Environ. Sci. Technol. 2002, 36, 3372-3377
Sol-Gel Glass Immunosorbent-Based Determination of s-Triazines in Water and Soil Samples Using Gas Chromatography with a Nitrogen Phosphorus Detection System CONSTANTINE STALIKAS,† D I E T M A R K N O P P , * ,‡ A N D REINHARD NIESSNER‡ Institute of Hydrochemistry and Chemical Balneology, Technical University Munich, Marchioninistrasse 17, D-81377 Mu ¨ nchen, Germany
A rapid and efficient method for the selective extraction of s-triazine herbicides in environmental samples was developed using an immunosorbent of monoclonal antiatrazine antibodies, which were encapsulated in a sol-gel glass matrix. The cross-reactivity of the antibody for analytes structurally related with atrazine enabled the simultaneous extraction of several s-triazine herbicides (atrazine, propazine, terbuthylazine, cyanazine, desethyl atrazine). After trace enrichment on the immunoextraction column, the s-triazines were desorbed by means of an acidic buffer (pH 2.5) and further extracted with ethyl acetate before being injected into the GC. Compared to liquid-liquid extraction and solid-phase extraction with a hydrophobic SDB-L support, the GC-NPD chromatograms obtained after immunoaffinity enrichment of surface water (river water) samples or soil extracts and analysis were free from matrix interferences. Nonspecific adsorption of humic acids was not observed. The method allows for the determination of the herbicides in linear ranges up to 1.5 µg/L with correlation coefficients higher than 0.99 and relative standard deviations between 4% and 7% (n ) 5). The LODs for 50mL water samples were in the range 0.02 µg/L (atrazine, propazine) to 0.1 µg/L (desethyl atrazine) (S/N ) 3). In addition to its high selectivity, the immunosorbent proved to be reusable for a significant number of preconcentration runs. However, the composition of samples may influence the lifetime of the column.
Introduction Triazines are released into the environment by agricultural and industrial processes. Because of their persistence, they may end up in the soil, crop, and water. Triazine residues may cause severe problems in the environment (1). The European Union legislation sets quality standards of 0.1 µg/L for the individual pesticides and 0.5 µg/L for the total of pesticides in drinking water (2). Notwithstanding its prohibi* Corresponding author phone: ++49 89 7095 7994; fax: ++49 89 7095 7999; e-mail:
[email protected]. † On leave from Department of Chemistry, University of Ioannina, Ioannina 451 10, Greece. ‡ Technical University Munich. 3372
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 36, NO. 15, 2002
tion in the European Union, especially atrazine (2-chloro4-(ethylamino)-6-isopropylamino-s-triazine) is repeatedly detected in bodies of water. There are numerous methods known to be used for monitoring triazines by liquid chromatography (LC) (3-6) and gas chromatography (GC) (7-11). Whatever system used, pesticide screening programs require extraction, cleanup, and preconcentration steps for the enrichment of target analytes prior to chromatographic analysis to obtain low detection limits. To this end, liquid-liquid (LLE) and solidphase extraction (SPE) have been widely used. In recent years, SPE has become the primary method of choice for many analytical applications (12-18). In addition to the standard chromatographic methods currently employed, immunoassays are increasingly applied as screening methods for the analysis of environmental samples. Nowadays, a growing interest can be noticed in immobilizing antibodies onto solid supports to provide selective immunoaffinity solid-phase extraction. Immunoassays are frequently employed because of their high selectivity, sensitivity, speed, and ease of use (19). Immunoaffinity chromatography (IAC) provides a selective method for sample preparation prior to liquid chromatography (LC) and gas chromatography (GC) analyses. This type of chromatography uses antibodies raised against a specific target analyte, which are immobilized on a solid support (the immunoadsorbent), thus allowing a highly selective SPE, the so-called immunoextraction. To date, this has been successfully utilized for pesticides and other trace organics in environmental samples, as well as for drug metabolites and endogenous compounds in biological fluids. Excellent reviews have been published recently (20-22). While the coupling of IAC with LC, in on-line or off-line mode, has been reported for several applications, less data are available in the literature concerning the combination of IAC with GC (23-26). Some of them are multistep cleanup methods with the final determination by GC. Several supporting matrixes have been chosen for the preparation of immunoadsorbents. Most commonly, the immobilization of antibodies takes place on silica, controlled pore glass, or agarose. Sol-gel technology which enables the incorporation of active biomolecules into glasses has been used mostly for enzymes (27-31). Distinct advantages such as large surface area, high porosity, inertness, and mild conditions of preparation are well-established for sol-gel materials. Furthermore, the trapped biomolecules are protected against proteolytic degradation, retain their activity, and react with ligands that can easily diffuse into the highly porous matrix. Only a few applications have been reported in the literature using either polyclonal or monoclonal antibodies (32-46). Even less is the number of related papers, which describe the usage of sol-gel glass (SGG) immunoadsorbents for the group-selective enrichment of small analytes from real samples. To our knowledge, so far, only polyclonal antibodies against polycyclic aromatic hydrocarbons (PAHs) were applied for the selective extraction of these analytes from river and rainwater, plant material, and human urine (47-50). In the present study, we focused on the preparation of a sol-gel glass immunoaffinity support using a monoclonal antiatrazine antibody and its use for cleanup of surface water and soil extracts followed by the determination of s-triazines by GC with a nitrogen phosphorus detector (NPD).
Experimental Section Reagents and Solutions. Ethyl acetate and acetone (GCgrade) were purchased from J.T. Baker (Deventer, The 10.1021/es020542b CCC: $22.00
2002 American Chemical Society Published on Web 07/03/2002
Netherlands). Tetramethoxysilane (TMOS) was obtained from Fluka (Buchs, Switzerland). Hydrochloric acid (HCl), acetonitrile (ACN), sodium chloride (NaCl), and glycine were purchased from Merck (Darmstadt, Germany). Glycine-HCl buffer (glycine buffer) was prepared as described previously (23), by dissolving 0.1 mol of glycine and 0.1 mol of NaCl in 500 mL of Milli-Q grade water; the pH was adjusted to 2.5 with HCl. The s-triazines were obtained from Riedel-de Hae¨n (Seelze, Germany) and Dr. Ehrenstorfer (Augsburg, Germany). Stock solutions of 1 mg/mL atrazine, propazine, terbuthylazine, cyanazine, and desethyl atrazine were prepared in acetone. Appropriate dilutions were made to fulfill the requirements for the construction of the calibration curves. Humic acid in the form of sodium salt was supplied by Carl Roth (Karlsruhe, Germany). Phosphate-buffered saline (PBS) was prepared by dissolving 12.46 g of disodium hydrogen phosphate dihydrate, 1.56 g of sodium dihydrogen phosphate dihydrate, and 8.5 g of NaCl in 1 L of double-distilled water. The purified monoclonal antibody K4E7 (IgG2b,κ) directed against atrazine and used for IAC were kindly donated by Professor B. Hock (Department of Cell Biology, WTZWeihenstephan, Technical University Munich). Lyophilized mouse immunoglobulin IgG2b,κ, purified from ascites fluid was obtained from Sigma (Deisenhofen, Germany) and used for the study of the nonspecific interactions. Styrene divinylbenzene (Strata SDB-L, 100 µm) columns (100 mg) for SPE cleanup were freely supplied by Phenomenex (Torrance, CA). The Micro BCA protein assay (Pierce, Rockford, IL) was employed, when necessary, to determine the protein content using bovine serum albumin for the preparation of protein standard solutions. Instrumentation. For GC-NPD, a Hewlett-Packard gas chromatograph HP 5890 series II was used, equipped with an autosampler. A DB-5 (5% phenyl/95% methyl) capillary column (30 m × 0.25 mm i.d., 0.25 µm coating thickness) obtained form J&W Scientific (Folsom, CA) was used with helium of 99.999% purity as a carrier gas. The detector gas flows were hydrogen at 5 mL/min, air at 90 mL/min, and nitrogen, as an auxiliary gas, at 35 mL/min. The operating signal of the NPD was kept at 30 pA. Immunoaffinity (IAC) Column. Sol-Gel Entrapment of Antiatrazine Antibody K4E7. The sol-gel glass was prepared by mixing under stirring the following: 1.7 mL of TMOS, 100 µL of methanol, 300 µL of glycerol 50%, 300 µL of Milli-Q water, and 100 µL of 0.04 M HCl. The mixture was chilled on ice for 25 min under occasional vortexing. Two milligrams of the antibody K4E7 were dissolved in 700 µL of PBS, mixed with an equal volume of the TMOS preparation, and poured into a Petri dish. Gelation occurred within 2 min; the gel was weighed and allowed to age at 4 °C until loss of weight of 55-60%. The resulting amount of silicate glass (∼0.6 g) was ground in a mortar and packed into 5-mL glass columns obtained from Merck. To avoid loss of immunoadsorbent during utilization, two PTFE frits (porosity 10 µm, Merck) were placed above and below the sorbent bed. The column was subsequently washed with 5 mL of Milli-Q water and 5 mL of PBS, and aliquots of these solutions were used for the estimation of antibody immobilization efficiency. The IAC column was stored in PBS at 4 °C, when not in use. Assessment of Antibody Leaching from Sol-Gel Immunosorbent. Two milligrams of the antiatrazine antibody K4E7 were entrapped in the sol-gel matrix as described previously. The column filled with the material was washed 5 times with 15 mL of 2% (v/v) ACN followed by additional rinse with 5 × 15 mL of the eluting buffer (glycine buffer, pH 2.5). The protein content in the individual fractions was measured by the Micro BCA protein assay. Determination of the Capacity of IAC Column. The column capacity was evaluated by overloading the adsorbent with 10-mL aqueous solutions containing 1.0, 3.0, 5.5, 7.0, and 10
µg of atrazine and analyzing the eluted fractions according to the method described. Selectivity of IAC Column. The selectivity of IAC was evaluated by percolating mixtures of four s-triazine herbicides and one atrazine metabolite (desethyl atrazine) through the IAC column at a concentration of 40 ng each in 10 mL of water. The eluted fractions were analyzed according to the method described in the following section. Recovery as a Function of Loading Volume. The volume breakthrough of the IAC column was illustrated by percolating through the column increasing volumes of sample solutions at a constant herbicide content. To this end, 10, 50, 100, and 250 mL of river water were preconcentrated containing 2% (v/v) ACN and spikes of 20 ng of each s-triazine. The eluent was analyzed by the analytical method described in the following section. Analytical Procedure. Immunoextraction. Before use, the IAC column was preconditioned successively with 5 mL of double-distilled water, 10 mL of glycine buffer, and 10 mL of ACN/water (2:98). Then, the sample solution consisting of 50 mL in 2% (v/v) ACN was applied by pumping through the column, followed by 10 mL of 2% (v/v) ACN as a cleanup step to remove nonspecifically retained constituents and residual sample matrix components. The elution of the trapped analyte(s) was performed by percolating through the column 25 mL of glycine buffer, which was then transferred into a separatory funnel. The desorbed analytes were further extracted with 2 × 5 mL ethyl acetate, and the organic extracts were concentrated to 1 mL, under a gentle stream of nitrogen, before they had been analyzed. After elution, the column was regenerated with 20 mL of PBS and stored at 4 °C until further use. The entire procedure was performed at a flow rate of 1 mL/min and negative pressure (MSP peristaltic pump, Ismatec, Wertheim-Mondfeld, Germany). SPE. Samples were preconcentrated in SDB-L columns by an automated off-line SPE system (J.T. Baker). The SDB-L commercial cartridge was conditioned with 2 mL of ethyl acetate and 10 mL of water. A drying step with nitrogen took place between the loadings of the two solvents. The sample was pumped through the cartridge, which was then flushed with 5 mL of water to remove extraneous matrix components. Subsequently, the copolymer cartridge was dried with nitrogen for 20 min to remove traces of water, and the adsorbed analytes were desorbed with 1 mL of ethyl acetate. Sample Preparation. A water sample was collected in a glass bottle from a river (Windach) close to Munich. Two milliliters of ACN per 100 mL of sample were added upon its arrival in the laboratory. Then, the sample was filtered through a 40-µm glass fiber filter following an extraction and preconcentration process, as described previously. Two soil samples were processed; one taken from the domain of the Institute of Hydrochemistry and another one (grassland soil) was kindly provided by G. Henkelmann from the Bayerische Landesanstalt fu ¨ r Bodenkultur und Pflanzenbau Mu ¨ nchen. For triazine analysis, soil samples were air-dried and sieved through a mesh of 2 mm to remove the coarse material. An amount of 10 g was extracted with 100 mL of double-distilled water by shaking for 1 min, followed by sonication for 30 min. The suspension was left stand at room temperature for 8 h, and the supernatant was filtered through a folded filter paper (Cat. No. 1202125; Whatman, Maidstone, U.K.). The filtrate was collected in a glass vial containing 2 mL of ACN and brought up to 100 mL with double-distilled water before it was further treated. An aliquot of 50 mL of the aqueous extract was analyzed to find s-triazines and used as a matrix to carry out the recovery studies. The organic carbon content of soils was determined by oxidizing the sample with a hot mixture of potassium dichromate and concentrated sulfuric acid. The excess VOL. 36, NO. 15, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3373
reagent was titrated with a standard solution of ammonium ferrous sulfate. Diphenylamine was used as an indicator (51). Chromatographic Analysis. An aliquot of 1 µL of the organic extract was injected into the GC-NPD. External calibration was established for the s-triazines studied. The oven temperature was programmed from 60 °C (0.5 min) to 160 °C (1 min) at 40 °C/min, from 160-220 °C (0.5 min) at 3 °C/min, and finally from 220-250 °C at 40 °C/min. Injection of standards and samples were performed in the splitless mode (for 1 min) at an injector temperature of 250 °C and a detector temperature of 270 °C. Signal acquisition and processing were carried out with HP 3365 Series II ChemStation, version A.03.01, chromatography software.
Results and Discussion Antibody Immobilization Efficiency. The presence of a significant fraction of antibodies on the solid phase was verified by determination of the immobilization yield of the procedure. The amount of the entrapped antibodies was estimated by spectrophotometric detection of protein in the washing fraction with the BCA protein assay at 562 nm. Best results for antibody entrapment were obtained with gels containing 6% glycerol. This finding is in accordance with earlier experiments done by M. Schedl in this laboratory using antifungicide antibodies (unpublished data). All of the column materials prepared by the described procedure showed that the immobilization efficiency was reproducible in the range of 92-95%. These experiments demonstrated that, under the conditions stated in the procedure, amounts of antibody between 1.8 mg (1.2 × 10-8 mol) and 1.9 mg (1.3 × 10-8 mol) can effectively be entrapped on the SGG-IAC column. Another important aspect related to the immunosorbent, which should be taken into account, is the possible leaching of biomolecules from the support matrix through consecutive preconcentration runs, which can dramatically affect the performance characteristics of the IAC with concomitant effects on method applicability. It is likely that part of the biological material may be lost because of incomplete enclosure within the pores of the glass due mainly to the fact that the biomolecules are noncovalently encapsulated. This should be observed mainly at the initial stages of column operation (i.e., directly after the immobilization process). The evaluation of the degree of leaching of the entrapped monoclonal antibodies was performed by treating a newly constructed column with ample amounts of washing solution and eluting buffer. The measurement of the protein content in the individual fractions showed negligible loss (nondetected by the BCA protein assay) of the biological material. Capacity of the IAC Column. The next step was to confirm that the IAC column could readily retain atrazine. Column capacity has been assessed in terms of analyte mass breakthrough. Taking into consideration that 1.9 mg of antibody correspond to 1.3 × 10-8 mol of monoclonal antibody and that if both binding sites of an IgG molecule are accessible, then we could estimate that the theoretical column capacity is about 5.5 µg of atrazine per ∼600 mg immunosorbent bed (52). Experimentally, a dose dependency was estimated up to an applied dose of atrazine equivalent to the theoretical capacity, which accounts for 760 ng of retained atrazine. Loading increasing amounts of atrazine, higher than 5.5 µg, the bound yield and, hence, the column capacity, did not ameliorate. The amount of retained atrazine (760 ng) corresponds to 14% of the theoretical maximum capacity. The found capacity was far lower than the theoretical ones and significantly reduced as compared to those which we have obtained in the past with polyclonal antibodies of different specificity. As yet, only two papers have been published which report on the encapsulation of a monoclonal antiatrazine antibody 3374
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 36, NO. 15, 2002
TABLE 1. Percent Recoveries of s-Triazines Using SDB-L-SPE and IACa analyte
SDB-L-SPE
IACb
cross-reactivityc
atrazine propazine terbuthylazine cyanazine desethyl atrazine
88 75 70 65 60
93 88 58 57 50
100 138 26 28 18
a Mixture of five triazines in 10-mL of double-distilled water; spiking level, 40 ng each. b After elution, a liquid-liquid extraction step follows with ethyl acetate. c Data obtained by ELISA (53).
in SGG (35, 39). In both studies the same antibody was used but different sol-gel preparation methods were applied. The capacities of the immunoadsorbents obtained were strikingly different to each other. However, at this point, it is not clear whether the conditions of the sol-gel preparation solely affect the capacity of bioceramic material or if the antibody molecule itself (e.g., its subclass) may also be of importance in this respect. With the limited data available, it is not possible to draw general conclusions about this issue. In the course of the experimental work and after running more than 20 preconcentrations, the capacity decreased to about 280 ng atrazine per column. Very complex samples inevitably accelerate the decrease in the capacity of the immunoaffinity column due to, for example, nonspecific adsorption onto the glass surface and clogging of the micropores. Despite this fact, column capacity, as reported in the present investigation, was high enough to retain atrazine on the IAC support for its use in environmental analysis. Selectivity of the IAC. Immunoaffinity columns can retain analytes structurally related to the target compound(s) because of the cross-reactivity of antibodies. As shown in Table 1, higher recoveries were obtained for atrazine and propazine, which are structurally similar to each other, whereas lower affinity was estimated for the rest of the studied compounds. These results are in accordance with the crossreactivity pattern of this antibody using the enzyme-linked immunosorbent assay (ELISA), as reported earlier (53). However, far from being a disadvantage, when IAC is combined with the GC, efficient separation and identification of structurally related s-triazines, recognized by the antibody, can be carried out in unknown samples. The decrease of the capacity, at the levels mentioned before, with increasing the number of experimental runs does not deteriorate the yields of the recoveries. Going further, the contribution of nonselective interactions (i.e., adsorption to all sites which are not the paratopes of the antibodies) to the retention on IAC column has been evaluated using different SGG columns and including control samples, as follows: (A) bare SGG without any protein, (B) SGG with immobilized mouse IgG2b,κ, and (C) SGG with immobilized specific K4E7 antibody. The results shown in Table 2 revealed that almost the entire amounts of the applied triazines onto the bare SGG were recovered in the initial fractions of loading and washing solutions. More specifically, less than 7% of the analytes was present in the eluted fraction, because there are no selective recognition sites available on the support. Interestingly, on the SGG column containing the immobilized nonspecific IgG2b,κ, the binding remained as low as in the case of bare SGG, ratifying the absence of nonspecific interactions with molecular regions of an IgG antibody. Thus, specific binding holds considerably high value. Optimization of the Protocol for Immunoaffinity Extraction. An optimal IAC protocol involves trapping of the target analyte(s) on the solid phase, washing off interferences less attracted to the sorbent, and finally, eluting analytes
TABLE 2. Nonselective Binding of s-Triazines (%)a analyte atrazine propazine terbuthylazine cyanazine desethyl atrazine
bare SGG 7 6 6 nd 6
SGG-mouse IgG2b, 7 7 6 nd nd
TABLE 3. Recoveries of the Studied s-Triazines as a Function of the Loading Volume (%)a SGG-K4E7 97 98 87 76 68
a The loading solution consisted of 20 ng/10 mL of each s-triazine in double-distilled water. The calculated fractions of nonspecifically bond s-triazines on bare SGG and SGG-mouse IgG2b,κ represent the difference between the total amount of the applied s-triazines and the amount which was found in the washing fraction. (nd) Not detected.
according to their affinity with the immobilized antibody. The challenge is, therefore, to choose the optimized loading, washing, and elution conditions. The IAC column was conditioned with 5 mL of double-distilled water, 10 mL of glycine buffer, and 10 mL of 2% (v/v) ACN. When using the SGG immunoaffinity columns, desorption in the smallest volume and under the mildest conditions possible, without damaging the antibody or the support, is the ideal case. Most common eluents that are used in IAC for the dissociation of antigen-antibody complexes are acidic or basic buffer solutions with high ionic strength and organic solvents. In general, the stability of the antibody varies with the kind of the eluent. Pure organic solvents can affect the activity of antibodies very severely. In the present investigation, it was found that the entrapped antibodies cannot tolerate high amounts of water-miscible organic solvents. For instance, using ACN/water (40:60), high recoveries were achieved in a rather small fraction (∼4 mL) of the eluent but, on the other hand, reusability of the column was decreased dramatically. It is also known that extreme pH values of aqueous eluents can cause irreversible denaturation of the protein. As stated in earlier reports, good results were obtained using this antibody for the elution of the bound triazines from beaded cellulose IAC columns with 0.2 M glycine buffer, pH 2.2 (23, 52). In the present study, when using a glycine buffer of pH 2.5, high recovery rates were obtained, accounting for 96% of atrazine and propazine, in a 20-mL fraction without affecting the quality of the immunosorbent which remained active for the next loadings. The interactions between analytes and the antibody are fading in the presence of the acidic buffer. The weakly bound triazines (i.e., terbuthylazine, cyanazine, and desethyl atrazine) were desorbed in the early fractions (∼10 mL) of eluent. Values of pH lower than 2.5 were not attempted in order to avoid irreversible denaturation of the immobilized antibodies. Buffer systems with higher pH values (e.g., pH 3.5) were inferior in terms of elution efficiency, as higher volumes were required (∼50 mL) to attain quantitative desorption of the strongly bound triazines. Except for the composition of the eluent which influences the performance of IAC column, the application of samples containing a high percentage of organic solvent to an IAC column may also cause damage to the antibody and severely affect the binding of the analyte. The s-triazines were bound to the entrapped antibodies in the presence of 2% (v/v) ACN which was added to prevent nonspecific adsorption of the analytes on the glass material during sampling (water sample) and treatment (soil samples). Higher concentrations of ACN interfered with the binding of analytes on the IAC column. As far as the pH of the loaded sample is concerned, the behavior of the immunosorbent on the retention of analytes was gauged by loading aqueous mixtures of s-triazines with pH values in the range 5-9. Our data showed no quantifiable differences between the experiments performed in this pH range. Values of pH out of this range were not tested because they are rather harsh for loading the immunosorbent.
volume (mL) analyte
10
50
100
250
atrazine propazine terbuthylazine cyanazine desethyl atrazine
98 97 83 70 66
96 90 61 60 54
94 82 52 52 50
60 73 44 41 40
a
River water spiked with 20 ng of each s-triazine.
The washing solution should be in keeping with the composition of the loading solution. So, the column can be washed with at least 10 mL of 2% (v/v) ACN without any loss of analyte. In most cases, it is desirable to concentrate the highest volume possible of sample on the IAC column. Therefore, the recovery of the studied s-triazines as a function of the loading volume was investigated (Table 3). Up to a loaded volume of 100 mL, the recovery of atrazine was almost constant, accounting for about 94%. When the applied volume increased up to 250 mL, the recovery declined to around 60%. Higher risk for analyte loss occurs for s-triazines with lower affinity for the antibody. The volume of 50 mL can be selected as the most suitable, if one aims to analyze the whole group of s-triazines and to prevent escape of them through the material. Glycine buffer is incompatible with GC requirements. Therefore, after desorption from the immunoaffinity column, the analytes were extracted into an organic solvent. Ethyl acetate was selected because of its compatibility with GCNPD system. Twice the volume of 5 mL of ethyl acetate is capable of extracting quantitatively the s-triazines as well as the relatively polar desethyl atrazine, the principal degradation product of atrazine. No loss of the analytes occurs during the condensation of the organic extract, even when conducted to dryness. The baseline noise is kept low, and reproducible results can be achieved if the final volume is mostly the same (1 mL) between the experiments. Naturally occurring organic substances such as humic acids may interfere with the column material through nonspecific adsorption. As a consequence, they can bring about possible reduction of column capacity or give rise to poor chromatographic separation after elution and extraction. To detect potential effects of humic matter on column behavior, standard solutions fortified at a spiking level of 10 ng/50 mL for each of five s-triazines along with humic acid in the range 0-10 µg/mL were applied onto the column. In all cases, the elution profiles for the whole group of the studied s-triazines were similar, regardless of the presence or the increasing content of humic acids, signifying a remarkable tolerance to samples containing varying amounts of humic acids. Figures of Merit. Spiked samples at a concentration level of 10 ng/50 mL of each of the five s-triazines showed relative standard deviations in the range of 4.0-7.0% for five successive replicates. Calibration curves were constructed with filtered surface water drawn from river Windach and spiked with the s-triazines. The method allows for the determination of the target pesticides in linear ranges up to 1.5 µg/L with satisfying correlation coefficients. For each experiment, several replicates were performed. Some of the analytical features of the method are summarized in Table 4. Linearities up to 1.1 and 1.5 µg/L for the selected s-triazines signify that the capacity of the prepared immunosorbent is marginally sufficient when the analytes are present each one at the aforementioned concentrations. This situation, though, is rarely encountered in real samples. The lower limits of VOL. 36, NO. 15, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3375
TABLE 4. Dynamic Linear Ranges, Correlation Coefficients (R2), Limits of Detection (LOD), and Relative Standard Deviations (RSD) for the Determination of s-Triazines Using IAC-GC-NPD
analyte
linear range (µg/L)
R2
atrazine propazine terbuthylazine cyanazine desethyl atrazine
0.06-1.1 0.06-1.1 0.20-1.5 0.20-1.5 0.25-1.5
0.9981 0.9955 0.9971 0.9962 0.9942
limits of detection RSD (µg/L)a (n ) 5)b(%) 0.02 0.02 0.08 0.08 0.10
a Corresponding to a signal-to-noise ratio of 3. mL spikes.
b
4.5 4.0 4.9 5.5 7.0
Data for 10 ng/50
detection (LOD) for the triazines at a signal-to-noise ratio (S/N) of 3 were variable in the range 0.02-0.1 µg/L. However, taking care not to exceed the column capacity and violate the volume breakthrough limits, lower detection limits can be obtained using larger sample volumes. Application to Real Samples. A river water sample spiked with the studied triazines was subjected to cleanup and preconcentration on the IAC column and was analyzed by GC-NPD after extraction with ethyl acetate. Simultaneously, external standards were run with s-triazine concentrations identical to those in the spiked real samples. The recoveries obtained were almost quantitative. For the first time, soil extracts have been applied to SGG immunoadsorbents. Depending on the soil source, the respective extracts may be very complex matrixes; therefore, they may contain constituents which could interfere with the IAC support in different manner. In the present investigation, two soil samples containing low (0.98%; soil from the Institute) to modest amounts of organic matter (1.4%; grassland soil) were subjected to the same cleanup and preconcentration process after extraction, according to the procedure given in the Experimental Section. To assess the
selectivity power of the proposed IAC method and to demonstrate its efficiency to remove interfering substances, three extraction procedures were adopted in pure and spiked soil extracts: LLE, SPE using the commercial SDB-L, and immunoaffinity extraction relying on the prepared SGG immunosorbent. None of the selected s-triazines was detected in the two soil samples. LLE provided, as expected, chromatograms with a multitude of peaks attributed to coextracted substances (Figure 1A,B). SDB-L-SPE provided a slightly improved chromatogram (Figure 1C) as compared to the LLE procedure, although interfering peaks can still hinder the identification of certain s-triazines (e.g., cyanazine). The SDB-L cartridge consists of a hydrophobic material; therefore, it is not surprising that it can retain nonpolar organic compounds, which are detected either as coeluting or as near-eluting peaks. When the IAC column is applied, no interfering material was observed with GC trace in the area in which the s-triazines appeared (Figure 1D). This column can relieve the chromatogram of undesired peaks and obviates further sample cleanup. Also, a peak eluting at the retention time of propazine (denoted as unknown in the chromatogram of Figure 1A) may erroneously be assigned to it if no confirmatory means is available. After applying the developed immunoaffinity enrichment/extraction procedure to the unspiked soil extract, this peak is completely eliminated. Relevant GC-MS analysis advocated the foregoing assumption for an extraneous peak and revealed a benzenedicarboxylic acid ester compound naturally existing in the soil. This, however, validates the absence of nonspecific interaction of soil components with the immunosorbent and suggests that no interference with the analytes of interest occurs. Finally, the soil samples tested did not deteriorate the efficiency of the IAC column to an extent greater than the s-triazine aqueous solutions may induce. However, no definite conclusions can be drawn on the number of soil samples which can effectively be treated by one column, because of the limited data at hand. It is certain that the
FIGURE 1. GC-NPD chromatograms obtained for a soil sample extract (grassland soil; organic carbon content 1.4%). Peak assignment and spiking level: 1 ) desethyl atrazine, 50 ng; 2 ) atrazine, 10 ng; 3 ) propazine, 10 ng; 4 ) terbuthylazine, 50 ng; 5 ) cyanazine, 50 ng. Identities of the chromatograms: (A) pure soil extract after LLE; (B) spiked soil extract after LLE; (C) spiked soil extract after SDB-L-SPE; (D) spiked soil extract after SGG-IAC-SPE. 3376
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 36, NO. 15, 2002
column can be reused for the cleanup of multiple samples but that their composition can influence the lifetime of the column. The developed IAC-GC-NPD method is reliable and easily applicable. Cross-reactivity of the used antiatrazine antibodies with structurally related s-triazine compounds can be exploited for extracting a group of herbicides subsequently separated and measured by gas chromatography. The SGGIAC yields excellent chromatograms, well-separated compounds, and the absence of interferences when using environmental samples such as surface water and soil. In summary, the antibody-doped sol-gel bioceramic support is effective on s-triazine cleanup and preconcentration and is particularly suited for their qualitative and quantitative measurements using GC-NPD. Moreover, immunoaffinity columns offer the advantage of reusability. The combined benefits of the present method can constitute convincing arguments to use it as a complement to the well-established ELISA method, which is most suitable for screening in environmental analysis. Additional investigations using different sol-gel preparation and antibody isotypes will be conducted with the objective to increase the IAC column capacity.
Acknowledgments C. Stalikas acknowledges the support of a DAAD research fellowship. The authors are grateful to Professor Dr. B. Hock and Dr. K. Kramer from the Department of Cell Biology, WTZWeihenstephan, Technical University Munich, for their valuable gift of the purified monoclonal antibody K4E7, Phenomenex (Aschaffenburg, Germany) for Strata SDB-L, and G. Henkelmann from the Bayerische Landesanstalt fu¨r Bodenkultur und Pflanzenbau Mu ¨ nchen for providing a grassland soil sample.
Literature Cited (1) Tomlin, C. D. S., Ed.; The Pesticide Manual, 11th ed.; BCPC: Farnham, Surry, U.K., 1997. (2) Council Directive 91/414/EEC; European Union: Brussels, Belgium, 1991. (3) Aguilar, C.; Ferrer, I.; Borrull, F.; Marce´, R. M.; Barcelo D. J. Chromatogr. A 1998, 794, 147. (4) Hogenboom, A. C.; Nissan, W. M. A.; Brinkman, U. A. Th. J. Chromatogr. A 1998, 794, 201. (5) Gong, A.; Ye, C. J. Chromatogr. A 1998, 827, 57. (6) Vandecasteele, K.; Gaus, I.; Debreuck, W.; Walraevens, K. Anal. Chem. 2000, 72, 3093. (7) Sabik, H.; Jeannot, R. J. Chromatogr. A 1998, 818, 197. (8) Sauret, N.; Millet, M.; Herckes, P.; Mirabel, P.; Wortham, H. Environ. Pollut. 2000, 110, 243. (9) Zambonin, C. G.; Palmisano, F. J. Chromatogr. A 2000, 874, 247. (10) Hankemeier, Th.; Steketee, P. C.; Vreuls, J. J.; Brinkman, U. A. Th. J. Chromatogr. A 1996, 750, 161. (11) Loos, R.; Niessner, R. J. Chromatogr. A 1999, 835, 217. (12) Lacorte, S.; Vreuls, J. J.; Salau, J. S.; Ventura, F.; Barcelo, D. J. Chromatogr. A 1998, 795, 71. (13) Louter, A. J. H.; van Beekvelt, C. A.; Cid Montanes, P.; Slobodnik, J.; Vreuls, J. J.; Brinkman, U. A. Th. J. Chromatogr. A 1996, 725, 67. (14) Ahmed, F. E. Trends Anal. Chem. 2001, 20, 649. (15) Martı´nez, D.; Cugat, M. J.; Borrull, F.; Calull, M. J. Chromatogr. A 2000, 902, 65. (16) Junker-Buchheit, A.; Witzenbacher, M. J. Chromatogr. A 1996, 737, 67.
(17) Wolska, L.; Wiergowski, M.; Galer, K.; Go´recki, T.; Namiesnik, J. Chemosphere 1999, 39, 1477. (18) Sabik, H.; Jeannot, R.; Rondeau, B. J. Chromatogr. A 2000, 885, 217. (19) Sherry, J. P. Crit. Rev. Anal. Chem. 1993, 23, 217. (20) Van Emon, J. M.; Gerlach, C. L.; Bowman, J. J. Chromatogr. B 1998, 715, 211. (21) Hage, D. S. Clin. Chem. 1999, 45, 593. (22) Delaunay, N.; Pichon, V.; Hennion, M.-C. J. Chromatogr. B. 2000, 745, 15. (23) Dallu ¨ ge, J.; Hankemeier, T.; Vreuls, R. J. J.; Brinkman, U. A. Th. J. Chromatogr. A. 1999, 830, 377. (24) Delahaut, Ph.; Jacquemin, P.; Colemonts, Y.; Dubois, M.; De Graeve, J.; Deluyker, H. J. Chromatogr. B. 1997, 696, 203. (25) Hooijerink, H.; Schilt, R.; van Bennekom, E. O.; Huf, F. A. J. Chromatogr. B. 1994, 660, 303. (26) Farjam, A.; Vreuls, J. J.; Cuppen, W. J.; Brinkman, U. A. Th; De Jong, G. J. Anal. Chem. 1991, 63, 2481. (27) Avnir, D.; Braun, S.; Lev, O.; Ottolenghi, M. Chem. Mater. 1994, 6, 1605. (28) Livage, J. C. R. Acad. Sci., Ser. IIb 1996, 322, 417. (29) Lin, J.; Brown, C. W. Trends Anal. Chem. 1997, 16, 200. (30) Collinson, M. M. Crit. Rev. Anal. Chem. 1999, 29, 289. (31) Gill, I.; Ballesteros, A. Tibtech 2000, 18, 282. (32) Venton, D. L.; Cheeseman, K. L.; Chatterton, R. T., Jr; Anderson, T. L. Biochim. Biophys. Acta 1984, 797, 343. (33) Wang, R.; Narang, U.; Prasad, P. N.; Bright, F. V. Anal. Chem. 1993, 65, 2671. (34) Zu ¨ hlke, J.; Knopp, D.; Niessner, R. Fresenius J. Anal. Chem. 1995, 352, 654. (35) Turniansky, A.; Avnir, D.; Bronshtein, A.; Aharonson, N.; Altstein, M. J. Sol-Gel Sci. Technol. 1996, 7, 135. (36) Jordan, J. D.; Dunbar, R. A.; Bright, F. V. Anal. Chim. Acta 1996, 332, 83. (37) Roux, C.; Livage, J.; Farhati, K.; Monjour, L. J. Sol-Gel Sci. Technol. 1997, 8, 663. (38) Shabat, D.; Grynszpan, F.; Saphier, S.; Turniansky, A.; Avnir, D.; Keinan, E. Chem. Mater. 1997, 9, 2258. (39) Bronshtein, A.; Aharonson, N.; Avnir, D.; Turniansky, A.; Altstein, M. Chem. Mater. 1997, 9, 2632. (40) Wang, J.; Pamidi, P. V. A.; Rogers, K. R. Anal. Chem. 1998, 70, 1171. (41) Cichna, M.; Knopp, D.; Niessner, R. Anal. Chim. Acta 1997, 339, 241. (42) Cichna, M.; Markl, P.; Knopp, D.; Niessner, R. Chem. Mater. 1997, 9, 2640. (43) Bronshtein, A.; Aharonson, N.; Turniansky, A.; Altstein, M. Chem. Mater. 2000, 12, 2050. (44) Doody, M. A.; Baker, G. A.; Pandey, S.; Bright, F. V. Chem. Mater. 2000, 12, 1142. (45) Lan, E. H.; Dunn, B.; Zink, J. I. Chem. Mater. 2000, 12, 1874. (46) Altstein, M.; Bronshtein, A.; Glattstein, B.; Zeichner, A.; Tamiri, T.; Almog, J. Anal. Chem. 2001, 73, 2461. (47) Scharnweber, T.; Knopp, D.; Niessner, R. Field Anal. Chem. Technol. 2000, 4, 43. (48) Spitzer, B.; Cichna, M.; Markl, P.; Sontag, G.; Knopp, D.; Niessner, R. J. Chromatogr. A 2000, 880, 113. (49) Cichna, M.; Markl, P.; Knopp, D.; Niessner, R. J. Chromatogr. A 2001, 919, 51. (50) Schedl, M.; Wilharm, G.; Achatz, S.; Kettrup, A.; Niessner, R.; Knopp, D. Anal. Chem. 2001, 73, 5669. (51) Gaudette, H. E.; Flight, W. R.; Toner, L.; Folger, D. W. J. Sediment Petrol. 1974, 44, 249. (52) Marx, A.; Giersch, T.; Hock, B. Anal. Lett. 1995, 28, 267. (53) Giersch, T. J. Agric. Food. Chem. 1993, 41, 1008.
Received for review January 21, 2002. Revised manuscript received May 28, 2002. Accepted May 28, 2002. ES020542B
VOL. 36, NO. 15, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3377
