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EVALUATION OF GAS CHROMATOGRAPHIC AND FOURIER TRANSFORM INFRARED SPECTROSCOPIC METHODS FOR THE DETERMINATION OF TRANS- FAT LEVELS IN THE DIETS OF LAYING HENS AND THEIR EGGS by ROBERT LOUIS FUSCO (Under the Direction of Ronald B. Pegg) Abstract Five groups of 10 single comb white leghorn hens were fed different diets (standard corn/soy laying rations supplemented with edible oils) to ascertain the effect of trans-fat deposition in eggs. Trans-fat contents of the feeds and eggs were determined by gas chromatography (GC) and Fourier transform infrared (FTIR) spectroscopy. Diets devoid of hydrogenated oils contained no detectable levels of trans-fats. Feeds supplemented with tallow, shortening, or their combination contained 2.78±0.08, 3.25±0.03, and 2.85±0.05 g/100-g dietary fat, and 3.33±0.32, 4.16±0.03, and 3.52±0.05 g/100-g dietary fat as assessed by GC and FTIR, respectively. Eggs from hens fed these latter diets contained 0.68±0.05, 0.81±0.05, and 0.76±0.04 g/100 g dietary fat, by GC, but were below the 1% detection limit of FTIR. Nearly 25% of trans-fats in the diets accumulated in the eggs. Supplementation of edible oils did not appreciably increase trans-fat levels in the eggs. Data acquired by these methods correlated well, r=0.98. INDEX WORDS: Trans-fat, GC-FID, FTIR-ATR, Silver-Ion TLC, SCWL Hen
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Page 1: EVALUATION OF GAS CHROMATOGRAPHIC AND FOURIER … · index words: trans-fat, gc-fid, ftir-atr, silver-ion tlc, scwl hen. evaluation of gas chromatographic and fourier transform infrared

EVALUATION OF GAS CHROMATOGRAPHIC AND FOURIER TRANSFORM

INFRARED SPECTROSCOPIC METHODS FOR THE DETERMINATION OF TRANS-

FAT LEVELS IN THE DIETS OF LAYING HENS AND THEIR EGGS

by

ROBERT LOUIS FUSCO

(Under the Direction of Ronald B. Pegg)

Abstract

Five groups of 10 single comb white leghorn hens were fed different diets (standard corn/soy

laying rations supplemented with edible oils) to ascertain the effect of trans-fat deposition in

eggs. Trans-fat contents of the feeds and eggs were determined by gas chromatography (GC) and

Fourier transform infrared (FTIR) spectroscopy. Diets devoid of hydrogenated oils contained no

detectable levels of trans-fats. Feeds supplemented with tallow, shortening, or their combination

contained 2.78±0.08, 3.25±0.03, and 2.85±0.05 g/100-g dietary fat, and 3.33±0.32, 4.16±0.03,

and 3.52±0.05 g/100-g dietary fat as assessed by GC and FTIR, respectively. Eggs from hens fed

these latter diets contained 0.68±0.05, 0.81±0.05, and 0.76±0.04 g/100 g dietary fat, by GC, but

were below the 1% detection limit of FTIR. Nearly 25% of trans-fats in the diets accumulated in

the eggs. Supplementation of edible oils did not appreciably increase trans-fat levels in the eggs.

Data acquired by these methods correlated well, r=0.98.

INDEX WORDS: Trans-fat, GC-FID, FTIR-ATR, Silver-Ion TLC, SCWL Hen

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EVALUATION OF GAS CHROMATOGRAPHIC AND FOURIER TRANSFORM

INFRARED SPECTROSCOPIC METHODS FOR THE DETERMINATION OF TRANS-

FAT LEVELS IN THE DIETS OF LAYING HENS AND THEIR EGGS

by

ROBERT LOUIS FUSCO

B.S., The University of Connecticut, 2007

A Thesis Submitted to the Graduate Faculty of the University of Georgia in Partial

Fulfillment of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2009

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©2009

Robert Louis Fusco

All Rights Reserved

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EVALUATION OF GAS CHROMATOGRAPHIC AND FOURIER TRANSFORM

INFRARED SPECTROSCOPIC METHODS FOR THE DETERMINATION OF TRANS-

FAT LEVELS IN THE DIETS OF LAYING HENS AND THEIR EGGS

by

ROBERT LOUIS FUSCO

Major Professor: Ronald B. Pegg

Committee: Rakesh K. Singh Michael P. Lacy

Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2009

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I would like to thank Dr. Ronald Pegg, Dr. Rakesh Singh, Dr. Michael Lacy, and Dr.

Ramesh Avula for their guidance throughout my time at the University of Georgia. I would also

like to thank the American Egg Board for funding this project.

iv

ACKNOWLEDGEMENTS

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TABLE OF CONTENTS

Page

LIST OF TABLES....................................................................................................................... viii

LIST OF FIGURES ....................................................................................................................... ix

CHAPTER

1 LITERATURE REVIEW ...........................................................................................1

Origins of Trans-Fats ..............................................................................................1

Health Effects Of Trans Fatty Acids......................................................................6

Recent Government Regulations ...........................................................................8

Trans-Fat Alternatives ............................................................................................9

Fatty acid supplementation of laying hen feeds..................................................13

Trans Fatty Acid Analysis: Gas Chromatography (GC) ...................................14

Trans Fatty Acid Analysis: FTIR.........................................................................16

2 MATERIALS, METHODS, RESULTS, AND DISCUSSION ..............................18

Chemicals ...............................................................................................................18

Feed Trials..............................................................................................................18

Acid Hydrolysis......................................................................................................19

Lipid Extraction ....................................................................................................19

Fatty Acid Methylation .........................................................................................21

Gas Chromatographic Analysis ...........................................................................21

Silver-Ion Thin Layer Chromatography (Ag+-TLC) .........................................23

Linearity: Gas Chromatography ........................................................................23

v

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Precision and Accuracy: Gas Chromatography ................................................24

Limit of Detection (LOD) and Limit of Quantification (LOQ): Gas

Chromatography .............................................................................................24

Recovery: Gas Chromatography ........................................................................24

Fourier Transform Infrared Spectroscopy (FTIR) Analysis ............................25

Linearity: Fourier Transform Infrared Spectroscopy (FTIR) Analysis .........26

Precision, Accuracy, and Limit of Detection (LOD): Fourier Transform

Infrared Spectroscopy.....................................................................................26

Statistical Methods ................................................................................................27

Results and Discussion ..........................................................................................27

Gas Chromatography............................................................................................27

Fourier Transform Infrared Spectroscopy .........................................................32

Conclusions ............................................................................................................34

References .....................................................................................................................................36

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LIST OF TABLES

Page

Table 1: Egg weight (g) of hens fed diets of varying levels of trans-fat for 21 days ...................42

Table 2: Response factors of FAMEs as determined by GC-FID using Supelco 37 component

FAME mixture ..............................................................................................................43

Table 3: Fatty acid profile of egg yolk lipids from hens consuming diets with varying levels of

trans-fat .........................................................................................................................45

Table 4: Percentage of fatty acids in egg yolk lipids from hens consuming diets with varying

levels of trans-fat...........................................................................................................47

Table 5: Fatty acid composition of feeds with and without added trans-fats ...............................49

Table 6: Recovery of triheptadecanoin and methyl heptadecanoate during GC-FID analysis .....50

Table 7: Precision and accuracy of trans-fat analysis using GC-FID...........................................51

Table 8: Precision and accuracy of trans-fat analysis using FTIR-ATR ......................................52

vii

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LIST OF FIGURES

Page

Figure 1: Partial hydrogenation reaction mechanism ...................................................................53

Figure 2: GC-FID Chromatogram of egg yolk lipids from diet I .................................................55

Figure 3: GC-FID Chromatogram of egg yolk lipids from diet II ................................................57

Figure 4: GC-FID Chromatogram of egg yolk lipids from diet III...............................................59

Figure 5: GC-FID Chromatogram of egg yolk lipids from diet IV ..............................................61

Figure 6: GC-FID Chromatogram of egg yolk lipids from diet V................................................63

Figure 7: GC-FID Chromatogram of diet I ...................................................................................65

Figure 8: GC-FID Chromatogram of diet II..................................................................................67

Figure 9: GC-FID Chromatogram of diet III ................................................................................69

Figure 10: GC-FID Chromatogram of diet IV.............................................................................71

Figure 11: GC-FID Chromatogram of diet V ...............................................................................73

Figure 12: Overlay of silver ion-TLC chromatogram and original chromatogram for

shortening ......................................................................................................................75

Figure 13: Chromatogram of silver ion-TLC separation of trans monounsaturated fraction of

shortening ......................................................................................................................77

Figure 14: Interference band of egg lipids during FTIR analysis .................................................79

Figure 15: Second derivative spectra of absorbance of trans band at 967 cm-1 ...........................81

Figure 16: Spectra of absorbance of trans band at 967 cm-1 ........................................................83

viii

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Figure 17: TQ Analyst calibrations of prepared standards of 0.5 to 10% (w/w) methyl elaidate in

methyl oleate .................................................................................................................85

Figure 18: Correlation of GC versus FTIR data ...........................................................................87

ix

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Chapter 1

LITERATURE REVIEW

Potential health effects of dietary trans fatty acids and the US Food And Drug Administration

(FDA) mandate that as of January 1, 2006, the Nutrition Facts panels on all packaged food labels

must indicate the quantity of trans fatty acids in a serving of the food product, have driven the

development of efficient methods for accurate measurements of isomeric fatty acids in foods.

Since this mandate food manufactures have reformulated many of their products and the general

public has had an increased awareness of the effects of dietary trans fatty acids.

Origins of Trans-Fats

Mono- and poly- unsaturated fatty acid molecules exist in either a cis or trans

configuration. Cis and trans isomers are geometric isomers of on another, i.e., they differ only in

the spatial configuration of the atoms in the compound. A cis-double bond contains hydrogen

atoms on the same side of the double bond, while a trans double bond contains hydrogen atoms

on opposite sides of the double bond. In nature, double bounds occur almost exclusively in the

cis configuration; it has been reasoned that this is because fatty acids are important structural

components of cell membranes, and the cis double bond imparts a bend in the molecule,

reducing the van der Waals forces between fatty acids, and as a result maintains the fluidity of

the membrane (Kodali and List, 2005).

1

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The principal source of natural trans fatty acids comes from biohydrogenation by rumen

microorganisms. In the gut of ruminant animals, bacteria convert cis-9,cis-12-octadecadienoic

acid into cis-9,trans-11-octadecadienoic acid, i.e., one of the conjugated linoleic acid (CLA)

isomers. A majority of the cis-9 double bonds in CLA are subsequently hydrogenated resulting

in trans-11-octadecenoic acid. These “natural” trans-fats derived from biohydrogenation

constitute 3-8% of the fat in beef and milk (Kodali and List, 2005).

The trans fatty acids of meat and milk of ruminant animals arising from

biohydrogenation is different from trans fatty acids arising from partial hydrogenation practiced

by the edible oil industry. The hydrogenation process generates many different cis and trans

isomers while biohydrogenation produces mainly trans-11-octadecenoic acid (Ratnayake,

2004). Trans-11-octadecenoic acid has been shown to form conjugated cis-9,trans-11-

octadecadienoic acid during metabolism in humans (Jiang and others 1996). CLA

supplementation in the diet did not change LDL or HDL cholesterol levels during clinical trials,

nor has an association been shown between the consumption of ruminant animal trans fatty acid

(in meat and milk) and cardiovascular disease (CVD) or coronary heart disease (CHD) (Huth,

2007; Jakobsen and others 2008). Therefore, the following discussion of the health related

effects of trans-fats will consider only those resulting from the partial hydrogenation of edible

oils.

The main source of dietary trans fatty acids comes from the partial hydrogenation of

vegetable oils. Hydrogenated vegetable oils can contain 10% to 40% trans fatty acids depending

on the reaction conditions and the degree of hydrogenation to which the starting material was

subjected (Kodali and List, 2005). Wilhelm Normann patented the process of hydrogenating

2

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liquid oils in 1902. In 1909 Proctor and Gamble purchased the patent from Wilhelm, for the

production of shortening from cottonseed oil (Crisco) as a replacement for lard.

During the hydrogenation of oils, hydrogen atoms are added across unsaturated carbon-

carbon double bonds, creating saturated carbon-carbon single bonds. This is done at high

temperatures and pressures in the presence of a metal catalyst. In industry, reduced nickel is the

most widely used catalyst because of its good activity, selection, filterability, reusability, and

economical use. For efficient conversion of double bonds to single bonds, the concentration of

hydrogen on the catalyst surface and the contact of triacylglycerols with that surface must be

maximized. Hydrogen is dispersed as small bubbles into the bulk oil; smaller bubbles have a

greater surface to mass ratio than larger bubbles. The catalyst may be fixed onto a support

thereby further increasing the surface area of the catalyst to mass of the catalyst. To promote the

diffusion of triacylglycerols and from the surface of the catalyst gentle agitation, at temperatures

between 250-300oC is employed (Akoh and Min, 2002).

Hydrogen atoms will attach to either of the carbons of the double bond. If there is

another hydrogen atom available, it will attack the other now negatively charged carbon to form

a carbon-carbon single bond. If, however, another hydrogen atom is not available, then the

double bond will reform as either a cis or trans double bond. This can occur on either side of the

carbon anion (Akoh and Min, 2002).

The isomerization of fatty acids will decrease when there is a higher concentration of

hydrogen present on the catalyst surface, so that the saturated single bond is formed before the

double bond reforms. This can be achieved by using low temperatures, high pressures, high

agitation rates, high gassing rates, and low catalyst concentrations. These conditions will

completely saturate the catalyst with hydrogen thereby allowing saturated bonds to form faster.

3

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A study by Evans et al. showed that the formation of trans isomers can be reduced to 0.4% trans

bond formation per IV unit reduction at 120oC and 689 kPa, compared to 0.7% trans formation

per IV unit reduction, at 170oC and 34 kPa (Kodali and List, 2005). The mechanism for

hydrogenation is depicted in Figure 1.

Despite the main reaction forming saturated fatty acids, there are also two side reactions

that occur: the isomerization of cis double bonds to trans double bonds and the migration of the

double bond along the carbon chain. The shift of the double bond creates fatty acid isomers that

are not commonly found otherwise. The dominant naturally-occurring isomer of octadecenoic

acid is C18:1 n-9 (oleic acid) where the position of the double bond is located at the 9th carbon

from the terminal methyl end. However during hydrogenation, a distribution of isomers is

generated ranging from the 4th carbon to the 16th carbon with the majority occurring at the 8th

thru 11th positions (Kramer and others 2002). This migration happens for both the cis and trans

isomers.

There are two main reasons for the partial hydrogenation of edible oils: to increase the

oxidative stability as well as the solid fat content (SFC) of the oil (Akoh and Min, 2002).

Autooxidation occurs when oxygen radicals interact with the double bond in a fatty acid

producing hydroperoxides which subsequently breakdown to odiferous secondary oxidation

products leading to rancidity. Trans fatty acids are more stable than cis fatty acids, but much

less stable than saturated fats. As such, they undergo oxidation at a slower rate than their cis-

monounsaturated counterparts, but at a faster rate than saturated fats. For example Oleic acid

undergoes oxidation 10 times faster than stearic acid and 10 times slower than linoleic acid

(Kodali and List, 2005). As a result, trans-monounsaturated fats and the products fabricated

4

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from them have a longer shelf life than those made with cis-monounsaturated and

polyunsaturated fatty acids (Gunstone, 2007).

Triacylglycerols can exist in various crystal structures because fatty acid chains pack into

different crystal lattices. Different polymorphic forms like α, β′, and β, impart different physical

attributes to solid fats. The α form is the least thermodynamically stable and has the lowest

melting point, followed by β′ and then β with the latter having the highest melting point. Fats

will initially crystallize into the α form, but it is unstable and will transform to either β′ or β

forms. The β′ form is the most functional for margarines and shortenings, because it forms very

small crystals and instills desirable characteristics into products such as a smooth mouth feel and

sharp melting point. It has been report that the β form contains larger crystals and can give fats a

grainy mouth feel. When comparing the crystaline structures of tristearin, trielaidin, and triolein,

Kodali showed that while tristearin and triolean will pack into a β′ form, trielaidin will only pack

into the β form (Kodali and List, 2005). The melting points of tristearin, trielaidin, and triolein

are 73oC, 42oC, and 5oC respectively.

Solid fat content adds important characteristics to baked goods and confections. A high

solid fat content adds snap and improved textural properties to these goods. For example cocoa

butter illustrates the importance of solid fat content and melting characteristics of fats. Cocoa

butter comprises primarily stearic, palmitic, and oleic acid. This unique set of fatty acids give

cocoa butter a sharp melting point between 20oC and 35oC, allowing the fat to melt at body

temperature (Vaclavik, 2007). When the fat melts in the mouth, it provides a cooling sensation

as it draws energy, in the form of heat, from the mouth. Most fats have more than three primary

fatty acids in them, causing them to have a broader melting point. Fatty acids that have a melting

point higher then body temperature generally result in a waxy mouth feel.

5

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Health Effects of Trans Fatty Acids

Heart disease is the leading cause of death for both men and women in the United States

(Kung and others 2008). Several studies have indicated that the consumption of trans-fats, even

at low levels (i.e., 1-3% of total energy intake), can substantially increase the risk for coronary

heart disease (CHD) (Mozaffarian and others 2006). The Nurses Health Study, an

epidemiological study conducted by Hu and colleagues, followed 120,000 female nurses for 14

years and concluded that when a 2 % increment in energy from trans unsaturated fat replaced

carbohydrates in the diet the risk of developing CHD was 1.93 times more likely compared to

only a 1.17 times when a 5 % increment in energy from saturated fat replaced carbohydrates in

the diet. (Hu and others 1997). Hu and coworkers estimated that the replacement of 2 % of

energy from trans-fat with energy from cis-unsaturated fats would reduce the risk of CHD by a

whopping 53% (Hu and others 1997).

Two of the main biomarkers for cardiovascular disease (CVD) are the levels of low

density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol. Because of its size

LDL cholesterol, “bad cholesterol”, is able to enter the capillaries of endothelial cells where it

can be oxidized. Oxidized LDL cholesterol leads to plaque formation on artery walls, one of the

symptoms of CVD. It has been shown that saturated fatty acids increase total and LDL

cholesterol levels but do not change HDL cholesterol levels, while trans fatty acids not only

increase total and LDL cholesterol but also decrease HDL cholesterol (Katan and others 1995).

The lowering of HDL cholesterol causes trans-fats to have a less favorable effect on the plasma

lipoprotein profile than saturated fats (Kyoko Hayakawa and others 2000). The increased risk of

CHD from increased trans-fat consumption is greater than what would be predicted by

cholesterol levels alone. Several studies point to a correlation between trans-fat consumption

6

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and an increase in systemic inflammation. In observational studies, an increased trans fatty acid

intake led to an increased activity of the tumor necrosis factor (TNF) system as well as increased

levels of C-reactive protein (CRP) and interleukin-6 (IL-6) (Lopez-Garcia and others 2005;

Micha and Mozaffarian, 2008; Mozaffarian, 2006; Mozaffarian and others 2004). All of which

are biomarkers of increased systemic inflammation and endothelial dysfunction. During an

experimental study of 50 healthy men fed a diet consisting of 8% of energy from trans fatty

acids, plasma levels of CRP and IL-6 increased when compared to a diet consisting of 8% of

energy of oleic acid (Baer and others 2004). Mozzofarin described a possible mechanism for

these increases: trans fatty acids consumed in the diet incorporate themselves into the cell

membrane of endothelial cells and macrophages, as is seen with n-3 and n-6 monounsaturated

fats. Endothelial cells and macrophages have various cell-specific pathways that activate the

TNF system (Mozaffarian and others 2004).

While most research regarding the adverse health effects of trans fatty acids has dealt

with CVD, trans fatty acids have been implicated as increasing the risk of several other diseases

including diabetes and Alzheimer’s disease. There has been less scientific consensus concerning

the role of trans-fats in these diseases than its apparent effects on CVD. A study by Salmeron

and coworkers showed that when energy from trans fatty acids was increased by 2%, the relative

risk for diabetes was increased by 1.39 (Salmeron and others 2001). In an epidemiological study

Hu and coworkers reported that trans fatty acids adversely affected glucose metabolism and

insulin resistance (Hu and others 2001). Investigations by van Dam et al. and Meyer et al.

however, showed no association between trans fatty acids and diabetes (van Dam and others

2002; Meyer and others 2001).

7

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A study of 815 people over the age of 65 implicated trans fatty acids as having a relative

risk of 2.4 for the onset of Alzheimer’s disease (Morris and others 2003). A recent study of mice

fed diets high in trans-fats showed changes in the fatty acid profile of the brain shifting from

docosahexaenoic acid (DHA) to docosapentaenoic acid (DPA), but did not show any of the

major brain neuropathological hallmarks of Alzheimer’s disease (Phivilay and others 2009).

Recent Government Regulations

Based upon growing scientific evidence relating trans-fats to an increased incidence of

CHD and a citizen petition’s in 1994, the FDA, proposed in 1999 that the quantity of trans-fats

in products be included on food labels, specifically, in the section dealing with the amount and

percent daily value for saturated fats, a footnote was included indicating the amount of trans

fatty acids per serving of the product (Services, 2003). Following the FDA’s proposal, the

Institute of Medicine issued a report indicating a direct association between trans fatty acids and

an increased incidence of CHD. They failed, however, to establish a daily reference value,

simply stating that trans-fat consumption should be as low as possible (Food and nutrition board,

2002). In 2003, the FDA issued its final ruling requiring the posting of trans-fat on the

nutritional labels of all products. This law went into effect on January 1st 2006. The trans-fat

column would be listed as a sub grouping of total fat and would not contain a %DV. Any

product that contained less then 0.5g of trans-fat per serving could be listed as 0 g trans-fat, or

alternatively would not be required to list trans-fat on the nutritional label (Services, 2003).

Many believe that 0.5 g is too high of a value to be listed as 0 g because a person consuming

multiple servings of products containing 0.5g trans-fats could easily consume over the maximum

recommended intake of 1% of energy or 2 g of trans fatty acids, as recommended by the

8

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American Heart Association, without knowing it (American Heart Association, 2008). The

FDA, unlike several other countries, did not decide to limit the amount of trans-fats that a

product could contain. In 2003, Denmark banned any product that contained over 2% trans-fat.

This regulation included commercial oils practically eliminating partially hydrogenated oils from

the food supply. Even fast food has low a level of trans-fats in Denmark. For example the

cooking oil used to fry McDonalds French fries has, on average, 24% trans-fats in the United

States while it has less than 1% in Denmark (Stender and others 2006). In 2008, Sweden

adopted the same laws as Denmark concerning trans-fat levels.

Several US cities have enacted laws limiting the quantity of trans-fats that can be used by

restaurants. As and example, in 2006, New York City banned the use of trans-fats in oils

employed for deep frying (Tucker and Markt, 2006). These regulations came with a public

education campaign to inform the consumers of the health effects attributed to trans-fats and

how to choose foods that are low in them. Other cities have enacted similar trans-fat regulations

including Philadelphia in 2007 (City of Philadelphia, 2007a) and San Francisco in 2008,

although San Francisco’s regulation is voluntary. As of 2010, the state of California will be the

first state to ban the use of trans-fats for deep frying, except for donuts, food businesses making

donuts will have until 2011 before the restriction of using partially hydrogenated oils will apply

(State of California, 2008).

Trans-Fat Alternatives

With these regulations two things have become apparent: the need for fats and oils free of

trans-fat, and an effective and efficient way to determine the amount of trans-fat contained in a

product. One concern while reformulating products is that while replacing trans-fats, the level of

9

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unsaturated fats should be maximized while providing the same sensory attributes and

maintaining a similar cost. There are several alternatives at hand to replace partially

hydrogenated oils. These include naturally stable oils and fats, interesterified oils, fractionated

oils, and trait-enhanced oils from new oilseed varieties. Palm, corn, and cottonseed oils are

relatively stable and do not require partial hydrogenation for most commercial applications.

Interesterification also produces relatively stable oils. Depending on what oil stocks are

used and the degree of interesterification, these oils can be utilized in many commercial

applications. This technology is highly developed in Europe for the production of margarines

and shortenings (Kodali and List, 2005). Industrially, interesterification can be accomplished by

batch or continuous methods. During a batch process, the raw lipid is sparged with nitrogen and

heated to 120-150oC under vacuum to remove any residual moisture. Trace amounts of water

will deactivate the catalyst. The mixture is then cooled to 70-100oC followed by the addition of

the catalyst, usually in the form of methylated or ethylated sodium or potassium at a

concentration of <0.4%. The reaction proceeds for 30-60 min, is then neutralized with CO2, and

then terminated by the addition of water. The water and catalyst are centrifuged out and the

catalyst is recovered.

During interesterification, fatty acids are exchanged within triacylglycerols and between

triacylglycerols resulting in a completely random distribution of fatty acids within the

triacylglycerol molecules and a random distribution of triacylglycerols within the lipid. If three

completely pure stocks in equal proportions of tristearin, triolein, and trilinolein, are

interesterified, the resulting composition of triacylglycerols will be as follows:

SSS, 3.7% SOO, 11.1%

OOO, 3.7% SLL, 11.1%

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LLL, 3.7% OOL, 11.1%

SSO, 11.1% OLL, 11.1%

SSL, 11.1% SOL, 22.2%

where S = stearic acid, L = linoleic acid, and O = oleic acid. These percentages can change if the

reaction temperature is below the melting point of the highest melting point triacylglycerol,

usually a trisaturate. This kind of interesterification, called directed interesterification, will result

in a product enriched in the highest melting point molecules. Directed interesterification is

useful to increase the solid fat content without affecting the content of unsaturated fats. During

the reaction when trisaturates form, they will crystallize out of the liquid phase. They can then

be removed and the reaction will proceed until all of the trisaturates have propagated out of the

mixture. The final product from equal proportions of tristearin, triolein, and trilinolein, will have

the following composition:

SSS, 33.3%

OOO, 8.3%

OOL, 24.99%

OLL, 24.99%

LLL, 8.3%

To increase the rate of settling, gentle agitation is employed. Interesterification can change the

physical properties such as solid fat content and melting point without affecting the content of

fatty acids. There is also no cis/trans isomerization, thereby making it a useful technique to

replace partial hydrogenation. It has also been used to produce zero-trans fat margarines and

shortenings.

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Fractionation is the process in which lower melting triacylglycerols, oleins, are separated

from higher melting triacylglycerols, stearines. It is commonly used on palm oil, palm kernel

oil, coconut oil, and animal fats. Fractionation of palm oil is accomplished at 20oC resulting in

70% palm olein, and 30% palm stearine. Palm stearine is used in the formulations of margarines

and shortenings. The palm olein may undergo a second fractionation at a lower temperature to

produce super palm olein used in frying applications, and palm mid-fraction, used in cocoa butter

substitutes. To increase the rate of settling, gentle agitation is employed. If too vigorous

agitation is used, it will cause the crystals to break, hindering the settling rate. To decrease

viscosity, fractionation is sometimes preformed in a solvent such as hexane.

The use of certain varieties of oil, such as high-oleic corn oil and low-linolenic canola oil

can replace partially hydrogenated oils in deep frying applications. These specialty oils,

however, tend to be higher in price than the partially hydrogenated oils previously, used because

many of them are not produced in commercially-viable quantities for large scale operations.

Increasing production of these specialty crops would cut down on cost and increase supply;

however, to develop oil at an industrially useful scale can take up to 6 or 7 years for a crop to be

planted until it is available for use by the consumer.

Oil seeds have been produced with modified fatty acid compositions by traditional seed

breeding procedures and by modern genetic methods to manufacture oils that meet specific

consumer needs. Monsanto is a leader in the latter approach. Mutants from oil plants have been

identified that produce oil with varying fatty acid compositions. Canola oil, which is widely

consumed as cooking oil, is in fact the result of a low eurcic rapeseed oil cultivar (Tarrago-Trani

and others 2006). High-oleic sunflower oils were produced in Russia in 1976 by a selective

sunflower breeding program (Fick, 1983). These oils are more stable in deep-frying applications

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and have an extended shelf life compared to traditional sunflower oil. The National Sunflower

Association released a mid-oleic oil NuSun in 1998 (NuSun, 2006).

Genetic engineering offers a more direct way to alter the fatty acid compositions of oil

seed crops (Flickinger and Huth, 2004; Hammond and Fehr, 1984). Genetic engineering of

soybeans has produced soybean oil with a lower content of α-linolenic acid (C18:3 n-3), which is

prone to rancidity, and higher amounts of oleic acid thereby making it more suitable for deep-

frying (Hammond and Fehr, 1984). Canola has been engineered to contain low amounts of α-

linolenic acid and higher amounts of oleic acid; soybean oil has been made with increased levels

of saturated fatty acids, such as stearic or palmitic acid for use in margarines and shortenings.

Soybean oil has also been produced with decreased amounts of saturated fatty acids to improve

its nutritional value (Hammond and Fehr, 1984).

Fatty acid supplementation of laying hen feeds

Laying hen diets are often supplemented with fat or oil to increase the weight of the eggs

produced (Sell and others 1987). This is especially important when sorghum or barley is

substituted for corn, which contains higher amounts of fat (Coon and others 1988). Adding fat to

laying hens’ diets can substantially increase the cost of the feed, so economically feed-grade fat

and oils come from many different places, including spent grease from restaurants, rendered

animal fats, and refuse from oil refining plants. All of these sources have the possibility of

containing high levels of trans fatty acids.

Lipids deposited into the egg yolk are formed in the liver with fatty acids acquired

through the diet or synthesized de novo. Supplying hens with dietary fat from the diet reduces

hepatic fatty acid synthesis, increasing yolk formation and egg weight (March and MacMillan,

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1990). The increased use of dietary lipids during yolk formation leads to a fatty acid composition

similar to that of the diet. Fat absorbed from the intestine is transported to the liver where some

desaturation and elongation of saturated fatty acids takes place. Linoleic acid is the only fatty

acid essential to hens, with a minimum requirement of 1% of dietary feed. Higher levels of

linoleic acid can produce heavier eggs (Subcommittee on Poultry Nutrition, 1994).

Recent research has focused on changing the n-3:n-6 ratio to better reflect the needs of

the human diet. Laying hens that were feed diets rich in omega-3 fatty acids, such as flaxseed,

showed a marked increase of omega-3 fatty acids in the yolk fat. (Leeson and Caston, 1990)

showed that a diet containing 30% flaxseed increased the linolenic acid composition from 0.38%

to 11.50% of total fat. Small increases in long chain n-3 fatty acids also occurred. Studies have

also shown that supplementation with fish oil will increase ALA and long chain n-3 fatty acids

but impart negative sensory attributes to the eggs.

Trans Fatty Acid Analysis: Gas Chromatography (GC)

The other concern that was triggered by the enactment of the FDA’s labeling mandate is

how to accurately measure the trans-fat content in a product. There are several methods that can

be used including GC-FID, Fourier transform infrared (FTIR) spectroscopy, Raman

spectroscopy, NMR spectroscopy, and reversed phase HPLC (Akoh and Min, 2002). The

American Oil Chemists’ Society (AOCS) has several official methods for the determination of

trans-fat, and these have been updated since the initial meeting by the FDA in 1999. Specifically,

AOCS method Ce 1f-96 (AOCS Official method Ce 1f-96, 2002) for the determination of trans

fatty acids for hydrogenated and refined oils by capillary GLC is used to identify and quantify

individual fatty acid methyl esters (FAMEs). Prior to GC analysis, fatty acid moieties of

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triacylglycerols must be released from the glycerol backbone and the resultant the non-volatile

fatty acids must then be derivatized to FAMEs. As and illustration, methyl oleate has a boiling

point of only 219oC compared to 360oC for oleic acid, allowing FAMEs to volatilize at

temperatures reasonable for GC (MSDS, 2007b; MSDS, 2005). The approved method for

derivitizing fatty acids into FAMEs is AOCS method Ce 2-66 (AOCS Official Method Ce 2-66,

1997). This method calls for the use of boron triflouride (BF3). BF3, as a derivatizing agent, has

some major disadvantages according to Cristie; the use of BF3, which has a relatively short shelf

life even when refrigerated, can cause artefacts to form during derivitization and can lead to a

significant loss of polyunsaturated fatty acids (Christie, 1989). Its major advantage, however, is

that substantial amounts of fatty acids can be derivatized at a given time (~1g) and it only takes

15 min for complete derivitization. Cristie recommends the use of 6% (v/v) sulfuric acid in

methanol at 65oC for 12 h (Christie, 1989), because it employs a low temperature and does not

generate artefacts. The time can be shortened if constant stirring is used. Other options for

derivitizing agents include BCl3 and 1% (v/v) HCl in water.

The challenging part of trans fatty acid analysis is resolving the cis and trans isomers

from each other. There are two columns reviewed for use in trans fatty acid analysis, the SP-

2560 and Cp-88 (Ratnayake and others 2006). Both columns have a very polar bis-cyanopropyl

stationary phase and are 100-m in length, have a 0.25-mm-inner diameter, and a film thickness of

0.20-μm. Even with optimized operating conditions, considerable overlap still exists especially

when there is a high concentration of oleic acid in the lipid. However, cis isomers are eluted

after the oleic acid peak while the trans isomers are eluted before (Buchanam, 2008). This

makes determination of total trans-fat applicable even if all the trans peaks are not resolved from

one another. Peaks are identified based on retention times of known standards.

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One way to isolate trans fatty acids, for GC analysis, from their cis counterparts is by

silver-ion TLC. By impregnating silica with AgNO3, trans FAMEs can be separated from cis

FAMEs. The silver ions form complexes with the double bonds in the FAMEs. Cis isomers

form more stable complexes with Ag+ because they are less sterically hindered than trans

FAMEs, therefore, trans FAMEs will migrate further than cis FAMEs. The separation of

FAMEs follows the order of polyunsaturated FAMEs (cc, ct, tt, for C18:2 in order of decreasing

Rf values), monounsaturated cis-isomers, monounsaturated trans-isomers, and then saturated

FAMEs. The TLC plates can be developed either in a mixture of heptane and diethyl ether or

toluene at -25oC. Silver-ion LC and silver-ion HPLC have also been used to isolate trans

FAMEs.

Trans Fatty Acid Analysis: Fourier Transform Infrared Spectroscopy

FTIR spectroscopy is widely used to determine the amount of trans-fat in foodstuffs.

There are several AOCS official methods available for the determination of trans fatty acids for

hydrogenated and refined oils. AOCS methods Cd 14-95 (AOCS Official method Cd 14-95,

2000) and Cd 14d-99 (AOCS Official method Ce 14d-99, 1999) are the official methods for

determining trans fatty acid content using FTIR spectroscopy. The most practical method, Cd

14d-99, uses a horizontal attenuated total reflectance (ATR) cell as this allows for direct analysis

of either triacylglycerols or FAMEs without having to weigh or quantitatively dilute the samples

in volatile solvents. This method is able to accurately detect trans fatty acids down to 1.0% by

weight (AOCS Official method Ce 14d-99, 1999). Instead of transmission or partial reflections

horizontal ATR cells utilize the internal reflection of a substance, in this case FAMEs, to

measure how much of the IR waves are internally reflected. The depth of penetration of the IR

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light is extremely small, so accurate weighing is not needed, only that the cell is completely

covered with no air bubbles (Mossoba and others 2007b). Many ATR cells can be temperature

controlled, because the lipid needs to be in the liquid state. To ensure that triacylglycerols are in

the liquid state the cell should be heated to around 65oC, but for FAMEs, the temperature should

be maintained at ~40oC. The temperature must not only keep the sample in a liquid state it must

also be kept relatively constant, as the reflective property of a liquid is temperature dependent.

The main problem with FTIR methodologies is that the detection limit is around 1% trans-fat per

gram total fat, so small quantities are difficult to detect (AOCS Official method Ce 14d-99,

1999; Mossoba and others 2007a; Sherazi and others 2009). This stems from the fact that the

trans-fat peak at 966 cm-1 lies on a sloping baseline. Some recent advances in methods have

improved the accuracy at low levels by eliminating the baseline problem. For instance, one

study showed that by using reference oils identical to the sample oil but devoid of trans-fat, the

detection limit was lowered to 0.1% (Mossoba and others 2007a). By using a reference oil

identical to the sample but devoid of trans-fat for the background, it produces a straight baseline

because it subtracts out the equivalent slope that is seen in the sample itself. However,

determining and finding reference oils to use is difficult when analyzing samples that were not

industrially processed oils. Another way of increasing the accuracy at low levels is by using the

second derivative absorbance spectroscopy (Mossoba and others 2007a). By using the 2nd

derivative, the slope of the baseline becomes straight. The 2nd derivative is also useful for

identifying peaks that are shoulders of other peaks. For regulatory practices, an FTIR reading of

3.57% trans-fat would translate into 0.5-g trans-fat per serving (14g) of oil. Consequently

readings below this value will result in a trans-fat content of 0-g on the nutrition label, making

the low limit of the detection a non-issue for regulatory procedures.

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Chapter 2

MATERIALS, METHODS, RESULTS, AND DISCUSSION

Chemicals

Soybean oil and vegetable shortening for feed trials were purchased from Kroger’s

(Athens, GA). Tallow was obtained from Welch, Holme & Clark Co., Inc. (Newark, NJ). ACS-

grade anhydrous methanol, hexanes, chloroform, carbon disulfide, silver nitrate, and sulfuric acid

were purchased from Fisher Scientific Ltd. (Suwanee, GA), whereas anhydrous sodium sulfate,

acetonitrile, and sodium hydroxide were acquired from VWR International Inc. (Suwanee, GA).

Hydroquinone, 14% (v/v) boron trifluoride in methanol, TLC plates (20 x 20 cm, 250 μm, on

polyester), diethyl ether, 2',7'-dichlorofluorescein, hydrochloric acid, and a Supelco 37

component FAME mixture were purchased from Sigma Chemical Company (St. Louis, MO).

Absolute (200 proof) enthanol was obtained from AAPER Alcohol and Chemical Co.

(Shelbyville, KY). Triheptadecanoin (>99%), methyl heptadecanoate (>99%), methyl oleate

(>99%), and methyl elaidate (>99%) were acquired from Nu-Chek Prep Inc. (Elysian, MN).

Industrial-grade nitrogen, scientific air, UHP helium, and UHP hydrogen were obtained from

Airgas National Welders (Toccoa, GA).

Feed Trials

Fifty adult Single Comb White Leghorn (SCWL) laying hens were maintained on a diet

free of animal by-products for a period of 30 days. After that time, hens were divided into five

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groups of ten hens each and fed diets containing different types of fats to ascertain the effect of

varied trans-fat levels and various types of lipids on the trans-fat levels in eggs. The five diets

were as follows: I - Standard corn/soy laying ration with 0% added fat/oil; II - Standard corn/soy

laying ration with 4% (w/w) added soybean oil; III - Standard corn/soy laying ration with 4%

(w/w) added tallow; IV - Standard corn/soy laying ration with 4% (w/w) added shortening; and

V - Standard corn/soy laying ration with 4% (w/w) added tallow shortening blend. During the

feeding period, both feed consumption and egg production were monitored, as was any gain or

loss in body weight. Each hen was individually leg banded so as to ensure proper identification.

Eggs from each of the five groups of hens were collected on day 7, 14, and 21. Five egg yolks,

from each of the five groups, were pooled and homogenized separately before analyzing for

trans-fats.

Acid Hydrolysis

Lipids that are covalently and ionically bonded to proteins and carbohydrates are released

by digestion with hydrochloric acid. Feed samples were ground, while still frozen to avoid

deterioration of the samples. Approximately 32 g of ground feed was transferred to a 250-mL

Erlenmeyer flask to which 60 mL of ethanol and 60 mL of 8 N HCl were added. The

Erlenmeyer flask was covered with a watch glass placed in an 80oC water bath for 40 min with

occasional swirling . After cooling to room temperature, the lipids were extracted as follows.

Lipid Extraction

Total lipids were extracted by the Bligh & Dyer method (Bligh and Dyer, 1959) with

slight modifications. Briefly, 25 g of separated egg yolks were weighed into a 250-mL

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Erlenmeyer flask, followed by 35 mL of deionized water to adjust the moisture content to 80%.

The contents were then blended with a Polytron® PT 3100 homogenizer for 1 min at a speed of

15,000 rpm (Kinematica, Inc. Bohemia, NY). Then, 50 mL of methanol and 25 mL of

chloroform were added creating a monophasic system. To create a diphasic system 25 mL of

chloroform were added to the egg yolk solution, followed by 25 mL of deionized water. The

lower organic phase was comprised of nearly 100% chloroform and extracted lipids, while the

upper phase was comprised of almost entirely methanol-water. Finally, an additional 35.5 mL of

chloroform were added to aid in separation. The egg yolk mixture was homogenized between

each addition for complete lipid extraction. A 2-min quiescent period facilitated separation

before filtering under slight vacuum through a Büchner funnel lined with Whatman #1 filter

paper. The filter paper and solids were returned to the Erlenmeyer flask. To this, 37.5-mL

chloroform and ~10 mg of hydroquinone (as an antioxidant) were added. Contents were blended

for 1 min and again filtered as described above. The supernatant containing any residual non-

polar lipids was transferred to a 250-mL separatory funnel and the phases were allowed to

separate overnight. The bottom chloroform layer was collected through anhydrous sodium

sulfate to remove any remaining moisture. The lipid extract was then transferred to a 250-mL

round bottom flask and concentrated with a Büchi Rotovapor R-210 using a V-700 vacuum

pump connected to a V-850 vacuum controller (Büchi Corporation, New Castle, DE) at 35oC and

25 kPa. Collected lipids were transferred to a 20-mL amber vial and the chloroform was

evaporated off with an N-EVAP 111 nitrogen evaporator (Organomation Associates, Inc. Berlin,

MA). Lipids were extracted in triplicates and were stored under a nitrogen headspace at -80oC to

retard lipid oxidation.

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Fatty Acid Methylation

Fatty acid profiles of egg lipids were determined by GC-FID after converting

triacylglycerols to their respective FAME (Christie, 1989). Approximately 80 mg of extracted

lipids were weighed into a 5-mL Reacti-vialTM (Pierce, Rockford, IL). Then, 100 µL of 20%

(w/v) triheptadecanoin in hexanes were added to each Reacti-vialTM as an internal standard. The

triacylglycerols were then hydrolyzed and derivatized with 2-mL transmethylation reagent

consisting of 6% (v/v) concentrated H2SO4 in anhydrous methanol with ~5 mg of hydroquinone.

Each Reacti-vialTM was incubated at 65°C overnight with stirring on a Pierce Reacti-Therm III

incubating block (Pierce, Rockford, IL, USA). The following day samples were removed from

the incubator and allowed to cool to room temperature. Then, 1 mL of deionized water was

added to each Reacti-vialTM and the contents were vortexed for ~30 s. FAMEs were extracted 3x

with 1.5-mL aliquots of hexanes and subsequently washed 2x with 1.5 mL of deionized water.

The extracted FAMEs were collected in a clean test tube and then dried under nitrogen gas using

the N-EVAP nitrogen evaporator. The FAMEs were dissolved in 3 mL of carbon disulfide, a

100-μL aliquot was transferred to a 2-mL amber GC vial and diluted with 900-μL carbon

disulfide; the vial was then capped and crimped for GC analysis.

Gas Chromatographic Analysis

FAMEs from diet rations and egg samples were analyzed for trans fatty acids using an

Agilent Technologies 6890N Network GC System with capillary split/splitless inlet with

electronic pneumatics control (EPC) and a flame ionization detector (FID) for packed and

capillary columns (Agilent Technologies, Wilmington, DE). A 100-m, highly polar,

biscyanopropyl column with an inner diameter of 0.25 mm and a film thickness of 2 μm (SP-

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2560, Supelco, Bellefonte, PA) was employed. The initial oven temperature was held at 140oC

for 5 min then ramped at 5oC/min to a final temperature of 240oC, which was maintained for 15

min resulting in a total runtime of 45 min. The injector temperature was set at 250oC. An

Agilent 7683 auto-sampler tray module equipped with a 7683B auto-injector module and a 10-

μL syringe was employed for injecting 1-μL samples through a pre-pierced 11-mm inlet septum

(P/N: 5183-4761-100) into the GC inlet containing a split liner packed with glass wool (P/N:

5183-4647). The GC was operated in split mode at a split ratio of 50:1 with helium as the carrier

and gas flow at 59.2 mL/min. The column pressure was 276.62 kPa. The detector was heated to

250oC and the FID flame was generated from hydrogen at a flow of 40 mL/min, air at 450

mL/min, and a nitrogen makeup gas flow of 23.9 mL/min. A Supelco 37 component FAME

mixture was used to identify and quantify individual FAMEs by retention time mapping. A

relative response factor was calculated for each FAME using methyl heptadecanoate as an

internal standard. Response factors are the concentration of an analyte divided by the response

to that analyte.

Ri = Wsi / Psi

where, Ri = response factor for FAME i; Wsi = mg of FAME i injected into the inlet; and Psi =

peak area corresponding to FAME i.

A relative response factor is defined as the response factor of the internal standard divided by the

response factor of the analyte.

RRfi = Rfis/Rfi

where, RRfi = relative response factor for FAME i; Rfis = response factor for the internal

standard; and Rfi = response factor for FAME i. Each FAME will have a different response to

the FID depending on chain length, saturation, and cis/trans configuration. For positively-

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identified compounds, employment of relative response factors improves the accuracy of the

data. For unidentified compounds the relative response factor for the closest positively identified

fatty acid was used.

Silver-Ion Thin Layer Chromatography (Ag+-TLC)

Silver Ion TLC provides a means to separate cis/trans isomers, and to increase the

resolution of trans fatty acids by GC-FID. Trans-fat isomers of C18:1 may be coeluted or

appear as shoulders off the much larger oleic acid peak. By eliminating the cis components from

the chromatogram all trans peaks are able to be visualized. A 20 x 20 cm 250-µm silica G TLC

plate was submerged into 20% (w/v) AgNO3 in CH3CN for 20 min. Before analysis, the silica

plate was activated at 120oC for 1 h. One hundred microliters of FAMEs were spotted as a long

contiguous band across the origin on the silica plate and developed in hexanes:diethyl ether

(80:10, v/v). The plates were sprayed with 0.1% (w/v) 2',7'-dichlorofluorescein in ethanol and

viewed under UV light to visualize the bands. The band corresponding to the eluted trans

FAMEs was scraped off the plate and the FAMEs were extracted 2x with 25-mL aliquots of

diethyl ether. The ether was washed 2x with water to remove any residual silica, and then dried

using the N-EVAP system to remove the organic solvent. Subsequently, 25 µL of methyl

heptadecanoate, as an internal standard and 0.5-mL carbon disulfide were added to the isolated

trans fraction, followed by GC-FID analysis as already described.

Linearity: Gas Chromatography

The linearity of the peak area response versus concentration for the standard

triheptadecanoin was studied from 75-600 μg/mL. The calibration curve was generated with

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eight different concentrations (i.e., 75, 150, 225, 300, 375, 450, 525, 600 μg/mL) using the least-

squares method.

Precision and Accuracy: Gas Chromatography

Assay precision was determined by repeatability (intra-day) and intermediate precision

(inter-day). Intra-day was evaluated by assaying samples at the same concentration and during

the same day. The inter-day was studied by comparing the assay on three different days

(Demirkaya and Kadioglu, 2007). The accuracy of this analytic method was evaluated by

checking three different concentrations of triheptadecanoin.

Limit of Detection (LOD) and Limit of Quantification (LOQ): Gas Chromatography

LOD and LOQ were determined by an empirical method that consisted of analyzing a

series of standard solutions containing decreasing amounts of triheptadecanoin. The LOD and

LOQ were defined as having a signal-to-noise (S/N) ratio of 3 to 1 and 10 to 1, respectively

(Bononi and Tateo, 2008).

Recovery: Gas Chromatography

The analytical recovery of triheptadecanoin was assessed by direct comparison of

concentrations of triheptadecanoin to methyl heptadecanoate. Three replicates at four different

concentration levels (i.e., 75, 150, 300, and 600 μg/mL) was employed.

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Analysis by Fourier Transform Infrared Spectroscopy

Extracted lipids from diet and egg samples were transmethylated according to AOCS

official method Ce 2-66. This method was used for FTIR analysis because it was completed in a

shorter amount of time, thereby, allowing the FTIR method to maintain its rapidity in analyzing

trans-fats. Moreover, a greater quantity of sample can be transmethylated by this method, which

is important, because a larger quantity of sample is needed for FTIR analysis compared with GC

analysis. One gram of egg lipids was transferred to a 250-mL flat bottom flask. Then, 10 mL of

0.5 N NaOH in anhydrous methanol and several glass boiling beads were added to the flask,

which was then connected to a condenser. The mixture was refluxed for 10 min, forming free

fatty acids from triacylglycerols. A subsequent 12-mL addition of 14% (v/v) boron trifluoride in

methanol, as the transmethylating reagent, was made through the top of the condenser. After 2

min, 5 mL of hexanes were introduced and the mixture was refluxed for an additional minute.

The contents were transferred to a 250-mL separatory funnel where FAMEs were extracted 2x

with 50-mL aliquots of hexanes. The organic layer was passed through anhydrous sodium

sulfate to remove any moisture and collected in a 100-mL round bottom flask. The hexanes were

evaporated off using the Büchi Rotovapor system, and solvent residue was removed under a

stream of nitrogen with the N-EVAP.

A 300-μL aliquot of FAMEs was pipetted onto a Thermo Smart Ark Horizontal

Attenuated Total Reflectance (ATR) cell installed in a Nicolet 6700 FTIR spectrophotometer

(Thermo Fisher Scientific, Rockford, IL). The ATR cell was maintained at a temperature of

45oC to ensure samples were in a liquid state. The ATR cell was cleaned with a dustless wipe

prior to each sampling. Thirty two scans over a wavenumber range of 4000 to 400 cm-1 were

taken at a resolution of 4 cm-1. A new background was collected prior to each sample, and the

25

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results were expressed as absorbances. A series of standards were made by accurately weighing

proportions of methyl elaidate and methyl oleate ranging from 0.5 to 10% methyl elaidate by

weight. Using these standards, a calibration curve was generated using the TQ Analyst software

program. Partial least squares regression analysis of second derivative spectra in the

wavenumber range of 978 cm-1 to 955 cm-1 was employed to regress the standards. The Norris

derivative filter was used to smooth the spectra, reducing random noise, thus decreasing the

random error in the calibration model.

Linearity: Fourier Transform Infrared Spectroscopy

The linearity of the peak area versus %trans-fat per g total fat for mixtures of methyl

elaidate and methyl oleate ranging from 0.5 to 10% methyl elaidate by weight was determined.

The calibration curve was generated with eight percentage proportions (0.5, 1, 2, 3, 4, 5, 6, 7, 8,

9, and 10%) using the least-squares method.

Precision, Accuracy, and Limit of Detection (LOD): Fourier Transform Infrared

Spectroscopy

Assay precision was determined by repeatability (intra-day) and intermediate precision

(inter-day). Intra-day was evaluated by assaying samples, at the same concentration and during

the same day. The inter-day was studied by comparing the assay on three different days

(Demirkaya and Kadioglu, 2007). The accuracy of this analytic method was evaluated by

assessing two different concentrations of methyl elaidate in methyl oleate (5 and 8%).

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Statistical Methods

Eggs were collected at 7, 14, and 21 days after initiation of the experimental diets. The

eggs for a diet were pooled on each day and analyzed in triplicate (n=9). The experimental unit

was the hen, and the measurement unit was the pool of five egg yolks. A one-way ANOVA for

egg composition and diet was performed using Minitab Statistical Software. Tukey’s multiple

range test, with a family error rate of five, was employed for the comparison of fatty acid profiles

in the diets to determine if significant (p < 0.05) differences existed.

Results and Discussion

The weights of whole eggs (g) including the shell, the whole liquid egg, the egg white,

and the egg yolk were recorded every week for each diet. Over the three-week period, the

average weights of the shells, egg whites, and yolks were recorded and found not to be

statistically (p > 0.05) different amongst the test diets. The average whole egg weight did not

vary over the three-week collection period, but was statistically (p=0.001) different between hens

fed different diets. Egg weights from hens fed diets I, II, III, and IV were not statistically (p >

0.05) different. This, however, was not the case for diet V; egg weights from hens fed diet V

showed the lowest average egg weight of 59.05 g. A summary of egg weights is provided in

Table 1.

Gas Chromatography

Figures 2 through 11 depict GC-FID chromatograms of egg yolk lipid extracts (i.e., after

derivatization to FAMEs) from hens fed diets I-V as well as chromatograms for each of the five

trial diets. The chromatograms were completely resolved (i.e., with excellent resolution between

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peaks, Rs > 2.0), and in some cases fully resolving the cis/trans isomers. The baseline was very

stable even with a temperature ramp of 140 to 240oC. The peaks indicate a Gaussian distribution

thereby indicating that the column was not overloaded with the sample.

A Supelco 37 component FAME mixture was used to identify and quantify individual

FAMEs by retention time mapping. Each of the 37 components in the standard was identified

and their retention times recorded. Samples’ FAMEs retention times were compared to those of

the Supelco 37 component FAME mixture. Table 2 lists the retention times, response factors

and relative response factors for each of the 37 compounds in the standard. Unidentified peaks

were tentatively identified based by retention times when compared to positively-identified

FAMEs. Peak 3, as shown in Figure 5, was not included in the Supelco 37 component FAME

mixture but eluted between palmitic acid, C16:0 (peak 2), and palmitoleic acid, C16:1 n-7 (peak

4), it is suspected that this peak corresponds to an isomer of palmitoleic acid, C16:1 n-9.

Silver-ion TLC was used to separate trans-monounsaturated FAMEs from cis-

monounsaturated FAMEs. The Rf value for cis-monounsaturated FAMEs was 0.28, whereas the

Rf value for trans-monounsaturated FAMEs was 0.34, and for saturated FAMEs it was 0.43.

The GC-FID chromatogram of the trans isomers contained in shortening compared with its full

fatty acid profile and is depicted in Figure 12. This figure indicates that a very small portion

(i.e., < 3% of all trans fatty acids present in the sample) of trans FAMEs coeluted with the oleic

acid peak. This chromatogram is consistent for all lipids containing trans fatty acids from this

study. Figure 13 identifies the five trans fatty acid peaks present at detectable levels from GC-

FID chromatograms analyzed by Juanéda and coworkers (Juanéda and others 2007). The first

peak, 6t-10t C18:1, eluted as a shoulder off the 11t C18:1 peak. This was followed by the

elution of the isomers 12t C18:1, 13t-14t C18:1, and finally 15t C18:1, which coeluted with the

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oleic acid peak in the full chromatogram. The major trans fatty acid in the sample was found to

be 11t C18:1 (vaccenic acid). Therefore, all fatty acids eluting directly before oleic acid are

trans fatty acids, while those eluting afterwards are cis isomers of C18:1. For calculations of

total trans-fats present, the peak areas of 6t-14t C18:1 were used.

The trans-fat contents of diet I (i.e., standard corn/soy-laying ration with 0% added

fat/oil) and diet II (i.e., standard corn/soy-laying ration + 4% (w/w) soybean oil) were below the

detection limit for the GC-FID system. It, however, cannot be assumed that the diets were

devoid of all trans-fats. Not unexpectedly, the trans-fat contents of egg samples collected from

the hens fed either diet I or II for 21 days were also below the limit of detection of the GC

system. Stating this another way: hens fed diets devoid of detectable levels of endogenous

trans-fats, produced eggs that did not contain detectable levels of trans-fats. Unlike diets I and

II, diets III, IV, and V contained some level of fats known to come from the partial

hydrogenation process of edible oils. The trans-fat levels in diets III, IV, and V (i.e., standard

corn/soy-laying ration + 4% tallow, shortening, and tallow-shortening blend, respectively) were

found to contain 2.78 ± 0.08, 3.25 ± 0.03, and 2.85 ± 0.05 g trans-fat/100-g dietary fat,

respectively, whereas, the egg yolk from hens fed these diets showed only 3.33 ± 0.32, 4.16 ±

0.03, and 3.52 ± 0.05 g trans-fat/100-g dietary fat, respectively. Therefore, approximately one

quarter of the trans-fats available in each ration was deposited in the egg yolks of hens

consuming this feed. The percentages correspond to under 0.1% trans-fats (g trans-fat/g whole

egg) in a serving of eggs. It was observed that eggs contained similar proportions of both elaidic

acid (9t C18:1) and vaccenic acid (11t C18:1) in the yolks despite a much higher concentration

of vaccenic acid in the diet. This may be a metabolic consequence, as desaturation of vaccenic

acid is seen in humans.

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Over 98% by weight of the lipids in the recovered lipid extracts comprised six fatty acids,

namely myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1 n-7), stearic acid

(C18:0), oleic acid (C18:1 n-9), linoleic acid (C18:2 n-6), γ-linolenic acid (C18:3 n-6), and

arachidic acid (C22:0). Of these, oleic acid is the dominant fatty acid contributing to greater than

40% (g/total g lipid). The weight percentages of the aforementioned fatty acids for each diet are

shown in Table 3, while saturated, monounsaturated, polyunsaturated, and trans-fatty acids are

summarized in Table 4. The myristic acid content was the highest, at 0.6% (w/w), in hens fed

diets III, IV, and V; diets III, IV, and V each contained approximately 2% myristic acid. A

summary of the fatty acid profile of the feeds is reported in Table 5. Although diets I and II

possessed no detectable levels of myristic acid, in egg yolks, a myristic acid content of 0.43%

was detected. Palmitic acid, on the other hand, remained relatively constant at around 26.2% and

there was no correlation determined between how much palmitic acid was present in the feed.

Eggs from hens fed diets I, IV, and V contained the highest levels of palmitic acid, 26.9, 26.4,

and 26.5% respectively, and were statistically (p < 0.01) different from eggs of hens fed diets II

and III, which contained 25.3 and 25.9% palmitic acid, respectively. Stearic acid showed the

opposite relationship towards diets compared to palmitic acid; that is, eggs from hens fed diets I,

IV, and V contained the least amount of stearic acid (i.e., 9.2, 9.0, and 9.0%), while eggs from

hens fed diets II and III contained the highest percentage of stearic acid (i.e., 10.4 and 9.7%).

Arachidic acid varied from 1.8% in eggs from hens fed diet V, to 2% in eggs from hens fed diets

II, III, and IV, and to 2.3% in eggs from hens fed diet I. None of the feeds contained a detectable

level of arachidic acid. Even though there were variations in the levels of the four primary

saturated fats – namely myristic, palmitic, stearic, and arachidic acid – a relatively constant

amount of saturated fatty acids, 38.1 ± 0.46%, of total fat resulted.

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Egg yolks contained low levels of palmitoleic acid ranging from 1.8% in eggs from hens

fed diet II, to 2.5% in eggs from hens fed diet III, to 2.9% in eggs from hens fed diets IV and V,

and finally to 3.2% in eggs from hens fed diet I. Oleic acid constitutes the major portion of

lipids in egg yolks. As the oleic acid content in the feed increased (i.e., 20.5, 21.1, 27.6, 29.2, and

29.6% for diets II, I, IV, V, and III, respectively), the oleic acid levels in the eggs also

systematically increased (i.e., 40.0, 43.0, 45.9, 46.9, and 47.6% for diets II, I, IV, V, and III,

respectively). Monounsaturated fatty acids showed a similar pattern as that of oleic acid likely

because oleic acid constitutes the bulk of the monounsaturated fatty acids.

Linoleic acid showed the most variation amongst the diets and eggs produced. Diets I

and II contained 54% linoleic acid and the hens gave eggs with a linoleic acid content ranging

from 14.5 to 18.8%. Eggs from hens fed diets III, IV, and V, which contained only 25% linoleic

acid, were found to have 10.2, 11.3, and 10.7% linoleic acid contents, respectively. Furthermore,

all eggs contained very small quantities of γ-linolenic acids ranging from 0.2 to 1.0%. Note,

however, that no α-linolenic acid (C18:3 n-3) was detected in any of the diets or eggs of this

study. The quantities of polyunsaturated and monounsaturated fats in the eggs varied depending

on how much of each was contained in the hens feed.

The GC method was evaluated for the recovery of FAMEs from triacylglycerols during

analysis, linearity, precision and accuracy, as well as LOD and LOQ. The recovery of FAMEs

was analyzed by comparing the peak areas of triheptadecanoin to that of methyl heptadecanoate

at four different concentrations (i.e., 75, 150, 300, and 600 μg/mL). It was determined that

triheptadecanoin had a recovery of only 81.33 ± 1.5% compared to methyl heptadecanoate which

had a 100% recovery; a summary of this data is presented in Table 6. It can be assumed that if

the triheptadecanoin had only an 81% recovery, then the triacylglycerols from the lipid extracts

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should also have a similar recovery. Based on this reasoning, triheptadecanoin was deemed to be

the better choice of an internal standard for GC analysis and was so used when applicable. The

linearity of triheptadecanoin was assessed by generating a calibration curve using eight different

concentrations of the standard (i.e., 75, 150, 225, 300, 375, 450, 525, 600 μg/mL) and the least-

squares method. The correlation coefficient, r, was found to be 0.999 (n = 24), the slope was

0.4033, and the intercept was -13.441. The method was analyzed for intra-day precision

[%relative standard deviation (%RSD)], and accuracy [percent relative error]. The %RSD was

0.86, 5.54, and 1.11%, whereas the percent relative error was -5.09, -3.32, and 5.40%, for

triheptadecanoin concentrations of 160, 320, and 520 μg/mL, respectively. The inter-day

precision was determined to be 8.64, 6.15, and 4.92%, while the inter-day accuracy was -11.03, -

4.71, -0.41%, for triheptadecanoin concentrations of 160, 320, and 520 μg/mL, respectively.

Table 7 summarizes the precision and accuracy results for GC analyses. The LOQ, defined as

the lowest concentration of measurable value of a standard solution of triheptadecanoin, was

determined to be 6 μg/mL while the LOD was approximately 3 μg/mL.

Fourier Transform Infrared (FTIR) Spectroscopy

Methods have been developed to determine trans-fat percent using neat oils mainly from

edible oil processing industries (Sherazi and others 2009). These methods have been examined

for use in identifying the trans-fat content in diet and egg samples. Initially, extracted egg lipids

were employed as such, which eliminated the need for their derivatization to FAMEs. The

extracted lipids, however, showed an interference band at the wavenumber = 966 cm-1 (Figure

14), which interferes with the quantification of the trans-fats. Therefore, the extracted lipids

were transmethylated to FAMEs in an effort to either reduce or eliminate the interference band.

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Transmethylated samples showed no interference at the characteristic trans absorption band of

966 cm-1.

Figures 15 and 16 depict the trans band at the 966 cm-1 of prepared samples as the second

derivative of absorbance versus wavenumber and absorbance versus wavenumber, respectively.

The trans band can be visualized better by taking the second derivative of absorbance

mathematically. This action removes the sloping baseline in the FTIR spectra; thus, making area

measurements more accurate at lower trans-fat levels and at the same time improves the

detection limit of the assay (Mossoba and others 2007a).

Figure 17 shows the results of the TQ Analyst software for the partial least squares (PLS)

regression with and without taking the second derivative of absorbance readings. It can be seen

that the PLS curve, using the second derivative spectrum, affords slightly less variance below 2%

trans-fat levels. This provides greater accuracy in the detection of trans-fat of samples that

possess trans-fat levels below 2%.

The trans-fat content of each diet, as well as pure tallow and shortening, was determined

by FTIR spectroscopy. Diets III, IV, and V had trans-fat concentrations of 3.33 ± 0.32, 4.16 ±

0.03, and 3.52 ± 0.05 g trans-fat/100-g dietary fat, respectively, while pure tallow and shortening

were measured as containing 5.44 ± 0.19 and 7.79 ± 0.56 g trans-fat/100-g dietary fat,

respectively. The intra-day precision for the FTIR method was 0.21 and 0.14%, and the intra-

day accuracy was -3.90 and 4.08% for concentrations of 5.11 and 7.88% methyl elaidate in

methyl oleate, respectively. Inter-day precision was determined to be 0.31% at both

concentrations whereas inter-day accuracy was -2.68 and -2.26% for concentrations of 5.11 and

7.88% methyl elaidate in methyl oleate, respectively. Precision and accuracy results are shown

in Table 8. The linearity was assessed using 11 different concentrations of methyl elaidate (i.e.,

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0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10%). The correlation coefficient, r, was found to be 0.995 (n =

22), the slope was 0.1016, and the intercept was 0.0104. Figure 15 depicts the absorbance of

trans band at the 966 cm-1 wavenumber of prepared standards of methyl elaidate in methyl

oleate. It can also be noted that below 1% trans-fat, the baseline has a significant amount of

noise associated with it compared to the trans peak signal, thus, making accurate measurements

of the peak area quite difficult. The LOD and LOQ for this FTIR spectroscopic method were

determined to be 1 and 2.4%, respectively.

The FTIR results were compared to those obtained by GC-FID and an attempt was made

to establish a correlation between these two analytical techniques. The FTIR data for trans-fat

content in egg yolk lipid extracts was below the detection limit of the method. FTIR methods

have been cited as having limits of detection of 1% trans-fat per gram total fat. A correlation

coefficient of 0.988 was determined when comparing GC-FID and FTIR data of the hen diets

and pure fat samples, as depicted in Figure 18. No correlation could be made using egg yolk

data because the % trans-fat could not be quantified by the FTIR method.

Conclusions

Based on these findings, approximately 25% of the trans-fat available in the ration was

deposited into the egg yolks giving trans-fat levels of about 0.1 g/100 g whole egg. Using fats

and oils with high levels of trans-fat, such as grease from restaurants, rendering of animal

carcasses, and by-products from vegetable oil refining, to supplement laying hen feed will not

result in appreciable levels of trans-fat in the hens’ eggs. However, public perception of this

practice (i.e., supplementing rations with trans-fat) is not favorable. The type of fat

supplemented into the feed should be chosen knowing that the diet can significantly effect the

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lipid composition of the egg mainly in its proportions of mono-to-polyunsaturated fatty acids. In

this study, saturated fatty acid levels remained relatively constant.

GC-FID analysis is a more sensitive technique at detecting low concentrations of trans-

fats or fatty acids in general compared with FTIR spectroscopy. It also provides information

about the types of fatty acids contained in the egg yolk, whereas FTIR analysis gives only the

percentage of trans-fat within the lipid extract. The FTIR method provided more precise and

accurate measurements of trans-fat at elevated concentrations compared to GC-FID. If extremely

low levels of trans-fat are not a concern, as is the case for nutrition labeling purposes (NB, if the

trans-fat content is less than 0.5 g per serving, then it is indicated as 0 g on the label), then the

FTIR approach offers a rapid and cost-effective means of determining trans-fat contents in

foodstuffs.

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Services HaH. 2003. Food labeling; trans fatty acids in nutrition labeling; consumer research to

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in relation to risk of type 2 diabetes in men. Diabetes Care 25:417-424.

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List of Tables

Table 1: Egg weights (g) of hens fed diets of varying levels of trans-fat for 21 days

Day

Diet*† 7 14 21

Ia 60.738 ± 3.12 62.30 ± 3.55 62.06 ± 4.42

IIa 61.327 ± 5.40 61.89 ± 4.56 63.30 ± 5.14

IIIa 59.123 ± 4.83 59.07 ± 4.23 61.50 ± 4.94

IVa 63.009 ± 3.89 62.27 ± 3.11 63.16 ± 4.12

Vb 57.945 ± 2.81 60.09 ± 3.80 59.11 ± 3.12 †Mean ± standard deviation (n = 12)

* Diet I – standard corn/soy-laying ration with 0% added fat/oil; Diet II – Standard corn/soy laying ration with 4% added soybean oil;

Diet III – Standard corn/soy laying ration with 4% added tallow; Diet IV - Standard corn/soy laying ration with 4% added shortening;

Diet V - Standard corn/soy laying ration with 4% added tallow/shortening blend

.a,b,c - Diets with different superscripts were significantly different (p<0.05) as determined by Tukey’s test

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Table 2: Response factors of FAMEs as determined by GC-FID using Supelco 37 component FAME mixture

Peak # RT* Compound Name Response Factor† Relative Response Factor‡

1 10.46 Methyl Butyrate 0.0042 0.53 2 11.06 Methyl Hexanoate 0.0030 0.73 3 12.12 Methyl Octanoate 0.0027 0.81 4 13.89 Methyl Decanoate 0.0025 0.88 5 15.08 Methyl Undecanoate 0.0024 0.92 6 16.45 Methyl Dodecanoate 0.0024 0.94 7 17.97 Methyl Tridecanoate 0.0023 0.98 8 19.59 Methyl Myristate 0.0022 0.99 9 21.04 Methyl Myristoleate 0.0023 0.97 10 21.24 Methyl Pentadecanoate 0.0021 1.04 11 22.71 Methyl cis-10-pentadecenoate 0.0023 0.97 12 22.91 Methyl Palmitate 0.0022 1.03 13 24.12 Methyl Palmitoleate 0.0022 1.00 14 24.54 Methyl Heptadecanoate 0.0022 1.00 15 25.72 Methyl cis-10-Heptadecenoate 0.0022 1.02 16 26.11 Methyl Stearate 0.0021 1.05 17 26.90 Methyl Elaidate 0.0021 1.05 18 27.13 Methyl Oleate 0.0021 1.04 19 27.92 Methyl Linoleaidate 0.0022 1.01 20 28.62 Methyl Linoleate 0.0022 1.03 21 29.09 Methyl Arachidate 0.0021 1.06

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22 29.72 Methyl y-Linolenate 0.0022 1.00 23 30.03 cis-11-Eicosenoic Acid Methyl Ester 0.0021 1.08 24 30.30 Methyl Linolenate 0.0022 1.01 25 30.49 Methyl Heneicosanoate 0.0020 1.10 26 31.47 cis-11,14-Eicosadienoic Acid Methyl Ester 0.0022 1.03 27 31.92 Methyl Behenate 0.0021 1.08 28 32.58 cis-8,11,14-Eicosatrienoic Acid Methyl Ester 0.0023 0.98 29 32.87 Methyl Eurcate 0.0021 1.04 30 33.16 cis-11,14,17-Eicosatrienoic Acid Methyl Ester 0.0025 0.90 31 33.32 Methyl Arachidonate 0.0020 1.09 32 33.46 Methyl Tricosanoate 0.0022 1.00 33 34.36 cis-13,16-Docosadienoic Acid Methyl Ester 0.0021 1.05 34 34.80 Methyl Tetracosanoate 0.0021 1.08 35 35.34 cis-5,8,11,14,17--Eicosapentaenoic Acid Methyl Ester 0.0023 0.97 36 35.84 cis-15-Tetracosenoate 0.0020 1.09 37 39.89 cis-4,7,10,13,16,19-Docosahexanoic Acid Methyl Ester 0.0024 0.91

*RT – Retention time

†Response factor: Ri = Wsi / Psi where, Ri = response factor for FAME i; Wsi = mg of FAME i injected into the inlet; and Psi = peak

area corresponding to FAME i.

‡ Relative response factor: RRfi = Rfis/Rfi where, RRfi = relative response factor for FAME i; Rfis = response factor for the internal

standard; and Rfi = response factor for FAME i.

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Table 3: Fatty acid profile of egg yolk lipids from hens consuming diets with varying levels of trans-fat

Fatty acid Diet* % Fatty acid† Fatty acid Diet* % Fatty acid†

Ia 0.48 ± 0.01 Ia,b 9.16 ± 0.48 IIb 0.49 ± 0.04 IIc 10.70 ± 0.19 IIIc 0.59 ± 0.06 IIIa 9.72 ± 0.66 IVc 0.62 ± 0.02 IVb 8.96 ± 0.34

Myristic Acid

(C14:0)

Vc 0.63 ± 0.03

Stearic Acid (C18:0)

Vb 8.99 ± 0.57

Ia 26.83 ± 0.48 Ia 2.30 ± 0.08 IIb 25.29 ± 0.24 IIb,c 2.07 ± 0.09

IIIb,c 25.85 ± 0.63 IIIb 1.97 ± 0.15 IVc 26.33 ± 0.39 IVb 1.97 ± 0.06

Palmitic Acid

(C16:0)

Va,c 26.40 ± 0.52

Arachidic Acid

(C20:0)

Vc 1.82 ± 0.10

Ia 3.16 ± 0.24 Ia 14.51 ± 0.27 IIb 1.85 ± 0.02 IIb 17.94 ± 0.33 IIIc 2.49 ± 0.33 IIIc 10.20 ± 0.57 IVd 2.93 ± 0.14 IVd 11.28 ± 0.38

Palmitoleic Acid

(C16:1 n-7)

Vd 2.86 ± 0.10

Linoleic Acid (C18:2 n-6)

Vc,d 10.72 ± 0.42

Ia 42.90 ± 0.62 Ia 0.44 ± 0.03 IIb 40.04 ± 0.20 IIb 0.92 ± 0.05 IIIc 47.44 ± 1.22 IIIc 0.27 ± 0.03 IVd 45.74 ± 0.67 IVa 0.39 ± 0.12

Oleic Acid (C18:1 n-9)

Vc,d 46.73 ± 0.74

γ-Linolenic Acid

(C18:3 n-6)

Vc 0.30 ± 0.02

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†Mean (g fatty acid / 100 g total lipid) ± standard deviation (n = 12)

* Diet I – standard corn/soy-laying ration with 0% added fat/oil; Diet II – Standard corn/soy

laying ration with 4% added soybean oil; Diet III – Standard corn/soy laying ration with 4%

added tallow; Diet IV - Standard corn/soy laying ration with 4% added shortening; Diet V -

Standard corn/soy laying ration with 4% added tallow/shortening blend

.a,b,c - Diets with different superscripts were significantly different (p<0.05) as determined by

Tukey’s test

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Table 4: Percentage of fatty acids in egg yolk lipids from hens consuming diets with varying levels of trans-fat

Fatty acid Diet* % Fatty acid† Fatty acid Diet* % Fatty acid†

Ia 0.00 ± 0.00 Ia 46.07 ± 0.42

IIa 0.00 ± 0.00 IIb 41.63 ± 0.54

IIIb 0.68 ± 0.05 IIIc 49.94 ± 0.97

IVb 0.81 ± 0.05 IVd 48.66 ± 0.63

Trans Fatty Acids

Vb 0.76 ± 0.04

Monounsaturated Fatty Acids

Vc 49.65 ± 0.73

Ia 38.90 ± 0.49 Ia 14.95 ± 0.28

IIb 38.54 ± 0.32 IIb 19.72 ± 0.75

IIIa,b 37.98 ± 0.21 IIIc 10.47 ± 0.60

Ivb 38.23 ± 0.39 IVd 11.67 ± 0.41

Saturated Fatty Acids

Vb 38.26 ± 0.73

Polyunsaturated Fatty Acids

Vc,d 11.01 ± 0.41

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†Mean (g fatty acid / 100 g total lipid) ± standard deviation (n = 12)

* Diet I – standard corn/soy-laying ration with 0% added fat/oil; Diet II – Standard corn/soy laying ration with 4% added soybean oil;

Diet III – Standard corn/soy laying ration with 4% added tallow; Diet IV - Standard corn/soy laying ration with 4% added shortening;

Diet V - Standard corn/soy laying ration with 4% added tallow/shortening blend

.a,b,c - Diets with different superscripts were significantly different (p<0.05) as determined by Tukey’s test

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Table 5: Fatty acid composition of feeds with and without added trans-fats

Feeds*

Fatty Acid Diet I Diet II Diet III Diet IV Diet V

Myristic acid (C14:0) 0.00 0.00 2.04 2.11 2.18

Palmitic acid (C16:0) 15.85 12.92 21.86 22.00 22.20

Palmitoleic acid (C16:1 n-7) 0.09 0.00 1.81 1.70 1.84

Stearic acid (C18:0) 2.78 3.79 11.57 12.39 12.22

Oleic acid (C18:1 n-9) 21.09 20.53 29.56 27.55 29.15

Linoleic acid (C18:2 n-6) 54.24 53.44 25.06 25.57 24.68

Arachidic acid (C20:0) 0.00 0.00 0.00 0.00 0.00

Trans fatty acid isomers (4t-16t C18:1)

0.00 0.00 2.78 3.25 2.85

* Diet I – standard corn/soy-laying ration with 0% added fat/oil; Diet II – Standard corn/soy laying ration with 4% added soybean oil; Diet III – Standard corn/soy laying ration with 4% added tallow; Diet IV - Standard corn/soy laying ration with 4% added shortening; Diet V - Standard corn/soy laying ration with 4% added tallow/shortening blend

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Table 6: Recovery of triheptadecanoin and methyl heptadecanoate during GC-FID analysis

Lipid Type Amount Injected (ng) Mass Recovered (ng) Percent Recovered Peak area

FAME* 600 600.07 100.01 370.70 FAME 300 298.82 99.61 183.40 FAME 150 151.59 101.06 91.87 FAME 75 74.55 99.40 43.97 TAG† 600 478.69 79.78 295.23 TAG 300 245.63 81.88 150.33 TAG 150 122.91 81.94 74.03 TAG 75 61.31 81.74 35.73

*FAME – Fatty Acid Methyl Ester

†TAG - Triacylglycerol

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Table 7: Precision and accuracy of trans-fat analysis using GC-FID

Concentration added* Intra-day (μg/mL) Observed Precision† Accuracy‡

160 151.85 ± 1.31 0.8640 -5.0939 320 309.47 ± 4.25 5.5438 -3.2919 520 548.08 ± 1.51 1.1098 5.4002 Concentration added * Inter-day (μg/mL) Observed Precision† Accuracy‡ 160 142.34 ± 12.31 8.6445 -11.0345 320 304.92 ± 18.76 6.1513 -4.7124 520 517.89 ± 25.46 4.9158 -0.4066

*Concentration of triheptadecanoin in CS2

†Precision as percent relative standard deviation (%RSD)

%RSD = 100 * standard deviation / mean

‡Accuracy as percent relative error

Percent relative error = 100 * (observed – added) / added

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Table 8: Precision and accuracy of trans-fat analysis using FTIR-ATR

Intra-day

%Methyl Elaidate* Observed Precision† Accuracy‡

5.11 4.91 ± 0.01 0.2139 -3.8990

7.88 7.56 ± 0.01 0.1377 -4.0812

Inter-day

%Methyl Elaidate* Observed Precision† Accuracy‡

5.11 4.97 ± 0.02 0.3084 -2.6791

7.88 7.70 ± 0.02 0.3088 -2.2632 *Methyl elaidate in methyl oleate

†Precision as percent relative standard deviation (%RSD)

%RSD = 100 * standard deviation / mean

‡Accuracy as percent relative error

Percent relative error = 100 * (observed – added) / added

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List of Figures

Figure 1: Partial hydrogenation reaction mechanism – Figure 1 shows the cis/trans

isomerization, double bond migration, and saturation reactions during partial hydrogenation of

oils.

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Figure 2: GC-FID Chromatogram of egg yolk lipid from diet I

1.Myristic Acid, 2. Palmitic Acid, 3. cis-9-Hexanoic acid, 4. Palmitoleic Acid 5. Heptadecanoic

Acid (IS), 6. Stearic Acid, 7. Oleic Acid, 8. cis-11-Octadecenoic Acid, 9. Linoleic Acid, 10.

Arachidic Acid

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Figure 3: GC-FID Chromatogram of egg yolk lipid from diet II

1.Myristic Acid, 2. Palmitic Acid, 3. cis-9-Hexanoic acid, 4. Palmitoleic Acid 5. Heptadecanoic

Acid (IS), 6. Stearic Acid, 7. Oleic Acid, 8. cis-11-Octadecenoic Acid, 9. Linoleic Acid, 10.

Linolenic Acid, 11. Arachidic Acid

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Figure 4: GC-FID Chromatogram of egg yolk lipid from diet III

1.Myristic Acid, 2. Palmitic Acid, 3. cis-9-Hexanoic acid, 4. Palmitoleic Acid 5. Heptadecanoic

Acid (IS), 6. Stearic Acid, 7. Trans Fatty Acids 8. Oleic Acid, 9. cis-11-Octadecenoic Acid, 10.

Linoleic Acid, 11. Arachidic Acid

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Figure 5: GC-FID Chromatogram of egg yolk lipid from diet IV

1.Myristic Acid, 2. Palmitic Acid, 3. cis-9-Hexanoic acid, 4. Palmitoleic Acid 5. Heptadecanoic

Acid (IS), 6. Stearic Acid, 7. Trans Fatty Acids 8. Oleic Acid, 9. cis-11-Octadecenoic Acid, 10.

Linoleic Acid, 11. Arachidic Acid

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Figure 6: GC-FID Chromatogram of egg yolk lipid from diet V

1.Myristic Acid, 2. Palmitic Acid, 3. cis-9-Hexanoic acid, 4. Palmitoleic Acid 5. Heptadecanoic

Acid (IS), 6. Stearic Acid, 7. Trans Fatty Acids 8. Oleic Acid, 9. cis-11-Octadecenoic Acid, 10.

Linoleic Acid, 11. Arachidic Acid

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Figure 7: GC-FID Chromatogram of diet I

1.Palmitic Acid, 2. Heptadecanoic Acid (IS), 3. Stearic Acid, 4. Linoleic Acid, 5 Oleic Acid, 6.

Linolenic Acid.

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Figure 8: GC-FID Chromatogram of diet II

1.Palmitic Acid, 2. Heptadecanoic Acid (IS), 3. Stearic Acid, 4. Linoleic Acid, 5 Oleic Acid, 6.

Linolenic Acid

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Figure 9: GC-FID Chromatogram of diet III

1. Myristic Acid, 2. Palmitic Acid, 3. Palmitoleic Acid 4. Heptadecanoic Acid (IS), 5. Stearic

Acid, 6. Trans Fatty Acids 7. Oleic Acid, 8 Linoleic Acid, 9. Linolenic Acid

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Figure 10: GC-FID Chromatogram of diet IV

1. Myristic Acid, 2. Palmitic Acid, 3. Palmitoleic Acid 4. Heptadecanoic Acid (IS), 5. Stearic

Acid, 6. Trans Fatty Acids 7. Oleic Acid, 8 Linoleic Acid, 9. Linolenic Acid

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Figure 11: GC-FID Chromatogram of diet V

1. Myristic Acid, 2. Palmitic Acid, 3. Palmitoleic Acid 4. Heptadecanoic Acid (IS), 5. Stearic

Acid, 6. Trans Fatty Acids 7. Oleic Acid, 8 Linoleic Acid, 9. Linolenic Acid

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Figure 12: Overlay of silver ion-TLC chromatogram and original chromatogram for shortening.

Figure 2 shows (1) the resulting chromatogram of silver ion-TLC isolation of trans fatty acids

for shortening and the full fatty acid profile in the same region.

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Figure 13: Chromatogram of silver ion-TLC separation of trans monounsaturated fraction of

shortening

1. 6t-10t C18:1, 2. 11t C18:1, 3. 12t C18:1, 4. 13t-14t C18:1, 5. 15t C18:1

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Figure 14: Interference band of egg lipids during FTIR analysis - Egg lipids were

transmethylated to remove the interference band at 967 cm-1 seen in (2) extracted egg lipids

compared to the spectra of (1) prepared FAMEs of extracted egg lipids.

80

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81

Page 92: EVALUATION OF GAS CHROMATOGRAPHIC AND FOURIER … · index words: trans-fat, gc-fid, ftir-atr, silver-ion tlc, scwl hen. evaluation of gas chromatographic and fourier transform infrared

Figure 15: Second derivative spectra of absorbance of trans band at 967 cm-1 - Prepared

standards of 0.5 to 10% methyl elaidate in methyl oleate. (1) 10; (2) 9; (3) 8; (4) 7; (5) 6; (6) 5;

(7) 4; (8) 3; (9) 2; (10) 1; (11) 0.5% methyl elaidate.

82

Page 93: EVALUATION OF GAS CHROMATOGRAPHIC AND FOURIER … · index words: trans-fat, gc-fid, ftir-atr, silver-ion tlc, scwl hen. evaluation of gas chromatographic and fourier transform infrared

A

bsor

banc

e (2

nd d

eriv

ativ

e)

Wavenumbers (cm-1)

83

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Figure 16: Spectra of absorbance of trans band at 967 cm-1 - Prepared standards of 0.5% to

10% methyl elaidate in methyl oleate. (1) 10%; (2) 9%; (3) 8%; (4) 7%; (5) 6%; (6) 5%; (7) 4%;

(8) 3%; (9) 2%; (10) 1%; (11) 0.5% methyl elaidate.

84

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85

Page 96: EVALUATION OF GAS CHROMATOGRAPHIC AND FOURIER … · index words: trans-fat, gc-fid, ftir-atr, silver-ion tlc, scwl hen. evaluation of gas chromatographic and fourier transform infrared

Figure 17: TQ Analyst calibrations of prepared standards of 0.5% to 10% (w/w) methyl elaidate

in methyl oleate - Comparison of TQ Analyst calibration curve of trans standards using the

second derivative of absorbance versus using absorbance.

86

Page 97: EVALUATION OF GAS CHROMATOGRAPHIC AND FOURIER … · index words: trans-fat, gc-fid, ftir-atr, silver-ion tlc, scwl hen. evaluation of gas chromatographic and fourier transform infrared

87

Page 98: EVALUATION OF GAS CHROMATOGRAPHIC AND FOURIER … · index words: trans-fat, gc-fid, ftir-atr, silver-ion tlc, scwl hen. evaluation of gas chromatographic and fourier transform infrared

Figure 18: Correlation of GC versus FTIR data – Fig. 17 shows a strong correlation between the

results obtained by GC-FID compared with FTIR

88

Page 99: EVALUATION OF GAS CHROMATOGRAPHIC AND FOURIER … · index words: trans-fat, gc-fid, ftir-atr, silver-ion tlc, scwl hen. evaluation of gas chromatographic and fourier transform infrared

y = 1.2735x - 1.8486R2 = 0.9759

0

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5 6 7 8 9

g trans -fat / 100 g total fat by FT-IR

g tra

ns-fa

t / 1

00 g

tota

l fat

by

GC

-FID

89


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