O RGANIC S OLVENT E XTRACTION

6.2.1 O RGANIC S OLVENT E XTRACTION

Extraction with an organic solvent using a blender or a homogenizer is still the most widely used approach for the separation of nonionic pesticides from the plant matrix. The most commonly used extraction solvents are acetonitrile, acetone, ethyl acetate, and methanol. While water-miscible solvents, such as acetonitrile and acetone, will effectively extract pesticide residues from high moisture fresh fruits and vegetables, they will not adequately extract pesticide residues from dry samples, such as grains or feeds. Bertuzzi et al. [9] first demonstrated that acetonitrile –water (65=35, v=v) will effectively extract pesticides from dry products. Later, Luke and Doose [10] likewise showed that acetone –water mixtures (65=35, v=v) could also be used to extract pesticides from dry products.

Traditionally, it has been accepted that pesticide extraction from solid foods must

be accomplished using some type of mechanical homogenization. Studies at the Florida Department of Agriculture and Consumer Services Laboratory showed that pesticides could be extracted from well-comminuted produce samples by shaking with acetonitrile [11]. The advantages of shaking over mechanical homogenization are mainly that it is faster and easier, less equipment is needed, and there is less chance of carryover from one sample to the next. A number of methods have been presented that employ organic solvent extraction by shaking instead of using mech- anical blenders or homogenizers [8,11 –20]. Shaking may not work as well as homogenizing for some of the more nonpolar OCs. Okihashi et al. [21] reported

that recoveries of incurred residues of nonpolar OCs like o,p 0 -DDT, dicofol, and endrin were lower when shaking instead of homogenizing with acetonitrile.

6.2.1.1 Acetonitrile Extraction and Liquid–Liquid Partitioning Acetonitrile was the extraction solvent used for one of the earliest multiclass MRMs,

the ‘‘Mills method,’’ developed in the1960s [3,22,23]. Even though the Mills method was developed when pesticide methods were primarily concerned with the recovery

of the nonpolar OCs, a water-miscible solvent, acetonitrile, was used. Polar solvents are needed for the extraction of nonpolar OC pesticides from the plant matrix.

154 Analysis of Pesticides in Food and Environmental Samples Mumma et al. [24] hypothesized that this is caused by the pesticides interacting with

surfactant phospholipids, sulfolipids, and glycolipids from the plant. Using the Mills method, water and NaCl are added to the sample extract, and pesticide residues are partitioned from the acetonitrile –water mixture into a very nonpolar solvent, petroleum ether.

While the Mills method worked very well with the nonpolar OC pesticides used in the 1960s, some of the more polar OPs that were developed in the 1970s were not easily recovered. An advantage of using acetonitrile as an extraction solvent is that while it is completely miscible with water, it can be readily separated from water not only by liquid –liquid partition with nonpolar solvents (Mills method) but also by the addition of salts (salting out). Acetonitrile extractions of produce samples followed by salting out were used at the California Department of Food and Agriculture in the early 1990s [25,26]. While the resulting extracts were not as clean as those obtained by the Mills method, both polar and nonpolar pesticides could be recovered. This approach of using acetonitrile extraction followed by salting out has been adopted by regulatory agencies in Florida, Canada, and New York [11,27,28].

In 2003, a new approach to the extraction of pesticides from fresh fruits and vegetables with acetonitrile, called quick, easy, cheap, effective, rugged, and safe (QuEChERS) was reported [12]. This method entailed shaking the sample with

acetonitrile, followed by shaking with sodium chloride (NaCl) and MgSO 4 to remove the water. The salts create an exothermic reaction with water, induce phase separation between water and acetonitrile, and bind water to drive the pesticide analytes into the acetonitrile phase, resulting in high recoveries, including the polar and water-soluble pesticide, methamidophos. A modified QuEChERS extraction, using 1% acetic acid –acetonitrile extraction solvent and sodium acetate rather than NaCl, was developed to facilitate the recovery of base-sensitive fungicides like chlorothalonil and captan [16].

Vegetal matrices containing a high lipid content present a challenge for cleanup because the fats and waxes would have an adverse effect on the GC columns and could interfere with the analysis and detection of the pesticides. In 1952, Jones and Riddick [29] found that even the most lipophilic, nonpolar pesticides

like p,p 0 -DDT could be separated from fats and waxes by liquid –liquid partition between hexane and acetonitrile. Pesticides have been extracted from olive oil by dissolving the oil in hexane, and then shaking with acetonitrile [30,31]. Similarly soya oil [32] and olive oil [33] have been dissolved in hexane and loaded onto diatomaceous earth columns, with subsequent elution with acetonitrile.

6.2.1.2 Acetone Extraction and Liquid–Liquid Partitioning The introduction of new water-soluble, very polar, OP insecticides, such as metha-

midophos and acephate in the 1970s, resulted in the development of the ‘‘Luke method,’’ at the FDA [34,35]. Produce samples were extracted with acetone, and water was removed from the extract by a series of liquid –liquid partition steps,

first with petroleum ether–dichloromethane, followed by dichloromethane–NaCl. Both polar and nonpolar pesticides could be recovered. Hopper [36] substituted

Determination of Pesticides in Food of Vegetal Origin 155 diatomaceous earth columns for separatory funnels used in the Luke method.

Acetone –water extracts are adsorbed onto the diatomaceous earth, and the pesticides are eluted from the column with dichloromethane. The dichloromethane used in the Luke method came to be recognized as an environmental hazard, so in Europe, a combination of ethyl acetate and cyclohexane was used instead of dichloromethane [37]. Recently, Luke et al. [38] demonstrated that salting out with a combination of

fructose, anhydrous MgSO 4 , and NaCl could be used to separate water from acetone in produce samples extracts.

6.2.1.3 Ethyl Acetate Extraction Ethyl acetate is only slightly miscible with water, which simplifies the problem of

separating water from the sample extract. Since ethyl acetate is more nonpolar than the other solvents discussed, the polar pesticides do not readily partition into ethyl acetate. Large amounts of sodium sulfate are usually added to bind the coextracted water and force the polar pesticides into the organic phase. Most methods using ethyl acetate extraction entail two extractions of the sample matrix rather than the single extraction commonly used with acetonitrile and acetone. Polar solvents like ethanol may be added to the extraction solvent to increase the recovery of polar compounds [39]. Ethyl acetate extraction will result in a cleaner extract as it will extract less of the polar plant matrix compounds, but more lipids and waxes [12,40].

6.2.1.4 Methanol Extraction Methanol has not been as commonly used as an extraction solvent. Krause [41]

found methanol to be the most effective solvent for extracting 14 C-labeled N -methylcarbamate insecticides from produce samples. Produce samples were extracted with methanol, and methanol was removed from the methanol –water extract using a vacuum rotary evaporator. Pang et al. [42] used methanol to extract pyrethroids from produce samples. Water and NaCl were added to the methanol – water extract, and the nonpolar pesticides were partitioned into toluene. Klein and Alder [43] and Alder et al. [44] extracted produce samples with methanol, added NaCl to the methanol –water extract, transferred the extract to a diatomaceous earth column, and eluted the pesticide residues with dichloromethane. Granby et al. [45] extracted produce and dry samples with a methanol –acetate buffer mixture. The extracts were filtered and determined directly by HPLC with tandem mass spectrometry (HPLC-MS=MS) with no further cleanup.