) or room

temp (Werner et al., 1992), decreases CLA isomerization somewhat (Park

et al., 2001), but under these milder conditions methylation may not be

complete (Kramer et al., 1997; Cruz-Hernandez et al., 2006).

1) Acid-catalyzed methylation (cont.):

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Derivatization

• Not-quantitative: converts only acyl moieties to

FAME, but not FFA, N-acyl lipids and alk-1-enyl

ethers (Kramer et al., 1997; Cruz-Hernandez et al., 2004, 2006).

Example:

• NaOCH3/methanol (0.5N methanolic base #33080, Sigma-Aldrich)

Rapid methylation (15min at 50ºC) without CLA isomerization

(Kramer et al., 1997).

2) Base-catalyzed methylation:

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Derivatization

Option A:

• Perform the two methylations in sequence.

Option B:

• Perform the 2 methylations separately and merge the results.

3) Combining acid- & base-methylations:

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Derivatization

Option B: Use acid-catalyzed results for quantitative purposes and

correct specific regions, such as CLA region, from the base-

catalyzed results (Cruz-Hernandez et al., 2006; Kramer et al., 2008).

Products of acid- and base-catalysts permits the identification of acyl

and plasmalogenic lipids.

3) Combining acid- & base-methylations (cont.):

X-CH2-O-CH=CHR

(alk-1-enyl ether lipids)

Lipid

Structure

NaOCH3

X

RCOOCH3

(FAME)

No

reaction

Base

catalyzed

X-CH2-O-OCR

(acyl lipids)

CH3OH/H2SO4

Acid

catalyzed

RCOOCH3

(FAME)

RCH2CH(OCH3)2

(DMA)

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Derivatization

Partial GC chromatogram of total sheep lipids methylated using a

base- or an acid-catalyzed procedure

base

acid

9c-18:1

DMA

18:2 DMA

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Derivatization

• Direct transesterification of the lipids, without prior extraction, with

acid or base catalysts.

Note of caution:

- Analysis of different lipid classes is not possible.

- Information is limited since it provides only the total FAME

composition.

- A base catalyst does not methylate a number of lipid classes.

4) Direct methylation: quick

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• Rumen digesta samples are unique in that

they contain high levels of FFAs.

Options:

Direct saponification (5% NaOH or 0.5M NaOCH3 in ethanol)

and :

1) acid-catalyzed methylation (HCl in methanol) or

2) methylation with trimethylsilyl-diazomethane (TMS-DAM), if

the sample contains acid labile components.

Derivatization– rumen samples

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FAME purification / clean up

• Examine the FAMEs for purity & completeness of methylation

(i.e., digesta & grass samples)

• By TLC (Thin Layer Chromatography) silica plates:

… will depend on the sample type

CE TG FFA C PL

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FAME purification / clean up

• Examine the FAMEs for purity & completeness of methylation

• By SPE (Solid Phase Extraction) silica cartridges :

… will depend on the sample type

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• Separate specific lipids: polar & neutral lipids, polar lipid classes,

3D separations of all lipid classes (Kramer et al., 1983), …

• With silver ion (Ag+-):

fractionation of FAMEs

TLC or SPE - other applications

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• The choice of GC column depends on the sample type but there is

general agreement among researchers to use:

100m highly polar (100% cyanopropyl polysiloxane) capillary

columns: SP-2560 (Supelco) or CP-Sil88 (Varian)

New 100m ionic liquid column (SLB-IL111, Supelco)

(significantly more polar)

Selection of a GC column:

GC/FID – improved separations

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• 100m highly polar column provides improved separation of:

18:1 region (Molekentin & Precht, 1995; Precht & Molkentin, 1996, 1997, 1999, 2000b, 2003;

Wolff et al., 1995; Ratnayake, 2001, 2004; Ratnayake et al., 2006, 2009; Kramer et al., 2001, 2002,

2008; Wolff & Precht, 2002; Cruz-Hernandez et al., 2004, 2006; Shingfield et al., 2006; Destaillats et

al. 2007)

16:1 region (Precht & Molkentin, 2000a; Kramer et al., 2008)

18:2 region (Precht & Molkentin, 1997, 2003; Kramer et al., 2008)

20:1-18:3 region (Wolf 1994; Precht & Molkentin, 1999; Kramer et al., 2008)

CLA region (Roach et al., 2000, 2002; Cruz-Hernandez et al., 2004, 2006; Shingfield et al.,

2006)

SP-2560 (Supelco) or CP-Sil88 (Varian):


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• Reduce sample load onto GC column if sample is too concentrated:

Adjustment of sample load:

11t

9c/10c/13t/14t

9c12c-18:2

18:0 concentrated

5t 4t

11c

14c

16t

9c13

t/8t

12

c 19:0

15c 11t1

5c 12c

16c

8t13

c/9t

12c

17cy

clo

13c

18:1 isomers

9t12

c

min 34 36 38 40

diluted

5t

10t 9t

6-8t 4t

13t/

14t/

6-8c

12

t

11t

9c

11c

13c

15t,10c

14c

16t

9c13

t/8t

13c

15c 9t12

c 11

t15c

12c 16c

17cy

clo

19:0 8t12

c/9c

12t


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Partial GC chromatogram of 9c-18:1

injected at different concentrations

Adjustment of sample load:


(Delmonte & Rader, 2007)

The relative retention time shifts

(peak maximum) with increased

sample load. However, the start of

the peak remains the same.

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• Depends largely on the nature of the samples. The analysis of

wide range of FAMEs is impossible under isothermal conditions.

• Temperature programs: incorporates specific needs to resolve

either short- or long-chain FAMEs, and at the same time include a

temperature plateau that provides maximum resolution of specific

FAME regions.

• Ruminant fats: at least 2 complementary GC temperature programs

are proposed with plateaus at 175ºC & 150ºC to improve the

resolution of the 18:1 & other FAME regions (Kramer et al., 2008).

GC conditions: temperature program vs isothermal?


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Details of the 3 temperature programs evaluated:

(Kramer et al., 2008; Lipids 43:259-273)


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Separation of 3 types of fat samples with different proportions of

cis- and trans-18:1 isomers affected by temperature

36 37 38

46 47 48 49 50

64 65 66 67

36 37 38

45 46 47 48 49

64 65 66 67

min 36 37 38

min 46 47 48 49

64 65 66 min

12t 10t

11t

11t

10t

13t/1

4t/6

-8c

10t

11t

11c/

15t

12t/6

-8c

12t/6

-8c

11c

12c

15t

11c

12c

10t

11t

9t 9t

10t

11t

9t

9c/1

0c/1

5t

12c

10t

9t

10t

11t

11c/

15t

11c/

15t

15t

11c

12c

15t

11c

12c

9c/1

0c/1

5t

9c/1

0c/1

5t

9c/10c

13t/14t 9c/10c

13t/14t

9c/10c

13t/14t

10t

11t

9t

175ºC

163ºC

150ºC

A B C

(Kramer et al., 2008)


9c/10c

13t/14t 9c/10c

13t/14t

9c/10c

13t/14t

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• Resulted in enhanced separation of:

- cis- & trans-FA isomers 14:1 to 20:1.

- 20:1 & 18:3 isomers.

- CLA isomers: the first GC column able to resolve the isomeric CLA

pair 9c,11t- and 7t,9c-CLA (previously achieved only by Ag+-HPLC).

Disadvantage:

• Elution of the SFAs in the region between the trans- & cis- FA isomers.

Ionic liquid column (SLB-IL111):

GC/FID – improved separations (Delmonte et al., 2011)

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Differentiation of missing

intermediate (10t,15c-18:2)

of 10t-shifted rumen bio-

hydrogenation pathway

from 11t,15c-18:2.

(Alves & Bessa, 2014;

Lipids 49:527-541)

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Differentiation of missing

intermediate (10t,15c-18:2)

of 10t-shifted rumen bio-

hydrogenation pathway

from 11t,15c-18:2 (cont).

GC/FID – improved separations (Alves & Bessa, 2014;

Lipids 49:527-541)

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Ag+-HPLC (DAD; 233nm): CLA isomer separation

min 30 35 40 45

12,14

11,13

9,11 11t,13c

9c,11t

7t,9

c

11,13 t,c c,t

10t,12c

8t,1

0c

7t,9c

10,12

9,11

9t,11c

0.7/58

0.5/69

0.7/73

0.5/39

1.4/74

CLA/RA

(%)

trans,trans

RA: rumenic acid

DAD: diode array detector


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Partial GC chromatogram of CLA isomers with double bond

positions from 6,8- to 13,15-18:2 using the ionic column.

(Delmonte et al., 2011)


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• Lack of authentic standards for some (or many) FAMEs.

Commercial suppliers:

• Nu-Chek Prep, Inc., MN, USA.

• Matreya, PA, USA.

• Larodan Fine Chemicals, Sweden.

• Sigma-Aldrich Co., USA.

• Need to use methods/references where ID has been verified Use

retention times & elution orders from literature.

Standards (STD):

GC/FID – peak ID

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• Trans FAME from a partially hydrogenated oil

• Vegetable & fish oils

• Simple synthesis: isomerization with P-toluene sulfinic acid or with I2, reduction with

hydrazene, conjugation with alkali, … (Delmonte et al., 2009).

• In-house mixtures:

Good STD:

#463 (Nu-Chek) + 21:0 + 23:0 + CLA mixture #UC-59M (Nu-Chek)

+ other LC-SFAs

Make your own standards:

GC/FID – peak ID

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• For large tissue portions gravimetric determination of the total lipids

extracted is just as accurate as adding large amounts of IS to the

whole sample, and is more economical.

• Ideally, IS should not be present in the sample and should be

within the range of FA chain length found in the sample.

1) Examine the total lipid profile without addition of an IS.

2) Finding a FA not present in ruminant fat is virtually impossible,

therefore a compromise is necessary.

Chose one with less interference.

When / which to add?

GC/FID – quantification (IS)

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TAG, FAME or FFA of 13:0, 15:0, 17:0, 19:0; 21:0, & 23:0.

• 13:0 – reasonable. Present in only minor amounts.

• 15:0, 17:0 – not recommended. Present in animal fats and elute in a

region that is already crowded with many other FAME isomers.

• 19:0 – not recommended. Overlaps with 15c-18:1 and some t,t-18:2.

• 21:0 – not recommended. Elutes among the CLA isomers.

• 23:0 – reasonable. Present only in trace amounts and well resolved

on SP-2560 & ionic columns, but there may be solubility problems.

Most commonly used IS:

GC/FID – quantification (IS)

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Silver ion fractionation (Ag+-SPE)

Confirmatory methods

• Rapid method to confirm structure based on number of double

bonds & geometric configuration of FAME fractions.

Kramer et al. (2008)

• Use Ag+-SPE cartridges with glass casings.

Belaunzaran et al. (2014, 2016)

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Confirmatory methods GC program:

45-175-215ºC (Bravo-Lamas et al., 2016)

min24 26 28 30 32 34

STD

GLC463 14:0

15:0

16:0

17:0

18:0

10c-

15:1

9c-1

4:1

9c-1

6:1

9t-1

6:1

10c-

17:1

SFA

14:0

-6M

e14

:0-4

Me

iso

-15:

0

15:0

14:0

iso

-16:

0 16:0

16:0

-6M

e16

:0-8

Me

17:0

18:0

iso

-17:

0

ai-1

7:0

“9c”

17:0

-12M

e

Ph

y

iso

-18:

0

ai-1

5:0

16:0

-2M

e

?

lamb

backfat

14:0

-8M

e

14:0

-10M

e

14:0

-2,6

di-

Me

9c-1

4:1 15

:0-8

Me

15:0

-4M

e? 9c

-15:

18c

-15:

1?16:0

-2M

e

16:0

-4M

e16

:0-1

2Me

ai- 17:0

ai-1

5:0

?

7c-/

13t-

16:1

iso

-17:

09t

-16:

1

9c-1

6:1

14t-

17:1

/12c

-16:

1

17:0

17:0

-12M

e

Ph

y

9t-1

7:1? is

o-1

8:0 9c

-17:

17c

-17:

15c

-17:

1

11c-

17:1is

o-1

6:0

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45-175-215ºC

min35 36 37 38 39 40 41 42

lamb

backfat

trans

cis

dienes

STD

GLC463

SFA11

t-

9t-

18

:0

6c

-

9c

-

12

t-1

3t-

/14

t-

6t-

/ 8t-

5t-

11c

-

12

c-

19

:0

7c

-19

:1

11t,

15

c-

11t-

19

:1

9c

,13

t/

8t,

12

c-

17

-cy

clo

16c-/9c,12t-

t,t-16

t-/1

4c

-

13

c-

15

c-

18

:2 n

-6/

9c

-19

:1

9c-/10c-

4t-

11t-

19

:0

9t-

10t- 15t-

11c

-

t,t-

9t ,

12

t-1

8:2

18

:0

17

-cy

clo

19

:0

i-1

9:0

ai-

19

:0“18:0”

“18:0”

12

t-13t-/14t-

6t-

/ 8t-

5t-

9t-

10t-

15

t-

16

t-

4t-

11t-

11t-

19

:1

“11t-”

“11t-”

12

c-

13

c-

11c

-

15

c-

14

c-

16

c-

9c

-19

:1

9c

-/1

0c

-

“9c-” t,

t-

t,t- t,t-

9c,13t-/

8t,12c-

8t1

3c

-8

t13

c-

9c

,12

t-

11t,

15

c-

18

:2 n

-6

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45-175-215ºC

min35 36 37 38 39 40 41 42

lamb

backfat

trans

cis

dienes

STD

GLC463

SFA11

t-

9t-

18

:0

6c

-

9c

-

12

t-1

3t-

/14

t-

6t-

/ 8t-

5t-

11c

-

12

c-

19

:0

7c

-19

:1

11t,

15

c-

11t-

19

:1

9c

,13

t/

8t,

12

c-

17

-cy

clo

16c-/9c,12t-

t,t-16

t-/1

4c

-

13

c-

15

c-

18

:2 n

-6/

9c

-19

:1

9c-/10c-

4t-

11t-

19

:0

9t-

10t- 15t-

11c

-

t,t-

9t ,

12

t-1

8:2

18

:0

17

-cy

clo

19

:0

i-1

9:0

ai-

19

:0“18:0”

“18:0”

12

t-13t-/14t-

6t-

/ 8t-

5t-

9t-

10t-

15

t-

16

t-

4t-

11t-

11t-

19

:1

“11t-”

“11t-”

12

c-

13

c-

11c

-

15

c-

14

c-

16

c-

9c

-19

:1

9c

-/1

0c

-

“9c-” t,

t-

t,t- t,t-

9c,13t-/

8t,12c-

8t1

3c

-8

t13

c-

9c

,12

t-

11t,

15

c-

18

:2 n

-6

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45-175-215ºC

min35 36 37 38 39 40 41 42

lamb

backfat

trans

cis

dienes

STD

GLC463

SFA11

t-

9t-

18

:0

6c

-

9c

-

12

t-1

3t-

/14

t-

6t-

/ 8t-

5t-

11c

-

12

c-

19

:0

7c

-19

:1

11t,

15

c-

11t-

19

:1

9c

,13

t/

8t,

12

c-

17

-cy

clo

16c-/9c,12t-

t,t-16

t-/1

4c

-

13

c-

15

c-

18

:2 n

-6/

9c

-19

:1

9c-/10c-

4t-

11t-

19

:0

9t-

10t- 15t-

11c

-

t,t-

9t ,

12

t-1

8:2

18

:0

17

-cy

clo

19

:0

i-1

9:0

ai-

19

:0“18:0”

“18:0”

12

t-13t-/14t-

6t-

/ 8t-

5t-

9t-

10t-

15

t-

16

t-

4t-

11t-

11t-

19

:1

“11t-”

“11t-”

12

c-

13

c-

11c

-

15

c-

14

c-

16

c-

9c

-19

:1

9c

-/1

0c

-

“9c-” t,

t-

t,t- t,t-

9c,13t-/

8t,12c-

8t1

3c

-8

t13

c-

9c

,12

t-

11t,

15

c-

18

:2 n

-6

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45-175-215ºC

min35 36 37 38 39 40 41 42

lamb

backfat

trans

cis

dienes

STD

GLC463

SFA11

t-

9t-

18

:0

6c

-

9c

-

12

t-1

3t-

/14

t-

6t-

/ 8t-

5t-

11c

-

12

c-

19

:0

7c

-19

:1

11t,

15

c-

11t-

19

:1

9c

,13

t/

8t,

12

c-

17

-cy

clo

16c-/9c,12t-

t,t-16

t-/1

4c

-

13

c-

15

c-

18

:2 n

-6/

9c

-19

:1

9c-/10c-

4t-

11t-

19

:0

9t-

10t- 15t-

11c

-

t,t-

9t ,

12

t-1

8:2

18

:0

17

-cy

clo

19

:0

i-1

9:0

ai-

19

:0“18:0”

“18:0”

12

t-13t-/14t-

6t-

/ 8t-

5t-

9t-

10t-

15

t-

16

t-

4t-

11t-

11t-

19

:1

“11t-”

“11t-”

12

c-

13

c-

11c

-

15

c-

14

c-

16

c-

9c

-19

:1

9c

-/1

0c

-

“9c-” t,

t-

t,t- t,t-

9c,13t-/

8t,12c-

8t1

3c

-8

t13

c-

9c

,12

t-

11t,

15

c-

18

:2 n

-6

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45-175-215ºC

min35 36 37 38 39 40 41 42

lamb

backfat

trans

cis

dienes

STD

GLC463

SFA11

t-

9t-

18

:0

6c

-

9c

-

12

t-1

3t-

/14

t-

6t-

/ 8t-

5t-

11c

-

12

c-

19

:0

7c

-19

:1

11t,

15

c-

11t-

19

:1

9c

,13

t/

8t,

12

c-

17

-cy

clo

16c-/9c,12t-

t,t-16

t-/1

4c

-

13

c-

15

c-

18

:2 n

-6/

9c

-19

:1

9c-/10c-

4t-

11t-

19

:0

9t-

10t- 15t-

11c

-

t,t-

9t ,

12

t-1

8:2

18

:0

17

-cy

clo

19

:0

i-1

9:0

ai-

19

:0“18:0”

“18:0”

12

t-13t-/14t-

6t-

/ 8t-

5t-

9t-

10t-

15

t-

16

t-

4t-

11t-

11t-

19

:1

“11t-”

“11t-”

12

c-

13

c-

11c

-

15

c-

14

c-

16

c-

9c

-19

:1

9c

-/1

0c

-

“9c-” t,

t-

t,t- t,t-

9c,13t-/

8t,12c-

8t1

3c

-8

t13

c-

9c

,12

t-

11t,

15

c-

18

:2 n

-6

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GC - online reduction x GC (GC-OR x GC)


Inj. port FID

1D 2D 30-100 m 1-4 m

Modulator

CO

LD

CO

LD

HO

T

HO

T

GC oven

Reducer

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GC–OR x GC

…………..

16:0, 16:1, 16:3, 16:4

18:0, 18:1, 18:3, 18:4

20:0, 20:1, 20:3, 20:4, 20:5, …

22:0, 22:1, 22:2, 22:3, 22:4, …

24:0, 24:1, …

…………..

1D separation Reduction 2D separation

Pd capillary reducer

(H2, carrier gas)

…………..

16:0

18:0

20:0

22:0

24:0

…………..

∆T

Inj. port

Principle:

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GC–OR x GC

Reference mixture GLC463 (Nu-chek Prep., Inc.)


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GC–OR x GC

Menhaden fish oil sample


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GC–OR x GC

Menhaden fish oil sample (14:0 to 18:3n-3 region)


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GC–OR x GC

Bovine muscle (solvent front-16:0 region)

9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 min 0.00

0.15

0.30

0.45

se

c

0.60

14:0

15:0 i-

15:0

ai-

15:0

i-

16:0

16:0

13:0

i-

14:0

14:0

15:0 i-

15:0

ai-

15:0

i-

16:0 16:0

c9-

14:1 17:0

ai-

17:0 i-

17:0 12:0

15:1

i-

13:0

ai-

13:0

i-14:0 16:1 15:1

pA

1.5

2.5

3.5

min 9 10 11 12 13 8

16:0 14:0 15:0

i-15:0 ai-

15:0 i-16:0

13:0

i-14:0

12:0

i-13:0

ai-

13:0

c9-14:1

15:1

(17:0)

ai-

17:0

i-

17:0

16:0 (15:0)

(i-15:0) ai-

15:0

(i-16:0)

15:1 i-14:0 (14:0)

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GC – QTOF/MS


• Column: SP2560, 100m x 0.25mm

• Carrier: Helium at 1.1mL/min constant flow

• Elution temperature: 180ºC isothermal

• Ionization: chemical ionization with isobutane (CI+)

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GC–QTOF/MS

22:0

22:1

20:2

(n-9)

20:3

20:4 22:1

20:3 20:3

20:3 20:2

22:0

20:4

3 x10

0

2

4

6 +CI BPC(150.0000-450.0000)

4 x10

0.5

1.5

+CI EIC(321.2788)

3 x10

0.5

1.5

2.5 +CI EIC(323.2945)

3

x10

1

3

+CI EIC(353.3414)

3 x10

2

6

+CI EIC(355.4056

4 x10

1

3

+CI EIC(319.3091)

Counts vs. Acquisition Time (min)

86 88 90 92 94 96 98 100 102 104 106 108 110 112 114 116

(n-6)

20:3

(n-3)

20:3

TIC

Menhaden fish oil

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GC–QTOF/MS

Bovine muscle (16:0 to 18:0 region)

17:0

18:1 c9 c11 c12 c13 c15 c14

t11

17:1 c9

c11

DMA FAME

4 x10

0

1 +CI EIC(283.3056)

4 x10

4

+CI EIC(285.3215)

3 x10

0

5

+CI EIC(281.3264)

i-17:0 ai-17:0

18:0

(18:0)

0 4 x10

0

1

+CI EIC(269.2889)

5 x10

0

2

+CI EIC(271.3058)

4 x10

0

1

+CI EIC(299.3390)

3 x10

0

2

+CI EIC(267.2682)

4 x10

0

1

2

+CI BPC(150.0000-450.0000)

Counts vs. Acquisition Time (min) 32 33 34 35 36 37 38 39 40 41 42 43 44

18:0 16:0

16:0

18:0 i-18:0

16:1 c9 c7 c11 c10 c12 c13

17:1 c9 c11

trans

i-18:0

17:0 i-17:0 ai-17:0 c9 (c7)

c11 c10 trans

16:1 18:0 18:1

c9

c11 (c14/c15)

17:1 c9

c11 c12 t11 c13

(c13)

17:0

17:0 TIC

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1) Perform a complete lipid extraction (requires

chloroform).

2) Methylate using both acid & base catalysts.

3) Purify FAMEs when necessary.

4) Apply proper sample load (concentration) onto GC

column.

5) Use 100m columns (SP2560, ionic).

Final remarks

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6) Ensure good separations of 16:1, trans-18:1, 18:3

and CLA regions, and specific critical pairs:

iso17:0 // 8t- & 9t-16:1

anteiso17:0 // 7c- & 9c-16:1

10t-18:1 // 11t-18:1

10t,15c-18:2 // 11t,15c-18:2

21:0 // CLA

EPA // 24:0, …

Final remarks (cont.)

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7) Remember 9c,11t- is not resolved from 7t,9c-CLA

using SP-2560 or CP Sil88 columns. However, these

and other CLA isomers can be resolved using ionic

GC column or Ag+ -HPLC.

8) Deal with plasmalogens and sphingomyelin in meat

and blood products.

9) Use confirmatory methods to establish peak ID.

Final remarks (cont.)

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-Sebedio & Christie, W.W. (1998) Trans Fatty Acids in Human Nutrition.

The Oily Press, Dundee, Scotland.

-Christie, W.W. (2003) Lipid Analysis. Isolation, Separation, Identification and Structural

Analysis of Lipids. 3rd Ed. The Oily Press, Bridgwater, England.

-Cruz-Hernandez, C., J.K.G. Kramer, J. Kraft, V. Santercole, M. Or-Rashid, Z. Deng,

M.E.R. Dugan, P. Delmonte, and M.P. Yurawecz. (2006) Systematic analysis of trans and

conjugated linoleic acids in the milk and meat of ruminants. Pages 45-93 in Advances in

Conjugated Linoleic Acid Research, Volume 3. M.P. Yurawecz, J.K.G. Kramer, O.

Gudmundsen, M.W., Pariza, and S. Banni, eds. AOCS Press, Champaign, IL.

-Mossoba, M.M. & Kramer, J.K.G. (2009) Official Methods for the Determination of Trans

fat. 2nd Ed. AOCS Press, Urbana, Illinois.

Bibliography

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Bibliography (cont.)

-Ratnayake, W.M.N. & Cruz-Hernandez, C. (2009) Analysis of trans fatty acids of partially

hydrogenated vegetable oils and dairy products. Pp. 105-146 in Trans Fatty Acids in

Human Nutrition, 2nd Ed. F. Destaillats, J.L. Sebedio, F. Dionisi & J.M. Chardigny, eds. The

Oily Press, Bridgwater, UK.

-Aldai N; Kramer JKG; Cruz-Hernández C; Santercole V; Delmonte P; Mossoba MM;

Dugan MER. (2012) Appropriate Extraction and Methylation Techniques for Lipid Analysis.

Pp. 249-290 in Fats and Fatty Acids in Poultry Nutrition and Health (Eds., G. Cherian & R.

Poureslami), Nottingham Press, UK.

http://www.cyberlipid.org

http://lipidlibrary.aocs.org

http://library.med.utah.edu/NetBiochem

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Research group www.ehu.es/lactiker

Thank you for the attention

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Research group www.ehu.es/lactiker

Documents

Methods for the fatty acid analysis and their confirmation