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Analytical Methods

Detection of olive oil adulteration with some plant oils by GLC analysis of

sterols using polar column

Khalid M. Al-Ismail a, Ali K. Alsaed a, Rafat Ahmad b, Maher Al-Dabbas a,*

a Department of Nutrition and Food Technology, Faculty of Agriculture, The University of Jordan, Amman 11942, Jordanb Industrial Chemistry Center, The Royal Scientific Society, Amman, Jordan

a r t i c l e i n f o

Article history:

Received 1 July 2008

Received in revised form 11 January 2010

Accepted 16 January 2010

Keywords:

Olive oil

Adulteration

Sterol, Vegetable oils

Gas liquid chromatography

Campesterol

Stigmasterol

a b s t r a c t

A new method was developed to determine the presence of some refined vegetable oils in olive oil based

on the sum of campesterol and stigmasterol percentages. Model systems of corn, soybean, sunflower and

cotton seed oils in olive oil at levels of 5%, 10% and 20% were prepared. The unsaponifiables of these

model systems were analysed by GLC using polar column with high thermal stability. An olive oil authen-

ticity factor based on the summation of campesterol and stigmasterol percentages was established as an

indicator of olive oil adulteration with vegetable oils. The results indicate the possibility to detect the

presence as little as 5% of these plant oils in olive oil.

2010 Elsevier Ltd. All rights reserved.

1. Introduction

Olive oil is usually more expensive than other edible oils,

which makes it a candidate for adulteration with other cheaper

oils. Therefore, different methods have been developed to

countertract the falsification that is being perpetrated. Some

of these methods based on the qualitative analysis of olive oils

such as colour, triglycerides and fatty acids. The colour based

methods such as Belier test may be unsatisfactory because

they are not able to detect the presence of all edible oils in

olive oil. Furthermore, the qualitative analysis of fatty acids

or triglycerides may be inadequate since most vegetable oils

contain the same fatty acids or triglycerides (Maria & Robert,

1987).

On contrary, the quantitative analysis of fatty acids, triglycer-

ides or sterols can be useful for detection of olive oil adultera-

tion. The HPLC quantitative analysis of triglycerides is

considered a potent method for detection of olive adulteration

(El-Hamdy & Perkins, 1981; Maria & Robert, 1987). The deter-

mination of sterols in olive oil or other vegetable oil is usually

carried out by gas liquid chromatography (GLC). This analysis

could be used to detect the presence of refined vegetable oils

in olive oil by determining the stigmastadienes or/and campes-

terol, since the former does not occur in olive and the later

should not be more than 4% of total sterols. However, using

stigmastadienes as an indicator for olive oil adulteration is

reliable only when their concentration lies between 0.01 and

4 mg/kg (EC Regulation, 1991). Furthermore, the campesterol

content in some olive oils exceeds the 4% set by the EC regula-

tion (Salvador, Aranda, & Fregapane, 1998). These two facts

limit the use of these two sterols in detecting the presence of

refined vegetable oils in olive oil. Moreover, the determination

of these two sterols is accomplished by separation of unsaponif-

iables from lipid matrix, isolation of sterols by TLC, separation

of stigmastadienes by alumina column and determination of

these sterol derivatives by gas chromatography using non-polar

capillary columns. However, the isolation of sterols by TLC and

alumina column is criticised for its time consumption and labo-

rious work (Bohacenko & Kopicova, 2001).

The use of thermo stable polar column is adequate to sepa-

rate sterols, methyl sterols and alcohol of the silylated unsapo-

nifiables of vegetable oils without any further purification

steps. Furthermore, this column allowed the separation of com-

ponents that are considered as fingerprint of natural matrix.

Therefore, it was used to detect the presence of small quantities

of husk oil in olive oil using erthrodyol as a fingerprint (Frega,

Bocci, & Lercker, 1993).

The maximum content of campesterol in olive oils as set by

Commission Regulation (EEC) No. 22568/1991 must be 64%,

while that of stigmasterol must be less than that of campester-

ol. On the other hand, the level of these sterols in the other

0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved.

doi:10.1016/j.foodchem.2010.01.016

*Corresponding author. Tel.: +962 777 160820; fax: +962 6 5355577.E-mail address: [email protected] (M. Al-Dabbas).

Food Chemistry 121 (2010) 12551259

Contents lists available at ScienceDirect

Food Chemistry

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o o d c h e m


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vegetable oils exceeds their levels in olive oil by more than two

times or more. Therefore, this property was used in this study

as a new method for determining the minimum detectable lev-

els of olive oil adulteration with soybean, corn, cottonseed and

sunflower oils by the GLC analysis using the thermo stable polar

column.

2. Materials and methods

2.1. Vegetable oils

Pure sunflower, corn, soybean and cotton seeds oil samples

were purchased from local markets, checked for their authenticity

and used as adulterant. Twelve olive oil samples of extra virgin and

virgin olive oils were obtained from different olive oil press in Jor-

dan. Mixtures of olive oil and these vegetable oils at levels of 5%,

10%, and 20% (w/w) were prepared.

2.2. Sterols analysis

Five grams of each of olive oil, vegetable oils and their prepared

mixtures, were saponified, and extracted according to the Euro-

pean Official Methods of Analysis described in Regulations EEC

2568/1991. A solution of the unsponifiable in hexane (10% w/v)

was prepared. An aliquot of 50 ll of this solution was evaporatedand the residue was silanized with a mixture of pyridinehexam-

ethyldilizanetrimethylchlorosilazane 9:3:1 (v/v/v) at 50 ll/mg ofthe unsaponifiables (Gutierrez, Varona, & Albi, 2000). The silanized

unsaponifiables were analysed by gas chromatography using Shi-

madzu gas chromatograph (Model 2010 Shimadzu Inc., Koyoto, Ja-

pan) supplied with splitsplitless injector port, flame ionisation

detector (FID) and digital integrator (Shimdzu C-R 7A). A RTX-65

TG (Restek, USA) capillary column (60 m 0.25 mm i.d.; filmthickness was 0.25lm and the active ingredients were 35% diphe-nyl65% dimethyl polysiloxane) was used. The carrier gas was he-

lium at 1 ml/min column flow and 1: 80 split ratio. Injector and

detector temperatures were 320 C. Oven Temperature was pro-

grammed from 200 to 300 C with a rate of 3 C/min.

Peak identification of campesterol, sitgmasterol and b- Sitos-

terol was carried out by the retention times of their standards pro-

vided by Sigma Chemical Co (St. Luis, MO). Peak identification of

D5-avenasterol was carried out by comparing its retention time

with those reported in literature (Frega, Bocci, & Lercker, 1992).

3. Results and discussion

It is well established that the campesterol and stigmasterol con-

tents in plant oils are much higher than their content in olive oil.

For example, the campesterol percentage in cottonseed, sunflower,

soybean and corn oils sterols are 6.414.15%, 513%, 15.824.2%

and 1624%, while that of stigmasterol are 2.16.8%, 613%,

14.919.1% and 4.38.0%, respectively (CAC, 2005). On the other

hand, the campesterol percentage in olive oil sterols must not ex-

ceed 4% and the stigmasterol percentage must be less than that of

campestrol (EC Regulation 2568, 1991). Therefore, these two ster-

ols could be used as indicators to detect the adulteration of olive oil

with vegetable oils.

Fig. 1 shows the GLC traces of the unsponifiable fractions of ol-

ive, corn, soybean, sunflower and cottonseed oils. The traces show

that campesterol, sitgmasterol, b- Sitosterol and D5-avenasterol

were well separated. These results agreed with the results of otherworkers who reported that this polar column is adequate for frac-

tionating sterols, methyl sterols and alcohols of the unsponifiable

matter (Frega et al., 1992). They concluded also that this column

could be used as alternative for the recommended non-polar ones

(SE52 and SE54).

The reproducibility, defined as the relative standard deviation

of retention time for 15 replicates, for campesterol, stigmasterol,

b-sitosterol andD5-avenasterol was less than 2% and the precision

expressed as the relative standard deviation of peak areas of 15replicate runs for these four sterols was less than 4%.

It is well known that 4 sterols aforementioned represent more

than 95% of total sterols in olive oils (EC Regulation, 1991), and

accordingly could be represent the total sterol in olive oil. The per-

centage of each sterol was calculated in the analysed olive oil sam-

ples and those mixed with the other vegetable oils by dividing

their peak areas by the total peak areas of these 4 sterols and the

results are reported in Tables 1 and 2. It is evident that the studied

4 sterols are present within the acceptable concentrations estab-

lished by EC Regulation (1991). Campestrol percentage was com-

parable with those of other published data, while that of

stigmasterol was slightly higher (Boggia, Evangelisti, Ross, Salva-

deor, & Zunin, 2005; Bohacenko and Kopicova, 2001; Salvador,

Aranda, Gomez-Alonso, & Fregapane, 2000).To evaluate the capability of detecting the olive oil adulteration

with corn, sunflower, soybean and cottonseed oils mixtures con-

taining 5%, 10% and 20% of these oils in olive oil (sample No. 6, Ta-

ble 1) were prepared and analysed for their content of sterols.

Results in Table 3 shows that addition of 520% vegetable oils to

olive oil (sample No. 6t in Table 1) caused a significant increase

in campesterol percentage of the mixed olive oil sample. Such in-

crease ranged from 160% in case of addition of 5% of sunflower

oil to 470% in case of addition of 20% corn oil. The increase in stig-

masterol due to the mixing process ranged from200% in case of the

addition of 5% cottonseed oil to 500% in case of the addition of 20%

soybean oil.

The stigmasterol percentage in olive oil sterols must be less

than the maximum acceptable limit for campesterol (4% of total

sterol). In contrary to campesterol, stigmasterol percentage of the

mixtures did not exceed this limit. Therefore, campesterol could

be used as indicator but not stigmasterol to detect the adulteration

of olive oil with these vegetable oils at levels of more than 5%.

These results agree with those reported by Bohacenko and Kopico-

va (2001) who found that the contents of campesterol and D7-sti-

gamstenol could be used to detect the adulteration of olive oil with

some vegetable oils at levels as low as 5%. However, the campester-

ol percentage of some olive oils is higher than 4% set by the EC Reg-

ulation (1991) as observed in case of Cornicabra olive oil (Salvador

et al., 1998). This fact may limit the use of campesterol percentage

to detect the olive oil adulteration with plant oils. Therefore, both

campesterol and stigmasterol percentages were used in this study

to detect adulteration of olive with these oils by measuring the

authenticity factor (Af) as follows.

Af 100 Campesterol% stigmasterol%=Campesterol%

stigmasterol%:

The results are reported in Tables 13. The Af factors of the ana-

lysed olive oil samples were in the range 19.725.45 with an aver-

age of 21.99 1.65 (Table 1), Whereas the Af factor for the plant

oils used in this study were in the range 2.042.9 (Table 2).

Table 3 shows that the addition of 5% of corn, sunflower, soy-

bean, and cottonseed oils to the olive oil decreased Af factor to

9.9, 13.5, 12.5, and 13.7, which represent 40.5%, 55.2%, 51.1% and

56.0%, respectively, of the Af factor of the olive oil sample used

in the preparation of the mixtures (24.45). These finding demon-strated that Af factor is a valid parameter that could be used to de-

tect the adulteration of olive oil at levels as low as 5%, even the

olive oil content of campesterol and stigmasterol are 4% and 3%,

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Fig. 1. Gas chromatographic of unsaponifiables fraction of: (A) virgin olive oil, (B) cotton seed oil, (C) sunflower oil, (D) corn oil, (E) soybean oil and (F) standards. 1,

campesterol; 2, stigmasterol; 3, b-sitosterol and 4, D5-aveasterol. The retention time of theses four sterols are 22.56, 23.18, 24.27, 25.24 min, respectively.

Table 1

Content of the major sterols (%) and Authenticity factor (Af) in 12 olive oil samples collected from the local markets.

Sample 1 2 3 4 5 6 7 8 9 10 11 12 Average

Campesterol 2.98 3.05 2.91 3.2 2.82 2.72 3.2 3.13 2.8 3.11 3.41 2.7 3.0 0.22

Stigmasterol 1.59 1.26 1.7 1.29 0.96 1.21 1.12 1.14 1.65 1.11 1.43 1.96 1.37 0.30b-Sitosterol 88 87.4 89 83.8 82.4 88.8 85.5 85.6 86.0 86.86 85.13 88.0 86.37 2.02

D5-Avenasterol 7.46 8.28 6.4 11.6 13.8 7.25 10.2 10.2 10.4 8.93 10.0 8.48 9.4 2.06

Af 20.88 22.20 20.69 21.27 25.45 24.45 22.15 22.42 21.47 22.70 19.7 20.46 21.99 1.65

K.M. Al-Ismail et al. / Food Chemistry 121 (2010) 12551259 1257


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respectively. In this rare case, the Af factor will be 13.3 which are

higher than those found for 5% mixtures of corn and soybean oils

and comparable to those of sunflower and cotton seed oils.

The linear calibration curves obtained by plotting the percent-

age of the added plant oils versus the sum of campesterol and stig-

masterol contents (Fig. 2) show the possibility of the rough

estimation of the extent of olive oil adulteration with these four

vegetable oils at levels equal or higher than 5% by their regression

equations which can be represented as follows:

Added oil% Campesterol% Stigmasterol% or a=b

where a and b are the regression equation constants. Theses con-

stant differ according to the linear calibration curves of the added

oils. Thus the added corn, soybean, sunflower and cotton seed oil

can be calculated from their regression equations as follows:

Corn oil% Campesterol% Stigmasterol% 0:6=4:72

Soybean oil% Campesterol% Stigmasterol% 0:35=3:5

Sunflower oil% Campesterol% Stigmasterol% 1:25=2:7

Cottonseed oil% Campesterol% Stigmasterol% 1:2=2:63

The adulteration of olive oil with these oils at 3% or less cannot be

detected or estimated (Fig. 3). At these levels of adulteration, the Af

factor will be within the Af range of the analysed olive oil samples

(19.725.45).

This method, which employs the determination of sterols

using polar column with high thermal stability without previous

isolation of sterols by TLC can be used to detect the adulteration

of olive oil with vegetable oils by determining only the four major

sterols in olive oils. Furthermore, these four sterol components

could be easily identified from their GLC traces. However, thismethod can not decide the type of vegetable oil added to the ol-

ive oil and also can not be used for the detection of hazelnut and

lampante oils because of their low contents of campesterol and

stigmasterol.

References

Boggia, R., Evangelisti, F., Ross, N., Salvadeor, P., & Zunin, P. (2005). Chemical

composition of olive oils of thecultivar, Colombaia. Grasa y Aceites, 65, 276283.Bohacenko, I., & Kopicova, Z. (2001). Detection of olive oils authenticity by

determination of their sterol content using LC/GC. Gzech Journal of FoodScience, 19, 97103.

CAC (2005). Codex Alimentarius. Official standard for vegetable oils No. 210.

EC Regulation 2568 (1991). The characteristics of olive oil and olive residue oil andrelevant methods of analysis. Official Journal, L, 248, 67.

El-Hamdy, A. H., & Perkins, E. G. (1981). High performance reversed-phase

chromatography of natural triglyceride mixtures. Journal of the American OilChemists, 57, 4953.

Table 3

Content of some sterols in model samples of olive oil mixed with other vegetable oils.

Sample % of vegetable oil in olive oil

Corn Sunflower Soybean Cottonseed

5 10 20 5 10 20 5 10 20 5 10 20

Campesterol 6.4 9.6 12.8 4.4 5.5 6.8 4.9 6.2 8.6 4.5 5.3 8.1

Stigmasterol 2.8 3.9 5.4 2.5 3.5 5.4 2.5 4.3 5.9 2.3 3.5 4.4

b-Sitosterol 83.9 79.7 75.7 86.4 84.7 81.9 85.6 83.7 80.3 86.4 84.9 81.7

D5-Avenasterol 6.9 6.5 6.1 6.7 6.3 5.9 6.2 5.8 5.2 6.7 6.3 5.8

Af 9.9 6.4 4.5 13.5 10.1 7.2 12.5 8.5 5.9 13.7 10.4 7.0

Table 2

Content of some sterols (%) and Authenticity factor (Af) in some vegetable oils.

Sample Corn Sunflower Soybean Cottonseed

Campesterol 20.53 12.26 18.16 20.14

Stigmasterol 8.74 8.90 14.74 11.62

b-Sitosterol 65.09 74.64 64.28 63.93D5-Avenasterol 5.64 4.20 2.8 4.31

Af 2.42 2.9 2.04 2.15

0

5

10

15

20

0 5 10 20%

% of vegetable oil mixed with olive oil

Campesterol%+

Stigmasterol%

Corn

Soybean

Sunflower

Cotton

Fig. 2. Changes in the sum of campesterol and stigmasterol percentages due to the

mixing of vegetable oils with olive oil.

0

5

10

15

20

25

30

0 5 10 20%

% of vegetable oil mixed with olive oil

Af

Corn

Soybean

Sunflower

Cotton

Fig. 3. Changes in Af factor due to the mixing of vegetable oils with olive oil.

1258 K.M. Al-Ismail et al. / Food Chemistry 121 (2010) 12551259


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Frega, N., Bocci, F., & Lercker, G. (1992). Direct gas chromatographic analysis of the

unsaponifiable fraction of different oils with a polar capillary column. Journal ofthe American Oil Chemists, 69, 447450.

Frega, N., Bocci, F., & Lercker, G. (1993). High-resolution gas chromatographic

determination of alkanols in oils extractedfrom olive.Journal of the American OilChemists, 70, 920921.

Gutierrez, F., Varona, I., & Albi, M. A. (2000). Relation of acidity and sensory quality

with sterol content of olive oil from stored fruit. Journal of Agriculture and FoodChemistry, 48(4), 11061110.

Maria, T., & Robert, M. (1987). Authentication of virgin olive oils using principal

component analysis of triglyceride and fatty acid profiles: Part 2-detection of

adulteration with other vegetable oils. Food Chemistry, 25, 251258.Salvador, M. D., Aranda, F., & Fregapane, G. (1998). Chemical composition of

commercial Cornicabra virgin olive oil from1995/96 and 1996/97 crops.Journalof the American Oil Chemists, 75, 13051311.

Salvador, M. D., Aranda, F., Gomez-Alonso, S., & Fregapane, G. (2000). Quality

characteristics of Cornicabra virgin olive oil. Research Advances in Oil Chemistry,1, 3239.

K.M. Al-Ismail et al. / Food Chemistry 121 (2010) 12551259 1259

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