Analysis of Pesticide Degradation Products by Tandem High

Chromatography. John G. Rollag, Melissa Beck-Westermeyer, and David S. Hage*. Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588...
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Anal. Chem. 1996, 68, 3631-3637

Analysis of Pesticide Degradation Products by Tandem High-Performance Immunoaffinity Chromatography and Reversed-Phase Liquid Chromatography John G. Rollag, Melissa Beck-Westermeyer, and David S. Hage*

Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304

The analysis of pesticide degradation in environmental samples is often difficult to perform due to the different polarities and lower concentrations of the degradation products versus the parent compound. This work examines the use of tandem high-performance immunoaffinity chromatography (HPIAC) and reversed-phase liquid chromatography (RPLC) for measuring degradation products of the herbicide atrazine in water at levels in the low nanogram per liter, or parts-per-trillion, range. An HPIAC column containing anti-triazine antibodies was first used to extract the degradation products of interest from samples, followed by the on-line separation of the retained components on a C18 RPLC analytical column. The final system had a total analysis time of 35 min for a 13 mL sample injection and gave good correlation versus GC/ MS. The limits of detection for hydroxyatrazine, deethylatrazine, and deisopropylatrazine were 20-30 ng/L for 13 mL samples and 6-10 ng/L for 45 mL samples. The use of this method was demonstrated in the analysis of both river water and groundwater samples. By changing the HPIAC column and reversed-phase separation conditions, the same approach could be modified for the detection of other pesticides and their degradation products. The analysis of pesticide residues in the environment is of great current interest due to the possible risks that may arise from the exposure of humans and animals to such agents.1-3 In this type of analysis, the processes involved in pesticide degradation should be considered, since these processes can play an important role in determining the parent compound’s persistence and long-term toxicological effects. A consideration of pesticide degradation products is also of interest because these products will often possess activities and lifetimes different from the parent compound.1,4 However, the analysis of such products is complicated by a number of factors. For example, these products often occur at lower levels than the parent agent and will probably have different polarities or chemical properties. This would ideally (1) Somasundram, L., Coats, J. R., Eds. Pesticide Transformation Products: Fate and Significance in the Environment: ACS Symposium Series 459; American Chemical Society: Washington DC, 1991; Chapter 1. (2) Agricultural Chemicals in Ground Water: Proposed Pesticide Strategy; U.S. Environmental Protection Agency: Washington, DC, 1987; pp 1-150. (3) Wallace, L. W. Environ. Health Perspect. 1991, 95, 7-13. (4) Hoar, S. K.; Blair, A.; Holmes, F. F.; Boysen, C. D.; Robel, R. J.; Hoover, R.; Fraumer, J. F. J. Am. Med. Assoc. 1986, 256, 1141-1147. S0003-2700(96)00416-7 CCC: $12.00

© 1996 American Chemical Society

Figure 1. Structures of atrazine and its major degradation products.

require the use of analytical methods that are not only sensitive and specific for a given class of compounds but are capable of dealing with a range of polar and nonpolar analytes. Atrazine [i.e., 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine] is one example of an environmental contaminant for which degradation processes can be important. This compound is among the most widely used herbicides in the world2,5 and is frequently found in environmental samples. Its primary degradation products in water include hydroxyatrazine, deethylatrazine, and deisopropylatrazine (see structures given in Figure 1), which are formed through a variety of biological and nonbiological pathways.6,7 These products not only differ in their overall polarity from atrazine but they can also have significantly different activities.1,6 The Environmental Protection Agency (EPA) currently has a maximum allowable limit of 3 µg/L for atrazine in drinking water, but no official limits have yet been set for its degradation products in the United States.8 However, these products have been reported to occur at concentrations in the nanogram to microgram per liter range, or parts-per-trillion to parts-per-billion levels, within groundwater and surface water samples.9-11 (5) Fielding, M.; Barcelo, D.; Helweg, A.; Galassi, S.; Torstenson, L.; van Zoonen, P.; Wolter, R.; Angeletti, G. In Pesticides in Ground and Drinking Water (Water Pollution Report 27); Commission of the European Communities: Brussels, 1989; pp 16-34. (6) Erickson, L. E.; Lee, K. H. Crit. Rev. Environ. Control 1989, 19, 1-14. (7) Winkelmann, D. A.; Klaine, S. J. Environ. Toxicol. Chem. 1991, 10, 347354. (8) National Survey of Pesticides in Drinking Water Wells, Phase II Report; EPA 570/9-91-020; U.S. Environmental Protection Agency: Washington, DC, 1992.

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Although many studies have examined atrazine degradation products in environmental samples,9-16 there is still a lack of analytical methods that can quickly and conveniently determine these compounds at the necessary levels. Many current methods rely on liquid/liquid or solid-phase extraction prior to analysis by gas chromatography (GC) or high-performance liquid chromatography (HPLC).10-18 But the relatively high polarity of the degradation products makes it difficult to extract these along with atrazine, and the low volatility of these products means that they must be derivatized prior to GC analysis. A simpler approach involves the use of antibodies as specific binding reagents in methods such as an enzyme-linked immunosorbent assay (ELISA).19-23 Although these assays can quickly provide information on the overall composition of atrazine-related solutes in a sample, they are not as useful in providing specific quantitation of a particular chemical species because of the cross-reactivity of the antibodies for atrazine, its degradation products, and other structurally similar compounds.19,23 This work will examine the development of an alternative method for the analysis of atrazine degradation products based on the combination of high-performance immunoaffinity chromatography (HPIAC) and reversed-phase liquid chromatography (RPLC). HPIAC is a separation method based upon the use of immobilized antibodies placed onto a chromatographic support for the selective extraction of analytes from samples. It has been recently shown that an HPIAC/RPLC system can be used for the selective determination of atrazine in water at microgram per liter levels with the direct injection of only 250 µL of sample.24 Similar results have been obtained in the use of the same general approach for the measurement of carbendazim in water.25 In the earlier work with atrazine, it was noted that several related triazine compounds could also be resolved by HPIAC/RPLC in spiked water samples, including the major degradation products of atrazine.24 However, no further work was reported regarding the (9) Pionke, H. B.; Glotfelty, A. W. Chemosphere 1990, 21, 813-822. (10) Lerch, R. N.; Donald, W. W.; Li, Y.-X.; Alberts, E. E. Environ. Sci. Technol. 1995, 29, 2759-2768. (11) Sabik, H.; Cooper, S.; Lafrance, P.; Fournier, J. Talanta 1995, 42, 717724. (12) Karalangis, G.; Von Arx, R.; Ammon, H. U.; Camenzind, R. J. Chromatogr. 1991, 549, 229-236. (13) Adams C. D.; Thurman, E. M. J. Environ. Qual. 1991, 20, 540-547. (14) Schewes, R.; Maidl, F. X.; Fischbeck, G.; von Gleissenthall, J. L.; Su ¨ ss, A. J. Chromatogr. 1993, 641, 89-93. (15) Rustum, A. M.; Ash, S.; Saxena, A.; Balu, K. J. Chromatogr. 1990, 514, 209-218. (16) Baluch, H. U.; Somasundram, L.; Kanwar, R. S.; Coats, J. R. J. Environ. Sci. Health B 1993, 28, 127-148. (17) Durand, G.; Barcelo, D. J. Chromatogr. 1990, 502, 275-286. (18) Pichon, V.; Chen, L.; Guenn, S.; Hennion, M.-C. J. Chromatogr. 1995, 711, 257-267. (19) Gascon, J.; Barcelo, D. Chromatographia 1994, 38, 633-636. (20) Dunbar, B. D.; Niswender, G. D.; Hudson, J. M. U.S. Patent 4,530,786, 1985. (21) Karu, A. E.; Harrison, R. O.; 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.; ACS Symposium Series 451; American Chemical Society: Washington, DC, 1990; Chapter 6. (22) Goh, K.; Hernandez, J.; Powell, S. J.; Garretson, C.; Troiano, J.; Ray, M.; Greene, C. Bull. Environ. Contam. Toxicol. 1991, 46, 30-36. (23) Lucas, A. D.; Jones, A. D.; Goodrow, M. H.; Saiz, S. G.; Blewett, C.; Seiber, J. N.; Hammock, B. D. Chem. Res. Toxicol. 1993, 6, 107-116. (24) Thomas, D. H.; Beck-Westermeyer, M.; Hage, D. S. Anal. Chem. 1994, 66, 3823-3829. (25) Thomas, D. H.; Lopez-Avila, V.; Van Emon, J. J. Chromatogr. 1996, 724, 207-218.

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detection of these products at environmentally relevant concentrations. The purpose of this paper is to modify the previous atrazine HPIAC/RPLC system for the measurement of atrazine’s degradation products at nanogram per liter concentrations (i.e., parts-pertrillion levels). Items to be considered in this work will include the binding and cross-reactivity of the HPIAC column for atrazine degradation products, the resolution of these products on a RPLC system, and techniques for improving the limits of detection for the HPIAC/RPLC method without the use of additional sample pretreatment steps. The final system will then be evaluated in terms of its precision, speed, response, and accuracy versus reference techniques. Specific applications to be examined for this approach will include its use in the analysis of atrazine degradation products in river water and groundwater samples. Based on these studies, guidelines can be obtained for the design of similar systems aimed at the measurement of other pollutants and their degradation products. EXPERIMENTAL SECTION Reagents. Atrazine, deethylatrazine, and deisopropylatrazine were supplied by Ciba-Geigy (Greensboro, NC). Hydroxyatrazine was obtained from Dr. James D. Carr at the Chemistry Department of the University of Nebraska (Lincoln, NE). The antitriazine monoclonal antibodies (cell line AM7B2) were produced at the Monoclonal Antibody Production Facility at the University of CaliforniasBerkeley (Berkeley, CA).21 HPLC-grade acetonitrile was obtained from Fisher Scientific (Plano, TX), and the Nucleosil Si-1000 (7 µm diameter, 1000 Å pore size) was from P. J. Cobert (St. Louis, MO). The rabbit immunoglobulin G (IgG) used as a protein standard was from Sigma Chemical Co. (St. Louis, MO). Reagents for the bicinchoninic acid (BCA) protein assay were from Pierce (Rockfield, IL). All other chemicals were of the highest grades available. The deionized water used for mobile phase and solution preparation was generated by a Nanopure purification system (Barnstead, Dubuque, IA). Apparatus. The HPIAC/RPLC system used for determination of atrazine degradation products was a modified version of that described in ref 24. This system consisted of two Model CM3200 pumps from LDC Analytical (Riviera Beach, FL), one Model 350 SSI pump from Scientific Systems, Inc. (State College, PA), and a Rheodyne 7010 injection valve (Cotati, CA). The injection valve was operated either manually or through the use of an LDC 713 autosampler. A Rheodyne 5701 tandem switching valve equipped with a DVI actuator (Chromtech, Eden Prairie, MN) was used to control application of the mobile phases to the HPIAC and RPLC columns, as discussed in ref 24. The chromatographic system included a 20 × 2.1 mm i.d. HPIAC column, a 50 × 4.6 mm i.d. RPLC precolumn, and a 100 × 4.6 mm i.d. RPLC analytical column. Both RPLC columns were packed with a 3 µm, 80 Å pore size C18 Spherisorb support from Alltech (Deerfield, IL). The chromatographic supports were downward slurry-packed at 3500 psi using an Alltech column packer. All studies were performed at room temperature. Elution of analytes was monitored by using an LDC SM3100x variablewavelength absorbance detector operated at 223 nm during the analysis of atrazine or at 216 nm during the analysis of its degradation products. These detection wavelengths were chosen since they represent the absorption maxima for atrazine and deethylatrazine/deisopropylatrazine, respectively.6,12 Hydroxyatrazine has an absorption maximum at 240 nm;17 however, its

absorption band is quite broad and has an intensity at 216 nm that is sufficient for hydroxyatrazine to be monitored along with the other primary degradation products at this particular wavelength. Methods. The immobilized antibody support and diol-bonded silica were prepared as described earlier,24 using Nucleosil Si1000 silica as the starting material. The amount of immobilized antibody was determined by a BCA protein assay,26 using rabbit IgG as the standard and diol-bonded silica as the blank. This gave a protein content of approximately 10 mg of antibody/g of silica. The binding activity of the HPIAC column was measured for each analyte by frontal analysis. In this procedure, solute concentrations of 6.25-100 µg/L were continuously applied to the HPIAC column at flow rates of 0.10 or 0.20 mL/min. The breakthrough times were determined from the resulting saturation curves after correcting for the void time of the system, as measured on a diol-bonded silica column made from the same initial support as used in the HPIAC column. From the resulting breakthrough times, the apparent association equilibrium constants and binding capacities for each analyte were determined, as described previously for other affinity chromatographic systems.27,28 In the final optimized HPIAC/RPLC method, samples were applied onto the HPIAC column with pH 7.0, 100 mM phosphate buffer at a flow rate of 0.5 mL/min. Extra purification of the Nanopure water used in preparing this mobile phase was required when samples in the low nanogram per liter range were analyzed in order to remove the small amounts of triazine-related compounds that were present. This additional purification was accomplished by passing the water through a separate anti-triazine HPIAC column prior to use in mobile-phase preparation. Analytes that were adsorbed onto the HPIAC column were later eluted by using pH 2.5, 50 mM phosphate buffer applied at a flow rate of 1.0 mL/min for 8.0 min. As the analytes eluted from the HPIAC column in this solvent, they were captured by an on-line C18 RPLC precolumn.24 This small precolumn not only served to reconcentrate the analytes back into a narrow plug but it also helped to avoid the large background peak that results when the HPIAC column is directly coupled with the RPLC analytical column.24 In order to provide optimum capture efficiency for hydroxyatrazine, the pH 2.5 phosphate elution buffer was mixed on-line with an equal volume of pH 7.0, 100 mM phosphate buffer prior to entry into the RPLC precolumn (see Results and Discussion). Analytes adsorbed onto the RPLC precolumn were next separated by switching the precolumn on-line with a larger RPLC analytical column and applying a mobile phase that contained a 25:75-20:80 mixture of acetonitrile and pH 7.0, 100 mM phosphate buffer at a flow rate of 0.5 mL/min. For studies examining atrazine alone, the mobile phase for the RPLC separation was a 50:50 mixture of acetonitrile and pH 2.5, 100 mM phosphate buffer, with no pH 7.0 buffer being added between the HPIAC column and RPLC precolumn.24 The application volumes and injection times for the samples tested throughout this work varied depending on the limits of (26) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Anal. Biochem. 1985, 150, 76-85. (27) Loun, B.; Hage, D. S. Anal. Chem. 1994, 66, 3814-3822. (28) Chaiken, I. M., Ed. Analytical Affinity Chromatography; CRC Press: Boca Raton, FL, 1987.

detection that were required. For analysis in the microgram per liter range, a 400 µL injection loop was used; but for detection at lower concentrations, an injection volume of 3-45 mL was employed. The total run times were 12 min for the 400 µL samples, 35 min for the 13 mL samples, and 60 min for the 45 mL samples. The GC/MS method used in the correlation studies29,30 was performed in the laboratory of Dr. James D. Carr (University of Nebraska, Lincoln, NE). The river water samples used in this study were collected from the Salt Creek and Platte River in eastern Nebraska between May 20 and June 4, 1995. The groundwater samples were supplied by Dr. Roy F. Spalding of the Water Center at the University of Nebraska (Lincoln, NE) and were collected from wells near Shelton, NE, during October 7-8, 1995. RESULTS AND DISCUSSION Optimization of HPIAC Conditions. A method for conveniently lowering the detection limit of the HPIAC/RPLC system was one item that had to be considered in using this system for the analysis of atrazine degradation products. In the earlier studies with atrazine, a detection limit of 0.1 µg/L was reported for a 250 µL sample injection, but it was also noted that this detection limit could potentially be lowered by simply using larger samples.24 This was confirmed in this work by comparing calibration curves obtained for small versus large sample volumes. As shown in Figure 2, an increase in sample volume from 0.4 to 3.0 mL gave a 10-fold increase in the best-fit slope, which in turn helped produce a 9-fold decrease in the estimated detection limit (i.e., from 170 to 20 ng/L). Such behavior is caused by the strong, essentially irreversible binding that occurs between analytes and HPIAC columns under typical application conditions and gives rise to a calibration curve that is more related to the moles of applied analyte than to sample concentration.24 Before using larger sample volumes to obtain low detection limits for atrazine degradation products, it was first desired to determine the degree and strength of binding that each of these degradation products had to the anti-atrazine HPIAC column. This was done by performing frontal analysis, which involved measuring the apparent moles of each solute (mLapp) that was required to saturate the HPIAC column at several different analyte concentrations. When the results were plotted in terms of 1/mLapp versus 1/[analyte], each compound gave a linear relationship characteristic of a solute with one class of binding sites.27,28 The total number of active sites in the column, as determined from the intercepts of these plots,27 was statistically identical for all analytes (i.e., a binding capacity of approximately 0.2 nmol). This result was not surprising since monoclonal antibodies were used in preparing this column and thus, either the same binding capacity or no measurable binding should have been seen for each solute. By using both the intercepts and slopes of these plots, it was also possible to estimate the apparent association equilibrium constants (Ka) for the solutes on the HPIAC column.27 The strongest binding was noted for atrazine (Ka ) 4 × 108 M-1), (29) Shepherd, T. R.; Carr, J. D.; Duncan, D.; Pederson, D. T. Ground Water Monit. Rev. 1991, 144-150. (30) Shepherd, T. R.; Carr, J. D.; Duncan, D.; Pederson, D. T. J. AOAC Int. 1992, 75, 581-583. (31) Morris, R. T. Simultaneous Determination of the Herbicide Atrazine and Two of Its Degradation Products, Deethylatrazine and Deisopropylatrazine, in Water and Air Samples by Gas Chromatography/Mass Spectrometry. Ph.D. Thesis, University of Nebraska, Lincoln, NE, 1995.

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Figure 2. Calibration curves obtained for atrazine on the HPIAC/ RPLC system at various sample volumes. Best-fit linear parameters: (a) slope (4.9 ( 0.1) × 10-3, intercept ) (3.5 ( 1.6) × 10-3, correlation coefficient 0.9990 (n ) 8); (b) slope (5.0 ( 1.6) × 10-2, intercept (2.0 ( 3.7) × 10-4, correlation coefficient 0.9995 (n ) 6).

followed by hydroxyatrazine, deethylatrazine, and deisopropylatrazine (Ka ) 6.9 × 107, 0.9 × 107, and 0.8 × 107 M-1, respectively). This trend is identical to that reported previously for the crossreactivity of the monoclonal antibodies used in making this particular HPIAC column.23 Preliminary frontal analysis studies were also performed with didealkylatrazine, a secondary degradation product of atrazine, which involves removal of both the isopropyl and ethyl groups.6,7 However, it was found that there was only weak binding for this solute to the anti-atrazine HPIAC column, indicating that this particular column was not appropriate for the quantitative analysis of this product. The relatively low affinities measured for some of the primary degradation products on the HPIAC column, particularly deisopropylatrazine and deethylatrazine, was of concern since it meant that a portion of these solutes might dissociate and leave the HPIAC column during sample application. This would result in a decrease in extraction efficiency and poor detection limits. To study whether or not this was a problem, each degradation product was injected as a narrow plug onto the HPIAC column in the application buffer, followed by washing of this column with the same buffer for various lengths of time prior to sample separation and analysis by RPLC. A 0.4 mL injection of a 10 µg/L solution was used for each solute (i.e., a load equal to 10% of the total column binding capacity). For hydroxyatrazine and atrazine, less than a 2% decrease in the final peak area was noted when wash times up to 60 min in length were used. For deethylatrazine and deisopropylatrazine, less than a 5% decrease in final peak area was observed for 60 min wash times. From these results, it was concluded that the dissociation of adsorbed solutes during sample application was not a problem for the typical injection times that were later used in this study (i.e., 4-45 min). 3634 Analytical Chemistry, Vol. 68, No. 20, October 15, 1996

Although using large sample volumes can increase the time required for sample injection, the corresponding increase in overall analysis time can be minimized by also increasing the flow rate used for sample application. However, care must be exercised in doing this since the binding efficiency of the analytes may decrease as less time is allowed for the sample to interact with the HPIAC column.32,33 The extent of this effect was examined by injecting fixed sample volumes to the HPIAC column at different flow rates and quantitating the amount of each analyte that was later seen eluting from the RPLC analytical column. For a 0.40 mL sample containing 10 µg/L each of hydroxyatrazine, deethylatrazine, and deisopropylatrazine, a decrease of 9% or less in the final peak areas was noted for all analytes when the application flow rate increased from 0.1 to 1.5 mL/min. This indicated that sample binding efficiency remained relatively constant over this range of flow rates. Optimization of RPLC Conditions. The greater polarities of hydroxyatrazine, deethylatrazine, and deisopropylatrazine versus atrazine means that these solutes show much weaker retention on RPLC columns than their parent compound. This creates two possible difficulties when HPIAC/RPLC is used for the analysis of these solutes. First, weak retention on the RPLC precolumn will result in a low capture efficiency as these analytes elute from the HPIAC support. Second, the same weak retention will cause these compounds to elute near the void volume of the RPLC analytical column and near its buffer switching peak, making detection difficult to perform. One system parameter that was altered in order to deal with these problems was the pH of the mobile phase used on the RPLC precolumn and analytical column. The main analyte of concern here was hydroxyatrazine, which has a pKa of 4.68,18,35 giving it a net positive charge at the mobile phase pH of 2.5 that was used in the previous HPIAC/RPLC system for atrazine.24 Since a pH 2.5 buffer was still required in order to desorb retained species from the HPIAC column, a method was developed in which this buffer was mixed with a second buffer stream (at a higher pH and higher molarity) before the dissociated analytes entered the RPLC precolumn. By doing this, it was possible to separately adjust the pH of the HPIAC elution buffer and the RPLC precolumn application solvent. In this work a pH 7.0, 100 mM phosphate buffer was added to the HPIAC elution buffer in a 1:1 (v/v) ratio before the entry of solutes onto the RPLC precolumn. This gave a final pH for the mixture of 6.8-7.0, which helped convert the degradation products into their neutral and least polar forms. The retention of degradation products on the RPLC analytical column was next adjusted by varying the organic modifier content of the mobile phase used during analyte separation on this column. Table 1 summarizes the best-fit parameters that were obtained by measuring the capacity factors (k′) of each analyte in mobile phases containing mixtures of pH 7.0, 100 mM phosphate buffer and various amount of acetonitrile. Acetonitrile was chosen as the organic modifier for this study because of its low absorbance at the detection wavelengths being used. From Table 1, it was (32) Hage, D. S.; Walters, R. R.; Hethcote, H. W. Anal. Chem. 1986, 58, 274279. (33) Hage, D. S.; Walters, R. R. J. Chromatogr. 1988, 436, 111-134. (34) Her, N. H. Structure and Properties of Hydroxyatrazine, M.S. Thesis, University Nebraska, Lincoln, NE, 1994. (35) Zongwei, C.; Sadagopa Ramanujam, V. M.; Gross, M. L.; Monson, S. J.; Cassada, D. A.; Spalding, R. F. Anal. Chem. 1994, 66, 4202-4209. (36) Poole, C. F.; Poole, S. K. Chromatography Today; Elsevier: Amsterdam, The Netherlands, 1991; Chapter 4.

Table 1. Best-Fit Parameters for the Change in Retention versus Mobile-Phase Composition for Atrazine and Its Major Degradation Products in Mixtures of Acetonitrile and pH 7.0, 100 mM Phosphate Buffer best-fit parametersa analyte

log k′w

a

b

atrazine hydroxyatrazine deethylatrazine deisopropylatrazine

2.42 ( 0.05 1.54 ( 0.23 2.06 ( 0.01 1.37 ( 0.02

-6.2 ( 0.3 -5.1 ( 2.7 -9.1 ( 0.1 -6.3 ( 0.2

4.1 ( 0.3 0.5 ( 7.0 10.8 ( 0.2 5.9 ( 0.5

a The best-fit parameters are for the relationship log k′ ) log k′ + w a(φ) + b(φ)2, where φ is the volume fraction of the organic modifier 36 in the mobile phase. The best-fit results for hydroxyatrazine, deethylatrazine, and deisopropylatrazine were obtained for data collected at 10%, 20%, 25%, and 30% acetonitrile. The results for atrazine were obtained for data collected at 20%, 30%, 40%, 50%, and 60% acetonitrile.

determined that all degradation products gave fairly high retention in the absence of any organic modifier (k′w ) 23-115), as required for reconcentration of these substances on the RPLC precolumn.24 However, when a 25:75-20:80 mixture of acetonitrile and buffer was used, the calculated k′ values for all of the degradation products fell within the range of 1.5-4.7. Atrazine had a k′ value of 13.1-22.1 under these same conditions and could either be monitored along with the degradation products or be allowed to wash from the RPLC analytical column between sample injections. The capture and retention of the degradation products on the RPLC precolumn was further examined by studying how long the retained analytes stayed on the precolumn after they had been captured. This was of interest since the desorption of these solutes from the HPIAC column at pH 2.5 takes a few minutes to complete and can result in broad peaks or a loss of signal if the analytes are not held strongly by the RPLC precolumn. To study this, the elution times for the HPIAC column were varied between 4 and 14 min while the sizes of the final analyte peaks were monitored on the RPLC analytical column. Only small changes in the peak heights and areas occurred over this time range (i.e., less than 6% variation), demonstrating that binding on the RPLC precolumn was sufficiently strong to avoid any large amounts of analyte loss or desorption when working under these mobile phase conditions. System Performance. An example of a chromatogram that was obtained with the HPIAC/RPLC system is shown in Figure 3. In this example, all of the major degradation products of atrazine were separated with baseline resolution and quantitated within 12 min of sample injection. No pretreatment steps were used in this analysis other than filtering of the samples prior to injection. Typical calibration curves that were obtained by this HPIAC/RPLC method are shown in Figure 4. Under the conditions used in both Figures 3 and 4 (i.e., a 13 mL sample injection), the detection limits at S/N ) 3 were 20-30 ng/L for each degradation product. The linear range (i.e., the assay range giving values within (5% of the best-fit linear response) extended up to approximately 240 ng/L for each analyte, and the dynamic range extended up to at least 1 µg/L. This range of detection was sufficient for the analysis of atrazine degradation products in river water samples. When a larger sample volume of 45 mL was used, the limit of detection was 6 ng/L for hydroxyatrazine and 10 ng/L for deethylatrazine or deisopropylatrazine. As will be shown later,

Figure 3. Typical chromatogram for a mixture of hydroxyatrazine (HA), deethylatrazine (DEA), and deisopropylatrazine (DIA), each injected onto the HPIAC/RPLC system at a concentration of 60 ng/L in a 13 mL sample. The mobile phase used on the RPLC analytical column was a 25:75 mixture of acetonitrile and pH 7.0, 100 mM phosphate buffer. All other conditions are given in the text.

Figure 4. Calibration curves obtained with hydroxyatrazine (]), deisopropylatrazine (9), and deethylatrazine (0) for a 13 mL sample injection. Best-fit linear parameters: hydroxyatrazine, slope (4.25 ( 0.04) × 10-3, intercept (-6 ( 3) × 10-6, correlation coefficient 0.9999 (n ) 5); deisopropylatrazine, slope (2.39 ( 0.13) × 10-3, intercept (-2 ( 1) × 10-5, correlation coefficient 0.9966 (n ) 5); deethylatrazine, slope (1.65 ( 0.03) × 10-3, intercept (-9 ( 20) × 10-7, correlation coefficient 0.9997 (n ) 5).

these conditions were more appropriate for the analysis of groundwater samples. The same HPIAC/RPLC system that was used for the trace analysis of these degradation products could also be used to determine atrazine at nanogram per liter levels (e.g., see Figure 2b). However, in this case, a smaller increase in sample volume was required in order to reach nanogram per liter detection limits because of the stronger retention of atrazine on both the HPIAC and RPLC columns. When a 3 mL sample was used, the detection limit for atrazine was 20 ng/L (S/N ) 3), the linear range extended from 0 to 800 ng/L, and the dynamic range extended up to at least 2.5 µg/L. When a 13 or 45 mL sample of atrazine was applied, there was a further decrease in the detection limits and calibration range, as demonstrated earlier in Figure 2. The within-day precision of the HPIAC/RPLC method at an analyte level of 50 ng/L in a 13 mL sample volume (i.e., 3.0-3.6 pmol of injected solute) was (12%, (8.2%, or (6.9% (1 RSD, n ) 6) for hydroxyatrazine, deethylatrazine, or deisopropylatrazine, respectively. The day-to-day precision over 9 days of operation for the same samples was (11%, (9.5%, or (6.2%. For atrazine, the within-day and day-to-day precisions for 3 mL injections of a 50 ng/L standard (i.e., 0.7 pmol of injected solute) were (3.2% (n ) 6) and (20% (n ) 9), respectively. Analytical Chemistry, Vol. 68, No. 20, October 15, 1996

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Figure 6. Typical chromatogram for atrazine degradation products in a 45 mL groundwater sample. The concentrations measured for deethylatrazine (DEA), hydroxyatrazine (HA), and deisopropylatrazine (DIA) were 210, 10, and 60 ng/L, respectively. The mobile phase used on the RPLC analytical column was a 20:80 mixture of acetonitrile and pH 7.0, 100 mM phosphate buffer. All other conditions are given in the text.

Figure 5. Correlation plots for river water samples containing (a) deethylatrazine and (b) deisopropylatrazine, as analyzed by both HPIAC/RPLC and a reference method based on solid-phase extraction followed by gas chromatography/mass spectrometry (SPE GC/ MS). The best-fit parameters for each plot are given in the text.

A previous report showed that the analysis of atrazine in water by HPIAC/RPLC gives results that show a high correlation with those obtained by GC reference methods.24 A series of river water samples were similarly used in this work in a double-blind study aimed at comparing the results measured by HPIAC/RPLC for deisopropylatrazine and deethylatrazine with those determined by GC/MS. The resulting correlation plots are shown in Figure 5. For deethylatrazine, the analysis of 19 samples by both methods (Figure 5a) gave a correlation plot with a slope of 1.11 ( 0.06 (1 SD), an intercept of 30 ( 60 and a correlation coefficient of 0.9790 over sample concentrations ranging from 0 to 590 ng/ L. For deisopropylatrazine, an analysis of 21 samples (Figure 5b) gave a slope of 1.01 ( 0.05, an intercept of 10 ( 30, and a correlation coefficient of 0.9768 over concentrations ranging from 0 to 580 ng/L. In further work, recovery studies were performed with two sets of five replicate water samples containing each degradation product at an initial level of 100 ng/L and spiked with 500 ng/L of each analyte. The average recoveries were 98%, 87%, and 87% for deisopropylatrazine, hydroxyatrazine, and deethylatrazine, respectively. In addition to the use of HPIAC/RPLC for analyzing river water samples, it was found that this method could also be adapted for the detection of atrazine degradation products in groundwater. This was achieved by raising the sample injection volume to 45 mL and increasing the sample application flow rate to 1.5 mL/ min, the latter being done in order to help minimize the increase in the overall analysis time. An example of a chromatogram that was obtained by this system during the analysis of a groundwater 3636 Analytical Chemistry, Vol. 68, No. 20, October 15, 1996

sample is given in Figure 6. During these analyses, the presence of each degradation product was detectable over the range of 10250 ng/L, in agreement with levels reported by other methods.9-11,35 Throughout this work no significant interferences in the HPIAC/RPLC method were seen from other classes of pesticides or pollutants, as noted earlier in the determination of atrazine by this technique.24 It was found that some triazine compounds besides atrazine and its degradation products also absorbed to the HPIAC column (e.g., cyanazine, simazine, and propazine). But these did not interfere with detection of the desired degradation products since these other compounds eluted at different times on the RPLC system. The HPIAC column used in this work was found to be quite stable throughout this study, with consistent performance being obtained over 1000 sample injections and over a 10 month period of time. This agrees with data reported previously for the analysis of atrazine by the same type of HPIAC support.24 CONCLUSIONS In this study, it was found that the combination of HPIAC and RPLC provides a useful tool for examining pesticide degradation in environmental samples. Using the determination of atrazine degradation products as a model, it was shown that HPIAC/RPLC could determine these products in river water and groundwater at parts-per-trillion levels. One advantage of this approach versus standard immunoassay methods is that it allows for the simultaneous and separate determination of several solutes within a given compound class. Advantages of HPIAC/RPLC versus GC methods include its ability to directly analyze solution-phase samples and its need for little or no sample pretreatment or derivatization prior to injection. HPIAC/RPLC also overcomes many of the current, general difficulties encountered during pesticide degradation studies, including the low concentrations of analytes that must be monitored and the large difference in polarities that may be present between the degradation products and their parent compound. Based on the techniques and guidelines developed in this work, it should be possible to extend this approach to other classes of pollutants and their degradation products by simply changing the HPIAC column and RPLC conditions that are used in the final chromatographic system.

ACKNOWLEDGMENT This work was supported by the U.S. Geological Survey under a Section 104 Grant through the Nebraska State Water Resources Program. M.B.-W. was supported under a REU fellowship from the National Science Foundation. The authors thank Drs. James D. Carr, Roy F. Spalding, and Zongwei Cai at the University of Nebraska for providing the samples and GC/MS data used in this work. Thanks also go to Dr. Alex Karu at the University of

CaliforniasBerkeley for donation of the AM7B2 monoclonal antibodies. Received for review April 25, 1996. Accepted August 5, 1996.X AC960416O X

Abstract published in Advance ACS Abstracts, September 15, 1996.

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