Beruflich Dokumente
Kultur Dokumente
JOURNAL OF
FOOD COMPOSITION
AND ANALYSIS
Original Article
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.
ARTICLE IN PRESS
618
M.V. Russo et al. / Journal of Food Composition and Analysis 18 (2005) 617624
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).
ARTICLE IN PRESS
M.V. Russo et al. / Journal of Food Composition and Analysis 18 (2005) 617624
619
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.
ARTICLE IN PRESS
M.V. Russo et al. / Journal of Food Composition and Analysis 18 (2005) 617624
620
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.
ARTICLE IN PRESS
M.V. Russo et al. / Journal of Food Composition and Analysis 18 (2005) 617624
621
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
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
ARTICLE IN PRESS
M.V. Russo et al. / Journal of Food Composition and Analysis 18 (2005) 617624
622
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
1.2
0.8
0.4
15
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.
ARTICLE IN PRESS
M.V. Russo et al. / Journal of Food Composition and Analysis 18 (2005) 617624
623
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.
References
Abidi, S.L., 2001. Chromatographic analysis of plant sterols in foods and vegetable oils. Journal of Chromatography A
935, 173201.
Alonso, L., Lozada, L., Fontecha, L.J., Juarez, M., 1995. Determination of cholesterol in milk fat by gas
chromatography with direct injection and sample saponication. Chromatographia 41, 2328.
Borge, H., 1985. Rapid separation of free sterols by reversed phase high-performance liquid chromatography. Journal
of the American Oil Chemists Society 62, 13441346.
Bortolomeazzi, R., Frega, N., Lercker, G., 1990. Rapid determination of free and esteried sterols using pre-packed
silica columns. Italian Journal of Food Sciences 2, 265268.
Brown, H.G., 1987. Adaptation of an HPLC system to quantify cholesterol. Journal of the American Oil Chemists
Society 64, 106108.
ARTICLE IN PRESS
624
M.V. Russo et al. / Journal of Food Composition and Analysis 18 (2005) 617624
Casiraghi, E., Lucisano, M., Pompei, C., Dellea, C., 1994. Cholesterol determination in butter by high performance
chromatography. Milchwissenschaft 49, 194197.
Cercaci, L., Lercker, G., 2000. Proposta di un metodo di determinazione degli steroli liberi e degli steroli totali nelle
sostanze grasse. Bollettino dei Chimici Igienisti 51, 177183.
Cercaci, L., Rodriguez-Estrada, M.T., Lercker, G., 2003. Solid phase extraction-thin layer chromatographygas
chromatography method for the detection of hazelnut oil in olive oils by determination of esteried sterols. Journal
of Chromatography A 985, 211220.
Chen, C.W., Sun, L.C., 1996. Determination of free and esteried cholesterol contents in lard. Food Science 23,
367376.
Choong, Y.M., Lin, H.J., Chen, C.W., Wang, M.L., 1999. A rapid gas chromatographic method for direct
determination of free sterols in animal and vegetable fats and oils. Journal of Food and Drug Analysis 7, 279290.
Du, M., Ahn, D.U., 2002. Simultaneous analysis of tocopherols, cholesterol and phytosterols by gas chromatography.
Journal of Food Science 67, 16961700.
European Communities, 1992. Commission regulation (EEC) No. 3942/92.
Fenton, M., 1992. Review: chromatographic separation of cholesterol in foods. Journal of Chromatography B 624,
369388.
Frega, N., Bocci, F., Lercker, G., 1992. Direct gas chromatographic analysis of the unsaponiable fraction of different
oils with a polar capillary column. Journal of the American Oil Chemists Society 69, 447450.
Grob, K., Lanfranchi, M., Mariani, C., 1989. Determination of free and esteried sterols and of wax ester in oils and
fats by coupled liquid chromatographygas chromatography. Journal of Chromatography 471, 397405.
Hoving, E.B., 1995. Chromatographic methods in the analysis of cholesterol and related lipids. Journal of
Chromatography B 671, 341362.
Hunter, I.R., Walten, M.K., Heftmann, E., 1978. Liquid column chromatography of free sterol. Journal of
Chromatography 153, 5761.
Itoh, I., Tamura, T., Matsumoto, T., 1973. Sterol composition of 19 vegetable oils. Journal of the American Oil
Chemists Society 50, 122125.
Kim, H.Y., Salem, N., 1990. Separation of lipid classes by solid phase extraction. Journal of Lipid Research 31,
22852289.
Nelson, G.J., 1993. In: Perkins, E.G. (Ed.), Analyses of Fats, Oils and Derivatives. AOCS Press, Champaign, IL, USA.
Newkirk, D.R., Sheppard, A.J., 1981. High pressure liquid chromatographic determination of cholesterol in food.
Journal of the Association of Ofcial Analytical Chemists 64, 5457.
Russo, M.V., 2002. Diol sep-pak cartridges for enrichment of polychlorobiphenyls and chlorinated pesticides from
water samples; determination by GC-ECD. Chromatographia 53, 9398.
Russo, M.V., Goretti, G., Nevigato, T., 1998. Separation of polychlorinated biphenyls from chlorinated pesticides
using aminopropyl bonded-phase cartridge and determination by GC-ECD. Chromatographia 48, 293298.
Slover, H.T., Thompson Jr., R.H., Merola, G.V., 1983. Determination of tocopherol and sterols by gaschromatography. Journal of the American Oil Chemists Society 60, 15241528.
Ulberth, F., Reich, H., 1992. Gas chromatographic determination of cholesterol in processed foods. Food Chemistry
43, 387391.
Worthington, R.E., Hitchcock, H., 1984. A method for the separation of seed oil steryl esters and free sterol,
application to peanut and corn oils. Journal of the American Oil Chemists Society 61, 10851088.