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ARTICLE IN PRESS

JOURNAL OF
FOOD COMPOSITION
AND ANALYSIS

Journal of Food Composition and Analysis 18 (2005) 617624


www.elsevier.com/locate/jfca

Original Article

Solid phase extractiongas-chromatographic method to


determine free cholesterol in animal fats
Mario Vincenzo Russo, Antonella De Leonardis, Vincenzo Macciola
Dipartimento di Scienze e Tecnologie Agro-Ambientali e Microbiologiche, Universita` degli Studi del Molise, Via De
Sanctis, 86100 Campobasso, Italy
Received 26 September 2003; received in revised form 14 June 2004; accepted 23 June 2004

Abstract
An analytical method to determine cholesterol in animal fats was developed. Cholesterol of four different
animal fats was analysed by solid phase extraction, using porous silica derivatized with NH2 groups. The
compound, puried from glycerides, was quantied as its trimethylsilyl derivative by gas chromatography
coupled with a ame ionization detector. The results obtained with the proposed method were compared
with those obtained with a saponication method. Proposed method repeatability and accuracy were
reasonable. Coefcient of variation relative to standard deviation (S.D.) of repeatability was p3.5%, while
recovery was on average 96.2%. The limit of detection was about 0.8 mg of cholesterol per mg of sample
loaded, with a S.D. of 0.01 mg. In conclusion, the proposed method is simple, fast and reproducible.
r 2004 Elsevier Inc. All rights reserved.
Keywords: Solid phase extraction; Gas chromatography; Cholesterol; Animal fats

1. Introduction
Traditional methods for analysis of sterols in food require a laborious procedure that consists
in: saponication of triglycerides, recovery of unsaponiable with an organic solvent, optional
purication of sterols by thin layer chromatography (Itoh et al., 1973; Worthington and
Corresponding author. Fax: +39-87-440-4652.

E-mail address: antomac@unimol.it (A. De Leonardis).


0889-1575/$ - see front matter r 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2004.06.014

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Hitchcock, 1984; European Communities, 1992) and, nally, gas-chromatography (GC) (Slover et
al., 1983; Fenton, 1992; Abidi, 2001) or high-performance liquid chromatography (Newkirk and
Sheppard, 1981; Brown, 1987; Grob et al., 1989; Casiraghi et al., 1994). In alternative, some
methods were developed to determine free sterols by direct GC analysis (Hunter et al., 1978;
Borge, 1985; Bortolomeazzi et al., 1990; Frega et al., 1992; Chen and Sun, 1996). In these abovementioned GC direct methods, the triglycerides are not previously separated and thus, must be
considered impurities regarding sterol compounds.
Cholesterol is the principal component of animal lipid sterol fraction and it is also
present in traces in some vegetable oils; its determination in food is very important in relation
to nutritional and health aspects. Cholesterol in animal fats is prevalently present in its
free form and therefore it can be fully determined by GC direct methods (Ulberth and
Reich, 1992; Alonso et al., 1995; Chen and Sun, 1996; Hoving, 1995; Choong et al., 1999; Du
and Ahn, 2002).
Solid phase extraction with polar adsorbents is usually used to split lipids into their components
(Kim and Salem, 1990; Nelson, 1993) and to separate the polychlorobiphenyls from the
chlorinated pesticides (Russo et al., 1998; Russo, 2002). In the method claried in this paper, the
SPE technique was applied to different animal-fat samples to clean up cholesterol by glycerides
before quantifying it by GC analysis. The proposed method is simple, rapid and reproducible and
it lends itself to other applications offering an alternative to saponication (Cercaci and Lercker,
2000; Cercaci et al., 2003).

2. Materials and methods


2.1. Materials
Methanol, ethanol, isopropanol, chloroform, acetone, n-hexane, pyridine, hesamethyldisilazane, trimethylchlorosilane, ethyl acetate, potassium hydroxide, anhydrous sodium sulphate were
purchased from Carlo Erba (Milan, Italy).
Cholesterol and stigmasterol were purchased from Sigma Chemical CO. (St. Louis, MO, USA),
while the Sep-Pak cartridges Strata-NH2 were from Phenomenex (Torrance, CA, USA).
Animal fats studied were butter, lard, buffalo milk fat and sh oil. Butter and lard were
commercial samples. In our laboratory, buffalo milk fat was extracted from fresh milk by
centrifugation at 3000g for 30 min, while sh oil was extracted from sardine llets with a mixture
of 3:2 (v/v) n-hexane:isopropanol (Nelson, 1993).
2.2. Saponification method
One hundred and fty milligrams of fats, exactly weighed, with 100 mL of stigmasterol standard
solution (5.4 mg mL1) were mixed with 1 mL of saturated KOH ethanol solution and saponied
in a water bath at 80 1C for 30 min, stirring at regular intervals. After cooling and adding 3 mL of
distilled water and 3 mL of n-hexane, the saponied sample was accurately stirred and centrifuged
at 3000 g for 15 min. Upper n-hexane layer was dried, while on the water layer the n-hexane

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extraction was, in the same way, repeated twice more. The n-hexane extract was dried at 50 1C; the
residue was silylated with 300 mL of pyridine:hexamethyldisilazane:trimethylchlorosilane, 40:36:24
(v/v), at 80 1C for 30 min and then analysed (1 mL) by GC.

2.3. Sterol purification by SPE


Fifteen milligrams of fats with 50 mL of stigmasterol (i.s.) standard solution (0.5 mg mL1) were
dissolved in 450 mL of chloroform and loaded to the Sep-Pak cartridge (Strata-NH2) that was rst
equilibrated after each elution with 3 mL of methanol, acetone and n-hexane. Triglycerides were
washed off the cartridge with 3 mL of mixture (95:5, v/v) of n-hexane:ethyl acetate and nally
cholesterol was eluted with 3 mL of chloroform:methanol (2:1, v/v). Sterol samples were dried at
50 1C, silylated with 50 mL of pyridine:hexamethyldisilazane:trimethylchlorosilane, 40:36:24 (v/v),
at 80 1C for 30 min and then analysed (1 mL) by GC.

2.4. Gas-chromatographic analysis


Gas-chromatographic analysis was performed by a gas-chromatograph Mod.8000 Finnigan
(Milano, Italy) equipped with a fused silica Rtx-65TG (Restek International, Bellefonte, PA,
USA) capillary column (30 m  0.32 mm), coated with 65% diphenyl35% dimethyl-polysiloxane, lm thickness 0.10 mm. Flame ionization detector (FID) was at 370 1C. Experimental
conditions were: carrier gas He: 50 kPa head pressure; initial column temperature: 100 1C running
to 150 and 360 1C at 30 and 15 1C min1, respectively; injector temperature: 300 1C; injection: split
mode (1:25).

2.5. Repeatability, accuracy and limit of detection


Repeatability for both the saponication and SPE-GC methods was evaluated measuring the
variability on four independent and consecutive analyses of each fat sample.
SPE method accuracy was estimated on triplicate recovery percentage of different amounts
(ranging from 11 and 182 mg) of pure cholesterol added to 15 mg of lard or butter samples. Then,
cholesterol was determined by SPE-GC method according to the above-described procedure and
using stigmasterol as internal standard (i.s.).
Limit of detection of SPE-GC methods was estimated on six determinations of the original
cholesterol present in decreasing amounts of lard sample (ranging from 3 to 15 mg) loaded on SPE
cartridge.

2.6. Statistical analysis


The mean, standard deviation (S.D.), coefcient of variation (CV), variance analysis and
linearity were analysed statistically by SPSS software (SPSS Inc. Headquarters, Chicago, IL).

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3. Results and discussion


Fig. 1 shows the GC-FID buffalo milk fat component prole performed by the different
techniques. In particular, chromatogram A refers to direct GC injection of the sample (without
i.s.) previously silanized; chromatogram B refers to the sterol fraction separated from buffalo milk
fat by the SPE-GC method; nally, chromatogram C is relative to unsaponiable fraction
obtained by the saponication method.
Fig. 1 highlights the fact that all performed methods were suitable to determine cholesterol in
animal fats. In all cases, in fact, as in the direct GC injection technique, cholesterol peak was well
dened and measured when its signal to noise ratio was considered. Purication grade of sterols
was the principal dissimilarity from direct GC injection to the other two methods. In
chromatogram A, also other matrix components, above all di- and tri-glycerides were eluted all
together with cholesterol. For this reason, when this technique was used, it was necessary to x
one nal GC step that remained at high temperatures long enough to elute triglycerides. For this
reason, GC direct injection analysis had a delay of about 15 min in comparison to the other two
methods studied.
In the chromatograms B and C, the complete absence of triglycerides is evident, while the
previous peaks of cholesterol were identied in free fatty acids and monoglycerides. In comparing
these two chromatograms, we noted that the sterol fraction purication grade pre-formed by SPE
technique was highly comparable with the saponication method. The mechanism of SPE
technique was as follows: cholesterol and stigmasterol (i.s.) were strongly bonded with Sep-Pak
cartridge, containing porous silica derivatized with NH2 groups, while triglycerides were removed
14

m Volt

m Volt

14

2
1

(A)

(B)
14

m Volt

0
(C)

Time

30 min

Fig. 1. GC-FID buffalo milk fat component chromatograms: (A) direct GC injection of sample; (B) sterol fraction by
CG-SPE method; and (C) unsaponiable fraction by saponication method. Peak number identication: 1
cholesterol; and 2stigmasterol.

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from sample during the clean-up with a mixture of n-hexane:ethyl acetate (95:5, v/v). Afterwards,
the recovery of sterols was performed by chloroformmethanol (2:1, v/v) elution.
Table 1 gives the mean values, S.D. and CV for cholesterol determination in fat samples, by both
saponication and SPE methods. The analysis of variance at 0.05 signicant grade was also
reported.
Comparing the fat samples, we noted that cholesterol content signicantly varied among all
samples, ranging from 0.86 mg mg1 in the lard to 4.89 mg mg1 in the sh oil. Comparing the
results obtained for each sample by the saponication and SPE methods, it is evident that the
cholesterol values found were signicantly similar in each fat sample test group, as the analysis of
variance shows. Considering that total cholesterol and free cholesterol by saponication and SPEGC method are in that order determined, the similar values found with both methods could
actually imply that in the fat samples cholesterol was prevalently in its free form. This observation
could be the subject of future investigation.
Finally, both methods were characterized by a good repeatability, measured by CV (relative to
S.D.) that, in fact, was generally signicantly low, p3.5%.
In order to evaluate the reliability of the procedure, fat samples to which amounts of pure
cholesterol had been added were analysed and the repeatability and recovery percentage were
determined. Accuracy of the proposed method was estimated on the recovery percentage of
increasing amounts of pure cholesterol added to lard and butter, after subtraction of their original
cholesterol content, previously determined and shown in Table 1.
In Table 2, the results relative to lard and butter samples, with different cholesterol
concentrations, are presented. The CV is especially lower in the fat samples with a higher
cholesterol concentration, whereas it is high in all other at low doses.
Variability observed on the single assay was caused by random errors. Nevertheless, accuracy of
the SPE-GC proposed method could be considered acceptable. In fact, recovery percentage of
cholesterol added (considered in absolute terms) was generally good and signicantly high,
ranging from 91.5% and 99.4%, with a mean of 95.3% and 97.1% for lard and butter,
respectively. Finally, into the cholesterol concentration interval tested, in both lard and butter,

Table 1
Repeatability of cholesterol determination by the saponication and SPE proposed methods
Fat samples
Butter

Buffalo milk fat

Lard

Fish oil

Saponication method
Mean* (mg mg1)
S.D.
CV (%)

2.99a
0.08
2.7

1.90b
0.06
3.2

0.86c
0.01
1.5

4.89d
0.17
3.5

SPE method
Mean* (mg mg1)
S.D.
CV (%)

2.93a
0.03
1.0

1.85b
0.04
2.2

0.83c
0.02
2.4

5.11d
0.08
1.6

n=6; different letters mean signicant difference at Pp0:05:

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Table 2
Accuracy and linearity of SPE-GC method proposed to determine cholesterol (n=3)
Cholesterol (mg)
Added

CV%

Accuracy (%)

Found

Linearity
Mean

Equation

R2

95.373.6

y=0.948x+0.06

0.999

97.172.2

y=0.947x+1.23

0.999

Lard
11
22
46

10.171.0
21.570.7
43.573.6

10.3
3.3
8.3

91.579.4
98.670.9
95.777.9

Butter
22
46
91
182

21.972.6
45.474.8
86.773.1
173.779.0

11.7
10.5
3.6
5.2

99.4711.7
98.6710.3
95.173.6
95.472.2

cholesterol dosed (g mg-1)

1.2

0.8

0.4

15

mg of lard used for cholesterol analysis

Fig. 2. SPE-GC determination of cholesterol in decreasing amounts of lard sample. Mean and S.D. (n 6).

there was a good linear correlation between the cholesterol added and found, as the R2 (0.999)
high values show.
In the end, the limit of detection of SPE-GC method was evaluated measuring cholesterol in
decreasing lard amounts diluted in 0.5 mL of chloroform and loaded on SPE cartridge.
Fig. 2 shows the mean and S.D. of six replicates of each assay.
Examining the graph of Fig. 2, it can be noticed that precision and accuracy of proposed
method progressively decreased in function of the lard sample amount used for analysis.

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Measuring the signal of cholesterol to noise ratio and considering that precision and accuracy
decreased, it was estimated that the sample loaded with SPE-GC proposed method should
approximately contain 0.8 mg of cholesterol per mg of sample loaded.

4. Conclusion
The SPE-GC method proposed here to determine cholesterol in animal fat gives good accuracy
and precision, especially if the fat sample contains at least 0.8 mg mg1 of cholesterol. It is a rapid
method that can be considered an alternative to traditional saponication procedures. This
technique presented several other advantages that in synthesis are
1. purication of cholesterol performed by SPE technique was satisfactory;
2. using the SPE technique, the quantity of fat sample was lower than saponication method (15
and 150 mg, respectively);
3. preparation time of sample was as short as 30 min for SPE technique, while the saponication
method required at least 2 h;
4. the nal solvent volume that must be evaporated was lower in SPE method (3 mL of
chloroform/methanol) than saponication (9 mL of n-hexane);
5. the GC analysis time and temperature program was lower using SPE method with respect to
direct injection method; for this fact the column life time is extended.
In conclusion, the SPE-GC method could be suggested for routine analysis. Nevertheless,
further investigations are necessary to verify if cholesterol in animal fat is exclusively in its free
form. In addition, the SPE-GC proposed method could be applied to vegetable fats to quickly
analyse free sterols.

Acknowledgements
This study was nancially supported by a University of Molise research fund for the year 2000.

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