methods for the fatty acid analysis and their confirmation
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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):
GC/FID – improved separations
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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
GC/FID – improved separations
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Partial GC chromatogram of 9c-18:1
injected at different concentrations
Adjustment of sample load:
GC/FID – improved separations
(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?
GC/FID – improved separations
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Details of the 3 temperature programs evaluated:
(Kramer et al., 2008; Lipids 43:259-273)
GC/FID – improved separations
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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)
GC/FID – improved separations
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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GC/FID – improved separations
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
GC/FID – improved separations
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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)
GC/FID – improved separations
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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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Confirmatory methods GC program:
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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Confirmatory methods GC program:
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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Confirmatory methods GC program:
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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Confirmatory methods GC program:
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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Confirmatory methods GC program:
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)
Confirmatory methods
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.)
(Delmonte et al., 2013)
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GC–OR x GC
Menhaden fish oil sample
(Delmonte et al., 2013)
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GC–OR x GC
Menhaden fish oil sample (14:0 to 18:3n-3 region)
(Delmonte et al., 2013)
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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
Confirmatory methods
• 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
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