E XTRACTION T ECHNIQUES For food samples of animal origin, the selection of the extraction technique depends

7.2.2 E XTRACTION T ECHNIQUES For food samples of animal origin, the selection of the extraction technique depends

on the nature of the matrix investigated; different procedures are used for solid and liquid samples. The amount of sample required varies largely depending on the contamination level anticipated in the sample and on the sensitivity provided by the detection technique. Table 7.3 summarizes relevant extraction methods related to pesticides in food samples of animal origin.

Solid samples can be extracted by any one of a number of techniques (Tables 7.1 and 7.2). The main points to consider here are the use of adequate solvent systems (e.g., low boiling solvents to facilitate concentration), adequate exposure time between solvents and the sample matrix, and limitation of sample handling steps, that is, avoid filtration steps by using Soxhlet, extraction columns (sample matrix eluted after soaking in solvent), or semiautomated extraction systems (e.g., pressur- ized liquid extraction, PLE). Cross-contamination from residues left behind by high levels in previous samples is a concern at this stage and equipment must be thoroughly cleaned and checked from batch to batch. Purity of extraction solvents

188 Analysis of Pesticides in Food and Environmental Samples

TABLE 7.3 Description of the Most Common Techniques Used for the Extraction of Pesticides from Solid Food Samples of Animal Origin

Technique

Overview

Conventional Soxhlet Sample þ desiccant mixture in glass or paper thimble is extracted with condensed (cold) solvent for 4 –12 h Automated Soxhlet

Sample þ desiccant mixture in extraction thimble is immersed in boiling (e.g., ‘‘Soxtec’’)

solvent (30 –60 min), then raised for Soxhlet extraction. Solvent can also be evaporated

Supercritical fluid Sample þ desiccant mixture is placed in high-pressure cartridge and CO 2 at extraction (SFE)

150 –450 atm at temperature of 408C–1508C passed through. After depressurization, analytes are collected in solvent trap

Column extraction Sample þ desiccant mixture is placed in large column with filter. Eluted with large volume of extraction solvent Sonication-assisted

Sample (þdesiccant) in open or closed vessel immersed in solvent and heated extraction

with ultrasonic radiation using ultrasonic bath or probe Pressurized liquid

Sample þ desiccant mixture is placed in extraction cartridge and solvent extraction (PLE)

(heated, pressurized) passed through, then dispensed in extraction vial Microwave-assisted

Sample (þdesiccant) is placed in open or closed vessel immersed in solvent and extraction (MAE)

heated with microwave energy

is also a major consideration here. Only pesticide-grade or high-purity glass-distilled solvents should be used because evaporation steps will later concentrate any con- taminants.

A generally used method is homogenization of the solid sample with a wide variety of solvents, such as dichloromethane

–hexane or ethyl acetate. Such technique allows quantitative extraction of pesticides directly from matrices or

after drying with anhydrous Na 2 SO 4 , but also uses large volumes of solvents. Alternatively, the use of matrix solid-phase dispersion (MSPD) in different variants is a suitable choice, which results in an intimate contact between the sample components and the sorbent particles and therefore in a more efficient retention of

impurities. 28 Moreover, lower solvent consumption and cleaner extracts can be expected using MSPD compared with the column extraction technique, in which the sample is packed above the sorbent.

Binary solvent mixtures typically containing acetone

31 –n-hexane or petroleum ether 32 have been preferred for Soxhlet-based extractions. In general, extraction with

a polar –nonpolar binary mixture has been found to be more efficient for recovering pesticides from fish tissues of low lipid content than a nonpolar solvent. 31,35 This

technique has a number of advantages, such as minimum sample pretreatment required, simplicity, and high recoveries obtained for most pesticides. The time- and solvent-consuming nature of Soxhlet extraction (or related techniques involving percolation of a solvent through the sample) is generally thought to be related to the slow diffusion and desorption of the analytes from the sample matrix. Semivolatile pesticides can also be lost from Soxhlet apparatus via volatilization.

Determination of Pesticides in Food of Animal Origin 189 Therefore, more automated extraction techniques, such as PLE or microwave-

assisted extraction (MAE), are an alternative to Soxhlet and column extraction methods. The use of microwave energy (for MAE) or elevated temperatures and pressure (as in PLE) increases the rates of diffusion and desorption and thus speeds up extraction. Although these techniques use less solvent, they suffer the disadvant- age of initial high cost. Moreover, the preparation of a homogeneous dry sample from wet tissue for PLE can be a challenge due to the limited size of PLE vials, typically <100 mL. The use of PLE-based extraction methods for organic

pollutants, including pesticides, has recently been reviewed by Björklund et al. 63 and Carabias-Martinez et al. 64 Supercritical fluid extraction (SFE) has also been used for the extraction of pesticides from biological samples. Due to the wide polarity range of pesticides, a

polar modifier was typically used during extraction with supercritical CO 8,19,37,38,41

2 . However, SFE is less popular due to the high number of variables which have to be optimized and to its lesser ability to accommodate a wide variety of analytes and matrices. 41

For liquid samples, such as milk, solid-phase extraction (SPE) has been estab- lished as a robust extraction method for a wide variety of pesticides and has been increasingly used compared with the classical liquid

40,47 –liquid extraction technique. 13 Various adsorbents, for example, silica-based C 18 or Carbograph 4 have

shown high recoveries of investigated pesticides from milk. Another sampling device, solid-phase microextraction (SPME) involves immersing a polydimethylsiloxane-coated syringe into or above liquid samples. 10,11 Hydrophobic compounds are adsorbed onto the coated fiber and the syringe is then placed into a hot injection liner which desorbs these compounds into the GC. The benefit of this approach is that it requires no solvent or multistepped cleanup=concentration procedures. A drawback is that the adsorption efficiency can

be affected by complex matrices, especially when the fiber is directly immersed in the sample.

Homogenization of a liquid sample with solvents, for example, acetonitrile

ethanol 46,49

is less used than for solid samples, while MSPD has found some –

applications for milk, 12,43 butter, 43 and eggs. 50,65,66

For honey, the first step consisted of transferring the analytes into an aqueous phase by shaking the samples with water. This phase can subsequently be extracted

as described earlier for milk samples, with SPE 29,52,54,56 and SPME as the most used techniques.

Lipid contents of biological samples should be determined during the pesticide analysis in food samples of animal origin. Most studies have determined total extractable lipid gravimetrically by drying a fraction of the sample extract to constant weight. However, results can vary widely among laboratories due to different extraction efficiencies of various combinations of solvent and extraction systems. 67 The benchmark method for total lipid is that of Bligh and Dyer.

Recently, Smedes 68 demonstrated that mixtures of isopropanol are an effective substitute for the Bligh and Dyer mixture of chloroform –cyclohexane–water (8:10:11)

water. The Smedes method gave more consistent results for extractable lipids in fish –methanol– tissue with low lipid content (<1% lipid).

190 Analysis of Pesticides in Food and Environmental Samples