Quantitative analysis of cholesterol by gas chromatography and gas
chromatography/mass spectrometry
Monica Culea, Gh. Ciurdaru, Lidia Luncian, Mihaela Apetri, Laura Fromondi, R. Podea
Natex S.R.L., 3400 Cluj-Napoca, P.O.Box 374, Romania
Abstract
A method for quantitative analysis of cholesterol in egg yolk and tissues is presented. The
internal standard was diosgenin, added 5mg/g of egg yolk or 1mg/g of tissue. The
extraction was performed in dichloroethane:methanol in an optimization process of the
amount of sample, extractant solvent volume, extraction temperature, extraction time. The
extraction was followed by methylation of fatty acids and gas chromatographic and gas
chromatography- mass spectrometric analysis. The extract was derivatized with methanol
HCl 1M, at 60oC, 1h for fatty acids. A HP-5 capillary column, 30mx0.32mm, 0.25µm film
thickness, in a temperature program: 70oC( 1 min) to 100oC with a rate of 20oC per minute,
then 30o C per minute to 310 oC (10min) was used. The method was validated showing
good analytical parameters. The method was applied for monitoring the cholesterol level in
egg yolk or tissues for testing different functional food diet to chicken.
Keywords: cholesterol, gas chromatography; gas chromatography-mass spectrometry;
egg yolk; tissue;
Introduction
Cholesterol, indispensable substance to our body present in our cell membrane, is healthy
to be intake in low quantities by food. It is present especially in egg yolk, meat and dairy
products. In the last years many articles show the health benefits of different ingredients
contained in foods. The influence of some of these functional foods could be observed in
the variation of cholesterol level in biological matrices [1].
Several methods have been used to quantitate total cholesterol in multicomponent foods;
eggs [2-6], meat [7-9], dairy products. Methods usually involve an organic extraction and
a quantitative technique such as gas chromatography (GC) or liquid chromatography
(LC). The organic extract is saponificated or not and the cholesterol sample is derivatized
or not before GC analysis. Many collaborative studies are made for the optimization of
the experimental design approach for obtaining the best recoveries, low solvent
consumption and reduced extraction times [2-10].
The aim of this paper is to present a rapid method for cholesterol quantitation in egg
yolk and tissues. Cholesterol was analysed without derivatization. The recovery of our
method was compared with the recovery obtained by other methods [2]. Saponification
was replaced by methylation for monitoring the total cholesterol and fatty acids levels
too. The analytical technique chosen is gas chromatography (GC) with flame ionization
detection (FID) and gas chromatography - mass spectrometry (GC/MS) for identification
of the separated compounds.
Experimental
Apparatus
A Hewlett Packard GC 5890 couplet with a MS engine 5989B in the EI mode was used
for compounds identification. The GC was equipped with an HP-5MS capillary column
30mx0.25mm diameter, 0,25µm film thickness, in the temperature program: 70 oC for 1
min, then increased to 100oC with a rate of 20 oC /min, followed by a rate of 30oC/min to
310oC(10min) at a helium flow rate of 1ml/min.
A Hewlett Packard 6890 gas chromatograph was equipped with a HP-5 fused silica
capillary column, 30mx0.32mm, 0.25µm film-thickness with the same temperature
program.
Deactivation by treating the injector glass liner with 5% dimethyldichlorosilane in
toluene gave a higher sensitivity. The GC/MS interface line and the ion source were
maintained at 200oC, and quadrupol analyser at 100 oC. Electron energy was 70eV and
electron emission 300µA.
Reagents and chemicals
Solvents (methanol, ethanol, methylen chloride, 1,2dichloroethane, purchased from
Chimopar (Bucharest, Romania) were purified by distillation, when necessary. Acetyl
chloride was purchased from Carlo Erba (Milano,Italy). Cholesterol (99% pure) and
diosgenin (99% pure) were purchased from Sigma-Aldrich ( Deisenhofen, Germany).
Procedure
Six aliquots containing 50, 100, 150, 200, 250 and 300 µg cholesterol and the same
quantity of 80 µg of the internal standard diosgen were prepared. 3 µl were injected two
times from each alliquot by using the autosampler injector to obtain a regression curve
for egg cholesterol determination in the range 0-300 mg. The relative integrated areas
were plotted against the injected amount as the calibration curve.
For tissue cholesterol analysis the chosen range was 0-70mg; seven aliquots of 10, 20, 30,
40, 50, 60 and 70 µg cholesterol and the same quantity of 100 µg of the internal standard
diosgen were prepared, then injected twice by using the autosampler injector. The relative
integrated areas were plotted against the injected amount as the calibration curve.
The weight of boiled and dried egg yolk corresponding to 1g of boiled egg yolk ( or 1 g
well mixed tissue) was placed in a 10 ml screw cap glass centrifuge tube with 5 ml 1,2
dichloroethane :methanol (1:1). The extraction was performed at 50oC, 30 minutes,
agitating well every 5 minutes. The extract was cooled, centrifuged and the supernatant
was removed. 0.5 ml were transferred was methylated with 0.5 ml methanol: acetyl
chloride (10:1,v/v) at 60oC for 1h. The reagent was removed under nitrogen. 0.5 ml
1,2dichloroethane and 5mg diosgenin/g in egg yolk or 1 mg diosgenin/g in tissues were
added. Three extracts were made and each sample and injected twice.
Experimental design
10 replicates egg yolk were analyzed by our method. 3 replicated dried egg yolk were
analyzed by our method and by saponification [2] to compare the recovery obtained by the
two methods.
Results and discussion
Method validation
Linearity in the range 0-300 mg cholesterol gave a good correlation coefficient of r=0.998
and in the range 0-70 mg cholesterol of r=0.999. The regression curve obtained for the two
ranges were: y=0.02x - 0.02 (Fig. 1) and y= 0.01x - 0.01 ; precision of the method
measured in the aliquots containing 50, 60 and 200 µg cholesterol gave R.S.D lower than
2% (n=9) and for egg yolk control 6.5% (n=10); accuracy showed better than 4 %; (Table
1) recovery gave 95% (Table 2); L.O.D was lower than 5ng at a signal-to-noise ratio of 10.
The chromatogram of separation of the compounds studied is presented in Fig.2. The
results of the precision, accuracy and recovery studies are presented in Tables 1 and 2. The
average values are resulted from three extraction procedures and two injections of each
extract. Precision and day-to-day reproducibility measured for 50 and 60 µg standards
(n=6) showed relative standard deviation values bellow 4%. The mean values for the
recoveries found for each method studied are compared. The best values for recovery were
found for dried egg yolk (Table 2). Significant test studies showed no significant
differences at the 0.05 level between the two methods (Table 3).
The mass spectra fot cholesterol and diosgenin are shown in Fig.3. Diosgenin was a
mixture of two isomers with the same mass spectrum and closed retention times.
Conclusions
This paper presents a method of cholesterol determination in egg yolk and tissues. The
comparison between two extraction methods gave similar recovery results without
significant differences of the mean values of cholesterol levels. The method was validated
showing good analytical parameters: linearity in the range 0-300 mg cholesterol gave a
good correlation coefficient of r=0.999; precision of the method gave R.S.D. 6.5% (n=10
in egg yolk; accuracy showed R.S.D. better than 3 %; recovery gave 95% comparable with
the recoveries obtained with other methods (99%) [2]; L.O.D was lower then 5ng. The
boiled and dried egg showed better analytical parameters.
The methods are suitable for the determination of cholesterol amount in food and
biological matrices. The method is simple, rapid, accurate and suitable for cholesterol
level variation study for testing the benefits of functional food diet.
The author thank the management, Director and colleagues, S. C. Natex s.r.l., for their
co-operation in conducting this study.
References
1. C. M. Hasler, “Functional foods: Their role in diseases prevention and heath promotion”
Food Tech. 52( 11) 1998, 63-70.
2. J J.D.Beyer, F.X. Milani, M. J. Dutelle, R.L. Bradley , “Gas Chromatographic determination
of cholesterol in egg products”J. Assoc. Off. Anal. Chem. 72( 5) 1979, 746-748.
3. M. T. Rodriguez- Estrada, M. F. Caboni, A. Costa, G. Lercker, “ Gas chromatographic
analysis of cholesterol oxidation products on a thermostable medium polarity capillary
column”, J. High Resolut. Chromatogr. 21(9) 1998, 509-512.
4. G. Pasin, G. M. Smith, M. O’Mahony, “Rapid determination of total cholesterol in egg using
commercial diagnostic cholesterol reagent”, Food Chem. 61(1-2) 1998, 255-259.
5. M. S. Lai, J. I. Gray, M. E. Zabik, “Evaluation of solid-phase extraction and gas
chromatography for determination of cholesterol oxidation products in spray-dried whole
egg”, J. Agric. Food Chem. 43(5) 1995, 1122-1126.
6. F. Guardiola, R. Codony, M. Rafecas, J. Boatella, “Selective gas-chromatographic
determination of cholesterol in eggs”, J. Am. Oil Chem Soc. 71(8) 1994, 867-871.
7. M. Fenton, J. S. Sim., “Determination of egg yolk cholesterol content by on-column capillary
gas chromatography”, J. Chromatogr. 540 (1-2) 1991, 323-329.
8. E. D. Naeemi, N. Ahmad, T. K. Al-Sharrah, M. Behbahani, “Rapid and simple method for
determination of cholesterol in processed food”, J. AOAC Int. 78(6) 1995, 1522-1525.
9. K. Duckett, D. G. Wagner, “ Effect of Laidlomycin proprionate on bovine longissimus
muscle fatty acid and cholesterol content”, J. Agric. Food Chem. 45, 1997, 1725-1728.
10. M. Van. Boven, P. Daenens, K. Maes, M. Cokelaere, “ Content and composition of free
sterol free fatty alcohols in jojoba oil”, J. Agric. Food. Chem. 45, 1997, 1180-1184.
Table 1
Precision and accuracy characterization for cholesterol in egg yolk (n=9)
____________________________________________________________________
Cholesterol(mg)
precision
accuracy
added
measured
R.S.D. (%)
R.S.D. (%)
____________________________________________________________________
200
201.85
0.04
0.92
50
48.27
1.39
3.49
60
59.36
1.16
2.71
_
_________________________________
R.S.D.= relative standard deviation
Table 2
Comparative recovery mean values for cholesterol in egg yolk (n=3)
____________________________________________________________________
Procedure
S
M
US
USM
(%)
(%)
(%)
(%)
____________________________________________________________________
boiled and dried
99.8
95.54
70.40
95.36
boiled
94.06
91.6
____________________________________________________________________
S = saponification[2]; M=methyilation(ours); US=unsaponification; USM=unsaponification and
methylation
Table 3
Comparative mean value for cholesterol by two different methods (n=3)
_____________________________________________________________________
Method
Cholesterol (mg)
S. D.
R.S.D.
M
193.41
6.58
3.40
S
209.56
1.25
0.59
_____________________________________________________________________
M = methylation; S = saponification; S. D. : standard deviation; R.S.D.: relative standard
deviation
Figures legend
Fig. 1. Correlation curve for egg yolk cholesterol calculation
Fig. 2 The separation chromatogram of lipids in egg yolk. Fatty acids methyl esters
(FAME) of: myristic acid (C14:0); palmitoleic acid (C16:1); palmitic acid C16:0); oleic
acid (C18:1); linoleic acid (C18:2) ;stearic acid (C18:0); arachidonic acid( C20:4);
Fig. 3 Diosgenin (M=414) and cholesterol (M=386) mass spectra