S OLID -P HASE E XTRACTION SPE, as LLE, is based on the different affinity of target analytes for two different

2.3.4 S OLID -P HASE E XTRACTION SPE, as LLE, is based on the different affinity of target analytes for two different

phases. In SPE, a liquid phase (liquid sample or liquid sample extracts obtained following the techniques mentioned earlier) is loaded onto a solid sorbent (polar, ion exchange, nonpolar, affinity), which is packed in disposable cartridges or enmeshed

46 Analysis of Pesticides in Food and Environmental Samples

FIGURE 2.4 Solid-phase extraction steps.

in inert matrix of an extraction disk. Those compounds with higher affinity for the sorbent 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 solvent with 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 by conditioning with same solvent in which analytes are dissolved. Then, the liquid sample or a liquid sample extract are loaded onto the cartridge. Usually, target analytes 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, by SPE, it is possible to obtain final sample extracts ideally free of coextractives; thanks to the cleanup performed, with high enrichment factors due to the low volume of solvent used for eluting target analytes. These aspects together with the simplicity of operation and the easy automation (see later) have made SPE a very popular technique 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 of the sorbent to be used. Understanding the mechanism of interaction between the sorbent 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 in the market.

2.3.4.1 Polar Sorbents The purification of organic sample extracts is usually performed by SPE onto

polar sorbents. Within this group, the sorbent mostly used is silica, which possesses active 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

Sample Handling of Pesticides in Food and Environmental Samples 47 character of silanol groups. Other common polar sorbents are alumina (commercially

available in its acid, neutral, and base form) and Florisil. In the loading step, analytes compete with the solvent for the adsorption active sites of the sorbent, and elution is performed by displacing analytes from the active sites by an appropriate solvent. In this sense, the more polar the solvent is, the higher elution power it gets. The elution power is established by the eluotropic strength («8), which is a measure of the adsorption energy of a solvent in a given sorbent. The eluotropic series of different common solvents in alumina and silica are shown in Table 2.3. In this way, by a careful selection of solvents (or mixture of them), analytes (or interferences) will be retained on the sorbent by loading in a nonpolar solvent subsequently eluted using a second solvent with a higher eluotropic strength. Obviously, the selection of these solvents will be determined by the polarity of the analytes. Thus, after loading, hydrophobic pesticides such as pyrethroids can

be eluted with a mixture of hexane:diethylether, whereas for eluting carbamates a more polar mixture such as hexane:acetone is necessary.

TABLE 2.3 Eluotropic Series Solvent

« 8 Al 2 O 3 « 8 SiOH Pentane

0.04 0.03 Carbon tetrachloride

0.32 0.25 Ethyl ether

Methylethyl ketone — 0.51 Acetone — 0.56

0.53 Methyl t-butyl ether

0.48 Ethyl acetate

0.38 –0.48 Dimethyl sulfoxide

0.7 — n -Propyl alcohol

— Isopropyl alcohol

0.6 Ethanol

0.88 — Methanol

48 Analysis of Pesticides in Food and Environmental Samples The number of developed methods based on SPE using polar sorbents for the

determination of pesticides in food and environmental solid samples is huge, and thus, for specific examples, the interested reader should consult Chapters 6 through 8 of this book.

2.3.4.2 Nonpolar Sorbents This kind of sorbent is appropriate for the trace-enrichment and cleanup of pesticides

in polar liquid samples (i.e., environmental waters). Traditionally, n-alkyl-bonded silicas, mainly octyl- and octadecyl-silica, both in cartridges or disks, have been used due to its ability of retaining nonpolar and moderate polar pesticides from liquid samples. Retention mechanism is based on van der Waals forces and hydrophobic interactions, which allows handling large sample volumes and the subsequent elution of target analytes in a small volume of a suitable organic solvent (i.e., methanol, acetonitrile, ethyl acetate) getting high enrichment factors. However, for more polar pesticides, the strength of the interaction is not high enough and low recoveries are obtained due to the corresponding breakthrough volume is easily reached.

An easy manner of increasing breakthrough volumes is to increase the amount of sorbent used, which will increase the number of interactions that take place. A second option is the addition of salts to the sample, diminishing the solubility of target analytes (salting-out effect) and thus favoring their interactions with the sorbent. Table 2.4 shows the obtained recoveries of several triazines by the SPE of 1 L of water spiked at 1 mg=L concentration level of each analyte in different experimental

conditions. It is clear that the combination of using two C 18 disks and the addition of

a 10% NaCl to the water sample allow the obtainment of quantitative recoveries for all the tested analytes including the polar degradation products of atrazine. However, these approaches do not always provide satisfactory results. In that case, the most direct way of increasing breakthrough volumes of most polar pesticides is the use of sorbents with higher affinity for target analytes. These sorbents include

TABLE 2.4 Recoveries (R%) and Relative Standard Deviations (RSD) of Several Triazines Obtained by SPE of 1 L of LC Grade Water Spiked with 1 mg=L of Each Triazine

2C 18 Disk Without NaCl

1C 18 Disk

Without NaCl 10% NaCl Triazine

10% NaCl

RSD R% RSD Desisopropylatrazine

94.6 8.7 98.3 4.9 104.3 4.6 97.0 4.1 Source: Adapted from Turiel, E., Fernández, P., Pérez-Conde, C., and Cámara, C., J. Chromatogr. A,

872, 299, 2000. With permission from Elsevier.

Sample Handling of Pesticides in Food and Environmental Samples 49 styrene

–divinylbenzene-based polymers with a high specific surface (~1000 m =g), which are commercialized by several companies under different trademarks (i.e.,

Lichrolut, Oasis, Envichrom). The interaction of analytes with these sorbents is also based on hydrophobic interactions, but the presence of aromatic rings within the polymeric network leads to strong p –p* interactions with the aromatic rings present in the chemical structure of many pesticides. Another alternative is the use of graph- itized carbon cartridges or disks, which have a great capacity for the preconcentration of highly polar pesticides (acid, basic, and neutral) and transformation products such as oxamyl, aldicarb sulfoxide, and methomyl; thanks to the presence of various functional groups, including positively charged active centers on its surface.

2.3.4.3 Ion-Exchange Sorbents Ionic or easily ionizable pesticides can be extracted by these sorbents. Sorption

occurs at a pH in which the analyte is in its ionic form and then it is eluted by a change of the pH value with a suitable buffer. The mechanism involved provides a certain degree of selectivity. Phenoxy acid herbicides can be extracted by anion-exchangers and amines or n-heterocycles using cation-exchangers. However, its use is rather limited due to the presence of high amount of inorganic ions in the samples, which overload the capacity of the sorbent leading to low recoveries of target analytes.

2.3.4.4 Affinity Sorbents The sorbents described earlier are able to extract successfully pesticides from a great

variety of samples. However, the retention mechanisms (hydrophobic or ionic interactions) are not selective, leading to the simultaneous extraction of matrix compounds, which can negatively affect the subsequent chromatographic analysis. For instance, the determination of pesticides (especially polar pesticides) in soil and water samples by liquid chromatography using common detectors is affected by the presence of humic and fulvic acids. These compounds elute as a broad peak or as a hump in the chromatogram, hindering the presence of target analytes and thus making difficult in some cases to reach the required detection limits. Even using selective detectors (i.e., mass spectrometry) the presence of matrix compounds can suppress or enhance analyte ionization, hampering accurate quantification.

The use of antibodies immobilized on a suitable support, so-called immuno- sorbent (IS), for the selective extraction of pesticides from different samples appeared some years ago as a clear alternative to traditional sorbents [8,9]. In this approach, only the antigen which produced the immune response, or very closely related molecules, will be able to bind the antibody. Thus, theoretically, when the sample is run through the IS, the analytes are selectively retained and subsequently eluted free of coextractives. The great selectivity provided by immunosorbents has allowed the determination of several pesticides in different matrices such as carbo- furan in potatoes, or triazines and phenylureas in environmental waters, sediments, and vegetables. However, this methodology is not free of important drawbacks. The obtainment of antibodies is time-consuming, expensive, and few antibodies for pesticides are commercially available. In addition, it is important to point out that after the antibodies have been obtained they have to be immobilized on an adequate

50 Analysis of Pesticides in Food and Environmental Samples

Prepolymerization complex Monomers

Template

Polymerization Imprinted polymer

Washing

FIGURE 2.5 Scheme for the preparation of molecularly imprinted polymers.

support, which may result in poor antibody orientation or even complete denatur- ation. Because of these limitations, the preparation and use of molecularly imprinted polymers (MIPs) has been proposed as a promising alternative.

MIPs are tailor-made macroporous materials with selective binding sites able to recognize a particular molecule [10]. Their synthesis, depicted in Figure 2.5, is based on the formation of defined (covalent or noncovalent) interactions between a template molecule and functional monomers during a polymerization process in the presence of a cross-linking agent. After polymerization the template molecule is removed, cavities complementary in size and shape to the analyte are found. Thus, theoretically, if a sample is loaded on it, in a SPE procedure, the analyte (the template) or closely related compounds will be able to rebind selectively the polymer subsequently eluted free of coextractives. This methodology, namely molecularly imprinted SPE (MISPE), has been successfully employed in the determination of pesticides such as triazines, phenylureas, and phenoxy acids herbicides, among others, in environmental waters, soils, and vegetable samples. As an example of the selectivity provided by MIPs, Figure 2.6 shows the chromatograms obtained in the analysis of fenuron in potato sample extracts with and without MISPE onto a fenuron-imprinted polymer. It is clear that the selectivity provided by the MIP allowed the determination of fenuron at very low concentration levels [11].

Because of their easy preparation and excellent physical stability and chemical characteristics (high affinity and selectivity for the target analyte), MIPs have received special attention from the scientific community not only in pesticide residue analysis but also in several fields. Besides, there are already MISPE cartridges

Sample Handling of Pesticides in Food and Environmental Samples 51

Without MISPE 2000

mAU (244 nm) 0

1000 ⫺5 0 1 2 3 4 5 mAU (244 nm)

Time (min) 500

With MISPE ⫺500 0 1 2 3 4 5

Time (min)

FIGURE 2.6 Chromatograms obtained at 244 nm with and without MISPE of potato sample extracts spiked with fenuron (100 ng=g). Graph insert shows the same chromatograms with different absorbance scale. (Reproduced from Tamayo, F.G., Casillas, J.L., and Martin- Esteban, A., Anal. Chim. Acta, 482, 165, 2003. With permission from Elsevier.)

commercially available for the extraction of certain analytes (i.e., triazines) and some companies offer custom synthesis of MIPs for SPE, which will ease the implemen- tation of MISPE in analytical laboratories.

The wide variety of available sorbents as well as the reduced processing times and solvent savings have made SPE to be a clear alternative against LLE. Besides, automation is possible using special sample preparation units that sequentially extract the samples and clean them up for automatic injections. However, the typical drawbacks associated to off-line procedures, such as the injection in the chromato- graphic system of an aliquot of the final extract or the necessity of including a evaporation step remain, which affects the sensitivity of the whole analysis.

The use of SPE coupled online to liquid and gas chromatography can sort out the previously mentioned drawbacks. The coupling of SPE to liquid chromatography is especially simple to perform in any laboratory and has been extensively described for the online preconcentration of organic compounds in environmental water samples [12]. The simplest way of SPE –LC coupling is shown in Figure 2.7, where a precolumn (1 –2 cm 3 1–4.6 mm i.d.) filled with an appropriate sorbent is inserted in the loop of a six-port injection valve. After sorbent conditioning, the sample is loaded by a low-cost pump and the analytes are retained in the precolumn. Then, the precolumn is connected online to the analytical column by switching the valve, so that the mobile phase can desorb the analytes before their separation in the chromato- graphic column. Apart from a considerable reduction of sample manipulation, the main advantage is the fact that the complete sample is introduced in the analytical column. Besides, there are equipments commercially available for the whole auto- mation of the process.

52 Analysis of Pesticides in Food and Environmental Samples

SPE column

HPLC column

Sample Detector

Waste P 2

Waste

HPLC solvents

FIGURE 2.7 SPE–LC coupling setup. Alkyl-bonded silicas (mainly C 18 -silica) have been widely used as precolumn

sorbent, although they are replaced by styrene –divinylbenzene copolymers, which offer higher affinity for polar analytes, so that permit the usage of larger sample volumes without exceeding the breakthrough volumes of analytes. Other materials successfully employed have been small extraction disks and graphitized carbons; and in order to provide selectivity to the extraction, precolumns packed with yeast cells

immobilized on silica gel [13] or with immunosorbents have been proposed for the extraction of polar pesticides from environmental waters [14,15].

The coupling of SPE to GC is also possible, thanks to the ability of injecting large volumes into the gas chromatograph using a column of deactivated silica (retention gap) located between the injector and the analytical column. SPE –GC uses the same sorbents employed in SPE –LC but, in this case, after the preconcen- tration step, the analytes are desorbed with a small volume (50 –100 mL) of an appropriate organic solvent, which is directly introduced into the chromatograph. In general, using only 10 mL of water sample, it is possible to reach detection limits at micrograms per liter level employing common detectors.