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Food Chemistry 121 (2010) 12551259

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

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 b

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

a r t i c l e

i n f o

a b s t r a c t
A new method was developed to determine the presence of some rened vegetable oils in olive oil based on the sum of campesterol and stigmasterol percentages. Model systems of corn, soybean, sunower and cotton seed oils in olive oil at levels of 5%, 10% and 20% were prepared. The unsaponiables of these model systems were analysed by GLC using polar column with high thermal stability. An olive oil authenticity 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.

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

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 falsication 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, triglycerides or sterols can be useful for detection of olive oil adulteration. The HPLC quantitative analysis of triglycerides is considered a potent method for detection of olive adulteration (El-Hamdy & Perkins, 1981; Maria & Robert, 1987). The determination 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 rened vegetable oils in olive oil by determining the stigmastadienes or/and campesterol, since the former does not occur in olive and the later
* Corresponding author. Tel.: +962 777 160820; fax: +962 6 5355577. E-mail address: m.aldabbas@ju.edu.jo (M. Al-Dabbas). 0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.01.016

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 regulation (Salvador, Aranda, & Fregapane, 1998). These two facts limit the use of these two sterols in detecting the presence of rened vegetable oils in olive oil. Moreover, the determination of these two sterols is accomplished by separation of unsaponifiables 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 laborious work (Bohacenko & Kopicova, 2001). The use of thermo stable polar column is adequate to separate sterols, methyl sterols and alcohol of the silylated unsaponiables of vegetable oils without any further purication steps. Furthermore, this column allowed the separation of components that are considered as ngerprint of natural matrix. Therefore, it was used to detect the presence of small quantities of husk oil in olive oil using erthrodyol as a ngerprint (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 campesterol. On the other hand, the level of these sterols in the other

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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 levels of olive oil adulteration with soybean, corn, cottonseed and sunower oils by the GLC analysis using the thermo stable polar column.

2. Materials and methods 2.1. Vegetable oils Pure sunower, 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 Jordan. 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 saponied, and extracted according to the European Ofcial Methods of Analysis described in Regulations EEC 2568/1991. A solution of the unsponiable in hexane (10% w/v) was prepared. An aliquot of 50 ll of this solution was evaporated and the residue was silanized with a mixture of pyridinehexamethyldilizanetrimethylchlorosilazane 9:3:1 (v/v/v) at 50 ll/mg of the unsaponiables (Gutierrez, Varona, & Albi, 2000). The silanized unsaponiables were analysed by gas chromatography using Shimadzu gas chromatograph (Model 2010 Shimadzu Inc., Koyoto, Japan) supplied with splitsplitless injector port, ame 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.; lm thickness was 0.25 lm and the active ingredients were 35% diphenyl 65% dimethyl polysiloxane) was used. The carrier gas was helium at 1 ml/min column ow and 1: 80 split ratio. Injector and detector temperatures were 320 C. Oven Temperature was programmed from 200 to 300 C with a rate of 3 C/min. Peak identication of campesterol, sitgmasterol and b- Sitosterol was carried out by the retention times of their standards provided by Sigma Chemical Co (St. Luis, MO). Peak identication 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 contents in plant oils are much higher than their content in olive oil. For example, the campesterol percentage in cottonseed, sunower, 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 exceed 4% and the stigmasterol percentage must be less than that of campestrol (EC Regulation 2568, 1991). Therefore, these two sterols could be used as indicators to detect the adulteration of olive oil with vegetable oils. Fig. 1 shows the GLC traces of the unsponiable fractions of olive, corn, soybean, sunower and cottonseed oils. The traces show that campesterol, sitgmasterol, b- Sitosterol and D5-avenasterol were well separated. These results agreed with the results of other workers who reported that this polar column is adequate for fractionating sterols, methyl sterols and alcohols of the unsponiable 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, dened as the relative standard deviation of retention time for 15 replicates, for campesterol, stigmasterol, b-sitosterol and D5-avenasterol was less than 2% and the precision expressed as the relative standard deviation of peak areas of 15 replicate 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 percentage of each sterol was calculated in the analysed olive oil samples 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 established by EC Regulation (1991). Campestrol percentage was comparable with those of other published data, while that of stigmasterol was slightly higher (Boggia, Evangelisti, Ross, Salvadeor, & Zunin, 2005; Bohacenko and Kopicova, 2001; Salvador, Aranda, Gomez-Alonso, & Fregapane, 2000). To evaluate the capability of detecting the olive oil adulteration with corn, sunower, soybean and cottonseed oils mixtures containing 5%, 10% and 20% of these oils in olive oil (sample No. 6, Table 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 signicant increase in campesterol percentage of the mixed olive oil sample. Such increase ranged from 160% in case of addition of 5% of sunower oil to 470% in case of addition of 20% corn oil. The increase in stigmasterol due to the mixing process ranged from 200% 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 Kopicova (2001) who found that the contents of campesterol and D7-stigamstenol could be used to detect the adulteration of olive oil with some vegetable oils at levels as low as 5%. However, the campesterol percentage of some olive oils is higher than 4% set by the EC Regulation (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 analysed olive oil samples were in the range 19.725.45 with an average 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, sunower, soybean, 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 nding demonstrated that Af factor is a valid parameter that could be used to detect 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 unsaponiables fraction of: (A) virgin olive oil, (B) cotton seed oil, (C) sunower 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 Campesterol Stigmasterol b-Sitosterol D5-Avenasterol Af 1 2.98 1.59 88 7.46 20.88 2 3.05 1.26 87.4 8.28 22.20 3 2.91 1.7 89 6.4 20.69 4 3.2 1.29 83.8 11.6 21.27 5 2.82 0.96 82.4 13.8 25.45 6 2.72 1.21 88.8 7.25 24.45 7 3.2 1.12 85.5 10.2 22.15 8 3.13 1.14 85.6 10.2 22.42 9 2.8 1.65 86.0 10.4 21.47 10 3.11 1.11 86.86 8.93 22.70 11 3.41 1.43 85.13 10.0 19.7 12 2.7 1.96 88.0 8.48 20.46 Average 3.0 0.22 1.37 0.30 86.37 2.02 9.4 2.06 21.99 1.65

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Table 2 Content of some sterols (%) and Authenticity factor (Af) in some vegetable oils. Sample Campesterol Stigmasterol b-Sitosterol D5-Avenasterol Af Corn 20.53 8.74 65.09 5.64 2.42 Sunower 12.26 8.90 74.64 4.20 2.9 Soybean 18.16 14.74 64.28 2.8 2.04 Cottonseed 20.14 11.62 63.93 4.31 2.15

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 5 Campesterol Stigmasterol b-Sitosterol D5-Avenasterol Af 6.4 2.8 83.9 6.9 9.9 10 9.6 3.9 79.7 6.5 6.4 20 12.8 5.4 75.7 6.1 4.5 Sunower 5 4.4 2.5 86.4 6.7 13.5 10 5.5 3.5 84.7 6.3 10.1 20 6.8 5.4 81.9 5.9 7.2 Soybean 5 4.9 2.5 85.6 6.2 12.5 10 6.2 4.3 83.7 5.8 8.5 20 8.6 5.9 80.3 5.2 5.9 Cottonseed 5 4.5 2.3 86.4 6.7 13.7 10 5.3 3.5 84.9 6.3 10.4 20 8.1 4.4 81.7 5.8 7.0

Campesterol % + Stigmasterol %

20 15 10 5 0

Corn Soybean Sunflower Cotton

30 25 20 Af 15 10 5

Corn Soybean Sunflower Cotton

10

20%

10

20%

% of vegetable oil mixed with olive oil

Fig. 2. Changes in the sum of campesterol and stigmasterol percentages due to the mixing of vegetable oils with olive oil.

% of vegetable oil mixed with olive oil

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

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 sunower and cotton seed oils. The linear calibration curves obtained by plotting the percentage of the added plant oils versus the sum of campesterol and stigmasterol 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 constant differ according to the linear calibration curves of the added oils. Thus the added corn, soybean, sunower and cotton seed oil can be calculated from their regression equations as follows:

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 identied from their GLC traces. However, this method can not decide the type of vegetable oil added to the olive 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 the cultivar, 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 Food Science, 19, 97103. CAC (2005). Codex Alimentarius. Ofcial standard for vegetable oils No. 210. EC Regulation 2568 (1991). The characteristics of olive oil and olive residue oil and relevant methods of analysis. Ofcial 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 Oil Chemists, 57, 4953.

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

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K.M. Al-Ismail et al. / Food Chemistry 121 (2010) 12551259 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, 69, 447450. Frega, N., Bocci, F., & Lercker, G. (1993). High-resolution gas chromatographic determination of alkanols in oils extracted from olive. Journal of the American Oil Chemists, 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 Food Chemistry, 48(4), 11061110.

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Maria, T., & Robert, M. (1987). Authentication of virgin olive oils using principal component analysis of triglyceride and fatty acid proles: 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 from 1995/96 and 1996/97 crops. Journal of 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.