AM. Chem. 1 ~ 9 0 62, , 2043-2048 (15)
Schoknecht, U.: Schwarze. E.
Ausgrabungen und Funde 1967, 72,
205-209.
(16)
Thoms, H. Handbuch der praktischen und wissenschsftlichen pharma l i e : Springer: Berlin, 1924-1931; Vol. 4, pari 2/1, pp 1608-1609, p
1766. (17) Hadzi, D.; Cvek, F. Archeoloski vestnik 1976, 2 7 , 128-134. (18) RottYnder, R. C. A. Archiiobg. Kwesmn&nzb&tt 1974. 4 . 95-98. (19) RottlBnder, R . C. A. I n Die Schussenriedersiedlung im “SchlGsslesfeM”:Luning, J.. Zurn. H., Eds.. 1977. (20) Heintzei, C. 2.Ethnologic 1880, 72, 375-378; 1881, 73, 241-242. (21) Sauter, F.; Hayek, E. W. H.; Moche, W.; Jordis, U. 2. Natudorsch. 1867, 42‘2, 1151-1152. (22) Evershed, R. P.; Jerman, K.; Eglinton. G. Nature 1885, 374, 528-530. (23) Chaffee,A. L.; Hoover, D. S.;Johns, R. E.; Schweighardt. F. K. In 6io/OgiceiMarkers h the Sedimentary Records: Johns, R. B.,Ed.; Elsevier: Amsterdam-Oxford-New York-Tokyo, 1986: pp 311-345. 124) Varmuza. K.: Werther. W.: Lohninaer. H. In Software-Entwic&/unoin der Chemie 3;Gauglk, G.. Ed.; Sphger: Berlin-Heidelberg, 1989,pp 285-287 ~I
2043
(25) Werther, W.; Varmuza, K. In Software-Development in Chemistry 4 ; Gasteiger, G., Ed.; Springer: Berlin-Heidelberg, in press. (26) Windholz. M., Ed. The Merck Index, 10th ed.: Merck 8 Co.: Rahway. NJ, 1983; pp 1204, 4153, 5421. (27) Steiner, M. In Moderne Methoden der H/anzenana/yse; Paech, K., Tracey, M. V., Eds.; Springer: Berlin, 1955; Vol. 3, pp 128-135. (28) Varmuza, K. fanern Recognitlon in Chemistry; Springer: Berlin-Heideiberg, 1980. (29) Kvalheim, 0. A. Anel. Chim. Act8 1965, 777, 71-79.
RECEIVED for review January 26,1990. Accepted May 31,1990. The authors wish to thank the Austrian “Fonds zur Forderung der wissenschaftlichen Forschung” for financial support (Project P 6811 C).
Enzyme-Linked Immunosorbent Assay Compared with Gas Chromatography/Mass Spectrometry for the Determination of Triazine Herbicides in Water E. M. Thurman,* Michael Meyer, Michael Pomes, Charles A. Perry, and A. Paul Schwab’
US.Geological Survey, Water Resources Division, 4821 Quail Crest Place, Lawrence, Kansas 66049
An enzyme-llnked lmmunosorbent assay (ELISA) was compared to a gas chromatography/mass spectrometry (GWMS) procedure for the analysis of triazine herbicldes and their metabolites in surface water and groundwater. Apparent recoveries from natural water and splked water by both methods were comparable at 0.2-2 pg/L. Soild-phase extraction (SPE) was examined also, and recoveries were determined for a suite of trlazine herbicides. A signfflcant correlatlon was obtained between the ELISA and GC/MS method for natural water samples that were extracted by SPE. Because ELISA was developed with an atrazine-like compound as the hapten with conJugationat the 2-position, It was selective for trlazines that contalned both ethyl and Isopropyl side chains. Concentrations for 50 % inhibition (IC,) were as follows: atrazine, 0.4 pg/L; ametryne, 0.45 pg/L; prometryn and propazlne, 0.5 pg/L; prometon, 0.7 pg/L; slmazlne and terbutryn, 2.5 pg/L; hydroxyatrazine, 28 pg/L; deethylatrazlne and delsopropylatrazlne, 30 pg/L; cyanarlne, 40 pg/L; dkbalkylatrarine had no response. The comMnath of screening analysis by ELISA, which requires no sample preparatkn and works on 160 pL of sample, and conlltmation by GC/MS was designed for rapld, inexpensive analysis of triazlne herbicides In water.
INTRODUCTION The enzyme-linked immunosorbent assay (ELISA) has been shown to be a useful residue analysis method for herbicides (1-5). ELISA has been used extensively in clinical chemistry but has been introduced only recently into environmental chemistry on a commercial basis (2,3,5). The conventional analysis method for triazine herbicides (by gas-liquid chromatography with nitrogen-phosphorus detection, GC-NPD) Department of Agronomy, Kansas State University, Manhattan, KS 66506.
is sensitive and well characterized (6-9). Furthermore, both liquid chromatography (6,1&12) and gas chromatography/ mass spectrometry (13-18) have been applied successfully to the analysis of triazine herbicides. A major reason for the numerous methods of analysis of triazines, which are used extensively as preemergent herbicides for corn (Zea mays L.) and sorghum (Sorghum bicolor L.) is the fact that these herbicides are common contaminants in surface and groundwater (19). Thus, the triazine herbicides provide a challenging target for ELISA for rapid inexpensive analysis of surface and groundwater. This study examined the ELISA analysis method for triazine herbicides and their metabolites to determine cross reactivity, and to verify accuracy and precision against a reference GC/MS method. The study also checked for interference from naturally occurring humic substances and acetanilide herbicides that commonly occur in surface water. Gas chromatography/mass spectrometry was chosen as a reference method to verify that the ELISA was responding to the triazines rather than other herbicides that may be present. Because the ELISA method was designed to screen large numbers of water samples without sample preparation, an automated solid-phase extraction (SPE) method was designed also for rapid GC/MS confirmation. Objectives of the study were to (1) compare ELISA to GC/MS analysis for accuracy, precision, and cross reactivity for herbicides in natural water, (2) streamline the S P E extraction and GC/MS analysis by selected ion monitoring (SIM) for rapid confirmation, and (3) determine if the combination of ELISA screening and GC/MS confirmation for triazine herbicides would be a viable and inexpensive method for large water-quality surveys.
EXPERIMENTAL SECTION Reagents. Methanol (Burdick and Jackson, Muskegon, MI) and ethyl acetate (Fisher, Springfield, NJ) were pesticide-grade solvents. Ametryn, atrazine, prometon, prometryn, propazine, simazine, and terbutryn were obtained from Supelco (Bellefonte,
This article not subject to U S . Copyright. Published 1990 by the American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 18, SEPTEMBER 15, 1990
Table I. Selected Ions for Mass Spectrometry of Triazine and Acetanilde Herbicides atomic mass unit (amu) compound no. (Figure 2)
retention time, compound
rnin (*0.04)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
deisopropylatrazine deethylatrazine simazine atrazine prometon propazine phenanthrene-dlo terbuthylazine metribuzin ametryn alachlor prometryn terbutryn cyanazine metolachlor
17.98 18.32 19.71 20.02 20.08 20.25 20.38 20.61 22.02 22.85 22.91 23.05 23.45 23.75 24.18
PA); alachlor, cyanazine, metolachlor, metribuzin, and terbuthylazine were obtained from the EPA Pesticide Chemical Repository (Research Triangle Park, NC); and the triazine metabolites, deethylatrazine, deisopropylatrazine, and didealkylatrazine, were from Ciba Geigy (Greensboro, NC). The CIS cartridges (Sep-Pak from Waters, Milford, MA) contained 360 mg of 40-pm C18bonded silica. Deionized water was charcoal filtered and glass distilled prior to use. Standard solutions were prepared in methanol, and phenanthrene-dlo (EPA, Cincinnati, OH) was used as an internal GC/MS quantitation standard. ELISA Procedure. Res-I-Mune kits (ImmunoSystems Inc., Scarborough, ME) were used for the immunoassay analysis of water samples. The kits use polyclonal antibodies coated to the walls of a polystyrene test tube and an enzyme conjugate that was prepared by covalently binding atrazine to horseradish peroxidase by a modified carbodiimide technique (2, 3). The procedure was similar to that described in the kit and by Bushway and others (2) with the exception that percent inhibition was measured. Briefly, 160 pL of sample was added to an antibody-coated tube with 160 pL of enzyme conjugate. After 5 min, the test tubes were rinsed 3 times with deionized water to remove excess unreacted sample and enzyme conjugate, and 160 pL of substrate was added followed by 160 pL of chromogen, sequentially. After 2 min, color was fixed with 40 pL of "stop" solution (2.5 N sulfuric acid). Samples and standards were analyzed by measuring percent inhibition, which is the difference in optical density at 450 nm (A450) between the negative control and the sample divided by the optical density (A450)of the negative control times 100. Cross reactivity of the herbicides was measured in triplicate as the percent inhibition of the compound a t a concentration of 1 pg/L and compared to ICM values in the kit literature (IC, is the amount required for 50% inhibition). Measurements were made on the atrazine standard curve using an Artel differential spectrophotometer (Windham, ME). Extraction Procedures. The C18cartridges were prepared by washing with 3 mL of methanol, 3 mL of ethyl acetate, 3 mL of methanol, and 2 mL of distilled water. Ethyl acetate eluate blanks of the Sep-Pak cartridges were analyzed by GC/MS to determine retention time of interfering peaks. Extraction and elution efficiencies of the cartridge were determined by comparing the recoveries of terbuthylazine and atrazine to an external standard curve of equivalent mass by GC/MS. The capacity of the cartridge for the triazine herbicides was determined by passing the herbicide mixture through a Sep-Pak cartridge and determining the volume required for breakthrough of the solute. Surface-water samples were filtered through 0.7-pm glass-fiber filters (Whatman GF/F, Maidstone, England). The sample (100 mL) was spiked with a recovery, surrogate standard (terbuthylazine) and was passed through the Sep-Pak cartridge using a Millipore Workstation (Waters, Milliford, MA) a t 20 mL/min. The cartridge was eluted first with air to remove residual water and then eluted with 2.0 mL of ethyl acetate. The ethyl acetate was spiked manually with phenanthrene-&, vortexed, dried over
molecular ion (M+) 173 187 20 1 215 225 229 188 229 214 227 269 241 24 1 240
none
base peak
ion 1
ion 2
173 172 201 200 210 214 188 214 198 227 188 241 226 225 162
158 145 186 173 168 172
145
173 199 212 160 184 185 212 238
176
182 170 237 226 170 240
sodium sulfate, evaporated under nitrogen at 50 "C to 100 pL, and analyzed by GC/MS. GC/MS Analysis. GC/MS analyses of the eluates were performed on a Hewlett-Packard Model 5890A GC (Palo Alto, CA) and a 5970A mass selective detector (MSD). Operating conditions were as follows: ionization voltage, 70 eV; ion source temperature of 250 "C; electron multiplier, 300 V above autotune; direct capillary interface a t 280 "C, daily tuned with perfluorotributylamine; and 50 ms dwell per ion. For sample analysis, the filament and multiplier were not turned on until 5 min into the run. Thirty-two ions were selectively monitored (Table I), and the base-peak ion current was measured for the quantification curve versus the response of the 188 ion of phenanthrene-&, Confirmation was based upon presence of the molecular ion, two confirming ions (with area counts *20%), and a retention time match of 4~0.2%relative to phenanthrene-dlo. A fused silica capillary column of methylsilicone with a film thickness of 0.33 pm, 12 m x 0.2 mm i.d., called the HP-1 (Hewlett-Packard, Palo Alto, CA), separated the herbicides with helium as a carrier gas a t 1 mL/min and a head pressure of 35 kPa. The samples were injected in the splitless mode by autoinjector. The column temperature was held at 50 "C for 1 min and programmed to 250 "C a t 6 "C/min and held for 10 min. Injector temperature was 280 "C.
RESULTS AND DISCUSSION ELISA. The standard curve was linear from 0.2 to 2.0 pg/L when percent inhibition was plotted versus concentration on a log scale. Plotting percent inhibition normalizes the absorbance readings (A450) of each set of samples to the variability of the negative control, which is a sample run with each set that contains no herbicide and has the maximum absorbance reading possible (absorbance is inversely related to concentration in this assay). If raw absorbance readings (A4501 are plotted rather than percent inhibition, then the variation among analyses may double. For example, Table I1 shows the coefficient of variation for the negative control and 0.5 and 1.0 pg/L atrazine standards. Both standards have a coefficient of variation of approximately 0.1 (AOD column in Table 11), a value that is affected by the absorbance of the negative control. This value may be decreased to 0.07 for the 0.5 pg/L standard and to 0.03 for the 1.0 pg/L standard by normalization and plotting of percent inhibition. Because concentration is plotted on a log scale, the decrease in the coefficient of variation makes an important difference in the precision of the method. The normalized values give a relative standard deviation in concentration units of 20% for the 0.5 p g / L standard and 15% for the 1.0 Fg/L standard. Plotting percent inhibition versus concentration is similar to the logit plots commonly used in enzymology (20).
ANALYTICAL CHEMISTRY, VOL. 62, NO. 18, SEPTEMBER 15, 1990
2045
0.4 pg/l Atrazine
0.5 pg/1 Prometryn
0.45 pg/l Ametryn
0.5 pg/1 Propazine
N2;
N'N H , /N A$?AH N, H3C-CH-CH3
A!A~,~
A~A,,.
N;H H&CH-CH3
HY
\
0.7 pg/1 Prometon
CH3-C-CHg
CH3-CH2
CH3-CH2
2.5 pg/l Simazine
2.5 pg/l Terbutryn
CH3
?
\
CH3-CH2
CI
?H
I
N A N
A!A
H,$-CH-CH3 H>N
"2
30 pq/1 Deisopropylatrazine
30 pg/1 Deethylatrazine
40 pgll Cyanazlne
2 8 pg/l Hydroxyatrazine H3CO
\
H3C O-H2C N ''
0 t-CH2CL
No response Didealkylatrazine
""-6" 2c' H3
H3(""'@'~CH3
H2N
H3C'
/CH2 0 HC kH2-CL N ''
No response Alachlor
No response Metolachlor
Flgure 1. Chemical structure and cross reactivity for triazines with ELISA. Expressed as concentration required for 50% inhibition in units of PB/L (IC50).
Table 11. Comparison of Absorbance Readings for ELISA Standards (n = 7) standard negative (0.5 rglL) control % A450b 'OD' mean std dev coeff of variance
1.70 0.20 0.12
0.71 0.09 0.12
42 3 0.07
standard (1.0rg/L) 'OD' 0.97 0.09 0.10
%
57 2 0.03
AOD, difference in optical density at 450 nm in absorbance units between sample and negative control determined with a differential spectrophotometer. A450 negative control, difference in optical density at 450 nm in absorbance units between negative control and deionized water, which is the maximum reading. CPercentinhibition = (absorbance negative control - absorbance of saple)/absorbance negativecontrol x 100 = (AoD sample/A450) x loo.
Multiple analyses of a deionized water blank containing color reagent against the negative control gave a mean value of 7% inhibition and a standard deviation of 7% inhibition.
If the recommendation of the Subcommittee on Analysis (22) is used, then the limit of detection is 3 standard deviations above the blank, or 21 % inhibition, which corresponds to a 0.2 pg/L detection limit. Cross reactivity was measured for the common triazine herbicides and metabolites and is expressed in Figure 1as the concentration required for IC,, (50% inhibition). It was found that the response of atrazine was greatest with the lowest ICw concentration of 0.4 pg/L, which is expected because it was similar to the hapten used to make the antibody (proprietary information). Ametryn's IC,, value was 0.45 Fg/L, prometrp and ProPazine were 0.5 %/L, and SO forth (Figure 1). The cross reactivity expressed as the IC,, of each triazine is related to the extent of binding that occurs between the antibody coated to the test tube and the analfie molecule. Note that the binding was strongest for compounds that have structures most atrazine, that a 4-ethy1amino and a 6-isopropylamino group. For example, ametryn (Figure 1) has a 0.45 pg/L IC5,, compared to atrazine (0.4 pg/L). Likewise, a substitution from an ethyl to an isopropyl group (propazine) increases the ICm to 0.5 pg/L. Simazine, with two ethyl groups, has a ICw of 2.5 pg/L. Substitution of hydrogen
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 18, SEPTEMBER 15, 1990
Table 111. Breakthrough Capacity (mL) of the Sep-Pak CL8 Cartridges for Triazine Herbicides compound
10% breakthrough4
100% breakthrough
didealkylatrazine deisopropylatrazine deethylatrazine metribuzin simazine atrazine cyanazine propazine ametryn prometon terbuth ylazine alachlor prometryn terbutryn metolachlor
-5 75 300 600 1200 2000 2000 4000 >7000 >7000 >7000 >7000 >7000 >7000 >7000
25 225 400 1800 1800 3200 3200 6000
"Flow rate was 4 mL/min, concentration, 1 pg/L of each herbicide; 25-mL increments were monitored from 0 to 500 mL, and 100 mL increments were monitored to 7000 mL by passing effluent through a clean Sep-Pak cartridge. Experiment terminated a t 7000 mL. *Refers to the concentration of the compound in the effluent as a Dercentane of influent concentration.
for either the ethyl (deethylatrazine) or the isopropyl (deisopropylatrazine) increases the ICm dramatically to 30 pg/L. Likewise, cyanazine, which has a bulky cyano group a t the 6-position had a ICm of 40 pg/L, which shows that its binding has been decreased by the cyano group. Because the immunizing hapten was bound at the 2-position on the triazine ring, it appears that the relative response of the immunoassay is related to antibody recognition and binding to the alkyl side chains on the triazine ring, especially the isopropyl group. Only hydroxyatrazine breaks this pattern in cross reactivity. Because it has the same alkyl structure as atrazine, one would predict an ICbosimilar to ametryn (0.45 pg/L). However, hydroxyatrazine has an ICso of 28 pg/L. A possible explanation is that the hydroxyl group decreases the binding energy at the specific antibody recognition site because of a difference in the spacer or perhaps the fact that hydroxyatrazine also occurs in the keto form that is different in structure than the immunizing hapten. Didealkylatrazine was nonreactive, again showing that the alkyl side chains are critical to binding. Neither alachlor nor metolachlor cross reacted with atrazine, which is consistent with a previous study ( 2 ) . ELISA was checked also for interference by naturally occurring humic and fulvic acids, which account for the majority of dissolved organic carbon in natural water (22). Atrazine was measured in water samples that contained from 5 to 100 mg/L of humic and fulvic acid from the Suwannee River (standard-reference surface-water sample, refs 23 and 24) and humic and fulvic acids from Biscayne aquifer near Miami, FL (25). In all cases, there was no difference between the immunoassay response in the presence and absence of the humic material. These data suggest that neither positive nor negative cross reactivity should occur from natural dissolved organic matter in surface or groundwater by ELISA analysis. Solid-Phase Extraction and GC/MS. Capacity and elution efficiency of solid-phase extraction were examined in two separate experiments. Table I11 shows the capacity determinations for the herbicide mixture. Didealkylatrazine has the least sorption capacity for the c,, cartridge with complete breakthrough of the compound in the effluent at 25 mL and is followed by deisopropylatrazine a t 225 mL. Next, deethylatrazine had complete breakthrough a t 400 mL. The removal of both the ethyl and isopropyl side chain increases the polarity of the molecule and greatly decreases sorption capacity on the resin (didealkylatrazine). Addition of the ethyl
Table IV. Recovery and Precision Analysis for Herbicides from CISSEP-Pak Cartridge and GC/MS Determination Using 100-mL Sample, 4 mL/min Flow Rate, and Ethyl Acetate as an Eluting Solvent, and Based on an External Standard Curve for Each Herbicide at 1 pg/L Concentration. compounds
% mean recovery f l std dev
deethylatrazine deisopropylatrazine didealkylatrazine simazine atrazine prometon propazine metribuzin terbuthylazine ametryn alachlor prometryn terbutryn metolachlor cyanazine
90 f 5 55 f 5 10 f 5 101 f 5 99 f 5 105 f 5 108 f 5 108 f 5 99 f 5 107 f 5 104 f 5 113 f 5 104 f 5 100 f 5 95 f 10
YO re1 std dev 6 10 50 5 5 5 5 5 5 5 5 4
5 5 10
group increases capacity with two added carbons (deisopropylatrazine), and addition of the isopropyl group with three carbons increases it slightly more (deethylatrazine). If ethyl groups are present on both amino groups, the capacity is considerably more (simazine at 1800 mL). Atrazine reached breakthrough at 3200 mL and propazine a t 6000 mL. Again, these results demonstrate that the replacement of an ethyl group with an isopropyl group increases sorption capacity (simazine to atrazine), and a second replacement further increases capacity (atrazine to propazine). After 7 L of solution, many of the herbicides were still being retained with no evidence of elution (ametryn, prometon, terbuthylazine, prometryn, terbutyrn, and alachlor and metolachlor), which indicates the large capacity of the herbicides for the C18cartridge. The experiment was terminated a t 7 L. Table IV shows the recovery data for 15 herbicides from the Sep-Pak cartridge. From 90 to 113% of the herbicides were recovered, with the exception of didealkylatrazine and deisopropylatrazine, which were recovered a t 10 and 55%, respectively. Analysis of a second elution aliquot of ethyl acetate indicated that the first 2 mL of solvent was sufficient for removal of the herbicides. Other studies of herbicide elution from C18resin showed that elution occurs with small volumes of solvent (26). The decreased recovery of both didealkylatrazine and deisopropylatrazine was caused by poor sorption capacity on the resin, which was shown in the prior experiment (Table 111). On the basis of these recovery results, terbuthylazine was chosen as a surrogate for the recovery of triazines in field samples because it recovers similar to atrazine, is easier to obtain and less costly than the deuterated analogues of the herbicides, and is not used as a herbicide commercially. Finally, the types of contaminants present on the solidphase cartridge were examined by elution of the cartridge both before and after cleaning with the elution solvent, ethyl acetate. The major contaminants included aliphatic hydrocarbons (C, to C18),dioctyl phthalate, and in some samples a large unidentified peak at 24.5 min, which may be a silylated compound from the resin (27). This peak may result from the hydrolysis of the cartridge by the water sample, itself, which was concluded from the fact that after cleaning the resin the ethyl acetate blank did not contain the large peak at 24.5 min. If the samples are allowed to remain on the cartridge for several hours before elution, this peak will appear; thus, cartridges should be eluted immediately. Detection limits (defined by Committee on Chemical
ANALYTICAL CHEMISTRY, VOL. 62, NO. 18, SEPTEMBER 15, 1990
, , , ,
IWOOO
,
1 Chromatogram 0 1
,
, , , ,
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3.0
Herbicide Mixture
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r
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2047
0
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23
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,
.
25
Flgure 2. Chromatogram for a 14-compound standard herbicide mix and phenanthrene4,o showing total ion chromatogram. Compound, name, number, and ions found in Table I .
Analysis, ref 18) for the herbicides varied from 0.01 to 0.05 pg/L, depending upon instrument response factors, and quantitation limits varied from 0.05 to 0.20 pg/L. Precision (relative standard deviation) varied from 4 to 50% (Table IV). Accuracy was established by checking both fortified distilled-water samples containing the herbicide mixture in Table IV and actual water samples with analyses from two independent laboratories using liquid-liquid extraction (LLE) and GC/MS and LLE with GC/NPD detection. In general the results by solid-phase extraction and GC/MS were within f10 to 20% of the independent laboratory analyses. Only cyanazine varied to &50%. This cyanazine result has recently been traced to losses in the injection port caused by a dirty injector sleeve. Thus, for reliable cyanazine response by GC/MS the injector sleeve should be changed frequently depending on sample throughput. Chromatographic separation was nearly baseline for all compounds, with the exception of atrazine/prometon (peaks 4 and 5 in Figure 3) and ametryn/alachlor (peaks 10 and 11). Because different basepeak ions were used for quantitation for each compound, neither of the sets of peaks interfered with one another (Table I), and the chromatogram of the blank did not contain interfering peaks. Several of the metabolites of the triazines were too polar to chromatograph; this includes didealkylatrazine and hydroxyatrazine. Derivatization procedures are required for these compounds before GC/MS analysis. Comparison of ELISA and GC/MS. The correlation between ELISA and GC/MS was 0.99 ( r ) ,and the slope of the regression line was 0.88 for spiked distilled water containing a mixture of herbicides (including atrazine, alachlor, cyanazine, metolachlor, propazine, and simazine). This selection of herbicides was chosen because they represent the six major herbicides found in a survey 450 surface water samples from the midwestern United States that were screened and assayed as part of this study. The cross reactivity (Figure 1)for each of the triazine herbicides was used for correlation, and samples were run in duplicate pairs. The acetanilide herbicides did not interfere, and there was no response to these compounds. Cyanazine, metolachlor, and alachlor essentially have no response to the ELISA but were included as part of the comparison because they are important herbicides used in row crops in the midwestern United States (19). For example, Bicep is the commercial name for the combination of atrazine and metolachlor, and commonly alachlor and atrazine are used on adjacent fields to kill weeks on soybeans and corn, respectively (19). Furthermore cyanazine is widely used in conjunction with these preemergent herbicides. The cross reactivity of the ELISA was sufficient for a semiquantative response for atrazine, ametryn, prometryn, propazine, prometon, simazine, and terbutryn. The response was too low for deethylatrazine, deisopropylatrazine, hydroxyatrazine, cyanazine, and didealkylatrazine to be a screening method for these triazines in surface or groundwaters because their concentrations are typically at the pg/L level
m
5
g
Surface-Water Samples
2.5
e
Q
Represents more than one value at this bcatbn
0 L
2.0 1 L
a m
2I 1.5 0
2
0
I E .10
mm
Intercept = 0.0058 r = 0.95 Slope = 0.84 n = 66 samples
m C
E’ 0.5
E -
1
I
I
I
1.o
1.5 2.0 25 G U M S Summed Response, in Mlcrograms Per Liter
0.5
0
Figure 3. Plot of 66 surface-water samples from the central United States showing ELISA concentration versus concentration by &/MS.
Ground-Water Samples 2,5
-
Represents more than one value at this location
-
L
m
c
i
;2.0
a
-
E
Intercept = 0.1 r=0.91 Slope
= 1.30
n - 6 4 samples
0’” 0
‘
I
I
I
I
0.5
1.o
1.5
2.0
-
2.5
GC/MS Summed Response, in Micrograms Per Liter
Figure 4. Plot of 64 groundwater samples from the center United States showing ELISA concentration versus concentration by GC/MS.
or below. At these concentrations the ELISA response is not detectable. With these considerations in mind, the ELISA was used as a screen for both surface waters and groundwaters from the central United States. Sixty-six surface-water samples from the central United States were analyzed for the triazine mixture shown in Figure 2, and their responses relative to atrazine were computed for the ELISA. This summed response was plotted on the GC/MS or x axis of Figures 3 and 4. The correlation was 0.95 (Figure 3), using cross-reactivity factors for each of the triazine herbicides. In all cases, the immunoassay detected the presence of triazine herbicides at the detection limit of 0.2 pg/L with no false negatives. Because the majority of these samples contained atrazine as the major herbicide, the ELISA response was quite similar to the atrazine concentration by GC/MS, with total triazines only slightly larger than atrazine concentrations. No false positives
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 18, SEPTEMBER 15, 1990
were detected at the 0.2 pg/L detection limit of ELISA. Also, the concentration of triazine herbicides was compared by ELISA and GC/MS for 64 groundwater samples from the central United States. The correlation was 0.91 (Figure 4). Again, the immunoassay detected the presence of triazine herbicides a t the detection limit of 0.2 pg/L with no false negatives. The ELISA method appears to be a reliable screening method for triazine herbicides (atrazine, propazine, and simazine) in both surface and groundwater at environmentally relevant concentrations (0.2-2 pg/L). Although other triazines, such as cyanazine, are not detected by the ELISA, the combined usage of these compounds with atrazine makes the ELISA a reliable screening tool for preemergent triazine herbicides in surface and groundwaters. CONCLUSIONS ELISA methodology showed a good correlation with conventional GC/MS analysis of spiked or field samples of water for triazine herbicides (specifically atrazine, simazine, and propazine). The ELISA has several positive aspects as a screening analysis for water-quality surveys for triazines. Firstly, it is an inexpensive technique ($15/sample) that can be done routinely and rapidly in both the field and laboratory. Secondly, it works a t environmentally significant concentrations from 0.2 to 2 pg/L (relative standard deviation of &15-20% compared to &5-10% for GC/MS a t 1 pg/L) with no false negatives observed. The lack of false negatives is typical for ELISA methods in general because to generate a response, physical sequesting or sorption must occur a t the antibody-binding sites, which is unexpected in most analytical situations. ELISA cross reacts significantly with several major triazine herbicides (atrazine, propazine, and simazine), which makes the method useful as a class-specific analysis; however, the antibodies used in this ELISA does not react significantly with triazine degradation products. Thirdly, ELISA works well on samples of limited volume (160 pL required) when other methods may have inadequate sensitivity. Precision of ELISA was improved by measuring percent inhibition rather than absorbance at 450 nm. Fourthly, this ELISA requires no sample preparation and is field portable. Solid-phase extraction and GC/MS SIM may be used in conjunction with ELISA as a confirmation technique. The GC/MS SIM method does not give an absolute confirmation, as defined by EPA regulations for identification by GC/MS (28). However, if molecular ion and two confirming ions are used with a 0.2% retention-time window corrected to phenanthrene-dIo,it is a reliable and sensitive method that is easily automated with conventional laboratory workstations. The combination of ELISA and GC/MS SIM provides a low-cost screening analysis for triazine herbicides in surface and groundwater and is suitable for large water-quality surveys. ACKNOWLEDGMENT We appreciate the help of Jeri Damman and Chris Wilcox
in our laboratory. We also appreciate the support of CibaGeigy, Inc. (Janice McFarland), in supplying standards of atrazine degradation products. LITERATURE C I T E D (1) Huber, S.J.; Hock, B. 2.Pflanzenkrankh. (Pflanzenpafhol.)Pfknzenschutz 1985, 92, 147-156. (2) Bushway, R. J.; Perkins, 6.; Savage, S. A.; Lekousi, S.L.; Ferguson, B. S . Bull. Environ. Contam. Toxicol. 1988, 4 0 , 647-654. (3) Bushway, R. J.; Perkins, 6.; Savage, S.A,; Lekousi, S.L.; Ferguson, B. S. Bull. Environ. Contam. Toxicol. 1989, 42. 699-904. (4) Li, Q. X.; Gee, s. J.; McChesney, M. M.; Hammock. B. D.; Seiber, J. N. Anal. Chem. 1989, 6 1 , 819-823. (5) Vanderlaan, Martin; Watkins, B. E.; Stanker, Larry, Environ. Sci. Technol. 1988, 22, 247-254. ( 6 ) Popl, M.; Voznakova, 2.; Tatar, V.; Strnadova, J. J. Chromafogf. Sci. 1983, 21, 39-42. (7) Steinheimer, T. R.; Brooks, M. G. I n f . J. Environ. Anal. Chem. 1984, 77, 97-111. (6) Kreindl, T. G.; Malissa, H.; Winsauer, K. Mikrochim. Acta 1988, 7 , 1-13. (9) Bardaiaye, P. C.; Wheeler, W. B. I n f . J. Environ. Anal. Chem. 1988, 2 5 , 105-113. (10) Smolkova, E.; Pacakova, V. Chromatographla 1978, 1 7 , 696. ( 11) Di Corcia, Antonio; Marchetti. Marcello; Samperi, Roberto J . Chromafogr. 1983, 405, 357-363. (12) Ferris, I. G.; Haigh, 8. M. J. Chromafogr. Sci. 1987, 25, 170-173. (13) Karihuber, B. A.; Hormann, W. D.; Ramsteiner, K. A. Anal. Chem. 1975, 47, 2450-2452. (14) Mangani, F.; Bruner, F. Chromafographk 1983, 77, 377-380. (15) Lopez-Avila, Vlorica; Hirata. Pat; Kraska, Susan; Flanagan. Michael; Taylor, J. H., Jr. Anal. Chem. 1985, 5 7 , 2797-2801. (16) Davoii, E.; Benfenati, E.; Bagnati, R.; Fanelii, R. Chemosphere 1987, 76, 1425-1430. (17) Pereira, W. E.; Rostad, C. E.; Leiker, T. J. Anal. Chim. Acta 1989, 228, 69-75. (18) Huang, L. Q. J. Assoc. Off. Anal. Chem. 1989, 72, 349-354. (19) Leonard, R. A. Environmental Chemistry of Herbicides; Grover, R., Ed., CRC Press: Boca Raton, FL, 1986; Chapter 3, pp 45-87. (20) Ritchie, D. G.; Nickerson, J. M.; Fuller, G. M. Methods Enzymol. 1983, 92, 577-585. (2 1) ACS Committee on Environmental Improvement and Subcommittee on Environmental Analytical Chemistry Anal. Chem. 1980, 52, 2242-2249. (22) Thurman, E. M. Organic Geochemistry of Natural Wafers; Martinus NijhoffIDr. W. Junk: Dordrechdt, 1985; p 496. (23) Malcom, R. L.; MacCarthy, P. Environ. Scl. Technol. 1986, 2 0 , 904-9 11. (24) Thurman, E. M.; Aiken. G. R., Ewa!d. M.;Fischer, W. R.; Forstner, U.; Hack. A. H.; Mantoura. R . F. C.; Parsons, J. W.; Pocklington, R.; Stevenson, F. J.; Swift, R. S . ; Szpakowska, B. Humic Substances and Their Role in the Environment; Frimmel, F. H., Christman, R. F., Eds.: John Wiley and Sons: New York, 1988; S.Bernard, Dahlem Konferenzen. (25) Thurman, E. M. Humic Substances in Soil, Sediment and Wafer; Aiken, G. R., and others, Eds.; Wiley-Interscience: New York, 1965. (26) Junk, G. A.; Richard, J. J. Anal. Chem. 1988, 60, 451-454. (27) Junk, G. A.; Avery, M. J.; Richard, J. J. Anal. Chem. 1988, 60, 1347-1350. (28) Guidelines establishing test procedures for the analysis of pollutants under the Clean Water Act, 40 CFR Part 136, Friday, October 26, 1964, Fed. Reglst. 1984, 1-210.
RECEIVED for review February 12, 1990. Revised manuscript received June 21, 1990. Accepted June 26, 1990. We appreciate the financial support of the Surface-Water and Ground-Water Toxics Program of the US. Geological Survey. Brand names are for identification purposes only and do not imply endorsement by the U S . Geological Survey.
