Anal. Chem. 1994,66, 3823-3829
Determination of Atrazine in Water Using Tandem High-Performance I mmunoaffinity Chromatography and Reversed-Phase Liquid Chromatography David H. Thomas, Melissa Beck-Westermeyer, and David S. Hage’ Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304
A fully automated HPLC method was developed for the analysis of atrazine in water. This method used a high-performance immunoaffinity column for the extraction of atrazineand other triazines from samples, followed by separation of the retained compounds with an on-line reversed-phase column. This technique used only 250 pL of sample and required minimal sample pretreatment. Atrazine determinationsby this method showed no significant interferences from the sample matrix, related triazines, or several common pesticidestested. Atrazine plus all of its major degradationproducts could be determined in 20 min, with a throughput of 10 min per injection. A more rapid scheme for measuring atrazine alone was also developed, with a total analysis time of 12 min and a throughput of 6 min per injection. The calibration curve for atrazine was linear over 2 orders of magnitude and had a lower limit of detection of 0.1 pg/L. The within-day precision was f5.4% for samples containing 1.1 pg/L atrazine. The results of this method showed good correlation with those obtained by GC/MS or CC/NPD. By using different immunoaffinity columns and elution conditions, this method could be adapted for use in the determination of other compounds of environmental interest. Atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-striazine) and related triazine herbicides are used throughout the world for the protection of crops from broadleaf weeds and for nonagricultural purposes, such as soil sterilization and road maintenance. Because of its widespread application, its solubility in water, and its persistence in the environment, atrazine has become a common pollutant in the United States’ and Europe.2 Since atrazine carryover is known to reduce yields when crop rotation is practiced, fields are often monitored for residual atrazine before vulnerable crops are planted.3 Although the threat to humans is less clear, the U S . Safe Drinking Water Act currently sets the maximum allowable level of atrazine in drinking water at 3 pg/L and requires that public water supplies be regularly monitored for its pre~ence.~ Figure 1 shows the structures of atrazine and its major degradation products in the environment. After application, atrazine that enters the soil, groundwater, and surface water (1) Agricultural Chemicals in Ground Water: Proposed PesticideStrategy; US. Environmental Protection Agency: Washington, DC, 1987; pp 1-150. (2) Fielding, M.; Barcelo, D.; Helweg, A.; Galassi, S.; Torstenson, L.; van Zoonen, P.; Wolter, R.; Angeletti, G. Pesticides in Groundand Drinking Water;Water Pollution Report 27; Commission of the European Communities: Brussels, Belgium, 1989; pp 16-34. (3) Pestemer, W.; Stalder, L.; Eckert, B. Weed Res. 1980, 20, 341-353. (4) National Survey of Pesticides in Drinking Water Wells, Phase 11 Report; EPA 570/9-91-020; US.Environmental Protection Agency, National Technical Information Service: Springfield, VA, 1992.
0003-2700/94/0366-3823$04.50/0
0 1994 American Chemical Society
CI
Atrazine ,A\
N
N
,A
HzN
NHCH,CH,
Deisopropylatrazine
(CH,I,CHNH OH
N
NY?
Deethylatrazine
Hydroxyatrazine
Figure 1. Structures of atrazine and its major degradation products in water.
is degraded by both biological and nonbiological processes. This leads to the formation of dealkylated triazines (e.g., deethylatrazine and deisopropylatrazine) as well as hydroxylated products (e.g., hydroxyatra~ine).~ The task of examining atrazine and related triazines in the environment can be both complex and expensive. Many of the procedures for the determination of these analytes are based on HPLC or GC.6 These methods achieve low detection limits for atrazine by using a large volume of sample (e.g., 100 mL to 1 L) taken through several extraction and sample cleanup steps. This helps to concentrate the sample while also increasing selectivity by limiting the analysis to a few compounds with similar polarity and/or acid-base properties. However, this type of sample pretreatment is time-consuming and labor intensive and generates waste organic solvents that may be dangerous or have high disposal costs. Some of these disadvantages can be reduced by using solid phase extraction (SPE). This can be performed either before GC analysis7J or on-line with HPLC by using automated column switching method~.~JOHowever, this approach still has several problems. For example, many solid phase extractions are based on simple polar or nonpolar stationary phases. These phases are generally nonselective and can lead (5) Cook, A. M . FEMS Microbiol. Rev. 1987, 46, 93-1 16. (6) Barcelo, D. J . Chromatogr. 1993, 643, 117-143. (7) Thurman, E. M.; Meyer, M.; Perry, C.; Schwab, P. Anal. Chem. 1990, 62, 2043-2048. (8) Junk, G. A.; Richard, J. J. Anal. Chem. 1988, 60, 451-454. (9) Slobodnik, J.; Groenewegen, M. G. M.; Brouwer, E. R.; Lingeman, H.; Brinkman, A. A. Th. J. Chromatogr. 1993, 642, 359-370. (10) Hennion, M.-C.; Coquart, V. J . Chromatogr. 1993, 642, 21 1-224.
Analytical Chemistry, Vol. 88, No. 21, November 1, 1994 3823
to difficulties with coextracted interferences. This is particularly a problem when working with large sample volumes. Also, the solid phase extraction cartridge itself can be a source of interferences, such as phthalate esters and various silicon comp~unds.~JTo avoid this, the extraction cartridge must be scrupulously cleaned before sample application. Many recent studies have examined the use of immunoassays as a simple and inexpensivealternative for routine atrazine measurements. This is generally performed using a competitive binding enzyme immunoassay, in which a sample containing atrazine is combined with a limited amount of atrazine-binding antibodies and a fixed amount of an enzymelabeled atrazine analog. After incubation of this mixture, the amount of atrazine in the sample is determined by measuring how much of the enzyme-labeled analog has bound to the antibodies.l2-'5 These immunoassay methods have good detection limits and correlate well with reference method^,^ but they also suffer from a number of disadvantages. For example, the procedures used in current atrazine immunoassays are based on manual techniques and have only moderate accuracy and precision.12-ls One factor limiting the accuracy of these methods is the crossreactivity of the antibodies with compounds similar in structure to atrazine, such as its degradation products or related triazine herbicides. The ability to discriminate between these compounds is important since different triazine compounds (e.g., atrazine and hydroxyatrazine) can vary significantly in their biological effects. These disadvantages have limited the application of current atrazine immunoassays mainly to their use in the on-site screening of field samples prior to quantitation by a second method, such as GC/MS.I6 High-performance immunoaffinity chromatography (HPIAC) is one approach that can be used to overcome some of these limitations. In HPIAC, a column containing antibodies immobilized onto silica or some other high-performance support is used for the rapid separation and determination of a given sample c0mponent.1~ Like immunoassays, this technique makes use of antibodies for the selective binding of an analyte or class of analytes. This method can be used al~ne,I~-~O or it can be coupled to other techniques, such as reversed-phase liquid chromatography (RPLC), in order to produce a multidimensional separation of closely related compounds.21 This work will examine the development of an automated HPIAC/RPLC system for the determination of atrazine in water samples. This method will use an immobilized antibody (11) Junk, G. A.;Avery, M. A.; Richard, J. J. Anal. Chem. 1988,60, 1347-1350. (12) Dunbar, B.D.;Niswender,G. D.;Hudson, J. M.U.S. Patent4,530,786,1985. (13) Bushway,R. J.;Perkins,B.;Savage,S.A.;Lekousi,S. J.;Ferguson,B.S.Bu//. Environ. Contam. Toxicol. 1988, 40, 647-654. (14) Schlaeppi, J.-M.: Fory, W.; Ramsteiner, K. J. Agric. Food Chem. 1989, 37, 1532-1538. (15) Karu, A. E.; Harrison, R. 0.;Schmidt, D. J.; Clarkson, C. E.; Grassman, J.; Goodrow, M. H.; Lucas, A.;Hammock, B. D.; Van Emon, J. M.; White, R. J. In Immunoassays for Trace Chemical Analysis; Vanderlaan, M., Stanker, L. H., Watkins, B. E., Roberts, D. W., Eds.; American Chemical Society: Washington, DC, 1990; Chapter 6. (16) Goh, K.; Hernandez, J.; Powell, S. J.; Garretson, C.; Troiano, J.; Ray, M.; Greene, C. Bull. Enuiron. Contam. Toxicol. 1991, 46, 30-36. (17) De Alwis, U.; Wilson, G. S. Anal. Chem. 1987,59, 27862789. (18) Hage, D. S.; Walters, R. R. J. Chromatogr. 1987, 386, 37-49. (19) Ohlson, S.; Gudmundsson, B.-M.; Wikstrom, P.; Larsson, P.-0. Clin. Chem. 1988, 34, 2039-2043. (20) Hage, D. S.; Kao, P. C. Anal. Chem. 1991, 63, 586595. (21) De Frutos, M.; Regnier, F. E. Anal. Chem. 1993, 65, 17A-25A.
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Analytical Chemistry, Vol. 66,No. 21, November 1, 1994
column to extract atrazine and related compounds from samples, followed by the separation and detection of these compounds on a reversed-phase HPLC column. Items to be considered in the design of this system will include the application and elution conditions for the HPIAC and RPLC columns and ways of minimizing background peaks associated with switching between these two types of columns. The developed system will then be characterized with respect to its speed, response, and precision. A comparison between the results of this technique and the results obtained by GC reference methods will also be made. Finally, the future application of this approach in the analyses of other compounds of environmental interest will be discussed.
EXPERIMENTAL SECTION Reagents. The anti-atrazine monoclonal antibodies (Line No. AM7B2) were a gift from the University of California-Berkeley Monoclonal Antibody Production Facility (Berkeley, CA). The atrazine, deethylatrazine, deisopropylatrazine, hydroxyatrazine, simazine, and terbuthylazine were generously provided by Ciba-Geigy (Greensboro, NC). The Nucleosil Si-1000 silica (7 pm particle diameter, 1000A pore size) was from Alltech (Deerfield, IL). The Nucleosil Si-100 CISsilica (3 pm particle diameter, 100 A pore size) was from P.J. Cobert Associates, Inc. (St. Louis, MO). The standard pesticide mixture used in the interference studies was obtained from Supelco (Bellefonte, PA). The rabbit immunoglobulin G (IgG) was from Sigma (St. Louis, MO). All other reagents were ACS grade or better. All buffer solutions were prepared using water from a NANOpure water system (Barnstead, Davenport, IA). Instrumentation. A schematic of the chromatographic system used in this study is shown in Figure 2. This system included two CM3000 isocratic pumps, one GM4000 gradient controller, and one SM3 100 UV/visible variable wavelength absorbance detector from LDC Analytical (Riviera Beach, FL). Samples were injected using an LDC/Milton Roy 715 autosampler and a Rheodyne 7 126 six-port switching valve with a 250 pL external sample loop (Rheodyne, Cotati, CA). The application, desorption, and separation buffers were routed through a Rheodyne tandem enrichment valve driven by a Vici DVI actuator (Chromtech, Apple Valley, MN) to control buffer switching and transfer of sample between columns. All HPLC columns were packed using a Model 1666slurry packer (Alltech, Deerfield, IL). Data were collected and analyzed using a Thermochrom Model I1 chromatography data system from LDC Analytical. Immobilized Antibody Column. Diol-bonded silica was prepared from the Nucleosil Si-1000as described previously.22 The anti-atrazine antibodies were purified from ascites fluid by ammonium sulfate pre~ipitation~~ and immobilized onto the diol-bonded support using the Schiff base method.24 After immobilization of the antibody, the silica was centrifuged, washed several times with 2 M sodium chloride and 0.10 M phosphate buffer (pH 7.0), and stored at 4 O C until further use. A portion of the silica was further washed with water, (22) Ruhn, P. F.; Gamer, S.; Hage, D. S. J. Chromatogr. 1994, 669, 9-19. (23) Harlow, E.; Lane, D., Eds. Antibodies: A Laboratory Manual; Cold Springs Harbor Laboratory: Cold Springs Harbor, NY, 1988; Chapter 8. (24) Larsson, P.-0. Methods Enzymol. 1984, 104, 212-223.
Injector
Application Buffer
l -I
I
Waste
C,, Analytical Column
I
HPIAC Elution Buffer Waste
Precolumn Waste
RPLC Buffer Flgure 2. Schematic of the atrazine HPIACIRPLC system.
vacuum dried at room temperature, and assayed for protein content using a commercial BCA assay (Pierce, Rockford, IL), with rabbit IgG as the standard and diol-bonded silica as the blank. The protein content of the support, as determined by triplicate analysis, was 8.7 f 1.4 (1 SD) mg of protein per gram of silica. Chromatography. The anti-atrazine immobilized antibody support was downward slurry packed at 3500 psi into a 6.35 mm X 2.1 mm i.d. column of a previously published design.25 A 1.O cm X 0.46 cm i.d. precolumn for trapping the desorbed triazines and a 10 cm X 0.46 cm i.d. analytical column were similarly packed with Nucleosil Si- 100C18 at 6000 psi. When not in use, the immobilized antibody column was stored at 4 OC in 0.1 M phosphate buffer (pH 7). All chromatography was performed at room temperature. Elution of atrazine and all other triazine compounds was monitored at 223 nm. Individual triazine stock standards were prepared by weighing 10 mg of each triazine into a 100 mL volumetric flask, adding about 10 mL of methanol or ethyl acetate, sonicating the solution to dissolve the atrazine, and bringing the flask to volume with methanol. Dissolution of hydroxyatrazine was aided by acidifying the solution with a few drops of dilute phosphoric acid. These standards were stored at 4 "C until use. Mixed controls and calibration standards were prepared daily from these stock solutions by dilution with NANOpure water. Samples were applied to the immunoaffinity column in 0.1 M phosphate buffer (pH 7.0) flowing at 0.5 mL/min. At 3 (25) Walters, R. R. Anal. Chem. 1983, 55, 591-592.
min after injection, the valve configuration shown in Figure 2 was switched to desorb analytes from the HPIAC column with 0.05 M phosphate buffer (pH 2.5) flowing at 1.5 mL/ min. The desorbed triazines were trapped on-line with the C18 precolumn. After 8 min, the switchingvalve was returned to its original position, coupling the precolumn to the C18 analytical column. This allowed the analytes retained by the precolumn to be eluted and separated on the analytical column using a 45:55 (v/v) mixture of 0.05 M phosphate buffer (pH 2.5) and methanol applied at a flow rate of 0.5 mL/min. During this step, pH 7.0 phosphate buffer was reapplied to the HPIAC column to regenerate the immobilized antibodies prior to the next application cycle. The binding capacity of the anti-atrazine column was estimated by frontal analysis. A 10 pg/L solution of atrazine in pH 7.0 phosphate buffer was applied to the immobilized antibody column at a flow rate of 0.1 mL/min, and the amount of atrazine required to saturate the column was determined by integration of the resulting breakthrough curves.26 Corrections for nonspecific binding and the void volume of the system were made by performing similar experiments on columns containing diol-bonded silica that had undergone the Schiff base procedure with no protein present. GC/MS analyses with solid phase extraction were performed in the laboratory of Dr. James D. Carr at the University of Nebraska (Lincoln, NE) using a previously described method.27 The samples used in correlation studies with this (26) Lund, U. J. Liq. Chromatogr. 1981, 4, 1933-1945. (27) Shepherd, T. R.; Carr, J. D.; Duncan, D.; Pederson, D. T. J.-Assoc. Chem. 1992, 75, 581-583.
Off.Anal.
Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
3825
3
Table 1. Initial Properties of the Anti-Atrazine Immunoaffinlty support
property
valuea
antibody immobilizedb antibody coverageC binding capacity specific activity
60(9) nmol antibody/g silica 0.14(0.02) monolayers 30(7) nmol atrazine/g silica OS(0.2) nmol atrazine/mol antibody
-,
I
i, 4
-
All numbers in parentheses represent f l SD. Determined using a molecular mass of 150 000 g/mol for rabbit IgG. Calculated using a surface area of 25 m2/g for Nucleosil Si1000 and a Stoke's diameter of 100 A for IgG.
'
0-
-1 -
30
method were collected in accordance with a published procedure28from the Elkhorn River near Elkhorn, NE, and from the Platte River and adjacent monitoring wells near Ashland, NE, during the months of July and August, 1993. A portion of each sample was transferred to a clean glass scintillationvial and stored at 4 "C before analysis by HPIAC/ RPLC. Analyses using GC with nitrogen-phosphorus detection (GC/NPD) and liquid/liquid extraction were performed at Midwest Laboratories, Inc. (Omaha, NE) using EPA Method 507.6 The samples used in correlation studies with this technique were collected and analyzed in accordance with standard procedures approved by the U.S. EPA.
RESULTS AND DISCUSSION Initial Design and Optimization of System. Table 1 summarizesthe binding properties of the immobilizedantibody support used in this work. On the basis of these data, the total binding capacity of the immunoaffinitycolumn was determined to be 6.3 X g of atrazine. For a 250 pL sample injection, the column had sufficient static capacity to handle atrazine concentrations up to 250 pg/L. At more typical atrazine levels (e.g., below 10 pg/L), only 4% or less of the column binding capacity was used per analysis. The rate of analyte binding to the immobilized antibody support was examined by injecting 250 pL of a 10 pg/L atrazine solution onto the HPIAC/RPLC system at various flow rates. The fraction of atrazine bound at each flow rate was determined by comparing the peak areas obtained under these conditions with those measured for direct injections of atrazine onto a column. A gradual decrease in atrazine binding from 95 to 60% was seen as the flow rate was increased from 0.1 to 4 mL/min. This behavior is in agreement with previous kinetic studies performed with other types of highperformance affinity c o l ~ m n s As . ~ a~compromise ~~~ between assay speed and extraction efficiency, an injection flow rate of 0.5 mL/min was selected for all further work. At this flow rate, 90% or more of the atrazine was extracted for sample levels at or below 10 pg/L. The amount of buffer required to desorb atrazine from the HPIAC column was studied by injecting a 10 pg/L solution of atrazine onto the HPIAC/RPLC system and desorbing the retained atrazine for 5 min with 0.05 M phosphate buffer (pH 2.5) at several flow rates. Dissociation of bound atrazine from the HPIAC column was fairly slow. Quantitative desorption required about 10 mL of pH 2.5 buffer flowing at (28) Shepherd, T. R.; Carr, J. D.; Duncan, D.; Pederson, D. T. Ground Water Monit. Rev. 1991, 144150. (29) Hage, D. S.; Walters, R. R. J. Chromatogr. 1988, 436, 111-135.
3826
Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
40
50
60
Percent Methanol (v/vl
Figure3. Retentionof hydroxyatrazine(B),deethylatrazine(V),simazine (0 ), atrazine (A),and terbuthylazine(0) on a CI8column using mobile phases containingdifferent relativeamounts of methanolas an organic modifier. The retention of each compound in this plot is expressed - 1. The values in terms of the capacity factor k', where k' = of t, and torepresent the analyte's retention time and the void time of the column, respectively.
1.5 mL/min. Slow desorption of analytes from immobilized antibody columns has been observed in other studies and can be the limiting step in an HPIAC method.*O This is important to consider since incomplete analyte desorption can lead to carryover from one sample to the next. An attempt was made to increase the rate of analyte release by using a stronger desorption buffer (Le., pH 2.3 phosphate buffer). This gave some improvement but also appeared to result in a more rapid loss of antibody activity. For this reason, pH 2.5 buffer was used as the HPIAC desorption solvent in all later work. The main difficulty produced by the slow desorption of atrazine from the HPIAC column was that the resulting peak was too broad and tailed for direct quantitation. This problem was overcome by using the pH 2.5 desorption buffer as a weak mobile phase for the reapplication and concentration of atrazine at the head of a c18 RPLC column. By later switching this RPLC column to a stronger mobile phase, such as a buffer containing methanol, the retained atrazine and other triazines could be eluted and separated from one another. A similar approach has been used in the design of other HPIAC/RPLC systems.2lJ0-32 The feasibility of using this dual column approach for atrazine was tested by measuring the retention of atrazine and other triazines on a C18 column in the presence of mobile phases containing various proportions of the pH 2.5 buffer and methanol. The results are shown in Figure 3. All compounds examinedgave linear plots for log k'versus percent methanol when intermediate amounts of organic solvent were used, in agreement with theory.33 When no methanol was used, very large values of k'were observedfor all of the triazines tested (i.e., k'> 240). This indicated that little or no elution of the triazines occurred on the c18 column when only pH 2.5 buffer was present. This confirmed that this buffer could be used for application and concentration of triazines on the RPLC column after their elution from the immobilized antibody support. (30) (31) (32) (33)
Johansson, B. J. Chromatogr. 1986, 382, 107-113. Janis, L. J.; Regnier, F. E. J. Chromatogr. 1988, 444, 1-11. Reh, F. J. Chromatogr. 1988, 433, 119-130. Poole, C. F.; Poole, S. K. Chromatography Today; Elsevier: Amsterdam, The Netherlands, 1991; pp 396-397.
Figure 3 was also used to choose mobile phase conditions for the separation of atrazine from other triazines on a C18 column. In this case, an amount of methanol was selected that caused all compounds to elute with capacity factors between 2 and 10, thus providing a good compromise between speed and resolution. This occurred when about a 4555 (v/ v) mixture of pH 2.5 buffer and methanol was used. One difficulty with using an RPLC column for the reconcentrationof atrazine and other analytes was that a large background peak was produced whenever this column was switched to the pH 2.5 buffer/methanol mobile phase. The area of this peak was proportional to the size of the RPLC column and was due to the plug of HPIAC desorption buffer present in this column at the time of the switching event. The main problem associated with this peak was that it obscured early eluting analytes, such as hydroxyatrazine and the dealkylatrazines. The same problem can be seen in earlier reports of HPIAC/RPLC application^.^^+^^^^ Reducing the size of the RPLC analytical column decreased the size of the background peak but also resulted in a decrease in analyte resolution. A better approach was to use a small c 1 8 precolumn for analyte concentration along with a larger analytical RPLC column for compound separation. The CIS precolumn was connected to a switching valve so that it could be placed on-line with either the HPIAC column or analytical Cls column (see Figure 2). A similar technique has been used to interface other low- and high-performance immunoaffinity extraction cartridges to HPLC analytical columns.34-36 In this study, the switching valve arrangement was used to trap analytes eluting from the HPIAC column and then to later release these analytes for separation on the c 1 8 analytical column. This resulted in a large reduction in the size of the background peak without sacrificing resolution. Because of these advantages, all later experiments were performed using this approach. An additional advantage of using this type of column switching was that the HPIAC column was decoupled from the analytical RPLC column during the triazine separation step. This allowed adequate time for regeneration of the HPIAC column without adding any extra time to the overall analysis. Two minutes of washing the HPIAC column with 0.1 M phosphate buffer (pH 7.0) proved sufficient to fully restore the atrazine binding capacity, since no difference in binding capacity was noticed after longer regeneration times. System Evaluation. Typical chromatograms obtained with the final HPIAC/RPLC system are shown in Figures 4 and 5. These results illustrate two different possible separation modes for the system. In the first mode (Figure 4), conditions were chosen so that atrazine and other triazine compounds could be resolved and quantitated in a single run. This used the 4555 (v/v) pH 2.5 buffer/methanol mixture for the separation of both atrazine and more weakly retained compounds, such as hydroxyatrazine. The total analysis time in this format was 20 min. However, the actual throughput was 10 min per injection since one sample could be applied (34) Farjam, A.; de Jong, G. J.; Frei, R. W.; Brinkman, U. A. Th. J . Chromafogr. 1988,452,419-433. (35) Haasnoot, W.; Ploum, M. E.; Paulusscn, R. J. A.; Schilt, R.; Huf, F. A. J . Chromatogr. 1990, 519, 323-335. (36) Rule, G. S.; Mordehai, A. V.; Henion, J. Anal. Chem. 1994, 66, 230-235.
,
HA
S
A
\
8
4
12
16
20
Time after Sample Injection (min)
Figure 4. Chromatogramforthe simultaneous determinationof atrazine, its degradation products, and related triazine herbicides by HPIAC/ RPLC. A 250 pL injection of Platte River water spiked with a triazine mixture was made under the conditionsstated inthe text. The Identified peaks represent hydroxyatrazine (HA), deisopropyiatrazlne (DIA), deethyiatrazine (DEA), simazine (S), and atrazine (A). The arrow indicates the time at which the final column switch was made, causing elution of these componentsonto the analytical Cle column. The solute concentrations were as follows: 45 pg/L HA, 150 pgIL DIA, 40 pgIL DEA, 5 pg/L S, and 20 pg/L A.
I 6
12
18
24
Time after First Sample Injection (min)
Figure 5. Chromatogram obtained with HPIAC/RPLC for the rapid determination of atrazine (A). The sample and the mobile phase conditions were the same as those in Figure 4, except that the RPLC mobile phase contained a 40:60 (v/v) mixture of 0.1 M phosphate buffer (pH 2.5) and methanol.
to the HPIAC column while another was being separated on the c18 analytical column. In the second separation mode, the HPIAC/RPLC system was optimized for the determination of atrazine but not for any other related compounds or metabolites. Examples of several runs performed in this mode are given in Figure 5. In this case, a stronger mobile phase &e., a 40:60 (v/v) pH 2.5 buffer/methanol mixture) was used for the more rapid elution of atrazine from the Cl8 analytical column. The total analysis time in this mode was 12 min. But, as in the first mode, the sample throughput was only 6 min per injection since one sample could be injected while compounds from the previous sample were being separated. Figure 6 shows a calibration curve obtained in the second separation mode. The linear range of the atrazine curve (i.e., the response within *5% of the best-fit line) extended from 0 to 32 pg/L. The correlation coefficient was 0.999 over the lower six data points shown in the graph. The lower limit of detection for atrazine at a signal-to-noise ratio (S/N) of 3 Analytical Chemistry, Vol. 66, No. 21, November 1. 1994
3827
Table 2. Composition of the Mixed Herbicide Sample Used In the Interference Studies’
15
compound E
.-0
lo
Y
a,
a a, .5 a a
,
5
0
20
40
80
60
100
Conc. Atrazine (cIg/L)
Figure 6. Calibration curve for atrazine on the HPIAC/RPLC system. The conditions were the same as those in Figure 5. The parameters of the best-fit line are given in the text.
0
2
4
6
8
Conc. Atrazine by GC/MS with SPE (pg/L) Figure 7. Correlation of HPIAWRPLC with a GC/MS method using solid phase extraction for the determination of atrazine in water. The parameters of the best-fit line are given in the text.
was 0.1 pg/L. With this particular column, the dynamic range extended up to at least 200 pg/L. The plateau in the calibrationcurve at high atrazine levels occurred as the amount of injected atrazine approached the number of available antibody binding sites. The accuracy of the HPIAC/RPLC method was evaluated by comparing it to two reference methods based on GC. Split samples of well water, river water, and surface water were tested in a double blind study, with one fraction being analyzed by HPIAC/RPLC and the other being measured by GC. Figure 7 shows the results obtained by HPIAC/RPLC and a GC/MS method using solid phasee~traction.~’In this study, 25 water samples with atrazine levels ranging from 0.2 to 9.1 pg/L were compared. The correlation coefficient between the two methods was 0.998, with a best-fit slope of 1.03 f 0.02 (1 SD) and an intercept of 0.07 f 0.20 pg/L. These results indicated that the two methods gave equivalent results over the concentration range examined. Similar results were obtained in a small scale study comparing the HPIAC/RPLC system with EPA Method 507, a technique based on GC/NPD and liquid/liquid extraction.6 A correlation coefficient of 0.998 was obtained between these methods for nine water samples with atrazine concentrations between 0.4 and 5 pg/L. The slope of the best-fit line was 1.35 f 0.06, and the intercept was -0.14 f 0.20 pg/L. These 3828
Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
bromoacil butylate cycloate EPTC hexazinone isopropalin metribuzin molinate oxyfluorfen pebulate terbacil trilfluralin
trade name Sutan Ro-Neet Eptam Velpar Paarlan Sencor Ordram Goal Tillam Treflan
CAS registry no. 314-40-94 2008-41-5 1137-23-2 759-94-4 51235t04-2 33820-53-0 21087-64-9 22 12-67-1 42874-03-3 1114-71-2 5902-5 1-2 1582-09-8
a All of the above herbicides and atrazine were injected at 100 pg/L, as prepared from Herbicides Mixt No. 1 (Supelco).
data indicated that there was also good agreement between the HPIAC/RPLC system and the EPA reference method. The within-day precision of the HPIAC/RPLC method was evaluated by making 26 injections of a spiked river water sample. A precision of f5.4% relative standard deviation was obtained at an atrazine level of 1.10 pg/L. The withinday precision obtained at other atrazine concentrations was 4.1% at 0.5 pg/L, 5.3% at 5.0 pg/L, and 7.8%at 20 pg/L (n = 3 for each level). The day-to-day precision was f13.6% relative standard deviation as evaluated by making injections of a 10 pg/L atrazine solution on 7 days over a 2 week period. The immobilized antibody column used in this study was stable for about 1 year. Over the course of this study, the column was used for more than 700 injection and desorption cycles. Only a gradual decrease in the activity of immobilized antibody was noted over this time when pH 2.5 phosphate buffer was used as the desorption solvent. This slow loss of activity was not a problem since the HPIAC column originally contained a large excess of binding sites relative to the amount of triazines injected. Also, adjustments for small decreases in activity were made automatically whenever a standard curve was prepared. During the course of a typical day (i.e., an 8-10 h run), no change in column response was ever noted. In the analysisof over 100samples from a variety of sources, including monitoring wells, production wells, river water, and soil extracts, it was found that this method enjoyed remarkable specificityand was virtually free from any sampleinterferences. The selectivity of this method was further tested by injecting a mixed sample containing 100 pg/L of atrazine plus 100 pg/L each of 11 other common herbicides. A list of the herbicides that were present in this mixture is given in Table 2. In the resulting chromatogram, no peaks were noted other than that due to atrazine. Injecting a 10-fold dilution of the same mixture also gave only an atrazine peak. The size of this peak was 97% of that obtained by injecting a 10 pg/L standard containing atrazine alone. These results indicated that none of the other pesticides present in the text mixture had any significant binding to the HPIAC column and showed no interferences in the atrazine determination. Some binding of other triazine herbicides, such as simazine and terbuthylazine, did occur on the HPIAC column due to antibody crossreactivity. These did not create any positive interferences since they were well resolved from atrazine on
the analytical Cl8 column (see Figures 3 and 4). However, it was possible for these compounds to cause a negative interference by competing with atrazine for antibody binding on the HPIAC column. This effect, which produced a low response for atrazine, became important only when the total amount of injected triazines approached the amount of active antibody sites. In routine use, this problem could easily be avoided by using a large capacity HPIAC column or by diluting and reanalyzing samples determined to have high amounts of triazines present. Another item which should be monitored with this type of system is the retention of any nonpolar buffer contaminants on the c18 precolumn during desorption of analytes from the HPIAC column. This can be important when large volumes of buffer are required for analyte desorption. In this study, two small peaks arising from the buffer were noted in the RPLC separation. Because one peak eluted from the analytical c 1 8 column after atrazine, the total analysis time had to be extended. This problem was minimized by using ultrapure water for buffer preparation. An alternative solution would be to use a desorption buffer which requires lower application volumes for removal of analytes from the HPIAC column. CONCLUSIONS This study examined the use of tandem HPIAC/RPLC in environmental analysis using the determination of atrazine in water as an example. The method was fully automated, showed high specificity for atrazine, and required only 250 pL of sample per analysis. With this method, a total analysis time of 12 min and a throughput of one sample every 6 min was obtained in the selective determination of atrazine. An analysis time of 20 min and a throughput of one sample every 10 min was achieved in the measurement of atrazine along with several related degradation products or other triazine compounds. The accuracy and precision of this technique compared favorably to those of methods based on GC/MS7,27 and GC/NPDS6 However, the HPIAC/RPLC method required less time, labor, and solvents than these reference techniques. Many of these advantages arose from the fact that no sample derivatization or extraction was required prior to sample injection on the HPIAC/RPLC system.
The use of HPIAC/RPLC for environmental testing also has several advantages over traditional immunoassay formats. One important advantage is the ability of this method to quantitateindividual members of a related class of compounds, such as atrazine and its degradation products. Achieving the same goal with standard immunoassays would require the use of several types of antibodies, each specific for a different member of the compound class to be studied. Other advantages of HPIAC/RPLC over traditional immunoassays include its speed and preci~ion.~ In addition, the use of serial sample introduction in the chromatographic system instead of the parallel processing used in most manual immunoassays may allow for easier quality control and faster reanalysis of samples.*O The system described in this work for atrazine represents a generic approach that may be applied to the determination of other compounds in water or soil samples. Many antibodies capable of binding common environmental contaminants have already been developed for use in traditional immunoassays. Immobilization of these antibodies to chromatographic supports and adjustment of the separation conditions are all that is necessary to adapt this approach to a particular application. The speed, selectivity, and flexibility of this approach should make HPIAC/RPLC a valuable tool for the analysis of a variety of components present in environmental samples. ACKNOWLEDGMENT The authors wish to thank Dr. James D. Carr and Robert Morris at the University of Nebraska and Candy Stock at Midwest Laboratories for the samples and GC results used in the correlation studies. This work was supported by the U S . Geological Survey under a Section 104 Grant to the Nebraska State Water Resources Program. D.H.T. was supported in part by a fellowship from the University of Nebraska Water Center. M.B.-W. was supported by a 1992 REU fellowship from the National Science Foundation. Received for review February 18, 1994. 1994." e Abstract
Accepted July 12,
published in Aduance ACS Abstracts, September 1, 1994.
Anaiytcal Chemistty, Vol. 66, No. 21, November 1, 1994
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