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Craig Aurand, Olga Shimelis, An Trinh, and Michael YeSupelco, Div. of Sigma-Aldrich, Bellefonte, PA 16823

Toshiro KanekoSigma-Aldrich Japan, Tokyo, Japan

Noriko Shionoya, Tomoji Igarashi, Yoshiaki Hirata, Hirofumi GotoJapan Food Research Laboratory, Nagoya, Japan

Fractionation of Cis and Trans Fatty Acid Isomers for Food Sample Analysis on Silver SPE Material

T407018

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• Trans-fats produced commercially when vegetable oils are hardened into shortenings and margarine. They are used in commercially baked products and fast foods for flavor preservation and spoilage prevention

• Health effects of trans-fats- Adverse effect on blood lipids levels – increase LDL (“bad”)

cholesterol- Increased risk of coronary heart disease

• Replacing trans-fats with naturally unhydrogenated vegetable oils could prevent up to 100,000 premature deaths annually (http://www.hsph.harvard.edu/reviews/transfats.html)

Importance of fat analysis for the presence of trans-fats

Introduction

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• US FDA issued a regulation that requires food manufacturers to list the TRANS FAT on the Nutrition Facts panel of foods. Trans Fats information is required since January 2006

• The specified analytical method is AOAC 996.06 “Fat (Total Saturated and Unsaturated) in Foods”

• Several cis and trans monoenes overlap under these chromatographic conditions (100 m GC column)

• This overlap reduces the accuracy of the method.

Introduction (contd.)

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The Limit of Current Analytical Method

Extracted fat from margarine

20 30 40 50Time (min)

35.0 36.0 37.0 38.0Time (min)

16:018:0

18:118:2

18:3

6t 9t10t

11t 12t

13t6c7c

9c

10c11c 12c13c

Peaks from 18:1 trans and cis isomers overlap

Overlap of trans/cis monounsaturated octadecenoic fatty acids on the GC chromatogram

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• An orthogonal method is needed to differentiate the cis and trans monoenes unresolved by the GC separation.

• Silver chromatography has traditionally been used to separate saturated and unsaturated compounds. The Silver chromatography technique can be applied to the separation of fatty acid methyl esters.

• Advantages of Ag-Ion SPE- More simple technique than Ag-Ion thin-layer chromatography- The resulting samples are not contaminated with silver ions- No sophisticated equipment is necessary as in Ag-HPLC- Simpler fractions from complex natural samples are more easily

identifiable

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Types of Fatty Acids

O

HO

Structure Com m on Source s Health Ef fects

Tran s Fatt y Acid s (≥ 1 tra n s d o u b le b o n d )

Mono and Pol yun saturat ed Fatt y Acid s (≥ 1 c is d o u b le b o n d )

O

HO

cis

O

HOt ran s P artia ll y H ydr oge nate d Oils ,

S hor te nin gs , Margar i nes , a nd Chips

Flui d/Li qui d oils s uch as S o ybean,Canola , Oliv e , S un flo wer a ndCorn oils .

P a lm kerne l, P a l m oi l, Coc on ut(tro pica l oils ), Bu tter , H ydro gena tedOils a nd S h or te nin gs

Ra ise LD L ch oles terol, a nd increase r isk o fcardiov ascula r dise ase

Lo wer LD L ch oles terol,assoc ia te d wit h red uced r isk of car diov ascular disease .

Rai se L DL ch o lest ero l l ik esatu rat ed fat, ma y also lo w e rHDL . A sso ciat ed w i thin cre as ed ris k o fca rd io va scu l ar d is ea se an dp o ssib ly typ e I I d iab ete s.

Saturated Fatt y Acid s (n o d o u b le b o n d s)

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SO3SPESupport

-Ag+

C4H8

C11H23

O OCH3

Charge- transfer complex between Ag+ and unsaturated bond

Interaction Mechanism• Charge – transfer• Unsaturated compound – electron donor, Silver - electron

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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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/transmonoene FAMEs

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Ag-Ion SPE Method for cis/trans Separation1. Condition 4 mL acetone2. Equilibrate 4 mL hexane3. Sample Load 1 mL of 1 mg/mL FAMEs in hexane at

5 mL/min.4. Elution Fraction 1 6 mL hexane:acetone (96:4 v/v)5. Elution Fraction 2 4 mL hexane:acetone (90:10 v/v)6. Elution Fraction 3 4 mL acetone7. Elution Fraction 4 4 mL acetone:acetonitrile (97:3)8. 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

Experimental

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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 even when an isothermal GC method is used.

oven: 180 °C, isothermalinj.: 220 °C

det.: FID, 220 °Ccarrier gas: hydrogen, 40 cm/sec. at 180 °C

injection: 0.5 µL, 100:1 splitliner: 4 mm I.D., split, cup design

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18:1 c

18:1 t

Fractionation of the Standard FAME MixtureStandard sample, total FAMEs at 1 mg/mL

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE Fraction 4

SPE Fraction 2

SPE Fraction 3

SPE Fraction 1

Sample before SPE

16:0 18:0

18:118:2 18:3

14:0

7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0Time (min)

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Results of Fractionation of Standard Mix of FAMEs (% recovery)

Elution 18:0 18:1t 18:1c 18:2tt 18:2 c/t 18:2cc 18:3ttt 18:31 6 mL

Hexane:acetone (96:4) 100 98.1 0.4

2 4 mLHexane:acetone (90:10) 1.90 99.60 100

3 4 mL Acetone 100 50 100 40

4 4 mL Acetone:acetonitrile (97:3)

50 55

TOTAL 100 100 100 100 100 100 100 95

Note: more polar elution solvent is needed to completely elute 18:3ccc isomer.

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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

20 30 40Time (min)

Margarine Fat Sample before Separation16:0 18:0

18:218:1

20 30 40Time (min)

20 30 40Time (min)

Fraction 1 after SPE

Fraction 2 after SPE

18:1 trans

18:1 cis

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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

6 8 10 12 14 16 18 20Time (min)

coun

ts

Untreated Extract

6 8 10 12 14 16 18 20

coun

ts

6 8 10 12 14 16 18 20Time (min)

coun

ts

6 8 10 12 14 16 18 20Time (min)

coun

ts

16:0 18:0

18:1

18:2 18:314:0

SPE Fraction 1

SPE Fraction 2

18:1 t

18:1 c

SPE Fraction 3

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FAMEs Extraction Procedure for Cheeses

• Homogenize 3.0 g of cheese with 10 mL isopropyl alcohol.• Extract homogenized sample twice with 10 mL each of hexane.• Evaporated and reconstituted into 16 mL toluene.• Trans-esterify 1.0 mL sample with 2.0 mL of 7% BF3/MeOH.

Heat at 80 °C for 15 minutes.• Cool to room temperature and quench with 1.0 mL water.• Extract twice with 1.0 mL each hexane.• Evaporate hexane extract, redissolved with 5 mL hexane. Dry

with 50 mg anhydrous Na2SO4.

• Dilute 1:2 with hexane and load 1 mL of the diluted extract into Ag-Ion SPE 750 mg/6 mLProcedure adopted from D. Precht et al. Lipids 2001, 36(8), pp.827-832

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18:1 c

18:1 t

SPE Fraction 4

SPE Fraction 2

SPE Fraction 3

SPE Fraction 1

GC Chromatograms for the Imitation Cheese Extract after SPE Fractionation

Sample before SPE

16:0 18:018:1

18:218:3

14:0

Time (min)

Time (min)

Time (min)

Time (min)

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• Ag-Ion SPE resolves completely the cis/trans 18:1 fatty acids, making it possible to accurately quantify the trans fat.

• 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 analysis.

Conclusions

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• SUPELCO scientists:Katherine Stenerson, Robbie Wolford, and Sumeer Kakar

Acknowledgements