Anal. Chem. 1994,66, 1495-1499
Determination of Alachlor and I t s Sulfonic Acid Metabolite in Water by Solid-Phase Extraction and Enzyme-Linked Immunosorbent Assay D. S. Aga,‘ E. M. Thurman, and M. L. Pomes U.S. Geological Survey, 482 I Quail Crest Place, Lawrence, Kansas 66049 Solid-phase extraction (SPE) and enzyme-linked immunosorbent assay (ELISA) were combined for the trace analysis of the herbicide alachlor and its major soil metabolite, ethanesulfonic acid (ESA). The anti-alachlor antibodycross-reacted with ESA, which produced false-positive detections of alachlor in water samples by immunoassayscreens. Alachlor and ESA were isolated from water by SPE on a CISresin and eluted sequentially with ethyl acetate and methanol. Alachlor is soluble in ethyl acetate while the anionic ESA is not. Thus ESA remained adsorbed on the CISresin and was eluted later with methanol. The combination of SPE withELISAeffectively separatedand quantifiedboth alachlor and ESA using the same antibody for two ELISA methods. The general method may have applicability for the separation of other herbicides and their ionic metabolites. The SPE-ELISA method has a detection limit of 0.01 pg/L for alachlor and 0.05 pg/L for ESA, with a precisionof *lo%. Analyses of surface and ground water samples were confirmed by gas chromatography/mass spectrometry and high-performance Liquid chromatography with photodiode-arraydetection. Results showed widespread occurrence of ESA in surface and ground water of the midwestern United States, with concentrations ranging from 10 pg/L. Solid-phase extraction (SPE) and .enzyme-linked immunosorbent assay (ELISA) are two analytical techniques that recently have found wide application in environmental chemistry. For example, ELISA has been applied to the analysis of herbicide residues in soil and water.l-I3 It is a (1) Van Emon, J. M.; Lopez-Avila, V. Anal. Chem. 1992, 64, 79A-88A. (2) Vanderlaan, M.; Stanker, L.; Watkins, B. In Immuncussaysfor Trace Chemical Analysis; Vanderlaan, M., Stanker, L. H., Watkins, B. E., Roberts, D. W., Eds.; ACS Symposium Series 45 1;American Chemical Society: Washington, DC, 1990; pp 2-13. (3) Hammock, B. D.; st al. In Immunochemlcal Methods for Enuimnmental Analysis; Van Emon, J. M., Mumma, R. O., Eds.; ACS Symposium Series 442; American Chemical Society: Washington, DC, 1990; pp 112-139. (4) Harrison, R. 0.; Gee, S. J.; Hammock, B. D. In Biotechnology in Crop Protection; Hcdin, P. A., Menn, J. J., Hollingworth, R. M., Eds.; ACS Symposium Series 379; American Chemical Society: Washington, DC, 1988; pp 316-330. ( 5 ) Vanderlaan, M.; Watkins, B. E.; Stanker, L. Enuiron. Sei. Technol. 1988,22, 247-254. (6) Lawruk, T. S.;Hottenstein, C. S.;Herzog, D. P.; Rubio, F. M. Bull. Enuiron. Conlam. Toxicol. 1992, 48, 643-650. (7) Thurman, E. M.; Meyer, M.; Pomes, M.; Perry, C. A.; Schwab, A. P. Anal. Chem. 1990,62, 2043-2048. (8) Leavitt, R. A.; Kells, J. J.; Bunkellmann, J. R.; Hollingworth, R. M. Bull. Enuiron. Conram. Toxicol. 1991, 46, 22-29. (9) Goh, K. S.;Hernandez, J.; Powell, S.J.; Garretion, C.; Troiano, J.; Ray, M.; Grccne, C. D. Bull. Enuiron. Contam. Toxicol. 1991, 46, 30-36. (10) Bushway. R. J.; Perkins, E.; Savage, S.A.; Lekousi, S.J.; Ferguson, B. S.Bull. Enuiron. Conram. Toxicol. 1988, 40, 647-654. (1 1) Schlaeppi, J. M.; Fory, W.; Ramsteiner, K. J . Agric. Food Chem. 1989, 37, 1532-1 538. This article not subJect to U S . Copyright. Pubilshed 1994 by the American Chemical Society
sensitive, fast, and cost-effective technique that can be conducted both in the laboratory and in the field. However, many of the applications of ELISA to complex environmental matrices are limited to screening of samples due to the potential bias of analysis toward false-positive results.12J3 Less commonly, false-negative results are observed when the detection limit of the assay is reached. Some immunoassay performance may be affected significantly, not only by cross-reacting compounds but also by matrix components that interfere with the assay detection system and the antibodylantigen interactions. SPE is used extensively as a cleanup procedure for clinical and environmental sample^.^"" While ELISA is used to detect known substances by using the specificity of the antibodies, SPE is used to separate unknown substances by mechanisms similar to that of high-performance liquid ~hromatography.’~ The complimentary features of these two techniques may be combined to providea selective and sensitive analytical method analogous to a chromatographic separation where the SPE cartridge is the column and ELISA is the detector. With properly designed SPE procedures, closely related compoundscan be separated, and proper interpretation of immunoassay results can be made. Moreover, because SPE is also a preconcentration technique, the overall detection limit of the SPE-ELISA method may be improved by several orders of magnitude. SPE can be automated easily for water analysis;18J9hence the reproducibility of immunoassay is not compromised. In addition, cross-reactivity of a structurally similar compound could become a positive aspect of ELISA because the same antibody may be used to quantify the crossreacting compound after it has been concentrated and separated from the specific analyte by SPE. Alachlor (2-chloro-2’,6’-diethyl-N-(methoxymethyl)acetanilide), a herbicide widely used in the United States, and its metabolite 2-[(2,6-diethylphenyl)(methoxymethyl)amino]S.J.; Horton, S.R.; Sharp, C. R.; Logusch, E. W. J. Agric. Food Chem. 1990, 38, 159-163. (13) Feng, P. C.; Wratten, S.J.; Logussch, E. W.; Horton, S.R.; Sharp, C . R. In Immunochemical Methods for Environmental Analysis; Van Emon, J. M., Mumma, R. O., Eds; ACS Symposium Series 442; American Chemical Society: Washington, DC, 1990; pp 18&192. (14) Appllcations Bibliography Sample Preparation Producrs; Varian Sample Preparation Products, Harbor City, CA, 1991. (IS) Tippins, E. Nature 1988, 334, 273-274. (16) McDonald, P. D. Waters SepPak Cartridge Applications Bibliography; Millipore Corp., Milford, MA, 1991. (17) Morris, 2.;Ruthann, K. Solid Phase Extraction for Sample Preparation; J. T.Baker, Inc.: Phillipsburg, NJ, 1988. (18) Castellani, W. J.;Lente, F.V.;Chou,D.J. Autom. Chem. 1990,12, 141-144. (19) Meyer, M.T.;Mills, M.S.;Thurman, E. M.J. Chromatogr. 1993,629,55-59. (12) Feng, P. C.; Wratten,
Analytical Chemistry, Vol. 66, No. 9, May 1, 1994
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2-oxoethanesulfonic acid (ESA)FO are important target compounds for the development of a sensitive method that combines SPE and ELISA. Alachlor has been classified as a possible human carcinogen and its maximum contaminant level (MCL) is established at 2.0 pg/L by the U.S.EPA.21 EPA's recent toxicological studies on FSA suggest that the metabolite is not mutagenic and does not bioaccumulate or undergo further metabolism at the levels commonly found in surface and ground water.22 However, ESA needs to be monitored constantly because it is relatively persistent and highly mobile23and may occur widely in ground water. The frequency of false positives observed in the ELISA screening kits for alachlor has been attributed to the significant crossreactivity of the ESA metabolite toward the anti-alachlor a n t i b ~ d y .The ~ ~specific ~ ~ ~ objectives of the research described herein were to (1) measure cross-reactivity of two alachlor metabolites toward anti-alachlor antibodies, (2)understand the principles involved in separating parent herbicides and ionic metabolites by SPE and couple SPE and ELISA for analysis of alachlor and ESA, and (3) apply the method to samples of surface and ground water and compare the results with gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography with photodiodearray detection (HPLC/PDA).
EXPERIMENTAL SECTION Reagents. Methanol (Burdick and Jackson, Muskegon, MI) and ethyl acetate (Fisher, Springfield, NJ) were HPLCgrade solvents. Alachlor and its metabolites were obtained from Monsanto Agricultural Co. (St. Louis, MO). The SPE cartridges that were used (SepPak from Waters-Millipore, Milford, MA) contained 360 mg of 40-pm C18-bonded silica. Phenanthrene-& (EPA, Cincinnati, OH) was used as the external standard for GC/MS quantification. Metribuzin (EPA PesticideChemical Repository, ResearchTrianglePark, NC) was used as the extemal standard for HPLC quantification. Standard stock solutions were prepared in methanol. Solid-Phase Extraction Procedure. The SPE procedure was automated with a Millipore Workstation (Waters, Milford, MA) as described previ~usly.~J~ (Brand names in this paper are for identification purposes only and do not constitute endorsement by the U.S.Geological Survey.) The c 1 8 cartridges were washed sequentially with 2 mL of methanol, 6 mL of ethyl acetate, 2mL of methanol, and 2 mL of distilled water. A 100-mL aliquot of sample was passed through the cartridge (Figure 1) at a flow rate of 10mL/min. The cartridge was eluted with 3 mL of ethyl acetate, followed by a transfer step to remove the ethyl acetate (top layer) containing the alachlor from the residual water (bottom layer) in the eluate. Then, the cartridge was eluted with methanol to remove ESA, which was collected in a separate test tube. Both ethyl acetate and methanol extracts were evaporated to (20) Baker, D. B.; Bushway, R. J.; Adam, S. A.; Macomber, C. S. Emiron. Sci. Technol. 1993,27,562-564. (21) U.S.EnvironmentalProtection Agency, Drinkingwater regulationsand health advisories. Office of Water, Washington, DC,1992. (22) Klein, A. J. Monsanto Co., personal communication, August 18, 1993. (23) Goolsby, D. A.; Battaglin, W. A.; Fallon, J. D.; Aga, D. S.; Kolpin, D. W.; Thurman, E. M. Absrracrs ofrhe Technical Meering, U.S.Geological Survey Toxic Substances Hydrology Program, Sept 1993; p 83. (24) Macomber, C.; Bushway, R. J.; Perkins, L. B.; Baker, D.; Fan,T. S.;Ferguson, B. S. J. Agric. Food Chem. 1992.40, 1450-1452.
1496 Ana!vticaIChemklry, Vd. 66, No. 9, May 1, 1994
AlPchlor and ESA metabolite ( 1 0 0 " sample)
n
#I El'fluent to waste
SPE Cia cartridge
Elute with3 mL of ethyl acetate (containsalachlor)
J.
a) Analysis by E L I S A evaporate to dryness, then reconstitutcwih I m ~of20 . s methanohater or
b) Analysis by GUMS: spike with phenanhnmcdio and evaporate to 100 @
Elute w i h 3 mL of methanol (contains ESA and OXA metabolilts)
4
a) Analysis by E L I S A evaporate to dryness. men econstitute with 5 mL
o f d i a i l ' d water or
b) Analysis by HPLC spike with meuibuzin and evaporateto dryness. then reconstitute with 100 pL of 20/80 huffer/methanol mixture
Flguro 1. Flow chart for SPE-ELISA method.
dryness under nitrogen at 45 OC using a Turbovap (Zymark, Palo Alto, CA). The ethyl acetate extracts (containing alachlor) were reconstituted with 1 mL of 20% methanol/ water and the methanol extracts (containing ionic metabolites) with 5 mL of distilled water for analysis by,ELISA. The preparation of samples for GC/MS and HPLC analyses was performed in a similar SPE procedure. The ethyl acetate extracts were spiked with phenanthrene-dlo and evaporated to about 100 pL for the analysis of alachlor by GC/MS. The methanol extract for HPLC analysis was spiked with metribuzin, evaporated to dryness, and then redissolved in 100 pL of 10 mM phosphate buffer/methanol (20:80) mixture. ELISA Procedure. The cross-reactivitiesof the two ELISA kits from different manufacturers toward acetanilide herbicides and metabolites were determined. The alachlor EnviroGard assay (Millipore, Bedford, MA) had antibodiescoated on the wellsof the microtiter plate, whereas the alachlor RaPID assay (Ohmicron Corp., Newtown, PA) had antibodies covalently attached to magnetic particles. Solutions of each compound were prepared at concentrations of 0 , l .O, 5.0,10, 100,and 1000pg/L in water, and each solution was analyzed in duplicate using both ELISA kits. For EnviroGard ELISA, 80 pL of sample and 80 pL of hapten-enzyme conjugate were mixed in the well, and the mixture was incubated at 30 OC in an orbital shaker (200rpm). After 1 h, the plate was rinsed five times with deionized water, and excess water was removed. Color reagent (160 pL, of 1:l mixture of 0.02% hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine) was added. The color was allowed to develop for 30min, and then stop solution (40 pL, 2 M sulfuric acid) was added. Optical densities were read on a Vmax microplate reader using a Softmax software (Molecular Devices, Menlo Park, CA). For the alachlor RaPID assay, 250pL of samplewas mixed with 250 pL of hapten-nzyme conjugate and 500 pL of paramagnetic particles. This mixture was incubated for 30 min at r c " temperature and then a strong magnetic field was applied to separate unbound analytes from the antibodybound analytes. The e x a s reagent was removed. A 500-pL aliquot of color reagent was added next, and the mixture was
incubated for 20min at room temperature,followed by addition of the stop solution (500 pL). The optical densities were read using the RPA-I RaPID photometric analyzer (Ohmicron). For the analysis of alachlor and ESA in actual water samples, the alachlor RaPID assay kit was used. The concentrations of alachlor were calculated from Ln/LogitB transformed data using alachlor standards of 0, 0.1, 1.0, and 5.0 pg/L. ESA analysis was performed using the alachlor RaPID assay, with the same procedure described for alachlor except that the kit was calibrated with ESA standards prepared in distilled water at concentrationsof 0 , l .O,5.0, and 20 pg/L. The accuracy and precision of the SPE-ELISA method using the RaPID assay kit was evaluated for several alachlor and ESA mixtures. Analyses of alachlor and ESA were done in duplicate. GUMS Analysis. GC/MS analysis of alachlor was performed on a Hewlett-Packard Model 5890A GC (Palo Alto, CA) and 5970A mass-selective detector (MSD). Operating conditions were identical to those described by Thurman et ala7 The detector was operated in a selected-ion monitoring (SIM) mode, and confirmation was based on the presence of the molecular ion peak, two confirmingions (with area counts 4=20%), and a retention/time match of f0.2% relative to phenanthrene-&,. The ratio of the areas of the base-peak ion of alachlor to the 188 amu ion of phenanthrenedlo was used to construct the calibration curve. A 12-m,HP- 1 capillary column made of cross-linked methylsilicone with a film thickness of 0.33 pm and 0.2 mm i.d. (Hewlett-Packard) was used for separation. Helium was used as the carrier gas with a flow rate of 1 mL/min and a head pressure of 35 kPa. The samples were injected in the splitless mode by an autoinjector. The column temperature was held at 60 OC for 1 min, then increased at 6 OC/min to 250 OC, and held at this temperature for 10 min. Injector temperature was 280 OC. HPLC/PDA Analysis. The HPLC analysisof the methanol extracts for the confirmation of ESA was performed in a HP Model 1090 series I1 liquid chromatograph with photodiodearray detector (Hewlett-Packard). The HPLC was equipped with a 2.1 mm X 100.0 mm narrow-bore, reversed-phase column packed with 5-pm Hypersil ODS (Hewlett-Packard). The mobile phase consisted of 38.5% HPLC-grade methanol and 61.5% 10 mM Na2HP04 (pH 7.0 buffer) prepared in Nanopure water. The flow rate of the mobile phase was 1.200 mL/min. The sample injection volume was 90 pL. ESA was monitored at a wavelength of 200 nm with a 4-nm bandwidth. The reference wavelength was set at 450 nm with a 80-nm bandwidth. To confirm the identity of the ESA, the ultraviolet (UV) spectra were scanned from 190to 400 nm and the spectra matched to standard spectra in an automated library search. RESULTS AND DISCUSSION Acetanilide Cross-Reactivity and E L S A Development. The cross-reactivities of the antibodies in the RaPID (magnetic particle-based) and EnviroGard (microtiter plate-based) alachlor-ELISA with other acetanilides and alachlor metabolites were measured (Figure 2). Cross-reactivities were expressed as the IC50, which is the concentration of the compound that causes 50% inhibition of absorbance, and as the least detectable dose (LDD), which is the concentration at 90% inhibition of absorbance. Both ELISAs were most
cHiocH%
CHiOC\.
cocHIcl
-$J-
""$JCh"
ElhmesuIfonlc add (ESA)wt.bdite
N d o r
IC.%=
COCHISOIH
0.89 ,0.75
LDLk0.19,0.12 15.4, 1.7
LDD=0.07,0.06
W"
m c " No aou-dvily 4.000 Wl.
Figure 2. Cross-reactivities of antibodies with chloroacetaniiide herbicides and alechior metabolites. Cross-reactivity Is expressed as the concentratlon required for 50% inhibition(ICm) in micrograms per titer and in least detectable dose (LDD) at 90% inhibbn. The first value Is for the alachlor RaPID assay kit (magnetic particle-based ELISA),andthesecondvakreIsfortheEnvlroOerdala~ kit(mlcrotlter plate-based ELISA).
sensitive for alachlor, with LDD of 0.07 pg/L for RaPID assay and 0.06 pg/L for EnviroGard assay. The RaPID assay had an IC50for alachlor of 0.89 pg/L whereas the EnviroGard assay had an ICs0 of 0.75 pg/L. Of the compounds tested for cross-reactivity with the alachlor-ELISA,ESA had the highest cross-reactivity, with an IC50 of 5.4 pg/L and a LDD of 0.19 pg/L for the RaPID assay and an ICs0 of 1.7 pg/L and LDD of 0.12 pg/L for the EnviroGard kit. 2-[2,6-Diethylphenyl)(methoxymethyl)amino]-2-oxoacetic acid (OXA) has an ICs0 of 335 and LDD of 21 pg/L (RaPID assay) and IC50 of 66 pg/L and a LDD of 2.7 pg/L (EnviroGard). The cross-reactivity of the anti-alachlor antibodies with ESA has caused problems in previous water-quality surveys for alachlor by ELISA because of false-positive detections of alachlor.20 Furthermore, it has been reported that the ELISA for alachlor does not correlate well with GC/MS and gives more than 10%false positives near the limit of detection, with concentration range of 0.10-0.20 pg/L.l29l3 This poor correlation has been attributed to the presence of high ESA levels as confirmed by liquid chromatography with tandem mass spectrometry (LC/MS/MS).24 ESA and OXA are two major soil metabolites of a l a c h l ~ r ;however, ~~ only ESA appears to have sufficient cross-reactivity (Figure 2) to cause interference with ELISA. Other soil metabolites and precursors of alachlor, such as hydroxydiethylacetanilideand chlorodiethylacetanilide, re(25) Sharp,D. B.Herbicides: Chentisry,DegradationandModeofAction.Kearney, P. C., Kaufman, D.D..Eds.: Dekker: N e w York, 1988; pp 301-333.
Analytical Chemlstry, Voi. 00, No. 9, May 1, 1994
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Table 1. Breakthrough Capacity (mL) of Sep-Pak C1, Cartridges for Alachlor and Other Ionlc Compounds
compound alachlor ESA OXA 2,4-D
10% breakthrough
100%breakthrough
>7000 175 150 10
750 750 20
Table 2. Recovery and Precldon of Alachlor and ESA from a CI8 RmIn by SrqumflaI E M h wtlh Ethyl Acetato and Methanol by SPE-ELISA Udng the RaPID A m y Klt ( n = 7) % recovered concn of fortified
distilled water (fig/L)
alachlofl
ESAb
A. 0.20ESA B. 0.050 alachlor + 0.10 ESA C. 0.10 alachlor + 0.050 ESA D. 0.025 alachlor 0.025 ESA E. 0.20 alachlor
0 100f 10 97 f 10 105 f 10 98 f 10
92 f 10 97 f 10 99 f 10 91 f 10 0
+
spectively, did not show any cross-reactivity up to a concentration of 1000pg/L. Metolachlor, another chloroacetanilide herbicide, showed low cross-reactivity, with an ICs0 of 109 and 27 pg/L for the RaPID and EnviroGard assay, respectively. This cross-reactivity pattern suggests that the binding of alachlor toward the antibody is affected by the presence of the methoxymethyl side chain and the alkyl group in the ring. These results are not surprising because the antibodies against alachlor are usually generated by the use of an alachlor-protein conjugate, formed through the chlorinebearing carbon of alachlor via a thioether bond.12J3 The antibodies develped for alachlor recognize ESA to a significant degree relative to the other metabolites because the sulfur atom in ESA is similar in size to the chlorine atom. Separation by SPE. The separation of alachlor and ESA was achieved by SPE using a CISresin. Both compounds adsorbed quantitatively from water onto the CIS cartridge (Table 1). Alachlor had a large capacity on the CISresin with more than 7 L of sample passing through the cartridge before a 10% breakthrough was ~bserved.~ In contrast, ESA had considerably less capacity with 10% and 100% breakthroughs observed after 175 and 750 mL of sample, respectively, had passed through the cartridge. This difference in capacity is caused by the greater solubility of ESA in water. Likewise, OXA had a small capacity on the c18 resin, with a 10% breakthrough beginning at 150 mL, and a 100% breakthrough observed after 750 mL of water had passed through the cartridge. A more soluble herbicide, 2,4dichlorophenoxyacetic acid (2,4-D), was also examined. It was found that 2,4-D had a very small capacity for sorption, with a 10% breakthrough observed after only 10 mL, and a 100%breakthrough observed after 20 mL of sample had passed through the cartridge. These results suggest that the isolation of ionic compounds on CISresin is possible, but this isolation technique is quite dependent on the aqueous solubility of the analyte. Thurman and others26reported that ionic character was critical in the sorption of organic acids onto a macroporous acrylic resin and was a function of chain length. The same concept appears to apply to the isolation of ionic metabolites on c18 resin because both isolations involve hydrophobic interactions. The separation of alachlor and ESA occurs in the elution step. Alachlor is eluted first with ethyl acetate without removal of ESA from the resin, and then ESA is eluted with methanol. The recovery of spiked ESA from distilled water when analyzed by SPE-ELISA ranged from 91% to 99% in the methanol extract and none in ethyl acetate (Table 2). Alachlor was recovered from 97% to 105% in the ethyl acetate extract and 0% in the methanol extract. Hence, there is a complete (26) Thurman, E.M.;Malcolm, R.L.;Aiken, G.R. A n d . Chem. 1978,50,775779.
1490
Analytical Chemism, Vol. 66,No. 9, M y 1, 1994
a
*
In ethyl acetate extract. In methanol extract.
separation of ESA and alachlor by SPE. After separation and concentration by SPE, it was possible to use the alachlorELISA for quantification of ESA due to the sufficient crossreactivity of the latter toward the anti-alachlor antibody. The OXA coeluted with ESA in methanol as observed from HPLC data, but because OXA has almost no cross-reactivity with the alachlor-ELISA, it does not interfere with the analysis of ESA. Themechanism of differential solubility in ethyl acetate for ESA and OXA is probably a result of the fact that the ionic organic molecules require a cation to be eluted with them, either a sodium or calcium ion for most natural waters. Apparently, the solubility of ESA-Ca or OXA-Ca in ethyl acetate is low, probably because of its ionic character and because of the necessity that its inorganic cation and water also must be present in solution. The result is a separation of parent compound and metabolites for ELISA. The importance of this finding is that many ionic compounds, particularly metabolites of herbicides, may be separated from parent compounds by this procedure. SPE-ELISA Method. The detection limit for alachlor of the RaPID assay using the SPE-ELISA method described in Figure 1 isO.01 pg/L. Thisis 10-foldlowerthan thedetection limit of the conventional ELISA (detection limit is 0.1 pg/L). ESA was eluted with methanol and was analyzed by ELISA using the alachlor-RaPID assay but calibrated with ESA standards. The calibration curve for ESA was linear for concentrationsof 1.O-20 pg/L. Because SPE preconcentrates the sample, the detection limit can go as low as 0.05 pg/L if a 100-mL sample is used and the dried extract is diluted to 5 mL with water for ELISA. Samples containing ESA were verified by HPLC. It should be noted that solvent exchange of the organic solvent to an aqueous medium was performed prior to ELISA because methanol may affect the antibody/ antigen interaction and produce an erroneous result. Although some antibodies can tolerate more than 50%methanol, others may be disrupted by minor amounts of methanol. The RaPID assay kit tolerates 20% methanol, but the reproducibility of the Enviroguard alachlor kit was affected, with increased 7% CV observed using 20% methanol in the sample matrix. The automated workstation for SPE acts as a liquid chromatograph with a fraction collectorby pumping the sample through the cartridge, separating the compounds by sequential elution, and collecting the fractions for analysis by immunoassay, GC/MS, and HPLC. The automated SPE procedure is capable of reproducing the separation within *lo%. The ability of SPE to separate alachlor and ESA was demonstrated with ground water samples from 12 locations. The presence of both alachlor and ESA in the ground water
Table 3. Analyrk of Ground Water Samples for Alachlor and ESA by ELISA, SPE-ELISA, and GC/MS
alachlor concn oCg/L) ESA concn 01 /L) direct anal. SPE-ELIS! sample by ELISA GUMS SPE-ELISA 1 2 3 4 5 6 7 8 9 10 11 12
0.6 0.16 0.92 0.33 1.83 1.10 0.22 8.60 0.22 2.85 1.79 4.63
0.45 C0.05 K0.05
