Analysis of Fatty Acid Methyl Esters in Egg Yolk Using GC-MS
Analysis of Fatty Acid Methyl Esters in Egg Yolk Using GC-MS
Analysis of Fatty Acid Methyl Esters in Egg Yolk Using GC-MS
Recently fatty acid methyl esters (FAMEs) have been in the In the experiment described here, students determine the
news because they are the chemicals in biodiesel. Articles in this relative fatty acid content of egg yolk lipids, in both store-brand
Journal have addressed biodiesel as an alternative, renewable, fuel large eggs and large eggs from a commercial supplier of eggs
(1–5) and have described the process of creating, identifying, stated to be higher in PUFA, using GC–MS. This experiment
and measuring FAMEs from natural sources, including vegetable could also be used in an instrumental analysis or food science
oil (3, 6–8), olive oil (4), peanut oil (2, 6), nutmeg (6), bacteria laboratory course.
(9), leaves (10, 11), and eggs (12–14). Previous articles in this
Journal examined the fatty acid content of egg yolk (14), or of Experimental Methods
phosphatidylcholine isolated from egg yolk (12, 13), but none
of these articles examined gas chromatography coupled with Eggs are purchased at a local chain supermarket and the
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mass spectrometry (GC–MS). In those experiments, FAMEs large eggs used in the experiment are the store brand. Students
were separated using gas chromatography and identified using obtain the egg yolk using an egg separator. They generate FAMEs
their retention times. using 100 mg of egg yolk and the acid-catalyzed transesterifi-
The hen egg is a convenient and inexpensive source of cation procedure of Schultz and Pugh (9). Working in pairs,
lipids for biochemistry laboratory experiments. Egg yolk students prepare two samples for GC–MS analysis: one from
contains phosphatidylcholine, phosphatidylethanolamine, a store-brand large egg yolk and one from a higher PUFA egg
triglycerides, sphingomyelin, and small quantities (1% or less) of yolk.
phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, Students inject 1 μL of each sample into the GC–MS, a
lysophosphatidylcholine, lysophosphatidylserine, lysophosphati- Hewlett-Packard 5890 GC interfaced with a 5971A mass se-
dylethanolamine, galactosylceramide, and cholesterol esters lective detector and equipped with the ChemStation software
(15); all of these contain fatty acids. The relative fatty acid ratio package and the NBS54k library. We purchased a Supelco
J. Chem. Educ. 2009.86:962.
in eggs can be altered by manipulating the hens’ diet (16–21). SP-2380 column [poly(90% biscyanopropyl/10% cyanopropy-
Two polyunsaturated fatty acids (PUFA) are essential in the lphenyl siloxane) phase, 100 m × 0.25 mm i.d. × 0.20 μm film
human diet, and these essential fatty acids and others synthe- thickness)] to separate FAMEs, but shorter and less expensive
sized in the body from them are linked to health benefits (22). columns can be used (6–9, 11–13). This column allows for
Foods containing these particular fatty acids, called omega-3 resolution, detection, and identification of small quantities of
and omega-6 fatty acids, “are believed to be beneficial for heart FAMEs, including methyl myristate, methyl palmitoleate, and
health, brain, and eye function, infant development, and alleviat- methyl arachidonate, not reported by Hodges (12).
ing hypertension” (23). Students identify individual FAMEs primarily by matching
mass spectral data to the library database but also by compar-
ing GC retention time with standards run by the instructor
previously (Figure 1). Not all molecules in the prepared egg
yolk sample are FAMEs, so if student data do not match a
library entry, students must examine the MS data to determine
whether the base peak and other fragment ions correspond
to those typically found in FAMEs. Using an electron ioniza-
18:1
tion source, saturated FAMEs produce a base peak, m/z = 74,
which results from a McLafferty rearrangement, a prominent
10 m/z = 87 fragment resulting from cleavage between the β and
γ carbons, as well as detectable m/z = 57 and 43 fragments
Response / (106 Count)
16:0
from fragmentation at the terminus of the fatty acid (Figure 2).
8 18:2
Unsaturated FAMEs produce a base peak from cleavage near
the terminus: monounsaturated FAMEs have a base peak at
6 m/z = 55, diunsaturated FAMEs at m/z = 67, and tri- and
tetraunsaturated FAMEs at m/z = 79. In addition, all FAMEs
4 18:0 exhibit the characteristic clumps of peaks resulting from series
of “losses of 14”, due to fragmentations differing in mass by a
methylene group, −CH2−, m/z = 14, as illustrated in Figure 3
2
16:1
18:3 20:4
by methyl palmitate.
14:0 Students integrate their peaks using the Hewlett-Packard
0 ChemStation software. They then convert the integration values
15 20 25 30 to a mass percent value and compare their data to the literature.
Elution Time / min Only peaks identified as FAMEs are included in the denomina-
Figure 1. Gas chromatogram of egg yolk FAMEs. tor for the mass percent calculations.
962 Journal of Chemical Education • Vol. 86 No. 8 August 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
Hazards γ α McLafferty
O rearrangement O
Raw eggs may contain Salmonella. Therefore after handling M ≡ R β
raw egg, students should wash their hands with soap. If sample O H O
preparation is carried out in a well-ventilated hood, there are
no significant hazards in using these chemicals. The instructor m/z ∙ 74
adds acetyl chloride. Toluene, methanol, and acetyl chloride are
flammable liquids and flammable vapors. Toluene and methanol
cause eye, skin, and respiratory tract irritation. Methanol is a O
poison and may be fatal or cause blindness if swallowed. Acetyl
chloride can cause eye, skin, digestive, and respiratory tract O
burns. It reacts violently with water, releasing toxic and corrosive m/z ∙ 43 m/z ∙ 57 m/z ∙ 87
fumes of hydrogen chloride and aqueous hydrochloric acid.
FAMEs may cause eye and skin irritation and respiratory and Figure 2. Common fragments produced from a saturated FAME.
digestive tract irritation.
ratios ranging from 1.5 (2004 data) to 1.1 (2006 data), while 143
227
the literature values for store-brand eggs have a ratio of 1.7. 25
Presumably this variation is due to changes in the diet local hens 185
129
are fed from year to year. 213 271
In lecture students learn the IUPAC, common, and simpli-
fied nomenclatures for fatty acids. In simplified nomenclature 0
arachidonic acid is 20:4(Δ5,8,11,14), which indicates a 20-carbon 100 200 300 400 500
fatty acid with 4 double bonds at carbons number 5, 8, 11, and m/z
14. In the lab students learn the alternative “omega minus” or Figure 3. Mass spectrum of methyl palmitate.
Methyl linolenate 18:3(Δ9,12,15) 0.76 ± 0.34 0.17 Not available 2.00 ± 0.46
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 8 August 2009 • Journal of Chemical Education 963
In the Laboratory
20:1(Δ11) — — — 0.20
20:3(Δ8,11,14) — — — 0.27
22:6(Δ4,7,10,13,16,19) — — — 0.27
Note: The literature data are from ref 24.
964 Journal of Chemical Education • Vol. 86 No. 8 August 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 8 August 2009 • Journal of Chemical Education 965
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