Anal. Chem. 1997, 69, 4508-4514
Immunosorbents Coupled On-Line with Liquid Chromatography/Atmospheric Pressure Chemical Ionization/Mass Spectrometry for the Part per Trillion Level Determination of Pesticides in Sediments and Natural Waters Using Low Preconcentration Volumes Imma Ferrer,† Marie-Claire Hennion,‡ and Damia` Barcelo´*,†
Department of Environmental Chemistry, CIDsCSIC c/Jordi Girona, 18-26, 08034 Barcelona, Spain, and Laboratoire de Environment et Chimie Analytique, Ecole Supe´ rieure de Physique et de Chimie Industrielles (ESPCI) de Paris, 10, rue Vauquelin, 75231 Paris, cedex 05, France
A new automated on-line immunosorbent phase extraction method was developed and validated for the analysis of triazine and phenylurea herbicides in environmental matrixes such as sediments and natural waters. This method is a first application and consists of trace analyte extraction using an immunosorbent columnscontaining either anti-atrazine or anti-chlortoluron antibodiess combined with a liquid chromatograph by use of an online sample preparation system coupled directly to an atmospheric pressure chemical ionization/mass spectrometer in positive mode of operation. After the percolation of 20 mL of water through the immunosorbent columns, high recoveries of extraction were obtained for all the compounds with the exception of deisopropylatrazine and diflubenzuron. Calibration curves were linear in the range between 0.01 and 0.2 µg/L in groundwater. The limits of detection ranged from 0.001 to 0.005 µg/ L, indicating good sensitivity achieved by both types of immunosorbents. Environmental sediment samples from the Ebre Delta area (Tarragona, Spain) containing several triazines and linuron were Soxhlet extracted with methanol, and the extracts were brought to a volume of 100 mL in water in order to perform the extraction with the immunosorbents. No significant interferences from the sample matrix were noticed, thus indicating a good selectivity of the immunosorbents used. Seawater samples from a marina nearby (Masnou, Barcelona, Spain) were also analyzed. An interlaboratory study using Aquacheckcertified groundwater samples was carried out in order to validate the application of the method for the analysis of environmental samples.
There is a considerable interest in developing selective and sensitive methods to extract and isolate components of complex environmental matrixes. Solid-phase extraction (SPE) using C18 or polymeric phases has been widely used for the determination of pesticides in water samples followed by gas chromatography † ‡
CIDsCSIC. ESPCI.
4508 Analytical Chemistry, Vol. 69, No. 22, November 15, 1997
(GC) or high-performance liquid chromatography (HPLC) methods.1-3 However, these stationary phases are generally nonselective and can lead to difficulties with coextracted interferences present in the environmental matrixes. Most of the polar pesticides cannot be determined due to their coelution with the matrix peak obtained at the beginning of the chromatogram when both complex water samples and soil extracts are analyzed by HPLC. This matrix peak is a coeluting interferent due to humic substances present in soil and natural waters. In this sense, an effort has been made toward the development of new selective sorbent materials for the analysis of surface water and soil samples.4 Recently there has been a growing interest in employing the highly selective analyte-antibody interactions achieved by immunosorbents.4-7 In the immunosorbent, the antibody is immobilized onto a silica support and used as an affinity ligand to extract the target analyte and other compounds with similar structures from the aqueous sample. In this way, any material not recognized by the antibody is not retained in the immunosorbent while the target analyte remains bound to the antibody, leading to a high selectivity. The development and the evaluation of two immunosorbents for the selective trace solid-phase extraction of phenylurea and triazine herbicides have been presented in previous works.4,5 Other immunosorbents for the analysis of single pesticides have been described in the literature.8-10 Many times, sample preparation procedures involve numerous steps. For soil samples, the coextraction of chemical interferents (1) Barcelo´, D.; Chiron, S.; Lacorte, S.; Martı´nez, E.; Salau, J. S.; Hennion, M.C. Trends Anal. Chem. 1994, 13, 352-361. (2) Oubin ˜a, A.; Ferrer, I.; Gasco´n, J.; Barcelo´, D. Environ. Sci. Technol. 1996, 30, 3551-3557. (3) Pichon, V.; Hennion, M.-C. J. Chromatogr., A 1994, 665, 265-281. (4) Pichon, V.; Chen, L.; Hennion, M-C.; Daniel, R.; Martel, A.; Le Goffic, F.; Abian, J.; Barcelo´, D. Anal. Chem. 1995, 67, 2451. (5) Pichon, V.; Chen, L.; Durand, N.; Le Goffic, F.; Hennion, M.-C. J. Chromatogr., A 1996, 725, 107-119. (6) Pichon, V.; Rogniaux, H.; Fischer-Durand, N.; Ben Rejeb, S.; Le Goffic, F.; Hennion, M.-C. Chromatographia 1997, 45, 289. (7) Ferrer, I.; Pichon, V.; Hennion, M.-C.; Barcelo´, D. J. Chromatogr., A 1997, 777, 91-98. (8) Rule, G. S.; Mordehal, A. V.; Henion, J. Anal. Chem., 1994, 66, 230-235. (9) Shahtaheri, S. J.; Katmeh, M. F.; Kwasowski, P.; Stevenson, D. J. Chromatogr., A 1995, 697, 131-136. (10) Marx, A.; Giersch, T.; Hock, B. Anal. Lett. 1995, 28, 267. S0003-2700(97)00843-3 CCC: $14.00
© 1997 American Chemical Society
necessitates a step in the procedure that isolates the analyte from the other components before the final analysis by GC or liquid chromatography (LC). Immunosorbents have been applied for the cleanup of river water and soil samples followed by liquid chromatography with diode array detection (LC/DAD).6 Using immunosorbents, extraction, trace enrichment, and cleanup are accomplished in one step when surface waters are analyzed. So chromatograms present a clear baseline, allowing the determination and quantification at the 0.01 µg/L level. To our knowledge no analytical methodology using on-line immunosorbent coupled to LC/MS has been applied to the determination of several pesticides in environmental samples. By this approach, a selective extraction method can be combined with a highly sensitive detection system such as the use of LC/MS with selected ion monitoring. Previous work from our group has applied the on-line SPE methodology using conventional sorbents (C18 and polymerics) followed by LC/MS for the analysis of pesticides11,12 and phenols.13 In the present work, the coupling of such a selective sorbents together with the high sensitivity achieved by LC/atmospheric pressure chemical ionization MS allows for a determination of organic pollutants at the lownanogram per liter level using small preconcentration sample volumes. The objectives of this study were as follows: (i) to develop a new analytical method combining immunosorbents on-line with LC/APCI/MS for the determination of several pesticides in environmental matrixes, (ii) to study the extraction efficiency of triazine and phenylurea pesticides upon anti-atrazine and antichlortoluron immunosorbents, respectively, after the preconcentration of groundwater samples, (iii) to analyze several sediments and seawater samples to prove the feasibility of such an on-line methodology, and (iv) to evaluate the performance of the IS for the determination of traces of pesticides in Aquacheck certified groundwater samples. EXPERIMENTAL SECTION Immunosorbent Columns. Preconcentration of the water samples was carried out through experimental on-line sample preparation (OSP-2) cartridges prepacked with 80 mg of silica and 2 mg of anti-atrazine and anti-chlortoluron antibodies. Polyclonal antibodies immobilized on this adsorbent were supplied by Prof. Le Goffic (ENSCP, Paris, France). Polyclonal antibodies were synthesized against atrazine and chlortoluron according to the procedure described in a previous study.4 Pesticides were modified by the introduction of a carboxylic group so that they could be linked to BSA before injection into rabbits. The antibodies were then covalently bound to a silica matrix in order to obtain a pressure-resistant sorbent. Chemicals. HPLC-grade solvents acetonitrile and methanol and LC-grade water were purchased from Merck (Darmstadt, Germany). The pesticide standards hydroxy-atrazine, atrazine, deisopropylatrazine, deethylatrazine, simazine, terbuthylazine, propazine, monuron, chlorotoluron, isoproturon, diuron, linuron, and diflubenzuron were obtained from Promochem (Wesel, Germany). Irgarol 1051 was a gift from Ciba Geigy (Barcelona, Spain). Deuterated atrazine was used as an internal standard and (11) Lacorte, S.; Barcelo´, D. Anal. Chem. 1996, 68, 2464-2470. (12) Aguilar, C.; Ferrer, I.; Borrull, F.; Marce´, R. M.; Barcelo´, D. J. Chromatogr., A, in press. (13) Puig, D.; Silgoner, I.; Grasserbauer, M.; Barcelo´, D. Anal. Chem., 1997, 69, 2756-2761.
was purchased from Cambridge Isotopes (Cambridge, U.K.). Sodium phosphate, sodium chloride, and sodium azide were obtained from Merck. Acetic acid was purchased from Panreac (Barcelona, Spain). Stock standard solutions of 500 µg/mL were prepared by weighing the solutes and dissolving them in methanol. A stock solution of 0.2 µg/mL was used to spike LC-grade and groundwater at themicrogram per liter level for the preconcentration through the cartridges and further determination of recoveries and construction of the calibration graphs. The final standard solutions did not contain more than 0.5% methanol. The phosphate-buffered solution (PBS) consists of a 0.01 M sodium phosphate buffer containing 0.15 M NaCl (pH 7.4) and 0.2% azide. Chromatographic Conditions. The eluent was delivered by a gradient system from Waters 616 pumps coupled to a Waters Model 600S controller (Waters, Milford, MA). The analytical column used was a 25 cm × 4.6 mm i.d. packed with 5-µm octylsilica gel from Shandon (Cheshire, England). The mobile phases used for the elution of the analytes were acetonitrile and water containing ammonium acetate (0.01 M) in the case of triazine determinations. For phenylurea detection, the mobile phase consisted of acetonitrile and water containing 0.5% acetic acid. These conditions were optimized for each family of compounds. It was observed that when acetic acid was used the ionization for diuron and linuron was enhanced as compared with the use of ammonium acetate. The gradient elution was performed as follows: from 30% A (acetonitrile) and 70%B (LC-grade water) to 100% A and 0% B in 40 min. Mass Spectrometric Analysis. LC/APCI/MS in positive mode of operation was used for the determination of triazine and phenylurea herbicides. A VG Platform mass spectrometer from Micromass (Manchester, U.K.) equipped with an APCI source interface was used. The VG Platform APCI interface consists of a heated nebulizer probe and the standard atmospheric pressure source configured with a corona discharge needle.11 The different operating parameters included a drying gas (N2) flow rate of 250300 L/h and a nebulizing gas flow rate of 10 L/h. The cone voltage was set at 20 and 40 V and the corona voltage at 3.5 kV. The ion source was set at 180 °C, and the probe temperature was 400 °C. The instrument control and data processing utilities included the use of the MassLynx application software installed in a Digital DEC PC 466. The MS conditions were optimized in a previous work.12 Chromatograms were recorded under selected ion monitoring (SIM) conditions, and in the case of the analysis of triazines, time-schedule SIM conditions using two acquisition windows were used. Full-scan (m/z ) 100-400) conditions were also used for the analysis of blank samples in order to detect possible interferences coextracted with the analytes. Two internal standardssdeuterated atrazine for the triazines and monuron for the phenylureasswere used for the quantitation of all the pesticides studied. Sampling. (a) Sediment Samples. Sediments containing linuron, atrazine, and their metabolites were collected from an estuarine area (Ebre Delta, Tarragona, Spain) during 1990-1991.14 They were collected from the top surface (10 cm), freeze-dried, and sieved through 120-µm mesh. The composition of the sediments was as follows: 8% clays, 28% silt, 64% sand, and 2.5% (14) Durand, G.; Barcelo´, D. Toxicol. Environ. Chem. 1992, 36, 225-234.
Analytical Chemistry, Vol. 69, No. 22, November 15, 1997
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organic matter and pH of 7.5. The sediments were stored at -20 °C in the dark in order to avoid degradation of the compounds. (b) Seawater Samples. Seawater sampling was carried out at a depth of 1 m from the surface layer of the Masnou marina, which is located at 20 km north of Barcelona. The samples (taken between April 1996 and March 1997) were collected in 2.5-L precleaned amber glass bottles and kept at 4 °C in the dark until analysis.15 Water sample pH varied from 7.9 to 8.3. Two of the samples collected corresponding to the months of November and December were chosen for their preconcentration through the immunosorbents and further determination by LC/APCI/MS. Sample Preparation. (a) Sediment Extraction. Sediment sample (10 g) was Soxhlet extracted for 12 h with 100 mL of methanol. The extracts obtained were preconcentrated in a rotary evaporator to 2 mL, then carefully evaporated to dryness with a gentle stream of nitrogen, and brought to a volume of 1 mL with methanol. Afterward, 50 µL of this extract was added to a volume of 100 mL of groundwater in order to get the sample into an aqueous phase and be able to perform the preconcentration through the immunosorbent, so that the sediment samples could be treated as water samples. Blanks of sediment samples were also extracted and analyzed under SCAN conditions. (b) Water Samples. Water samples were filtered through a 0.45-µm filter (Millipore, Bedford, MA) before use. Preconcentration of the samples was performed on-line with an automated sample preparation. The automated SPE device used (OSP-2, Merck) was connected on-line with the gradient pumps. A LiChroGraph Model L-600A intelligent pump (Merck-Hitachi) was used to deliver the solvents to condition the precolumns and the water containing the pesticides. The general scheme of the system was described previously.16 The first step of the solid-phase extraction consisted of conditioning the immunosorbent (80 mg of bonded silica) with 6 mL of PBS and then with 3 mL of LC-grade water. Afterward, 20 mL of the sample was percolated through the immunosorbent at a flow rate of 1 mL/min followed by 1 mL of LC-grade water. The compounds trapped on the immunosorbent were eluted with the chromatographic mobile phase by switching the valve into the elute position. For recovery studies, 20 mL of LC-grade and groundwater sample spiked at 0.2 µg/L was percolated through the immunosorbent. This experiment was performed in triplicate for all the pesticides studied. Blanks of sediment samples, seawater, LC-grade water, and groundwater were percolated through the immunosorbent and then analyzed by LC/APCI/MS in SCAN conditions in order to evaluate the possible interferences present in the cartridge and eluted after. The calibration curves were obtained by percolating 20 mL of groundwater sample spiked in the trace level range of 0.01-0.2 µg/L in order to have the same matrix as in the environmental water samples. Validation of the immunosorbent was carried out by performing an interlaboratory calibration study for herbicide compounds organized by Aquacheck (WRC, Medmenham, U.K.). A certified standard solution containing an unknown concentration of pesticides and a 2-L bottle of groundwater were provided by the (15) Ferrer, I.; Ballesteros, B.; Marco, M. P.; Barcelo´, D. Environ. Sci. Technol., in press. (16) Chiron, S.; Papilloud, S.; Haerdi, W.; Barcelo´, D. Anal. Chem. 1995, 67, 1637-1643.
4510 Analytical Chemistry, Vol. 69, No. 22, November 15, 1997
Table 1. Major Ions and Relative Abundances (RA) of the Triazines and the Phenylureas Studied after Analysis by LC/APCI/MS in the PI Operation Modea compound
Mnb
m/z of main ions
RA
hydroxyatrazine deisopropylatrazine deethylatrazine simazine atrazine deuterated atrazine propazine terbuthylazine
197 173 187 201 215 220 229 229
irgarol monuron chlortoluron isoproturon diuron linuron diflubenzuron
253 198 212 206 232 248 310
198 [M + H]+ 174 [M + H]+ 188 [M + H]+ 202 [M + H]+ 216 [M + H]+ 221 [M + H]+ 230 [M + H]+ 230 [M + H]+ 174 [M - C4H7]+ 254 [M + H]+ 199 [M + H]+ 213 [M + H]+ 207 [M + H]+ 233 [M + H]+ 249 [M + H]+ 158 [M - C6H4NHCOCl + H]+ 311 [M + H]+
100 100 100 100 100 100 100 100 15 100 100 100 100 100 100 100 45
a Cone voltage set at 20 V and corona at 3.5 kV. Carrier stream: acetonitrile/water containing 0.01 M of ammonium acetate for the triazine analysis or 0.5% of acetic acid for the phenylurea analysis at a flow rate of 1 mL/min. b Mn, nominal mass.
organization. The aim was to spike the groundwater with the solution provided in order to determine the levels of these pesticides in water; 500 mL of the groundwater sample was spiked with 50 µL of the certified standard solution. When the immunosorbent was not in use, it was stored at 4 °C in a solution of PBS containing 0.2% azide after a washing step using 70% methanol (5 mL). RESULTS AND DISCUSSION APCI Characteristics. Table 1 reports the typical ions of all the pesticides studied in this work in PI mode of operation. Cone voltages of 20 and 40 V were studied in order to assess the best conditions of detection under LC/APCI/MS. It was observed that almost all the pesticides studied did not present a major fragmentation at 40 V, and moreover, the sensitivity decreased under those conditions, so all the determinations were carried out at a cone voltage of 20 V, which gave both good structural information and the best sensitivity for the pesticides studied. All the triazines studied gave only the molecular ion plus a proton as a base peak except terbuthylazine, which gave a slight fragmentation corresponding to the lose of the terbutyl group. For phenylurea herbicides the same behavior was observed with the exception of diflubenzuron, which gave as a major ion m/z ) 158, corresponding to the fragmentation between the C-N bound of the two amide groups present in the molecule. It was observed that diuron and linuron did not suffer ionization when ammonium acetate was used as a buffer in the mobile phase. The reason for this behavior can be attributed to the fact that APCI is basically a gas-phase chemical ionization process and so the addition of salts can affect this ionization. Recoveries. The affinity of the immunosorbents for compounds other than the antigen is achieved because of the similarity in the chemical structures of compounds in the same family of pesticides. This is the case of the anti-atrazine and anti-chlorotoluron immunosorbents evaluated in the present work; triazines and phenylureas with structures similar to atrazine and chlorotoluron, respectively, can be retained in the immunosorbent as their affinity for selective antibodies is increased (see Figure 1).
Figure 1. Chemical structures of the compounds studied.
Table 2. Recoveries of Extraction (%) and Repeatability (Relative Standard Deviation among Replicates, n ) 5)a compound
recoveries (%)
repeatability (%)
deisopropylatrazine deethylatrazine simazine atrazine deuterated atrazine propazine terbuthylazine irgarol monuron chlortoluron isoproturon diuron linuron diflubenzuron
0 87 89 102 103 97 101 86 80 90 90 91 88 42
10 1 3 4 5 4 11 4 6 8 4 4 2
a Obtained after the percolation of 20 mL of groundwater spiked at 0.2 µg/L with a mixture of triazines and phenylureas through the antiatrazine and anti-chlortoluron immunosorbent, respectively.
Table 2 presents the extraction recoveries obtained for all the pesticides analyzed after their extraction in 20 mL of groundwater spiked at 0.02 µg/L on the anti-atrazine and anti-chlorotoluron immunosorbents. For triazine pesticides, high recoveries are obtained, ranging from 86 to 103%, with the exception of deisopropylatrazine, which was not retained at all in the anti-atrazine immunosorbent. In previous work,5 it was assessed that this compound showed a better recovery on anti-simazine immunosorbent rather than on the anti-atrazine immunosorbent, due to the presence of the ethyl group in the molecular moiety. Simazine contains two ethyl groups in its structure, so an immunosorbent
containing this antibody can recognize a similar compound such as the case for deisopropylatrazine. On the other hand, antiatrazine immunosorbents are expected to trap those triazines that contain an isopropyl group in their structure. For phenylurea pesticides, the recoveries ranged from 42 to 91%, showing good affinity of the immunosorbents except in the case of diflubenzuron, which gave the lowest recovery. In previous work,7 the recoveries of phenylurea pesticides on anti-isoproturon immunosorbents were studied, showing low recoveries for chlorotoluron and linuron herbicides. For that reason, in the present work, the performance of anti-chlortoluron immunosorbent together with mass spectrometry detection was evaluated and compared to that from the previous work. Chlortoluron presents a disubstituted phenyl ring, so that anti-chlorotoluron immunosorbent is more suitable for trapping those phenylureas containing disubstituted phenyl rings in their chemical structure such as chlorotoluron, diuron, and linuron. Although monuron presents a monosubstituted ring it is also retained on anti-chlorotoluron immunosorbents due to the presence of the dimethylamide group in the chemical structure. Incomplete recoveries may be due either to the low affinity presented by the antibodies toward the analytes or to overloading of the capacity of the immunosorbent when related compounds are competing for the same binding sites. In the first case, the recovery is limited by the retention factor of the analyte by the immunosorbent, whereas in the second case, a decrease in the competition process will represent an improvement in the recovery. Repeatability of the method was calculated from five independent extractions of triazine and phenylurea pesticides from groundwater samples on anti-atrazine and anti-chlorotoluron immunosorbents, respectively. The repeatability ranged from 1 to 11%, indicating good performance of the method developed in Analytical Chemistry, Vol. 69, No. 22, November 15, 1997
4511
Table 3. Calibration Data Obtained with LC/APCI/MS in Time-Scheduled SIM-PI Mode for the Pesticides (Spiked from 0.01 to 0.2 µg/L) after On-Line Preconcentration of 20 mL of Groundwater compound
calibration eqa
R2
LODb (µg/L)
deethylatrazine simazine atrazine propazine terbuthylazine irgarol chlortoluron isoproturon diuron linuron
Y ) 0.15 + 6.36x Y ) 0.05 + 11.04x Y ) 0.15 + 10.84x Y ) 0.22 + 9.73x Y ) 0.05 + 10.71x Y ) -0.05 + 21.72x Y ) 0.01 + 14.28x Y ) 0.18 + 13.67x Y ) 0.01 + 10.84x Y ) -0.02 + 5.96x
0.980 0.996 0.995 0.994 0.992 0.993 0.998 0.981 0.996 0.998
0.003 0.003 0.001 0.001 0.002 0.003 0.002 0.002 0.005 0.005
a Least squares regression equation. b LODs were calculated by using a signal-to-noise ratio of 3 (the ratio between the peak intensity and the noise).
this work. One advantage of automation in an on-line preconcentration is that more reproducible results are expected, provided that manipulation of the samples is avoided as compared with an off-line methodology. Moreover, the use of internal standards represents an improvement in the precision of measurements when quantitation of analytes by LC/MS is performed since the signal variations of this system are quite common. Variations in the solvent pumps and plugging problems encountered in the source are the main causes for the lack of reproducibility in many measurements.17 The use of internal standards, either deuterated or not, is, therefore, a good analytical quality tool when quantification with LC/MS is performed. Calibration Curves and LODs. Calibration graphs were constructed by percolating 20 mL of groundwater sample spiked with the solution containing either triazines or phenylureas through anti-atrazine and anti-chlorotoluron immunosorbents, respectively. Calibration data are summarized in Table 3. The curves were linear in the range studied from 0.01 to 0.2 µg/L and the correlation coefficients were higher than 0.98 for all the pesticides studied. In previous work,5 it was seen that the calibration curves were linear for the lowest concentration levels and reached a plateau for the highest concentration levels (3 µg/ L) due either to the overloading of the immunosorbent or to the competition process of related compounds. In this paper, the concentrations studied are lower and this behavior is not observed. From a qualitative point of view, it is important to note that, when the immunosorbent is not overloaded, the slope of the calibration curve for a compound is constant. The linear range of a calibration curve can be then increased by decreasing the competitive binding between the antibodies and the analytes and decreasing the levels of concentration studied. The limits of detection were calculated using a signal-to-noise ratio of 3 (the ratio between the peak intensity under SIM conditions and the noise). Low detection limits in the parts-pertrillion (ppt) level can be obtained due to the high selectivity achieved by the immunosorbents and the high sensitivity encountered by the APCI/MS system. The interaction of the matrix of water and the antibodies is low, thus leading to a high selectivity of the immunosorbent for the analytes studied. The limits of (17) Ferrer, I.; Thurman, E. M.; Barcelo´, D. Anal. Chem. 1997, 69, 4547-4553 (in this issue).
4512 Analytical Chemistry, Vol. 69, No. 22, November 15, 1997
Table 4. Concentration Values (µg/kg) of the Pesticides Analyzed in Sediment Samples and in Seawater Samples (µg/L) after Analysis by Solid-Phase Immunosorbent Extraction Followed by LC/APCI/MSa compound samples
deethylatrazine
atrazine
linuron
diuron
irgarol
sedimentc 1 sediment 2 sediment 3 sediment 4 sediment 5 seawatere 1 seawater 2
19.5 6.5 15.4 -
33.6 19.2 39.2 -
-d 139.0 59.2 -
0.04 0.02
0.04 0.03
DARb 0.58 0.34 0.39
a Experimental conditions as described in the text. b DAR, deethylatrazine-to-atrazine ratio.18 c Sediment samples were collected in the Ebre Delta area during 1990-1991. d -, not detected. e Seawater samples were collected in Masnou area during 1996.
detection depend on the recovery of extraction achieved by the immunosorbent and, therefore, depend on the affinity developed by the immobilized antibodies. The higher is the affinity for a compound the lower is the detection limit. On the other hand, APCI/MS detection has been proved to be very selective for all the compounds studied since few interferences are encountered under SIM conditions.7 So the combination of the two selective and highly sensitive steps has been demonstrated to be a powerful technique for the preconcentration and detection of traces of organic contaminants in environmental samples, respectively. Environmental Samples. (a) Sediment Samples. Atrazine, deethylatrazine, and linuron were found in the sediment samples analyzed with the methodology developed in this work. The herbicides atrazine and linuron were used in the rice crop fields of the Ebre Delta area in amounts of 1 ton/year active compound during 1990-1991. These herbicides are used for weed control of corn, wheat, barley and sorghum. In Table 4, the environmental concentrations found in all the sediments analyzed by LC/APCI/ MS are presented. Concentrations were consistent with those encountered in a previous work using GC/NPD and LC/DAD techniques.14 The concentrations of atrazine are lower than those for linuron and it can be explained by the difference in the respective KOC. The value of this parameter for atrazine is 160 whereas for linuron it is 400, indicating that this compound is more likely to remain in the sediment than in the water. Deethylatrazine was the major metabolite of atrazine found in the sediment samples. The degradation of the herbicide atrazine in soils has lead to numerous investigations. Dealkylation of atrazine is the most significant biotic degradation pathway for atrazine in soil environments. The conversion of atrazine to its main metabolite, deethylatrazine, is primarily due to the metabolic activity of soil bacteria and fungi, deethylatrazine is the major degradation product of atrazine in soils. The DAR (deethylatrazine-to-atrazine ratio) is a valid indicator of a soil-mediated transport of atrazine to an aquifer.18,19 A DAR of unity or greater may be an indicator of non-point-source contamination of an aquifer whereas a small DAR may be an indicator of point-source contamination of an aquifer. In the case of non-point-source (18) Adams, C. D.; Thurman, E. M. J. Environ. Qual. 1991, 20, 540-547. (19) Thurman, E. M.; Fallon, J. D. Int. J. Environ. Anal. Chem. 1996, 65, 203214.
Table 5. Mean Concentration (ng/L) and Mean Difference (%) (n ) 3) in Relation to Reference Values of Pesticides from Two Interlaboratory Studiesa October 1996
Figure 2. On-line solid-phase extraction of 20 mL of water containing the extract of a sediment sample through an anti-atrazine immunosorbent followed by LC/APCI/MS in positive ion (PI) mode of operation and under Time-Schedule SIM-PI conditions. Analytical conditions as described in the Experimental Section. Peak: (1) deethylatrazine; (2) atrazine + deuterated atrazine.
contamination, as atrazine is normally stored in sediments, soil microorganisms can convert significant quantities of atrazine to deethylatrazine, thereby increasing the DAR. On the other hand, point-source contamination, resulting from a direct entry of atrazine into an aquifer, would not involve prolonged contact of the applied atrazine to soil microorganisms and then the concentration of deethylatrazine would be lower than the concentration of atrazine. In this case, the DAR value would be smaller than unity. In the present study, we obtained a DAR value smaller than unity (see Table 4), thus indicating a point-source contamination of the aquifer that contained the sediment samples analyzed in this work. This can be easily explained if we take into account that samples were collected a few months after the application period of the pesticides and that atrazine is currently applied over this area.14 As can be seen in Figure 2, the chromatograms obtained from the sediment extracts analyzed after their preconcentration through the anti-atrazine immunosorbent presented a clear baseline and the compounds could be easily identified and detected. The selectivity of the preconcentration through the immunosorbent is so high that small sample volumes, such as 20 mL, can be used as compared with those normally analyzed by nonselective sorbents. Normally, a cleanup step before the GC or HPLC detection is needed due to the complex matrix present in these kind of samples. However, since selective interactions by the antibodies are achieved using immunosorbents, this additional step is avoided, allowing lower coefficients of variation in the measurements to be obtained. Blanks of sediment samples were also preconcentrated through both immunosorbents and analyzed by LC/APCI/MS under SIM and SCAN conditions, showing no significant interferences with the pesticides studied. (b) Seawater Samples. Seawater samples were preconcentrated on the anti-atrazine and anti-chlortoluron immunosorbents and analyzed by LC/APCI/MS under SIM conditions. Irgarol and diuron concentrations in the ppb level (see Table 4) were found in the two seawater samples collected from a marina and were correlated well with those analyzed in the previous study carried out with these samples.15 Both compounds are used in antifouling paints as biocide agents in substitution for the tributyltin (TBT) and copper-based agents. These compounds are used in tin-free antifouling paint formulations that are mainly based on copper and zinc metal oxides. The herbicides are added in order to
January 1997
compound
concn
error (%)
concn
error (%)
simazine atrazine propazine chlortoluron isoproturon diuron linuron
86.7 141.9 294.0 55.1 119.1 96.5 62.9
14.1 21.3 15.6 0.2 36.9 3.7 -3.3
99.4 174.8 74.7 46.3 30.2 55.3 98.3
-7.1 1.0 0.9 10.2 25.7 20.3 6.8
a Results are obtained after preconcentrating 20 mL of groundwater sample spiked with the certified solution from Aquacheck). The relative standard deviation varied between 5 and 16% (n ) 3).
inhibit the primary growth of copper-resistant fouling organisms such as algal slimes and the growth of seaweeds in sea boats. Irgarol and diuron have a good recovery on anti-atrazine and antichlortoluron immunosorbents, respectively; therefore their extraction from a seawater sample as accomplished by means of selective interaction between the antibodies and the pesticides is feasible. This result confirms again the high selectivity of the immunosorbent for the compounds present in any type of water. Validation of the Method. The automated on-line solid-phaseimmunosorbent extraction followed by LC/APCI/MS was validated by participating in the Aquacheck interlaboratory exercise organized by the Water Research Center at Medmenham, U.K. Every 2-3 months, certified samples of groundwater containing herbicides (atrazine, simazine, propazine, MCPA, MCPB, mecoprop, chlortoluron, isoproturon, diuron, linuron) were distributed. In Table 5, the results obtained in two of the interlaboratory exercises are reported. The percentage of error of the target values reported by Aquacheck is also indicated. According to the Association of Official Analytical Chemists (AOAC), the generally maximum accepted errors between laboratories is 22%.20 Most of the compounds gave acceptable values according to this accepted error with the exception of isoproturon, which gave a higher value than the real one in both interlaboratory exercises. This phenomenon was also observed in previous work using an on-line methodology with C18 cartridges and APCI/MS detection.12 Since in immunosorbents a high selectivity for compounds is achieved, this result may be related to the affinity for a similar compound in the water sample. Some interferent compound present in the certified standard solution could be the cause of this overestimation in the concentration of isoproturon since blanks of groundwater did not show any interferences. Figure 3 corresponds to the percolation of 20 mL of a groundwater sample spiked with the Aquacheck certified solution through the antichlortoluron immunosorbent. As it can be observed in this figure, all the phenylurea herbicides present in the water sample could be identified and quantified easily. Blanks of the groundwater sample provided by Aquacheck were also analyzed with this method showing no interferences present in the water matrix. We have demonstrated in this work that the coupling of immunosorbents with LC/APCI/MS detection is a powerful technique for the determination and quantitation of polar pesticides in environmental matrixes at the low ppt level without the (20) Mesley, R. J.; Pocklington, W. D.; Walker, R. F. Analyst 1991, 116, 975.
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is well-known that polyclonal antibodies can be produced and prepared in SPE cartridges and this takes approximately one year at research laboratories. Only when industry is involved are the costs reduced due to the production of larger amounts. However, the costs at this stage are high as compared to conventional systems and are difficult to estimate. Also, the half-life of the columns depend on many parameters and can vary from 3-6 months up to 2 years. On the other hand, industry has demonstrated interest in such columns and most probably they will be available at reasonable prices in the near future.
Figure 3. On-line SPE of 20 mL of groundwater sample certified by Aquacheck with a mixture of herbicides through an anti-chlortoluron immunosorbent followed by LC/APCI/MS in PI mode of operation and under SIM conditions. Peaks: (1) monuron; (2) chlortoluron; (3) isoproturon; (4) diuron; (5) linuron.
need of additional cleanup steps. By using a small sample volume, very low detection limits can be reached due to the enhanced selectivity and high sensitivity obtained with this new methodology. Overall, the reported methodology represents a new approach to sample handling in environmental analysis. Some questions remain in regard to costs and availability of immunosorbents. It
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ACKNOWLEDGMENT This work has been supported by the Commission of the European Communities, Environment & Climate Program 199498 (ENV4-CT95-0016 and ENV4-CT97-0384) and CICYT (AMB961600-CE). We thank E. M. Thurman from USGS (Lawrence, Kansas) for detailed discussion on the DAR results. Rosi Alonso and Roser Chaler are gratefully acknowledged for HPLC/MS technical support. Received for review August 5, 1997. Accepted September 1, 1997.X AC970843H X
Abstract published in Advance ACS Abstracts, October 15, 1997.
