**2. Sample pre-treatment**

The accurate determination of trace metals in olive oil is an analytical challenge due to their low concentration and the difficulties that arise because of the high organic content. Due to the high organic content, sample pretreatment is a critical step and frequently necessary in olive oil analysis. Sample pretreatment step provides the decomposition of organic matrix or the extraction of metals without matrix decomposition. On the other hand, oil sample can be diluted in a suitable solvent or emulsified with an appropriate emulsifier in a rapid pretreatment for direct determinations. The atomic spectrometers are the most commonly used devices but have some problems such as the reduced stability of the analytes in the solution, requirement of organometallic standards, the use of dangerous organic solvents or sample digestion with an acid or acid mixture (Nunes et al., 2011).

#### **2.1 Acid digestion**

Digestion procedures are regularly carried out with either open vessels using acid, acid mixture or basic reagents on hot plates or open- and closed-vessel microwave ovens. The decomposition in open system is hard, time consuming and prone to systematic error sources, i.e. contamination or analyte losses. In case of using microwave radiation, the high cost of instrumentation and dilution of the sample can be considered as disadvantages in the microwave assisted digestion system. Although the amount of sample in vessels is limited due to the generation of gaseous reaction products that can increase of pressure, the use of closed high-pressure vessels is appropriate for efficient sample digestion. On the other hand, in the use of open-focused microwave ovens, the advantages are decreasing the risk to the operator, possible introduction of reagents during procedure, opportunity to digest larger amounts of sample and low cooling time (Sant'Ana et al., 2007).

Microwave-assisted digestion has been performed to dissolve the oil sample for elemental analysis in a large number of papers (Angioni et al., 2006; Ansari et al., 2009; Levine et al., 1999; Llorent-Martinez et al., 2011a, 2011b; Mendil et al., 2009; Sant'Ana et al., 2007), while focused microwave assisted digestion for the same purpose has been employed in a few papers (Sant'Ana et al., 2007). As shown in Table 1, some investigation have been done on microwave digestion for olive oil using various procedures.

#### **2.2 Dry ashing**

In general, ashing methods may provide lower analyte recovery and exhibit poorer accuracy compared to acid digestion methods. Although dry ashing procedures are effective, they are time consuming and can often result in loss of analyte species that could occur during the preparation of the sample. Oil is decomposed by high-temperature dry ashing, subsequently the ash is dissolved in an acidic aqueous medium and the metal content of the aqueous phase can be measured by various detection techniques such as AAS, adsorptive stripping voltammetry (AdSV) and derivative potentiometric stripping analysis (dPSA) (Abbasi et al., 2009; Carbonell et al., 1991; Lo Coco et al., 2003). There are limited researches for metal determinations in oils after dry ashing of olive oil (Lo Coco et al., 2003).

The accurate determination of trace metals in olive oil is an analytical challenge due to their low concentration and the difficulties that arise because of the high organic content. Due to the high organic content, sample pretreatment is a critical step and frequently necessary in olive oil analysis. Sample pretreatment step provides the decomposition of organic matrix or the extraction of metals without matrix decomposition. On the other hand, oil sample can be diluted in a suitable solvent or emulsified with an appropriate emulsifier in a rapid pretreatment for direct determinations. The atomic spectrometers are the most commonly used devices but have some problems such as the reduced stability of the analytes in the solution, requirement of organometallic standards, the use of dangerous organic solvents or

Digestion procedures are regularly carried out with either open vessels using acid, acid mixture or basic reagents on hot plates or open- and closed-vessel microwave ovens. The decomposition in open system is hard, time consuming and prone to systematic error sources, i.e. contamination or analyte losses. In case of using microwave radiation, the high cost of instrumentation and dilution of the sample can be considered as disadvantages in the microwave assisted digestion system. Although the amount of sample in vessels is limited due to the generation of gaseous reaction products that can increase of pressure, the use of closed high-pressure vessels is appropriate for efficient sample digestion. On the other hand, in the use of open-focused microwave ovens, the advantages are decreasing the risk to the operator, possible introduction of reagents during procedure, opportunity to digest larger amounts of sample and low cooling time

Microwave-assisted digestion has been performed to dissolve the oil sample for elemental analysis in a large number of papers (Angioni et al., 2006; Ansari et al., 2009; Levine et al., 1999; Llorent-Martinez et al., 2011a, 2011b; Mendil et al., 2009; Sant'Ana et al., 2007), while focused microwave assisted digestion for the same purpose has been employed in a few papers (Sant'Ana et al., 2007). As shown in Table 1, some investigation have been done on

In general, ashing methods may provide lower analyte recovery and exhibit poorer accuracy compared to acid digestion methods. Although dry ashing procedures are effective, they are time consuming and can often result in loss of analyte species that could occur during the preparation of the sample. Oil is decomposed by high-temperature dry ashing, subsequently the ash is dissolved in an acidic aqueous medium and the metal content of the aqueous phase can be measured by various detection techniques such as AAS, adsorptive stripping voltammetry (AdSV) and derivative potentiometric stripping analysis (dPSA) (Abbasi et al., 2009; Carbonell et al., 1991; Lo Coco et al., 2003). There are limited researches for metal determinations in oils after dry ashing of olive oil (Lo Coco et

sample digestion with an acid or acid mixture (Nunes et al., 2011).

microwave digestion for olive oil using various procedures.

**2. Sample pre-treatment**

**2.1 Acid digestion**

(Sant'Ana et al., 2007).

**2.2 Dry ashing**

al., 2003).

i: initial ; f: final

Table 1. The summary of microwave digestion procedures for various metals in olive oil.

#### **2.3 Extraction**

Sample preparation involves acid extraction (Anwar et al., 2004; De Leonardis et al., 2000; Dugo et al., 2004; Jacob & Klevay, 1975), solid phase extraction (SPE) (Bat & Cesur, 2002) or extraction with special agents (Köse Baran & Bağdat Yaşar, 2010).

After the extraction of metals from oil with nitric acid, hydrochloric acid or acid mixture, the extracts are analyzed. Despite the fact that extraction method has the same advantage both in the separation and preconcentration of metals in oil samples, the recoveries are not satisfactory for many metals in most cases. Bat and Cesur described another method for the preconcentration and separation of copper in edible oils, based on using a solid Pbpiperazine-dithiocarbamate complex for extraction and a potassium cyanide solution for back extraction (Bat & Cesur, 2002).

Anwar et al. reported a simple acid-extraction method for the determination of trace metals in oils and fats. The method has been performed with the use of ultrasonic intensification and successfully applied for accurate determination of iron, copper, nickel and zinc in oils (Anwar et al., 2004). Many extraction procedures are available in literature, the summary of these is given in Table 2.

Metal Determinations in Olive Oil 93

al., 2003; Tantaru et al., 2002; Ziyadanoğullar et al., 2008). The chemists have attended to the Schiff bases and their metal complexes because of their widespread applications in biological systems and industrial uses (Issa et al., 2005; İspir, 2009; Kurtaran et al., 2005; Li et al., 2007; Mohamed, 2006; Neelakantan et al., 2008; Prashanthi et al., 2008; Sharaby, 2007).

Although most techniques for metal determinations in edible oils require sample digestion, dilution or emulsification, the improved method can be employed for the same purpose without digestion. The procedure is based on efficient extraction of metals from oil to aqueous solution, and the determination of metals in aqueous phase by FAAS. The proposed approach has been applied for Fe, Cu, Ni and Zn successively. This method includes two main steps. Metal complexes with Schiff bases shown in Fig. 1 were investigated spectrophotometrically as a first step. In this step, the investigation of the complexation reaction as a driving force for the extraction is necessary to decide the

> X Y

Z -H ; -OCH3 ; -Br Q -H ; -CH3

P -H ; -CH3 ; -OH

Levels -1.682 -1 0 +1 +1.682

appropriate pH and the equilibrium time in terms of complexation efficiency.

C H

As a second step, the experimental conditions affecting the extraction efficiency of metals should be researched. In the procedure of metal extraction with a Schiff base, the optimization of parameters -the ratio of Schiff base solution volume to oil mass, the stirring time and the temperature- for the metal extractions has been achieved by carrying out

As shown in Table 3, the CCD consisting of a combination of 23 full factorial design and a star design was used, in which three independent factors were converted to dimensionless

*x2* (2nd factor) Stirring time (minute) 9.56 30 60 90 110.46 *x3* (3rd factor) Temperature (C) 13.18 20 30 40 46.82

Table 3. Variables, levels and the values of levels used in CCD (Köse Baran & Bağdat Yaşar,

(mL g-1) 0.159 0.5 1 1.5 1.841

z OH HO z

Fig. 1. Chemical structure of Schiff base used in the extractions

central composite design (CCD) as an optimization method.

ones (*x1, x2, x3*) with the coded values at 5 levels: -1.682, -1, 0, +1, +1.682.

X X

Q P

Y Y

N

C N H

Factors

*x1* (1st factor) VLDM / moil ratio

2010)

1Acc.: Accuracy; 2Rec.: Recovery; 3LOD: Limit of detection; 4RSD: Relative standard deviation

Table 2. The extraction methods used for metal determination in vegetable oils

A Schiff base has been suggested for the extraction of metals from oils as an appropriate chelating agent under the optimum extraction conditions (Köse Baran & Bağdat Yaşar, 2010). In recent analytical applications, Schiff bases have been used in order to form complexes due to their good complexing capacity with metals (Afkhami et al., 2009; Ashkenani et al., 2009; Ghaedi et al., 2009; Khedr et al., 2005; Khorrami et al., 2004; Köse Baran & Bağdat Yaşar, 2010; Kurşunlu et al., 2009; Mashhadizadeh et al., 2008; Shamspur et 92 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

**Detection**

Extraction with conc. HCl Cu Ad-SSWV 3LOD: 0.49 ng mL-1 Galeano Diaz

ICP-AES

SPE and KCN back-extraction Cu FAAS Rec.% 91-97 Bat & Cesur,

SPE Cd FAAS Rec.% 93.1-100 Yağan Aşç

Cu, Fe, Ni FAAS and ETAAS

1Acc.: Accuracy; 2Rec.: Recovery; 3LOD: Limit of detection; 4RSD: Relative standard deviation Table 2. The extraction methods used for metal determination in vegetable oils

A Schiff base has been suggested for the extraction of metals from oils as an appropriate chelating agent under the optimum extraction conditions (Köse Baran & Bağdat Yaşar, 2010). In recent analytical applications, Schiff bases have been used in order to form complexes due to their good complexing capacity with metals (Afkhami et al., 2009; Ashkenani et al., 2009; Ghaedi et al., 2009; Khedr et al., 2005; Khorrami et al., 2004; Köse Baran & Bağdat Yaşar, 2010; Kurşunlu et al., 2009; Mashhadizadeh et al., 2008; Shamspur et

Cu, Ni UV-Vis spec. Rec.% 90-118 (Cu);

Cu, Pb SCP Rec.% 82-107 (Cu);

Fe, Cu FAAS Rec.% 100.2±5.6

**technique Notes Reference**

97±12 (Fe)

2Rec.% 92-98 (Fe); 91-100 (Cu); 92-97 (Ni); 93-101 (Zn)

Rec.% 96.5±2.1 (Cd); 97.0±2.7 (Cu); 95.0±1.8 (Pb); 93.5±1.7 (Zn)

96-100 (Ni)

4RSD%: < 10 (Cu); 5 (Fe); 15 (Mn); 8 (Co); 10 (Cr); 20 (Pb); 5 (Cd); 16 (Ni); 11 (Zn)

Rec.% 96.5-97.5 (Fe); 96.5-97.1 (Cu); 95.8-97.5 (Ni); 96.0-

Rec.% 95.9-98.3 (Cu); 95.7-98.2 (Fe); 95.2-97.5 (Ni)

84-105 (Pb)

(Fe); 99.4±2.8 (Cu)

97.8 (Zn)

De Leonardis et al., 2000

Anwar et al., 2004

Dugo et al., 2004

et al., 2006

Hussain Reddy et al., 2003

Pehlivan et al., 2008

Anwar et al., 2003

2002

et al., 2008

Ansari et al., 2008

Cypriano et al., 2008

2010

Köse Baran & Bağdat Yaşar,

**determined**

Cu, Fe, Mn, Co, Cr, Pb, Ni, Cd, Zn

Fe, Cu, Zn, Ni FAAS

Extraction with 10% HNO3 Fe, Cu GF-AAS 1Acc.% 94±23 (Cu);

Fe, Cu, Ni, Zn FAAS

**Extraction method Metals**

Extraction with CCl4 + 2 N

Extraction with 35% H2O2 and

Extraction with conc. HNO3

Extraction with 10% HNO3

Extraction with CCl4 and 2 N HNO3 after pretreatment with conc. HNO3 (ultrasonic bath,

Pb-piperazinedithiocarbamate

Zn-piperazinedithiocarbamate

Ultrasonic-Assisted extraction with conc. HNO3 and H2O2

Ultrasonic-Assisted extraction with conc. HCl and 30% H2O2

*N,N'*-bis(salicylidene)-2,2*'*-

propanediaminato (LDM)

and 6% H2O2

(50 Hz, 60 s)

30 C)

(35 kHz)

Extraction with

dimethyl-1,3-

36% HCl (30 min., 90 C) Cd, Cu, Pb, Zn dPSA

HNO3 (ultrasonic intensification)

al., 2003; Tantaru et al., 2002; Ziyadanoğullar et al., 2008). The chemists have attended to the Schiff bases and their metal complexes because of their widespread applications in biological systems and industrial uses (Issa et al., 2005; İspir, 2009; Kurtaran et al., 2005; Li et al., 2007; Mohamed, 2006; Neelakantan et al., 2008; Prashanthi et al., 2008; Sharaby, 2007).

Although most techniques for metal determinations in edible oils require sample digestion, dilution or emulsification, the improved method can be employed for the same purpose without digestion. The procedure is based on efficient extraction of metals from oil to aqueous solution, and the determination of metals in aqueous phase by FAAS. The proposed approach has been applied for Fe, Cu, Ni and Zn successively. This method includes two main steps. Metal complexes with Schiff bases shown in Fig. 1 were investigated spectrophotometrically as a first step. In this step, the investigation of the complexation reaction as a driving force for the extraction is necessary to decide the appropriate pH and the equilibrium time in terms of complexation efficiency.

Fig. 1. Chemical structure of Schiff base used in the extractions

As a second step, the experimental conditions affecting the extraction efficiency of metals should be researched. In the procedure of metal extraction with a Schiff base, the optimization of parameters -the ratio of Schiff base solution volume to oil mass, the stirring time and the temperature- for the metal extractions has been achieved by carrying out central composite design (CCD) as an optimization method.

As shown in Table 3, the CCD consisting of a combination of 23 full factorial design and a star design was used, in which three independent factors were converted to dimensionless ones (*x1, x2, x3*) with the coded values at 5 levels: -1.682, -1, 0, +1, +1.682.

Table 3. Variables, levels and the values of levels used in CCD (Köse Baran & Bağdat Yaşar, 2010)

Metal Determinations in Olive Oil 95

New corresponding equations were obtained by equalization of the derivatives of *y* equation in terms of *x1, x2, x3* to zero and solved using software to provide optimum extraction conditions. Optimum conditions are variable depending on the structure of Schiff base and significant metal. The found optimum conditions are given in Table 5 when LDM (Q and P = CH3; X, Y and Z = H) was used as a Schiff base. The recovery values for the extraction of Cu and Fe from oil under the optimum experimental conditions were found to be 99.4(±2.8) and 100.2(±5.6)%, respectively (n=10). To test the applicability of the improved procedure, it was applied on spiked olive, sunflower, corn and canola oils. The recovery percentages were varied between 97.2-102.1 for Cu and 94.5-98.6 for Fe (Köse Baran &

> VLDM / moil ratio (mL g-1)

emulsifiers such as Triton X-100 or a solid sampling strategy.

Cu 0.76 73 31 Fe 1.19 67 28

Table 5. Optimum extraction conditions for determination of Cu and Fe in edible oils (Köse

The improved determination strategy after the extraction with Schiff bases has main advantages like independency from hard oil matrix, elimination of explosion risk during decomposition, no requirement for expensive instruments, high accuracy, sensitivity,

The direct determination of metals in oils can be carried out by sample solubilization in an organic solvent, an emulsification procedure in aqueous solutions in the presence of

The procedure of the dilution with organic solvents is an easy way to sample pretreatment before detection, but has some requirements: special devices for sample introduction e. g. for FAAS (Bettinelli et al., 1995), the addition of oxygen as an auxiliary gas in ICP-OES or ICP-MS (Costa et al., 2001). The volatile organic solvents have been directly introduced into ICPs for many years, but this can cause plasma instability, less sensitivity, less precision and high cost. Al, Cr, Cd, Cu, Fe, Mn, Ni and Pb contents of olive oil were investigated using diethyl ether, methyl isobutyl ketone (MIBK), xylene, heptane, 1,4-dioxane as solvent and N,N-hexamethylenedithiocarbamic acid, hexamethyleneammonium (HMDC-HMA) salt as a modifier by ETAAS (Karadjova et al., 1998). A transverse heated filter atomizer (THFA) was employed for the direct determination of Cd and Pb in olive oil after sample dilution with nheptane (Canario & Katskov, 2005). Moreover, Martin-Polvillo et al. (1994) and List et al. (1971) determined trace elements in edible oils based on the direct aspiration of the samples, diluted in MIBK. In another research, the mixture of 2%lecithin-cyclohexane was used to introduce the oil samples to a polarized Zeeman GFAAS (Chen et al., 1999). Van Dalen was

Optimum Conditions

Temperature (C)

Stirring time (min.)

Bağdat Yaşar, 2010).

Baran & Bağdat Yaşar, 2010)

rapidity and cheapness.

**3. Direct determination**

**3.1 Dilution with organic solvent**

Metal

Fifteen experiments should be done in a CCD. Additionally, to estimate the experimental error, replications of factor combinations are necessary at the center point (the level, 0). Experiment at the center point has been repeated five times. The total number of experiments in the CCD with three factors then amounts to 20 (Morgan, 1991; Otto, 1999). Accordingly, 20 experiments given in Table 4 were carried out in the extent of the CCD optimization procedure.

Table 4. The coded values of levels for the experiments in the extent of CCD

Organo-metallic standards in oil (Conostan code number; 354770 for iron, 687850 for copper) were used in CCD and metal concentrations of oil standards were fixed to be a certain concentration. The metal concentrations of the extracts gained from each experiment were determined by FAAS. The obtained results were used in order to establish recovery values for the extraction of metals from oil. The response values (y) were calculated from experimentally obtained recovery percentages. The empirical equations were developed by means of response values (Morgan, 1991; Otto, 1999). The following y equations were constructed based on the b values which were calculated by applying to the appropriate matrixes.

$$y = b\_1X\_1 + b\_2X\_2 + b\_3X\_3 + b\_{11}X\_1^2 + b\_{22}X\_2^2 + b\_{33}X\_3^2 + b\_{12}X\_1X\_2 + b\_{13}X\_1X\_3 + b\_{23}X\_2X\_3 + b\_{123}X\_1X\_2X\_3 \tag{1}$$

Fifteen experiments should be done in a CCD. Additionally, to estimate the experimental error, replications of factor combinations are necessary at the center point (the level, 0). Experiment at the center point has been repeated five times. The total number of experiments in the CCD with three factors then amounts to 20 (Morgan, 1991; Otto, 1999). Accordingly, 20 experiments given in Table 4 were carried out in the extent of the CCD

Coded values of levels

Stirring time (min.) *x2*

Temperature (C) *x3*

optimization procedure.

matrixes.

Experiment no. VLDM / moil ratio

(mL g-1) *x1*

Table 4. The coded values of levels for the experiments in the extent of CCD

Organo-metallic standards in oil (Conostan code number; 354770 for iron, 687850 for copper) were used in CCD and metal concentrations of oil standards were fixed to be a certain concentration. The metal concentrations of the extracts gained from each experiment were determined by FAAS. The obtained results were used in order to establish recovery values for the extraction of metals from oil. The response values (y) were calculated from experimentally obtained recovery percentages. The empirical equations were developed by means of response values (Morgan, 1991; Otto, 1999). The following y equations were constructed based on the b values which were calculated by applying to the appropriate

*y = b1X1 + b2X2 + b3X3 + b11X12 + b22X22 + b33X32 + b12X1X2 + b13X1X3 + b23X2X3 + b123X1X2X3* (1)

New corresponding equations were obtained by equalization of the derivatives of *y* equation in terms of *x1, x2, x3* to zero and solved using software to provide optimum extraction conditions. Optimum conditions are variable depending on the structure of Schiff base and significant metal. The found optimum conditions are given in Table 5 when LDM (Q and P = CH3; X, Y and Z = H) was used as a Schiff base. The recovery values for the extraction of Cu and Fe from oil under the optimum experimental conditions were found to be 99.4(±2.8) and 100.2(±5.6)%, respectively (n=10). To test the applicability of the improved procedure, it was applied on spiked olive, sunflower, corn and canola oils. The recovery percentages were varied between 97.2-102.1 for Cu and 94.5-98.6 for Fe (Köse Baran & Bağdat Yaşar, 2010).

Table 5. Optimum extraction conditions for determination of Cu and Fe in edible oils (Köse Baran & Bağdat Yaşar, 2010)

The improved determination strategy after the extraction with Schiff bases has main advantages like independency from hard oil matrix, elimination of explosion risk during decomposition, no requirement for expensive instruments, high accuracy, sensitivity, rapidity and cheapness.