The faster extraction process (5-10 min) is obtained by the use of high pressures (500-3000 psi) and temperatures (50-200 °C). This technique requires smaller amounts of solvent compared with traditional extraction, this is a very positive advantage because reduces the dilution of the sample [38]. The main factor that's affects de extraction time and efficiency of extraction is the temperature meanwhile the sample mass [12]. There are three ways to perform PLE: static, dynamic (flow-through) and mixed mode. In static mode, the sample is enclosed in a stainless steel vessel filled with an extraction solvent, and following extraction the remaining solvent is purged with N2 into a collection vial. Flow-through systems continuously pump solvent through the sample, but this has the disadvantage of using larger volumes of solvent and of diluting the extract. A desiccant, such as sodium sulphate, diatomaceous earth or cellulose can be added directly to the extraction cell or sorbent materials can be used to provide in situ cleanup [39, 40]. When compared with LLE, the PLE show as main advantage the low solvent consumption, but requires highly expensive and specialized equipment. The limitation of PLE is the presence of large amounts of coextracted lipids when working with fatty matrices, which means that post-cleanup of the

The effect of sample matrix depends on sample composition. Food samples can differ significantly in their physical-chemical properties, type of compounds present, or granulation (particle diameter). The factor that mainly affects the extraction of trace compounds in the presence of extractable major sample components are lipids, that offers special problems in the subsequent determination by gas or liquid chromatography [41].

Carabias-Martinez *et al*. [37] developed a method based on pressurized liquid extraction and liquid chromatography–electrospray ionization mass spectrometry (LC-ESI-MS) for the determination in cereal samples of seven endocrine-disrupting compounds: bisphenol A (BPA), 4-tert-butylbenzoic acid (BBA), 4-nonylphenol (NP), 4-tert-butylphenol (t-BP), 2,4-

samples, like cereals, forces its penetration into the pores of the sample [12].

QuEChERS in other food and environmental samples [7, 36].

**2.3. Pressurized-liquid extraction (PLE)**

extract is required to carry out lipid elimination [12].

Kolberg *et al*. [18] developed and validated a multiresidue approach also based on QuEChERS method for the determination of 24 pesticides in wheat, white flour and bran using triplequadrupole GC-MS in negative chemical ionization mode. The method was validated evaluating the following parameters: linearity, limit of detection, limit of quantification, matrix effect as well as precision and accuracy, evaluating the percentage of recovery at four different spike levels. The linear range used in the calibration curves was from 1.0 to 100 µg L-1 for wheat and 2.0 to 200 µg L-1 for flour and bran, both with values of r2≥ 0.99. The recoveries had been considered satisfactory presenting values between 70 and 120% with RSD < 20% for the majority of compounds.

Koesukwiwat *et al*. [26] modified the original QuEChERS method for the analysis of phenoxy acid herbicide residues in rice samples. The new approach was based on the extraction with 5% (v/v) formic acid in acetonitrile and inclusion of citrate buffer for helped partitioning of all the analytes into the acetonitrile phase. The extract was then cleaned up by d-SPE using C18 and alumina neutral as selective sorbents. Further optimization of sample preparation and determination allowed recoveries between 45 and 104% for all 13 phenoxy acid herbicides with RSD ≤ 13.3% at 5.0 µg kg-1 concentration level. Limit of detections (LODs) of 0.5 µg kg-1 or below were attained for all 13 phenoxy acids. Quantitative analysis was done by UHPLC-MS/MS in the multiple-reaction monitoring (MRM) mode using two combinations of selected precursor ion and product ion transition for each compound. This developed method when compared with original QuEChERS method produced relatively higher recoveries of the acid herbicides with a smaller range of variation and less susceptibility to matrix effects.

Otherwise, the main disadvantage of the QuEChERS method compared to other common methods is that the 1 g mL-1 final extract concentration is lower than the 2–5 g mL-1 concentrated extract of most traditional methods. If matrix is not the limiting source of noise in the analysis, this leads to a higher LOQ for the same injection volume in the QuEChERS method [3]. This method is adaptable and can be easily tailored to cope with new matrices through the selection of alternative sorbents. In fact the initial extract can be divided across tubes containing different sorbents to cater for problem analytes. Work in progress indicates that the developed extraction conditions will recover the majority of food contaminants [35]. Although QuEChERS has mainly been used for the determination of pesticides, some other compounds, such as pharmaceuticals, or veterinary drugs have been determined using QuEChERS in other food and environmental samples [7, 36].

#### **2.3. Pressurized-liquid extraction (PLE)**

138 Pesticides – Recent Trends in Pesticide Residue Assay

120% with RSD < 20% for the majority of compounds.

variation and less susceptibility to matrix effects.

In a new work Walorczyk [1] improved his previous method. The most important aspects considered during the present work were (i) extension of the scope of the previous method to include as many as 144 target analytes and (ii) re-design of the GC-MS/MS acquisition method. The linearity of the calibration curves was excellent in matrix-matched standards, and yielded the coefficients of determination r2 ≥ 0.99 for approximately 96% of the target analytes. Average recoveries of the pesticides spiked at 0.01 mg kg-1 into a feed mixture and wheat grain were in the range 70–120% with associated RSD values ≤ 20% for approximately 60 and 67% of the compounds, respectively. Base on these results, the proposed approach has been proven to be highly efficient and suitable for routine determinations of multiclass pesticides in a range of cereal and related matrices. In this study, 145 samples of matrices of differing complexity including cereals grain, bran, whole ears, straw, hay, feed mixtures and other samples such as malt, starch and dry vegetables have been analyzed. A total of 15 different compounds have been detected, among which pirimiphos methyl, deltamethrin, tolylfluanid, dichlofluanid and tebuconazole were the most frequently encountered ones.

Kolberg *et al*. [18] developed and validated a multiresidue approach also based on QuEChERS method for the determination of 24 pesticides in wheat, white flour and bran using triplequadrupole GC-MS in negative chemical ionization mode. The method was validated evaluating the following parameters: linearity, limit of detection, limit of quantification, matrix effect as well as precision and accuracy, evaluating the percentage of recovery at four different spike levels. The linear range used in the calibration curves was from 1.0 to 100 µg L-1 for wheat and 2.0 to 200 µg L-1 for flour and bran, both with values of r2≥ 0.99. The recoveries had been considered satisfactory presenting values between 70 and

Koesukwiwat *et al*. [26] modified the original QuEChERS method for the analysis of phenoxy acid herbicide residues in rice samples. The new approach was based on the extraction with 5% (v/v) formic acid in acetonitrile and inclusion of citrate buffer for helped partitioning of all the analytes into the acetonitrile phase. The extract was then cleaned up by d-SPE using C18 and alumina neutral as selective sorbents. Further optimization of sample preparation and determination allowed recoveries between 45 and 104% for all 13 phenoxy acid herbicides with RSD ≤ 13.3% at 5.0 µg kg-1 concentration level. Limit of detections (LODs) of 0.5 µg kg-1 or below were attained for all 13 phenoxy acids. Quantitative analysis was done by UHPLC-MS/MS in the multiple-reaction monitoring (MRM) mode using two combinations of selected precursor ion and product ion transition for each compound. This developed method when compared with original QuEChERS method produced relatively higher recoveries of the acid herbicides with a smaller range of

Otherwise, the main disadvantage of the QuEChERS method compared to other common methods is that the 1 g mL-1 final extract concentration is lower than the 2–5 g mL-1 concentrated extract of most traditional methods. If matrix is not the limiting source of noise in the analysis, this leads to a higher LOQ for the same injection volume in the QuEChERS method [3]. This method is adaptable and can be easily tailored to cope with new matrices Also known as accelerated solvent extraction (ASE), pressurized fluid extraction (PFE), pressurized hot solvent extraction (PHSE), subcritical solvent extraction (SSE) and hot water extraction (HWE), the PLE utilizes solvents that are raised to the near supercritical region, where they show better extraction properties [37]. This effect is due the decreasing of surface tension and increasing of solubility and diffusion rate into the sample showed by the solvent at high temperatures. Pressure keeps the solvent below its boiling point and, in solid samples, like cereals, forces its penetration into the pores of the sample [12].

The faster extraction process (5-10 min) is obtained by the use of high pressures (500-3000 psi) and temperatures (50-200 °C). This technique requires smaller amounts of solvent compared with traditional extraction, this is a very positive advantage because reduces the dilution of the sample [38]. The main factor that's affects de extraction time and efficiency of extraction is the temperature meanwhile the sample mass [12]. There are three ways to perform PLE: static, dynamic (flow-through) and mixed mode. In static mode, the sample is enclosed in a stainless steel vessel filled with an extraction solvent, and following extraction the remaining solvent is purged with N2 into a collection vial. Flow-through systems continuously pump solvent through the sample, but this has the disadvantage of using larger volumes of solvent and of diluting the extract. A desiccant, such as sodium sulphate, diatomaceous earth or cellulose can be added directly to the extraction cell or sorbent materials can be used to provide in situ cleanup [39, 40]. When compared with LLE, the PLE show as main advantage the low solvent consumption, but requires highly expensive and specialized equipment. The limitation of PLE is the presence of large amounts of coextracted lipids when working with fatty matrices, which means that post-cleanup of the extract is required to carry out lipid elimination [12].

The effect of sample matrix depends on sample composition. Food samples can differ significantly in their physical-chemical properties, type of compounds present, or granulation (particle diameter). The factor that mainly affects the extraction of trace compounds in the presence of extractable major sample components are lipids, that offers special problems in the subsequent determination by gas or liquid chromatography [41].

Carabias-Martinez *et al*. [37] developed a method based on pressurized liquid extraction and liquid chromatography–electrospray ionization mass spectrometry (LC-ESI-MS) for the determination in cereal samples of seven endocrine-disrupting compounds: bisphenol A (BPA), 4-tert-butylbenzoic acid (BBA), 4-nonylphenol (NP), 4-tert-butylphenol (t-BP), 2,4-

dichlorophenol (DCP), 2,4,5-trichlorophenol (TCP) and pentachlorophenol (PCP). Methanol was selected as the extraction solvent. The recoveries achieved for the all seven compounds were in the 81 to 104% range, with relative standard deviations of 4 to 9%. The detection limits achieved in corn breakfast cereals were in the 0.003 to 0.043 µg g-1 range.

An Overview About Recent Advances in Sample

Preparation Techniques for Pesticide Residues Analysis in Cereals and Feedstuffs 141

which allows the extraction of thermally labile or easily oxidized compounds. The main drawback of SCCO2 is its low polarity, and some modifiers as required to change the polarity of supercritical fluid and increase de solvent power. The modifiers can also reduce the

In a typical commercial equipment, the fluid is pumped, at a pressure above its critical point, with the sample placed in an inert extraction cell. The temperature of the cell is increased to overcome the critical point of the fluid. After depressurization, the analytes are collected in a small volume of organic solvent or on a solid-phase filled cartridge (solid adsorbent trap). Extraction can be performed in three ways: static, dynamic or recirculating (mixed) mode: in the static extraction mode, the cell containing the sample is filled with the supercritical fluid, pressurized and allowed to equilibrate; using the dynamic mode, the supercritical fluid is passed through the extraction cell continuously; finally in the recirculating mode the same fluid is repeatedly pumped through the sample and, after the

SFE is usually an efficient extraction method, primarily applicable to solid samples. However, as well as its numerous advantages (efficacy, selectivity, short extraction times, low solvent volumes) it also has serious drawbacks: difficult optimisation, high apparatus and maintenance cost, high blank and noise levels, co-extraction of lipidic content of samples [48]. With commercially equipments available SFE has been used not only for analytical applications in samples preparation, but mostly for food science, pharmaceutical and environmental science in lab or pilot scale [45]. Recently, only a few applications describing

Aguilera et al. [49] used the SFE method followed by a cleanup step with aminopropyl SPE for the analysis of 22 pesticides in white and wild rice. The authors tested different experimental conditions for extraction with CO2: temperature, volume and pressure in order to optimize the extraction method. The authors noticed that fat extraction from rice was appreciable and the use of higher pressures might also increase extraction of fat and other non-fat material in this matrix. For the final procedure validated pesticide mean recoveries obtained from rice samples, at fortification levels around 0.5 mg kg-1, ranged between 74 and 98%, except for captafol and dimethoate for which mean recoveries lower

Solid-phase based extraction techniques are widely applied to many matrices, like foods and include: matrix solid-phase dispersion (MSPD), solid-phase extraction (SPE), solid-phase microextraction (SPME) and stir-bar sorptive extraction (SBSE). In this technique a sorbent will retain and concentrate some target analytes from the sample solution due strong affinity between sorbent and analytes [12]. Solid-phase extraction involves the use of disposable cartridges and disks to trap analytes. As the sample solution passes through the activated sorbent bed, analytes concentrate on its surface, while the other sample

analyte–matrix interactions improving their quantitative extraction [45].

required number of cycles, it is pumped out to the collection system [47].

sample preparation for determination of pesticides in cereals were published.

than 21% were determined.

**2.5. Solid-phase extraction (SPE)**

A method based on pressurized liquid extraction and LC-MS/MS has been developed for determining nine benzoylureas in fruit, vegetable, cereals and animal products. Samples (5 g) were homogenized with diatomaceous earth and extracted in a 22 mL cell with 22 mL of ethyl acetate at 80 °C and 1500 psi. Method LOQs were between 0.002 and 0.01 mg kg-1. Excellent linearity was achieved over a range of concentrations from 0.01 to 1 mg kg-1. Validation of the method was performed in seven different commodities (milk, eggs, meat, rice, lettuce, avocado, and lemon). The recoveries ranged from 58 to 97% and the RSDs from 5 to 19% [42].

In order to extract eight acetanilide herbicides from cereal crops, Zhang *et al*. [43] evaluated the effect of four parameters for PLE efficiency (temperature, static time, static cycles and solvent). The results of final method, based on accelerated solvent extraction (ASE) and solid-phase extraction (SPE) for cleanup using graphitized carbon black/primary secondary amine (GCB/PSA), GCB, Florisil and alumina-N were compared with shake-ask extraction method. At 0.05 mg kg-1 spiked level, recoveries and precision values for rice, wheat and maize were from 82.3 to 115.8% and from 1.1 to 13.6%, respectively. For all the herbicides, LOD and LOQ ranged from 0.8 to 1.7 µg kg-1 and from 2.4 to 5.3 µg kg-1, respectively.

#### **2.4. Supercritical-fluid extraction (SFE)**

From 1960, when applications of supercritical fluid extraction have been extensively examined until 90 years, few researchers investigated the use of supercritical fluids in analytical, non-chromatographic applications. After that, commercial supercritical fluid extraction (SFE) instruments have been developed and the studies began to be published using SFE as extraction technique in combination with chromatographic techniques for analytical applications for analysis of pesticide residues [44, 45].

The unique properties exhibited by supercritical fluids have been applied for the analysis of pesticide residues in solid samples SFE is selective and Less solvent consuming, thus it is environmental friendly. The critical step in the off-line SFE methods is evaporation of solvent at the end of extraction to acquire high pre-concentration factor. This procedure is time-consuming and can contaminate the environment. Another negative factor is loses or degradation of collected analytes [46].

Carbon dioxide (critical conditions = 30.9 ºC and 73.8 bar) is the most used supercritical solvent, because is cheap, environmentally friendly and safe. Supercritical CO2 (SCCO2) had high diffusivity and easily tuneable solvent strength. Another advantage is that CO2 is gaseous at room temperature and pressure, which makes analyte recovery very simple and provides solvent-free analytes. Also, important for food and natural products sample preparation, is the ability of SFE using CO2 to be operated at low temperatures using a non-oxidant medium, which allows the extraction of thermally labile or easily oxidized compounds. The main drawback of SCCO2 is its low polarity, and some modifiers as required to change the polarity of supercritical fluid and increase de solvent power. The modifiers can also reduce the analyte–matrix interactions improving their quantitative extraction [45].

In a typical commercial equipment, the fluid is pumped, at a pressure above its critical point, with the sample placed in an inert extraction cell. The temperature of the cell is increased to overcome the critical point of the fluid. After depressurization, the analytes are collected in a small volume of organic solvent or on a solid-phase filled cartridge (solid adsorbent trap). Extraction can be performed in three ways: static, dynamic or recirculating (mixed) mode: in the static extraction mode, the cell containing the sample is filled with the supercritical fluid, pressurized and allowed to equilibrate; using the dynamic mode, the supercritical fluid is passed through the extraction cell continuously; finally in the recirculating mode the same fluid is repeatedly pumped through the sample and, after the required number of cycles, it is pumped out to the collection system [47].

SFE is usually an efficient extraction method, primarily applicable to solid samples. However, as well as its numerous advantages (efficacy, selectivity, short extraction times, low solvent volumes) it also has serious drawbacks: difficult optimisation, high apparatus and maintenance cost, high blank and noise levels, co-extraction of lipidic content of samples [48].

With commercially equipments available SFE has been used not only for analytical applications in samples preparation, but mostly for food science, pharmaceutical and environmental science in lab or pilot scale [45]. Recently, only a few applications describing sample preparation for determination of pesticides in cereals were published.

Aguilera et al. [49] used the SFE method followed by a cleanup step with aminopropyl SPE for the analysis of 22 pesticides in white and wild rice. The authors tested different experimental conditions for extraction with CO2: temperature, volume and pressure in order to optimize the extraction method. The authors noticed that fat extraction from rice was appreciable and the use of higher pressures might also increase extraction of fat and other non-fat material in this matrix. For the final procedure validated pesticide mean recoveries obtained from rice samples, at fortification levels around 0.5 mg kg-1, ranged between 74 and 98%, except for captafol and dimethoate for which mean recoveries lower than 21% were determined.

#### **2.5. Solid-phase extraction (SPE)**

140 Pesticides – Recent Trends in Pesticide Residue Assay

**2.4. Supercritical-fluid extraction (SFE)**

degradation of collected analytes [46].

analytical applications for analysis of pesticide residues [44, 45].

5 to 19% [42].

dichlorophenol (DCP), 2,4,5-trichlorophenol (TCP) and pentachlorophenol (PCP). Methanol was selected as the extraction solvent. The recoveries achieved for the all seven compounds were in the 81 to 104% range, with relative standard deviations of 4 to 9%. The detection

A method based on pressurized liquid extraction and LC-MS/MS has been developed for determining nine benzoylureas in fruit, vegetable, cereals and animal products. Samples (5 g) were homogenized with diatomaceous earth and extracted in a 22 mL cell with 22 mL of ethyl acetate at 80 °C and 1500 psi. Method LOQs were between 0.002 and 0.01 mg kg-1. Excellent linearity was achieved over a range of concentrations from 0.01 to 1 mg kg-1. Validation of the method was performed in seven different commodities (milk, eggs, meat, rice, lettuce, avocado, and lemon). The recoveries ranged from 58 to 97% and the RSDs from

In order to extract eight acetanilide herbicides from cereal crops, Zhang *et al*. [43] evaluated the effect of four parameters for PLE efficiency (temperature, static time, static cycles and solvent). The results of final method, based on accelerated solvent extraction (ASE) and solid-phase extraction (SPE) for cleanup using graphitized carbon black/primary secondary amine (GCB/PSA), GCB, Florisil and alumina-N were compared with shake-ask extraction method. At 0.05 mg kg-1 spiked level, recoveries and precision values for rice, wheat and maize were from 82.3 to 115.8% and from 1.1 to 13.6%, respectively. For all the herbicides,

LOD and LOQ ranged from 0.8 to 1.7 µg kg-1 and from 2.4 to 5.3 µg kg-1, respectively.

From 1960, when applications of supercritical fluid extraction have been extensively examined until 90 years, few researchers investigated the use of supercritical fluids in analytical, non-chromatographic applications. After that, commercial supercritical fluid extraction (SFE) instruments have been developed and the studies began to be published using SFE as extraction technique in combination with chromatographic techniques for

The unique properties exhibited by supercritical fluids have been applied for the analysis of pesticide residues in solid samples SFE is selective and Less solvent consuming, thus it is environmental friendly. The critical step in the off-line SFE methods is evaporation of solvent at the end of extraction to acquire high pre-concentration factor. This procedure is time-consuming and can contaminate the environment. Another negative factor is loses or

Carbon dioxide (critical conditions = 30.9 ºC and 73.8 bar) is the most used supercritical solvent, because is cheap, environmentally friendly and safe. Supercritical CO2 (SCCO2) had high diffusivity and easily tuneable solvent strength. Another advantage is that CO2 is gaseous at room temperature and pressure, which makes analyte recovery very simple and provides solvent-free analytes. Also, important for food and natural products sample preparation, is the ability of SFE using CO2 to be operated at low temperatures using a non-oxidant medium,

limits achieved in corn breakfast cereals were in the 0.003 to 0.043 µg g-1 range.

Solid-phase based extraction techniques are widely applied to many matrices, like foods and include: matrix solid-phase dispersion (MSPD), solid-phase extraction (SPE), solid-phase microextraction (SPME) and stir-bar sorptive extraction (SBSE). In this technique a sorbent will retain and concentrate some target analytes from the sample solution due strong affinity between sorbent and analytes [12]. Solid-phase extraction involves the use of disposable cartridges and disks to trap analytes. As the sample solution passes through the activated sorbent bed, analytes concentrate on its surface, while the other sample

components pass through the bed or vice versa, if necessary for cleanup [50]. When compared with LLE, the advantages of SPE are: simultaneous removal of interfering substances and concentrations of analytes, multiple samples can be treated in parallel and the use of a relatively small quantity of solvent [12]. Before SPE can be applied to a solid matrix (soil, vegetables and fruits), a separate homogenization step and, often, filtration, sonication, centrifugation, and liquid/liquid cleanup are required. However, the presence of interfering substances, such as salts, humic acids, and other humic substances in water or proteins, lipids and carbohydrates in food makes the determination of polar or early-eluted pesticides, difficult or almost impossible [51].

An Overview About Recent Advances in Sample

Preparation Techniques for Pesticide Residues Analysis in Cereals and Feedstuffs 143

the matrix and extract the analytes, but also has some functionality which can retain the fat/lipids [56]. MSPD can combine the procedures of homogenization, disruption, extraction and cleanup into one simple process. In fact, it is a sample preparation strategy that consists of a manual blending of samples with a bulk dispersing agent, to produce complete disruption of the original matrix structure, thus providing an enhanced surface area for subsequent sample extraction. Usually, the blended material is then transferred and packed into a column to perform sequential extraction and eventual cleanup with an appropriate

Most applications have utilized 5–10 mL of solvent to perform analyte extraction. Evidence from some studies indicates that most target analytes are eluted in the first 4 mL of extractant, provided that 0.5 g of the sample is mixed with 2 g of the solid support [9]. In general, the choice of the adsorbent used depends on the polarity of the analyte and on the possible coextracted components of the matrix. The most usual adsorbents are Florisil and C18; nevertheless, there are others (e.g., diatomaceous earth, alumina, silica and C8) that

The main advantages of MSPD procedure compared to other extraction techniques are as follows: (1) the analytical protocol is simplified and shortened; (2) the possibility of emulsion formation is eliminated; (3) solvent consumption is substantially reduced; and (4) the extraction efficiency of the analytes is enhanced as the entire sample is exposed to the extractant. An interesting feature of the MSPD technique is that it can be used for extracting analytes from both solid and liquid foods. The main disadvantage is the lack of automation

Tsochatzis *et al*. [58] development and validation of such a method for the determination of eight rice pesticides penoxsulam, tricyclazole, propanil, azoxystrobin, molinate, profoxydim, cyhalofop-butyl and deltamethrin) and 3,4-dichloroaniline, the main metabolite of propanil. Pesticide extraction and cleanup was performed by an optimized MSPD protocol on neutral alumina (5 g) using acetonitrile as the elution solvent. Samples were analyzed by highperformance liquid chromatography with diode array detection (HPLC-DAD). Method validation was performed by means of linearity, intra-day accuracy, inter-day precision and sensitivity. Linear regression coefficients (r2) were always above 0.9948. Limits of detection (LOD) and quantification (LOQ) varied from 0.002 to 0.200 mg kg−1 and 0.006 to 0.600 mg kg−1, respectively. Recoveries were investigated at three fortification levels and were found to be acceptable (74–127%) with relative standard deviations (RSD) below 12%. Application of the method for the analysis of five commercial rice grain samples showed that the

Michel and Buszewski [58] proposed MSPD for extraction of carbendazim residue from wheat grain and determination by column-switching HPLC-DAD. For extraction, a representative portion (200 g) of sample grain was transferred into a mill, and sample material disintegrated by high speed blending. A subsample of 5 g was weighed into a mortar of ca. 10 cm of diameter, 10 g of acidic silica gel was added and ground to obtain a homogeneous mixture. The extraction column was fitted with polyethylene frit, the

solvent or a sequence of solvents [57].

of the procedure [9].

pesticide levels were below the LOD.

have been used for the analysis of pesticides in food [11].

An analytical method for the determination imazaquin residues in soybeans was proposed by Guo *et al*. [52] based on liquid/liquid partition strong anion exchange solid-phase extraction. This technique was used, in order to achieve an effective cleanup, removing the greatest number of sample matrix interferences. In this procedure, the combination between optimized chromatographic conditions and detection by ultraviolet the procedure was showed to be sensitive and reliable for determining the imazaquin residues in soybean samples. This method is characterized by recovery >88.4%, precision 6.7% RSD, and sensitivity of 0.005 mg kg-1. The proposed method was successfully applied to the analysis of imazaquin residues in soybean samples grown in an experimental eld after treatments of imazaquin formulation.

A sensitive and simple method for simultaneous analysis of acetochlor and propisochlor in corn and soil has been developed by Hu *et al*. [53]. The extraction of pesticides from soil and corn matrices was performed with methanol/water and acetone, respectively, followed by solid phase extraction (SPE) to remove co-extractives, prior to analysis by gas chromatography with electron capture detection (GC-ECD). Primary secondary amine (PSA) SPE cartridges (500 mg, 3 mL) were used for sample preparation. The elution was made with 5 mL petroleum ether-acetic ether (95/5, v/v) and 3 mL petroleum ether-acetic ether (95/5, v/v), respectively. The recoveries of two pesticides ranged from 73.8% to 115.5% with relative standard deviations (RSD) less than 11.1% and sensitivity of 0.01 mg kg-1. The method was successfully applied to determine acetochlor and propisochlor in real corn and soil samples. The authors related that residues of acetochlor and propisochlor residues were detected (<0.01 mg kg-1) in corn at harvest time with holding period of 2.5 months after treatments of the pesticides.

The use of SPE in combination with HPLC-DAD was employed to determine bispyribacsodium residues in rice. The liquid–liquid partition and anion exchange solid phase procedures that were developed provide effective extraction and cleanup methods for analysis feasibility, with recoveries between 83.98 to 98.51% with a RSD from 0.56 to 6.36% and sensitivity of 0.01 mg kg-1, with main advantages of high precision, accuracy and good selectivity. Another favorable feature is the reduction of sample processing time [54].

#### **2.6. Matrix solid-phase dispersion (MSPD)**

In 1989, Barker *et al*. [55] introduced the MSPD, as a sample preparation technique for solid or semi-solid samples. It provides both a porous structure to enable the solvent to penetrate the matrix and extract the analytes, but also has some functionality which can retain the fat/lipids [56]. MSPD can combine the procedures of homogenization, disruption, extraction and cleanup into one simple process. In fact, it is a sample preparation strategy that consists of a manual blending of samples with a bulk dispersing agent, to produce complete disruption of the original matrix structure, thus providing an enhanced surface area for subsequent sample extraction. Usually, the blended material is then transferred and packed into a column to perform sequential extraction and eventual cleanup with an appropriate solvent or a sequence of solvents [57].

142 Pesticides – Recent Trends in Pesticide Residue Assay

pesticides, difficult or almost impossible [51].

treatments of the pesticides.

components pass through the bed or vice versa, if necessary for cleanup [50]. When compared with LLE, the advantages of SPE are: simultaneous removal of interfering substances and concentrations of analytes, multiple samples can be treated in parallel and the use of a relatively small quantity of solvent [12]. Before SPE can be applied to a solid matrix (soil, vegetables and fruits), a separate homogenization step and, often, filtration, sonication, centrifugation, and liquid/liquid cleanup are required. However, the presence of interfering substances, such as salts, humic acids, and other humic substances in water or proteins, lipids and carbohydrates in food makes the determination of polar or early-eluted

An analytical method for the determination imazaquin residues in soybeans was proposed by Guo *et al*. [52] based on liquid/liquid partition strong anion exchange solid-phase extraction. This technique was used, in order to achieve an effective cleanup, removing the greatest number of sample matrix interferences. In this procedure, the combination between optimized chromatographic conditions and detection by ultraviolet the procedure was showed to be sensitive and reliable for determining the imazaquin residues in soybean samples. This method is characterized by recovery >88.4%, precision 6.7% RSD, and sensitivity of 0.005 mg kg-1. The proposed method was successfully applied to the analysis of imazaquin residues in soybean samples grown in an experimental eld after treatments of imazaquin formulation.

A sensitive and simple method for simultaneous analysis of acetochlor and propisochlor in corn and soil has been developed by Hu *et al*. [53]. The extraction of pesticides from soil and corn matrices was performed with methanol/water and acetone, respectively, followed by solid phase extraction (SPE) to remove co-extractives, prior to analysis by gas chromatography with electron capture detection (GC-ECD). Primary secondary amine (PSA) SPE cartridges (500 mg, 3 mL) were used for sample preparation. The elution was made with 5 mL petroleum ether-acetic ether (95/5, v/v) and 3 mL petroleum ether-acetic ether (95/5, v/v), respectively. The recoveries of two pesticides ranged from 73.8% to 115.5% with relative standard deviations (RSD) less than 11.1% and sensitivity of 0.01 mg kg-1. The method was successfully applied to determine acetochlor and propisochlor in real corn and soil samples. The authors related that residues of acetochlor and propisochlor residues were detected (<0.01 mg kg-1) in corn at harvest time with holding period of 2.5 months after

The use of SPE in combination with HPLC-DAD was employed to determine bispyribacsodium residues in rice. The liquid–liquid partition and anion exchange solid phase procedures that were developed provide effective extraction and cleanup methods for analysis feasibility, with recoveries between 83.98 to 98.51% with a RSD from 0.56 to 6.36% and sensitivity of 0.01 mg kg-1, with main advantages of high precision, accuracy and good selectivity. Another

In 1989, Barker *et al*. [55] introduced the MSPD, as a sample preparation technique for solid or semi-solid samples. It provides both a porous structure to enable the solvent to penetrate

favorable feature is the reduction of sample processing time [54].

**2.6. Matrix solid-phase dispersion (MSPD)**

Most applications have utilized 5–10 mL of solvent to perform analyte extraction. Evidence from some studies indicates that most target analytes are eluted in the first 4 mL of extractant, provided that 0.5 g of the sample is mixed with 2 g of the solid support [9]. In general, the choice of the adsorbent used depends on the polarity of the analyte and on the possible coextracted components of the matrix. The most usual adsorbents are Florisil and C18; nevertheless, there are others (e.g., diatomaceous earth, alumina, silica and C8) that have been used for the analysis of pesticides in food [11].

The main advantages of MSPD procedure compared to other extraction techniques are as follows: (1) the analytical protocol is simplified and shortened; (2) the possibility of emulsion formation is eliminated; (3) solvent consumption is substantially reduced; and (4) the extraction efficiency of the analytes is enhanced as the entire sample is exposed to the extractant. An interesting feature of the MSPD technique is that it can be used for extracting analytes from both solid and liquid foods. The main disadvantage is the lack of automation of the procedure [9].

Tsochatzis *et al*. [58] development and validation of such a method for the determination of eight rice pesticides penoxsulam, tricyclazole, propanil, azoxystrobin, molinate, profoxydim, cyhalofop-butyl and deltamethrin) and 3,4-dichloroaniline, the main metabolite of propanil. Pesticide extraction and cleanup was performed by an optimized MSPD protocol on neutral alumina (5 g) using acetonitrile as the elution solvent. Samples were analyzed by highperformance liquid chromatography with diode array detection (HPLC-DAD). Method validation was performed by means of linearity, intra-day accuracy, inter-day precision and sensitivity. Linear regression coefficients (r2) were always above 0.9948. Limits of detection (LOD) and quantification (LOQ) varied from 0.002 to 0.200 mg kg−1 and 0.006 to 0.600 mg kg−1, respectively. Recoveries were investigated at three fortification levels and were found to be acceptable (74–127%) with relative standard deviations (RSD) below 12%. Application of the method for the analysis of five commercial rice grain samples showed that the pesticide levels were below the LOD.

Michel and Buszewski [58] proposed MSPD for extraction of carbendazim residue from wheat grain and determination by column-switching HPLC-DAD. For extraction, a representative portion (200 g) of sample grain was transferred into a mill, and sample material disintegrated by high speed blending. A subsample of 5 g was weighed into a mortar of ca. 10 cm of diameter, 10 g of acidic silica gel was added and ground to obtain a homogeneous mixture. The extraction column was fitted with polyethylene frit, the powdery sample was transferred through a wide mouth polypropylene funnel (10 cm top i.d.). Mortar and pestle were rinsed with 20 mL of methanol-dichloromethane (1:5, v/v), and the rinsings were carefully poured into the column. The carbendazim residues were extracted with total volume of 120 mL eluent and collected in round-bottomed flasks. The mean recovery rate for fortified samples was 87.3% with a RSD of 2.9%. The achieved method LOQ was 0.04 µg g-1. The method was applied to the determination of carbendazim residues in wheat grain samples from a treated field.

An Overview About Recent Advances in Sample

Preparation Techniques for Pesticide Residues Analysis in Cereals and Feedstuffs 145

acetate extraction, GPC and d-SPE cleanup steps, additionally determination by comprehensive two-dimensional gas chromatography (GC×GC) with TOF-MS detection in the full scan mode. The method was demonstrated to give recoveries between 70 and 110% and RSDs below 20% at two levels of 0.01 and 0.1 mg kg-1 for the majority of the studied compounds. Zhang *et al*. [63] used GPC for the determination of pesticides in unpolished rice. GPC provides ideal cleanup for high molecular weight components. Otherwise, low molecular weight components may result in serious interferences that can

GPC is highly effective as a post-extraction cleanup procedure in removing interferences with high molecular weight (e.g. lipids, proteins and pigments) prior to the analysis by GC or LC. GPC also increases analytical precision and accuracy. It also ex-tends the column life and lifetime of the instrument. This technique can provide very good results. However, GPC, use large columns, need long analysis times and large amounts of solvents. Some pesticides with high molecular mass (e.g. pyrethroids) need not be sufficiently separated from wide elution band of co-extractants that can result in lower

The two main analytical techniques used in food analysis are gas chromatography (GC) and liquid chromatography (LC), which allow identification and quantitation of pesticide

GC is combined with different kinds of detection methods, mainly depending on the class of pesticides to be quantified [48]. Electron capture detection (ECD), nitrogen-phosphorus detection (NPD), flame-ionization detection (FID), flame-photometric detection (FPD) and mass-selective detection (MSD) have been employed for pesticide residue determination in cereal samples. GC-MS is also frequently used for determination pesticide residues in cereal samples and can be done by electron impact (EI), and Positive or negative chemical ionization (PCI, NCI). The Table 2 summarizes details of several methods developed for the

LC has been used for the analysis of polar and/or non-volatile and/or thermally labile pesticides for which GC conditions were not suitable [65]. Most used detectors are UV, diode array detector (DAD) and MSD, as we can see the Table 2. Traditional UV detectors and DADs are often less selective and less sensitive than GC instruments but, in recent years, the commercial availability of atmospheric pressure ionization (API), in combination with MS instruments, has increased the sensitivity by several orders of magnitude [66]. LC-MS/MS applications reported for the analysis of pesticides residues in cereal samples are performed with two different ionization techniques: electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). Although ESI is the more often used

**3. Instrumental identification and quantification techniques**

determination of pesticide and others analytes in different cereal samples.

affect the final result.

recoveries [64].

residues in complex matrices.

when compared with APCI source.

MSPD method have been published also for determination of other residues and contaminants e.g. mycotoxins in cereal samples. Rubert *et al*. [60] reported a reliable and rapid method for the determination of 14 mycotoxins in flour samples (with different cereals composition). MSPD was performed weighing 200 g which were prepared using a food processor and mixed thoroughly. Portions of 1 g were weighed and placed into a glass mortar (50 mL) and were gently blended with 1 g of C18 for 5 min using a pestle, to obtain homogeneous mixture. The homogeneous mixture was introduced into a 100×9 mm i.d. glass column, and eluted dropwise with 20 ml of acetonitrile/methanol (50/50, v/v) 1 mM ammonium formiate by applying a slight vacuum. In a total of 49 samples investigated, 9 mycotoxins were identified. Nivalenol and beauvericin were the mycotoxins found most frequently in the studied samples. The samples that presented major contamination were wheat flours and bakery preparations.

Juan *et al*. [61] determinate ochratoxin A (OTA) in organic and non-organic cereals and cereal products from Spain and Portugal. A method based on extraction with matrix solid phase dispersion (MSPD) using octylsilica (C8) followed by liquid chromatography coupled with fluorescence detection (LC–FD) was used to determine OTA from the selected samples. Recoveries of OTA from the studied samples spiked at 10 ng g-1 level ranged from 78 to 89% with a RSD of 3.7%. The limits of detection and quantification of this method were 0.05 and 0.19 ng g-1, respectively. Furthermore, LC–FD after OTA methylation was used to confirm the identity of OTA in all positive samples. This procedure was applied to 83 organic and non-organic samples including rice, wheat, barley, rye, oats and maize from Spain and Portugal.

#### **2.7. Gel-permeation chromatography (GPC)**

In the last years polystyrene divinylbenzene type gel with mixture of elution solvents as ethyl acetate and cyclohexane has been used in pesticide residue analysis of cereals samples taking advantage of GPC to isolate pesticides from co-extractants with higher molecular weight (600–1500 g mol-1). GPC is able to separate the pesticides from lipids, waxes and other low volatile larger non-polar co-extractives. Generally, in pesticide residue analysis in fatty food, addition of sulfuric acid or additional SPE cleaning (avoiding decomposition of several pesticides occurring in the former case) are desirable to offer satisfactory protection for the gas chromatographic capillary column [3].

Regarding analysis of pesticide residues in cereals and feedstuffs, Lee *et al*. [62] developed a method for the analysis of 106 compounds in cereal-based animal feed applying ethyl acetate extraction, GPC and d-SPE cleanup steps, additionally determination by comprehensive two-dimensional gas chromatography (GC×GC) with TOF-MS detection in the full scan mode. The method was demonstrated to give recoveries between 70 and 110% and RSDs below 20% at two levels of 0.01 and 0.1 mg kg-1 for the majority of the studied compounds. Zhang *et al*. [63] used GPC for the determination of pesticides in unpolished rice. GPC provides ideal cleanup for high molecular weight components. Otherwise, low molecular weight components may result in serious interferences that can affect the final result.

144 Pesticides – Recent Trends in Pesticide Residue Assay

wheat flours and bakery preparations.

**2.7. Gel-permeation chromatography (GPC)**

for the gas chromatographic capillary column [3].

Portugal.

residues in wheat grain samples from a treated field.

powdery sample was transferred through a wide mouth polypropylene funnel (10 cm top i.d.). Mortar and pestle were rinsed with 20 mL of methanol-dichloromethane (1:5, v/v), and the rinsings were carefully poured into the column. The carbendazim residues were extracted with total volume of 120 mL eluent and collected in round-bottomed flasks. The mean recovery rate for fortified samples was 87.3% with a RSD of 2.9%. The achieved method LOQ was 0.04 µg g-1. The method was applied to the determination of carbendazim

MSPD method have been published also for determination of other residues and contaminants e.g. mycotoxins in cereal samples. Rubert *et al*. [60] reported a reliable and rapid method for the determination of 14 mycotoxins in flour samples (with different cereals composition). MSPD was performed weighing 200 g which were prepared using a food processor and mixed thoroughly. Portions of 1 g were weighed and placed into a glass mortar (50 mL) and were gently blended with 1 g of C18 for 5 min using a pestle, to obtain homogeneous mixture. The homogeneous mixture was introduced into a 100×9 mm i.d. glass column, and eluted dropwise with 20 ml of acetonitrile/methanol (50/50, v/v) 1 mM ammonium formiate by applying a slight vacuum. In a total of 49 samples investigated, 9 mycotoxins were identified. Nivalenol and beauvericin were the mycotoxins found most frequently in the studied samples. The samples that presented major contamination were

Juan *et al*. [61] determinate ochratoxin A (OTA) in organic and non-organic cereals and cereal products from Spain and Portugal. A method based on extraction with matrix solid phase dispersion (MSPD) using octylsilica (C8) followed by liquid chromatography coupled with fluorescence detection (LC–FD) was used to determine OTA from the selected samples. Recoveries of OTA from the studied samples spiked at 10 ng g-1 level ranged from 78 to 89% with a RSD of 3.7%. The limits of detection and quantification of this method were 0.05 and 0.19 ng g-1, respectively. Furthermore, LC–FD after OTA methylation was used to confirm the identity of OTA in all positive samples. This procedure was applied to 83 organic and non-organic samples including rice, wheat, barley, rye, oats and maize from Spain and

In the last years polystyrene divinylbenzene type gel with mixture of elution solvents as ethyl acetate and cyclohexane has been used in pesticide residue analysis of cereals samples taking advantage of GPC to isolate pesticides from co-extractants with higher molecular weight (600–1500 g mol-1). GPC is able to separate the pesticides from lipids, waxes and other low volatile larger non-polar co-extractives. Generally, in pesticide residue analysis in fatty food, addition of sulfuric acid or additional SPE cleaning (avoiding decomposition of several pesticides occurring in the former case) are desirable to offer satisfactory protection

Regarding analysis of pesticide residues in cereals and feedstuffs, Lee *et al*. [62] developed a method for the analysis of 106 compounds in cereal-based animal feed applying ethyl GPC is highly effective as a post-extraction cleanup procedure in removing interferences with high molecular weight (e.g. lipids, proteins and pigments) prior to the analysis by GC or LC. GPC also increases analytical precision and accuracy. It also ex-tends the column life and lifetime of the instrument. This technique can provide very good results. However, GPC, use large columns, need long analysis times and large amounts of solvents. Some pesticides with high molecular mass (e.g. pyrethroids) need not be sufficiently separated from wide elution band of co-extractants that can result in lower recoveries [64].

## **3. Instrumental identification and quantification techniques**

The two main analytical techniques used in food analysis are gas chromatography (GC) and liquid chromatography (LC), which allow identification and quantitation of pesticide residues in complex matrices.

GC is combined with different kinds of detection methods, mainly depending on the class of pesticides to be quantified [48]. Electron capture detection (ECD), nitrogen-phosphorus detection (NPD), flame-ionization detection (FID), flame-photometric detection (FPD) and mass-selective detection (MSD) have been employed for pesticide residue determination in cereal samples. GC-MS is also frequently used for determination pesticide residues in cereal samples and can be done by electron impact (EI), and Positive or negative chemical ionization (PCI, NCI). The Table 2 summarizes details of several methods developed for the determination of pesticide and others analytes in different cereal samples.

LC has been used for the analysis of polar and/or non-volatile and/or thermally labile pesticides for which GC conditions were not suitable [65]. Most used detectors are UV, diode array detector (DAD) and MSD, as we can see the Table 2. Traditional UV detectors and DADs are often less selective and less sensitive than GC instruments but, in recent years, the commercial availability of atmospheric pressure ionization (API), in combination with MS instruments, has increased the sensitivity by several orders of magnitude [66]. LC-MS/MS applications reported for the analysis of pesticides residues in cereal samples are performed with two different ionization techniques: electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). Although ESI is the more often used when compared with APCI source.

An Overview About Recent Advances in Sample

Preparation Techniques for Pesticide Residues Analysis in Cereals and Feedstuffs 147

must be similar to common analytical techniques, in order to minimize errors. For these reasons, modern trends in analytical chemistry are towards the simplification and miniaturization of sample preparation, and the minimization of sample size and organic solvent used. The development of such procedures combined with modern chromatographic-mass spectrometric techniques will enable analysis at the low levels now required by legislation for many pesticides, but more importantly, result in methods which produce more reliable data to support food safety monitoring programs. This is the trend although it is impossible to develop a unique protocol covering such a wide range of compounds. Finally, multiclass multiresidue methods covering large number of pesticides are highly desirable, although the different nature, classes and physico-chemical properties

of pesticides hamper the development of such methods.

Manoel Leonardo Martins and Martha Bohrer Adaime

*Universidade Federal de Santa Maria, Santa Maria - RS, Brazil*

Renato Zanella, Osmar Damian Prestes, Caroline do Amaral Friggi,

*Laboratório de Análises de Resíduos de Pesticidas (LARP), Departamento de Química,*

do Sul (FAPERGS, Porto Alegre, Brazil) for financial support and fellowships.

The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, Brazil) and the Fundação de Amparo a Pesquisa do Estado do Rio Grande

[1] Walorczyk S (2008) Development of a multi-residue method for the determination of pesticides in cereals and dry animal feed using gas chromatography–tandem quadrupole mass spectrometry II. Improvement and extension to new analytes. J.

[2] Wang J, Leung D (2009) Applications of Ultra-performance Liquid Chromatography Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry on Analysis of 138 Pesticides in Fruit- and Vegetable-Based Infant Foods. J. Agric. Food Chem. 57:

[3] Hercegová A, Dömötörová M, Matisová E (2007) Sample preparation methods in the analysis of pesticide residues in baby food with subsequent chromatographic

[4] Beddington J (2010) Food security: contributions from science to a new and greener

[5] Nguyen TD, Yu JE, Lee DM, Lee GH (2008) A multiresidue method for the determination of 107 pesticides in cabbage and radish using QuEChERS sample

**Author details**

**Acknowledgement**

**5. References**

2162–2173.

Chromatogr. A 1208: 202-214.

determination. J. Chromatogr. A 1153: 54-73.

revolution. Phil. Trans. R. Soc. B 365: 61-71.

**Table 2.** Different methods applied to the determination of pesticide and others analytes in different cereal samples.

### **4. Conclusions**

Although detection equipment's are becoming more specific and sensitive, there is still a need for efficient sample preparation methods for pesticides residues analysis in cereals and feedstuffs, which are compatible with modern analytical techniques. Despite the advances in separation and detection of the chromatographic systems, cleanup remains important for obtaining reliable data. There is still a need for effective, environmentally friendly and fast methods for sample treatment and determination of pesticide residues in fatty food matrices e.g. cereals and feedstuffs. Modern sample preparation techniques should be not only simple, reliable, cheap and take into account chemical laboratory waste problems, but also must be similar to common analytical techniques, in order to minimize errors. For these reasons, modern trends in analytical chemistry are towards the simplification and miniaturization of sample preparation, and the minimization of sample size and organic solvent used. The development of such procedures combined with modern chromatographic-mass spectrometric techniques will enable analysis at the low levels now required by legislation for many pesticides, but more importantly, result in methods which produce more reliable data to support food safety monitoring programs. This is the trend although it is impossible to develop a unique protocol covering such a wide range of compounds. Finally, multiclass multiresidue methods covering large number of pesticides are highly desirable, although the different nature, classes and physico-chemical properties of pesticides hamper the development of such methods.

## **Author details**

146 Pesticides – Recent Trends in Pesticide Residue Assay

bisphenol A (BPA), 4-tert-butylbenzoic acid (BBA), 4 nonylphenol (NP), 4-tert-butylphenol (t-BP), 2,4 dichlorophenol (DCP), 2,4,5-trichlorophenol (TCP) and pentachlorophenol (PCP)

benzoylureas

acetochlor and

Penoxsulan, tricyclazole, propanil, azoxystrobin, molinate, profoxydim, cyhalofop-butyl, deltamethrin and 3,4-dichloroaniline (the main metabolite of propanil)

cereal samples.

**4. Conclusions**

acetanilide herbicide rice, wheat

Bispyribac sodium rice

22 pesticides white and

Analytes Sample Extraction

breakfast

fruit, vegetable, cereals and animal products

Procedure Cleanup Analytical

cereals PLE - LC–ESI-

wild rice SFE SPE GC-ECD

liquid–liquid partition and anion exchange solid phase

rice MSPD HPLC-

**Table 2.** Different methods applied to the determination of pesticide and others analytes in different

Although detection equipment's are becoming more specific and sensitive, there is still a need for efficient sample preparation methods for pesticides residues analysis in cereals and feedstuffs, which are compatible with modern analytical techniques. Despite the advances in separation and detection of the chromatographic systems, cleanup remains important for obtaining reliable data. There is still a need for effective, environmentally friendly and fast methods for sample treatment and determination of pesticide residues in fatty food matrices e.g. cereals and feedstuffs. Modern sample preparation techniques should be not only simple, reliable, cheap and take into account chemical laboratory waste problems, but also

carbendazim wheat grain MSPD HPLC-

propisochlor Soil and corn methanol/water/SPE GC-ECD 10.0 73.8 - 115.5

technique

PLE - LC-MS/MS 2.0 – 10.0 58 - 97

and maize PLE SPE GC-ECD 2.4 - 5.3 82.3 - 115.8

HPLC-

LOQ (µg kg−1)

MS 3.0 – 43.0 81 - 104

GC-TSD - <sup>74</sup>

DAD 10.0 83.98 - 98.51

DAD 6.0 - 600.0 74 – 127

DAD 40.0 87.3

Recovery (%)

(RSD %) References

(4 - 9) <sup>37</sup>

(5 - 19) <sup>42</sup>

(1.1 - 13.6) <sup>43</sup>

<sup>98</sup><sup>49</sup>

(< 11.1) <sup>53</sup>

(0.56 - 6.36) <sup>54</sup>

(< 12) <sup>58</sup>

(3.3) <sup>59</sup>

Renato Zanella, Osmar Damian Prestes, Caroline do Amaral Friggi, Manoel Leonardo Martins and Martha Bohrer Adaime *Laboratório de Análises de Resíduos de Pesticidas (LARP), Departamento de Química, Universidade Federal de Santa Maria, Santa Maria - RS, Brazil*

## **Acknowledgement**

The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, Brazil) and the Fundação de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS, Porto Alegre, Brazil) for financial support and fellowships.

#### **5. References**

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An Overview About Recent Advances in Sample

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Food Chem. 59: 8589-8608.

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[31] Wang J, Chow W, Cheung W (2011) Application of a Tandem Mass Spectrometer and Core–Shell Particle Column for the Determination of 151 Pesticides in Grains. J. Agric.

[32] Anagnostopoulos CJ, Sarli PA, Liapis K, Haroutounian SA, Miliardis GE (2011) Validation of Two Variations of the QuEChERS Method for the Determination of Multiclass Pesticide Residues in Cereal-Based Infant Foods by LC–MS/MS. Food Anal.

[33] Marchis D, Ferro GL, Brizio P, Squadrone S, Abete MC (2012) Detection of pesticides in

[34] Wang P, Yang X, Wang J, Cui J, Dong AJ, Zhao HT, Zhang LW, Wang ZY, Xu P, Li RB, Zhang WJ, Zhang YC, Jing H (2012) Multi-residue method for determination of 7 neonicotinoid insecticides in grains using dispersive solid-phase extraction and dispersive liquid-liquid micro-extraction by High performance liquid chromatography.

[35] Wilkowska A, Biziuk M (2011) Determination of pesticide residues in food matrices

[36] Prestes OD, Friggi CA, Adaime MB, Zanella R (2009) QuEChERS - A modern sample preparation method or pesticide multiresidue determination in food by chromatographic methods coupled to mass spectrometry. Quim. Nova 32: 1620-1634. [37] Carabias-Martinez R, Rodriguez-Gonzalo E, Revilla-Ruiz P (2006) Determination of endocrine-disrupting compounds in cereals by pressurized liquid extraction and liquid chromatography–mass spectrometry Study of background contamination. J.

[38] Richter BE, Jones BA, Ezzell JL, Porter NL (1996) Accelerated Solvent Extraction: A

[39] Pallaroni L, Von Holst C (2003) Determination of zearalenone from wheat and corn by pressurized liquid extraction and liquid chromatography-electrospray mass

[40] Von Holst C, Serano F, Sporring S, Björklund E, Muller A (2005) Optimisation of pressurized liquid extraction for the determination of seven selected polychlorinated

[41] Sun H, Ge X, Lv Y, Wan A (2012) Application of accelerated solvent extraction in the analysis of organic contaminants, bioactive and nutritional compounds in food and

[42] Brutti M, Blasco C, Pico Y (2010) Determination of benzoylurea insecticides in food by

[43] Zhang Y, Yang J, Shi R, Su Q, Yao L, Li P (2011) Determination of acetanilide herbicides in cereal crops using accelerated solvent extraction, solid-phase extraction and gas

[44] Lehotay SJ (1997) Supercritical fluid extraction of pesticides in foods. J. Chromatogr. A

[45] Herrero M, Mendiola JA, Cifuentes A, Ibanez E (2010) Supercritical fluid extraction:

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biphenyls in feed samples. Chromatographia 61: 391-396.

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	- [61] Juan C, Moltó JC, Lino CM, Mañes J (2008) Determination of ochratoxin A in organic and non-organic cereals and cereal products from Spain and Portugal. Food Chem. 107: 525-530.

**Chapter 8**

© 2012 Tan and Abdulra'uf, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Tan and Abdulra'uf, licensee InTech. This is a paper distributed under the terms of the Creative Commons

**Recent Developments and Applications**

Guan Huat Tan and Lukman Bola Abdulra'uf

Additional information is available at the end of the chapter

pesticide residues in fruit and vegetable samples [2].

http://dx.doi.org/10.5772/46843

**1. Introduction**

**of Microextraction Techniques for the Analysis**

**of Pesticide Residues in Fruits and Vegetables**

Analysis of pesticide residues and other contaminants in fruit and vegetable samples is becoming increasingly important due to the health hazards caused by their accumulation in human tissue. The body requires some important nutrients which can be provided by the consumption of fruits and vegetables. The purpose of any analytical study is to obtain information about substances and analytes present in the sample. Analytical process involves several steps: sampling, sample preparation, separation, quantification and data analysis [1]. Sample preparation is a very important step and indeed the bottleneck of analytical methodologies, in the analysis of fruits and vegetables for the presence of

The first step in any instrumental analysis is sample preparation, which involves the isolation or extraction of the desired analytes from the sample matrix, since they are present at trace concentration (usually µg.kg or less). This helps in the elimination of any interferences and also reduces the volume of extracts, thereby concentrating the analytes [2]. The type, nature and composition of sample and the nature and concentration of analytes to be isolated or extracted determines the choice of separation and detection method to be used, and this also dictates the type of sample preparation to be employed [3, 4], since the efficiency of any analysis is determined by the sample preparation step. The current trend of microextraction techniques is aimed at a reliable and accurate analysis of contaminants from complex samples. It is focused on the reduction of sample and solvent volume, with the automation/coupling of the sampling step to the analytical instruments, while maintaining the high throughput performance, low cost operation and improvement of the sample preparation, such as extraction, concentration, isolation of analytes, and clean-up [5, 6].

## **Recent Developments and Applications of Microextraction Techniques for the Analysis of Pesticide Residues in Fruits and Vegetables**

Guan Huat Tan and Lukman Bola Abdulra'uf

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46843

## **1. Introduction**

152 Pesticides – Recent Trends in Pesticide Residue Assay

Commun. Mass Spectrom. 20: 609-617.

Chromatographica 3: 51-64.

525-530.

A 1186: 325-339.

[61] Juan C, Moltó JC, Lino CM, Mañes J (2008) Determination of ochratoxin A in organic and non-organic cereals and cereal products from Spain and Portugal. Food Chem. 107:

[62] Lee MK, Weg G, Traag WA, Mol HGJ (2008) Qualitative screening and quantitative determination of pesticides and contaminants in animal feed using comprehensive twodimensional gas chromatography with time-of-flight mass spectrometry. J. Chromatogr.

[63] Zhang WG, Chu XG, Cai HX, An J, Li CJ (2006) Simultaneous determination of 109 pesticides in unpolished rice by a combination of gel permeation chromatography and Florisil column purification, and gas chromatography/mass spectrometry. Rapid

[64] Presta MA, Kolberg DIS, Wickert C, Pizzutti IR, Adaime MA, Zanella R. (2009) High Resolution Gel Permeation Chromatography Followed by GC-ECD for the

[66] Alder L, Greulich K, Kempe G, Vieth B (2006) Residue analysis of 500 high priority pesticides: Better by GC–MS or LC–MS/MS? Mass Spectrom. Rev. 25: 838-865.

Determination of Pesticide Residues in Soybeans. Chromatographia 69: 237-241. [65] Prestes OD, Adaime MB, Zanella R (2011) QuEChERS: possibilities and trends in sample preparation for multiresidue determination of pesticides in food. Scientia

> Analysis of pesticide residues and other contaminants in fruit and vegetable samples is becoming increasingly important due to the health hazards caused by their accumulation in human tissue. The body requires some important nutrients which can be provided by the consumption of fruits and vegetables. The purpose of any analytical study is to obtain information about substances and analytes present in the sample. Analytical process involves several steps: sampling, sample preparation, separation, quantification and data analysis [1]. Sample preparation is a very important step and indeed the bottleneck of analytical methodologies, in the analysis of fruits and vegetables for the presence of pesticide residues in fruit and vegetable samples [2].

> The first step in any instrumental analysis is sample preparation, which involves the isolation or extraction of the desired analytes from the sample matrix, since they are present at trace concentration (usually µg.kg or less). This helps in the elimination of any interferences and also reduces the volume of extracts, thereby concentrating the analytes [2]. The type, nature and composition of sample and the nature and concentration of analytes to be isolated or extracted determines the choice of separation and detection method to be used, and this also dictates the type of sample preparation to be employed [3, 4], since the efficiency of any analysis is determined by the sample preparation step. The current trend of microextraction techniques is aimed at a reliable and accurate analysis of contaminants from complex samples. It is focused on the reduction of sample and solvent volume, with the automation/coupling of the sampling step to the analytical instruments, while maintaining the high throughput performance, low cost operation and improvement of the sample preparation, such as extraction, concentration, isolation of analytes, and clean-up [5, 6].

© 2012 Tan and Abdulra'uf, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Tan and Abdulra'uf, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The conventional solvent-based sample preparation methods: liquid-liquid extraction (LLE), solid phase extraction (SPE), accelerated solvent extraction (ASE), matrix solid phase dispersion (MSPD) [7-11], usually require various matrix pretreatment steps, which are tedious, time consuming, contains multistep techniques, require large volumes of sample and toxic solvents which impose environmental pollution and health hazards with high operation cost [5]. Therefore it is necessary to reduce the number of sample preparation steps in order to reduce the sources of error. Microextraction techniques are recently developed sample preparation methods which are vital as well as effective and efficient ways to save time, reduce solvent use and operation cost, and can efficiently measure the trace analytes embedded with high molecular mass compounds in complex sample matrices [12].

Recent Developments and Applications of Microextraction Techniques for the Analysis of Pesticide Residues in Fruits and Vegetables 155

on the sample matrix composition and the nature and volatility of the analytes. In the headspace sampling mode, the analyte is transported through a layer of gas before reaching the coating. The HS-SPME involves the exposure of the fiber to the vapour phase above a liquid or solid sample [5], where the analytes are extracted from the gas phase equilibrated with the sample matrix [20]. It could be effectively used for the extraction of analytes from complex liquid or solid samples [21]. This method helps to protect the coated fiber from the effects of any non-volatile high molecular weight compounds in the sample matrix, which binds irreversibly to the coating and often cause interference in the extraction process. In the DI-SPME, the coated fiber is completely inserted into the samples, where the transport of the analyte from the sample matrix into the extracting phase is achieved [22]. It is used for the

The DI-SPME can also be applied for the extraction of analytes from complex samples, provided optimum dilution is achieved [18]. The HS-SPME is suitable for the extraction of volatile or semi-volatile and low-to medium-polar analytes, but the headspace volume should always be taken into consideration. The DI-SPME is used for the extraction of nonvolatile analytes or analytes with low volatility and high-to-medium polar analytes [18, 21]. The advantage of HS-SPME over DI-SPME is the shorter equilibrium time, due to the higher diffusion coefficients in gaseous state, higher concentration of the analytes in the headspace prior to extraction and also allows for the variation of sample matrix properties without any effect on the fiber. Extraction mode is an important parameter which should be considered and optimized in the experimental design (described later) of SPME for a particular analyte

There are two distinct steps in SPME; partitioning of the analytes between the extracting stationary phase and the sample matrix in DI-SPME mode and partitioning between fiber, headspace and sample matrix in the HS-SPME mode, followed by the desorption of concentrated extract [23], into analytical instruments, thermally when coupled to GC or with a mobile phase solvent when coupled to LC, and in the offline and online mode when combined with CE [12]. The technique is based on the partitioning of analyte and establishment of equilibrium between the analyte in the sample matrix and the stationary phase of the coated fused silica, which can either be liquid or solid particles suspended in a liquid polymer or a combination of both [14, 17, 24]. Recently, new SPME microextraction techniques have been developed, which make use of a microsyringe and is called in-needle

The experimental design of SPME for the analysis of pesticide residues from fruits and vegetables involves several steps and parameters which must be considered and optimized in order to achieve an efficient extraction. Parameters such as fiber type, extraction mode (described earlier), extraction and desorption time and temperature, separation and detection system, agitation method, ionic strength, sample pH, water and organic solvent content and method of calibration are considered and optimized based on the nature of analyte and constituent of sample matrix. Optimization of these parameters in the extraction of pesticide residues from food samples have also been reviewed in the literatures [5, 17, 22, 25, 26]. The first step in the development and optimization of an SPME method is the

extraction of gaseous or simple liquid samples.

SPME and with a pipette tip, known as in-tip SPME [12].

under investigation.

The microextraction techniques have been developed by different researchers, to corroborate the recent advances in the development of highly sensitive and efficient analytical instrumentation. Instrumental techniques like gas and liquid chromatography and capillary electrophoresis which are compatible with the microextraction technique, and coupled to different detectors (mass spectrometry, diode array detector, ultraviolet detector, *etc*), have been developed for qualitative and quantitative analysis of pesticide residues and other contaminants from foods. Prior sample preparation is necessary in order to extract, isolate and concentrate the analytes of interest from the complex fruits and vegetables matrices, which contain high molecular mass compounds. The low cost and ease of hyphenation of these microextraction techniques to analytical instruments helps to reduce errors, due to contamination and sample losses.

In this review, recent advances in different microextraction techniques used in the analysis of pesticide residues from fruit and vegetables are discussed, with a major focus on their methodologies, advantages, limitations and future trends.

## **2. Solid phase microextraction (SPME)**

Solid phase microextraction, a solvent-free sample preparation technique, was developed by Pawliszyn and his co-workers in 1990 [13], and its applications has been examined, optimized and automated [14, 15]. It eliminates the need for solvents and combines sampling, isolation, concentration and enrichment in one step and offers the benefit of short sample preparation step, solvent-free extraction, small sample volumes and analyte concentration from solid, liquid or gaseous samples. It employs a chemically inert fusedsilica optical fiber or metal alloys (Fiber SPME) coated on the outside with a thin film of sorbent [14] and a thin film internally coated fused silica capillary column (In-tube) [5, 16], as the extraction stationary phase, containing a polymeric organic compound. The fiber SPME has been widely used for the extraction of pesticide residues in fruits and vegetable samples, and will be discussed extensively in this review.

There are two major types of extraction modes used in the analysis of pesticides residues in fruit and vegetable samples. These are the headspace SPME (HS-SPME) and direct immersion SPME (DI-SPME). [5, 17-19]. The efficiency of each extraction mode is dependent on the sample matrix composition and the nature and volatility of the analytes. In the headspace sampling mode, the analyte is transported through a layer of gas before reaching the coating. The HS-SPME involves the exposure of the fiber to the vapour phase above a liquid or solid sample [5], where the analytes are extracted from the gas phase equilibrated with the sample matrix [20]. It could be effectively used for the extraction of analytes from complex liquid or solid samples [21]. This method helps to protect the coated fiber from the effects of any non-volatile high molecular weight compounds in the sample matrix, which binds irreversibly to the coating and often cause interference in the extraction process. In the DI-SPME, the coated fiber is completely inserted into the samples, where the transport of the analyte from the sample matrix into the extracting phase is achieved [22]. It is used for the extraction of gaseous or simple liquid samples.

154 Pesticides – Recent Trends in Pesticide Residue Assay

errors, due to contamination and sample losses.

**2. Solid phase microextraction (SPME)**

methodologies, advantages, limitations and future trends.

samples, and will be discussed extensively in this review.

[12].

The conventional solvent-based sample preparation methods: liquid-liquid extraction (LLE), solid phase extraction (SPE), accelerated solvent extraction (ASE), matrix solid phase dispersion (MSPD) [7-11], usually require various matrix pretreatment steps, which are tedious, time consuming, contains multistep techniques, require large volumes of sample and toxic solvents which impose environmental pollution and health hazards with high operation cost [5]. Therefore it is necessary to reduce the number of sample preparation steps in order to reduce the sources of error. Microextraction techniques are recently developed sample preparation methods which are vital as well as effective and efficient ways to save time, reduce solvent use and operation cost, and can efficiently measure the trace analytes embedded with high molecular mass compounds in complex sample matrices

The microextraction techniques have been developed by different researchers, to corroborate the recent advances in the development of highly sensitive and efficient analytical instrumentation. Instrumental techniques like gas and liquid chromatography and capillary electrophoresis which are compatible with the microextraction technique, and coupled to different detectors (mass spectrometry, diode array detector, ultraviolet detector, *etc*), have been developed for qualitative and quantitative analysis of pesticide residues and other contaminants from foods. Prior sample preparation is necessary in order to extract, isolate and concentrate the analytes of interest from the complex fruits and vegetables matrices, which contain high molecular mass compounds. The low cost and ease of hyphenation of these microextraction techniques to analytical instruments helps to reduce

In this review, recent advances in different microextraction techniques used in the analysis of pesticide residues from fruit and vegetables are discussed, with a major focus on their

Solid phase microextraction, a solvent-free sample preparation technique, was developed by Pawliszyn and his co-workers in 1990 [13], and its applications has been examined, optimized and automated [14, 15]. It eliminates the need for solvents and combines sampling, isolation, concentration and enrichment in one step and offers the benefit of short sample preparation step, solvent-free extraction, small sample volumes and analyte concentration from solid, liquid or gaseous samples. It employs a chemically inert fusedsilica optical fiber or metal alloys (Fiber SPME) coated on the outside with a thin film of sorbent [14] and a thin film internally coated fused silica capillary column (In-tube) [5, 16], as the extraction stationary phase, containing a polymeric organic compound. The fiber SPME has been widely used for the extraction of pesticide residues in fruits and vegetable

There are two major types of extraction modes used in the analysis of pesticides residues in fruit and vegetable samples. These are the headspace SPME (HS-SPME) and direct immersion SPME (DI-SPME). [5, 17-19]. The efficiency of each extraction mode is dependent The DI-SPME can also be applied for the extraction of analytes from complex samples, provided optimum dilution is achieved [18]. The HS-SPME is suitable for the extraction of volatile or semi-volatile and low-to medium-polar analytes, but the headspace volume should always be taken into consideration. The DI-SPME is used for the extraction of nonvolatile analytes or analytes with low volatility and high-to-medium polar analytes [18, 21]. The advantage of HS-SPME over DI-SPME is the shorter equilibrium time, due to the higher diffusion coefficients in gaseous state, higher concentration of the analytes in the headspace prior to extraction and also allows for the variation of sample matrix properties without any effect on the fiber. Extraction mode is an important parameter which should be considered and optimized in the experimental design (described later) of SPME for a particular analyte under investigation.

There are two distinct steps in SPME; partitioning of the analytes between the extracting stationary phase and the sample matrix in DI-SPME mode and partitioning between fiber, headspace and sample matrix in the HS-SPME mode, followed by the desorption of concentrated extract [23], into analytical instruments, thermally when coupled to GC or with a mobile phase solvent when coupled to LC, and in the offline and online mode when combined with CE [12]. The technique is based on the partitioning of analyte and establishment of equilibrium between the analyte in the sample matrix and the stationary phase of the coated fused silica, which can either be liquid or solid particles suspended in a liquid polymer or a combination of both [14, 17, 24]. Recently, new SPME microextraction techniques have been developed, which make use of a microsyringe and is called in-needle SPME and with a pipette tip, known as in-tip SPME [12].

The experimental design of SPME for the analysis of pesticide residues from fruits and vegetables involves several steps and parameters which must be considered and optimized in order to achieve an efficient extraction. Parameters such as fiber type, extraction mode (described earlier), extraction and desorption time and temperature, separation and detection system, agitation method, ionic strength, sample pH, water and organic solvent content and method of calibration are considered and optimized based on the nature of analyte and constituent of sample matrix. Optimization of these parameters in the extraction of pesticide residues from food samples have also been reviewed in the literatures [5, 17, 22, 25, 26]. The first step in the development and optimization of an SPME method is the

selection of fiber coating, since the efficiency of SPME technique is dependent on the fiber coating/sample matrix distribution constant [18], because the extraction process involves the diffusion of analyte from sample matrix to the fiber/headspace, through the interface between the fiber/headspace and sample matrix.

Recent Developments and Applications of Microextraction Techniques for the Analysis of Pesticide Residues in Fruits and Vegetables 157

The most widely used mixed phase fiber for fruits and vegetables analysis is the 65 µm PDMS/DVB fiber. It was employed in the extraction of pyrethroid pesticides from tomato and strawberry [64], and 70 multiclass pesticides from cucumber, pepper and tomato [65]. The 60 µm PDMS/DVB fiber was employed for the extraction of 25 multiclass pesticides from tomato [61], carbamate pesticides from strawberry [66] and apple [67] and pyrethroid pesticides from cucumber and water melon and 7 multiclass pesticides from tomato [68], the 65 µm PDMS/DVB coupled to multi-dimensional GC/MS was recently used for the analysis of multiclass pesticides in peach, orange and pineapple juices [69]. The less frequently used fibers in the extraction of pesticides from fruit and vegetable samples include the 65 µm CW/DVB, used for the extraction of carbamate pesticides from apple and grape juice [70], the 50 µm CW/TPR used for the extraction of postharvest fungicides from cherry, orange and peach [71], and multiclass pesticides in lettuce [72] and activated carbon/polyvinylchloride (AC/PVC) used for the extraction of organophosphorus pesticides from grape [73]. The extractions gave good repeatability and reproducibility with better

efficiency than the single phase coated fibers.

comes in contact with the sample matrix. 1. Single Drop Microextraction (SDME)

2. Hollow Fiber Liquid Phase Microextraction (HF-LPME) 3. Dispersive Liquid Liquid Microextraction (DLLME)

**Microextraction (SME)**

**3. Liquid phase microextraction (LPME) also called Solvent**

This is also a newly developed microextraction technique, which drastically reduces the quantity of solvent used for extraction [74]. It is rapid and less expensive, and is performed between a small amount of water-immiscible solvent called the acceptor phase, and an aqueous phase (donor phase) containing the analytes of interest [75]. The acceptor phase can either be immersed directly in the sample matrix or suspended above the sample for headspace extraction [12]. The volume of the receiving phase is in microliter (droplet of organic solvent). It is also known as liquid-liquid microextraction (LLME) and can be divided into three major categories and their difference is the way the extraction solvent

The development of the methods for the analysis of pesticides by the LPME technique as in SPME, requires the optimization of the parameters related to the donor and acceptor phases. The first and the most important is the selection of an organic solvent which is of high purity, less volatile, high boiling point, so as to prevent solvent evaporation and ensure analyte concentration. The solvents should also be water immiscible for extraction of analytes from an aqueous sample. The SDME extraction like SPME is a non-exhaustive extraction method, thus an appropriate drop volume must be selected to prevent shrinking, and volumes are usually between 0.9 and 1.6 µL. Although, the use of a large volume of solvent drop yields better instrumental response and improve extraction efficiency, they are difficult to manipulate and could be easily dislodged from the syringe. Agitation increases the extraction efficiency, by decreasing the extraction time. It also leads to an increase in

The commercial fibers are coated with different polymeric materials (single or mixed), which extract by either absorption or adsorption. Due to their relative low recommended operating temperature (200 – 300 OC) of the commercially available fiber, the recent trend is focused on the development of new fibers using sol-gel technology, with high thermal stability (>320 OC) [27-37] and molecular imprinted polymers (MIPs) [38-42]. Ionic liquids and sol-gel prepared ionic liquid coated fibers have also been recently developed and have been used extensively for the SPME extraction of pesticide residues in water samples [43- 46], but their use for the extraction of analytes from food samples is a potential research area to be explored in the future.

The PDMS is the most commonly used SPME fiber coating in fruit and vegetable analysis. Although, it is suitable for the extraction of non-polar pesticides, it has also been widely used for the extraction of more polar pesticides, after the extraction conditions have been optimized. [5]. The mixed coating: PDMS/divinylbenzene (DVB), DVB/PDMS/carboxen (CAR), carbowax (CW)/PDMS, CW/DVB and CW/template resin (TPR) extract by adsorption and possess complementary properties of each constituent polymeric coating.

The fiber SPME has been used successfully for the analysis of pesticide residues from fruit and vegetable samples. The 65 µm PA fiber was employed for the extraction of 54 multiclass pesticides from orange and peach in the DI mode [47]. The fiber coating was used for the extraction of phenyl urea pesticides from carrot, onion and potato in the DI mode [48], organophosphorus pesticides from orange, grape and lemon juice in the DI mode [49], triazole in strawberry in the DI mode [50], 14 multiclass pesticides from mango in the DI mode [51], while 80 µm PA fiber was used for the extraction of strobilurin fungicides from grape, ketchup, strawberry and tomato in the HS mode [52]. The 100 µm PDMS was used by Chai et al for the extraction of multiresidue organochlorine and organophosphorus pesticides from cabbage, tomato and guava in HS mode [53] and from strawberry, guava, pak-choi, tomato, star fruit and cucumber in HS mode [15, 54]. Organochlorine, organophosphorus and carbamate pesticides from tomato and cucumber in DI mode [55] has been employed. Organophosphorus pesticides was extracted from pear and apple in DI mode [56], strawberry, cherry and cherry juice in HS mode [57, 58], *C. coronaium* plant in DI mode [59] and 13 different vegetables in HS mode [60] using 100 µm PDMS. Multiclass pesticides were also extracted from tomato in the DI mode with 100 µm PDMS (Guillet [61].

A pre-extraction method, involving the use of matrix-assisted extraction followed by SPME extraction using the 100 µm PDMS was developed. The method was used for the extraction of organophosphorous pesticides from strawberry, tomato and pakchoi in the HS mode [62] and extraction of pyrethroid pesticides from strawberry in the DI mode [63]. The 100 µm PDMS gave better extraction efficiency, good linear range, lower detection limit and good recovery with relative standard deviation lower than 10%.

The most widely used mixed phase fiber for fruits and vegetables analysis is the 65 µm PDMS/DVB fiber. It was employed in the extraction of pyrethroid pesticides from tomato and strawberry [64], and 70 multiclass pesticides from cucumber, pepper and tomato [65]. The 60 µm PDMS/DVB fiber was employed for the extraction of 25 multiclass pesticides from tomato [61], carbamate pesticides from strawberry [66] and apple [67] and pyrethroid pesticides from cucumber and water melon and 7 multiclass pesticides from tomato [68], the 65 µm PDMS/DVB coupled to multi-dimensional GC/MS was recently used for the analysis of multiclass pesticides in peach, orange and pineapple juices [69]. The less frequently used fibers in the extraction of pesticides from fruit and vegetable samples include the 65 µm CW/DVB, used for the extraction of carbamate pesticides from apple and grape juice [70], the 50 µm CW/TPR used for the extraction of postharvest fungicides from cherry, orange and peach [71], and multiclass pesticides in lettuce [72] and activated carbon/polyvinylchloride (AC/PVC) used for the extraction of organophosphorus pesticides from grape [73]. The extractions gave good repeatability and reproducibility with better efficiency than the single phase coated fibers.

## **3. Liquid phase microextraction (LPME) also called Solvent Microextraction (SME)**

This is also a newly developed microextraction technique, which drastically reduces the quantity of solvent used for extraction [74]. It is rapid and less expensive, and is performed between a small amount of water-immiscible solvent called the acceptor phase, and an aqueous phase (donor phase) containing the analytes of interest [75]. The acceptor phase can either be immersed directly in the sample matrix or suspended above the sample for headspace extraction [12]. The volume of the receiving phase is in microliter (droplet of organic solvent). It is also known as liquid-liquid microextraction (LLME) and can be divided into three major categories and their difference is the way the extraction solvent comes in contact with the sample matrix.

1. Single Drop Microextraction (SDME)

156 Pesticides – Recent Trends in Pesticide Residue Assay

to be explored in the future.

between the fiber/headspace and sample matrix.

recovery with relative standard deviation lower than 10%.

selection of fiber coating, since the efficiency of SPME technique is dependent on the fiber coating/sample matrix distribution constant [18], because the extraction process involves the diffusion of analyte from sample matrix to the fiber/headspace, through the interface

The commercial fibers are coated with different polymeric materials (single or mixed), which extract by either absorption or adsorption. Due to their relative low recommended operating temperature (200 – 300 OC) of the commercially available fiber, the recent trend is focused on the development of new fibers using sol-gel technology, with high thermal stability (>320 OC) [27-37] and molecular imprinted polymers (MIPs) [38-42]. Ionic liquids and sol-gel prepared ionic liquid coated fibers have also been recently developed and have been used extensively for the SPME extraction of pesticide residues in water samples [43- 46], but their use for the extraction of analytes from food samples is a potential research area

The PDMS is the most commonly used SPME fiber coating in fruit and vegetable analysis. Although, it is suitable for the extraction of non-polar pesticides, it has also been widely used for the extraction of more polar pesticides, after the extraction conditions have been optimized. [5]. The mixed coating: PDMS/divinylbenzene (DVB), DVB/PDMS/carboxen (CAR), carbowax (CW)/PDMS, CW/DVB and CW/template resin (TPR) extract by adsorption and possess complementary properties of each constituent polymeric coating.

The fiber SPME has been used successfully for the analysis of pesticide residues from fruit and vegetable samples. The 65 µm PA fiber was employed for the extraction of 54 multiclass pesticides from orange and peach in the DI mode [47]. The fiber coating was used for the extraction of phenyl urea pesticides from carrot, onion and potato in the DI mode [48], organophosphorus pesticides from orange, grape and lemon juice in the DI mode [49], triazole in strawberry in the DI mode [50], 14 multiclass pesticides from mango in the DI mode [51], while 80 µm PA fiber was used for the extraction of strobilurin fungicides from grape, ketchup, strawberry and tomato in the HS mode [52]. The 100 µm PDMS was used by Chai et al for the extraction of multiresidue organochlorine and organophosphorus pesticides from cabbage, tomato and guava in HS mode [53] and from strawberry, guava, pak-choi, tomato, star fruit and cucumber in HS mode [15, 54]. Organochlorine, organophosphorus and carbamate pesticides from tomato and cucumber in DI mode [55] has been employed. Organophosphorus pesticides was extracted from pear and apple in DI mode [56], strawberry, cherry and cherry juice in HS mode [57, 58], *C. coronaium* plant in DI mode [59] and 13 different vegetables in HS mode [60] using 100 µm PDMS. Multiclass pesticides were also extracted from tomato in the DI mode with 100 µm PDMS (Guillet [61]. A pre-extraction method, involving the use of matrix-assisted extraction followed by SPME extraction using the 100 µm PDMS was developed. The method was used for the extraction of organophosphorous pesticides from strawberry, tomato and pakchoi in the HS mode [62] and extraction of pyrethroid pesticides from strawberry in the DI mode [63]. The 100 µm PDMS gave better extraction efficiency, good linear range, lower detection limit and good

The development of the methods for the analysis of pesticides by the LPME technique as in SPME, requires the optimization of the parameters related to the donor and acceptor phases. The first and the most important is the selection of an organic solvent which is of high purity, less volatile, high boiling point, so as to prevent solvent evaporation and ensure analyte concentration. The solvents should also be water immiscible for extraction of analytes from an aqueous sample. The SDME extraction like SPME is a non-exhaustive extraction method, thus an appropriate drop volume must be selected to prevent shrinking, and volumes are usually between 0.9 and 1.6 µL. Although, the use of a large volume of solvent drop yields better instrumental response and improve extraction efficiency, they are difficult to manipulate and could be easily dislodged from the syringe. Agitation increases the extraction efficiency, by decreasing the extraction time. It also leads to an increase in aqueous mass transfer and exposes the analytes to the extraction solvent, and should be optimized to avoid the dislodgment of the solvent drop.

Recent Developments and Applications of Microextraction Techniques for the Analysis of Pesticide Residues in Fruits and Vegetables 159

extraction and injected directly into the GC system [81]. Another limitation is the instability of the microdrop of solvent in the DI-mode at high stirring rate and long extraction times [3]. Therefore, in the development of SDME technique, there is need for the optimization of factors such as selection of extraction solvent, solvent drop volume, agitation rate, ionic strength, extraction temperature and time, sample and headspace volume and automation

Few studies have been published regarding its application for the extraction of pesticide residues from fruit and vegetable samples. The techniques was used for the analysis of 20 multiclass pesticides in apple [84], and in tomato, using toluene [85, 86], analysis of triazole pesticides in water and grape juice samples, with extraction by n-hexanol/n-hexane (50:50,v/v%) [87]. The presence of organochlorine pesticides was also investigated in vegetable samples (cabbage, cauliflower and Chinese cabbage) using SDME-GC/MS using a mixture of 1 µL p-xylene/acetone (80:20, v/v %) [88] and organophosphorus pesticides in fruit juices. [89], and in apple, pear and orange juice samples [90], using toluene as the extraction solvents. Organophophorus and organochlorine pesticides were also determined in cucumber and strawberry using toluene (1.5 µL) [91], while a mixture of octanone and undecanone was used for the extraction of oxazole fungicides from fruits samples [92]. The choice of toluene by most authors was based on the fact that it is stable, less toxic, possesses a higher extraction efficiency and its suitability for direct GC injection. All the developed

HF-LPME is a multi-phased microextraction system, which was developed to enhance the stability of the organic solvent in 1999 [93, 94]. It is based on the principle of a supported liquid membrane [95], in which the organic solvent is used to fill both the wall pores and the HF lumen. It makes use of a polymeric membrane which forms a barrier between the solvent and the sample [96], and acts as a support for the small volumes of extracting solvents. It involves filling of a few microliters of solvent inside the pores and lumen of a semi-permeable polypropylene hollow fiber attached to a syringe. The HF-LPME can be carried out in either the static or dynamic mode. In the static mode, the acceptor phase is introduced in the lumen followed by the immersion of the fiber into the aqueous sample by a syringe, while in the dynamic mode the HF is attached to a syringe connected to

The extraction can be carried out in two- or three phases [95]. In the two-phase mode, the aqueous sample (donor) makes contact directly with the organic solvents (acceptor) through the membrane pores, by a repeated pushing and pulling of the microsyringe plunger [97], and the mass transfer of the analytes is driven by the diffusion of the analytes from the sample matrix into the organic solvent. When the pores are prefilled with an organic solvent, which provides a supported liquid membrane, a three-phase system is formed, and the sample solution and organic solvent are separated by the hollow fiber membrane. The solvent used must be compatible with the membrane, so as to ensure that the pores in the

methods gave comparable analytical figures of merit compared to SPME.

**3.2. Hollow fiber liquid phase microextraction (HF-LPME)**

programmable pump [75, 78].

[76].

Salt addition enhances extraction efficiency by reducing the solubility of the analytes in the sample, especially for the moderately polar and low molecular weight analytes. The optimization of extraction temperature is important especially in the headspace mode, but a higher temperature may affect the stability of the microdrop, and for this reason most of the reported analyses have been carried out by DI- mode at room temperature. The sample and headspace volume should be maintained constantly at minimal level, since larger sample volumes will require longer extraction time and affect the stability of the microdrop. In the headspace mode, the optimized volume should be at a level which allows the microdrop to be suspended over the stirred sample, while in the direct immersion mode, the sample volume should only be enough to avoid the sample coming in contact with the septum [3, 76, 77]. The presence of air bubbles should also be avoided in the syringe to avoid errors in the analysis.

#### **3.1. Single drop microextraction (SDME)**

SDME is a microextraction technique, which is based on the suspension of a single droplet of water-immiscible organic solvents (typically 0.5–3 µL) from the tip of a microsyringe needle in an aqueous solution [12, 75], thus drastically reducing the volume of organic solvents used. The analytes are distributed between the microdrop of the solvent at the tip of the syringe and the sample solution [78]. The analyte which is extracted by passive diffusion onto the droplet is retracted back into the syringe and injected directly into the analytical instruments (GC, HPLC or CE). The evaporation and reconstitution of analytes before injection is eliminated and the technique provides highly enriched extracts of the analytes [12].

The SDME extraction can be carried out in direct immersion (in which the droplet is suspended directly into the aqueous sample), and is most suitable for the analysis of medium polar or non-polar analytes or headspace mode (where the droplet is suspended in the headspace of the sample solution) and is suitable for the analysis of volatile or semivolatile analytes. The technique was first reported by Liu and Dasgupta, using a 1.3 µL chloroform suspended in a large aqueous solution containing a methylene blue active substance [79], and later by Jeannot and Cantwell for the extraction of 4-methyacetophenone in aqueous solution, using 8 µL of n-octane suspended at the end of a Teflon rod [80]. The extraction technique was found to be simple, flexible and can easily be coupled to chromatographic instruments. Its coupling with GC and HPLC has been reported and reviewed extensively, while it has also been successfully coupled with ICP-MS, ET-AAS, FI-AAS, MALDI-MS, CE and MS [3, 81]

The use of a Teflon rod as a microdrop holder implies that extraction from the sample and injection of analytes into chromatographic instruments are performed separately with different apparatus [75, 82]. This limitation was overcome by the introduction of a microsyringe as the microdrop holder [83], and the organic solvent can be withdrawn after extraction and injected directly into the GC system [81]. Another limitation is the instability of the microdrop of solvent in the DI-mode at high stirring rate and long extraction times [3]. Therefore, in the development of SDME technique, there is need for the optimization of factors such as selection of extraction solvent, solvent drop volume, agitation rate, ionic strength, extraction temperature and time, sample and headspace volume and automation [76].

Few studies have been published regarding its application for the extraction of pesticide residues from fruit and vegetable samples. The techniques was used for the analysis of 20 multiclass pesticides in apple [84], and in tomato, using toluene [85, 86], analysis of triazole pesticides in water and grape juice samples, with extraction by n-hexanol/n-hexane (50:50,v/v%) [87]. The presence of organochlorine pesticides was also investigated in vegetable samples (cabbage, cauliflower and Chinese cabbage) using SDME-GC/MS using a mixture of 1 µL p-xylene/acetone (80:20, v/v %) [88] and organophosphorus pesticides in fruit juices. [89], and in apple, pear and orange juice samples [90], using toluene as the extraction solvents. Organophophorus and organochlorine pesticides were also determined in cucumber and strawberry using toluene (1.5 µL) [91], while a mixture of octanone and undecanone was used for the extraction of oxazole fungicides from fruits samples [92]. The choice of toluene by most authors was based on the fact that it is stable, less toxic, possesses a higher extraction efficiency and its suitability for direct GC injection. All the developed methods gave comparable analytical figures of merit compared to SPME.

#### **3.2. Hollow fiber liquid phase microextraction (HF-LPME)**

158 Pesticides – Recent Trends in Pesticide Residue Assay

the analysis.

analytes [12].

optimized to avoid the dislodgment of the solvent drop.

**3.1. Single drop microextraction (SDME)**

AAS, MALDI-MS, CE and MS [3, 81]

aqueous mass transfer and exposes the analytes to the extraction solvent, and should be

Salt addition enhances extraction efficiency by reducing the solubility of the analytes in the sample, especially for the moderately polar and low molecular weight analytes. The optimization of extraction temperature is important especially in the headspace mode, but a higher temperature may affect the stability of the microdrop, and for this reason most of the reported analyses have been carried out by DI- mode at room temperature. The sample and headspace volume should be maintained constantly at minimal level, since larger sample volumes will require longer extraction time and affect the stability of the microdrop. In the headspace mode, the optimized volume should be at a level which allows the microdrop to be suspended over the stirred sample, while in the direct immersion mode, the sample volume should only be enough to avoid the sample coming in contact with the septum [3, 76, 77]. The presence of air bubbles should also be avoided in the syringe to avoid errors in

SDME is a microextraction technique, which is based on the suspension of a single droplet of water-immiscible organic solvents (typically 0.5–3 µL) from the tip of a microsyringe needle in an aqueous solution [12, 75], thus drastically reducing the volume of organic solvents used. The analytes are distributed between the microdrop of the solvent at the tip of the syringe and the sample solution [78]. The analyte which is extracted by passive diffusion onto the droplet is retracted back into the syringe and injected directly into the analytical instruments (GC, HPLC or CE). The evaporation and reconstitution of analytes before injection is eliminated and the technique provides highly enriched extracts of the

The SDME extraction can be carried out in direct immersion (in which the droplet is suspended directly into the aqueous sample), and is most suitable for the analysis of medium polar or non-polar analytes or headspace mode (where the droplet is suspended in the headspace of the sample solution) and is suitable for the analysis of volatile or semivolatile analytes. The technique was first reported by Liu and Dasgupta, using a 1.3 µL chloroform suspended in a large aqueous solution containing a methylene blue active substance [79], and later by Jeannot and Cantwell for the extraction of 4-methyacetophenone in aqueous solution, using 8 µL of n-octane suspended at the end of a Teflon rod [80]. The extraction technique was found to be simple, flexible and can easily be coupled to chromatographic instruments. Its coupling with GC and HPLC has been reported and reviewed extensively, while it has also been successfully coupled with ICP-MS, ET-AAS, FI-

The use of a Teflon rod as a microdrop holder implies that extraction from the sample and injection of analytes into chromatographic instruments are performed separately with different apparatus [75, 82]. This limitation was overcome by the introduction of a microsyringe as the microdrop holder [83], and the organic solvent can be withdrawn after

HF-LPME is a multi-phased microextraction system, which was developed to enhance the stability of the organic solvent in 1999 [93, 94]. It is based on the principle of a supported liquid membrane [95], in which the organic solvent is used to fill both the wall pores and the HF lumen. It makes use of a polymeric membrane which forms a barrier between the solvent and the sample [96], and acts as a support for the small volumes of extracting solvents. It involves filling of a few microliters of solvent inside the pores and lumen of a semi-permeable polypropylene hollow fiber attached to a syringe. The HF-LPME can be carried out in either the static or dynamic mode. In the static mode, the acceptor phase is introduced in the lumen followed by the immersion of the fiber into the aqueous sample by a syringe, while in the dynamic mode the HF is attached to a syringe connected to programmable pump [75, 78].

The extraction can be carried out in two- or three phases [95]. In the two-phase mode, the aqueous sample (donor) makes contact directly with the organic solvents (acceptor) through the membrane pores, by a repeated pushing and pulling of the microsyringe plunger [97], and the mass transfer of the analytes is driven by the diffusion of the analytes from the sample matrix into the organic solvent. When the pores are prefilled with an organic solvent, which provides a supported liquid membrane, a three-phase system is formed, and the sample solution and organic solvent are separated by the hollow fiber membrane. The solvent used must be compatible with the membrane, so as to ensure that the pores in the wall of the membrane is completely filled [75]. The technique can also be carried out in the headspace mode, but most researchers have used the direct immersion mode for efficient pre-concentration of the analytes. The limitation of this technique is the issue of carry-over [98], therefore a new membrane should be used for each extraction.

Recent Developments and Applications of Microextraction Techniques for the Analysis of Pesticide Residues in Fruits and Vegetables 161

an extraction solvent with acetone (1 ml) as the dispersive solvent [109], and in water melon using chlorobenzene (27 µL) and acetonitrile (2.5 ml). Carbamate pesticides were also analyzed in banana, pineapple and tomato juice using trichloromethane (800 µL) and methanol (1.5 ml) [110] and in apple using trichloromethane (60 µL) and acetone (1 ml) [111].

Neonicotinoid insecticides was also analyzed in apple using chlorobenzene (9 µL) and acetone (1 ml) [112], and in tomato and cucumber using tricholoromethane (200 µL) and acetonitrile (2.5 ml) [113]. Multiclass pesticides were determined in banana [114], table grape and plum [115], using ionic liquid ([C6MIm][PF6]) based extraction solvent and methanol (714 µL) and in apple juice using tetrachloromethane (100 µL) and acetone (500 µL). It was also used for the analysis of triazophus and carbaryl from apple, grape and peach [116], fungicides from pear, grape and strawberry [117], imidacloprid from toamato [118], using tetrachloroethane and acetonitrile, cypermethrin and permethrin from pear juice using tetrachloroethene and methanol, diethofencarb and pyrimethanil from apple pulp and peel using 1-undecanol and acetonitrile as the extraction and dispersion solvents respectively. The technique was also found to yield a better enrichment factor and low detection limit.

SBSE is also a microextraction technique similar to SPME but with a greater extraction capacity. It helps to overcome the small volume of the coated SPME fiber for a better enrichment factor. In the SBSE technique, a 10 to 40 mm long magnetic stir bar coated with 50 – 300 µL of polydimethylsiloxane (PDMS) is used as the extracting phase [12]. The extraction mechanisms are similar to those of SPME, but the enrichment factor, which is determined by the amount of extractive phase is higher. The analytes are adsorbed on a PDMS coated magnetic rod, by stirring the sample solution with the rod for a given time. The stir bar can then be thermally desorbed into GC or by organic solvent to be subsequently injected to an LC system [119, 120]. The limitation of this technique include the need for reconstitution in a solvent before chromatographic analysis, since the stir bar fiber cannot be directly injected into the split/splitless injection port of a GC [96, 120]. The tedious reconstitution step can lead to introduction of contaminants and loss of analyte. It also

The extraction process like SPME is dependent on the partition coefficients between the analyte and the coated stir bar. The PDME coated fiber is commercially available, and has widely been used, while other polymeric phases such as restricted access materials, sol-gel prepared coatings and carbon adsorbents have also been recently developed. In the SBSE method development, factors such as extraction time and temperature, pH, salt and organic solvent addition, sample volume, agitation speed and volume of acceptor phase are very important and need to be optimized [96, 120] for higher extraction efficiency, recovery and low detection limit. The technique has successfully been applied for the extraction of pesticides residues in fruit and vegetable samples. An organophosphorus pesticide (OPP) was analyzed in cucumber and potato using sol-gel coating stir bar [121]. It has also been used for the extraction of multiclass pesticides in different fruits and vegetable samples [122-

involves longer desorption time, due to the higher amount of stir bar coating.

**4. Stir bar sorptive extraction (SBSE)**

The HF-LPME membranes are capable of accommodating larger volumes of organic solvents which increases the efficiency of the extraction, while the pore acts as a filter which prevents interference of large molecular weight molecules present in the sample matrix. The technique has not been used extensively for pesticides analysis in food samples, therefore only a few studies have been reported for its applications in fruit and vegetable samples. It has been used for extraction of pesticide residues in vegetables using dihexyl ether [99], triazines in watermelon using toluene immobilized on MIP-HF [100], fungicides from orange juice using 2-octanone [101], organochlorine pesticides from tomato and strawberry using a mixture of toluene and hexane [102] and pesticides from grapes with pressurized hot water [103]. The choice of the water-immiscible solvents was based on their ease of immobilization into the HF, with low volatility and high partition coefficients.

#### **3.3. Dispersive liquid-liquid microextraction (DLLME)**

DLLME is a microextraction technique introduced by Assadi and co-workers [104] which also makes use of microliter volume of a mixture of extraction solvent and dispersive solvent. This helps to minimize dislodgement of the organic solvent drop inherent in SDME. It involves the formation of a cloudy solution when an appropriate mixture of high density water-immiscible extraction solvent and dispersive solvents is injected rapidly into an aqueous solution [104, 105], containing the analytes of interest. The hydrophobic solutes are then enriched in the extraction solvents, which are dispersed into the bulk aqueous solution and the mixture is centrifuged, thus, making DLLME a two-step extraction technique. The extractive solvent accumulates at the bottom of the extraction vessel and can be injected with or without further treatment into analytical instruments. The choice of the type and volume of dispersive solvent is as important as that of the extraction solvent, because, it helps the extraction solvent to form fine droplets in the sample matrices. The nature of the fine droplet has been found to enhance extraction efficiency, because of the abundant surface contact between the droplet and the analytes, thus the mass transfer of analytes into the extraction solvent is speeded up [75].