**5. Sample preparation**

The first step required in the analysis of herbicides is to separate the analytes from the matrix and other interfering compounds, therefore isolating the herbicides from the rest of the sample. This separation includes extraction, pre-concentration and clean-up steps, although sometimes they can be performed at the same time (Gilbert-López et al., 2009; Mukherjee & Gopal, 1996).

This part of the analysis is critical, because the final results will be completely related to the success of the separation process. The sample has to come into contact with an extractant (solid, liquid or supercritical fluid) in optimized conditions in order to weaken the analytematrix interactions and increase the analyte-extractant interactions.

There is a continuous development of sample-treatment procedures for the isolation of herbicides in samples with relatively high fat content, such as olive oil and olives. The preparation of oil samples for their determination by chromatographic techniques requires the removal of the fatty components from the sample. The main problem associated when working with this kind of samples is that dirty extracts may harm the instruments employed. For this reason, proper extraction and clean-up steps are required. In addition, the different nature and physicochemical properties of the classes of herbicides to be analyzed makes it more complex to select the appropriate extraction methodology.

There are numerous extraction and clean-up procedures. However, the most common ones are solid-liquid or liquid-liquid extraction, gel permeation-chromatography (GPC) and solid-phase extraction (SPE) (Gilbert-López et al., 2009; Mukherjee & Gopal, 1996; Walters, 1990).

#### **5.1 Solid-liquid extraction**

In this case, the sample comes to contact with an appropriate solvent. After that, different procedures can be applied to homogenize both phases: ultrasonic agitation and microwave extraction.

*Ultrasonic agitation*: the solvent interacts with the sample simply by a shaking process. Ultrasonic radiation (frequency of 25-40 Hz) causes the vibration of the molecules, increasing the collision between them. Hence, the contact between sample and solvent is enhanced. After the analyte is dissolved in the extractant, a filtration step and the evaporation of the solvent are required.

*Microwave extraction*: samples are enclosed in high quality Teflon vessels together with the solvent and heated to a controlled temperature with microwave power, being the extract filtered when the process finishes. Electromagnetic irradiation is used to heat the solvent that acts as extractant, which is in most cases a mixture between hexane and acetone.

#### **5.2 Liquid-liquid extraction**

Usually, it has been the chosen method for the extraction, preconcentration and clean-up of liquid samples. It is based on the relative solubility of the analyte in two different immiscible liquids; therefore, it is an extraction of a substance from one liquid phase into another liquid phase. The liquid sample comes to contact with an appropriate solvent (immiscible with the sample) and, after a mixing time, both phases (sample and extractant) are separated. During the mixing period, the analyte is distributed between both phases until it reaches the equilibrium.

The KO/W value is critical to decide the appropriate extractant. If this value is high, it indicates that the analyte tends to dissolve in the organic phase instead of in water. As a general rule, the organic solvents selected are volatile substances that present high affinity for the analytes and are immiscible with the sample.

## **5.3 Gel-permeation chromatography**

216 Herbicides – Properties, Synthesis and Control of Weeds

The first step required in the analysis of herbicides is to separate the analytes from the matrix and other interfering compounds, therefore isolating the herbicides from the rest of the sample. This separation includes extraction, pre-concentration and clean-up steps, although sometimes they can be performed at the same time (Gilbert-López et al., 2009;

This part of the analysis is critical, because the final results will be completely related to the success of the separation process. The sample has to come into contact with an extractant (solid, liquid or supercritical fluid) in optimized conditions in order to weaken the analyte-

There is a continuous development of sample-treatment procedures for the isolation of herbicides in samples with relatively high fat content, such as olive oil and olives. The preparation of oil samples for their determination by chromatographic techniques requires the removal of the fatty components from the sample. The main problem associated when working with this kind of samples is that dirty extracts may harm the instruments employed. For this reason, proper extraction and clean-up steps are required. In addition, the different nature and physicochemical properties of the classes of herbicides to be

There are numerous extraction and clean-up procedures. However, the most common ones are solid-liquid or liquid-liquid extraction, gel permeation-chromatography (GPC) and solid-phase extraction (SPE) (Gilbert-López et al., 2009; Mukherjee & Gopal, 1996; Walters,

In this case, the sample comes to contact with an appropriate solvent. After that, different procedures can be applied to homogenize both phases: ultrasonic agitation and microwave

*Ultrasonic agitation*: the solvent interacts with the sample simply by a shaking process. Ultrasonic radiation (frequency of 25-40 Hz) causes the vibration of the molecules, increasing the collision between them. Hence, the contact between sample and solvent is enhanced. After the analyte is dissolved in the extractant, a filtration step and the

*Microwave extraction*: samples are enclosed in high quality Teflon vessels together with the solvent and heated to a controlled temperature with microwave power, being the extract filtered when the process finishes. Electromagnetic irradiation is used to heat the solvent

Usually, it has been the chosen method for the extraction, preconcentration and clean-up of liquid samples. It is based on the relative solubility of the analyte in two different immiscible liquids; therefore, it is an extraction of a substance from one liquid phase into another liquid phase. The liquid sample comes to contact with an appropriate solvent (immiscible with the

that acts as extractant, which is in most cases a mixture between hexane and acetone.

analyzed makes it more complex to select the appropriate extraction methodology.

matrix interactions and increase the analyte-extractant interactions.

**5. Sample preparation** 

Mukherjee & Gopal, 1996).

**5.1 Solid-liquid extraction** 

evaporation of the solvent are required.

**5.2 Liquid-liquid extraction** 

1990).

extraction.

It is probably the most extensively used technique for the analysis of pesticide residues in olive oil, usually after a liquid-liquid extraction. In this technique, an aliquot of an olive oil extract (obtained from the extraction step) is injected into the GPC system. The selected fraction is collected and, after a solvent-exchange step, the sample is analyzed by GC. A GPC system is composed of a chromatographic pump, a fraction collector and a detector. The columns are made from polymeric porous microspheres that enable the separation of compounds according to their molecular weights (which are related to the size of the compound). Using this principle, the herbicide fraction is separated from the triglyceride fraction, which presents higher molecular weight. In GPC, the compounds are eluted from higher to lower molecular weight.

#### **5.4 Solid-phase extraction**

It is a separation process by which compounds that are dissolved or suspended in a liquid mixture are separated from other compounds in the mixture according to their physical and chemical properties. SPE is used to concentrate and purify samples for analysis and to isolate the analytes of interest from a wide variety of matrices.

SPE uses the affinity of the analytes dissolved or suspended in a liquid (known as the mobile phase) for a solid through which the sample is passed (known as the stationary phase) to separate a mixture into desired and undesired components. The result is that either the desired analytes of interest or undesired impurities in the sample are retained on the stationary phase. The portion that passes through the stationary phase is collected or discarded, depending on whether it contains the desired analytes or undesired impurities. If the portion retained on the stationary phase includes the desired analytes, they can then be removed from the stationary phase for collection in an additional step, in which the stationary phase is rinsed with an appropriate eluent.

The stationary phase comes in the form of a packed syringe-shaped cartridge that can be mounted on its specific type of extraction manifold. The manifold allows multiple samples to be processed by holding several SPE media in place and allowing for an equal number of samples to pass through them simultaneously. A typical cartridge SPE manifold can accommodate up to 24 cartridges and is equipped with a vacuum port. Application of vacuum speeds up the extraction process by pulling the liquid sample through the stationary phase. The analytes are collected in sample tubes inside or below the manifold after they pass through the stationary phase.

Evaluation of the Contamination by Herbicides in Olive Groves 219

In a GC analysis, a known volume of gaseous or liquid sample is injected into the head of the column using a micro syringe. As the carrier gas sweeps the analyte molecules through the column, this motion is inhibited by the adsorption of the analyte molecules either onto the column walls or onto packing materials in the column. The rate at which the molecules progress along the column depends on the strength of adsorption, which in turn depends on the type of molecule and on the stationary phase materials. Since each type of molecule has a different rate of progression, the various components of the analyte mixture are separated as they progress along the column and reach the end of the column at different times. A detector is used to monitor the outlet stream from the column; thus, the time at which each

component reaches the outlet and the amount of that component can be determined.

our research.

compounds.

Different detectors can be used in GC. The most common ones until recent years were the flame ionization detector (FID), the thermal conductivity detector (TCD) and the electron capture detector (ECD). However, nowadays most of the developed analytical methods use GC coupled to mass spectrometry (MS) detectors. In the analytical methods that will be described later, only ECD, thermoionic specific detector (TSD) and MS have been used in

ECD is used for detecting electron-absorbing compounds. It uses a radioactive beta particle (electron) emitter. The electrons are formed by collision with a nitrogen molecule because it exhibits low excitation energy. The electron is then attracted to a positively charged anode, generating a steady current. Therefore, there is always a background signal present in the chromatogram. As the sample is carried into the detector by the carrier gas, analyte molecules absorb the electrons and reduce the current between the collector anode and a cathode. The analyte concentration is thus proportional to the degree of electron capture. ECD is particularly sensitive to halogens, organometallic compounds, nitriles, or nitro

TSD is a very sensitive but specific detector that responds almost exclusively to nitrogen and phosphorous compounds. It contains a rubidium or cesium silicate (glass) bead situated in a heater coil, at little distance from the hydrogen flame. The heated bead emits electrons by thermionic emission. These electrons are collected under a potential of few volts by an appropriately placed anode, and provides a background current. When a solute containing nitrogen or phosphorous is eluted from the column, the partially combusted nitrogen and phosphorous materials are adsorbed on the surface of the bead. The adsorbed material reduces the work function of the surface and, as consequence, the emission of electrons is

Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles. It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules. The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios. In MS detection: 1) the analytes undergo vaporization; 2) they are ionized by one of a variety of methods (e.g., by impacting them with an electron beam), which results in the formation of charged particles (ions); 3) the ions are separated according to their mass-to-charge ratio in an analyzer by electromagnetic fields; 4) the ions are detected, usually by a quantitative

increased, raising the current collected at the electrode.

method; and 5) the ion signal is processed into mass spectra.

Solid phase extraction cartridges are available with a variety of stationary phases, each of which can separate analytes according to different chemical properties. Most stationary phases are based on silica that has been bonded to a specific functional group.

## **6. Detection techniques for the analysis of pesticides. Gas chromatography**

Pesticides (herbicides, fungicides or insecticides) are the most abundant environmental pollutants found in soil, water, atmosphere and agricultural products, and may exist in harmful levels and pose an environmental threat. Even low levels of these contaminants can cause adverse effects on humans, plants, animals and ecosystems. As it has been commented in the Introduction, there are several alternatives for the determination of pesticides in different kinds of samples. In this sense, automated systems of analysis with spectroscopic or electrochemical detection have been developed (Llorent-Martínez et al., 2011). In particular, the development of sensors have been specially useful. Electrochemical sensors present the advantages of miniaturization, simplicity, possibility of in-situ measurements and low-cost. In general, the main contribution of these sensors to pesticide analysis has consisted of the development of methods of analysis for determining a whole family of compounds (Du et al., 2009; Halámek et al., 2005; Liu & Lin, 2006). In spectroscopic methods, fluorescence (Calatayud et al., 2006; Mbaye et al., 2011) or chemiluminescence (Catalá-Icardo et al., 2011; López-Paz & Catalá-Icardo, 2011) detections have been usually employed due to their intrinsic sensitivity and selectivity. Among the spectroscopic methods of analysis, the design of flow-through optosensors has also been paid particular attention (Llorent-Martínez et al., 2011). These sensors present enhanced sensitivity and selectivity and have been applied to the determination of a small number of pesticides in different kind of samples (Llorent-Martínez et al., 2005; Llorent-Martínez et al., 2007; López Flores et al., 2007).

In general, electrochemical sensors can provide a useful tool when a whole family of compounds is targeted, while optosensors provide an interesting approach in order to quantify a small number of analytes in a particular sample. However, chromatographic techniques are still the chosen ones when a multi-residue analysis is required, being GC the most common one for the analysis of pesticides.

GC is an analytical technique that is used for separating and analysing compounds that can be vaporized without decomposition (Harris, 2007; Skoog et al., 1996). Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture. In GC, the mobile phase is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen. The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called column. A gas chromatograph uses a flow-through narrow tube known as the column, through which different chemical constituents of a sample pass in a gas stream (carrier gas, mobile phase) at different rates depending on their various chemical and physical properties and their interaction with a specific column filling, called the stationary phase. As the chemicals exit the end of the column, they are detected and electronically identified. The function of the stationary phase in the column is to separate different components, causing each one to exit the column at a different time (retention time). Other parameters that can be used to alter the order or time of retention are the carrier gas flow rate, column length and the temperature.

Solid phase extraction cartridges are available with a variety of stationary phases, each of which can separate analytes according to different chemical properties. Most stationary

**6. Detection techniques for the analysis of pesticides. Gas chromatography**  Pesticides (herbicides, fungicides or insecticides) are the most abundant environmental pollutants found in soil, water, atmosphere and agricultural products, and may exist in harmful levels and pose an environmental threat. Even low levels of these contaminants can cause adverse effects on humans, plants, animals and ecosystems. As it has been commented in the Introduction, there are several alternatives for the determination of pesticides in different kinds of samples. In this sense, automated systems of analysis with spectroscopic or electrochemical detection have been developed (Llorent-Martínez et al., 2011). In particular, the development of sensors have been specially useful. Electrochemical sensors present the advantages of miniaturization, simplicity, possibility of in-situ measurements and low-cost. In general, the main contribution of these sensors to pesticide analysis has consisted of the development of methods of analysis for determining a whole family of compounds (Du et al., 2009; Halámek et al., 2005; Liu & Lin, 2006). In spectroscopic methods, fluorescence (Calatayud et al., 2006; Mbaye et al., 2011) or chemiluminescence (Catalá-Icardo et al., 2011; López-Paz & Catalá-Icardo, 2011) detections have been usually employed due to their intrinsic sensitivity and selectivity. Among the spectroscopic methods of analysis, the design of flow-through optosensors has also been paid particular attention (Llorent-Martínez et al., 2011). These sensors present enhanced sensitivity and selectivity and have been applied to the determination of a small number of pesticides in different kind of samples (Llorent-Martínez et

In general, electrochemical sensors can provide a useful tool when a whole family of compounds is targeted, while optosensors provide an interesting approach in order to quantify a small number of analytes in a particular sample. However, chromatographic techniques are still the chosen ones when a multi-residue analysis is required, being GC the

GC is an analytical technique that is used for separating and analysing compounds that can be vaporized without decomposition (Harris, 2007; Skoog et al., 1996). Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture. In GC, the mobile phase is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen. The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called column. A gas chromatograph uses a flow-through narrow tube known as the column, through which different chemical constituents of a sample pass in a gas stream (carrier gas, mobile phase) at different rates depending on their various chemical and physical properties and their interaction with a specific column filling, called the stationary phase. As the chemicals exit the end of the column, they are detected and electronically identified. The function of the stationary phase in the column is to separate different components, causing each one to exit the column at a different time (retention time). Other parameters that can be used to alter the order or time of retention are the carrier gas flow rate, column length and the

phases are based on silica that has been bonded to a specific functional group.

al., 2005; Llorent-Martínez et al., 2007; López Flores et al., 2007).

most common one for the analysis of pesticides.

temperature.

In a GC analysis, a known volume of gaseous or liquid sample is injected into the head of the column using a micro syringe. As the carrier gas sweeps the analyte molecules through the column, this motion is inhibited by the adsorption of the analyte molecules either onto the column walls or onto packing materials in the column. The rate at which the molecules progress along the column depends on the strength of adsorption, which in turn depends on the type of molecule and on the stationary phase materials. Since each type of molecule has a different rate of progression, the various components of the analyte mixture are separated as they progress along the column and reach the end of the column at different times. A detector is used to monitor the outlet stream from the column; thus, the time at which each component reaches the outlet and the amount of that component can be determined.

Different detectors can be used in GC. The most common ones until recent years were the flame ionization detector (FID), the thermal conductivity detector (TCD) and the electron capture detector (ECD). However, nowadays most of the developed analytical methods use GC coupled to mass spectrometry (MS) detectors. In the analytical methods that will be described later, only ECD, thermoionic specific detector (TSD) and MS have been used in our research.

ECD is used for detecting electron-absorbing compounds. It uses a radioactive beta particle (electron) emitter. The electrons are formed by collision with a nitrogen molecule because it exhibits low excitation energy. The electron is then attracted to a positively charged anode, generating a steady current. Therefore, there is always a background signal present in the chromatogram. As the sample is carried into the detector by the carrier gas, analyte molecules absorb the electrons and reduce the current between the collector anode and a cathode. The analyte concentration is thus proportional to the degree of electron capture. ECD is particularly sensitive to halogens, organometallic compounds, nitriles, or nitro compounds.

TSD is a very sensitive but specific detector that responds almost exclusively to nitrogen and phosphorous compounds. It contains a rubidium or cesium silicate (glass) bead situated in a heater coil, at little distance from the hydrogen flame. The heated bead emits electrons by thermionic emission. These electrons are collected under a potential of few volts by an appropriately placed anode, and provides a background current. When a solute containing nitrogen or phosphorous is eluted from the column, the partially combusted nitrogen and phosphorous materials are adsorbed on the surface of the bead. The adsorbed material reduces the work function of the surface and, as consequence, the emission of electrons is increased, raising the current collected at the electrode.

Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles. It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules. The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios. In MS detection: 1) the analytes undergo vaporization; 2) they are ionized by one of a variety of methods (e.g., by impacting them with an electron beam), which results in the formation of charged particles (ions); 3) the ions are separated according to their mass-to-charge ratio in an analyzer by electromagnetic fields; 4) the ions are detected, usually by a quantitative method; and 5) the ion signal is processed into mass spectra.

Evaluation of the Contamination by Herbicides in Olive Groves 221

diuron levels were higher than the MRL established by the European Union (0.2 mg kg-1); however, none of the olive oil samples presented levels higher than the corresponding MRL (0.8 mg kg-1). Terbuthylazine was also quantified in many samples with values over the MRL in some of them, being the established MRLs 0.05 and 0.2 mg kg-1 for olives and olive oil, respectively. Although other herbicides were also detected, in most cases the levels were below the quantification limit of the system. In general, the levels of herbicides found in soil olives have been significantly higher than those ones found in flight olives, which have not been in contact with the soil. These results suggest that herbicide residues are mainly caused by the contamination of the olives when they come to contact with the soil after falling

**Olives Olive oil** 

**Linear Range (µg kg-1)** 

**DL (µg kg-1)** 

**DL (µg kg-1)** 

down (Guardia-Rubio et al., 2006b; Guardia-Rubio et al., 2006c).

**Linear Range (µg kg-1)** 

Simazine 11.971 0.75-125 0.25 3-2000 1

Atrazine 12.095 1.25-250 0.50 5-1000 2

Trietazine 12.621 2.50-200 1.25 10-800 5

Terbuthylazine 12.666 0.25-250 0.12 1-1000 0.5

Terbutrine 18.125 5.00-250 1.25 20-1000 5

Diuron 6.908 1.25-250 0.12 5-1000 0.5

Oxyfluorfen 25.918 2.50-250 1.25 10-1000 5

Table 2. Analytical parameters for olives and olive oil determination.

**Herbicide tR (min)** 
