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Pesticides Pesticides

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AbstractIn this chapter we discuss some important aspects

related to the context of the analytical determination of pesticides in food by chromatographic techniques. Top-ics explored a brief introduction to the topic, emphasizing the importance of monitoring of pesticides in foods. Next we discuss the major sample preparation methods used, the gas chromatography technique (GC) and liquid (LC) in the detection and quantification and finally, the prob-lem of pesticide metabolites and breakdown products.

IntroductionThe determination of pesticides, especially in food

matrices, is a topic of great relevance. Since hundreds of pesticides are applied in several crops to combat pos-sible pests that may affect agricultural production [1-3]. Whereas that the presence of amounts in levels of traces, of both pesticide residues and their degradation products are potential causes of health risks, these must be con-trolled and monitored. Therefore, many countries include these analytes as hazardous pollutants to human health. Therefore, monitoring of pesticide residues in food is of great interest to ensure food security. For this reason, nu-merous regulations of various organizations such as the European Union directives established maximum residue limits (MRL) of pesticides in fruits, vegetables, cereals, water and other foods [2-5].

Usually, we can refer to pesticides through a common name or a business name. The formulated product, which

Chapter 1

Relevant Aspects in the Determination of Pesticides in FoodsRonaldo Ferreira do Nascimento*, Fátima Itana Chaves Custódio Martins, Jhonyson Arruda Carvalho Guedes, Vítor Paulo Andrade da Silva and Pablo Gordiano Alexandre Barbosa

Department of Chemical analytical and physical-chemical, Federal University of Ceará, Brazil

*Corresponding Author: Ronaldo Ferreira do Nascimento, Department of Chemical analytical and physical-chemical, Federal University of Ceará, Brazil, Email: [email protected]

First Published July 02, 2016

Copyright: © 2016 Ronaldo Ferreira do Nascimento, et al.

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which per-mits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source.



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is marketed, comprises one or more active ingredients and formulates or excipient substances.The active ingredient is the main component of the product and whose effect is attached to the pesticide. That is, the substance that has an effect on the target organism. The formulates substances are other compounds added as solvents to dilute the ac-tive ingredient function.They also serve to improve some physical and chemical characteristics of the active ingredi-ent, such as the solubility (by cosolvence), spraying capac-ity or stability.

Pesticides can be applied in the form of powder (sol-id), liquid (spray, emulsion, solution) or in gas form (fu-migant). Some are sold ready for use, others require prior preparation (e.g.: dilution).

Pesticides can be classified based on several criteria, including the chemical nature, the chemical group to which it belongs, the target organism, the level of toxicity, time or mode of action, persistence, etc.

As for chemical nature, pesticides can be classified into two major groups: inorganic and organic.

The inorganic usually present as fine powder of crys-talline appearance are stable in the environment and can easily dissolve in water. Are derived from minerals, may be made based on boron, antimony, lime, sulfur, lead or cadmium, for example. The use of inorganic pesticides re-mote antiquity. Its use has been reduced considerably with the rise of organic.

Organic pesticides are characterized by the presence of carbon in their molecular structure and can be natu-rally occurring (extracted from sources like plants) or synthetic (produced in our laboratory).

According to the chemical structures, these com-pounds can be divided into chemical groups such as: tria-zines, carbamates, neonicotinoid, organochlorines, or-ganophosphates, pyrethrins, acetanilides, dinitroanilines, phenylureas, etc. An example for organophosphates and triazines can be seen in Figure 1.

Figure 1: Example for triazines and organophosphates.Organic pesticides have varied and relatively complex

molecular structures, and may present in the same mole-cule many functional groups. Consequently, it is expected



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a great variability in physicochemical properties and how these compounds interact with the components that sur-round (matrix).

Usually, the physical and chemical properties most used to characterize a pesticide are: vapor pressure, wa-ter solubility (S), octanol/water partition coefficient (Kow), dissociation constant (pKa) and DT 50 (degradation time) [7,8].

In the table below (Table 1) we can see the wide varia-tion in these characteristics between pesticides[1].

Table 1: Physical and chemical properties of some pesticides.

VP: Vapour pressure at 25 °C; S: Solubility in water at 20 °C; Kow: Octanol/water partition coefficient at pH7,20 °C; pKa:dissociation constant at 25 °C; DT 50: typical degra-

dation time (aerobic).

Taking as an example the solubility, we observed val-ues ranging from 9x10-3 to 6,2x104mg L-1corresponding to paraquat and cypermethrin respectively. In some cases, the pesticide is liable suffer dissociation and not in others.

Because of great diversity of structures and phys-icochemical characteristics, the nature of intermolecular interactions involved with matrix may also vary widely. These interactions can be ionic, hydrogen bond, covalent bonds, dipole, and van der Waal forces, hydrophobic in-teractions or partitioning. Two or more types of interac-tion can simultaneously occur between the same molecule and the matrix [9].

These are particular characteristics and often so di-vergent that hinder the laboratory analysis of monitoring pesticide residues in food.

Another factor is the very different chemical compo-sition among the many types of food (e.g.: are each acids, other fatty, others have many pigments) that interferes with the extraction and analysis by matrix effect.

Thus, methods of analysis should be assessed and ap-propriate case by case in order to obtain reliable results.

Methods of Sample Preparation for Determination of Pesticide Residues in Food Matrices

The determination of pesticides residues in food ma-trices is a difficult task because of the analytes usually

Pesticide Group VP (mPa) S (mg mL-1)

Kow at pH 7, 20 °C

pKa (25°C) DT 50 in soil (day)

Atrazine Triazine 0.039 35 5.01x102 1.7 752,4-dichlorophenoxya-cetic acid

Alkylchlorophenoxy

0.009

24300 1.51x10-1 3.40 4.4

Paraquat Bipyridylium 0.01

620000 3.16x10-5

No dissoci-ation

3000

Methiocarb Carbamate 1.50x10-2 27 1.51x103 No dissoci-ation

1.4

Chlorpyrifos Organophosphate 1.43

1.05 5.01x104

No dissoci-ation

50

Malathion Organophosphate 3.1

148 5.62x102 No dissoci-ation

0.17

MCPA Aryloxyalkanoicacid 0.4 29390 1.55x10-1 3.73 24Cypermethrin Pyrethroid 0.00023 0.009 2.00x105 No dissoci-

ation60



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present very low concentrations, distinct chemical prop-erties, complexity of matrices. Thus is necessary a prelimi-nary stage of sample preparation. Moreover, given the fact that the measurements are typically made at low concen-tration levels, interferences are frequent issues that should be considered[10-12] Thus, samples typically cannot be directly analyzed by analytical instruments requiring a prior sample preparation. Therefore, the sample prepara-tion aims to isolate/concentrate/extract analytes in a given matrix, moreover, it is important to obtain a fraction of the sample interfering free.

Mills and co-workers developed the first method for pesticides multiresidue extraction in 1963, in the labora-tory of the Food and Drug Administration (FDA). The method is based on an extraction with acetonitrile is used basically in the determination of nonpolar organochlorine compounds in non-greasy samples[13-14]. Through the years, there were many sample preparation procedures for pesticide extraction, among the most common include: Storherr Method[10,13], Luke method [10,13,15], mini-Luke extraction method, this last one has a miniaturiza-tion of the original Luke method [11].

Many of sample preparation procedures are carried out by conventional techniques such as liquid-liquid ex-traction (LLE) and other techniques mentioned above. However, they have the disadvantages of being expensive and uses large amounts of organic solvents, which are tox-

ic for the analyst and may contaminate the environment, since they are generated large amounts of waste. These limitations leading to the development of new and more convenient techniques, as they consume less organic sol-vents and have the ability to detect analytes at low concen-tration levels [10,16-18]. Thus, during the 1990s due to the strong environmental pressures and the factors associated with the human health, efforts in the field of analytical chemistry are focused in the miniaturization of the sample preparation associated with increased selectivity and sen-sitivity. Therefore, there was a great development of alter-native extraction methods based on the reduction of the volume of solvent used in the extraction step[10,11,13]. Among these alternative methods, we can mention the Solid Phase Extraction (SPE),Matrix Solid Phase Disper-sion (MSPD) and Solid Phase microextraction (SPME) which were developed with the aim of simplifying steps. Furthermore, we mention yet: Stir Bar Sorptive Extrac-tion (SBSE), which provides low detection limits, espe-cially for hydrophobic analytes; Supercritical Fluid Ex-traction (SFE);Accelerated solvent extraction (ASE) and Micro¬wave Assisted Extraction (MAE) [10-13]. The lit-erature report several studies on the applications of these sample preparation techniques for the extraction and de-termination of pesticide residues in various foods such as fruits, juices, vegetables, milk, grains, etc. Moreover, it is important to mention that each sample preparation meth-od has its advantages and disadvantages, and the choice of



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the most appropriate method for analysis depends on the type of sample and/or the type of analyte to be determined [12]. The disadvantage often presented in these sample preparation methods is the limited scope of pesticides that may be extracted under certain conditions. Therefore, these procedures can be employed in some applications, but they are distant from being considered ideal for mul-tiresidue method [11-13].

Thereby, in 2003 Anastassiades, Lehotay, Štajnbaher and Schenck, reevaluated the conditions often practiced in multiresidue pesticides analysis, therefore, aiming to over-come limitations of multiresidues methods available at the time, proposed a new staging procedure samples prepara-tion called by the acronym QuEChERS, which represent-ing details of the method assigned by the authors: “Quick”, “Easy”, “Cheap”, “Effective”, “Rugged” and “Safe” [20-22]. Currently, the sample preparation technique most used multiresidues extraction of pesticides in food matrices is the QuEChERS method. Since the method omit or replace many complicated analytical steps that are com-monlyused in traditional multiresidues extraction meth-ods. Thus, the method provides high-quality results with a minimum number of analytical steps, low consumption of organic solvents, non-chlorinated solvent use and extrac-tion of different polarities and different chemical classes pesticides [20,21,23]. The method QuEChERS ability to extract various compounds of different chemical classes is a major advantage over traditional methods that typically

possible to extract only one analyte or multiple analytes of the same chemical class [10]. Furthermore, proficiency testing employing QuEChERS method demonstrate that the method is highly robust, and successfully transferred between the participating laboratories [24].

The procedures involved in applying the QuEChERS method are characterized as a sequence of the following steps: extraction, phase separation and cleanup. First of all, an extraction step is performed with acetonitrile fol-lowed by separation of the phases promoted by addition of the salt mixture (MgSO4 and NaCl). Subsequent to extrac-tion with acetonitrile and the phase separation with the mixture of salts is execute the cleanup. A new method of the cleanup called Dispersive Solid Phase Extraction (SPE-D) has been suggested together with QuEChERS method, which is added to extract the mixture of sorbent Primary Secondary Amine (PSA) and MgSO4[3,22].Although the original QuEChERS method has proven to be efficient for hundreds of analytes in a wide variety of matrices, since it was very robust, particularly for pesticides in various food matrices. Modifications were accomplished with the in-tention to improve the method of characteristics for some analytes and certain complex matrices [25-36]. Many of these changes arose from the need to obtain better recov-ery rates for some pesticides in matrices of different com-plexity, to prevent degradation of pesticides and to reduce the effects of matrix [3-10].



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The first and maybe the most significant modifica-tions were developed to expand the method to some pes-ticides that are ionized and/or degraded during the ex-traction, depending on the pH of the matrix [3]. Thus, the first modification proposed for QuEChERS method was the addition of a buffering step aimed at improving the percentage recovery for some pesticides. Therefore, it was developed acetate QuEChERS method, where the buffer-ing effect (pH 4.8) is promoted by addition of sodium ac-etate. This method was adopted in 2007 by the Association of Official Analytical Chemists (AOAC) as official method for the determination of pesticide residues [3,10,37,38]. It was also developed citrate QuEChERS method, where the buffering effect (pH 5.0 to 5.5) is caused by the addition mix of sodium citrate dihydrate and hidrogenocitrato so-dium sesquihydrate. In 2008, the European Committee for Standardization (CEN) officiated the citrate QuEChERS method as the reference method of the European Union [3,13,39]. Versions of the method QuEChERS (citrate and acetate) allow satisfactorily extract the pesticides which are sensitive to acidic or basic conditions (e.g.:folpet, di-chlofluanid, pymetrozine, chlorothalonil), independently of the matrix. These versions have been extensively evalu-ated and adopted as a routine method in many laborato-ries around the world [3,40]. The procedures involved in the execution of the method QuEChERS (original, acetate and citrate) are summarized in Figure 2. Another impor-tant change in QuEChERS method is in the cleanup step, which changes included the use of different amounts of

PSA and the addition of C18 in order to obtain extracts cleaner [25,30,40].

Figure 2: Summary of the procedures involved in the ex-ecution of QuEChERS method (a) Original, (b) acetate (c)

citrate.Initially, the development of the QuEChERS meth-

od aimed to multiresidues extraction of pesticides in food. Thus, most of the applications of the QuEChERS method reported in the literature is the determination of pesticides in food matrices, as can be seen in Table 2 [25,28,30,32,33,36,41]. However, since its development the method has undergone several modifications for ex-traction of different analytes in various matrices, this fact can be attributed to the simple steps for applying the method. Therefore, a wide applicability of the method has been observed in several publications for various matrices



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and analytes for different techniques of the determination (Table 2), such publications denote the versatility of the method with regard to its various applications.

Table 2: Several applications of the QuEChERS method reported in the literature.

GC and LC Application in the Detec-tion and Quantification

The chromatography is an analytical technique that was used for the first time, the Russian botanical Mikhail Semenovich Tswett (1901) which separated color pig-ments obtained from extracts of green leaves using a glass column packed with calcium carbonate [45]. The word “chromatography” derives from the Greek χρώµα: “chro-ma” meaning “color” and γραφειν: “grafein” which means “writing”, and thus a separation technique that can be con-veniently used to identify chemical compounds, among which we can highlight the pesticides [46].

Pesticides have been widely used on crops to control the spread of pests and increase the quality and quantity of food needed to supply the population. However, this may result in persistence of the setoxic substances in food and other matrices, which may cause harm to humans and the environment [47,48]. These active substances (herbicides, insecticides, fungicides, acaricides), are applied to fruit and vegetables at various stages of cultivation and post harvest, to protect them from pests attack and keep them fit for consumption [48].

Chromatography is one of the techniques used cur-rently and better performance in the determination and control of organic substances in different matrices, among the advantages associated with its use, can be highlighted high selectivity, separation efficiency and good peak res-olution. For a long time, gas chromatography (GC) has

Analyte Matrix Analysis Technique Reference

107 Pesticides Cabbage GC-MS 20

21 Pesticides Guava GC-MS 25

Tocopherols and sitosterols

Seeds and nuts HPLC 26

Macrocycliclactones Bovine liver HPLC 27

14 Pesticides Tamarind GC–ECD 28

20 Pesticides Cashew LC-ESI-MS/MS 30

14 PAHs Ambient air and emission samples

HPLC 31

36 Pesticides Lotus seeds GC–ECD 32

35 Pesticides Melons GC-MS 33

Volatile

Phenols

Beer, Wines and fruit juices

HPLC 34

Benzodiazepines Water, blond and urine

GC-MS 35

14 Pesticides Milk GC–ECD 36

9 Pesticides Shrimp GC-MS 41

Sulfonamides Fish LC-ESI-MS/MS 42

Pharmaceuticals and personal care

products

Drinking-water treatment sludge

UPLC-ESI-MS/MS 43

5 Mycotoxins Popcorn GC-MS 44



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been the most applied technique for the determination of residues of organic pollutants in food, such as pesticides [11,48,49]. However, in recent years, one can observe a tendency to use more polar pesticides, which have lower persistence and toxicity as compared with non polar tra-ditionally used. The ionic polar compounds does not show good response when analyzed by GC, thereby liquid chro-matography (LC) is now used as an alternative technique for determining these compounds [11,50]. The Table 3 show some studies using GC and LC in the identification and quantification of some pesticides.

Table 3: Analytical methods used to determine the differ-ent pesticides in food samples.

* SPE= liquid-liquid extraction; d-SPE= solid phase ex-traction dispersive; ET-DLLME = Elevated temperature dispersive liquid-liquid microextraction; VLDS–SD–DLLME = vortex-assisted low density solvent based sol-

vent demulsified dispersive liquid–liquid micro extraction.

To ensure the safety of the population and food con-sumption, government agencies around the world per-form a control of the presence of contaminants that do not generate damage to the health of the consumer setting, therefore, maximum residue limits (MRL). The analyti-cal methods proposed for the determination of toxic sub-stances in food must be able to quantify residues of these substances in very low concentrations, as well as identify them unequivocally to ensure that MRLs are respected. Research on methods of development field allying analyti-cal techniques to previous stages of sample preparation, allow to obtain increasingly selective and sensitive meas-urements with detection and quantification limit values (LOD and LOQ) increasingly smaller, It makes it possible to establish MRLs increasingly lower [50].

Martins (2015) [58] developed a method for the de-termination of pesticide residues in7 different classes in mango fruits using GC/MS. The compounds analyzed were: (1) etridiazole, (2) chloroneb, (3) propachlor, (4) trifluralin, (5) hexachlorobenzene, (6) chlorotha-lonil, (7) Chlorpyrifos, (8) DCPA, (9) α-chlordane, (10) γ-chlordane, (11) chlorobenzilate, (12) trans-permethrin and (13) cis-permethrin.

In this paper, the authors conducted a sample clean-ing step using QuEChERS as extraction method, due to the notable presence of pigments and other components of the matrix. They also evaluated the use of gas chroma-tography-GC, and detection of compounds performed by mass spectrometer(MS).

Pesticide class Matrix Sample Preparation

Analytical Method LD Ref.

26 pesticidas: multiclasses

Cocoa Solvent extraction

GC-MS/MS and LC-MS/MS

10 µg kg-1 51

5 pesticides multiclasses

tomato, orange juice, grapefruit

juice, lemon and

tangerine

LLE HPLC-DAD 0.32-12 µg L-1 52

Triazoles honey ET-DLLME* GC-NPD 0.05-0.21 ng g-1 5346 pesticides cashew, apple,

guava, kaki and peach

LLE + d-SPE GC-ECD, GC-FPD and LC-MS/MS

1-8 μgkg-1 54

Organophosphates water VLDS–SD–DLLME

HPLC-DAD 0.25-1 ng mL-1 55

160 pesticides grape LLE + d-SPE GC×GC-ToFMS ≤10 µg L-1 56107 pesticides:

multiclassesCabbageand

radishQuEChERS GC-MS 0.002 mg kg-1 57



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For detection, was used a quadrupole mass analyzer, the ionization mode by electron impact (EI). Thus, when making the analysis of different compounds using MS, several parameters must be optimized for each, such that fragments are selected for quantitation and confirmation of peaks and the retention time window.

The identification of the pesticides was performed based on retention time window and comparison with reference spectra obtained from a mango extract fortified with uncontaminated concentration calibration curve. Limits of Detection were obtained (LOD) and Quantita-tion (LOQ) of 0.003 and 0.010 mgkg-1 and between 0.008 and 0.020 mgkg-1, respectively, for the 13 pesticides. An analytical curve was plotted to determine linearity of matrix free extracts using the analyte and fortified with standard solutions of compounds, where in the first point of the curve was determined at a concentration between LOQ and the lowest MRL found for the pesticides. In the validation of the developed method, the authors showed good linearity in the studied concentration range and also good levels of precision and accuracy. This analysis proce-dure was applied to real samples, which makes it suitable for routine analysis. In Figure 3 can be seen from the chro-matogram of a spiked sample with 1mg kg-1of analytes.

Figure 3: Ion chromatogram of totals - ICT pattern 13 Pesticides (1 mg kg-1) obtained by GC-SQ / MS, Full Scan Mode (m/z 50-430). Column: DB-5 30m x 0.25mm ID x 0:25 µm; carrier gas: helium (1mL min-1); gun: splitless

(1min), 250 °C; Temperature Program.

As in the example shown above, and the data provided in the Table3, many jobs involving multiresidue analysis of pesticides have been performed in different matrices. The complexity of arrays and high diversity of compounds that is (mixture of water, proteins, lipids, carbohydrates, vitamins and minerals), creates a critical problem in the quantification of pesticides by chromatography, this prob-lem is called matrix effect. Thus the matrix effect can be understood as the result of the sample coextractives in the reference parameters that affect analytical performance such as precision and accuracy, reducing the quality of the proposed method [47,59,60].



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During the analysis, the effect matrix can influence the separation, chromatographic identification and quan-tification of pesticides, this effect is also known as increas-ing or decreasing the chromatographic response induced matrix, allowing the explanation of the recovery levels that exceed 100% for some pesticides in analysis using calibra-tion by external standard in pure solvents. The matrix ef-fect can also cause other changes in the chromatographic analysis, for example:(i) masking the peak of the analyte of interest, generating a false negative result; (ii) the oc-currence of false positive due to ambiguous identification matrix components as the compound of interest, in the absence thereof; (iii) errors in measurement due to the in-crease of the detector signal, leading to over estimation of the result [47].

It is well known that the main problem associated with dealing with these kinds of analysis arrays is that the dirty extracts, even with a small amount off at, can damage the analytical column used in experiments and adversely affect the instrumental source analysis and detection of ions, and finally affect analyte determination suppression or enrichment signal. Thus, the development of analyti-cal methods increasingly sensitive, selective, reproducible, and fast has always been a prerequisite for achieving high-quality results monitoring samples [11].

Chromatographic techniques can be combined with different detection systems. Therefore, currently, they are among the most widely used analytical tools and better performance. Liquid chromatography coupled to Mass

spectrometry (LC/MS) is one of the most used techniques for the analysis of pesticide residues ionic polar, low vola-tility or thermal instability. LC is very effective in the sepa-ration of analytes while the MS enables identification and/or verification level dashes.

For a complex mixture of analytes, one time of flight (ToF) analyzer can perform simultaneous analysis of a large number of compounds in a reasonably short time and with good accuracy in analyzing. This becomes a bit more complicated when use mass analyzers with type slower scan as quadrupoleor ion trap (IT). In case of mul-tiresidue analysis by simple GC/MS, separation of a mix-ture of many components is often a challenging task in view of the limited capacity and co-elution of interfering compounds resulting matrix. To ensure the desired level of selectivity and sensitivity analytical, checking out better quality and reliability of the results, the monitoring and analysis laboratories generally adopt more selective detec-tion techniques such as tandem mass spectrometry, mass spectrometry/mass spectrometry or MS/MS.

Furthermore, this detection method tandem mass spectrometry becomes possible individual compounds, if they have different molecular weights or masses gen-erate different spectrums. Thanks to the high selectivity, the effects of interfering matrix components of the signal obtained are minimized, so that simpler procedures for preparation of the samples may be employed, eliminating often the need for various sample cleanup steps. This re-



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duces the cost and time required to perform the analysis, so that they can be applied as routine procedures in the quality of food control laboratories.

Another way to increase the quality of pesticide mon-itoring methods is chromatography application in two-dimensional gas phase (GC × GC). This technique was developed in 1991 by Liuand Phillips, and involves the coupling of two columns with different retention mecha-nisms, allowing thereby adding benefits such as increased resolution; increased sensitivity due to the solute band reconcentration in the second column; structuring the two-dimensional chromatogramin accordance with the chemical characteristics of the compound and increase the peak capacity [56,61].

The Problem of Metabolites and Degradation Products in the Pesticides Analysis

In analytical determination and evaluation of pes-ticide residues in food or even other matrices there is a complicating factor to consider, the existence of pesti-cides metabolites and degradation products. Metabolites and degradation products are compounds derived from the pesticide, which considered the parent compound (precursor). However, it must distinguish conceptually both. A metabolite is a chemical intermediate or a prod-uct result from metabolic processes, or biologically pro-duced. A degradation product is a compound generated by environmental effects, such as temperature, pH, among

others, not necessarily produced by metabolic processes [62,63].In some cases, products produced from biological processes are similar to degradation products [64].

A classic example is the case of the pesticides that have thionophosphate groups (P=S) that can be oxidized to biologically active oxons (P=O group). The pesticide parathion-methyl illustrates this case, so that its main toxic effect is attributed to its metabolite methyl paraoxon (Figure 4) [63].

Figure 4: Structural representations of the pesticide para-thion-methyl and its metabolite paraoxon-methyl.

The organochlorine pesticide dicofol shows signifi-cant instability when exposed to conditions of high tem-perature, light and basic pH, readily converting to its deg-radation product 4,4’-dichlorobenzophenone (Figure 5) [64]. Thus, in the case of oxidation of parathion-methyl to paraoxon-methyl, by biological means, we have a metabo-lite, as in the case of dicofol converting 4,4’-dichloroben-zophenone by physical or chemical ways (environmental conditions), we have a product degradation.

The existence of metabolites and degradation prod-ucts become more complex the task of the establishment



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and regulation of maximum residue level for pesticide (MRLs) by the government agencies and regulatory bod-ies. The maximum residue level (MRL) of a pesticide can be defined as the maximum amount of this pesticide, or the like, officially accepted in food, expressed in mg kg-1.Considering this concept, from a toxicological point of view, is not enough to determine the concentration only target pesticide (parent compound) in the food sample, but also of its major metabolites or degradation products [63].

Figure 5: Structural representations of dicofol and its deg-radation product 4,4’-dichlorobenzophenone formed by

the action of light, high pH or high temperatures.

In the case of a pesticide having two or three metab-olites/degradation products known to be important, the evaluating of MRL must be based on the sum of the con-centrations of the target pesticide and its metabolites (re-lated substances) detected in the sample. The captan pes-ticide, for example, has recognized instability, with some of its main metabolites/degradation products the com-pounds tetrahydrophtalamide and cyclohex-4-en-1,2-di-

carboxylic acid (Figure 6) [63].

Another important aspect related to metabolites and MRLs is the fact that for each crop or food will be a par-ticular MRL value for a given pesticide. For pesticide cap-tan the European Commission (EC) establishes the MRL of 0.02 mg kg-1 in citrus fruits (orange, tangerine, lemon), as for apples and pears this value is less restrictive of 3.0 mg kg-1.Once determined the active ingredient captan to a level of 2.50 mg kg-1 in a sample of apples is important to assess whether the total concentration of its metabolites in this sample does not exceed 0.5 mg kg-1, therefore, if it occurs, the sample would not be appropriate as the con-centration of captan.

Figure 6: Structural representations of the captan pesti-cide molecules and their metabolites and tetrahydroph-

talamida and cyclohex-4-en-1,2-dicarboxylic acid.As there is a very wide variety of pesticides, the lit-

erature also reports a variety of metabolites, degradation



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products known for many of these compounds. In addi-tion to the compounds already mentioned Table 4 reports some important pesticides and some of its metabolites.

Table 4: Important pesticides and some of its main metabolites.

aThis compound can also be metabolite of other pyrethroids.

There are recognized problematic pesticides groups with regard to its analytical determination due to its deg-

radation products and metabolites, mainly by analytical methods based on gas chromatography (GC).Among them, we can highlight compounds with functional group N-trihalomethylthio, which includes captan, quoted above, and others like dichlofluanid, folpet, tolylfluanid. The dichlofluanid has as its major degradation product N’, N’-dimethyl-N-phenylsulfonyldiamide and folpetthe compound phthalimide, for example [64,65].

Pesticides thioether group (-S-) are another class that tend to form degradation products/metabolites, as they may undergo oxidation processes usually converting to sulfoxides and subsequently to sulfones, so that these degradation products are also included in the pesticide residue definition. Compounds such as organophosphate fenthion and fenamiphos and the carbamate methiocarb are relevant representatives of this group (Figure 7) [64].

Figure 7: Structural representation of fenthion and their sulfoxides and sulfones metabolites.

Within a perspective of the development of analytical methods for the determination of pesticides listed above, in food samples, it is important to emphasize the fact that a method developed to detect the three target com-pounds, fenthion, fenaminfós and methiocarb should not

Pesticide Chemical group Metabolites

Cypermethrin Pyrethroid

3-phenoxibenzoic acida;

3-(2,2-dichloroethenyl(-2,2-di-methyl cyclopropane carboxylic acid

Chlorpyrifos Organophosphate 3,5,6-trichloro-2-pyridinol

Chlorothalonil

Chloronitrile

4-hydroxy-2,5,6-trichloroiso-phtalonitrile;

2-amido-3,5,6-trich-lo-4-cyanobenzenesulphonic acid;

3-carbamyl-2,4,5-trichloroben-zoic acid

Cyproconazole Triazole 1,2,4-triazole

Etridiazole Thiodiazolic ether

Dichloroetridiazole;

Etridiazole acid



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only be able to detect these three compounds. In fact, the developed method should have adequate analytical per-formance for determining nine compounds, comprising the three pesticides in their native form and the sulfoxides and sulfones metabolites associated.

Given the significantly similar molecular structures, it expected that the chromatographic separation of this group of compounds is difficult. This case is an interesting example of the challenges that metabolites and degrada-tion products can provide to researchers and professionals working in the determination of pesticides in foods.

ConclusionThe determination and monitoring of residue levels

of pesticides and other toxic components in food are in-dispensable tools for the control of food safety and public health. The carry out of research in development of ana-lytical methods more efficient and reliable for the detec-tion and quantification of pesticides and their metabolites in food is essential to the supervisory institutions and governments can control more effectively the application of these products in agriculture, as well as to assess more realistically the level of intake of these compounds by so-ciety through food.

The field of multiresidue determination of pesticides in food, despite advances over recent decades as the ad-vent of the QuEChERS technique still has major challeng-es, given the significant pesticide universe still difficult to detect in some food samples by analytical methods mod-ern.

As discussed briefly in this chapter, the question of metabolites is also shown as one of the points to be ex-plored more broadly, given that most of the methods cur-rently developed is focused only on the detection of target pesticides.

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