Enzyme Immunoassay Analysis Coupled with Supercritical Fluid

Chapter 6

Downloaded by SWINBURNE UNIV OF TECHNOLOGY on May 11, 2018 | https://pubs.acs.org Publication Date: October 23, 1996 | doi: 10.1021/bk-1996-0646.ch006

Enzyme Immunoassay Analysis Coupled with Supercritical Fluid Extraction of Soil Herbicides G. Kim Stearman, Martha J. M . Wells, Scott M . Adkisson, and Tadd E. Ridgill Center for the Management, Utilization and Protection of Water Resources, Tennessee Technological University, Box 5033, Water Center, North Dixie Avenue, Cookeville, TN 38505 Enzyme immunoassay analysis (EIA) was coupled with supercritical fluid extraction (SFE) for the analysis of herbicides 2,4-D, simazine, atrazine and alachlor in soil. Five soils, ranging in texture from sandy loam to silty clay were fortified with 500 ng/g of herbicide, allowed to air dry, and extracted using supercritical fluid or liquid vortex extraction. Field weathered soils with incurred residues were also extracted. EIA of herbicides using a microtiter plate format were in good agreement with GC or H P L C results (mean r of 0.95). SFE was performed using a Dionex model 703 extractor in the dynamic mode at 200 atm and 66°C for 3 min, followed by 340 atm extraction for 17 min. SFE recoveries with unmodified CO were 7, 56, 57, and 83%, respectively for 2,4-D, simazine, atrazine and alachlor. Recoveries improved to 101, 79, 90, and 88% for 2,4-D, simazine, atrazine and alachlor, respectively, by adding an acetone:water:triethylamine modifier (90:10:1.5, v:v:v). Collection of analytes by SFE was improved by using C solid-phase traps (90% recovery) compared to liquid acetone collection (65% recovery). There were differences in extraction recoveries based on soil type. 2

2

18

Enzyme immunoassay analysis (EIA) has gained acceptance as a technique for the rapid determination of pesticides. EIA can be used both as a screening method and as a semiquantitative method under different conditions. EIA microtiter plate techniques are easy to use and allow many samples to be run. In many cases EIA is also less expensive than traditional GC or H P L C methods. The major problem with using the EIA technique is the cross reactivity of similar compounds. This is not a problem with soil that contains no cross reacting compounds and that is spiked and extracted shortly after spiking. However with field weathered samples, the 0097-6156/96/0646-0056$15.00/0 © 1996 American Chemical Society

Van Emon et al.; Environmental Immunochemical Methods ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


6.

STEARMAN ET AL.

EIA Coupled with SFE of Soil Herbicides

57

metabolites can in some cases be more sensitive to the EIA than the parent compound. Supercritical fluid extraction (SFE) of organics from various environmental matrices has been utilized recently to avoid using large amounts of hazardous organic solvents, commonly used in traditional extractions. SFE, when coupled with enzyme immunoassay analysis (EIA) of the extracted pesticides, requires negligible organic solvent consumption and offers an alternative, inexpensive, safe and environmentally compatible method for determining pesticides in soil samples. The purpose of this study was to develop a SFE method, and couple it with EIA for the analysis of the herbicides 2,4-D, simazine, atrazine and alachlor in soil. C 0 is the most commonly used supercritical fluid because it is readily available and can be converted to the supercritical state at a relatively low pressure (72 atm) and temperature (31°C). SFE extraction of pesticides from soil often requires addition of polar organic modifiers, such as acetone or methanol, to the supercritical C 0 . The purpose of the modifier can be twofold; to increase the solubility of the analyte and/or to increase the surface area of the soil, by swelling the matrix (soil) or to competitively adsorb with the analyte to the soil. Extraction temperature must be increased as the modifier percentage is increased, in order to maintain the mixture in the supercritical state. Modifiers can also be added directly to the soil in the extraction cell. The concentration of the pesticides in the soil can be important in detennining extraction recoveries, as there may be differences in recovery between pesticide spikes of 10 ppm versus 50 ppb, under identical conditions. This may be due to the fact that at lower analyte concentrations, a larger percentage of the total pesticide concentration is less accessible to the extraction solvent than at higher pesticide concentrations. In addition to the actual extraction of the analyte from the matrix, the mode of sample collection plays an important role. Collection can be achieved either by directly eluting the sample into a liquid or by trapping on a solid phase, followed by solvent desorbtion. In the current study, EIA is compared with GC or H P L C for the analysis of 2,4-D, simazine, atrazine and alachlor in soil. The EIA is coupled with a SFE extraction method that has been optimized with respect to modifier addition and collection of analyte. 2

2

MATERIALS AND METHODS Soil Fortification. Properties of the herbicides used in this study are listed in Table I in order of increasing values of the soil adsorption coefficient (K^). The five soils described in Table II were fortified with 500 ng/g each of simazine and 2,4-D or with 500 ng/g each of atrazine and alachlor. Atrazine and alachlor were also added to give soil concentrations of 50 ng/g each. Soils were fortified by adding 50 mL of a herbicide-reagent grade water solution to 100 g of soil and allowing soils to air dry in a fume hood for several days.


58

ENVIRONMENTAL IMMUNOCHEMICAL METHODS

Table I.

Soil Adsorption Coefficient

Octanol-Water Partition Coefficient

900

20

443

Simazine

3

138

88

Atrazine

33

149

226

Alachlor

242

190

434

Herbicide

2,4-D Downloaded by SWINBURNE UNIV OF TECHNOLOGY on May 11, 2018 | https://pubs.acs.org Publication Date: October 23, 1996 | doi: 10.1021/bk-1996-0646.ch006

Herbicide properties*

Water Solubility (ppm)

'SOURCE: Adapted from ref. 2

Enzyme Immunoassay Analysis. Commercial EIA 96-well microtiter plate kits (Millipore, Inc., New Bedford, MA) were used for simazine, atrazine, alachlor and 2,4-D. Eight standards including a blank were made up in the same matrix as the diluted soil extracts and were analyzed on the microtiter plate in duplicate. The procedure followed that described previously (1). Soil extracts were diluted 25:1200:1 before pipetting into microtiter wells, dependant upon herbicide concentrations. H P L C and G C Analysis. A l l confirmatory analyses by either G C or H P L C were carried out on the same extracts used in the EIA analysis. Atrazine, alachlor, and 2,4-D were analyzed by GC; simazine was analyzed by H P L C . Atrazine and alachlor analysis was performed using a Hewlett Packard 5890 Gas Chromatograph equipped with a nitrogen-phosphorus detector (NPD) and a 30 m χ 0.32 mm i.d. (0.25 μηι film thickness) HP-5 capillary column. The helium carrier gas was maintained at a flow rate of 1.5 mL/minute. Helium was also used as the makeup gas at a flow rate of 10-15 mL/minute. Hydrogen and air were introduced at flow rates of 3.5 mL/min and 100-120 mL/min, respectively for operation of the N . D . The total gas flow through the detector was between 120-130 mL/min. Analysis of 2,4-D was performed using a Hewlett-Packard 5880 GC equipped with an electron capture detector (ECD) and a 30 m χ 0.53 mm i.d. (0.5 /xm film thickness) SPB-5 column (Supelco, Inc., Bellefonte, PA). The analysis was carried out on the 2,4-D methyl ester. Injector and detector temperatures were maintained at 250°C and 275°C, respectively. The carrier gas was helium at a flow rate of 10 mL/minute. Nitrogen was used as the makeup gas for the E C D resulting in a total flow rate of 60 mL/minute through the detector. Simazine analysis was performed by HPLC using a Hewlett-Packard 1090M liquid chromatograph equipped with a diode array detector, Chem Station data


u so 00

24.0 49.3

silty clay

Iberia

Η l>

silt loam

Tf

1 Maury

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silt loam

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Cation Exchange Capacity cmol kg" w

sandy loam

Texture

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Soil Properties Ο

Lindale

Soil Series


6. STEARMAN ET AL. EIA Coupled with SFE of Soil Herbicides

Ο

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59

60


processing software, a Hypersil ODS (250mm χ 4 mm i.d., 5 μπι) analytical column, and a Hypersil ODS (20 mm χ 4 mm i.d., 5 μτη) guard column (Hewlett-Packard Co., Wilmington, DE). The column was maintained at 40°C, the mobile phase flow rate was 1.5 mL/minute, and the injection volume was 25 /xL. The isocratic mobile phase consisted of acetonitrile/0.1M phosphoric acid, (30:70 v:v) p H . 2

SFE Extraction. A l l SFE extractions were conducted with a Dionex Model 703 system (Dionex Corp., Sunnyvale, CA). High purity C 0 (SFC grade with 2000 psi helium head pressure) was used throughout (Scott Specialty Corp., Plumsteadville, PA). Extractions were performed using a 3.5 mL extraction vessel. Glass wool was packed in both ends and 3 g of air-dried, ground (less than 2 mm diameter) soil was tightly packed into the extraction vessels. After several preliminary studies using various temperatures and pressures, SFE extractions were conducted at 200 atm for 3 min, followed by a 340 atm extraction for 17 min, both at 66°C. Restrictors were heated to 150°C and the collection vials were maintained at 4°C. Extractions were conducted in quadruplicate, both with and without modifiers. The modifier was acetone:water:triemylamine, 90:10:1.5, v:v:v. Analytes were collected either in 15 mL of acetone or by using solid-phase C traps (Dionex Corp., Sunnyvale, C A ) . The C traps were desorbed with 2 mL of acetone following SFE extraction.


2

1 8

1 8

Liquid Vortex Extraction. Simazine and 2,4-D were extracted from 10 g of soil with 20 mL of acetonitrile:water:acetic acid (80:20:2.5, v:v:v). Atrazine and alachlor were extracted from ten g of soil with 20 mL of acetonitrile:water (9:1, v:v). Samples were vortexed 3 times for 2 minutes each and allowed to sit overnight. They were then vortexed 4 times for 10 seconds, centriruged, and the supernatant saved for analysis. The liquid vortex extraction achieved equal or higher recoveries of atrazine compared to the automated Soxhlet (Soxtec) extraction as described in a previous study utilizing different soils (1). Field Incurred Samples. Soil samples containing simazine and 2,4-D were obtained from field plots located in Cookeville, Tennessee. Alachlor and atrazine containing soils were collected from field plots located in Cross ville, Tennessee. The samples in both cases were collected within 1 month of herbicide application. RESULTS AND DISCUSSION EIA Comparison with HPLC and GC. Linear regression analysis of the EIA versus G C or H P L C results was performed. For 2,4-D, the following relationship was obtained: EIA = 1.15 GC + 22.2, r = 0.81, η = 24. For simazine, the following relationship was obtained: EIA = 0.78 H P L C + 66.0, r = 0.92, η = 23. For atrazine the following relationship was obtained: EIA = 1.04 G C + 22.4, r = 0.98, η = 9. For alachlor, the following relationship was obtained: EIA = 0.79 G C + = 0.96, η = 9. Thus, in all cases, the EIA results agreed closely with G C and H P L C results. The EIA results are probably more accurate at the low end of analyte concentration, because of sensitivity, while the chromatographic results may 2

2

2

3 5 6 , 1 2


6. STEARMAN ET AL.


61

be more accurate at the high end of analyte concentration, due to increased error caused by dilution with the EIA. The EIA has a narrow standard curve range, (the standard curve range for atrazine is from 0.1-2.0 ppb), so significant dilution is necessary for extracts from soils containing high concentrations of the analyte. Effect of Modifier on SFE Extraction. Modifier addition to SFE C 0 improved recovery for 2,4-D (from 7 to 101 %), simazine (from 56 to 79%) and atrazine (from 57 to 90%). There was no significant difference for alachlor recovery with (88%) or without modifier (78%). Alachlor is very soluble in supercritical C 0 , therefore, it is not surprising that its recovery was not improved by adding modifier. For the other herbicides both recovery and relative standard deviations (RSD) were improved by using C 0 modified with acetone: water: triethy lamine (90:10:1.5, ν:ν:ν). In preliminary studies no difference, in extraction recoveries between 15% and 20% acetone modifier were observed. Consequently, 15% acetone:water:triethylamine (90:10:1.5) (ν:ν:ν) or 15% acetone:water (9:1) modifier was used. Addition of triethylamine (TEA) improved recoveries significantly, as it raised the pH, and formed a salt complex with compounds, such as 2,4-D. The use of water as a modifier has been shown to increase the surface area of clay-containing soils as a result of swelling, especially montmorrillonitic soils, such as Iberia and Maury soils. Therefore, the addition of water improves recovery of some pesticides from soils, such as the triazines in this study. Consequently, water was added to enhance the effectiveness of the acetone modifier.


2

2

2

Effect of Collection Method. The use of C traps gave complete recovery for sand and soil samples, spiked with herbicides in the extraction vessel and immediately extracted using SFE. Collection of the sample in liquid acetone under otherwise identical SFE conditions gave recoveries of only 55-78%. C traps improved collection because the analyte is deposited on the C trap and is not eluted into the collection vial until it is washed off the trap. This prevents formation of aerosols or volatilization of the analyte, that may have occurred with liquid collection. Extraction recoveries and RSDs for SFE (with acetone: water: triethy lamine modifier as described) using both liquid and solid phase collection are shown in Table III. Both spiked and field weathered soils are represented by this data. Herbicide recovery was improved using C traps rather than liquid acetone collection, except for simazine, where no difference was observed for either the spiked or the field weathered soils (p = 0.05, 95% confidence level). Simazine is the least water soluble herbicide (Table I) and has the lowest vapor pressure of the herbicides studied, which could explain its relatively high recovery using liquid acetone collection compared to the other herbicides. The mean liquid vortex extraction recoveries were 78% for 2,4-D, 95% for simazine, 90% for atrazine, and 93% for alachlor. Use of the C traps resulted in acceptable recoveries (79-123%) for all of the soil herbicides. The 2,4-D recoveries were higher using SFE (101 %) compared to the liquid vortex (78%) extraction, while the simazine recoveries were lower using the SFE extraction procedure (79%) compared to liquid vortex extraction (95%). No difference between simazine and 1 8

1 8

1 8

1 8

1 8



90 ± 7.7 80 ± 7.2

62 ± 9.2 66 ± 10.4

atrazine

alachlor

•SOURCE: Adapted from ref. 2

alachlor

-

123 + 9.8

103 ± 7.5

81 ± 13.0

73 ± 24.9

simazine

atrazine

94 ± 20.0

66 ± 24.8

2,4-D

Field-Weathered Soil

79 ± 14.8

78 ± 8.0

simazine

1B

C Trap

101 ± 28.8

Liquid Collection

Recovery (+ RSD%)

55 ± 18.9

18

Comparison of SFE herbicide recoveries and relative standard deviations (RSD) using collection in liquid acetone (15mL) versus trapping on C sorbent for both spiked and field weathered soils with incurred residue. SFE parameters were 200 atm at 66°C for 3 min, followed by 340 atm for 17 min using 15% (acetone:water:TEA)(90:10:1.5) (v:v:v) modifier.

2,4-D

Spiked Soil

Table III.


6. STEARMAN ET AL.


63


atrazine SFE recoveries was observed when no modifier was used. However, SFE extraction of atrazine using modifier achieved more complete recovery than it did with simazine under the same conditions. No differences between SFE and liquid vortex extraction were observed for atrazine and alachlor. SFE Extraction Versus Liquid Extraction of Field Weathered Soils . For the field weathered soils, the overnight liquid vortex extraction was assumed to give 100% recovery and all other techniques were compared to that procedure. SFE recovery for 25 field weathered 2,4-D samples was improved using solid-phase C collection (94% recovery) compared to the acetone liquid trap (66% recovery). Atrazine and alachlor extraction recoveries on 15 field weathered samples were similar for SFE with C trapping and the liquid vortex extraction. In fact, the alachlor recoveries were slightly higher using the SFE procedure (Table III). 1 8

l g

Extraction Efficiency and Soil Properties. Soil properties, especially clay and organic matter content, determine the soil surface area and cation exchange capacity, and hence, the efficiency of extraction of the pesticides. Recoveries for SFE extraction with C trapping were compared for five soils spiked with 50 and 500 ng/g atrazine and alachlor. Alachlor recovery was lower for the Iberia silty clay soil (57%) than for the four other soils (average 94%). The Iberia soil is 50% clay which is more than double the amount of clay in any of the other soils. Also, the Iberia clay is predominately montmorillonitic clay, which readily shrinks and swells, and has a large surface area and ion binding capacity. Therefore, soil properties are important and impact the effectiveness of SFE. Achievement high recovery on one soil may not occur with another soil. Care must also be taken when extrapolating recoveries from spiked soils to other soils. Although the Iberia soil had lower numerical recoveries than the other soils they were not significant at the 0.05 level for the 500 ng/g atrazine spike. At the 50 ng/g atrazine spike level, the Iberia (75%) and Lindale (80% recovery) soil exhibited lower herbicide recoveries than the three other soils (105% average recovery). An extraction kinetics study for the interval from 0-20 minutes showed that the majority of herbicides were extracted in the first 5 minutes. After 15 minutes, about 80-90% of the herbicide had been extracted. This study showed that one type of extraction protocol does not necessarily achieve high recoveries for all soil types. High extraction efficiencies on one soil were not necessarily achieved on another soil having different properties. Clay content and type, were important soil properties that seemed to influence recovery. In this study coupling of EIA and SFE resulted in increased analytical output and lower costs. EIA compared closely with GC and HPLC results for soil extracted herbicides 2,4-D, simazine, atrazine and alachlor. 1 8

Acknowledgments The financial support of the Cooperative State Research Service, U.S. Department of Agriculture-Water Quality Grant, under Agreement no. 93-34214-8844, and the


64


U.S. Geological Survey seed money grant through the University of TennesseeKnoxville Water Resource Research Center are gratefully acknowledged. Devon Sutherland's and Anna Bryant's assistance in herbicide analysis by G C and H P L C is gratefully acknowledged. Literature cited


1. 2.

Stearman, G.K., and V . D . Adams. 1992. Bull. Environ. Contam. Toxicol. 48:144-151. Stearman, G . K . , M . J . M . Wells, S . M . Adkisson and T . E . Ridgill. 1995. The Analyst 120:2617-2621.


Enzyme Immunoassay Analysis Coupled with Supercritical Fluid

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