FOOD COMPOSITION
AND ANALYSIS
Journal of Food Composition and Analysis 18 (2005) 617624
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 NH
2
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
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doi:10.1016/j.jfca.2004.06.014
g
m
g
-
1
)
Fig. 2. SPE-GC determination of cholesterol in decreasing amounts of lard sample. Mean and S.D. (n 6).
M.V. Russo et al. / Journal of Food Composition and Analysis 18 (2005) 617624 622
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 mg
1
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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