Supercritical fluid extraction for the analysis of pesticide residues in miscellaneous samples

J. Biochem. Biophys. Methods 43 (2000) 313–328 www.elsevier.com / locate / jbbm

Review

Supercritical fluid extraction for the analysis of pesticide residues in miscellaneous samples ´ ´ c Noboru Motohashi a , *, Hideo Nagashima b , Cyril Parkanyi a

b

Meiji Pharmaceutical University, 2 -522 -1 Noshio, Kiyose, Tokyo 204 -8588, Japan Setagaya Public Health Center, M.K. Earth Building, 1 -11 -18 Setagaya Setagaya-ku, Tokyo 154 -0017, Japan c Department of Chemistry and Biochemistry, Florida Atlantic University, 777 Glades Road, P.O. Box 3091, Boca Raton, FL 33431 -0991, USA

Abstract Supercritical fluid extraction (SFE) procedures for pesticide residue analysis are reviewed and discussed. A variety of applications were classified, on matrices such as fruits, vegetables, soils, biological tissues, and other materials. Emphasis is placed on analysis of samples with a high water content containing polar pesticides, with particular attention paid to the multiresidue analyses.  2000 Elsevier Science B.V. All rights reserved. Keywords: Supercritical fluid extraction; Pesticides; Fruits and vegetables; Reviews

1. Introduction The analytical-scale supercritical fluid extraction (SFE) was introduced in the late 1980s and started a new field of research. SFE paved the way not only for a reduction in use of organic solvents but also for automation of analytical procedures. There is a reason to consider the elimination of organic solvent use in chemical analysis. The exposure of laboratory personnel to harmful solvents can be reduced. At present, SFE is beginning to play an important role in analytical chemistry. The fluid and analyte diffusion coefficients are better in supercritical fluid media than in organic solvents. For the extraction of thermally labile and oxygen-sensitive analytes, the influence of oxygen in the analytical procedure can be avoided. The determination of pesticides by SFE has already been reviewed in detail [1–4]. King proposed a strategy for the development of SFE methods [5]. *Corresponding author. 0165-022X / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0165-022X( 00 )00052-X

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This review covers the literature on pesticide analysis published between January 1, 1997 and July 31, 1999. Main sources of information were Chemical Abstracts, Journal of Chromatography, Analytical Chemistry, Analyst, Journal of AOAC International, Journal of Agricultural and Food Chemistry, and Journal of Chromatographic Science. The review is devoted basically to methods for the determination of residues of pesticides in fruits, vegetables, soil, biological tissues, and other matrices.

2. Development of methods in SFE

2.1. Supercritical media The supercritical parameters of carbon dioxide are easily accessible (72.8 bar, 31.18C), and supercritical carbon dioxide (supercritical CO 2 ) has played a major role in analytical SFE. It would be hard to find another supercritical fluid with such convenient characteristics. Supercritical CO 2 is non-toxic, non-flammable, and practically benign toward most analytes. Recently, water has been used in subcritical state, applied to analysis of polar metabolites of pesticides and polar pesticides [6–8]. Subcritical water extraction is similar to pressurized liquid extraction, also known as accelerated solvent extraction, and the solubility behavior can be changed by adjusting the extraction temperature of water under pressure, associated with the solvents such as ethyl acetate and diethyl ether. Incidentally, water has a critical point of 3748C, 218 atm. The supercritical water shows very corrosive characteristics, and successfully degrades many chemicals. Therefore, supercritical water is not appropriate as an extraction fluid for pesticides [9]. In a unique approach, Liang and Tilotta described an application to SFE with argon by inductively coupled plasma atomic emission spectroscopy [10].

2.2. Drying agents for SFE Supercritical CO 2 has already been applied to the analysis of pesticide residues in different samples. The excessive water in high-water-content samples such as fruits and vegetables gives rise to restrictor plugging by ice, which carries water into the collection trap. There are basically two different approaches to the solution of this problem. One is to lyophilize the sample prior to extraction and the second is to mix the sample prior to SFE with an appropriate material to adsorb water. Initially, the researchers into SFE used Hydromatrix, a pelletized diatomaceous earth, as a drying agent to control water in moist samples. ´ demonstrated the use of magnesium sulfate, and Lehotay and Valverde-Garcıa reported high recovery of the problematic pesticides such as methamidophos, as well as other diverse pesticides [20]. Eller and Lehotay reported an evaluation for a mixture of Hydromatrix with magnesium sulfate as drying agent. Additionally, magnesium sulfate was shown to be

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applicable to the extraction of the polar pesticides, methamidophos, acephate and omethoate [11]. Obana et al. demonstrated that a super-adsorbent polymer was superior to diatomaceous earth as a drying agent [12]. Although the recoveries of methamidophos and acephate were 3–5% when diatomaceous earth was used as drying agent, the recovery of these analytes was 22–43% when the super-adsorbent polymer was used without any modifier added to supercritical CO 2 .

2.3. Modifiers for SFE Although supercritical CO 2 is considered to be a nonpolar solvent with a polarity similar to hexane, most pesticides have moderately or highly polar characteristics. Usually, it is difficult to extract polar pesticides, such as methamidophos and acephate, with supercritical CO 2 alone. Therefore, an organic solvent, sometimes called a modifier, is used to increase the polarity of supercritical CO 2 . There are two ways to add the modifier to supercritical CO 2 . One is addition of an organic solvent like methanol, acetonitrile or acetone, directly to supercritical CO 2 , and the other is the addition of a polar solvent, methanol, acetonitrile, acetone or water, to the sample in the extraction thimble [13].

2.4. Extraction parameters The selection of operating conditions in SFE is still an important task for the researchers. The traditional approach to study analyte–matrix interactions has involved keeping all the variables constant except one, which is examined across the typical range. This strategy is time-consuming and rarely effective for the determination of the supercritical parameters. Zhou et al. reported a multivariate optimization scheme (MOS), which was a highly efficient approach to study the different variables and to identify optimal SFE parameters, such as supercritical density, temperature, fluid flow-rate and extraction time [14].

2.5. Trapping Generally, SFE can be operated in on-line mode or off-line mode. In the on-line mode, the outlet of the SFE instrument is interfaced directly to an analytical instrument. On the other hand, in the off-line mode, the extracted analytes are determined via trapping and elution and are analyzed by an analytical instrument. About a decade ago, supercritical CO 2 was thought to be a very selective extraction medium, and the extracts relatively free from coextractives. Therefore, many researchers advocated methods to analyze target analytes using instruments which were connected directly to the supercritical fluid chromatography system. However, when the on-line mode was applied to the matrices which contained appreciable quantities of lipids, problems arose with the co-extracted substances.

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Furthermore, the on-line extraction method had a drawback because of limited sample sizes. For off-line SFE, trapping of the extracted analytes is an important aspect. Analyte trapping after the extraction step can be carried out with either small amounts of collection solvents or into an adsorbent trap. In solvent trapping, supercritical CO 2 containing analytes is depressurized and the analytes are directly collected into liquid solvents, while in adsorbent trapping, the analytes contained in supercritical CO 2 are adsorbed on solid materials, for example, glass bead cartridges, C 18 bonded silica, or Florisil, and subsequently eluted with an appropriate solvent. McDaniel and Taylor investigated the effect of improvement by adding methanol as a modifier to the collection solvent, using liquid trapping with direct restrictor immersion after SFE for volatile analytes [15]. Chaudot et al. demonstrated the trapping efficiencies of various adsorbents for SFE by supercritical CO 2 which was modified with methanol. Five polymeric phases with a high specific surface (greater than 800 m 2 / g) and a C 18 adsorbent were evaluated for the efficiency of a solid trap using supercritical CO 2 alone, and with 2.5, 5, 10 and 20% methanol-modified supercritical CO 2 . A polymeric adsorbent was selected, because the effect of this trap was shown to be quantitative on all examined analytes, such as tetracosane, naphthalene, fluoranthene, acetophenone, N,N9-dimethylaniline, 2-naphthol and decanoic acid, with modified supercritical CO 2 containing even 10% methanol. On the other hand, a solid trap filled with C 18 adsorbent gave a quantitative collection only at a methanol content lower than 2.5% [16].

2.6. Detection Most detection methods for pesticides depend on gas chromatographic detectors and dual-column conformation to identify the pesticides. Some methods have used gas chromatography–mass spectrometry (GC–MS) for detection of the pesticides. Recently, the usefulness of gas chromatography with atomic emission detection (GC–AED) for selective detection of pesticides has been reported. Cook et al. screened over 400 pesticides by GC–AED and created an experimental database. A technique called retention time locking was used to match GC–AED and GC–MS retention times to those of the database. Samples were analyzed for sulfur, nitrogen, phosphorus and chlorine by GC–AED. Possible pesticides were suggested by database search and identified by GC–MS [17].

3. Publications In the past few years, publications have appeared on SFE pesticide analysis applied to different matrices. In this review, the publications were classified as follows: fruits and vegetables (Table 1), soils (Table 2), biological tissues (Table 3) and other applications (Table 4).

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Table 1 Applications of SFE to pesticides in fruits and vegetables Analyte(s)

Matrices

Pre-treatment

SFE conditions

Collection and analysis

Fluazinam

Fruits

Grinding (5 g)

CO 2 , 120 atm, 708C, 180 ml/min, 3 min dynamic

Water (ca. 258C). 97.9% The extract was dissolved in 50 ml of MeOH. Cleaned-up by liquid–liquid partition Off-line GC

25 pesticides organochlorine, organophosphorus, organonitrogen, pyrethrinoids

Spiked cereals

Grinding

CO 2 1 10% MeOH, Glass beads 200 atm, 608C, Cartridge (80/100 mesh). 2 ml/min Off-line GC

62.6–99.8% [19]

56 pesticides

Spiked vegetables 100 g of sample were stored in a freezer. A drying agent consisting of 100 g of MgSO 4 ? H 2 O 1 50 g of hydromatrix was mixed and homogenized with a small amount of dry ice

CO 2 , 350 bar, 508C, 0.90 g/ml, 2 min static, 20.3 min dynamic, 2.0 ml/min

C 18 bonded silica. Eluted with acetone. Off-line GC-ion trap MS.

–

[20]

92 pesticides

Spiked apple

Grinding, mixed with Celite and anhyd. Na 2 SO 4

CO 2 , 0.8 g/ml, 189 bar, 2.5 ml/ min, 458C, 1 min static, 10 min dynamic

C 18 bonded silica trap. Extracted with hexaneacetone (1:1). step I: 1 ml; step II: 0.5 ml. Off-line GC, HPLC

–

[21]

Ground and mixed with anhydrous Na 2 SO 4 or lyophilized

For lyophilized strawberries, CO 2 1 10% acetone, 0.86 g/ml, 10 min static 3.0 ml/min, For strawberries mixed with anhyd. Na 2 SO 4 CO 2 1 10% acetone/MeOH, 1.01 g/ml, 10 min static 3.0 ml/min

Silanized glass beads cartridge (80/100 mesh, Suprex). Adsorption temp. 08C. Desorption temp. 458C. Eluted with n-hexane. Off-line GC

. 80%

[23]

Vinclozolin, Spiked tolclofos-methyl, strawberries malathion chlorpyrifos, 4,49-dichlorobenzophenone, procymidone, 4,49-DDE, chlorbenzilate, b-endosulfan, bromopropylate, tetradifon

Mean recovery

Refs.

[18]

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Table 1. Continued Analyte(s)

Matrices

Pre-treatment

SFE conditions

Collection and analysis

Mean recovery

Refs.

a-BHC, b-BHC, g-BHC, d-BHC, 4,49-DDT, 4,49-DDE, 4,49-DDD, aldrin, dieldrin, heptachlor, heptachlor epoxide, endrin, endrin aldehyde, a-endosulfan, b-endosulfan, endosulfan sulfate

Chinese herbal medicines

Grinding, 2 g of Florisil on top of 0.1 g sample

CO 2 , 250 atm, 508C, 5 min static, 20 min dynamic

Deactivated fused-silica beads at 2 308C trap. Eluted with hexane at 308C. Off-line GC

78–121%

[24]

Hexachlorobenzene, aldrin, dieldrin, endrin,

Spiked garlic

Grinding, mixed with anhyd. MgSO 4 in an ice-water bath

CO 2 , 30.3 MPa, 408C, 1 min static, CO 2 volume 25 ml

5 ml hexane, the extract was transferred onto AgNO 3 -loaded Florisil (pre-washed with hexane), and eluted with 20% ethyl acetate in hexane

85.0–110.0%

[22]

35 organophosphorus pesticides including acephate and methamidophos

Spiked fruits and vegetables

Grinding, diatomaceous earth or superadsorbent polymer was mixed

CO 2 , 0.80 g/ml, 214 bar, 508C, 5 min static, 10 min dynamic, 1.2 ml/min

C 18 at 408C, eluted with acetone

1. Diatomaceous earth as drying agent, methamidophos: 3–5%, acephate: 3–5%, other pesticides: 83–103% 2. super-adsorbent polymer as drying agent. methamidophos: 21–41%, acephate: 29–48%, other pesticides: 52–93%

[12]

3.1. Fruits and vegetables Fluazinam, a systemic fungicide preventing cucumber gray mold (Botrytis cinerea) on fruits was extracted by supercritical CO 2 alone. The trapped analyte with water was dissolved in methanol and cleaned up by liquid–liquid partition [18]. Faugeron et al. studied 25 pesticides by an analytical method applied to cereals and cereal products. Organochlorine, organophosphorus, organonitrogen and pyrethroids, i.e., fonofos, pirimiphos-methyl, phosalone, dichlorvos, thiometon, chlorpyriphos, malathion, pyrazophos, hexaconazole, cyproconazole, tebuconazole, flusilazole, pirimicarb, iprodione, chlorothalonil, a-endosulfan, b-endosulfan, cypermethrine, cyhalothrine, deltamethrine, bifenthrine, fluvalinate, fenvalerate and cyfluthrine were

N. Motohashi et al. / J. Biochem. Biophys. Methods 43 (2000) 313 – 328 Table 2 Applications of SFE to pesticides of soils

319

Analyte(s)

Matrices

Metribuzine

Spiked soils (Lowell silt loam)

Carbaryl, pirimicarb, aldicarb

Spiked soils

Trichloropyridinol (metabolite of chlorpyrifos)

Spiked soils

Soils

Pre-treatment

SFE conditions

Collection and analysis

Mean recovery

Refs.

Initial conditions: CO 2 1 2%CH 2 Cl 2 MeOH (1:2), CO 2 , 0.85 g/ml, 508C, 5 min, static, 20 min, dynamic Final conditions: CO 2 1 15% water: CH 2 Cl 2 :EtOH (1:5:10) (0.05% triethylamine added) 0.75 g/ml, 408C, 5 min static, 10 min, dynamic

C 18 bonded silica. Initial: 758C; Final: 1058C, Off-line GC–NPD, GC–MS

. 90%

[25]

Exposed overnight to come into equilibrium with the laboratory atmosphere moisture

CO 2 1 10%DMSO, 300 atm, 708C, 10 min static, 30 min dynamic

Liquid collection in vial packed with glass wool. Off-line HPLCpostcolumn OPA fluorescence reaction

91.5–107.8%

[26]

0.5 g of soil 1 0.5 ml of 0.1 M(IR)(2)-10-camphorsulfonic acid ammonium salt in MeOH 1 1 ml MeOH 0.1 g of soil 1 0.04 g of diatomaceous earth

CO 2 , 408C, 383 bar, 0.8 g/ml, 1.0 ml/min, 30 min dynamic

A packing of small stainless steel beads. Off-line immunoassay

94.7–102.8%

[6]

Subcritical water, 2508C, 200 bar, 4 ml/ min, 15 min dynamic

Off-line immunoassay

95.7–99.9%

[6]

Fenpropimorph, pirimicarb, parathion-ethyl, triallate, fenvalerate

Spiked soils, spiked field soil

Grinding and mixing with Hydromatrix

CO 2 1 5%MeO 3.8 3 107 Pa, 608C, 2 ml/min

Diol-modified silica gel trap. Extracted with ethyl acetate. Off-line GC

93–104%

[27]

Chlorsulfuron, tribenuronmethyl (sulfonyl ureas)

Spiked soils

100 ml of MeOH added to sample (chlorsulfuron)

CO 2 , 0.85 g/ml, 2 ml/min, 508C, 15 min dynamic

Trapped with 550– 650 mm stainless steel balls at 108C. Eluted with acetonitrile. Off-line HPLC

Chlorsulfuron 68.3–76.6% tribenuron-methyl 67.7–80.9%

[28]

CO 2 , 508C 0.90 g/ml, 6 min static 26 min dynamic

C 18 trap Off-line HPLC and TLC

Cyanzine 98% cyanzine amide 36.7% cyanzine hydroxy acid , 5%

[29]

50 ml of MeOH added to sample (tribenuron-methyl) Cyanzine and its metabolites

Spiked soils (Dundee silty clay loam soil)

Air-dried (moisture content 3.5%, w/w), added 20% (v/w) MeOH–water (1:1) as a modifier

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Table 2. Continued Analyte(s)

Matrices

Pre-treatment

SFE conditions

Collection and analysis

Mean recovery

Refs.

Chlorothalonil

Soils

Dried

CO 2 1 5% MeOH 400 atm, 408C 10 min static 20 min dynamic 0.7 ml/min

25 ml of hexane Heated at 58.58C. Off-line GC

92.9% ,

[30]

Dacthal and its metabolites, monoacid metabolite, diacid metabolite

Spiked soil and contaminated soil

Dried

For dacthal CO 2 , 1508C 400 bar, 15 min dynamic

5 ml acetonitrile

100%

[7]

For metabolites subcritical water, 508C, 200 bar, 10 min dynamic

SAX disk trap ethyl iodide added for modification Heated at 1008C for 1 h. Off-line GC

99.7–100%

Subcritical water 908C 5 min extracted with 2.5 ml of water at a flow rate of 0.4 ml/min and 22.5 ml of water at a flow rate of 1 ml/min

Carbograph 4 soild phase extraction cartridge trap. Rinsed with water. The cartridge was turned upside down and eluted with 1.5 ml of MeOH and 8 ml of CH 2 Cl 2 /MeOH (95:5, v/v) (nonacidic herbicides). Subsequently eluted with 10 ml of CH 2 Cl 2 /MeOH (80:20, v/v) acidified with 50 mmol/l of formic acid (LC–MS (negative-ion acquisition mode)

81–93% except 2,4-DB, MCPB 63%

Phenoxy acid herbicides nonacidic analytes: cyanzine monuron simazine atrazine isoproturon diuron sec-butylazine linuron acidic analytes clopyralid picloram dicamba bentazone MCPA 2,4-D mecoprop dichlorprop bromoxynil ioxynil 2,4-DB MCPB

Spiked soils

3 g of soil was mixed with 2 g of sand

[8]

examined. The pesticides were extracted with supercritical CO 2 with 10% methanol added and trapped on a glass bead cartridge. The SFE method was compared to a standard multiresidue method based on solid–liquid extraction. The mean recovery of the SFE method was better than the recovery which was obtained by the standard multiresidue method [19]. A determination method for 56 diverse pesticides was reported by Lehotay and ´ [20]. The sample was frozen and a drying agent consisting of Valverde-Garcıa magnesium sulfate and Hydromatrix was mixed and homogenized with a small amount

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of dry ice. The sample was extracted with supercritical CO 2 , trapped with C 18 bonded silica, eluted with acetone, and subsequently analyzed by GC ion-trap mass spectrometry. Magnesium sulfate as a drying agent was mixed with Hydromatrix to control water, and gave a high recovery of methamidophos as well as of other pesticides. This paper was devoted more to trapping than to about other aspects. Some highly polar pesticides from the groups of phosphorothioates and phosphoramidothioates showed very low recoveries by the supercritical CO 2 extraction method (e.g., acephate, omethoate and vamidothion). Generally, a modifier was added to supercritical CO 2 to improve the extraction yield. Stefani et al. did not choose the addition of a polar solvent to supercritical CO 2 . They worked on many extraction steps using two steps, such as two subsequent extractions of the same sample. The two steps were similar except for the volume of trap washing solvent. Celite and anhydrous calcined sodium sulfate were examined as drying agents added to samples [21]. A SFE method was applied to organophosphorus pesticides in foods. Thirty-five pesticides such as dichlorvos, methamidophos, acephate, ethoprophos, thiometon, dioxabenzofos, terbufos, diazinon, etrimfos, iprobenfos, dichlofenthion, cyanophos, dimethoate, tolclofos-methyl, pirimiphos-methyl, chlorpyrifos, parathion-methyl, fenthion, malathion, fenitrothion, parathion, bromophos-methyl, isofenphos, phenthoate, mecarbam, prothiofos, methidathion, butamifos, ethion, trithion, edifenphos, EPN, pyridaphenthion, phosmet and phosalone were examined. Although acephate and methamidophos were hardly recovered when diatomaceous earth was used as a drying agent, the recoveries of these analytes were improved by super-adsorbent polymer Arasorb S100J (Arakawa Chemical Industries, Tokyo, Japan) as a drying agent [12]. Wang et al. demonstrated an approach using SFE followed by clean-up with a AgNO 3 -loaded Florisil column which was used for the analysis of organochlorine pesticides in garlic. An active enzyme in garlic converts the sulfur-containing compound alliin into allicin. Allicin degrades to form the secondary products consisting of various sulfides, and these sulfur-containing products give strong electron-capture detection responses. AgNO 3 -loaded Florisil was developed as an SFE adsorbent to remove the sulfur-containing compounds. The extracted solution (SFE method) was transferred onto a column packed with AgNO 3 -loaded Florisil, and the column was eluted with 20% ethyl acetate in hexane [22]. The optimization of SFE of several organochlorine and organophosphorus pesticides in high-water-content samples was performed. The pesticides were as follows: vinclozolin, tolclofos-methyl, malathion, chlorpyrifos, 4,49-dichlorobenzophenone, procymidone, 4,49-DDE, chlorbenzilate, b-endosulfan, bromopropylate and tetradifon. Lyophilization and addition of anhydrous sodium sulfate were examined to solve the problem caused by the water content of vegetable samples [23]. A method involving the simultaneous extraction and clean-up of 13 organochlorine pesticides from Chinese herbal medicines was developed using SFE followed by gas chromatography–electron capture detection and mass spectrometric confirmation. The examined pesticides were as follows: a-BHC, b-BHC, g-BHC, 4,49-DDT, 4,49-DDE, 4,49-DDD, aldrin, dieldrin, heptachlor, heptachlor epoxide, endrin, endrin aldehyde, a-endosulfan, b-endosulfan, and endosulfan sulfate [24].

Corn oil and butter fat

Honeybees

Spiked meat

117 nonpolar to moderately polar organochlorine and organophosphate pesticides

Organophosphate, carbamates

Organophosphorus pesticides: chlorpyrifos, chlorpyrifus-methyl, malathion, pirifos-methyl, prothiofos

Other pesticides: carbofuran, phorate, procymidone, vinclozolin

Matrices

Analyte(s)

Table 3 Applications of SFE to pesticides in biological tissues

Hydromatrix was filled in the thimble

Blending and mixing with Hydomatrix at a honeybee/ Hydromatrix ratio of 1/2

4–5 g of fatty sample, 2–2.7 g of Hydromatrix added and mixed 0.5 ml of water added

Pre-treatment

CO 2 ,0.40 g/ml, 958C 2 ml/min 2 h

CO 2 , 608C, 187 bar (0.7 g/ml), 2 min static, 25 min dynamic

CO 2 , 27.58 MPa, 608C, 5 min static CO 2 1 3% (v/v) acetonitrile, 5 min dynamic, 1.4 ml/min

SFE conditions

Florisil trap at 358C Eluted with heptane (Fraction 1) and acetone at 508C (Fraction 2). Off-line GC

C 18 bonded silica trap. Off-line GC

C 1 silica-based preparative column trap (250 min 3 10 mm [) Eluted with acetone2-propanol (70:30, v/v)

Collection and analysis

[34]

[32]

. 75% except omethoate ( , 65%)

78–95%

[31]

Ref.

–

Mean recovery

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11 PCBs

15 organochlorine pesticides: a-BHC, b-BHC, g-BHC, d-BHC, aldrin, dieldrin, heptachlor, heptachlor epoxide, 4.49-DDT, 4.49-DDE, 4.49-DDD, a-endosulfan, endosulfan sulfate, endrin, endrin aldehyde

Spiked mussels, real samples

Lyophilized Aliquots of Florisil were added on top of the spiked mussel tissues

Co 2 , 250 atm, 508C, 5 min static, 20 min dynamic

Deactivated fusedsilica beads trap at 2 308C. Eluted with hexane at 308C Off-line GC

44–118%

[33]

N. Motohashi et al. / J. Biochem. Biophys. Methods 43 (2000) 313 – 328 323

Matrices Spiked wool wax

Recycled plastics (low density polyethylene, 90%, w/w) and ethylene– vinyl acetate copolymer (10%, w/w) supplied as a film of 90 mm thickness and as pellets

Spiked to diatomaceous earth (Celite)

Analyte(s)

Propetamphos diazinon, chlorfenvinphos, carbophenothion, cyhalothrin, coumaphos, cypermethrin, deltamethrin

Vinclozin, tolclofosmethyl, malathion, chlorpyrifos, 4,49-dichlorobenzophenone, procymidone, 4,49-DDE, chlorobenzilate, a-endosulfan, bromopropylate, tetradifon

88 pesticides, 16 organochlorine 33 organophosphorus, 8 pyrethroid 12 carbamate and 19 others containing acephate methamidophos and propamocarb

Table 4 Applications of SFE to others

Modifiers, water

Modifier: 400 ml toluene ‘sandwhich’ mode, i.e., silanized glass wool, anhydrous Na 2 SO 4 , sample and more silanized glass wool

Wool wax was dissolved in hexane–diethyl ether (6:4) to give a 10% (w/v) solution, 2 ml of wool wax solution was added to 2 g of Chromosorb W-HP in the extraction cell, a gentle stream of dry air was drawn through the cell

Pre-treatment

CO 2 , 508C, 0.70 g.ml 2.0 ml/min 3.0 min static 20 min dynamic

CO 2 , 400 atm, 758C, 2.0 ml/min, 5 min static, 10 min dynamic

CO 2 , 808C, 250 atm

SFE conditions

C 18 trap. Off-line GC

Silanized glass bead cartridge (80/100 mesh, Suprex). Off-line GC

6 ml toluene containing chlorpyrifos-ethyl as an internal standard

Collection and analysis

79 pesticides: . 90%; Others: . 70% except acephate, methamidophos, propamocarb

[13]

[36]

[35]

. 85%

90% for plastic film 40% for plastic pellets

Ref.

Mean recovery

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3.2. Soils Recently, supercritical fluids have been shown to yield good extraction efficiency for the triazine herbicides. These analytical procedures did not include metribuzine, one of the polar triazine compounds. The effect of several SFE parameters on the extraction efficiency of metribuzine was examined. The extraction efficiency of SFE was compared to the results obtained by traditional Soxhlet extraction methods [25]. For the determination of three carbamates, carbaryl, primicarb, and aldicarb for fruits, a suitable analysis was developed. The optimal fluid condition for the extraction of the carbamates from the majority of soil types was supercritical CO 2 with 10% DMSO added [26]. The extraction of trichloropyridinol, a metabolite of chlorpyrifos in soil, was performed with supercritical CO 2 and subcritical water. Owing to the polarity of the analyte, addition of both methanol and ion pair reagent [(IR)-(2)-10-camphorsulfonic acid ammonium salt] was necessary for the extraction with supercritical CO 2 . Subcritical water enabled the extraction without additives [6]. The applicability of SFE for residue analysis of selected pesticides in soil was investigated. A wide spectrum of different pesticides with physico-chemical properties was examined. Fenpropimorph (morpholine), pirimicarb (carbamate), triallate (thiocarbamate) and fenvalerate (pyrethroid) were applied to soil samples. Best efficiency was achieved at 608C extraction temperature and CO 2 pressure of 3.8 3 10 7 Pa using 5% methanol as modifier, a diol-modified silica gel trap and ethyl acetate as eluent. Recoveries of the target compounds ranged from 93 and 104% [27]. SFE-HPLC methods for the determination of tribenuron-methyl and chlorsulfuron (sulfonyl urea herbicides) residues in different types of solids were proposed. Methanol was added in advance to the spiked sample and then extracted with supercritical CO 2 , trapped with stainless steel balls at 108C, and eluted with acetonitrile [28]. Efficiency of supercritical fluid extraction for the recovery of cyanazine and its metabolites from soil was investigated. The extractability of cyanazine in soil without any modifier was poor, while the addition of methanol–water (1:1) mixed solvent improved the recovery [29]. The development of an analytical method for chlorothalonil from cranberry bog soil was reported. Chlorothalonil is used as a fungicide on fruits, vegetables, and ornamental plants. Soil samples were dried, extracted by supercritical CO 2 with 5% methanol added, and trapped in hexane. The results obtained by this method were then compared to a Soxhlet extraction procedure [30]. Dacthal and its mono- and diacid metabolites were consecutively extracted from soil by first performing the supercritical CO 2 extraction to recover dacthal, followed by subcritical water extraction step to recover the metabolites. Dacthal is a widely used pre-emergent herbicide, applied to many crops for the control of annual weeds [7].

3.3. Biological tissues An automated supercritical extraction and in-line clean-up system was developed for organochlorine and organophosphorus pesticide residues contained in fats. In all, 117

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nonpolar (to moderately polar) pesticides were examined. Eighty-six percent of the 117 pesticides were recovered by the method, and 31 pesticides were not recovered at all. Pesticide residues could be separated from butter fat and corn oil. The fatty sample was mixed with Hydromatrix and just a little water was added. The analytes were first extracted with supercritical CO 2 alone and then with supercritical CO 2 containing 3% acetonitrile. The analytes were trapped with C 1 silica-based preparative column [31]. An improved SFE method combining GC with ion-trap MS was performed for organophosphorus and carbamate insecticides, such as dichlorvos, heptenophos, demeton-S-methyl, omethoate, thiometon, diazinon, disulfoton, dimethoate, pirimiphosmethyl, chlorpyrifos, fenitrothion, quinalphos, vamidothion, triazophos and azinphosmethyl in honeybees [32]. For the determination of 15 organochlorine pesticides, lyophilized mussel tissue was ground to obtain homogeneous powder. Aliquots of Florisil which were activated in advance by drying at 1508C were added on top of the mussel tissues. The examined pesticides were as follows: a-BHC, b-BHC, g-BHC, d-BHC, aldrin, dieldrin, heptachlor, heptachlor epoxide, 4,49-DDT, 4,49-DDE, 4,49-DDD, a-endosulfan, endosulfan sulfate, endrin and endrin aldehyde [33]. A method for extraction of pesticides in meat and fatty matrices was developed. Hydromatrix was added to the sample, extracted with supercritical CO 2 , trapped with Florisil and eluted with heptane (Fraction 1) and acetone (Fraction 2). The polarity range covered by the SFE method was demonstrated using organophosphorus pesticides, chlorpyrifos, chlorpyrifos-methyl, malathion, pirimifos-methyl and prothiofos. Additionally, the method was evaluated for several (some) other pesticides — carbofuran, phorate, procymidone, and vinclozoline [34].

3.4. Other applications For the gas chromatographic analysis of pesticides in wool wax, SFE was investigated as a sample clean-up procedure [35]. From recycled plastics, several organochlorine and organophosphorus pesticides and their metabolites were extracted: vinclozolin, tolclofos-methyl, malathion, chlorpyrifos, procymidone, chlorbenzilate, b-endosulfan, bromopropylate, tetradifon, 4,49-dichlorobenzophenone, and 4,49-DDE [36]. SFE conditions for multiresidue analysis of pesticides were evaluated using diatomaceous earth (Celite) spiked with 88 pesticides (16 organochlorine, 33 organophosphorus, eight pyrethroid, 12 carbamate and 19 other pesticides). The SFE parameters were investigated on CO 2 density, CO 2 flow rate, extraction temperature, static and dynamic extraction times, trap temperature, and addition of modifier. Although SFE without any modifier was insufficient to extract polar pesticides from fortified Celite, the addition of water to Celite showed most effective enhancement of the recovery for almost all pesticides. Acephate, methamidophos and propamocarb were not recovered when water was used as a modifier. To investigate the effect of modifiers, other solvents such as methanol, ethanol, 2-propanol, acetonitrile, acetone, ethyl acetate, dichloromethane and hexane were also evaluated. For triadimenol, bitertanol, acephate and methamidophos, the highest recoveries were achieved with the addition of methanol.

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Consequently, water was chosen as a modifier because of its wide range of optimum amounts for addition to Celite [13].

4. Conclusions The main merit of SFE lies in the simplicity of the analytical procedures. In most cases, the extracts by SFE contain less co-extractives in comparison to the extracts which are obtained by other extraction methods and need less clean-up procedures. Secondly, although the low polarity of supercritical CO 2 , almost the same as hexane, seems to limit the range of analytes that can be extracted with SFE, almost moderately polar and nonpolar pesticides can be extracted with supercritical CO 2 alone. Water derived from the sample is valuable as a modifier and enables the extraction of relatively polar pesticides. Sometimes, an organic solvent, such as methanol, acetone or acetonitrile, is added to supercritical CO 2 or into the sample in the extraction thimble to improve the recoveries of some problematic polar analytes, such as methamidophos, acephate or omethoate. The addition of an appropriate mole fraction of the second fluid, e.g., 30 mol% nitrogen in supercritical CO 2 at 8000 psi, and 60–808C, plays a key role in reducing the lipid content of the extracts for the determination of organochlorine and organophosphorus pesticides in fatty samples [5]. Third, SFE can be applied to high-water-content samples by the addition of a drying agent such as Hydromatrix, magnesium sulfate or super adsorbent polymer. The use of a super-adsorbent polymer improved the recoveries of highly polar analytes such as methamidophos and acephate. The addition of a sufficient amount of adsorbent prevents restrictor plugging by water which is extruded by supercritical CO 2 during the dynamic extraction step. Also, the amount of water (approximately 0.3%) which is dissolved in supercritical CO 2 is not influenced by the use of adsorbents, which can only bind water physically. Finally, for the future automation of analysis, one-step supercritical fluid extraction and the clean-up system are introduced and examined [25].

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