1.

Introduction

The determination of trace elements in edible oils has gained more

importance during the last few years because of the fact that the quality of
edible oils is directly related to the concentration of trace metals in oils (Souza,
Mathias, Silveira, & Aucelio, 2005). Although some trace elements, such as Ca,
Co, Fe, Mg, Mn, and Ni, can promote the oxidative degradation of the oil, such
as As, Cd, Hg, and Pb, might present toxic effects in humans, depending on
their concentration in the oil (Lepri et al., 2011). The content of trace elements
and their chemical forms can be naturally present in vegetable oils that were
absorbed by the vegetable mainly from the soil where it was grown. A possibility
of trace elements’ entry into edible oils other than the technological one is the
environmental exposure to a large variety of elements. They can be also
incorporated during the extraction and refining process to which the oil is
submitted. Metals arrive in the plant via deposition as well as bioaccumulation
from the soil via the natural metal sources and/or environmental pollution. In
addition to these facts, the agricultural habits of the farmers play an important
role in the metal contents of their products, such as the application of fertilisers
or metal containing plant protection agents. There are examples of lead and
copper that are potentially present in oil samples caused by environmental
contamination (Allen, Siitonen, & Thompson, 1998; Juranovic, Breinhoelderb, &
Steffan, 2003). Additionally metals can be introduced during into oil from outer
sources the production process (by processing actions such as bleaching,
hardening, refining and deodorization) or by contamination from the metal
processing equipment (de Leonardis, Macciola, & de Felice, 2000). The oil
characterization in relation to the trace element composition is the basis for
further nutritional and technological researches such as adulteration detection
(Cordella, Moussa, Martel, Sbirrazzuoli, & Lizzani-Cuvelier, 2002). These
aspects, among others, make trace element determination in edible oils
important both an economy and a health wise and their concentration is an
important criterion for the evaluation of their quality concerning freshness,
stability, and storage. Atomic spectrometric methods like flame and graphite
furnace atomic absorption spectrometry (F-AAS and GF-AAS), as well as
inductively coupled plasma optical emission and mass spectrometry (ICP-OES
and ICP-MS), can be used for metal determinations in oil samples (Anthemidis,
Arvanitidis, Stratis, 2005; Ieggli, Bohrer, Do Nascimento, De Carvalho, 2011;
Mendil, Uluözlü, Tüzen, Soylak, 2009; Zhu, Fan, Wang, Qu, Yao, 2011).
However, the determination of trace elements in edible oils is difficult due to its
low concentration in the sample, requiring sensitive instrumental methods.
Moreover, their high viscosity makes it difficult to introduce the sample in the
instrument, and the high organic content of the oil matrix increases the
possibility of interference during analysis. For instance direct determination
method like dilution with an appropriate organic solvent followed by direct
aspiration into a flame atomic absorption or an ICP spectrometry excitation unit
is sometimes not sensitive enough. In most cases, the analytical method shows
changes in the excitation power for organic media compared to aqueous
solutions (Tserovsky Arpadjan, 1991). In addition, calibration is more difficult in
organic media (Boumans, 1987, chap. 4). To overcome these difficulties,
different sample preparation procedures like: wet or dry digestion, microwave
digestion, and some extraction methods and dilution are required, in order to
eliminate the organic content of the sample before its analysis (Ansari et al.,
2009; Juranovic et al., 2003; Mendil et al., 2009; Sahan, Basoglu, Gucer, 2007).
Sample preparation is a critical step in oil analysis and because of the high
organic content, sample pretreatment is frequently necessary. In this study, the
concentration of trace elements in some edible oils samples (sunflower,
hazelnut, canola, corn and olive oils) that were produced in Turkey and
collected from Turkish supermarkets were determined by using inductively
coupled plasma optical emission spectrometry (ICP-OES) after ultrasonic
extraction, wet digestion, and extraction induced by emulsion breaking
procedures.
 2. Experimental

2.1. Instrumentation

All metal measurements were carried out using a Varian (Varian Inc.,
Victoria, Australia) Vista-MPX; CCD Simultaneous inductively coupled optical
emission spectrometer (ICP-OES). The operating conditions are listed in Table
1. The analytical emission lines (nm) chosen were as follows: Cd (214.4); Cr
(267.7); Cu (327.4); Fe (238.2); Mn (257.6); Ni (231.6); Pb (220.3) and Zn
(213.9). A Bandelin Ultrasonic Bath-Sonorex Digital (BANDELIN electronic
GmbH Co.) was used during sample preparation. A Nüve ST 402 thermostatic
bath, maintained at the desired temperature, was used for temperature
experiments. A water bath with temperature control, model Wise Bath WB-22,
furnished by Daihan Scientific Co., Ltd., and was used for the heating of the
emulsions in order to induce their breaking.

2.2. Reagents and solutions

All reagents used were of the highest available purity and of at least
analytical reagent grade. The water (18 MX cm resistivity) was purified in an
ELGA Elgastat Maxima system. A multi-elemental standard solution of 100 g/L
containing 24 elements (Al, B, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, Ga, K, Li, Mg,
Mn, Na, Ni, Pb, Se, Sr, Te, Ti and Zn) dissolved in 5% HNO3 supplied by Merck
(Darmstadt, Germany) was used as stock solutions for calibration. Hydrochloric
acid (37%), nitric acid (65%) and hydrogen peroxide (35%) were supplied from
Merck (Darmstadt, Germany). The laboratory glassware was kept overnight in a
10% (v/v) nitric acid solution. Afterwards, it was rinsed thoroughly with ultra-
pure water and dried. The surfactant Triton X-114 was obtained from Sigma–
Aldrich (Milwaukee, USA). The acidic Triton X-114 solution used for the
extraction was induced by emulsion breaking (EIEB) procedure of the edible oil
and prepared by dissolving 7.0 g of Triton X-114 (Sigma–Aldrich, USA) in
exactly 100 mL of a 10% v/v HNO3 solution. Multi-element Standard Oiled stock
solutions of 100 lg/g were purchased from Alfa Aesar (Johnson Matthey
Company, USA). Diluted oiled standard solutions were prepared by diluting the
oil stock standard solutions in hexane. Total 50 samples and five varieties of
edible oils that were produced various plants in Turkey (ten of each sunflower,
hazelnut, canola, corn olive oil samples) were collected in Turkish supermarkets
during 2011. The collected oil samples were packed in polyethylene bags and
stored below 20 C until analysis.

2.3. Sample preparation procedure

Three types of sample preparation procedures were applied to determine

metal contents in edible oil samples; wet digestion, ultrasonic extraction and
extraction induced by emulsion breaking. In order to calculate precision, three
replicate measurements were performed for all sample preparation procedures.

2.3.1. Wet digestion

Triplicate samples (0.5 g) of each sunflower oil variety were accurately

weighed in 100-mL conical flasks. About 10 mL of a freshly prepared mixture of
concentrated HNO3–H2O2 (2:1, v/v) was added to each flask and kept for 10
min at room temperature. Then, the samples were heated on a hot plate at 80
C, until clear solutions were obtained. Then, the samples were evaporated, and
the semidried mass was dissolved in 5 mL 0.2 M HNO3, filtered through
Whatman No. 42 filter paper, and made up to final volume of 10 mL in
volumetric flasks with ultrapure water and metal contents were determined in
the diluted solutions by ICP-OES (Camin et al., 2010).

2.3.2. Ultrasonic extraction

About 0.1 g of sample were weighed into a 50 mL conical vial of

polypropylene (PP) and 10 mL of 1% HNO3/0.2% HCl (1:1) water solution was
added. The mixture was thoroughly shaken for 30 s using a vortex mixer and
then immediately placed in an ultrasonic bath (170 W 5 min) to extract the
metals from the oil to the acid solution. Then, the mixture was centrifuged (4000
rpm 5 min) to separate the two phases. The upper oil phase was accurately
removed by aspiration and the lower aqueous phase transferred into a clean PP
vial and subjected to ICP-OES analysis of Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn
(Ricardo, Cassella, Brum, de Paulaa, Lima, 2010). Extraction and analysis were
carried out in triplicate.

2.3.3. Extraction induced by emulsion breaking (EIEB) procedure

The extraction of Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn from edible oil
samples was performed by the extraction induced by emulsion breaking (EIEB)
procedure. The first step of the EIEB procedure was the formation of stable
water-in-oil emulsions of edible oil, which was always achieved by vigorous
mixing of 2 mL of edible oil with 2 mL of the acidic Triton X-114 solution
(solution containing 7% w/v Triton X-114 and 10% v/v HNO3) in a capped
plastic tube with 15 mL of capacity. Just after the formation of the emulsions,
the tube was transferred to the temperature-controlled water bath kept at 80 ± 2
C, where it was heated until the emulsion breaking. After the emulsion breaking,
three well-separated phases were formed: (i) the upper phase: an organic
phase containing only the edible oil, (ii) the intermediary phase: an acidic
aqueous phase, which is containing the extracted metals, and (iii) the lower
phase: a surfactant-rich phase. The total volume of the aqueous phase was
equal to 2 mL however approximately 1.5 mL could be collected by using a
micropipette. An exact volume of this solution was diluted (when necessary)
with ultra-pure water and the obtained solution was analysed by ICP-OES
regarding the concentration of the Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn metals
(Ansari et al., 2008; Cassellaa, Brumb, Limab, Caldasa, de Paulaa, 2011).

2.4. Calibration and statistics

A liqouits of an ICP multi-element standard solution (100 mg/L Merck)

containing the analysed elements (Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn) was used
in the preparation of calibration solutions. Working standard solutions were
prepared by dilution of the stock standard solutions to desired concentration in
1% HNO3. The ranges of the calibration curves (5 points) were selected to
match the expected concentrations (0–4.0 mg/L) for all the elements of the
sample studied by ICP-OES. The correlation coefficient r 2 obtained for all
cases was 0.9998. ANOVA was used for comparison of different digestion
procedures in oil samples. Statistically the different results were evaluated by
using TUKEY test. A value of P < 0.05 was considered statistically significant.
The results were given as mean ± standard deviation.

3. Results and discussion

In this work, the concentration levels of the elements in five typical edible
oil samples (Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn) were analysed by using ICP-
OES after ultrasonic extraction, wet digestion, and extraction induced by
emulsion breaking procedures. The results of the analyses are given in Tables
2a and b. As can be seen, the metal concentrations differed between oil types
and sample preparation procedures. Metal concentrations in edible oils were
found by using EIEB procedure between as 0.022–0.058, 0.126–7.106, 0.570–
4.504, 8.004–12.588, 0.035–0.054, 0.908– 2.182, 0.099–0.134 and 2.206–
8.982 mg kg1 for cadmium, chrome, copper, iron, manganese, nickel, lead and
zinc, respectively. The lowest and highest metal concentrations were observed
in manganese and iron in all samples. In general, the presence of these metals
is undesirable because they can be both toxic for the health of the consumers
and oxidative which facilitates degration of the oil decreasing their shelf life. It is
known sufficient amount of iron in a diet is very important for decreasing the
incidence of anaemia (Ashraf Mian, 2008), reduces labour capacity and
impaired intellectual development (Schümann, Ettle, Szegner, Elsenhans,
Solomons, 2007). High concentrations of iron may lead to tissue damage,
because of the formation of free radicals. The minimum amount of Fe (8.004 mg
kg1 ) was observed in canola oil, while the maximum level of Fe (12.588 mg
kg1 ) was observed in hazelnut oil (Table 2a). Fe is a strong oxidant, and a high
content in hazelnut oil may be due to a high amount of available Fe in the
agricultural soils where the hazelnut oil crops were grown. Iron concentration
was reported as 15.31 and 291.0–52.0 mg kg1 in the literature (Mendil et al.,
2009; Onianwa, Adeyemo, Idowu, Ogabiela, 2001). Copper is essential element
for good health but very high intake can cause adverse health problems such as
liver and kidney damage. Copper deficiency leads to hypochromic anaemia,
leucopenia, and osteoporosis in especially for children (Kanumakala, Boneh,
Zacharin, 2002). The amount of Cu in various sunflower oil varieties was found
by using the EIEB method to be in the range of 0.570–4.504 mg kg1. The
highest and lowest levels of copper were found in hazelnut oil and canola oil.
Copper may enter the food materials from soil through mineralization by crops,
as in the application of agricultural inputs, such as copper-based pesticides that
are in common use in farms in some countries, food processing, or
environmental contamination (Ajayi, Oderinde, Kajogbola, Uponi, 2006; Koc et
al., 2008). Copper contents of edible oil samples in the literature have been
reported in the range of 21.0– 31.0 mg kg1 (Joint FAO/WHO, 1999). Our copper
values in the investigated oil samples are in agreement with reported in the
literature. The FAO/WHO has set a limit for heavy metal intake based on body
weight. For an average adult (60 kg body weight), the provisional tolerable daily
intake (PTDI) for lead, iron, copper and zinc are 214 lg, 48 mg, 3 mg and 60
mg, respectively (Nunes et al., 2011). Metals such as copper, zinc, iron,
manganese are essential elements since they play an important role in
biological systems. Zinc is a nutritionally essential metal, and a deficiency
results in severe health consequences. Zinc deficiency can lead to loss of
appetite, growth retardation, skin changes, and immunological abnormalities.
Manganese and zinc concentrations were reported as 0.06 and 3.39 lg/g in the
literature (Onianwa et al., 2001). In this study, the highest content of
manganese and zinc were 0.054 and 8.982 mg kg1 in hazelnut oil, whereas the
lowest manganese and zinc concentration were 0.035 and 2.206 mg kg1 in
olive oil (Table 2b). The presence of Ni in hydrogenated oils and fats is
important from health and safety standpoints. The nickel values have been
obtained in the range of 0.908–2.182 mg kg1 ; the high level of Ni was observed
in hazelnut oil, while lowest concentration was observed in olive oil. Nickel
concentration was reported as 1.9– 2.74 mg kg1 in literature. Our nickel values
are lowest than literature values (Kowalewska, Izgi, Saracoglu, Gücer, 2005).
The analysis of elements such as Pb, and Cd in foodstuff is of general concern
since they pose toxicological risks to humans because children are more
sensitive to these metals than adults are. Cadmium is a highly toxic metal with a
natural occurrence in soil, but it is also expand in the environment due to human
activities. Excessive cadmium exposure may cause renal, pulmonary, hepatic,
skeletal, reproductive effects and cancer. In addition, cadmium is related to
kidney, liver damage, and anaemia. The European Community sets limit only
for Pb in edible oils, which is 0.1 mg kg1 (European Commission, 2006).
Furthermore, the levels of Cd and Pb in oil samples have been reported 0.05
and 0.1 mg kg1 (Kowalewska et al., 2005). In the present study, cadmium and
lead concentrations from 0.022–0.058 mg kg1 to 0.099–0.134 mg kg1 were
detected. The presence of lead, at such low levels could be due to
environmental contamination. Specifically, the content of Pb would be justified
by the presence of streets, highways, or metallurgic industries near plantations.
Generally, cadmium and lead levels in analysed edible oil samples were found
to be lower than legal limits.
Chromium is considered as essential trace element because chromium and
insulin act together to control rising blood sugar (Bratakos, Lazos, Bratakos,
2002). The levels of chromium found in this study oil for oil samples range
between 0.126 and 7.106 mg kg1 (Table 2a). The recommended daily intake of
chromium is 50–200 lg (National Research Council, 1989). It can be thought
that the technology used in oil processing can increase the natural levels of Cr
in raw material due to the transfer of the element from apparatus, utensils,
containers (i.e. ceramic containers), and packages. The presence of analysed
metals was observed in all the types of oils. Taking into account that these
analysed metals have been the most frequently, found so special attention has
been given to the discussion of the results of these elements. This presence
can be due to the factors such as species, soil used for the cultivation, irrigation
water, variety, and stage of maturity, and treatment processes or packaging
procedures. From these experiments, it can be concluded that the content of
trace metals is directly related to their provenance and the production, which
has been an important issues. The mean values obtained from EIEB were
significantly higher than two other methods. That is, 13 oil samples showed
statistically significant results regarding EIEB. On the other hand, UE had only
six statistically significant values compared to EIEB and wet methods.
Moreover, wet method gave only one significantly higher result than other two
methods in the 20 significant ANOVA tests. Accordingly, results for EIEB
strongly support that this method overrides the other methods. As a result, EIEB
may be preferred as a reliable method for such kind of studies. In addition, a
recovery test was done by spiking the edible oil samples with metals in the form
of organometallic compounds (oiled stock solutions) in order to verify the
efficiency of the EIEB extraction procedure and the accuracy of the EIEB
method. The recovery percentages verified in this experiment are shown in
Table 3. For all metals under study, the recovery values were suitable for
quantitative purposes, being always situated between 96% and 109%. In the
light of these results, EIEB procedure was chosen, as the best for the
preparation of all the oil samples as shorter required time and smaller
deviations than ultrasonic extraction and wet digestion procedures were
included. This procedure also finds application for trace elements determination
in vegetable and biodiesel oils by atomic spectrometric methods.

4. Conclusions

The extraction induced by emulsion breaking procedure for the

determination of Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn in edible oil by using ICP-
OES is useful for routine analysis and it can be considered as an excellent
alternative to other sample preparation procedures such as ultrasonic extraction
and wet digestion. This procedure avoids the laborious and time-consuming
digestion of the samples. It was carried out by simply formation of stable water-
in-oil emulsions of edible oil, which was always achieved by vigorous mixing of
edible oil with the acidic Triton X-114 solution for formation of emulsions, and
the sample was heated until the emulsion breaking. After the emulsion
breaking, three wellseparated phases were formed. The acidic aqueous phase
determined by ICP-OES. Sample preparation is a critical stage in the oil
analysis. Procedure requiring a pretreatment of the samples to destroy the
organic matrix involves certain manipulations and the subsequent risk of sample
contamination and/or analyte loss. Finally, The EIEB procedure was applied in
the determination of the elements of interest in real samples of edible oil. The
results observed in the analysis of the spiked samples clearly showed that the
method could be used very reliably in the elemental analysis of edible oil by
ICP-OES. The results obtained in this present work indicate that the extraction
induced by emulsion breaking can be very useful procedure for simple and
sensitive metal determination in edible oil samples by ICP-OES. These
suggestions given here are also supported by statistical analysis.