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Chapter 35

Supercritical Fluid Extraction—Enzyme-Linked Immunosorbent Assay Applications for Determination of Pesticides in Soil and Food 1

1

2

Viorica Lopez-Avila , Chatan Charan , and Jeanette Van Emon 1

Midwest Research Institute, California Operations, 625-B Clyde Avenue, Mountain View, CA 94043 Characterization Research Division, National Exposure Research Laboratory, U.S. Environmental Protection Agency, 944 East Harmon Avenue, Las Vegas, NV 89119 2

This paper describes the use of off-line supercritical fluid extraction (SFE) and enzyme-linked immunosorbent assay (ELISA) for the determination of nine pesticides (alachlor, aldicarb, atrazine, carbaryl, carbendazim, carbofuran, cyanazine, 2,4-D, and metolachlor) in soil and food matrices. Soil samples (freshly spiked or spiked and aged soils) were extracted with supercritical carbon dioxide containing 10 percent methanol (as modifier) at a flow rate of approximately 2 mL/min. The extraction conditions were: pressure, 450 atm; temperature, 80 °C; and extraction time, 30 min (dynamic). Food samples consisting of baby food and Food and Drug Administration (FDA) Total Diet Study (TDS) samples were extracted at lower pressures and temperatures (150 atm, 70 °C) but for a longer period of time (15 min static and 60 min dynamic) and without the modifier to avoid extraction of the fat present in these samples. Acetonitrile was used as matrix modifier in the food extractions. In both cases, the extracted material was collected in reagent water. The benefits of SFE-ELISA include replacement of harmful organic solvents used in extraction, quick extractions with a relatively inexpensive extractant, reduced number of steps in the determination of the target compounds, and sensitive and relatively inexpensive assays. The U . S. Environmental Protection Agency (EPA), National Exposure Research Laboratory (formerly U . S. E P A Environmental Monitoring Systems Laboratory) in Las Vegas, N V , is involved in an ongoing program that addresses sample preparation and instrumental analysis techniques that are faster and less expensive than the current methodologies and that prevent or mirrimize pollution from analytical laboratories. Two such techniques that have been evaluated in this study include supercritical fluid extraction (SFE) and enzyme-linked immunosorbent assay (ELISA). The potential 0097-6156/96/0621-0439$15.00/0 © 1996 American Chemical Society


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IMMUNOASSAYS FOR RESIDUE ANALYSIS

benefits of using SFE and ELISA have been described before by Nam and King (7, See chapter by King and Nam, this volume) and Lopez-Avila et al (2,5). The SFE technique has been successfully applied to the extraction of pesticides from various environmental samples (7-7) as well as food matrices (8-10). A multiresidue method for the determination of 46 pesticides in foods and vegetables by SFE and gas chromatography/mass spectrometry has been recently published by Lehotay and coworkers (10). Despite the wide applicability of SFE to the extraction of pesticides from various matrices, only a few studies (7-5) actually considered ELISA for quantitative analysis of the target analytes. However, immunoassays have been recognized as alternative techniques to chromatographic methods for analyzing environmental contaminants, and are being used in field studies (77). This paper describes the use of SFE-ELISA for the determination of nine pesticides in soil and 10 pesticides in food matrices. Experimental Reagents. Nine ELISA test kits (Ohmicron Corporation, Newtown, PA) including those for alachlor, aldicarb, atrazine, carbaryl, carbendazim, carbofuran, cyanazine, 2,4-D, and metolachlor were tested for soil and the TDS samples. In addition, we used the cWorpyrifos ELISA kit from Ohmicron to analyze SFE extracts of baby food (Gerber beef vegetable, Gerber Products, Fremont, MI) spiked with cMorpyrifos. A l l immunological reagents used in this study, including paramagnetic particles coated with anti-pesticide antibody (suspended in buffered saline with preservatives and stabilizers), pesticide enzyme conjugate (horseradish peroxidase labeled pesticide analog), phosphate buffer, hydrogen peroxide solution (0.02% in a citric buffer), chromogen solution (3,3',5,5'-tetramethylbenzidine 0.4 g/L in an organic base), stopping solution (2 M sulfuric acid), and washing solution (preserved deionized water) were obtained from Ohmicron Corporation. Materials. The soil samples used in this study are identified in Table I. The soil containing humic acid was prepared by mixing 3 g topsoil with 60 mg of humic acid sodium salt (Aldrich lot No. 00186HH, Aldrich Chemical Co., Milwaukee, WI). The topsoil and clay soil were obtained from Sandoz Crop Protection Co. (Gilroy, CA). The three soils identified as RT-801, RT-802, and RT-803 were obtained from R T Corporation (Laramie, WY). These samples contained various chlorophenoxy acid herbicides at levels ranging from 700 to 70,000 pg/kg. These samples were spiked only with the other eight pesticides since 2,4-D was already present in the samples at either 700, 7000, or 70,000 pg/kg. Several composite solutions containing the nine pesticides in methanol were used for spiking. For example, to prepare the 10-pg/kg spiked soil sample, 300 pL of a 100-ng/mL solution was added to the 3-g soil sample. The 50- and 500-pg/kg spiked soil samples were prepared using 150 pL of a l-pg/mL or a 10-pg/mL solution, respectively; the 700- and 7000-pg/kg spiked soil samples were prepared using 210 pL of a 10-pg/mL or a 100-pg/mL solution, respectively; and the 25,000pg/kg spiked soil samples were prepared using 750 pL of a 100-pg/mL solution. For spiking the soil matrix, 3-g portions of the soil matrix were weighed into an aluminum cup, and a concentrated solution containing the pesticides in methanol was


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LOPEZ-AVILA ET AL.

SFE-ELISA

To Detect Pesticides in Soil & Food

441

added with a syringe. Losses were minimized by ensuring that the solution did not contact the aluminum cup. Mixing was performed by gently shaking the aluminum cup by hand. The spiked samples were then loaded into the extraction vessel within 100 min of spiking and sandwiched between two plugs of silanized glass wool. The TDS sample used in this study is identified as sample RTP92A-007-DFPH (fatty); this is a composite solid sample from a diet pilot study that was conducted by Research Triangle Institute (Research Triangle Park, NC). The sample was kept frozen at -10 °C for approximately 5 months. Immediately prior to extraction, it was allowed to thaw at room temperature; 4-g portions were removed after thoroughly mixing the contents of the glass container, in which the sample was kept, with a glass rod. Each portion was spiked with a composite solution of the nine pesticides in methanol (spike level was 2 ng/g), and was then mixed with 1.5 g Hydromatrix, a pelletized diatomaceous earth (Varian, Harbor City, CA), and placed in the extraction vessel between two 2-g plugs of basic alumina. The baby food was Gerber beef vegetable (Gerber Products); it was spiked with the nine pesticides or chlorpyrifos and then mixed with Hydromatrix, as indicated above, for the TDS sample. SFE Procedure. The SFE operating conditions for soil samples are summarized in Table II. Soil samples (freshly spiked or spiked and aged soils) were extracted with a Dionex Model 703M SFE system. This system consisted of two pumps; one pump delivered the carbon dioxide; the second pump delivered the methanol at a constant volume ratio. The extracted material was collected in reagent water (pH 4.5) and was diluted with phosphate buffer immediately prior to analysis by ELISA. The dilution factors were chosen in such a way that the analyte concentration would fall within the linear range of the ELISA test kit. The SFE operating conditions for food samples are summarized in Table ΙΠ. TDS samples were extracted with an Isco SFX2-10 extractor and the spiked baby food samples were extracted with the Dionex Lee Scientific SFE system. In this case, different conditions were used to minimize the extraction of fat from the food matrix. Acetonitrile (300 μL) was used as a matrix modifier. All SFE experiments were performed with SFE/SFC-grade carbon dioxide (Air Products, Allentown, PA). E L I S A Procedure. For the ELISA, 100-250 of the soil extract (diluted with pH4.5 reagent water to 10 mL and subsequently with phosphate buffer as indicated above), 250 μΐ. of pesticide enzyme conjugate, and 500 μL of the anti-pesticide antibody coated paramagnetic particle solution was combined in a test tube. After vortexing for 1 to 2 s, the test tube was incubated at room temperature for 15-30 min. The mixture was separated using a magnetic separation rack, and was washed twice with the washing solution. The rack containing the tubes was removed from the magnet, and 500 μL of a freshly prepared chromogenic solution (hydrogen peroxide and 3,3\5,5'-tetramethylbenzidine) was added to each test tube and allowed to develop color. The reaction was stopped after 20 min with 500 μί, stopping solution (2 M sulfuric acid), and the color intensity in each test tube was determined at 450 nm using an RPA-1 photometric analyzer (Ohmicron Corporation).


442

Table I. Physico-chemical properties of the soil samples used in this study.


Parameter

Units

pH Cation exchange mequiv/100 g capacity

Topsoil

Clay Soil RT-801

RT-802

RT-803

7.5

7.4

8.1

8.0

7.4

14.6

21.3

3.8

10.0

10.5 2.48

0.4

0.76

Organic carbon content

%

0.1

1.8

Sand

%

57.6

33.6

66.0

29.0

63.0

Silt

%

21.8

35.4

26.0

50.0

19.0

Clay

%

20.6

31.0

21.0

18.0

8.0

Table Π. SFE operating conditions for the soil samples. Parameter Instrument

Dionex-Lee Scientific Model 703M with Cosolvent Addition Module; vessel size 10 mL

Operating conditions Fluid Pressure Temperature Flow rate Extraction time Restrictor temperature

Carbon dioxide with 10 % methanol 450 atm 80 °C 1.6-2.2 mL/min 30 min (dynamic) 100 °C

Collection solvent

Reagent water at pH 4.5 (5 mL)

Temperature of collection vial Sample size

2°C

a

a

3g

The SFE extract was first diluted to a 10-mL final volume with reagent water (pH 4.5). The diluted extract was subsequently diluted prior to E L I S A to bring the analyte concentration within the linear range of the ELISA test kit.


35.

LOPEZ-AVILA ET AL.

SFE-ELISA


Table III. SFE operating conditions for food samples.

Parameter Instrument

Operating conditions Fluid Pressure Temperature Flow rate Extraction time Restrictor temperature Collection solvent Temperature of collection vial Sample size

443

3

Isco SFX 2-10 equipped with 260-mL syringe pump and extraction module for simultaneous extraction of two samples; restrictor 24-cm length χ 50-pm ID fused-silica capillary; vessel size 10 mL Carbon dioxide 150 atm 70 °C 1-1.3 mL/min 15 min (static), 60 min (dynamic) 100 °C Reagent water (5 mL) Room temperature

4 g food sample was dispersed with 1.5 g Hydromatrix and placed between two 2-g portions of basic alumina Matrix modifier Acetonitrile (300 pL) A Dionex-Lee Scientific extractor was used for the extraction of chlorpyrifos from spiked baby food samples. The extraction conditions for chlorpyrifos are as specified above except that the extraction time was 30 min (dynamic). a

444



Concentrations of the nine pesticides were determined by comparing the results to a linear regression line using a ln/logit standard curve of the particular pesticide obtained at different concentrations as follows: 0, 0.1, 1.0, and 5.0 ng/mL for alachlor, atrazine, carbofuran, and metolachlor; 0, 0.25, 1.0, and 5.0 ng/mL for carbendazim; 0, 0.4, 1.5, and 5.0 ng/mL for carbaryl; 0, 0.1, 1.0, and 3.0 ng/mL for cyanazine; 0, 1, 10, and 100 ng/mL for aldicarb; and 0, 1, 10, and 50 ng/mL for 2,4D. Results and Discussion Despite the fact that supercritical carbon dioxide can dissolve a wide range of nonpolar and moderately polar compounds, preliminary experiments indicated that the nine compounds listed in Table IV were not extracted from freshly spiked soil with supercritical carbon dioxide. Therefore, we used supercritical carbon dioxide modified with 10% methanol and operated the SFE system at 450 atm; the temperature was set at 80 °C resulting in a supercritical fluid density of 0.86 g/mL. In addition, we verified the potential for cross-reactivity of each antibody with the nine pesticides in the spiking mixture and present the data in Table IV. Recoveries greater than 80% (Table V) were achieved for alachlor, atrazine, carbofuran, cyanazine (except for topsoil spiked at 10 μg/kg and soil mixed with humic acid spiked at 50 μg/kg), and metolachlor. The other four compounds exhibited recoveries ranging from 55.0 to 227% for aldicarb, 50.0 to 99.3% for carbaryl, 34.8 to 96.7% for carbendazim, and 40.3 to 99.3% for 2,4-D. Recoveries in excess of 130% were likely to be due to interferences from the matrix and not to quantification errors since we have repeated these experiments with separate portions of the spiked soil and obtained the same results. With the exception of carbendazim, recoveries of the target compounds from the spiked and aged soils were higher than those from the freshly spiked soils and also were less variable. This may be due to the fact that two of the aged soils were sandy-loam type soils that are less adsorptive than the clay-type soils. Overall, the precision of the SFE-ELISA technique, as established from the percent relative standard deviations (RSDs) of triplicate or quadruplicate determinations, is 20% or better for 56 of the 81 RSD values (or 69% of the total determinations) given in Table V and 67 of the 74 RSD values (or 91% of the total determinations) given in Table VI. For the food matrix, the approach taken in this study was quite different than that for the soil matrix. In the latter, we performed the extraction with supercritical fluid at relatively high pressure and high methanol content of the supercritical carbon dioxide and somewhat moderate temperature. For the food matrix, we knew that if we performed the extraction under these conditions, we would extract the fatty acids and triglycerides that would interfere with the ELISA determination. To miriirnize the amount of fat extracted by SFE, we chose to perform the extraction at 150 atm and 70 °C (density 0.51 g/mL). Under these conditions, only six of the target compounds (Table Vu) were recovered from spiked Hydromatrix (recoveries were at least 70% with the exception of aldicarb at 69.3%). Carbaryl recovery was 49.4%, carbendazim was poorly recovered and 2,4-D was not recovered at all. The Hydromatrix was

35.

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SFE—ELISA To Detect Pesticides in Soil & Food


Table IV. E L I S A results for composite standards.

Compound Alachlor

Aldicarb

Atrazine

Carbaryl

Carbendazim

Carbofuran

Cyanazine

2,4-D

Metolachlor

a

True Concentration (ng/mL) 0.1 1.0 3.0 5.0 0.1 1.0 3.0 5.0 0.1 1.0 3.0 5.0 0.1 1.0 3.0 5.0 0.1 1.0 3.0 5.0 0.1 1.0 3.0 5.0 0.1 1.0 3.0 0.1 1.0 3.0 5.0 0.1 1.0 3.0 5.0

N D = not detected.

Measured Concentration (ng/mL) 0.1 1.2 3.4 5.8 0.1 0.7 2.1 5.5 0.2 1.4 2.4 4.5 0.1 0.8 2.5 4.3 0.1 1.2 3.4 5.5 0.1 1.1 2.9 5.1 0.1 1.4 3.0 ND 1.7 2.5 6.4 0.1 1.3 3.0 6.8 a

445

446


Table V. SFE recoveries of the target pesticides from freshly spiked topsoil, clay soil, and soil with humic acid.


a

Compound

% Average Recovery Spike Soil Mixed Level with Humic (pg/kg) Topsoil Clay Soil Acid

Alachlor

10 50 500

Aldicarb

10 50 500

86.0 80.9 103 193b

96.7 79.3 112 b

95.6 81.2 87.7

78.0 61.5

187 55.0 69.0

225 96.7 85.1 119 104 124

b

% RSD Soil Mixed with Humic Acid Topsoil Clay Soil 15 7.9 5.2

16 24 11

8.3 10 11

36 34 9.3

26 30 26

29 33 32

1.9 10 8.6

8.4 6.8 12

Atrazine

10 50 500

106 82.1 89.1

104 86.0 107

Carbaryl

10 50 500

50.0 56.5 71.9

64.0 60.0 73.3

73.0 99.3 93.9

11 17 22

17 21 11

21 8.5 12

Carbendazim

10 50 500

37.8 40.0 34.8

75.6 56.3 52.5

96.7 55.7 61.3

12 12 24

35 24 17

18 27 29

Carbofuran

10 50 500

99.4 94.5 85.0

108 90.0 88.5

119 113 116

11 25 8.8

12 7.9 8.7

21 12 6.5

Cyanazine

10 50 500

75.6 92.5 86.0

84.4 105 89.0

121 76.0 103

20 12 8.0

9.8 11 17

18 9.8 12

2,4-D

10 50 500

61.3 40.3 43.3

99.3 60.0 69.0

81.3 59.0 50.4

20 15 30

34 26 34

27 34 6.8

Metolachlor

10 50 500

109 85.0 101

155 85.3 102

19 15 12

17 5.5 4.8

6.1 12 17

a

b

189 132 136

The number of deterrninations was four. conditions are given in Table Π. Cannot explain high recovery.

b

8.4 8.5 5.0

b

b

b

The sample size was 3 g. The SFE

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LOPEZ-AVILA ET AL.

SFE—ELISA To Detect Pesticides in Soil & Food

447

Table VI. SFE recoveries of the target pesticides from spiked and aged soil from R T corporation.*


Spike Level Compound

fog/kg)

% Average Recovery RT-802 RT-801 RT-803

% RSD RT-802 RT-801 RT-803

Alachlor

700 7000 25000

115 101 94.7

80.5 89.3 100

110 86.0 117

23 11 29

16 11 3.8

2 5.8 9.6

Aldicarb

700 7000 25000

149 111 116

122 116 126

120 110 117

15 6.2 7.6

11 4.4 11

33 6.7 _b

Atrazine

700 7000 25000

116 103 128

106 96.5 119

105 115 136

5.5 0.4 5.8

13.5 6.8 13

8.2 6.4 22

Carbaryl

700 7000 25000

75.9 75.2 99.1

109 88.5 102

158 89.1 122

12 8.8 14

19 10 29

11 15 20

210 2100 7500

76.3 81.6 89.7

37.7 36.7 60.5

44.9 30.9 31.3

6.6 8.5 24

13 13 14

13 10 15

Carbofuran

700 7000 25000

74.6 88.8 101

78.8 101 113

89.1 101 104

6.6 3.7 3.8

7.4 10 14

5.4 3.0 16

Cyanazine

700 7000 25000

88.3 75.9 90.1

84.4 82.4 106

87.1 78.8 105

7.4 7.8 25

4.2 1.2 14

16 6.2 17

2,4-D

700 7000 70000

69.9

700 7000 25000

98.4 86.0 97.6

Carbendazim

Metolachlor

a

14 61.3

17 46.2

98.4 91.2 120

101 94.8 118

16 12 13 15

12 2.8 16

11 3.5 17

The number of determinations was three. The sample size was 3 g. The SFE conditions are given in Table II. Duplicate determinations.

b

448


Table ΥΠ. Pesticide recovery from spiked TDS sample and hydromatrix.* Spike Level


Compound

b

TDS Sample % Average %RSD Recovery

Hydromatrix % Average Recovery

Alachlor

2

108.0

13.0

75.9

Aldicarb

2

86.5

29.0

69.3

Atrazine

2

74.6

14.0

118.0

Carbaryl

2

68.5

20.0

49.4

Carbendazim

2

10.5

14.0

16.7

Carbofuran

2

79.5

10.0

97.5

Cyanazine

2

30.4

24.0

70.0

2,4-D

2

ND

Metolachlor

2

72.6

0

ND

d

8.7

76.9

a

The extractions were performed by SFE using the conditions given in Table ΙΠ. The extracts were diluted twofold prior to ELISA. ^Triplicate determinations, duplicate determinations. N D = not detected. d

added to the food matrix to disperse it and make it into a free-flowing powder. Recoveries of the target compounds from the spiked food matrix were comparable to those from the spiked Hydromatrix with the exception of cyanazine (Table Vu). A manuscript describing the optimization of the SFE conditions using different baby foods is in preparation. Very encouraging results were obtained using SFE-ELIS A to extract chlorpyrifos from spiked baby food (Table VOX). The applications presented here indicate that SFE-ELISA is a promising technique for the determination of pesticides in soil as well as in food matrices. Use of multivessel SFE systems will result in increased sample throughput, and use of supercritical carbon dioxide in place of organic solvents will reduce pollution resulting from the laboratory. As the SFE technology matures and the various parameters that affect the extraction efficiency are better understood, then more SFE applications will be developed. Further developments in ELISA include multianalyte immunoassays and the automation of both the plate and tube assays. Acknowledgments The U.S. Environmental Protection Agency (EPA, through its Office of Research and Development (ORD), funded and collaborated in the research described here. It has been subjected to the Agency's peer review and has been approved as an E P A publication. Neither the E P A nor ORD endorses or recommends any trade names or

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LOPEZ-AVILA ET AL.

SFE-ELISA


Table VIII. Chlorpyrifos recoveries from spiked baby food.


Spike Level (μβ/kg) 1

% Average Recovery 134.0

% RSD 14.0

10

98.3

7.4

1000

90.3

12.0

449

a

a

Gerber beef vegetable. The number of determinations was three. The SFE conditions are given in Table III.

commercial products mentioned in this article; they are noted solely for the purpose of description and clarification. Literature cited 1. Nam, K.-S.; King, J. W. J. Agric. Food Chem. 1994, 42, 1469-1474. 2. Lopez-Avila, V.; Charan, C.; Beckert, W. F. Trends Anal. Chem. 1994, 13, 118-126. 3. Lopez-Avila, V.; Charan, C.; Van Emon, J. M. Environ. Testing Anal. 1990, 3, 34-39. 4. Lopez-Avila, V., Dodhiwala, N. S.; Beckert, W. F. J. Chromatogr. Sci. 1990, 28, 468-476. 5. McNally, M. E. P.; Wheeler, J. R. J. Chromatogr. 1988, 447, 53-63. 6. Janda, V.; Steenbeke, G.; Sandra, P. J. Chromatogr. 1989, 479, 200-205. 7. Engelhardt, H.; Zapp,J.;Kolla, P. Chromatographia 1991, 32, 527-537. 8. Hopper, M. L.; King, J. W. J. Assoc. Off. Anal. Chem. 1991, 74, 661-666. 9. Lehotay, S.J.;Aharonson, N.; Pfeil, E.; Ibrahim, M. A. J. AOAC Int. 1995, 78, 831-840. 10. Lehotay, S. J.; Eller, Κ. I. J. AOAC Int. 1995, 78, 821-830. 11. Van Emon, J. M.; Lopez-Avila, V. Anal. Chem. 1992, 64, 78A-88A. RECEIVED

October 11, 1995

Immunoassays for Residue Analysis - ACS Publications - American

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