Analysis of Pesticides in Food and Environmental Samples - Chapter 2 pdf

Pesticides from liquidsamples i.e., environmental waters are preferably extracted using solid phases bysolid-phase extraction SPE or solid-phase microextraction SPME procedures,although

2 Sample Handling of

Pesticides in Food and Environmental Samples Esther Turiel and Antonio Martín-Esteban

CONTENTS

2.1 Introduction 36

2.2 Sample Pretreatment 36

2.2.1 Drying 37

2.2.2 Homogenization 37

2.3 Extraction and Purification 38

2.3.1 Solid–Liquid Extraction 38

2.3.1.1 Shaking 39

2.3.1.2 Soxhlet Extraction 41

2.3.1.3 Microwave-Assisted Extraction 42

2.3.1.4 Pressurized Solvent Extraction 42

2.3.2 Supercritical Fluid Extraction 43

2.3.3 Liquid–Liquid Extraction 45

2.3.4 Solid-Phase Extraction 45

2.3.4.1 Polar Sorbents 46

2.3.4.2 Nonpolar Sorbents 48

2.3.4.3 Ion-Exchange Sorbents 49

2.3.4.4 Affinity Sorbents 49

2.3.5 Solid-Phase Microextraction 52

2.3.5.1 Extraction 53

2.3.5.2 Desorption 54

2.3.6 Solid–Solid Extraction: Matrix Solid-Phase Dispersion 54

2.3.7 Other Treatments 55

2.3.7.1 Stir Bar Sorptive Extraction 55

2.3.7.2 Liquid Membrane Extraction Techniques 55

2.4 Future Trends 56

References 56

2.1 INTRODUCTION

The determination of pesticides in food and environmental samples at low trations is always a challenge Ideally, the analyte to be determined would be already

concen-in solution and at a concentration level high enough to be detected and quantified

by the selectedfinal determination technique (i.e., HPLC or GC) Unfortunately, thereality is far from this ideal situation Firstly, the restrictive legislations fromEuropean Union and World Health Organization devoted to prevent contamination

of food and environmental compartments by pesticides make necessary the ment of analytical methods suitable for detecting target analytes at very low concen-tration levels Besides, from a practical point of view, even when the analyte isalready in solution (i.e., water or juice), there are several difficulties related to therequired sensitivity and selectivity of the selected determination technique that must

develop-be overcome, since the concentration of matrix-interfering compounds is muchhigher than that of the analyte of interest Consequently, the development of anappropriate sample preparation procedure involving extraction, enrichment, andcleanup steps becomes mandatory to obtain afinal extract concentrated on targetanalytes and as free as possible of matrix compounds

In this chapter, the different sample treatment techniques currently availableand most commonly used in analytical laboratories for the analysis of pesticides infood and environmental samples are described Depending on the kind of sample(solid or liquid) and the specific application (type of pesticide, concentration level,multiresidue analysis), thefinal procedure might involve the use of only one or thecombination of several of the different techniques described later

2.2 SAMPLE PRETREATMENT

Generally, sampling techniques provide amounts of sample much higher (2–10 L ofliquid samples and 1–2 kg of solid samples) than those needed for the final analysis( just few milligrams) Thus, it is always necessary to carry out some pretreatments toget a homogeneous and representative subsample Even if the sample is apparentlyhomogeneous, that is, an aqueous sample, it will be at least necessary to perform afiltration step to remove suspended particles, which could affect the final determin-ation of target analytes However, some hydrophobic analytes (i.e., organochlorinepesticides) could be adsorbed onto particles surface and thus, depending on theobjective of the analysis, might be necessary to analyze such particles This simpleexample demonstrates the necessity of establishing clearly the objective of the ana-lysis, since it will determine the sample pretreatments to be carried out, and high-lights the importance of this typically underrated analytical step

Usually, environmental water samples just require filtration, whereas liquidfood samples might be subjected to other kinds of pretreatments depending on theobjective of the analysis However, solid samples (both environmental and foodsamples) need to be more extensively pretreated to get a homogeneous subsample.The wide variety of solid samples prevents an exhaustive description of the differentprocedures in this chapter; however, some general common procedures will bedescribed later

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2.2.1 DRYING

The presence of water or moisture in solid samples has to be taken into accountsince it might produce alterations (i.e., hydrolysis) of the matrix and=or analytes,which will obviously affect thefinal analytical results Besides, water content variesdepending on atmospheric conditions and thus, it is recommended to refer thecontent of target analytes to the mass of dry sample

Sample drying uses to be carried out before crushing and sieving steps, although

it is recommended drying again beforefinal determination since rehydration processmight occur Typically, sample is dried inside an oven at temperatures about 1008C

It is important to stress that higher temperatures can be used to decrease the timedevoted to this step but losses of volatile analytes might occur In this sense, it isimportant to know a priori the physicochemical properties of target analytes topreserve the integrity of the sample A more conservative approach, using lowtemperatures, can be followed but it will unnecessarily increase the drying time.Alternatively, lyophilization is recommended if a high risk of analytes losses existsand it is an appropriate procedure for food, biological material, and plant samplesdrying However, even following this procedure, losses of analytes might occurdepending on their physical properties (i.e., solubility, volatility)

The results are evident that it is not possible to establish a general rule on how toperform sample drying Thus, studies on stability of target analytes in spiked samplesshould be carried out to guarantee the integrity of the sample beforefinal determin-ation of the analytes

to minimize sample heating In addition, due to heating, water content may varymaking necessary to recalculate sample moisture

Food samples use to be cut down to small pieces with a laboratory knife beforefurther homogenization with automatic instruments (i.e., blender) Sample freezing is

a general practice to ease blending, especially recommended for samples with highfat content (i.e., cheese) and for soft samples with high risk of phase separationduring blending (i.e., liver, citrus fruits)

Apart from these general guidelines, especially in food analysis, the ation of pesticides might be restricted to the edible part of the sample or to samplespreviously cooked and thus, sample pretreatments will vary depending on theobjective of the analysis

determin-Finally, it is important to point out that, in most of the cases, samples need to bestored for certain periods of time before performing the analysis In this sense,although sample storage cannot be considered a sample pretreatment, the addition

of preservatives as well as the establishment of the right conditions of storage (i.e., atroom temperature or in the fridge) to minimize analyte=sample degradation aretypical procedures carried out at this stage of the analytical process and need to betaken into account to guarantee the accuracy of thefinal result.

2.3 EXTRACTION AND PURIFICATION

The main aim of any extraction process is the isolation of analytes of interest fromthe selected sample by using an appropriate extracting phase Pesticides from liquidsamples (i.e., environmental waters) are preferably extracted using solid phases bysolid-phase extraction (SPE) or solid-phase microextraction (SPME) procedures,although for low volume samples, liquid–liquid extraction (LLE) can also be carriedout Extraction of pesticides from environmental or food solid samples is usuallyperformed by mixing the sample with an appropriate extracting solution, where themixture is subjected to some process (agitation, microwaves, etc.) to assist migration

of analytes from sample matrix to the extracting solution For certain applications,matrix solid-phase dispersion (MSPD) can also be a good alternative In all cases,once a liquid extract has been obtained, it is subsequently subjected to a purificationstep (namely cleanup), which is usually performed by SPE or LLE In some cases,extraction and cleanup procedures can be performed in a unique step (i.e., SPE withselective sorbents), which enormously simplifies the sample preparation procedure.2.3.1 SOLID–LIQUIDEXTRACTION

As mentioned earlier, solid–liquid extraction is probably the most widely usedprocedure in the analysis of pesticides in solid samples Solid–liquid extractionincludes various extraction techniques based on the contact of a certain amount ofsample with an appropriate solvent Figure 2.1 shows a scheme of the different steps

Solvent Organic matter

5 4

1 2 3 A

6

FIGURE 2.1 Scheme of the different steps involved in the extraction of a target analyte

A from a solid particle

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that take place in a solid –liqu id extra ction procedu re and will infl uence the fi nalextractio n ef ficiency In the fi rst stage (step 1), the solve nt must pene trate inside thepores of the samp le particula tes to achiev e desorp tion of the analyt es bound to mat rixactive sites (ste p 2) Su bsequen tly, analytes ha ve to diff use throu gh the mat rix(step 3) to be d issolved in the extra cting solve nt (step 4) Again, the analytes mustdiffuse through the solve nt to leave the samp le po res (step 5) an d be finally swe ptaway by the exter nal solve nt (ste p 6) Obvi ously, the proper selection of the solve nt

to be used is a key factor in a soli d–liquid extractio n procedu re However , otherparameter s such as press ure and temperat ure have an imp ortant in fluence on theextractio n ef ficiency Worki ng at high pressure facil itates the solvent to penetr atesample pores (ste p 1) and, in general, incre asing temperat ure incre ases solub ility ofthe analytes on the solve nt Moreo ver, high temperat ures incre ase diffusion coef fi-cients (steps 3 and 5) and the ca pacity of the solve nt to disr upt mat rix–analyt einteractions (step 2) Depending on the strength of the interaction between theanalyte and the sample matrix, the extraction will be performed in soft, mild, oraggres sive condit ions Table 2.1 shows a summary and a compa rison of drawbacksand advantages of the different solid–liquid extraction techniques (which will bedescribed later) most commonly employed in the analysis of pesticides in food andenvironmental samples

2.3.1.1 Shaking

It is a very simple procedure to extract pesticides weakly bound to the sample and isvery convenient for the extraction of pesticides from fruits and vegetables It justinvolves shaking (manually or automatically) the sample in presence of an appro-priate solvent for a certain period of time The most commonly used solvents areacetone and acetonitrile due to their miscibility with water making ease the diffusion

of analytes from the solid sample to the solution, although immiscible solvents such

as dichloromethane or hexane can also be used for the extraction depending on theproperties of target analytes In a similar manner, the use of mixtures of solvents is atypical practice when analytes of different polarity are extracted in multiresidueanalysis Once analytes have been extracted, the mixture needs to befiltered beforefurther treatments Besides, since volume of organic solvents used following thisprocedure is relatively large, it is usually necessary to evaporate the solvent beforefinal determination

However, shaking might not be effective enough to extract analytes stronglybound to the sample In order to achieve a more effective shaking, the use ofultrasound-assisted extraction is recommended Ultrasound radiation provokesmolecules vibration and eases the diffusion of the solvent to the sample, favoringthe contact between both phases Thanks to this improvement, both the time and theamount of solvents of the shaking process are considerable reduced

An interesting and useful modification for reducing both the amount of sampleand organic solvents is the so-called ultrasound-assisted extraction in small columnsproposed by Sánchez-Brunete and coworkers [1,2] for the extraction of pesticidesfrom soils Briefly, this procedure just involves placing the sample (~5 g) in a glasscolumn equipped with a polyethylene frit Subsequently, samples are extracted with

TABLE 2.1

Solid–Liquid Extraction Techniques

Shaking Samples and solvent are placed in a glass vessel.

Shaking can be done manually or mechanically

. Simple . Filtration of the extract is necessary. Fast (15–30 min) Dependent of kind of matrix. Low cost Moderate solvent consumption (25–100 mL) Soxhlet Sample is placed in a porous cartridge and

solvent recirculates continuously by distillation –condensation cycles

. Standard method . Time-consuming (12–48 h)

. No furtherfiltration of the extract necessary High solvent volumes (300–500 mL)

. Independent of kind of matrix . Solvent evaporation needed. Low cost

USE Samples and solvent are placed in a glass vessel and

introduced in an ultrasonic bath

Fast (15–30 min) Filtration of the extract is necessary. Low solvent consumption (5–30 mL) Dependent of kind of matrix. Bath temperature can be adjusted

. Low cost

MAE Sample and solvent are placed in a reaction vessel.

Microwave energy is used to heat the mixture

. Fast (~15 min) . Filtration of the extract is necessary. Low solvent consumption (15–40 mL) Addition of a polar solvent is required. Easily programmable . Moderate cost

PSE Sample is placed in a cartridge and pressurized

with a high temperature solvent

Fast (20–30 min) Initial high cost. Low solvent consumption (30 mL) . Dependent on the kind of matrix. Easy control of extraction parameters

(temperature, pressure)

High temperatures achieved

. High sample processing

Note: USE, Ultrasound-assisted extraction; MAE, microwave-assisted extraction; PSE, pressurized solvent extraction.

around 5–10 mL of an appropriate organic solvent in an ultrasonic water bath Afterextraction, columns are placed on a multiport vacuum manifold where the solvent isfiltered and collected for further analysis.

2.3.1.2 Soxhlet Extraction

As indicated earlier, in some cases shaking is not enough for disrupting interactionsbetween analytes and matrix components In this regard, an increase of the tempera-ture of the extraction is recommended The more simple approach to isolate analytesbound to solid matrices at high temperatures is the Soxhlet extraction, introduced bySoxhlet in 1879, which is still the more used technique and of reference of the newtechniques introduced during the last few years

Sample is placed in an apparatus (Soxhlet extractor) and extraction of analytes isachieved by means of a hot condensate of a solvent distilling in a closed circuit.Distillation in a closed circuit allows the sample to be extracted many times withfresh portions of solvent, and exhaustive extraction can be performed Its weakpoints are the long time required for the extraction and the large amount of organicsolvents used

In order to minimize the mentioned drawbacks, several attempts toward mation of the process have been proposed Among them, Soxtec systems (Foss,Hillerød, Denmark) are the most extensively accepted and used in analytical labora-tories and allow reducing the extraction times aboutfive times compared with theclassical Soxhlet extraction

auto-Table 2.2 shows a comparison of the recoveries obtained for several pesticides

in soils after extraction using different techniques In this case, it is clear thatultrasound-assisted extraction allows the isolation of target analytes, whereas the

Ultrasound-Assisted Extraction

simple shaking is not effective enough to extract the selected pesticides quantitatively.

It is important to stress that recoveries after Soxhlet extraction were too high, whichmeans that a large amount of matrix components were coextracted with targetanalytes At this regard, it is clear that an exhaustive extraction is not always requiredand a balance between the recoveries obtained of target analytes and the amount ofmatrix components coextracted needs to be established

2.3.1.3 Microwave-Assisted Extraction

Microwave-assisted extraction (MAE) has appeared during the last few years as aclear alternative to Soxhlet extraction due to the ability of microwave radiation ofheating the sample–solvent mixture in a fast and efficient manner Besides, theexistence of several instruments commercially available able to perform the sequen-tial extraction of several samples (up to 14 samples in some instruments), allowingextraction parameters (pressure, temperature, and power) to be perfectly controlled,has made MAE a very popular technique

Microwave energy is absorbed by molecules with high dielectric constant In thisregard, hexane, a solvent with a very low dielectric constant, is transparent tomicrowave radiation whereas acetone will be heated in few seconds due to its highdielectric constant However, solvents with low dielectric constant can be used if thecompounds contained in the sample (i.e., water) absorb microwave energy

A typical practice is the use of solvent mixtures (especially for the extraction ofpesticides of different polarity) combining the ability of heating of one of thecomponents (i.e., acetone) with the solubility of the more hydrophobic compounds

in the other solvent of the mixture (i.e., hexane) As an example, a mixture ofacetone:hexane (1:1) was used for the MAE of atrazine, parathion-methyl, chlorpy-riphos, fenamiphos, and methidathion in orange peel with quantitative recoveries in

<10 min [3]

As a summary, in general, the recoveries obtained are quite similar to thoseobtained by Soxhlet extraction but the important decrease of the extraction time(~15 min) and of the volume of organic solvents (25–50 mL) have made MAE

to be extensively used in analytical laboratories

2.3.1.4 Pressurized Solvent Extraction

Pressurized solvent extraction (PSE), also known as accelerated solvent extraction(ASE), pressurized liquid extraction (PLE), and pressurizedfluid extraction (PFE),uses solvents at high temperatures and pressures to accelerate the extraction process.The higher temperature increases the extraction kinetics, whereas the elevatedpressure keeps the solvent in liquid phase above its boiling point leading to rapidand safe extractions [4]

Figure 2 2 shows a schem e of the inst rumentat ion and the procedu re used inPSE Experimentally, sample (~10 g) is placed in an extraction cell andfilled up with

an appropriate solvent (15–40 mL) Subsequently, the cell is heated in a furnace

to the temperatures below 2008C, increasing the pressure of the system (up to a

20 Mpa) to perform the extraction After a certain period of time (10–15 min),

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the extract is directly transferred to a vial without the necessity of subsequentfiltration of the obtained extract Then, the sample is rinsed with a portion of puresolvent andfinally, the remaining solvent is transferred to the vial with a stream ofnitrogen The whole process is automated and each step can be programmed,allowing the sequential unattended extraction of up to 24 samples.

This technique is easily applicable for the extraction of pesticides from anykind of sample and the high temperature used allows to perform very efficientextraction in a short time In addition, the considerable reduction in the amount oforganic solvents used makes PSE a very attractive technique for the extrac-tion of pesticides The main limitations of this technique are the high cost of theapparatus and the unavoidable necessity of purifying obtained extracts, which iscommon to other efficient extraction techniques based on the use of organic solvents

as mentioned earlier

2.3.2 SUPERCRITICALFLUIDEXTRACTION

Supercriticalfluid extraction (SFE) has been widely used for the isolation of a greatvariety of organic compounds from almost any kind of solid samples Supercriticalfluids can be considered as a hybrid between liquids and gases, and possess idealproperties for the extraction of pesticides from solid samples Supercritical fluidshave in common with gases the ability to diffuse through the sample, whichfacilitates the extraction of analytes located in not easily accessible pores In add-ition, the solvation power of supercriticalfluids is similar to that of liquids, allowingthe release of target analytes from the sample to thefluid

Carbon dioxide has been widely used in SFE because it can be obtained withhigh purity, it is chemically inert, and its critical point (31.18C and 71.8 atm) is easily

Oven

Collection vial

Extraction cell Solvent

Pump

Static valve Purge valve

Nitrogen

Load sample into cell.

Fill cell with solvent.

Heat and pressurize cell.

Hold sample at pressure

Total 12-14

Time (min) 0.5-1

FIGURE 2.2 Pressurized solvent extraction equipment (Courtesy of Dionex Corporation.With permission.)

accessible Its main drawback is its apolar character, limiting its applicability to theextraction of hydrophobic compounds In order to overcome, at least to a certainextent, this drawback, the addition of a small amount of an organic solvent modifier(i.e., methanol) has been proposed and permits varying the polarity of thefluid, thusincreasing the range of extractable compounds However, the role of the modifierduring the extraction is not well understood Figure 2.3 shows schematically thepossible mechanisms taking place during the SFE of the herbicide diuron form soilsamples using CO2as supercriticalfluid modified with methanol [5] Some authorspropose that methanol molecules are able to establish hydrogen bonds with thephenolic moieties of the humic and fulvic acids present in soil samples and thus,diuron is displaced from active sites However, other authors consider that themodifier is able to interact with target analyte releasing it from the sample.

Once target analytes are in the supercriticalfluid phase, they have to be isolatedfor further analysis, which is accomplished by decompression of thefluid through arestrictor by getting analytes trapped on a liquid trap or a solid surface With a liquidtrap, the restrictor is immersed in a suitable liquid and thus, the analyte is graduallydissolved in the solvent while CO2is discharged into the atmosphere In the solidsurface method, analytes are trapped on a solid surface (i.e., glass vial, glass beads,solid-phase sorbents) cryogenically cooled directly by the expansion of the super-criticalfluid or with the aid of liquid N2 Alternatively, SFE can be directly coupled

to gas chromatography or to supercriticalfluid chromatography and is successful ofsuch online coupling dependent of the interface used, which determines the quanti-tative transfer of target analytes to the analytical column [6]

As mentioned earlier, SFE has been widely used for the extraction of pesticidesfrom solid samples; thanks to the effectiveness and selectivity of the extractionand to the possibility of online coupling to chromatographic techniques However,

H Supercritical fluid

FIGURE 2.3 Mechanisms of the extraction of the herbicide diuron from sediments by SFE(CO2þ methanol) (Reproduced from Martin-Esteban, A and Fernandez-Hernando, P., Toma

y tratamiento de muestra, Cámara, C., ed., Editorial Síntesis S.A., Madrid, 2002, Chap 6.With permission from Editorial Síntesis.)

ß 2007 by Taylor & Francis Group, LLC.

the costs of the instrumentation and the apparition in the market of new lesssophisticated extraction instruments is making SFE to be displaced by other extrac-tion techniques, especially by PSE.

2.3.3 LIQUID–LIQUIDEXTRACTION

LLE has been widely used for the extraction of pesticides from aqueousliquid samples and, although to a lesser extent, for the purification of organicextracts LLE is based on the partitioning of target analyte between two immiscibleliquids The efficiency of the process depends on the affinity of the analyte for thesolvents, on the ratio of volumes of each phase, and on the number of successiveextractions

Most of the LLE applications deal with the extraction of pesticides from mental waters Hexane or cyclohexane are typical organic solvents used for extract-ing nonpolar compounds such as organochlorine and organophosphorus pesticides;and dichoromethane or chloroform for medium polarity organic compounds such astriazines or phenylurea herbicides However, quantitative recoveries for relativelypolar compounds by LLE are difficult to achieve As an example, a recovery of 90%atrazine was obtained by LLE of 1 L water with dichloromethane, whereas therecoveries for its degradation products desisopropyl-, desethyl-, and hydroxyatrazinewere 16%, 46%, and 46%, respectively [7]

environ-In order to increase the efficiency and thus, the range of application, the tion coefficients may be increased by using mixtures of solvents, changing the pH(preventing ionization of acids or bases), or by adding salts (‘‘salting-out’’ effect)

parti-At this regard, the recoveries for the atrazine degradation products of the previouslymentioned example were 62%, 87%, and 63%, respectively, by carrying outthe extraction with a mixture of dichloromethane and ethyl acetate with 0.2 Mammonium formate

The high number of possible combinations of solvents and pHs makes ideallypossible the isolation of any pesticide from water samples by LLE, which has beentraditionally considered a great advantage of LLE However, LLE is not exempt ofimportant drawbacks One of the most important drawbacks is the toxicity of theorganic solvents used leading to a large amount of toxic residues In this sense, thecosts of the disposal of toxic solvents are rather high However, it is important tomention that this problem is minimized when LLE is used for cleanup steps wherelow volumes are usually employed Besides, the risk of exposure of the chemist totoxic solvents and vapors always exists From a practical point of view, the formation

of emulsions, which are sometimes difficult to break up, the handling of large watersamples and the difficulties for automation of the whole process make LLE to beconsidered a tedious, time-consuming, and costly technique

2.3.4 SOLID-PHASEEXTRACTION

SPE, as LLE, is based on the different affinity of target analytes for two differentphases In SPE, a liquid phase (liquid sample or liquid sample extracts obtainedfollowing the techniques mentioned earlier) is loaded onto a solid sorbent (polar, ionexchange, nonpolar, affinity), which is packed in disposable cartridges or enmeshed

in inert matrix of an extraction disk Those compounds with higher affinity for thesorbent will be retained on it, whereas others will pass through it unaltered Sub-sequently, if target analytes are retained, they can be eluted using a suitable solventwith a certain degree of selectivity.

The typical SPE sequence involving several steps is depicted in Figure 2.4.Firstly, the sorbent needs to be prepared by activation with a suitable solvent and byconditioning with same solvent in which analytes are dissolved Then, the liquidsample or a liquid sample extract are loaded onto the cartridge Usually, targetanalytes are retained together with other components of the sample matrix Some

of these compounds can be removed by application of a washing solvent Finally,analytes are eluted with a small volume of an appropriate solvent In this sense, bySPE, it is possible to obtainfinal sample extracts ideally free of coextractives; thanks

to the cleanup performed, with high enrichment factors due to the low volume ofsolvent used for eluting target analytes These aspects together with the simplicity ofoperation and the easy automation (see later) have made SPE a very populartechnique widely used in the analysis of pesticides in a great variety of samples.The success of a SPE procedure depends on the knowledge about the properties

of target analytes and the kind of sample, which will help the proper selection ofthe sorbent to be used Understanding the mechanism of interaction between thesorbent and the analyte is a key factor on the development of a SPE method, since

it will ease choosing the right sorbent from the wide variety of them available inthe market

2.3.4.1 Polar Sorbents

The purification of organic sample extracts is usually performed by SPE ontopolar sorbents Within this group, the sorbent mostly used is silica, which possessesactive silanol groups in its surface able to interact with target analytes This inter-action is stronger for pesticides with base properties due to the slightly acidic

Conditioning Loading Washing Elution

FIGURE 2.4 Solid-phase extraction steps

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