Detection of Chemlali Extra-Virgin Olive Oil Adulteration Mixed with Soybean Oil, Corn Oil, and...

Detection of Chemlali Extra-Virgin Olive Oil Adulteration Mixed withSoybean Oil, Corn Oil, and Sunflower Oil by Using GC and HPLCHazem Jabeur,† Akram Zribi,† Jamel Makni,‡ Ahmed Rebai,§ Ridha Abdelhedi,†

and Mohamed Bouaziz*,†,#

†Laboratoire d’Electrochimie et Environnement, Ecole Nationale d’Ingenieurs de Sfax, Universite de Sfax, B.P. 1173, 3038 Sfax,Tunisia‡Laboratoire de l’Office National de l’Huile a Sfax, 3000 Sfax, Tunisia§Laboratoire de Microorganismes et Biomolecules, Equipe des Procedes de Criblage Moleculaires et Cellulaires, Centre deBiotechnologie de Sfax, B.P. 1177, 3018 Sfax, Tunisia#Institut Superieur de Biotechnologie de Sfax, Universite de Sfax, B.P. 1175, 3038 Sfax, Tunisia

ABSTRACT: Fatty acid composition as an indicator of purity suggests that linolenic acid content could be used as a parameterfor the detection of extra/virgin olive oil fraud with 5% of soybean oil. The adulteration could also be detected by the increase ofthe trans-fatty acid contents with 3% of soybean oil, 2% of corn oil, and 4% of sunflower oil. The use of the ΔECN42 proved tobe effective in Chemlali extra-virgin olive oil adulteration even at low levels: 1% of sunflower oil, 3% of soybean oil, and 3% ofcorn oil. The sterol profile is almost decisive in clarifying the adulteration of olive oils with other cheaper ones: 1% of sunfloweroil could be detected by the increase of Δ7-stigmastenol and 4% of corn oil by the increase of campesterol. Linear discriminantanalysis could represent a powerful tool for faster and cheaper evaluation of extra-virgin olive oil adulteration.

KEYWORDS: extra-virgin olive oil adulteration, vegetable oils, triglycerides, fatty acids, sterols, linear discriminant analysis

■ INTRODUCTION

Olive cultivation is widespread throughout the Mediterraneanregion and is important for the rural economy, local heritage,and environment. In Tunisia, the olive oil sector plays animportant role in the economy, providing both employmentand export revenue. Olive trees in Tunisia cover an area of1.7 million ha and account for >4% of the olive oil produced inthe world. Indeed, with an annual production of 170,000 tons,Tunisia is the fourth largest producer and exporter of olive oilin the world.1 More than 50 different olive oil cultivars exist inTunisia such as Chemlali, Chetoui, Meski, Chemchali, Oueslati,and Zalmati. However, two important varieties dominate mostof the arable land: Chetoui and Chemlali.2,3 In fact, Chemlali isthe main cultivated variety, covering 60% of the olive-growingsurface, spread from the northeast of the country to theextreme south.4,5 In addition, despite its ordinary organolepticand taste characteristics, olive oil composition is differentaccording to the geographical origin.6

Extra-virgin olive oil is usually more expensive than othervegetable oils for several reasons. In fact, it is the oil obtainedfrom the fruit of the olive tree solely by mechanical means. Ithas not undergone any other treatment than washing,decantation, centrifugation, and filtration.7 Therefore, differentmethods have been developed to control the falsification of theproduct authenticity and quality that is being perpetrated. Thedeterminations of fatty acids, triacylglycerols (TAGs), andsterols through chromatographic methods allow the detectionof oil adulteration and the definition of the blend’scomposition.8−12

Fatty acid methyl esters (FAME), obtained by alkali/catalyzedtransesterification of vegetable oils, can be characterized by gas

chromatography (GC).13−16 The methyl esters of the fattyacids under investigation are usually the molecules used forthe chemical analyses establishing the limits pertaining tothe content of fatty acids in olive oil that can be used for thedifferentiation between genuine olive oil and other vegetableoils (soybean, sunflower, and corn).TAGs are the main component of vegetable oils as they are

generally found between the 97 and 99% range of the whole oilcomposition. The high-performance liquid chromatography(HPLC) quantitative analysis of TAGs is considered to be aneffective method for the detection of extra-virgin olive oil(EVOO) adulteration.17−19 The advantage of using the TAGsprofile includes the distribution of fatty acids between thedifferent stereospecific positions on the glycerol molecule.TAGs are separated according to the equivalent carbon number(ECN) and the positions of double bond(s). Until recently, themost prominent methods to detect the adulteration of extra-virgin olive oil with other vegetable oils have been thetrilinolein (LLL) content and the difference between thetheoretical value of TAGs with an equivalent carbon number of42 (ECN42theor). Appropriate software is used to compute theΔECN42 on the basis of fatty acid composition data andanalytical triglyceride results (ECN42HPLC).

20−22

Zeng et al.23 suggested the applicability of liquid chromatog-raphy electrospray tandem mass spectrometry (LC-ESI-MS2)for the structural elucidation of TAGs in vegetable oils.

Received: February 1, 2014Revised: May 6, 2014Accepted: May 8, 2014Published: May 8, 2014

Article

pubs.acs.org/JAFC

© 2014 American Chemical Society 4893 dx.doi.org/10.1021/jf500571n | J. Agric. Food Chem. 2014, 62, 4893−4904

pubs.acs.org/JAFC

With regard to GC, the International Union of Pure andApplied Chemistry (IUPAC) has adopted a method to resolve

triglycerides according to the number of equal carbon atoms bygas−liquid chromatography (GLC).24 However, the technique is

Figure 1. Gas chromatogram of standards solution containing the fatty acid methyl esters.

Table 1. Fatty Acid and Triglyceride Compositions of Extra-Virgin Olive Oil (EVOO), Soybean Oil (SO), and Mixtures ofEVOO and SOa

EVOO SOEVOO +1% SO

EVOO +2% SO

EVOO +3% SO

EVOO +4% SO

EVOO +5% SO

EVOO +10% SO

C16:0 15.82 ± 0.19 11.00 ± 0.13 15.59 ± 0.19 15.53 ± 0.19 15.49 ± 0.18 15.37 ± 0.18 15.30 ± 0.18 14.89 ± 0.18C16:1 1.99 ± 0.02 0.10 ± 0.00 1.98 ± 0.02 1.98 ± 0.02 1.97 ± 0.02 1.95 ± 0.02 1.94 ± 0.02 1.91 ± 0.02C17:0 0.04 ± 0.00 0.10 ± 0.00 0.05 ± 0.00 0.05 ± 0.00 0.05 ± 0.00 0.06 ± 0.00 0.06 ± 0.00 0.06 ± 0.00C17:1 0.09 ± 0.00 0.05 ± 0.00 0.09 ± 0.00 0.09 ± 0.00 0.09 ± 0.00 0.08 ± 0.00 0.08 ± 0.00 0.08 ± 0.00C18:0 2.18 ± 0.02 4.66 ± 0.06 2.24 ± 0.03 2.26 ± 0.03 2.29 ± 0.03 2.31 ± 0.02 2.34 ± 0.03 2.44 ± 0.03C18:1 62.37 ± 0.76 21.83 ± 0.26 62.13 ± 0.76 61.75 ± 0.75 61.34 ± 0.75 61.12 ± 0.74 60.70 ± 0.74 59.07 ± 0.72C18:2 16.21 ± 0.19 53.93 ± 0.66 16.60 ± 0.20 17.01 ± 0.20 17.44 ± 0.21 17.76 ± 0.21 17.91 ± 0.21 19.71 ± 0.24C18:3 0.69 ± 0.01 7.77 ± 0.09 0.71 ± 0.01 0.72 ± 0.01 0.74 ± 0.01 0.76 ± 0.01 1.08 ± 0.01 1.25 ± 0.01C20:0 0.41 ± 0.00 0.39 ± 0.00 0.41 ± 0.00 0.41 ± 0.00 0.40 ± 0.00 0.40 ± 0.00 0.40 ± 0.00 0 0.40 ± 0.00C20:1 0.20 ± 0.00 0.17 ± 0.00 0.20 ± 0.00 0.20 ± 0.00 0.19 ± 0.00 0.19 ± 0.00 0.19 ± 0.00 0.19 ± 0.00TC18:1 0.01 ± 0.00 0.04 ± 0.00 0.03 ± 0.00 0.03 ± 0.00 0.03 ± 0.00 0.03 ± 0.00 0.03 ± 0.00 0.03 ± 0.00∑(TC18:2 +TC18:3)

0.02 ± 0.00 0.66 ± 0.01 0.04 ± 0.00 0.04 ± 0.00 0.06 ± 0.00 0.06 ± 0.00 0.08 ± 0.00 0.10 ± 0.00

LLL 0.49 ± 0.00 8.27 ± 0.10 0.54 ± 0.00 0.62 ± 0.00 0.90 ± 0.01 1.17 ± 0.01 1.24 ± 0.01 1.86 ± 0.02ECN40 (LLLn) 0.00 ± 0.00 1.23 ± 0.01 0.27 ± 0.00 0.34 ± 0.00 0.41 ± 0.00 0.48 ± 0.00 0.70 ± 0.01 0.76 ± 0.01ECN42 1.12 ± 0.01 8.58 ± 0.10 1.42 ± 0.02 1.57 ± 0.02 1.88 ± 0.02 2.08 ± 0.02 2.71 ± 0.03 3.59 ± 0.04ECN44 10.03 ± 0.12 25.88 ± 0.31 10.15 ± 0.12 10.25 ± 0.12 10.46 ± 0.13 10.65 ± 0.13 10.70 ± 0.13 11.77 ± 0.14ECN46 34.95 ± 0.42 31.96 ± 0.39 34.90 ± 0.43 34.86 ± 0.43 34.78 ± 0.42 34.74 ± 0.42 34.74 ± 0.42 34.04 ± 0.41ECN48 50.14 ± 0.61 22.08 ± 0.27 49.49 ± 0.60 49.18 ± 0.60 48.62 ± 0.59 48.15 ± 0.59 47.24 ± 0.57 45.75 ± 0.56ECN50 3.76 ± 0.04 10.27 ± 0.12 3.77 ± 0.05 3.80 ± 0.05 3.85 ± 0.04 3.90 ± 0.04 3.91 ± 0.04 4.09 ± 0.05ECN42theor 1.19 ± 0.01 24.00 ± 0.29 1.32 ± 0.02 1.43 ± 0.02 1.58 ± 0.02 1.67 ± 0.02 1.79 ± 0.02 1.82 ± 0.02ΔECN42 0.07 ± 0.00 15.42 ± 0.19 0.10 ± 0.00 0.14 ± 0.00 0.30 ± 0.00 0.41 ± 0.00 0.93 ± 0.01 1.77 ± 0.02ECN44 + ECN46 44.98 ± 0.54 57.84 ± 0.70 45.05 ± 0.55 45.11 ± 0.55 45.24 ± 0.55 45.39 ± 0.55 45.44 ± 0.55 45.81 ± 0.55ECN46/LLL 71.32 3.86 64.62 56.22 38.64 29.69 28.01 18.30(ECN44 + ECN46)/LLL

91.79 6.99 83.42 72.75 50.26 38.79 36.64 24.62

aEach value represents the mean of four determinations of two successive crop seasons (n = 4; two different samples for each crop season).

Journal of Agricultural and Food Chemistry Article

dx.doi.org/10.1021/jf500571n | J. Agric. Food Chem. 2014, 62, 4893−49044894

Table 2. Fatty Acid and Triglyceride Compositions of Extra-Virgin Olive Oil (EVOO), Corn Oil (CO), and Mixtures of EVOOand COa

EVOO COEVOO +1% CO

EVOO +2% CO

EVOO +3% CO

EVOO +4% CO

EVOO +5% CO

EVOO +10% CO

C16:0 15.82 ± 0.19 12.03 ± 0.14 15.83 ± 0.19 15.82 ± 0.19 15.80 ± 0.19 15.77 ± 0.19 15.60 ± 0.19 14.81 ± 0.18C16:1 1.99 ± 0.02 0.13 ± 0.00 1.98 ± 0.02 1.98 ± 0.02 1.98 ± 0.02 1.97 ± 0.02 1.95 ± 0.02 1.90 ± 0.02C17:0 0.04 ± 0.00 0.06 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00C17:1 0.09 ± 0.00 0.03 ± 0.00 0.08 ± 0.00 0.08 ± 0.00 0.08 ± 0.00 0.08 ± 0.00 0.08 ± 0.00 0.08 ± 0.00C18:0 2.18 ± 0.02 1.88 ± 0.02 2.18 ± 0.02 2.17 ± 0.02 2.17 ± 0.02 2.15 ± 0.02 2.15 ± 0.02 2.13 ± 0.02C18:1 62.37 ± 0.76 28.48 ± 0.35 62.16 ± 0.76 61.87 ± 0.75 61.39 ± 0.75 60.78 ± 0.74 60.74 ± 0.74 59.52 ± 0.73C18:2 16.21 ± 0.19 55.89 ± 0.68 16.42 ± 0.20 16.72 ± 0.20 17.23 ± 0.21 17.89 ± 0.22 18.11 ± 0.22 20.17 ± 0.24C18:3 0.69 ± 0.01 0.88 ± 0.01 0.69 ± 0.01 0.70 ± 0.01 0.70 ± 0.01 0.71 ± 0.01 0.72 ± 0.01 0.74 ± 0.01C20:0 0.41 ± 0.00 0.39 ± 0.00 0.41 ± 0.00 0.41 ± 0.00 0.40 ± 0.00 0.40 ± 0.00 0.40 ± 0.00 0.40 ± 0.00C20:1 0.20 ± 0.00 0.23 ± 0.00 0.21 ± 0.00 0.21 ± 0.00 0.21 ± 0.00 0.21 ± 0.00 0.21 ± 0.00 0.21 ± 0.00TC18:1 0.01 ± 0.00 0.05 ± 0.00 0.02 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 0.11 ± 0.00∑(TC18:2 + TC18:3) 0.02 ± 0.00 1.74 ± 0.02 0.05 ± 0.00 0.06 ± 0.00 0.07 ± 0.00 0.09 ± 0.00 0.10 ± 0.00 0.18 ± 0.00LLL 0.49 ± 0.00 20.15 ± 0.24 0.52 ± 0.00 0.54 ± 0.00 0.90 ± 0.01 1.17 ± 0.01 1.24 ± 0.01 1.86 ± 0.02ECN40 (LLLn) 0.00 ± 0.00 1.11 ± 0.01 0.11 ± 0.00 0.17 ± 0.00 0.24 ± 0.00 0.29 ± 0.00 0.36 ± 0.00 0.69 ± 0.01ECN42 1.12 ± 0.01 20.63 ± 0.25 1.33 ± 0.01 1.38 ± 0.01 1.49 ± 0.02 1.86 ± 0.02 1.98 ± 0.02 2.87 ± 0.03ECN44 10.03 ± 0.12 38.05 ± 0.46 10.24 ± 0.12 10.73 ± 0.13 11.16 ± 0.13 11.13 ± 0.13 11.67 ± 0.14 12.20 ± 0.15ECN46 34.95 ± 0.42 27.94 ± 0.34 34.85 ± 0.43 34.83 ± 0.43 34.80 ± 0.43 34.77 ± 0.42 34.68 ± 0.42 34.22 ± 0.42ECN48 50.14 ± 0.61 11.08 ± 0.13 49.72 ± 0.61 49.17 ± 0.60 48.58 ± 0.59 48.24 ± 0.59 47.60 ± 0.58 46.36 ± 0.56ECN50 3.76 ± 0.04 1.19 ± 0.01 3.75 ± 0.04 3.72 ± 0.04 3.73 ± 0.04 3.71 ± 0.04 3.71 ± 0.04 3.66 ± 0.04ECN42theor 1.19 ± 0.01 18.39 ± 0.22 1.22 ± 0.01 1.23 ± 0.01 1.28 ± 0.01 1.36 ± 0.01 1.30 ± 0.01 1.61 ± 0.02ΔECN42 0.07 ± 0.00 2.24 ± 0.03 0.11 ± 0.00 0.15 ± 0.00 0.21 ± 0.01 0.50 ± 0.01 0.68 ± 0.01 1.26 ± 0.01ECN44 + ECN46 44.98 ± 0.54 65.99 ± 0.80 45.09 ± 0.55 45.56 ± 0.56 45.96 ± 0.56 45.90 ± 0.55 46.35 ± 0.56 46.42 ± 0.57ECN46/LLL 71.32 1.38 67.01 64.50 38.66 29.71 27.96 18.39(ECN44 + ECN46)/LLL

91.79 3.27 86.71 84.37 51.06 39.23 37.37 24.95


Table 3. Fatty Acid and Triglyceride Compositions of Extra-Virgin Olive Oil (EVOO), Sunflower Oil (SfO), and Mixtures ofEVOO and SfOa

EVOO SfOEVOO +1% SfO

EVOO +2% SfO

EVOO +3% SfO

EVOO +4% SfO

EVOO +5% SfO

EVOO +10% SfO

C16:0 15.82 ± 0.19 6.53 ± 0.08 15.68 ± 0.19 15.56 ± 0.19 15.41 ± 0.18 15.15 ± 0.18 15.00 ± 0.18 14.72 ± 0.18C16:1 1.99 ± 0.02 0.13 ± 0.00 1.99 ± 0.02 1.99 ± 0.02 1.99 ± 0.02 1.98 ± 0.02 1.98 ± 0.02 1.94 ± 0.02C17:0 0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00C17:1 0.09 ± 0.00 0.03 ± 0.00 0.09 ± 0.00 0.09 ± 0.00 0.09 ± 0.00 0.08 ± 0.00 0.08 ± 0.00 0.08 ± 0.00C18:0 2.18 ± 0.02 3.64 ± 0.04 2.22 ± 0.03 2.24 ± 0.03 2.25 ± 0.02 2.26 ± 0.02 2.27 ± 0.02 2.33 ± 0.03C18:1 62.37 ± 0.76 27.58 ± 0.33 61.91 ± 0.76 61.47 ± 0.75 61.23 ± 0.75 61.07 ± 0.74 60.82 ± 0.74 59.03 ± 0.72C18:2 16.21 ± 0.19 61.54 ± 0.75 16.78 ± 0.20 17.35 ± 0.21 17.73 ± 0.21 18.17 ± 0.22 18.56 ± 0.23 20.67 ± 0.25C18:3 0.69 ± 0.01 0.11 ± 0.00 0.68 ± 0.01 0.67 ± 0.01 0.67 ± 0.01 0.66 ± 0.01 0.66 ± 0.01 0.61 ± 0.00C20:0 0.41 ± 0.00 0.26 ± 0.00 0.41 ± 0.00 0.40 ± 0.00 0.40 ± 0.00 0.40 ± 0.00 0.40 ± 0.00 0.39 ± 0.00C20:1 0.20 ± 0.00 0.14 ± 0.00 0.20 ± 0.00 0.19 ± 0.00 0.19 ± 0.00 0.19 ± 0.00 0.19 ± 0.00 0.19 ± 0.00TC18:1 0.01 ± 0.00 0.05 ± 0.00 0.02 ± 0.00 0.02 ± 0.00 0.02 ± 0.00 0.02 ± 0.00 0.02 ± 0.00 0.02 ± 0.00∑(TC18:2 + TC18:3) 0.02 ± 0.00 0.73 ± 0.01 0.05 ± 0.00 0.05 ± 0.00 0.05 ± 0.00 0.06 ± 0.00 0.07 ± 0.00 0.15 ± 0.00LLL 0.49 ± 0.00 23.79 ± 0.29 0.76 ± 0.01 1.04 ± 0.01 1.11 ± 0.01 1.45 ± 0.02 1.50 ± 0.02 2.46 ± 0.03ECN40 (LLLn) 0.00 ± 0.00 0.21 ± 0.00 0.05 ± 0.00 0.06 ± 0.00 0.07 ± 0.00 0.07 ± 0.00 0.09 ± 0.00 0.11 ± 0.00ECN42 1.12 ± 0.01 23.91 ± 0.29 1.26 ± 0.01 1.54 ± 0.02 1.84 ± 0.02 2.16 ± 0.02 2.32 ± 0.03 3.39 ± 0.04ECN44 10.03 ± 0.12 35.66 ± 0.43 10.80 ± 0.13 10.97 ± 0.13 11.12 ± 0.14 11.35 ± 0.14 11.50 ± 0.14 13.00 ± 0.16ECN46 34.95 ± 0.42 23.31 ± 0.28 34.76 ± 0.42 34.69 ± 0.42 34.52 ± 0.42 34.28 ± 0.41 34.07 ± 0.41 33.41 ± 0.40ECN48 50.14 ± 0.61 14.61 ± 0.18 49.37 ± 0.60 48.99 ± 0.60 48.71 ± 0.59 48.42 ± 0.59 48.33 ± 0.59 46.47 ± 0.57ECN50 3.76 ± 0.04 2.30 ± 0.03 3.76 ± 0.04 3.75 ± 0.04 3.74 ± 0.04 3.72 ± 0.04 3.69 ± 0.04 3.62 ± 0.04ECN42theor 1.19 ± 0.01 23.47 ± 0.28 1.28 ± 0.01 1.34 ± 0.01 1.40 ± 0.02 1.45 ± 0.02 1.50 ± 0.02 1.76 ± 0.02ΔECN42 0.07 ± 0.00 0.44 ± 0.01 0.21 ± 0.00 0.29 ± 0.01 0.44 ± 0.00 0.72 ± 0.00 0.82 ± 0.01 1.64 ± 0.02ECN44 + ECN46 44.98 ± 0.54 58.97 ± 0.71 45.56 ± 0.55 45.66 ± 0.55 45.64 ± 0.56 45.63 ± 0.55 45.57 ± 0.55 46.41 ± 0.56ECN46/LLL 71.32 0.97 45.73 33.35 31.09 23.64 22.71 13.58(ECN44 + ECN46)/LLL

91.79 2.47 59.94 43.90 41.11 31.46 30.38 18.86




able to resolve triglycerides both by atom carbon number and byunsaturation using phenyl-methyl-silicone stationary phases.25−27

Sterols, which comprise a major portion of the unsaponifiablematter, are found in almost all fats and oils, and they are alsocharacteristics of the genuineness of vegetable oils. Whereassunflower, for example, contains significant levels of Δ7-stigmastenol, extra-virgin olive oil has high levels of β-sitosteroland Δ5-avenasterol, and soybean and corn oils have high levelsof campesterol and stigmasterol. These apparent differencesmake them the most suitable for determining the botanicalorigin of oils and, hence, for detecting the adulteration of oliveoil with other cheaper or lower quality vegetable oils.28−30

Many publications report the application of chemometricssuch as the linear discriminant analysis (LDA) and other toolsto find the best approach for the authenticity and traceability ofextra-virgin olive oils.31−35

The utmost objective of the present research study is thedetection of Chemlali EVOO adulteration with soybean, corn,and sunflower oils. For this purpose, analyses of fatty acid,sterol, and triacylglycerol profiles are performed using gas andliquid chromatography. This leads to the determination of theminimum detectable levels of vegetable oils (soybean, corn, andsunflower).

■ MATERIALS AND METHODSChemicals. Methanol (99.8%), diethyl ether (99.7%), ethanol

(≥99.9%), n-heptane (99.0%), chloroform (99.1%), acetone(≥99.8%), and acetonitrile (≥99.9%) HPLC grade solvents werepurchased from Riedel-de-Haen (Buchs SG, Switzerland). Potassiumhydroxide (88.5%), pyridine (99.7%), hexamethyldisilazane (98.5%),and trimethylchlorosilazane (99.0%) were obtained from Sigma-Aldrich, Supelco (Bellefonte, PA, USA). FAME multistandards

(>99.0%) were purchased from Sigma Chemical Co. (St. Louis,MO, USA).

Vegetable Oils. Four different extra-virgin olive oil samples wereobtained from a Tunisian olive variety, which is the Chemlali cultivarharvested from the Sfax region (southern Tunisia) during two cropseasons (2011/2012 and 2012/2013) (n = 4; two different samples foreach crop season).

The ripening degree was the same for the four Chemlali olive sam-ples (maturation indices were 4.5). The olive samples were collected atthe beginning of December from orchards in the same neighborhoodcarried out with the same cultural practices. The olive ripening index(RI) was determined according to the method developed by Boskou,36

on the basis of the evaluation of the olive skin and pulp colors. RIvalues range from 0 (100% intense green skin) to 7 (100% purple fleshand black skin).

The pure soybean, corn, and sunflower oils, purchased from thelocal market in Tunisia and used as adulterants, were checked for theirauthenticity by classical tests such as iodine and refraction indices andother technical procedures, most of which are based on GC (fatty acidand sterol compositions), with some being based on HPLC(triglyceride composition). The different mixtures of extra-virginolive oil with the aforementioned vegetable oils (at levels of 1, 2, 3, 4,5, and 10% w/w) were prepared.

Analytical Methods. Determination of Fatty Acid Composition.The methyl esters for the determination of the cis- and trans-fatty acidsby the use of capillary column GC were prepared according to astandard protocol (vigorous shaking of the solution of oil in n-heptane(0.1 g in 2 mL) with 0.2 mL of 2 N methanolic potassiumhydroxide).37,38 The GC analysis of FAMEs was performed on anAutoSystem gas chromatograph equipped with a FID detector (HP6890N, Agilent (Palo Alto, CA, USA)). The column used was acapillary Agilent CP-Sil88 (cyanopropyl polysiloxane) (length = 50 m,i.d. = 0.25 mm, and film thickness = 0.20 μm), and the analysisconditions were as follows: the initial column temperature was settledat 165 °C for 25 min, then raised at a gradient of 5 °C/min to 195 °C;

Table 4. Sterol Composition (Percent) in Extra-Virgin Olive Oil (EVOO), Soybean Oil (SO), Corn Oil (CO), and SunflowerOil (SfO) and Mixtures of EVOO with Different Percentages of Each Seed Oil (SO, CO, and SfO)a

cholesterol24-methylenecholesterol campesterol campestanol stigmasterol

apparentβ-sitosterolb

Δ7-stigmastenol

Δ7-avenasterol

EVOO 0.15 ± 0.00 0.12 ± 0.00 3.17 ± 0.03 0.07 ± 0.00 0.49 ± 0.00 94.94 ± 1.16 0.34 ± 0.00 0.72 ± 0.00SO 0.41 ± 0.00 0.07 ± 0.00 19.95 ± 0.24 0.17 ± 0.00 17.61 ± 0.21 58.21 ± 0.71 2.31 ± 0.03 1.27 ± 0.01EVOO + 1% SO 0.15 ± 0.00 0.12 ± 0.00 3.35 ± 0.04 0.07 ± 0.00 0.60 ± 0.00 94.62 ± 1.15 0.36 ± 0.00 0.73 ± 0.00EVOO + 2% SO 0.16 ± 0.00 0.12 ± 0.00 3.54 ± 0.04 0.07 ± 0.00 0.69 ± 0.00 94.31 ± 1.15 0.37 ± 0.00 0.74 ± 0.00EVOO + 3% SO 0.17 ± 0.00 0.11 ± 0.00 3.72 ± 0.04 0.08 ± 0.00 0.84 ± 0.01 93.95 ± 1.15 0.38 ± 0.00 0.75 ± 0.00EVOO + 4% SO 0.17 ± 0.00 0.11 ± 0.00 3.90 ± 0.05 0.08 ± 0.00 1.11 ± 0.01 93.47 ± 1.14 0.40 ± 0.00 0.76 ± 0.00EVOO + 5% SO 0.17 ± 0.00 0.11 ± 0.00 4.02 ± 0.05 0.08 ± 0.00 1.30 ± 0.02 93.11 ± 1.14 0.43 ± 0.00 0.78 ± 0.00EVOO +10% SO

0.19 ± 0.00 0.11 ± 0.00 6.17 ± 0.07 0.09 ± 0.00 2.81 ± 0.03 89.21 ± 1.09 0.59 ± 0.00 0.83 ± 0.01

CO 0.14 ± 0.00 0.12 ± 0.00 22.06 ± 0.27 0.05 ± 0.00 4.96 ± 0.06 70.50 ± 0.86 0.55 ± 0.00 1.62 ± 0.02EVOO + 1% CO 0.15 ± 0.00 0.12 ± 0.00 3.27 ± 0.04 0.07 ± 0.00 0.52 ± 0.00 94.81 ± 1.16 0.34 ± 0.00 0.72 ± 0.00EVOO + 2% CO 0.15 ± 0.00 0.12 ± 0.00 3.45 ± 0.04 0.06 ± 0.00 0.54 ± 0.00 94.61 ± 1.16 0.35 ± 0.00 0.72 ± 0.00EVOO + 3% CO 0.15 ± 0.00 0.12 ± 0.00 3.70 ± 0.04 0.06 ± 0.00 0.66 ± 0.00 94.23 ± 1.15 0.35 ± 0.00 0.73 ± 0.00EVOO + 4% CO 0.15 ± 0.00 0.11 ± 0.00 4.06 ± 0.05 0.06 ± 0.00 0.70 ± 0.00 93.82 ± 1.15 0.36 ± 0.00 0.74 ± 0.00EVOO + 5% CO 0.15 ± 0.00 0.11 ± 0.00 4.24 ± 0.05 0.06 ± 0.00 0.74 ± 0.01 93.60 ± 1.14 0.36 ± 0.00 0.74 ± 0.00EVOO +10% CO

0.15 ± 0.00 0.11 ± 0.00 5.82 ± 0.07 0.06 ± 0.00 0.87 ± 0.01 91.86 ± 1.12 0.38 ± 0.00 0.75 ± 0.01

SfO 0.08 ± 0.00 0.08 ± 0.00 8.30 ± 0.10 0.08 ± 0.00 5.74 ± 0.07 61.24 ± 0.75 17.60 ± 0.21 6.88 ± 0.08EVOO + 1% SfO 0.14 ± 0.00 0.12 ± 0.00 3.21 ± 0.04 0.07 ± 0.00 0.57 ± 0.00 94.60 ± 1.16 0.54 ± 0.00 0.75 ± 0.00EVOO + 2% SfO 0.14 ± 0.00 0.12 ± 0.00 3.29 ± 0.04 0.07 ± 0.00 0.64 ± 0.00 94.05 ± 1.15 0.89 ± 0.01 0.80 ± 0.01EVOO + 3% SfO 0.14 ± 0.00 0.11 ± 0.00 3.38 ± 0.04 0.07 ± 0.00 0.70 ± 0.00 93.70 ± 1.15 1.02 ± 0.01 0.88 ± 0.01EVOO + 4% SfO 0.14 ± 0.00 0.11 ± 0.00 3.45 ± 0.04 0.07 ± 0.00 0.75 ± 0.01 93.36 ± 1.14 1.17 ± 0.01 0.95 ± 0.01EVOO + 5% SfO 0.14 ± 0.00 0.11 ± 0.00 3.56 ± 0.04 0.07 ± 0.00 0.81 ± 0.01 92.89 ± 1.14 1.39 ± 0.02 1.03 ± 0.01EVOO +10% SfO

0.13 ± 0.00 0.11 ± 0.00 3.69 ± 0.05 0.07 ± 0.00 1.06 ± 0.01 91.62 ± 1.12 2.05 ± 0.02 1.27 ± 0.01

aEach value represents the mean of four determinations of two successive crop seasons (n = 4; two different samples for each crop season). b[Δ5,23-Stigmastadienol + cholerosterol + β-sitosterol + sitostanol + Δ5-avenasterol + Δ5,24-stigmastadienol.



the temperature of the injector and detector was set at 250 °C; heliumwas used as the carrier gas at a flow rate of 1 mL/min and 1:100 splitratio; the injection volume was 1 μL.cis- and trans-FAMEs were identified through a comparison of their

retention times versus pure standards analyzed under the sameconditions. They were quantified according to their percentage area,obtained by the integration of the peaks (Figure 1). The results wereexpressed as the percentages of individual fatty acids in the lipidfraction. The 10 fatty acids (cis) considered in this study were palmitic(C16:0), hypogeic (C16:1n-9) + palmitoleic (C16:1n-7), margaric(C17:0), margaroleic (C17:1), stearic (C18:0), oleic (C18:1n-9) +Z-vaccenic (C18:1n-7), linoleic (C18:2), linolenic (C18:3), arachidic(C20:0), and gondoic (C20:1n-9) acids. The trans-fatty acid (TFA)content considered in this study included trans-octadecenoic (tC18:1),trans−trans-octadecadienoic (tt C18:2), cis-trans- and trans-cis-octadecadienoic [(ct + tc) C18:2], and trans-cis-trans-, cis-cis-trans-,cis-trans-cis-, and trans-cis-cis-octadecatrienoic [(tct + cct + ctc + tcc)C18:3] acids expressed as percentages of fatty acid methyl esters.Determination of Triglyceride Composition. Five percent of the

sample to be analyzed was prepared by weighing 0.25 ± 0.001 g into a5 mL graduated flask and dissolved in 5 mL with acetone.22 A HP1100chromatograpic system from Agilent (Waldbronn, BW, Germany)equipped with a differential refractometer detector was employed.Next, the separation was carried out with a Spherisorb analyticalcolumn (250 × 4.6 mm, 5 μm particle size) from Supelco (Bellefonte,PA, USA). The optimized separation conditions were conducted byisocratic elution with a 60:40 acetone/acetonitrile mixture; column

temperature, 30 °C; flow rate, 1.5 mL/min; and injection volume, 20 μLof the sample solution prepared as indicated above.

For the identification of TAGs, the retention times plotted inaccordance with alternative reference chromatograms obtained fromsoybean oil, a 30:70 mixture of soybean oil/olive oil, and pure olive oilas described by COI.22 It was assumed that the sum of the areas of thepeaks corresponding to the various TAGs was equal to 100%, and therelative percentage of each TAG was calculated.

It is worthwhile noting that the theoretical value of ECN42triacylglycerols was calculated by the computer program.

Determination of Sterol Composition. A mixture of each extra-virgin olive oil sample and vegetable oils (5 g) was dissolved by 50 mLof 2 N ethanolic potassium hydroxide solution, saponified, andextracted according to the official International Olive Oil Council(IOOC) method.39 The unsaponifiable fraction was dissolved inchloroform, and approximately 20 mg was loaded on a basic silica TLCplate. The sterol and triterpene diol fraction was separated by elutionwith a mixture of hexane and diethyl ether 65:35 (v/v). Thecorresponding band was visualized under UV light after being sprayedwith a 2,7-dichlorofluorescein in 0.2% ethanolic solution, than scrapedoff with a spatula, and extracted with chloroform. After the extract wasevaporated to dryness, sterols and diols were converted to tri-methylsilyl ethers by the addition of pyridine/hexamethyldisilazane/trimethylchlorosilane (9:3:1, v/v/v), left for 15 min, and thencentrifuged. The analysis of sterols was performed on a 7890A Agilentgas chromatograph (Pudong, Shanghai, China) equipped with a flameionization detector (FID). The column used was a capillary HP-5(5% phenyl; 95% dimethylpolysiloxane) (length = 30 m, i.d. = 0.32 mm,

Figure 2. Gas chromatograms showing the FAMEs profiles of Chemlali EVOO (a) and binary mixtures containing 90% EVOO and 10% of eithersoybean (b), sunflower (c), or corn (d) oils using GC-FID.



and film thickness = 0.25 μm), and the conditions for the analysiswere as follows: the oven temperature was isothermal at 260 °C, thetemperature of the injector was 280 °C, and the detector was set at290 °C; helium was the carrier gas, with a flow through the column of1 mL/min and a 1:50 split ratio, and the injection volume was 3 μL.To identify the individual peaks of sterols, the determination of

relative retention times (RRT) for sterols was carried out according tothe majority compound of sterols (β-sistosterol), knowing that RRT(β-sistosterol) is equal to 1 as described by the COI.40

Statistical Analysis. The results were expressed as mean ±standard deviation (SD) of four measurements for the analyticaldetermination. LDA was applied to discriminate Chemlali EVOO andthe extra-virgin olive oils adulterated with different percentages ofsoybean, corn, and sunflower oils (1, 2, 3, 4, 5, and 10%) according toall of the parameters investigated. LDA plots were performed usingSPSS Statistics 17.0 for Windows (SPSS Inc., 2008).

■ RESULTS AND DISCUSSIONFatty Acid and Triglyceride Compositions in Vegeta-

ble Oils. The experimental results of fatty acid and triglyceridecompositions in extra-virgin olive oil, soybean, corn, andsunflower oils are summarized in the Tables 1−3.In the present research work, the fatty acid (FA) composition

(%) of Chemlali extra-virgin olive oil was studied within theofficial limits established for olive oil referred to in the IOOCtade standard applied to olive oils and olive pomace oils.7 Theacid limits in olive oil are palmitic acid (C16:0), 7.50−20.00%;palmitoleic acid (C16:1), 0.30−3.50%; stearic acid (C18:0),0.50−5.00%; oleic acid (C18:1), 55.00−83.00%; linoleic acid(C18:2), 3.50−21.00%; and linolenic acid (C18:3), ≤1.00%.In the studied extra-virgin olive oil, the percentages of

palmitic, oleic, linoleic, and linolenic acids are 15.82, 62.37,

16.21, and 0.69%, respectively. The vegetable oils, soybean,corn, and sunflower oils, exhibited considerably higher linoleicacid percentages than oleic acid percentages. Soybean oil hadconsiderably high linolenic acid (7.77%). These differences,related to the linoleic and linolenic acid percentages, aredepicted in Tables 1−3.The detection of a triglyceride, which is not present in extra-

virgin olive oil and has a number of equivalent carbons equal to40 (LLLn), is an indicator of the presence of seed oil in olive oil.This result is in agreement with that reported by Rezanka et al.41

In all vegetable oils, the trilinolein (LLL) content was higherthan that of the extra-virgin olive oil.42 Actually, largedifferences pertaining to trilinolein contents between extra-virgin olive oil and soybean, corn, and sunflower oils, depictedin Tables 1−3, were observed.The absolute value of the differences between theoretical and

experimental ECN42 contents (ΔECN42) in all seed oils ishigher than that in the extra-virgin olive oil. It ranges from15.42 in soybean oil to 0.07 in extra-virgin olive oil. Thesedifferences are depicted in Tables 1−3.According to the established limits, the ΔECN42 value must

not exceed 0.20 in the case of edible virgin olive oil, 0.30 in thecase of lampante olive oil, 0.50 in the case of olive pomace oil,and 0.60 in the case of crude olive pomace oil.The above results reveal that there is a correlation between

the linoleic acid percentage, trilinolein content, and ΔECN42.In other words, whereas soybean, corn, and sunflower oils havehigh linoleic acid percentages, high LLL contents, and highΔECN42, extra-virgin olive oil exhibits low LLL content and

Figure 3. Chromatograms showing the TAGs profiles of Chemlali EVOO (a) and binary mixtures containing 90% EVOO and 10% of either soybean(b), sunflower (c), or corn (d) oils using HPLC-RID.



low ΔECN42. These results are in good agreement with lit-erature data.9

Sterol Compositions in Vegetable Oils. Sterols are notonly found in almost all fats and oils but also characteristic ofthe genuineness of vegetable oils. Indeed, the determination ofthe sterol composition of a vegetable oil is a very importantcriterion for identifying the botanical origin.Sterol compositions (%) show higher contents in campester-

ol and stigmasterol in other vegetable oils compared to EVOO(Table 4). In fact, campesterol, which does not exceed 4.00% inEVOO, reaches values of 19.95, 22.06, and 8.30% in soybean,corn, and sunflower oils, respectively. Besides, stigmasterol,which always remains less than campesterol in EVOO, reachesvalues of 17.61, 4.96, and 5.74% in soybean, corn, andsunflower oils, respectively. Therefore, these two sterols couldbe used as an indicator of the adulteration of olive oil withvegetable oils. These results are in agreement with thosereported by Al-Ismail et al.,43 who suggested the presence ofsome refined vegetable oils in olive oil based on the sum ofcampesterol and stigmasterol percentages.Moreover, sunflower oil has a high level of Δ7-stigmastenol

(17.60%), whereas this sterol percentage in extra-virgin olive oilmust not exceed 0.50%.The model systems of corn, soybean, and sunflower oils at

levels of 1, 2, 3, 4, 5, and 10% were prepared. These apparentdifferences, as shown in Table 4, make them the most suitablefor the determination of the botanical origin of oils andtherefore for the detection of the adulteration or contaminationamong vegetable oils.Identification of EVOO Adulteration with Other Low-Cost

Oils. This research study is meant to detect adulteration of EVOOby lower cost seed oils. Consequently, various blends of EVOO andsoybean, corn, or sunflower oil were prepared and analyzed for fatty

acid, triglyceride, and sterol compositions. The adulteration per-centages ranged from 1 to 10% to determine a threshold of detection.

Use of the Fatty Acid and Triglyceride Compositionsfor the Detection of Fraud. Fatty acid and TAGcompositions of the adulterated extra-virgin olive oil mixedwith 1−10% (w/w) quantities of soybean, corn, and sunfloweroils are summarized in Tables 1−3 and Figures 2 and 3.Taking into account the results presented in Tables 1−3,

it could be concluded that the analysis of fatty acids does notproduce satisfactory results with regard to the levels of adul-teration investigated in this research study. The most effective

Table 5. Parameters Used for the Detection of Extra-VirginOlive Oil (EVOO) Adulteration with Vegetable Oils

type of seed oil

parameter used for thedetection ofadulteration

COIthresh-old value

percentage of detectableseed oil exceeding theCOI threshold value

soybean (SO) C18:3 1% EVOO + 5% SO (1.08%)ECN40 0 EVOO + 1% SO (0.27)ΔECN42 0.20 EVOO + 3% SO (0.30)campesterol 4% EVOO + 10% SO

(6.17%)Δ7-stigmastenol 0.50% EVOO + 10% SO

(0.59%)apparent β-sitosterol 93.00% EVOO + 10% SO

(89.21%)∑(TC18:2 + TC18:3) 0.05% EVOO + 3% SO (0.06%)

corn (CO) ECN40 0 EVOO + 1% CO (0.11)ΔECN42 0.20 EVOO + 3% CO (0.21)campesterol 4% EVOO + 4% CO (4.06%)apparent β-sitosterol 93.00% EVOO + 10% CO

(91.86%)∑(TC18:2 + TC18:3) 0.05% EVOO + 2% CO (0.06%)

sunflower (SfO) ECN40 0 EVOO + 1% SfO (0.05)ΔECN42 0.20 EVOO + 1% SfO (0.21)Δ7-stigmastenol 0.50% EVOO + 1% SfO (0.54%)apparent β-sitosterol 93.00% EVOO + 5% SfO

(92.89%)∑(TC18:2 + TC18:3) 0.05% EVOO + 4% SfO (0.06%)

Figure 4. trans-Fatty acids of mixtures of extra-virgin olive oil (EVOO)with (a) soybean, (b) corn, and (c) sunflower oils. Each valuerepresents the mean of four determinations of two successive cropseasons (n = 4; standard deviation < 0.001%).



parameters for the detection of adulteration are mentionedbelow.The linolenic acid percentage could be used as a parameter

for the detection of EVOO fraud with 5% of soybean oil as wellas by the increase of trans-fatty acid contents with 3% ofsoybean oil, 2% of corn oil, and 4% of sunflower oil (Table 5and Figure 4). None of the other fatty acids is effective for thedetection of the added vegetable oil in an extra-virgin olive oil.Although the composition of fatty acids in the examined seedoils is different from that of olive oils, the fatty acids experimentcould not be satisfactorily used as discriminatory parametersbetween olive oil and the respective vegetable oil in most cases.According to Tables 1−3, it can be noted that the appearance

of a specific triacylglycerol, normally not present in the olive oil,in oils adulterated with seed oils has a number of equivalentcarbons equaling 40, known by LLLn. Thus, this triacylglycerolis an indicator of the presence of seed oil in extra-virgin oliveoil.Indeed, LLLn increases with the increase in the adulteration

percentage of vegetable oils (from 1 to 10%). It ranges from0.27 to 0.76% for the extra-virgin olive oil adulterated withsoybean oil, from 0.11 to 0.69% for the extra-virgin olive oiladulterated with corn oil, and from 0.05 to 0.11% for the extra-virgin olive oil adulterated with sunflower oil. The adulterationwith soybean oil produces a large increase of the areas of peak

LLLn because the soybean oil is the only one rich in linolenicacid.The addition of small quantities of seed oil can be identified

in the extra-virgin olive oil by the determination of the rate ofLLL because the three seed oils are rich in linoleic acid(C18:2). Similarly, the presence of soybean, sunflower, andcorn oils was proven by an increase of the percentage of LLLand ECN42. The extra-virgin olive oils were adulterated bysunflower, corn, and soybean oils (from 1 to 10%). Having acontent of LLL, they varied from 0.76 to 2.46% with sunfloweroil, from 0.52 to 1.86% with corn oil, and from 0.54 to 1.86%with soybean oil. Besides, having a practical ECN42, they variedfrom 1.26 to 3.39% with sunflower oil, from 1.33 to 2.87% withcorn oil, and from 1.42 to 3.59% with soybean oil. It is to benoted that olive oil is largely compared to the seed oils knownby their highest linoleic acid percentages.The use of the ΔECN42 proved to be more effective in

detecting even low levels of adulteration of EVOO with most ofthe examined vegetable oils. According to the data on thefraudulent mixtures presented in Tables 1−3, the determinationof the ΔECN42 can be used as a parameter for the detectionof fraud of extra-virgin olive oils with each one of the studied seedoils: 1% of sunflower (0.21 > 0.20), 3% of soybean (0.30 > 0.20),and 3% of corn (0.21 > 0.20) (Table 5).

Figure 5. Typical gas chromatograms for sterols obtained in the analysis of Chemlali EVOO (a), EVOO adulterated with 10% soybean oil (b),EVOO adulterated with 10% sunflower oil (c), or EVOO adulterated with 10% corn oil (d).



This finding can be attributed to the fact that the ΔECN42is a number calculated by the combination of fatty acids andtriglyceride composition. Therefore, the difference in thecomposition of the triglycerides and the six fatty acids, takeninto account for the calculation of the theoretical ECN42,between the initial samples and their mixtures can beexpected. However, these differences do not have theexpected effect on the values of the theoretical ECN42 andΔECN42. A consequence of this is the complete lack ofcorrelation between linoleic acid or LLL and ΔECN42 ofvegetable oils and their admixtures with olive oil. These resultsare in agreement with those reported by Christopoulou et al.,9

who claimed the parameter ΔECN42 is very useful andeffective in the detection of adulteration of olive oils withvegetable oils.Use of the Sterol Compositions for the Detection of

Fraud. The composition of the sterol fraction of olive oil is avery useful parameter for detecting adulterations or to checkauthenticity, because it can be considered as a fingerprint.44,45

The comparison between the extra-virgin olive oil and thestudied seed oils reveals that there are high differencesrelating to sterol compositions as shown in Table 4 andFigure 5.As stated below (Table 4), the profile of sterols is almost

decisive in clarifying the contamination of some vegetable oils(EVOO) with other cheaper ones. Thus, the addition of 10% ofsoybean oil to EVOO can be detected by the increase ofcampesterol and Δ7-stigmastenol percentages, which exceed themaximum acceptable limits (6.17 > 4.00% and 0.59 > 0.50%,respectively) and also by the decrease of the apparentβ-sitosterol (89.21 < 93.00%, minimal value limited by the COI).The value of the Δ7-stigmastenol in sunflower oil is much

higher (17.60%) than that in the EVOO (0.34%). This sterol isvery effective for the detection of adulteration, even at the levelof 1% of sunflower oil (0.54) in the extra-virgin olive oil. Forexample, a mixture consisting of 99% of EVOO (with 0.34% ofΔ7-stigmastenol percentage) and 1% of sunflower oil (with17.60% of Δ7-stigmastenol percentage) caused a significantincrease in Δ7-stigmastenol percentage (from 0.34 to 0.54). Infact, the determined Δ7-stigmastenol percentage of the mixturewas 0.54% of the total sterols (>0.50%, the maximumacceptable limit for Δ7-stigmastenol). Besides, the contami-nation of EVOO with 5% of sunflower oil could also bedetected by the decrease of the apparent β-sitosterol (92.89%of total sterols). Similar results were reported by Aparicio andAparicio-Ruiz.8

In addition, the adulteration of EVOO with 4% of corn oilincreases the concentration of campesterol (4.06 > 4%), andfalsification with 10% of corn oil decreases the concentration ofapparent β-sitosterol (91.86% of the total sterols).In the first conclusion, the parameters used for the detection

of adulteration of extra-virgin olive oil with the studied seed oilsas well as the percentages of detectable seed oil in extra-virginolive oil mixtures are summarized in Table 5. It should be notedthat the conclusions for the detection of fraud are drawn fromthe analyses performed on the samples examined in thisresearch study. However, as has already been mentioned, theeffectiveness of a parameter was based on the comparison ofthe determined value in each fraudulent sample with themaximum established limit of this parameter in EVOO.Consequently, the findings of this research study can beapplied to the detection of adulteration of extra-virgin olive oilwith the only reservation that the percentage of seed oil that is

Figure 6. Values of the parameters ECN46/LLL (a) and (ECN44 +ECN46)/LLL (b) of Chemlali extra-virgin olive oil (EVOO), seed oils,and their mixtures: (■) 100% EVOO; (●) EVOO + 1% SO, CO, orSfO; (▲) EVOO + 3% SO, CO, or SfO; (×) EVOO + 5% SO, CO, orSfO; (+) EVOO + 10% SO, CO, or SfO; (◆) 100% SO, CO, or SfO.SO, soybean oil; CO, corn oil; SfO, sunflower oil.

Figure 7. LDA score plot of pure extrav virgin olive oil (EVOO) andadulterated EVOO based on all of the analyses performed with fourdeterminations: (1) EVOO; (2) EVOO + 1−10% of soybean oil; (3)EVOO + 1−10% of corn oil; (4) EVOO + 1−10% of sunflower oil.



detectable in olive oil mixtures may be somewhat different fromthat proposed in the present study. Overall, this table could beused as a simple guide for analysts working on EVOO analysis.Use of Other Parameters for the Detection of Fraud.

The parameters calculated by the triglycerides and reported inTables 1−3 are ECN46/LLL and (ECN44 + ECN46)/LLL.Certain researchers have pointed out the importance of thesefactors in the detection of adulteration of extra-virgin olive oilwith certain vegetable oils.9,46

These parameters are based on the low LLL content of theextra-virgin olive oil in comparison with that of the othervegetable oils such as soybean, corn, and sunflower oils. Thesimilar ECN46 and ECN44 + ECN46 values of the extra-virginolive oil are also compared with that of the studied seed oils.Then, the values of these parameters in the seed oils are muchlower than that in the extra-virgin olive oil. Besides, the valuesof these parameters in the mixture of extra-virgin olive oil andsunflower oil (1%) were greatly lower than that in extra-virginolive oil, whereas in the other mixtures of extra-virgin olive oil

with soybean and corn oils, these values were much lower onlyat 3%. All of the mixtures in the other percentages (5 and 10%)of EVOO and each seed oil type were well separated as shownin Figure 6.The use of the two parameters ECN46/LLL and (ECN44 +

ECN46)/LLL is very effective in the detection of the seed oilsin percentages <5% and produced very satisfactory results withregard to the studied vegetable oils, even in percentages lowerthan 3%, especially in the case of sunflower oil (1%). Theseresults agree with the study performed by Christopoulou et al.,9

which confirmed the effectiveness of these parameters for thedetection of EVOO adulteration with vegetable oils inpercentages lower than 5%.

Chemometric Analysis. LDA is probably the mostfrequently used supervised pattern recognition method. Inprinciple, LDA determines linear discriminant functions, whichmaximize the ratio of between-class variance and minimize theratio of within-class variance. It should be noted that, whereasthe principal component analysis (PCA) selects a direction that

Figure 8. LDA score plot of pure extra-virgin olive oil (EVOO), pure seed oils, and adulterated EVOO at the same level based on all of the analysesperformed with four determinations: (a) EVOO + 1% of seed oil; (b) EVOO + 2% of seed oil; (c) EVOO + 5% of seed oil; (d) EVOO + 10% ofseed oil; (1) EVOO; (2) soybean oil; (3) corn oil; (4) sunflower oil; (5) EVOO + % of soybean oil; (6) EVOO + % of corn oil; (7) EVOO + % ofsunflower oil.



retains maximal structure among the data in a lower dimension,LDA selects a direction that achieves maximum separationamong the given classes.47

Because the data structure analysis gave a good samplecharacterization, a classification model was built. LDA wasapplied to find a predictive classification model able todifferentiate the pure extra-virgin olive oil and the adulteratedolive oils.The plot of the discriminant functions (Figure 7) obtained

by LDA showed a clear discrimination between the ChemlaliEVOO and the adulterated extra-virgin olive oils mixed withdifferent percentages of soybean, corn, and sunflower oils (1, 2,3, 4, 5, and 10%). Discriminant function 1, which was highlyrelated to cholesterol, campestanol, stigmasterol, campesterol,Δ7-avenasterol, apparent β-sitosterol, Δ7-stigmastenol, ECN50,and C20:1, represents the function of minor compounds, alreadyconsidered as a fingerprint for a specific oil. Discriminantfunction 2 is highly related to C17:0, ECN42theor, LLLn, andC18:3.In particular, by discriminant function 1 it was possible to

discriminate Chemlali EVOO and the adulterated extra-virginolive oils whatever the percentage of seed oil added (Figure 7).All samples were correctly discriminated using the twofunctions.Application of LDA, after feature selection, was sufficient to

differentiate Chemlali EVOO and all adulterated extra-virginolive oils. The success was 100% in classification and close to100% in prediction.It is difficult to discriminate the adulterated EVOO with 1%

of sunflower oil and the pure EVOO, whereas those adulteratedwith other vegetables oils are clearly separated at the same level(Figure 8a). The separation improves when the percentage ofadulteration increases (Figure 8b−d).Compared to classical methods, this new approach of using

LDA application could represent an alternative and innovativetool for faster and cheaper evaluation of EVOO adulteration.In conclusion, the present study has demonstrated that the

threshold of the Chemlali extra-virgin olive oils adulterationby some cheaper vegetable oils such as soybean, corn, andsunflower oils was detected using gas chromatography incombination with liquid chromatography. According to thesetechniques, certain compounds found in the studied oils(triacylglycerols, trilinoleins, fatty acids, and sterols) areidentified, analyzed, and used for the detection of theadulteration of extra-virgin olive oils. It should also be notedthat this research work aims to facilitate the work of analystsby providing a simple guide for the detection of the adul-teration of extra-virgin olive oil with the most common veg-etable oils.

■ AUTHOR INFORMATION

Corresponding Author*(M.B.) Phone: +216 98 667 581. Fax: +216 74 674 364.E-mail: [email protected].

FundingWe thank the Ministere de l’Enseignement Superieur et de laRecherche Scientifique and the Ministere de l’Agriculture(ONH-Sfax), Tunisia, for their support of this research work.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank Leila Mahfoudhi, teacher of English at the SfaxFaculty of Science, for having proofread this paper.

■ ABBREVIATIONS USEDCO, corn oil; EC, equivalent carbon number; EVOO, extra-virgin olive oil; FAMEs, fatty acid methyl esters; GC, gaschromatography; GLC, gas−liquid chromatography; HPLC,high-performance liquid chromatography; IOOC, InternationalOlive Oil Council; IUPAC, International Union of Pure andApplied Chemistry; LC-ESI-MS2, liquid chromatographyelectrospray mass spectrometry; LDA, linear discriminantanalysis; LLL, trilinolein; PCA, principal component analysis;SfO, sunflower oil; SO, soybean oil; TAGs, triacylglycerols

■ REFERENCES(1) Issaoui, M.; Flamini, G.; Brahmi, F.; Dabbou, S.; Ben Hassine, K.;Taamali, A.; Chehab, H.; Ellouz, M.; Zarrouk, M.; Hammami, M.Effect of the growing area conditions on differentiation betweenChemlali and Chetoui olive oils. Food Chem. 2010, 119, 220−225.(2) Haddada, F. M.; Manaï, H.; Oueslati, I.; Daoud, D.; Sanchez, J.;Osorio, E.; Zarrouk, M. Fatty acid, triacylglycerol, and phytosterolcomposition in six Tunisian olive varieties. J. Agric. Food Chem. 2007,55, 10941−10946.(3) Tena, N.; Lazzez, A.; Aparicio-Ruiz, R.; García-Gonzalez, D. L.Volatile compounds characterizing Tunisian Chemlali and Chetouivirgin olive oils. J. Agric. Food Chem. 2007, 55, 7852−7858.(4) Ammar, S.; Zribi, A.; Gargouri, B.; Flamini, G.; Bouaziz, M. Effectof addition of olive leaves before fruits extraction process to somemonovarietal Tunisian extra-virgin olive oils using chemometricanalysis. J. Agric. Food Chem. 2014, 62, 251−263.(5) Gargouri, B.; Zribi, A.; Bouaziz, M. Effect of containers on thequality of Chemlali olive oil during storage. J. Food Sci. Technol. 2014,DOI: 10.1007/s13197-014-1273-2.(6) Gargouri, B.; Ammar, S.; Zribi, A.; Ben Mansour, A.; Bouaziz, M.Effect of growing region on quality characteristics and phenoliccompounds of Chemlali extra-virgin olive oils. Acta Physiol. Plant.2013, 35, 2801−2812.(7) International Olive Council, COI/T, 15/NC No. 3/Rev. 7. Tradestandard applying to olive oils and olive-pomace oils, 2013.(8) Aparicio, R.; Aparicio-Ruiz, R. Authentication of vegetable oils bychromatographic techniques. J. Chromatogr., A 2000, 881, 93−104.(9) Christopoulou, E.; Lazaraki, M.; Komaitis, M.; Kaselimis, K.Effectiveness of determinations of fatty acids and triglycerides for thedetection of adulteration of olive oils with vegetable oils. Food Chem.2004, 84, 463−474.(10) Gurdeniz, G.; Ozen, B. Detection of adulteration of extra-virginolive oil by chemometric analysis of mid-infrared spectral data. FoodChem. 2009, 116, 519−525.(11) Cordella, C.; Moussa, I.; Martel, A. C.; Sbirrazzuoli, N.; Lizzani-Cuvelier, L. Recent developments in food characterization andadulteration detection: technique-oriented perspectives. J. Agric. FoodChem. 2002, 50, 1751−1764.(12) Skevin, D.; Kraljic, K.; Miletic, L.; Obranovic, M.; Neđeral, S.;Petricevic, S. Adulteration of Oblica virgin olive oil with ediblesunflower and refined olive pomace oil. Croatian J. Food Technol.Biotechnol. Nutr. 2011, 6, 117−122.(13) Frankel, E. N. Chemistry of extra-virgin olive oil: adulteration,oxidative stability, and antioxidants. J. Agric. Food Chem. 2010, 58,5991−6006.(14) Aparicio, R.; Aparicio-Ruíz, R. Authentication of vegetable oilsby chromatographic techniques. J. Chromatogr., A 2000, 881, 93−104.(15) Garces, R.; Mancha, M. One-step lipid extraction and fatty acidmethyl esters preparation from fresh plant tissues. Anal. Biochem.1993, 211, 139−143.(16) Zribi, A.; Gargouri, B.; Jabeur, H.; Rebaı, A.; Abdelhedi, R.;Bouaziz, M. Enrichment of pan-frying refined oils with olive leaf



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phenolic-rich extract to extend the usage life. Eur. J. Lipid Sci. Technol.2013, 115, 1443−1453.(17) De la Mata-Espinosa, P.; Bosque-Sendra, J. M.; Bro, R.;Cuadros-Rodríguez, L. Olive oil quantification of edible vegetable oilblends using triacylglycerols chromatographic fingerprints and chemo-metric tools. Talanta 2011, 85, 177−182.(18) Tsimidou, M.; Macrae, R. Authentication of virgin olive oilsusing principal component analysis of triglyceride and fatty acidprofiles: Part 2 − Detection of adulteration with other vegetable oils.Food Chem. 1987, 25, 251−258.(19) Fellatzarrouck, K.; Bouteiller, J. C.; Maurin, R. Detection ofolive oil adulteration by HPLC in seed oils. Rev. Fr. Corps Gras 1988,35, 383−386.(20) García-Gonzalez, D. L.; Viera, M.; Tena, N.; Aparicio, R.Evaluation of the methods based on triglycerides and sterols for thedetection of hazelnut oil in olive oil. Grasas Aceites 2007, 58, 344−350.(21) Cert, A.; Moreda, W. Algorithms for the detection of hazelnutoil in olive oil. Grasas Aceites 2000, 51, 143−149.(22) International Olive Council, COI/T, 20/Doc. No. 20 Rev. 3.Determination of the difference between actual and theoretical contentof triacylglycerols with ECN42, 2010.(23) Zeng, Y. X.; Araujo, P.; Du, Z. Y.; Nguyen, T. T.; Frøyland, L.;Grung, B. Elucidation of triacylglycerols in cod liver oil by liquidchromatography electrospray tandem ion-trap mass spectrometry.Talanta 2010, 82, 1261−1270.(24) International Union of Pure and Applied Chemistry (IUPAC).Method 2.323. In Standard Methods for the Analysis of Oils, Fats andDerivatives, 7th ed.; Paquot, C., Hautfenne, A., Eds.; Blackwell: Oxford,UK, 1987.(25) Antoniosi Filho, N. R.; Carrilho, E.; Langas, E. M. Quantitativeanalysis of soybean oil in olive oil by high-temperature capillary gaschromatography. J. Am. Oil Chem. Soc. 1993, 70, 1051−1053.(26) Russo, M. V.; Goretti, G.; Soriero, A. Triglyceride analysis withmicrocapillary gas chromatography columns. Ann. Chim. 1996, 86,281−291.(27) Geeraert, E.; Sandra, P. Capillary GC of triglycerides in fats andoils using a high temperature phenylmethylsilicone stationary phase.Part II. The analysis of chocolate fats. J. Am. Oil Chem. Soc. 1987, 64,100−105.(28) Woodbury, S. E.; Evershed, R. P.; Rossel, J. B.; Griffith, R. E.;Farnell, P. Detection of vegetable oil adulteration using gaschromatography combustion/isotope ratio mass spectrometry. Anal.Chem. 1995, 67, 2685−2690.(29) Galeano Diaz, T.; Duran Meras, I.; Sanchez Casas, J.; AlexendreFranco, M. F. Characterization of virgin olive oils according to itstriglycerides and sterols composition by chemometric methods. FoodControl 2005, 16, 339−347.(30) Bohacenko, I.; Kopicova, Z. Detection of olive oils authenticityby determination of their sterol content using LC/GC. Czech J. FoodSci. 2001, 19, 97−103.(31) Longobardi, F.; Ventrella, A.; Napoli, C.; Humpfer, E.; Schutz,B.; Schafer, H.; Kontominas, M. G.; Sacco, A. Classification of oliveoils according to geographical origin by using 1H NMR fingerprintingcombined with multivariate analysis. Food Chem. 2012, 130, 177−183.(32) Roman Falco, I. P.; Grane Teruel, N.; Prats Moya, S.; MartínCarratala, M. L. Kinetic study of olive oil degradation monitored byFourier transform infrared spectrometry. Application to oil character-ization. J. Agric. Food Chem. 2012, 11800−11810.(33) Bucci, R.; Magrí, A. D.; Magrí, A. L.; Marini, D.; Marini, F.Chemical authentication of extra-virgin olive oil varieties by supervisedchemometric procedures. J. Agric. Food Chem. 2002, 50, 413−418.(34) Pizarro, C.; Rodríguez-Tecedor, S.; Perez-del-Notario, N.;Esteban-Díez, I.; Gonzalez-Saiz, J. M. Classification of Spanish extra-virgin olive oils by data fusion of visible spectroscopic fingerprints andchemical descriptors. Food Chem. 2013, 138, 915−922.(35) Kalua, C. M.; Allen, M. S.; Bedgood, D. R., Jr.; Bishop, A. G.;Prenzler, P. D. Discrimination of olive oils and fruits into cultivars andmaturity stages based on phenolic and volatile compounds. J. Agric.Food Chem. 2005, 53, 8054−8062.

(36) Boskou, D. Olive Oil: Chemistry and Technology; AOCS Press:Champaign, IL, USA, 1996; pp 85−127.(37) International Olive Council, COI/T, 20/Doc. No. 24.Preparation of the fatty acid methyl esters from olive oil and olivepomace oil, 2001.(38) International Olive Council, COI/T, 20/Doc. No. 17 Rev. 1.Determination of trans unsaturated fatty acids by capillary column gaschromatography, 2001.(39) International Olive Council, COI/T, 20/Doc. No. 10 Rev. 1.Determination of the composition and content of sterols by capillary-column gas chromatography, 2001.(40) International Olive Council, COI/ T, 20/ Doc. No. 30/Rev. 1.Determination of the composition and content of sterols andtriterpene dialcohols by capillary column gas chromatography, 2013.(41) Rezanka, T.; Rezankova, H. Characterization of fatty acids andtriacylglycerols in vegetable oils by gas chromatography and statisticalanalysis. Anal. Chim. Acta 1999, 398, 253−261.(42) Lerma-Garcia, M. J.; Lusardi, R.; Chiavaro, E.; Cerretani, L.;Bendini, A.; Ramis-Ramos, G.; Simo-Alfonso, E. F. Use oftriacylglycerol profiles established by high performance liquidchromatography with ultraviolet−visible detection to predict thebotanical origin of vegetable oils. J. Chromatogr., A 2011, 1218, 7521−7527.(43) Al-Ismail, K. M.; Alsaed, A. K.; Ahmad, R.; Al-Dabbas, M.Detection of olive oil adulteration with some plant oils by GLCanalysis of sterols using polar column. Food Chem. 2010, 121, 1255−1259.(44) Cert, A.; Moreda, W.; Garcia-Moreno, J. Determination ofsterols and triterpenic dialcohols in olive oils using HPLC separationand GC analysis. Standardization of the analytical method. GrasasAceites 1997, 48, 207−218.(45) Ballesteros, E.; Gallego, M.; Valcarcel, M. Simultaneousdetermination of sterols in edible oils by use of a continuousseparation module coupled to a gas chromatograph. Anal. Chim. Acta1995, 308, 253−260.(46) Gigliotti, C.; Daghetta, A.; Sidoli, A. Indagine conoscitiva sulcontenuto trigliceridico di oli extra vergini di oliva di varia provenienza.Riv. Ital. Sostanze Grasse 1993, 70, 483−489.(47) Berrueta, L. A.; Alonso-Salces, R. M.; Heberger, K. Supervisedpattern recognition in food analysis. J. Chromatogr., A 2007, 1158,196−214.



Detection of Chemlali Extra-Virgin Olive Oil Adulteration Mixed with Soybean Oil, Corn Oil, and...

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