Fractionation of cis and trans Fatty Acid Isomers
for Food Samples Analysis with Hexane/Acetone
on Silver SPE Material
Craig Aurand, Olga Shimelis, An Trinh, Len Sidisky, and Michael Ye
Supelco, Div. of Sigma-Aldrich, Bellefonte, PA 16823
Toshiro Kaneko
Sigma-Aldrich Japan K.K, Tennoz Central Tower 4F,
2-2-24 Higashi Shinagawa, Shinagawa-ku Tokyo 140-0002, JAPAN
Noriko Shionoya, Tomoji Igarashi, Yoshiaki Hirata, and Hirofumi Goto
Japan Food Research Laboratories, 4-5-13,
Osu, Naka-ku, Nagoya, 460-0011, JAPAN
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T406099
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Introduction
It is well-known when vegetable oils are hardened into
shortenings and margarine, some of the naturally occurring cisfats will be converted into trans-fats. Shortenings and margarine
are used in commercially baked products and fast foods for
flavor preservation and spoilage prevention. As study showed,
trans-fats will adversely affect blood lipids levels – increase LDL
(“bad”) cholesterol, thus increasing risk of coronary heart
disease. It was estimated that replacing trans-fats with natural
vegetable oils could prevent up to 100,000 premature deaths
annually (http://www.hsph.harvard.edu/reviews/transfats.html)
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Types of Fatty Acids
Stru c tu re
C o m m o n So u rc e s
Sa tu ra te d F a tt y A c id s
( n o d o u b le b o n d s)
P a lm k e r ne l, P a l m oi l, C oc on ut
(tr o pic a l oils ), B u tte r , H y dr o ge na te d
O
HO
Oils a nd S h or te nin gs
M o n o a n d Po l yu n sa tu ra t e d F a tt y A c id s
c is
O
HO
Flui d/Li qui d oils s uc h a s S o y be a n,
C a nola , Oliv e , S un flo we r a nd
C or n oils .
Tra n s F a tt y A c id s
O
t r an s
HO
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H e a lth Ef f e c ts
R a is e LD L c h ole s te r ol, a nd
inc r e a s e r is k o f
c a r diov a s c ula r dis e a s e
( ≥ 1 c is d o u b le b o n d )
Lo we r LD L c h ole s te r ol,
a s s oc ia te d wit h r e d uc e d
r is k of c a r diov a s c ula r
dis e a s e .
( ≥ 1 tra n s d o u b le b o n d )
P a r tia ll y H ydr oge na te d Oils ,
S hor te nin gs , Ma r ga r i ne s , a nd
C hips
Rai se L DL ch o l est ero l l i k e
satu rat ed fat, m a y al so l o w e r
HDL . A sso ci at ed w i th
i n cre as ed ri s k o f
ca rd i o va scu l ar d i s ea se an d
p o ssi b l y typ e I I d i ab ete s.
Introduction (contd.)
US FDA issued a regulation that requires food manufacturers
to list the TRANS FAT on the Nutrition Facts panel of foods,
effect on January first of 2006.
AOAC has an analytical method (AOAC 996.06) for “Fat
(Total Saturated and Unsaturated) in Foods”. The method
specifies a sample is prepared by saponification, liquid-liquid
extraction and trans-esterification and analyzed on a 100 m
GC column.
However, there are several cis and trans monoenes that
overlap under the GC conditions, reducing the accuracy of the
method (see following chromatograms).
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The Limit of Current Analytical Method
Overlap of trans/cis monounsaturated octadecenoic fatty
acids on the GC chromatogram
Extracted Fat from Margarine
18:2
18:1
16:0
18:0
18:3
20
30
40
Time (min)
9c
6t 9t
10t
11t 12t
35.0
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13t
6c
7c
10c 11c
50
Peaks from 18:1
trans and cis isomers
overlap
12c
13c
36.0
37.0
Time (min)
38.0
Introduction (contd.)
Silver chromatography has traditionally been used to separate
saturated and unsaturated compounds. W. Christie first
introduced Ag-Ion SPE to separate fatty acid methyl esters by
the degree of unsaturation in 1989. Japanese Food Research
Laboratory developed a method on an in-house prepared Ag
SPE to separate fatty acid isomers by cis/trans configuration up
to trienes in 2005. Thus it opens a door for a pre-separation of
cis/trans isomers prior to GC analysis.
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Results and Discussion
Properties of Ag-Ion SPE from Supelco
• Stable silver-loading
- No silver bleed when common organic
solvents are used
• Stable color
- No effect of light-exposure on the
Ag-Ion packing material
- Long shelf-life
• Capacity of one 750 mg SPE tube –
up to 1 mg of total FAMEs
• Reproducible resolution of cis/trans
monoene FAMEs
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Interaction Mechanism
• Charge – transfer
• Unsaturated compound –
electron donor, Silver - electron
O
acceptor
• One silver = 2 double bonds OR
• One silver = one double bond
and one carboxyl group
• Cis-fatty acid isomers form
stronger complexes than trans
• Conjugated polyenes form less
stable complexes
• Strength of interactions
increases with the number of
double bonds
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OCH 3
C4H 8
SPE
Support
SO3 Ag +
C11H 23
Charge-transfer complex
between Ag + and unsaturated bond
Ag-Ion SPE Method for cis/trans Separation
1. Condition
2. Equilibrate
3. Sample Load
4.
5.
6.
7.
8.
4 mL acetone
4 mL hexane
1 mL of 1 mg/mL FAMEs in hexane at
5 mL/min.
Elution Fraction 1 6 mL hexane:acetone (96:4 v/v)
Elution Fraction 2 4 mL hexane:acetone (90:10 v/v)
Elution Fraction 3 4 mL acetone
Elution Fraction 4 4 mL acetone:acetonitrile (97:3)
Evaporate fractions, reconstitute in hexane for GC injection
•Fraction 1 targets saturated FAMEs and trans monoenes
•Fraction 2 targets cis monoenes and T/T dienes
•Fraction 3 targets C/C, C/T, T/C dienes, most triens
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Moisture Exposure Testing of Ag-Ion SPE
• Cartridge was washed with 1 mL water prior to conditioning step to simulate
residual moisture effect on separation.
• Loading and elution were done under normal phase conditions
• Fractionation of cis/trans isomers was not affected by residual moisture
Margarine Fat Sample before Separation
16:0
20
Fraction 1 after SPE
18:1
18:2
18:0
30
40
Time (min)
18:1 trans
20
30
40
Time (min)
18:1 cis
Fraction 2 after SPE
20
30
40
Time (min)
10
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Fractionation of the Standard FAME Mixture
Standard sample, total FAMEs at 1 mg/mL
Standard Mixture
18:1
18:2
14:0
18:0
16:0
6
18:3
8
10
12
14
16
18
20
Time (min)
18:1 t
SPE Fraction 1
6
8
10
12
14
16
18
20
Time (min)
18:1 c
SPE Fraction 2
7.0
8.0
9.0
10.0
11.0
12.0
13.0
Time (min)
14.0
15.0
16.0
17.0
18.0
19.0
20.0
SPE Fraction 3
6
8
10
12
14
16
18
20
Time (min)
SPE Fraction 4
6
8
10
12
14
Time (min)
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18
20
GC Conditions using SP-2560, 75 m x 0.18 mm I.D.
Use of shorter GC column (SP-2560, 75 m) with hydrogen
carrier gas significantly decreased the time required for
the analysis.
oven: 180 °C, isothermal
inj.: 220 °C
det.: FID, 220 °C
carrier gas: hydrogen, 40 cm/sec. at 180 °C
injection: 0.5 µL, 100:1 split
liner: 4 mm I.D., split, cup design
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Results of Fractionation of Standard Mix of FAMEs
(% recovery)
Elution
1
18:1t
18:1c
100
98.1
0.4
1.90
99.60
18:2tt
18:2 c/t
18:2cc
18:3ttt
100
50
100
18:3
6 mL
Hexane:acetone (96:4)
2
18:0
4 mL
Hexane:acetone (90:10)
3
4 mL Acetone
4
4 mL
100
Acetone:acetonitrile
(97:3)
TOTAL
50
100
100
100
100
100
Note: more polar elution solvent is needed to completely elute 18:3ccc isomer.
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100
40
55
100
95
Fat Extraction Procedure for Potato Chips
• Ground and extract with 4 x 4 mL petroleum ether
• Evaporated and reconstituted into toluene
• Trans-esterified using 7% BF3/MeOH
• Re-extracted into hexane after completion of reaction, dried
over anhydrous Na2SO4
• Loaded into Ag-Ion SPE 750 mg/6 mL
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Fractionation of the Fat from Potato Chips
Untreated Extract
18:1
18:0
counts
16:0
18:2
18:3
14:0
6
8
10
12
14
16
18
20
Time (min)
18:1 t
counts
SPE Fraction 1
6
8
10
12
8
counts
16
18
20
18:1 c
SPE Fraction 2
6
14
10
12
Time (min)
14
16
18
20
counts
SPE Fraction 3
6
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8
10
12
Time (min)
14
16
18
20
Conclusion
• Ag-ION SPE completely resolves the cis/trans 18:1 fatty acids,
making it possible to accurately quantify the trans fat.
• More simple technique than Ag-Ion thin-layer chromatography
with no contamination from silver ions.
• Simpler fractions from complex natural samples are more
easily identifiable.
• The elution protocol was proved to be robust and reproducible
for variety of samples.
• The conditioning step sufficiently removed any traces of water
that may affect the separation.
• Use of shorter GC column (SP-2560, 75 m) with hydrogen
carrier gas significantly decreased the time required for the GC
analysis.
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Acknowledgements
• SUPELCO Scientists: Katherine Stenerson and Robbie Wolford
• R. Adlof, USDA, Peoria, IL
• Dr. P. Delmonte, FDA, Bethesda, MD
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