JFS:
Food Chemistry and Toxicology
Simultaneous Analysis of Tocopherols,
Cholesterol, and Phytosterols Using
Gas Chromatography
M. DU AND D.U. AHN
Food Chemistry and Toxicology
ABSTRACT: A method for the simultaneous analysis of tocopherols and sterols was developed. Lipids were
extracted with the Folch method, saponified by KOH-ethanol, and then tocopherols, cholesterol, and phytosterols were extracted with hexane. The extracted samples were dried under a nitrogen stream, derivatized using
trimethylsilyl compounds, and then subjected to a gas chromatography. The recovery rates for cholesterol,
stigmasterol, and sitosterol were about 100%, but recovery rates for tocopherols were low (25% for d-tocopherol
and 66% for g-tocopherol) and varied according to compound structures. However, the recovery rates for d- and
g-tocopherols increased to about 100% when the amounts of water and hexane were increased to 15 mL at the
extraction step after saponification.
Keywords: tocopherols, cholesterol, phytosterols, gas chromatography, food products
Introduction
T
OCOPHEROLS ARE FREQUENTLY
used in foods as antioxidants. The oxidative stability of meat was improved by
feeding tocopherols to animals (Sanders
and others 1997; Cabedo and others
1998). Dietary tocopherols, especially atocopherol, can prevent oxidative stress in
vivo and are good for overall health
(Haffner 2000; Factor and others 2000).
Phytosterols, which naturally exist in
plants, might inhibit the absorption of
cholesterol, lower serum LDL cholesterol
level and, thus, can help prevent circulatory and heart diseases in humans ( Jones
and others 1997). Since both tocopherols
and phytosterols are good for health and
coexist in food sources from plant origin, it
will be useful to have a simple method for
measuring both tocopherols and phytosterols simultaneously.
Cholesterol is a constituent of animal
tissues that is involved in the development of atherosclerosis and coronary
heart diseases (Weggemans and others
2001). Analyzing tocopherols and cholesterol simultaneously can significantly reduce time, expense, and effort. Furthermore, the amount of sample needed for
analyzing these 2 compounds can be
greatly reduced. This is especially important for the samples that are very difficult
to obtain in large quantities, such as blood
serum samples of laboratory mice.
Numerous reports on the separate
analysis of tocopherols and sterols are
available (Beyer and others 1989; Smidt
1696
and others 1989; Thompson and Merola
1993; Botsoglou and others 1998; Choong
and others 1999). However, no report for
analyzing tocopherols, cholesterol, and
phytosterols simultaneously in a sample
analysis is available. The objective of this
study was to establish a sample preparation method for tocopherols, cholesterol,
and other sterols and analyze them by GC
in a single run.
Materials and Methods
Chemicals and reagents
a-, d-, g-Tocopherols, cholesterol, 5acholestane, stigmasterol, sitosterol, and
stripped corn oil were purchased from Sigma Chemical Co. (St. Louis, Mo., U.S.A.).
Sylon BFT [Bis-(trimethylsilyl) trifluoroacetamide (BSTFA) + trimethylchlorosilane
(TMCS) = 99:1] was obtained from Supelco
(Bellefonte, Pa., U.S.A.). Hexane (HPLC
grade) and pyridine was obtained from
Fisher Scientific (Fair Lawn, N.J., U.S.A.).
Saponification
Ground meat (1 to 2 g) or 0.1 to 0.2 g of
oil was accurately weighed into a 50-mL
screw-cap test tube, and then 10 mL of saponification reagent prepared by freshly
mixing ethanol and 33% (w/v) KOH solution at a ratio of 94:6, 0.5 mL of 20% ascorbic acid (to prevent oxidation of tocopherols during saponification), and 50 mL
of 5a-cholestane solution (1 mg/mL in hexane) was added immediately (Fenton and
Sim 1991). The sample was homogenized
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with a polytron for 5 s at full speed,
capped, and then incubated for 1 hour at
50 °C. After cooling in ice water for 10 min,
5 mL deionized distilled water and 5 mL
hexane were added (for d- and g-tocopherols analysis, the volume of water and
hexane was increased to 15 mL). Tubes
were capped tightly and then the contents
were mixed thoroughly by shaking. After
15 hours for phase separation, the hexane
layer containing unsaponifiables was
carefully transferred to a scintillation vial
and dried under nitrogen flow. To the
dried sample 200 mL pyridine and 100 mL
Sylon BFT (99% BSTFA + 1% TMCS) were
added. The sample was derivatized either
at 50 °C in a water bath for 1 hour or overnight at room temperature, and then analyzed using a GC or GC-MS (Du and Ahn
2000).
Repeatability and reproducibility
in model system
Repeatability was measured using the
variation of 4 consecutive analysis of the
same sample, and reproducibility was determined by the 3 repeated measurements of the same sample on 3 different
days.
Recovery of standards in model
system
For recovery analysis, known amounts
of tocopherol and sterol standards were
added into 2 sets of test tubes. One set of
samples was prepared using the proposed
method that included homogenization,
© 2002 Institute of Food Technologists
7/9/2002, 4:28 PM
Table 1—Repeatability of measurement at different concentrations of tocopherols and sterols
1 mg
Detected
d-Tocopherol
g-Tocopherol
a-Tocopherol
Cholesterol
Stigmasterol
Sitosterol
10 mg
C.V.
Detected
100 mg
C.V.
Detected
C.V.
------------------------------------- mg/mL sample --------------------------------------0.29 ± 0.04
13.79
2.61 ± 0.12
4.60
25.28 ± 0.79 3.13
0.69 ± 0.06
8.70
6.69 ± 0.29
4.33
65.80 ± 2.12 3.22
0.92 ± 0.07
7.61
9.31 ± 0.32
3.44
92.07 ± 1.97 2.14
1.02 ± 0.05
4.90
9.71 ± 0.34
3.50
98.34 ± 2.11 2.15
0.98 ± 0.04
4.08
9.82 ± 0.23
2.34
98.06 ± 1.83 1.87
0.98 ± 0.04
4.08
9.99 ± 0.22
2.42
100.21 ± 2.31 2.31
n = 4
Table 2—Reproducibility of measurement at different concentrations of tocopherols and sterols
1 mg
Detected
d-Tocopherol
g-Tocopherol
a-Tocopherol
Cholesterol
Stigmasterol
Sitosterol
10 mg
C.V.
Detected
100 mg
C.V.
Detected
C.V.
------------------------------------- mg/mL sample --------------------------------------0.24 ± 0.05
20.83
2.29 ± 0.18
7.86
25.4 ± 2.03 7.99
0.68 ± 0.11
16.18
6.61 ± 0.29
4.39
65.3 ± 2.29 3.51
0.98 ± 0.11
11.22
9.35 ± 0.20
2.14
95.13 ± 2.15 2.26
1.03 ± 0.08
7.77
9.79 ± 0.39
3.98
97.78 ± 2.68 2.74
0.98 ± 0.07
7.14
9.74 ± 0.34
3.49
97.99 ± 1.88 1.92
1.05 ± 0.05
4.76
10.03 ± 0.30 2.99
99.68 ± 2.54 2.55
n = 3
saponification, extraction, drying, and derivatization, while the other set was directly derivatized and analyzed by GC. The recovery rates were calculated by comparing
the amounts of standards detected with or
without sample preparation steps.
Recovery of standards from spiked
meat and oil samples
Ground pork lion (1 g) and corn oil (0.2
mL) were accurately weighted into 20
screw-cap test tubes. To each of the sample, 0, 25, 50, 100, or 200 mg of a-tocopherol, g-tocopherol, cholesterol, stigmasterol, and sitosterol standards were
added. Samples were extracted, saponified, derivatized, and analyzed for tocopherol, cholesterol, and sterol contents as
described above.
GC analysis of tocopherols and
sterols
Analysis of tocopherols and sterols was
performed with a Hewlett-Packard (HP)
6890 GC equipped with an on-column
capillary injector and a FID detector
(Hewlett-Packard Co., Wilmington, Del.,
U.S.A.). A 0.25-mm (i.d.) x 30-m bondedphase 5% phenylsilicon column with a
0.25-mm film thickness (HP-5MS column)
was used. A splitless inlet was used to inject samples (5 mL) into the capillary column; and ramped oven temperature was
used (from 180 °C increased to 260 °C @
8 °C/min, increased to 280 °C @ 2 °C/min,
and held for 13 min). Inlet temperature
was 290 °C, and the detector temperature
was 300 °C. Helium was the carrier gas at
constant flow of 1.2 mL/min. Detector
(FID) air, H 2, and make-up gas (He) flows
were 136 mL/min, 35 mL/min, and 45 mL/
min, respectively. The identification of
sterols and tocopherols was confirmed using both a Mass Selective Detector (HP
5973) and the retention times of standards. The area of each peak (pA*sec) was
integrated using the ChemStation software (Hewlett-Packard Co., Wilmington,
Del., U.S.A.) and the amounts of tocopherols and sterols were calculated using
an internal standard, 5a-cholestane (Du
and Ahn 2000).
Statistical analysis
The means, standard deviations, and
covariance (R2) were analyzed statistically
by GLM using SAS â software (SAS Institute Inc. 1989). Mean values, standard deviation, and coefficients of variation (CV )
were reported.
Results and Discussion
T
ABLE 1 SHOWS THE REPEATABILITY
of the proposed method in measuring
tocopherols and sterols at 3 different concentrations. As low as 1 mg/mL of a-tocopherol, g-tocopherol, cholesterol, and
phytosterols in samples (raw materials)
could be detected. The variation for d-tocopherol was a little large, with a coeffi-
cient of variation (CV) more than 10%, but
was still acceptable. Therefore, the detection limit for this method should be
around 1 mg/mL sample. If the original
sample amount used for analysis is 1 g,
the threshold for detection is near 1 ppm.
As the amount of standards added increased to 10 and 100 mg, the variation
was reduced and CV decreased to within
the 10% level (Table 1). The variation
could come from inaccurate handling during preparation for analysis, and careful
handling would reduce the variation further. Also, when the amounts of analytes
in samples are very low, small changes in
baseline selection can have a significant
effect on the area of a peak.
The direct saponification method is
employed frequently for the sample preparation of cholesterol and cholesterol oxides (Kovacs and others 1979; King and
others 1998). Several extraction solvents
were used for extracting unsaponifiables,
including petroleum ether, hexane, and
ether acetate (Beyer and others 1989; King
and others 1998). Hexane is the most commonly used extraction solvent for nonpolar lipids and, thus, hexane was chosen as
an extraction solvent in this study. Adding
ascorbic acid can prevent tocopherols
from oxidation during saponification (Taylor and others 1976).
The hexane layer could contain some
moisture and impurities, so it has been
suggested that the end point washing and
filtration through anhydrous sodium sulfate was important due to the sensitivity
of the silylating reagent to any residual alcohol and moisture (Yan and White 1990;
King and others 1998). In our study, we
found that the washing step was not necessary since there was no contamination
of the saponification solution in the hexane layer. Using a pipette, we could collect
most of the hexane layer without contaminating it with visible droplets of saponification solution. The hexane layer then was
dried in scintillation vials under nitrogen
flow. Since tocopherols can be oxidized
during drying, it is important to prevent
the sample from exposure to air before derivatization.
Direct saponification has been used for
cholesterol and vitamin E measurement in
meats (Adams and others 1986; Liu and
others 1996; King and others 1998). The
common solvent used for saponification is
KOH-ethanol solution, but sometimes
KOH-methanol solution has also been
used (Naeemi and others 1995). One possible chemical change during sample homogenization and saponification is that
tocopherols may be oxidized. This oxida-
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Food Chemistry and Toxicology
GC analysis of tocopherols and sterols . . .
GC analysis of tocopherols and sterols . . .
Table 3—Percent (%) recovery rates of tocopherols and sterols
Tocopherols
Recovery rate (%)
Sterols
Recovery rate (%)
d-Tocopherol
g-Tocopherol
a-Tocopherol
25.27 ± 1.95
66.12 ± 1.59
92.10 ± 1.63
Cholesterol
Stigmasterol
Sitosterol
97.87 ± 1.11
98.47 ± 1.07
99.77 ± 1.25
n = 4
Table 4—Repeatability, reproducibility, and recovery rates of d- and g-tocopherols after adding 15 mL of water and hexane*
Food Chemistry and Toxicology
d-Tocopherol
g-Tocopherol
Repeatability 1
CV
Reproducibility 2
CV
Recovery rate
97.32 ± 3.78
99.12 ± 1.76
3.88
1.78
97.41 ± 4.95
98.95 ± 4.58
5.08
4.63
98.91 ± 4.95
99.07 ± 3.58
*The total amount of tocopherols added for analysis was 100 mg.
1n = 4
2n = 3
tion can be prevented by excessive
amounts of ascorbic acid (Taylor and others 1976). Without adding ascorbic acid,
large amounts of tocopherols might be oxidized and could not be detected after saponification. Taylor and others (1976)
used 0.5 mL of 25% ascorbic acid to prevent the oxidation during saponification.
Leray and others (1997) used hydroxytoluene instead of ascorbic acid to prevent oxidation during saponification. In this
study, 0.5mL of 20% ascorbic acid solution
was used.
Leray and others (1997) used butylated hydroxytoluene instead of ascorbic
acid to prevent oxidation during saponification. 5a-Cholestane is often used as an
internal standard in measuring cholesterol contents in meat and eggs (Fenton and
Sim 1991), and it was also used in this
study as an internal standard.
Some researchers indicated that cholesterol could be analyzed by gas chromatography (GC) without derivatization
(Beyer and others 1989; Naeemi and others 1995; Klatt and others 1995). Our earlier studies, however, indicated that derivatization was necessary for cholesterol
analysis in order to get good separation
and, thus, simultaneous derivatization of
tocopherols, cholesterol, and phytosterols
with Sylon BFT, which was the key point in
sample preparation used in this study.
Derivatization of tocopherols, cholesterol,
and phytosterols with Sylon BFT replaces
active hydrogen of these compounds with
trimethylsilyl group and improves the volatility and thermal stability of the compounds and, thus, improves the analysis
of these compounds by GC.
Table 2 shows the reproducibility of our
proposed method by triplicate analysis on
3 different days. As indicated by the rela1698
tively low CV, the reproducibility was good.
Without using derivatization, Botsoglou
and others (1998) reported that the within-day and between-days variation gave
overall relative standard deviations of
2.0% for cholesterol and 7.0% for a-tocopherol which was similar to our results.
Tables 1 and 2 indicate that the detected amounts of d-tocopherol and g-tocopherol were much lower than the spiked
amounts. This means that the recovery
rates for d-tocopherol and g-tocopherol
were low (25% for d-tocopherol, 66% for gtocopherol, and 92% for a-tocopherol),
which are unacceptable (Table 3). In the
determination of a-tocopherol content in
beef, Liu and others (1996) obtained a recovery rate of 91% for a-tocopherol, which
was similar to our result. The difference in
recovery rates among the tocopherols
could be due to the different chemical
structures of these 3 kinds of tocopherols.
g-Tocopherol has one more methyl group
and a-tocopherol has 2 more methyl
groups than d-tocopherol and, thus, a-tocopherol is the most hydrophobic and dtocopherol is the most hydrophilic among
the 3 tocopherols. b-Tocopherol, which
was not measured for recovery rate in this
study, should have about the same recovery rate as g-tocopherol, since they have
the same number of methyl groups on the
benzene ring of tocopherol. The low recovery rates for g-tocopherol and d-tocopherol
made it inevitable to adjust the analytical
procedure to increase recovery rates. The
recovery rates of tocopherols depend
upon the relative solubility of tocopherols
between hexane and saponification solution. Increasing the polarity of saponification solution by adding more water can reduce the solubility of tocopherols in the
saponification solution and thus improve
recovery rates. Further, increasing the
amount of hexane and extraction time will
also increase the recovery rates of tocopherols. Therefore, both the volumes of
hexane and water added into the saponified sample were increased to 15 mL. As
expected, the recovery rates of d- and g-tocopherols increased to near 100% (Table
4). However, one shortcoming associated
with the increased hexane and water was
that the phase separation between hexane layer and aqueous layer became difficult. This problem was solved by centrifuging the samples at 3200 x g for 30 min.
Employing 5a-cholestane as internal standards, Maraschiello and Regueiro (1998)
reported that absolute and relative recovery ranges of a-tocopherol was between 80
and 95%. The relative recovery rates for
cholesterol, stigmasterol, and sitosterol in
this study were close to 100% ( Table 3).
Therefore, it was not necessary to increase
water and hexane used for extraction in
the sterol analysis. Choong and others
(1999) reported that the recovery rates of
cholesterol, campesterol, stigmasterol,
and b-sitosterol from lard were in the
range of 89% to 106% with a CV less than
12%. Botsoglou and others (1998) reported
that the overall recoveries were 98.8 and
99.2% for cholesterol and a-tocopherol, respectively.
Table 5 shows the amounts of tocopherols, cholesterol, and sterols recovered
from the spiked meat and corn oil samples. In corn oil, the linearities (R2) for aand d-tocopherol were 0.987 and 0.989,
and those for cholesterol, stigmasterol,
and sitosterol were 0.997, 0.993, and 0.910,
respectively. Similar linearities were observed for meat: the R2 values for a- and dtocopherols were 0.998 and 0.997, and
those for cholesterol, stigmasterol, and sitosterol were 0.968, 0.996, and 0.994, respectively. The high R2 values for all these
analytes indicate that the linearities of
this method for both tocopherols and sterols were acceptable. Botsoglou and others
(1998) suggested a method for the simultaneous determination of cholesterol and
a-tocopherol in eggs. The linearity for both
analytes was r = 0.9964 for cholesterol and
0.9996 for a-tocopherol. Compared with
model system, the recovery rates of spiked
d-tocopherol in meat and oil were somewhat lower than that in model system, but
those for sterols were about the same as
that in model system. The reason for the
lower recovery rates for spiked tocopherols
in meat and oil could be due to the interaction of tocopherols with food components, which makes them difficult to be
extracted, or part of the spiked toco-
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GC analysis of tocopherols and sterols . . .
Table 5—Recovery of a- tocopherol, d-tocopherol, cholesterol, stigmasterol, and sitosterol from spiked meat and oil
samples 1
Amount of standards added (mg)
a-tocopherol
cholesterol
stigmasterol
sitosterol
N.D.2
19.82 ± 1.84
46.51 ± 2.93
74.53 ± 3.12
183.02 ± 15.59
0.989
91.51 ± 7.80
78.22 ± 9.80
102.41 ± 9.34
123.44 ± 2.77
177.78 ± 5.98
273.22 ± 11.20
0.987
97.50 ± 5.60
N.D.
24.04 ± 2.48
49.77 ± 1.37
99.90 ± 6.98
200.31 ± 6.26
0.997
100.16 ± 3.13
88.17 ± 2.23
118.64 ± 3.67
139.31 ± 9.57
189.29 ± 7.27
289.64 ± 6.91
0.993
100.73 ± 3.45
701.16
733.56
790.69
833.80
912.85
0.910
105.85
N.D.
19.65 ± 1.97
47.08 ± 2.38
91.02 ± 6.16
185.89 ± 5.21
0.997
92.95 ± 2.60
1.20 ± 0.16
23.23 ± 1.91
47.94 ± 3.59
89.28 ± 3.30
183.43 ± 4.87
0.998
91.12 ± 2.43
535.44
567.16
590.87
647.30
736.14
0.968
100.35
N.D.
25.16 ± 0.53
50.69 ± 3.28
100.80 ± 6.66
199.52 ± 8.16
0.996
99.76 ± 4.08
N.D.
27.91 ± 8.80
49.26 ± 3.20
98.72 ± 6.00
199.27 ± 5.60
0.994
99.64 ± 2.80
±
±
±
±
±
13.02
12.86
8.74
15.47
16.94
± 8.47
±
±
±
±
±
14.78
16.65
19.05
34.69
33.24
± 16.62
Food Chemistry and Toxicology
Corn oil
0
25
50
100
200
Linearity (R2)
Recovery rate (%) 3
Pork lion
0
25
50
100
200
Linearity (R2)
Recovery rate (%) 3
d-tocopherol
1 Spiked samples were extracted and washed with 15 mL hexane and 15 mL water. Each point represents the average of 8 replications.
2 Means non-detected.
3 The recovery rates of samples adding 200mg standards.
Table 6—␣-Tocopherol and cholesterol content (mg/mL) in turkey blood serum1
Components
0 IU
50 IU
100 IU
200 IU
a-Tocopherol
Cholesterol
--------------------------mg/mL serum----------------------------------------0.28 ± 0.04
0.75 ± 0.03
1.41 ± 0.33
2.22 ± 0.16
491.9 ± 25.4
514.4 ± 38.02
495.9 ± 23.49
518.2 ± 60.68
1 Serum collected from turkeys fed diets supplemented with 4 levels of a-tocopherol (0, 50, 100, and 200
IU/kg feed).
pherols were oxidized during sample
preparation. Also, the variations of recov-
ery rates in spiked samples were greater
than those of model system (Table 3 and
4). This was expected due to the complexity of foods compared with the model system.
Figure 1 shows the gas chromatogram
of tocopherols, cholesterol, and phytosterol standards. Internal standard a-cholestane was first eluted, followed by
tocopherols, cholesterol, and then phytosterols. The separation of these standards was excellent.
Table 6 shows the a-tocopherol and
cholesterol contents in the blood serum of
turkeys fed diets supplemented with 0,
50, 100, and 200 IU a-tocopheryl acetate.
The content of a-tocopherol increased as
the dietary tocopherol level increased,
while the content of cholesterol in serum
remained unchanged at 500 mg/mL.
Conclusions
T
HROUGH SAPONIFICATION, HEXane extraction, derivatization with trimethylsilyl and then GC separation, tocopherols, cholesterol, and phytosterols
could be analyzed by a single sample
preparation. The detection limit for these
compounds was below 1 mg, the linearity
between actual amount and detected
amount was high (R 2 > 0.99), and the repeatability and reproducibility of the
method was also satisfactory. This method
allows simultaneous analysis of tocopherols, cholesterol, and phytosterols in
various food products using GC.
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MS 20010439 Submitted 8/14/01, Accepted 11/19/
01, Received 11/19/01
MThe authors are with the Dept. of Animal Science, Iowa State Univ., Ames, Iowa 50011-3150.
Direct inquiries to author Ahn (E-mail:
duahn@iastate.edu).
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