Identification and Quantification of 77 Pesticides in Groundwater Using

as styrene-divinylbenzene16 and graphitized carbon black,26 which allow .... The detector was set at the following wavelengths: 225, 245, 254, 280...
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Anal. Chem. 2000, 72, 3093-3101

Identification and Quantification of 77 Pesticides in Groundwater Using Solid Phase Coupled to Liquid-Liquid Microextraction and Reversed-Phase Liquid Chromatography Karel Vandecasteele, Irina Gaus, William Debreuck, and Kristine Walraevens

Laboratory for Applied Geology and Hydrogeology, Ghent University, Krijgslaan 281, 9000 Gent, Belgium

This paper describes a method for the extraction, separation, identification, and quantification of 77 pesticides (neutral, acidic, and basic) including some s-triazine metabolites. The method is appropriate for organically (e.g. with humic acids) highly loaded groundwater samples. A comparative study of a pH-controlled mixed solid phase (LiChroprep RP18/LiChrolut EN) extraction with different desorption solvents (acetonitrile or acetonitrile and dichloromethane/methanol) is elaborated. A subsequent liquidliquid microextraction reduces matrix effects. The pesticides in the sample are separated using RP-HPLC, detected, and identified by diode array. The efficiency is illustrated on a natural groundwater sample from a phreatic aquifer. The contamination of groundwater by pesticides is becoming an increasing problem. Nowadays monitoring of vulnerable aquifers has become an essential aspect of environmental policy. This is related to the carcinogenic and health risks caused by the presence of pesticides in drinking water.1-2 European Community (EC) restrictions with respect to pesticide concentrations in drinking water (EC limit, 0.1 µg/L) therefore are defined near their limit of detection (LOD), indicating their presence is not sustained.3 Multiresidue methods for the measurement of pesticides in groundwater reduce analysis costs and make a quick and total screening of the sample possible.4 Their elaboration has therefore developed into an important research topic in chromatographic applications for the environment.5-8 (1) Merlo, F.; Bolognesi, C.; Reggiardo, C. J. Exp. Clin. Cancer Res. 1994, 13(1), 5-20. (2) Canter, K. Cancer-causes-and-control 1997, 8(3), 292-308. (3) Isenbeck-Schro ¨ter, M.; Bedbur, E.; Kofod, M.; Ko¨nig, B.; Schramm, T.; Matthess, G. Berichte, Fachbereich Geowissenschaften, Universita¨t Bremen, 1997, 91, 1-65. (4) Tekel, J.; KovaC ˇ iC ˇ ova´, J.; Brandsterova, E. J. Chromatogr. 1993, 643, 291303. (5) Di Corcia, A.; Marchetti, M. Environ. Sci. Technol. 1992, 26, 66-77. (6) Huen, J. M.; Gillard, R.; Mayer, A. G.; Baltensperger, B.; Kern H. Fresenius’ J. Anal. Chem. 1994, 348, 606-14. (7) Sennert, S.; Volmer, D.; Levsen, K.; Wu ¨ nsch, G. Fresenius’ J. Anal. Chem. 1995, 351, 642-60. (8) Slobodnı´k, J.; Groenewegen, M.; Brouwer, E.; Lingeman, H.; Brinkman, H. U. J. Chromatogr. 1993, 642, 359-70. 10.1021/ac991359c CCC: $19.00 Published on Web 06/20/2000

© 2000 American Chemical Society

The determination of pesticides, as with many other environmental pollutants, in water requires a sample pretreatment step which includes an analyte enrichment as well as the removal (complete or partial) of matrix compounds.9 An overview of sample preparation and chromatographic separation techniques is given in several reviews.10-13 Solid-phase extraction (SPE) has become the most appropriate technique, and constantly improved materials (both as cartridges or as filter disks) are tested. However, most of the multiresidue methods are applicable for one or similar pesticide groups (e.g., triazines, dinitroanilines, phenoxy acids, phenylureas, etc.). Neutral or basic pesticides are extracted in their natural or in neutral conditions.6,14-17 Acidic pesticides are, in general, extracted after acidification of the sample.6,13-19 Acidification (down to pH 3) of the sample has no influence on the recovery of neutral or basic pesticides.20-22 However, independent from the used SPE material, acidification of natural groundwaters (or other “real world” waters) generally leads to enlarged matrix peaks disturbing adequate identification and quantification.14,20,23 Peak interference and low recovery are attributed to the formation of pesticide-humic complexes that cannot be sufficiently extracted by the sorbents used.24 Indeed, humic acids, having a pKa of 5.5,25 generally are in the molecular form at pH values (such as pH 3.00 or lower) (9) Biziuk, M.; Przyjazny, A.; Czerwinski, J.; Wiergowski, M. J. Chromatogr., A 1996, 754, 103-23. (10) MacCarthy, P.; Klusman, R. W.; Cowling, S. W.; Rice, J. A. Anal. Chem. 1995, 67, 525-82. (11) McGarvey, B. D. J. Chromatogr., A 1993, 642, 89-105. (12) Sherma, J. Anal. Chem. 1993, 65(12), 40-54. (13) Hatrik, Sˇ .; Tekel, J. J. Chromatogr., A 1996, 733, 217-33. (14) Schu ¨ lein, J.; Martens, D.; Spitzauer, P.; Kettrup, A. Fresenius’ J. Anal. Chem. 1995, 352, 565-71. (15) Guenu, S.; Hennion, M.-C. J. Chromatogr., A 1996, 737, 15-24. (16) Pichon, V.; Cau Dit Coumes, C.; Chen, L.; Guenu, S.; Hennion, M.-C. J. Chromatogr., A 1996, 737, 25-33. (17) Dean, J.; Wade, G.; Barnabas, I. J. Chromatogr., A 1996, 733, 295-335. (18) Hodgeson, J.; Collins, J.; Bashe, W. J. Chromatogr., A 1994, 659, 395-401. (19) Heberer, Th.; Butz, S.; Stan, H.-J. Int. J. Environ. Anal. Chem. 1995, 58, 43-53. (20) Marce´, R. M.; Prosen, H.; Crespo, C.; Calull, M.; Borrull, F.; Brinkman, U. A. Th. J. Chromatogr., A 1995, 696, 63-74. (21) Balinova, A. J. Chromatogr. 1993, 643, 203-7. (22) Aquilar, A.; Borrull, F.; Marce´, R. M. J. Chromatogr., A 1996, 754, 77-84. (23) Chiron, S.; Martinez, E.; Barcelo´, D. J. Chromatogr., A 1994, 665, 283-93. (24) Balinova, A. J. Chromatogr., A 1996, 754, 125-35. (25) Wells, M.; Riemer, D.; Wells-Knecht, M. J. Chromatogr., A 1994, 659, 33748.

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which are optimal for the determination of acidic pesticides. Recently developed SPE materials such as styrene-divinylbenzene16 and graphitized carbon black,26 which allow extraction of basic, neutral, and acidic pesticides at neutral pH, only partly solve the problem.16 Also at neutral pH some of these materials seem to lead to a major “hump” in the chromatogram which is correlated to the amount of dissolved organic matter (DOC) in the water sample.27 In some publications acidification is recommended after a prior removal of fulvic and humic acids using liquid membranes or dialyses28 or a post SPE cleanup is proposed.16,27 In this paper, a method is presented for the simultaneous extraction of basic/neutral and acidic/phenolic pesticides (18 triazines, 11 phenylureas, eight phenoxy acids, six carbamates, one acylalanine, one benzimidazole, one oxazolidin, two pyridinecarboxylic acids, one methoxybenzoic acid, one quinolinecarboxylic acid, one pyridazinone, one oxobenzothiazoline, two aryloxy acids, two arylureas, one thiadiazone, three uracils, four anilides, two hydroxybenzonitriles, six azoles, one aryloxyalkanamide, two organophosphoruses, and two phenols) in one run of organically relatively highly loaded groundwater samples. The off-line extraction method consists of two steps. In the first step large amounts (1 L) of the sample are concentrated by a pH-controlled SPE in acidic conditions, using an optimized combination of SPE materials and maximized desorption of the pesticides during elution. The second step consists of a micro-LLE as a cleanup for disturbing organic material. The SPE applied in this method is based on a combination of LiChroprep RP18 and LiChrolut EN. According to Schu¨lein et al.,14 C-18 shows good recoveries with most of the basic/neutral and acidic/phenolic pesticides, but lower recovery rates with compounds such as desisopropylatrazine (DIA). This has been attributed to early breakthrough of polar and watersoluble analytes.15,29 LiChrolut EN has the best retention properties for s-triazines in humic-containing waters30 and quantitative recovery of polar components (such as desisopropylatrazine or desethylatrazine (DEA)).31 The acidification leads to an increase in matrix effects, as expected. To reduce the influence of coeluted humic and fulvic acids after SPE, a subsequent cleanup is carried out. This cleanup consists of a pH-controlled liquid-liquid microextraction (micro-LLE). It leads to a highly reduced matrix with an almost complete pesticide recovery. Micro-LLE executed after SPE eliminates most of the disadvantages of normal LLE as cited in the literature9,32-33 and can also easily be automated.32 Although this method has the disadvantage of including two extraction steps, it is unique in the fact that a wide variety of pesticides and metabolites can be extracted and analyzed in one run. For other published multiresidue analyses of pesticides,5 elution and HPLC analysis had to be performed separately for basic/neutral and for acidic pesticides. The present method is also able to cope with (26) Slobodnı´k, J.; O ¨ ztezkizan, O ¨ .; Lingeman, H.; Brinkman, U. A. Th. J. Chromatogr., A 1996, 750, 227-38. (27) Dupas, S.; Scribe, P.; Dubernet, J. F. J. Chromatogr., A 1996, 737, 117-26. (28) Van De Merbel, N. C.; Lagerwerf, F. M.; Lingeman, H.; Brinkman, U. A. Th. Int. J. Environ. Anal. Chem. 1994, 54, 105-18. (29) Meyer, M. T.; Mills, M. S.; Thurman, E. M. J. Chromatogr. 1993, 629, 559. (30) O ¨ nnerfjord, P.; Barcelo´, D.; Emne´us, J.; Gorton, L.; Marko-Varga, G. J. Chromatogr., A 1996, 737, 35-45. (31) Junker-Buchheit, A.; Witzenbacher, M. J. Chromatogr., A 1996, 737, 6774. (32) van der Hoff, G. R.; Baumann, R. A.; Brinkman, U. A. T.; van Zoonen, P. J. Chromatogr., A 1993, 644, 367-73.

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the interference of organic material, which may reach high concentrations in natural groundwater. Analysis is carried out with reversed-phase high-performance liquid chromatography (RP-HPLC) and diode array detection (DAD). This is the most appropriate analytical tool suited for nonvolatile, thermally labile, and polar (ionic) compounds which are not amenable to gas chromatography (GC).21 The combination of the extraction and the analysis method allows the screening of groundwater samples for a considerable amount of pesticides at the nanogram per liter level.

EXPERIMENTAL SECTION Reagents and Chemicals. HPLC-grade water was purchased from J. T. Baker (Deventer, The Netherlands), acetonitrile was from Lab-scan (Super gradient, Dublin, Ireland), dichloromethane SupraSolv and phosphoric acid p.a. were from Merck (Darmstadt, Germany), and the various pesticide standards were supplied by Riedel-de Hae¨n (Seelze, Germany). The SPE materials LiChrolut EN (40-120 µm) and LiChroprep RP-18 (25-40 µm) were provided by Merck (Darmstadt, Germany). Stock standard solutions of selected solutes were prepared by weighing and dissolving them in water/acetonitrile (3/1; v/v). The standard solutions were stored at 6-8 °C. No change in the chromatogram of the standard solutions was observed during 4 months of study. Humic acids, to prepare the organically loaded test samples, were obtained from Fluka (Buchs, Switzerland). Solid-Phase Extraction (SPE) Procedure. Two SPE methods (SPE1 and SPE2) were tested. For calculating the recovery rate, 1 L of deionized water was spiked with a pesticide standard (1.4-4 µg) and adjusted to pH 3.00 (using phosphoric acid). A glass filter (i.d., 20 mm) packed with 1 g of solid-phase material (LiChroprep RP-18 and LiChrolut EN (3/1)) was conditioned with 10 mL of acetonitrile and 10 mL of water (pH adjusted to pH 3.00). The sample was adsorbed on the SPE material using a weak vacuum (constant flow rate, 0.3 L/h). Any residual water was removed during vacuum-drying for 30 minutes. For SPE1, batches of (5×) 1.5 mL of acetonitrile were used to perform the elution. For SPE2, the SPE material was at first eluted with (5×) 1.5 mL of acetonitrile; subsequently an elution ((5×) 1.5 mL) with a mixture of methanol/dichloromethane (1:1, v/v) was executed. The eluted fractions (SPE1) or the combined eluted fractions (SPE2) were concentrated at 30 °C with nitrogen until dry. Liquid-Liquid Microextraction (Micro-LLE) Procedure. A volume of 200 µL of pesticide standard solution (1.4-4 µg) (after concentrating at 30 °C with nitrogen until dry) was taken in 1.5 mL of water (pH adjusted with phosphoric acid to pH 3.00) and subsequently extracted with 5× 1.5 mL of dichloromethane. The dichloromethane fractions were combined and concentrated at 30 °C with nitrogen until dry. HPLC Analysis. A Liquid Chromatograph (HP 1090 series II) equipped with a computer-operated (Chemstation HP 9000 series 300) photodiode array (HP 1040 A) High-Speed Spectrophotometric detector was used. The pre- and analytical LiChroCART column system (4 × 4 mm; 250 × 4 mm) packed with LiChrospher 60 RP-select B (5 µm) was obtained from Merck (33) Mukherjee, M.; Gopal, M. J. Chromatogr., A 1996, 754, 33-42.

Figure 1. RP-HPLC separation of a standard of 39 pesticides and some s-triazine metabolites (among them a majority of herbicides; for details of chromatographic protocol and peak identification see the experimental part). 1. desisopropyldesethylatrazine: 3.06 min; 2. desisopropylatrazine: 7.72 min; 3. quinmerac: 8.83 min; 4. metamitron: 9.74 min; 5. chloridazon: 10.69 min; 6. benazolin: 11.55 min; 7. carbendazim: 12.50 min; 8. 4-CPA: 13.29 min; 9. fluroxypyr: 13.96 min; 10. hexazinone: 14.51 min; 11. bromacil: 14.97 min; 12. terbacil: 15.48 min; 13. simazine: 16.13 min; 14. 2,4-D: 16.98 min; 15. MCPA: 18.30 min; 16. metabenzthiazuron: 19.12 min; 17. trichlopyr: 19.86 min; 18. bromoxynil: 20.77 min; 19. atrazine: 21.33 min; 20. monolinuron: 21.71 min; 21. isoproturon: 22.55 min; 22. diuron: 23.20 min; 23. mecoprop: 23.85 min; 24. metazachlor: 24.84 min; 25. ioxynil: 26.61 min; 26. sebuthylazine: 27.92 min; 27. triadimenol: 29.71 min; 28. 2,4-DB: 30.91 min; 29. terbuthylazine: 32.31 min; 30. phendimedipham 34.25 min; 31. terbumeton: 36.99 min; 32. chlorpropham: 41.24 min; 33. napropamide: 43.11 min; 34. metolachlor: 43.93 min; 35. alachlor: 45.14 min; 36. terbutryn: 48.27 min; 37. bitertanol (1RS, 2SR): 48.90 min; 38. bitertanol (1RS, 2RS): 49.49 min; 39. dinoseb: 50.29 min.

(Darmstadt, Germany). The separation runs were executed at constant oven temperature (35 °C) and a constant flow rate of 0.9 mL/min. The detector was set at the following wavelengths: 225, 245, 254, 280, and 335 nm (optical bandwidth, 4 nm) with a reference signal of 480 nm (optical bandwidth, 60 nm). A linear gradient profile was established using three eluents: solvent A (0.015% phosphoric acid in water), solvent B (water), and solvent C (acetonitrile). The gradient range contained the following steps. 0-1 min: isocratic 18% C + 67% B in A. 1-5 min: gradient from 18% C + 67% B in A to 25% C + 65% B in A. 5-11 min: gradient from 25% C + 65% B in A to 36.5% C + 53.5% B in A. 11-34 min: gradient from 36.5% C + 53.5% B in A to 38% C + 52% B in A. 34-44 min: gradient from 38% C + 52% B in A to 43% C + 52% B

in A. 44-52 min: gradient from 43% C + 52% B in A to 95% C + 0% B in A. 52-53 min: isocratic 95% C + 0% B in A. 53-57 min: gradient from 95% C + 0% B in A to 18% C + 67% B in A. 57-60 min: isocratic 18% C + 67% B in A. An automatic injection system injected 40 µL of the pesticide stock solution or the extracts. For the recovery calculations of the groundwater samples the dried extracts (from SPE, micro-LLE, or SPE + micro-LLE) were taken in 200 µL of acetonitrile/water (1:3, v/v) and filtered using a membrane 3-mm syringe filter (0.2-µm nylon) (Anglo-Euro Scientific, Nottingham, U.K.) before injection of 40 µL. Groundwater Samples. Samples (1 L) were taken from piezometers using a peristaltic pump. Extraction took place immediately after sampling. Prior to the extraction, the sample Analytical Chemistry, Vol. 72, No. 14, July 15, 2000

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Table 1. Characteristics of the Pesticides (Their Retention Time (Tr (min)), Wavelength Used for Quantification (λ Select (nm)), Pesticide Class, Group, and pKa Value Specified in the Literature) compound

Tr (min)

λ select (nm)a

pesticide classb

groupc

desethyldesisopropylatrazine clopyralid picloran desisopropylatrazine dicamba quinmerac metamitron chloridazon fenuron desethylatrazine benazolin carbendazim 4-CPA 4-nitrophenol fluroxypyr tebuthiuron hexazinone bentazone metoxuron bromacil oxadixyl terbacil metribuzin simazine monuron 2,4-D propoxur lenacil carbofuran MCPA pirimicarb metabenzthiazuron trichlopyr chlortoluron metalaxyl bromoxynil atrazine fluometuron fluatriafol 2,4,5-T monolinuron dichlorprop isoproturon diuron mecoprop paraoxon-ethyl desmetryn metazachlor ioxynil sebuthylazine propazine propanil triadimenol 2,4-DB MCPB prometon terbuthylazine linuron ametryn phenmedipham terbumeton chloroxuron trietazine chlorpropham triadimefon napropamide metolachlor prometryn alachlor hexaconazole terbutryn barban bitertanol (1RS,2SR) bitertanol (1RS,2RS) neburon dinoseb parathion-ethyl

3.06 3.06 3.50 7.72 8.11 8.83 9.74 10.69 10.70 10.82 11.55 12.50 13.29 13.67 13.96 14.12 14.51 14.75 14.88 14.97 15.29 15.48 15.86 16.13 16.26 16.98 17.32 17.32 17.96 18.30 18.94 19.12 19.86 20.30 20.77 20.77 21.33 21.41 21.48 21.70 21.71 22.26 22.55 23.20 23.85 23.93 24.02 24.84 26.61 27.92 28.42 28.89 29.71 30.91 31.82 32.07 32.31 33.12 33.36 34.25 36.99 39.96 40.96 41.24 41.41 43.11 43.93 44.07 45.14 47.07 48.27 48.65 48.90 49.49 50.11 50.29 51.39

225 225 225 225 225 225 225 225 245 225 225 225 225 335 225 254 245 225 245 280 225 280 225 225 245 225 225 225 225 225 245 225 225 245 225 225 225 245 225 225 245 225 245 254 225 280 225 225 225 225 225 254 225 225 225 225 225 245 225 245 225 245 225 245 225 225 225 225 225 225 225 245 254 254 280 280 280

triazine (Me) pyridinecarboxylic acid (H) pyridinecarboxylic acid (H) triazine (Me) methoxybenzoic acid (H) quinolinecarboxylic acid (H) triazine (H) pyridazinone (H) phenylurea (H) triazine (Me) oxobenzothiazolin (H) benzimidazole (F) phenoxy acid (Pl) phenol (Me) aryloxy acid (H) arylurea (H) triazine (H) thiadiazinone (H) phenylurea (H) uracil (H) oxazolidin (F) uracil (H) triazine (H) triazine (H) phenylurea (H) phenoxy acid (H) carbamate (I) uracil (H) carbamate (I) phenoxy acid (H) carbamate (I) arylurea (H) aryloxy acid (H) phenylurea (H) acylalanine (F) hydroxybenzonitrile (H) triazine (H) phenylurea (H) azole (F) phenoxy acid (H) phenylurea (H) phenoxy acid (H) phenylurea (H) phenylurea (H) phenoxy acid (H) organophosphorus (Me; I) triazine (H) chloroacetanilide (H) hydroxybenzonitrile (H) triazine (H) triazine (H) anilide (H) azole (F) phenoxy acid (H) phenoxy acid (H) triazine (H) triazine (H) phenylurea (H) triazine (H) bis-carbamate triazine (H) phenylurea (H) triazine (H) carbamate (Pl) azole (F) aryloxyalkanamide (H) chloroacetanilide (H) triazine (H) chloroacetanilide (H) azole (F) triazine (H) carbamate (H) azole (F) azole (F) phenylurea (H) phenol (H) organophosphorus (I)

b/n a/ph a/ph b/n a/ph a/ph b/n b/n b/n b/n a/ph b/n a/ph a/ph a/ph b/n b/n a/ph b/n b/n b/n b/n b/n b/n b/n a/ph b/n b/n b/n a/ph b/n b/n a/ph b/n b/n a/ph b/n b/n b/n a/ph b/n a/ph b/n b/n a/ph b/n b/n b/n a/ph b/n b/n b/n b/n a/ph a/ph b/n b/n b/n b/n b/n b/n b/n b/n b/n b/n b/n b/n b/n b/n b/n b/n b/n b/n b/n b/n a/ph b/n

pKa34-37 2.3 2.3 1.3-1.58 1.87 4.31

1.3-1.65 3.04

2.94 3.3

1.0 1.65-1.8 2.64 3.7 3.07

3.86 1.68-1.85 2.85 3.0 3.78 3.1-3.93 3.96 1.1-1.85 4.8 4.84 1.94-2.0 4.1 4.6

4.3-4.4

4.62

a Key: quantification is performed at one of the four preselected wavelengths, which corresponds to maximum absorbance. b Key: F, fungicide; H, herbicide; Me, metabolite; I, insecticide; Pl, plant growth regulator. c Key: b/n, basic/neutral; a/ph, acidic/phenolic pesticides.

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Figure 2. DAD spectra for hexazinone, bentazone, metoxuron and bromacil.

was filtered on a glassfiber filter (type A/E, 47 mm) from Gelman (Ann Arbor, Michigan) to eliminate particles. After SPE, the dried extract was taken in 1.5 mL of water (pH adjusted with phosphoric acid to pH 3.00) and submitted to micro-LLE before analysis. DISCUSSION AND RESULTS Separation. The separation of 39 pesticides including some s-triazine metabolites is shown in Figure 1. During the 53 min of the RP-HPLC analysis, several basic/neutral and acidic/phenolic pesticides were separated as narrow and symmetrical peaks. The pesticide group, class, retention time (Tr), wavelength for quantification (λ), and pKa value34-37 of 77 investigated pesticides are indicated in Table 1. Identification of the different pesticides was based on their retention time in combination with their UV spectrum. Quantification took place on the maximum absorbance of the preselected wavelengths (225, 245, 254, 280, or 335 nm). Pesticides with almost identical retention times form critical groups. The peaks for these pesticides coincide on the chromatogram. However, they can be identified by fundamental differences in their UV spectra. This is illustrated in Figure 2 for pesticides with retention times between 14.51 min (hexazinone) and 14.97 (34) Tomlin, C., Ed. The Pesticide Manual; Crop Protection Publications; The British Crop Protection Council and The Royal Society of Chemistry: London, U.K., 1994. (35) Hornsby, A. G.; Wauchope, R. D.; Herner, A. E. Pesticide Properties in the Environment; Springer Publishing: New York, 1996. (36) Schmitt, Ph.; Garrison, A. W.; Freitag, D.; Kettrup, A. J. Chromatogr., A 1996, 723, 169-77. (37) Paca´kova´, V.; Sˇ tulik, K.; Jistra, J. J. Chromatogr., A 1996, 754, 17-31.

min (bromacil) (Table 1). In this case, retention times, in combination with UV spectra, allow a unique identification of each compound. The chance that pesticides with the same retention time are present in the same water sample is rather small. Solid-Phase Extraction. Initial experiments (not described) on groundwater samples taken at different locations indicate that the organic content in the samples often clogs the available prepacked cartridges. To make this method applicable to a wide variety of groundwater samples with large differences in organic content, a self-made glass filter was used and the SPE material was packed in these filters. Because of the larger filter diameter, clogging was minimized. Since it was expected that, during the extraction of samples with high organic content, the capacity of the SPE material was influenced by the adsorption of humic and fulvic acids to the SPE material, a sufficient amount (1 g) of SPE material was used to pack the columns. This avoided early breakthrough of the pesticides. Comparison of the Recoveries for SPE1 and SPE2. In terms of recovery (Rec) and relative standard deviation (RSD), the efficiency of the SPE1 procedure (SPE using LiChrolut EN/ LiChroprep RP 18, elution with acetonitrile) was compared with that of the SPE2 procedure (SPE using LiChrolut EN/LiChroprep RP 18, elution with acetonitrile, and additional elution with dichloromethane/methanol). Data are reported in Table 2. Since SPE2 differs from SPE1 only in an extra elution step, recoveries for SPE2 must be higher or equal to those of SPE1. This also implies that increased recoveries using SPE2 are the result of a Analytical Chemistry, Vol. 72, No. 14, July 15, 2000

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Table 2. Recoveries of the Basic/Neutral and Acidic/Phenolic Pesticides (Rec, %), Relative Standard Deviations (RSD, %), Quantification Limit 1 (LOQ, µg/L) Obtained by Off-Line Preconcentration SPE(1) + Micro-LLE, Quantification Limit 2 (LOQ, µg/L) Obtained by Off-line Preconcentration SPE(2) + Micro-LLE (both of 1000 mL of Spiked Water) (n ) 5), Limit of Detection (LOD, nanograms per RP-HPLC Analysis)a SPE1

SPE2

SPE1 + micro-LLE

micro-LLE

SPE2 + micro-LLE

compound

Rec

RSD

Rec

RSD

Rec

RSD

Rec

RSD

LOQ (µg/L)

Rec

RSD

LOQ (µg/L)

LOD (ng)

desethyldesisopropylatrazine clopyralid picloram desisopropylatrazine dicamba quinmerac metamitron chloridazon fenuron desethylatrazine benazolin carbendazim 4-CPA 4-nitrophenol fluroxypyr tebuthiuron hexazinone bentazone metoxuron bromacil oxadixyl terbacil metribuzin simazine monuron 2,4-D propoxur lenacil carbofuran MCPA pirimicarb metabenzthiazuron trichlopyr chlortoluron metalaxyl bromoxynil atrazine fluometuron fluatriafol 2,4,5-T monolinuron dichlorprop isoproturon diuron mecoprop paraoxon-ethyl desmetryn metazachlor ioxynil sebuthylazine propazine propanil triadimenol 2,4-DB MCPB prometon terbuthylazine linuron ametryn phenmedipham terbumeton chloroxuron trietazine chlorpropham triadimefon napropamide metolachlor prometryn alachlor hexaconazole terbutryn barban bitertanol (1RS,2SR) bitertanol (1RS,2RS) neburon dinoseb parathion-ethyl

17 31 70 83 65 10 56 88 100 86 53 3 54 87 81 101 94 102 101 98 103 102 38 103 99 78 82 97 94 76 82 62 61 90 99 82 99 96 87 18 89 55 97 83 79 95 0 99 78 46 81 84 82 59 84 0 39 93 9 104 0 61 79 60 85 70 75 0 74 16 0 51 67 70 46 41 53

7 4 9 4 4 4 4 8 1 3 3 1 5 2 1 2 3 6 1 0 2 1 3 3 2 8 7 4 1 4 7 9 2 4 1 5 5 2 4 6 4 2 2 7 7 3

40 90 108 87 91 85 81 99 100 98 91 97 85 89 94 101 101 101 103 100 101 102 38 96 100 83 91 101 94 85 100 100 87 96 97 82 96 93 100 69 81 79 98 99 87 99 93 104 78 90 83 86 96 84 90 98 81 94 85 99 94 76 79 56 87 68 77 82 74 74 77 62 83 81 59 37 63

3 3 4 1 2 2 3 2 2 3 2 3 2 3 1 2 2 5 2 2 4 1 2 2 2 3 3 1 1 5 1 1 5 2 1 2 1 2 4 5 5 4 2 1 2 3 4 3 5 6 5 4 3 6 3 2 6 2 4 2 3 2 3 8 2 4 10 4 10 5 6 3 8 11 2 9 2

19 65 101 100 91 92 85 101 100 95 97 63 96 92 98 97 99 100 102 99 95 101 93 103 95 95 83 95 70 92 68 101 93 96 88 68 97 98 97 85 81 93 101 97 95 95 94 94 76 96 90 94 96 93 90 90 93 97 92 97 98 84 66 60 86 95 90 84 88 84 91 67 94 93 75 50 71

3 3 3 3 1 2 2 2 1 4 0 4 1 6 1 3 1 6 1 0 4 0 1 4 3 2 4 3 8 13 8 1 2 2 3 3 6 1 4 6 2 1 2 1 9 1 4 1 6 1 2 2 2 2 8 3 1 1 5 3 1 2 3 3 4 1 2 3 4 3 2 5 2 2 4 4 5

3 20 71 83 60 9 47 89 101 81 52 2 52 80 80 98 92 102 103 97 99 103 35 106 94 74 68 92 66 70 56 63 57 87 87 56 96 94 84 15 73 51 98 80 75 90 0 93 59 45 73 79 79 55 76 0 36 91 9 101 0 51 52 37 73 66 67 0 65 14 0 34 63 66 35 20 38

2 4 11 6 4 4 5 9 2 6 3 0 5 7 2 5 4 12 1 1 6 2 3 7 4 9 9 7 8 14 11 9 3 5 4 6 11 2 8 7 5 3 4 8 14 4

4.43 0.50 0.07 0.06 0.16 0.39 0.26 0.05 0.06 0.06 0.08 2.42 0.15 0.10 0.10 0.10 0.09 0.05 0.07 0.20 0.22 0.15 0.31 0.03 0.05 0.18 0.33 0.27 0.30 0.17 0.12 0.06 0.27 0.06 0.32 0.09 0.04 0.07 0.71 0.80 0.07 0.26 0.06 0.09 0.16 0.20

5 11 13 6 7 5 6 8

0.15 0.36 0.07 0.05 0.08 0.16 0.26 0.18

13 2 6 5

0.10 0.07 0.39 0.05

7 5 7 6 11 10

0.25 0.06 0.24 0.22 0.06 0.25

15 2

0.28 1.29

7 8 8 7 7 11

0.34 0.12 0.11 0.24 0.58 0.55

8 59 109 87 83 78 69 100 100 93 89 61 82 82 92 98 100 101 105 99 96 103 35 99 95 79 76 96 66 78 68 101 81 92 85 56 93 92 97 58 65 74 99 96 83 94 88 97 59 86 74 81 92 78 81 89 75 91 78 96 92 64 52 34 75 65 69 68 65 63 70 42 78 76 44 19 45

1 5 7 4 3 3 4 4 3 7 2 6 3 7 2 5 3 11 2 2 8 1 2 6 5 5 5 4 8 15 8 2 6 4 5 4 7 2 8 9 6 4 5 2 10 4 7 4 8 7 6 5 4 7 10 5 7 3 8 5 4 4 4 6 6 5 11 7 12 6 7 5 9 12 4 6 4

1.66 0.17 0.05 0.06 0.11 0.04 0.17 0.04 0.06 0.05 0.04 0.08 0.10 0.10 0.09 0.10 0.08 0.05 0.07 0.20 0.22 0.15 0.31 0.04 0.05 0.17 0.30 0.26 0.30 0.15 0.10 0.04 0.19 0.06 0.33 0.09 0.04 0.07 0.61 0.21 0.08 0.18 0.06 0.07 0.14 0.19 0.11 0.14 0.36 0.03 0.05 0.07 0.14 0.19 0.17 0.06 0.05 0.07 0.04 0.06 0.05 0.20 0.06 0.26 0.21 0.06 0.25 0.04 0.28 0.29 0.05 0.27 0.10 0.10 0.19 0.61 0.46

27 20 10 10 19 7 24 9 12 9 8 10 16 16 16 20 16 11 14 39 43 31 22 7 9 27 45 50 39 24 13 8 31 11 56 10 7 13 119 24 10 27 11 14 24 35 19 27 43 6 8 12 25 29 27 11 7 12 7 11 10 26 6 18 32 8 34 6 37 36 7 23 15 15 17 23 41

a

4 8 13 6 5 4 5 1 13 2 6 2 7 5 8 4 11 10 13 2 6 7 7 7 10 12

For the description of SPE1, SPE2 and micro-LLE see experimental part.

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Table 3. Structure and Retention Time of the Selected s-Triazines

S-TRIAZINES pesticide

Tr (min)

R1

R2

R3

desethyldesisopropylatrazine desisopropylatrazine desethylatrazine simazine atrazine sebuthylazine propazine terbuthylazine desmetryn ametryn prometryn terbutryn prometon terbumeton

3.06 7.72 10.82 16.13 21.33 27.92 28.42 32.31 24.02 33.36 44.07 48.27 32.07 36.99

Cl Cl Cl Cl Cl Cl Cl Cl SCH3 SCH3 SCH3 SCH3 OCH3 OCH3

H C2H5 H C2H5 C2H5 C2H5 CH(CH3)2 C2H5 CH3 C2H5 CH(CH3)2 C2H5 CH(CH3)2 C2H5

H H CH(CH3)2 C2H5 CH(CH3)2 CH(CH3)(C2H5) CH(CH3)2 C(CH3)3 CH(CH3)2 CH(CH3)2 CH(CH3)2 C(CH3)3 CH(CH3)2 C(CH3)3

Chart 1

more complete desorption of the pesticides from the SPE material due to the optimized choice of elution solvents. Recoveries are not dependent on the additional elution step with dichloromethane/methanol for the tested uracils, phenylureas, anilides, and phenolic compounds. This is also the case for the 6-chloro-substituted s-triazines, with the exception of sebuthylazine and terbuthylazine. The R3-substituted group, which is a branched C4-chain for both sebuthylazine and terbuthylazine, increases the sorptive strength of these pesticides to the SPE material, rendering desorption in an elution solvent such as acetonitrile incomplete. The higher selectivity of the methanol/ dichloromethane mixture increases desorption and therefore the recoveries of these compounds. The same effect is observed for the 6-methylthio and 6-methoxy s-triazines (desmetryn, ametryn, prometryn, terbutryn, prometon, terbumeton). Recoveries are very low or zero if elution is only executed with acetonitrile according to SPE1. Consecutive elution with methanol/dichloromethane (SPE2) increases recovery from 0 up to 98% in the case of prometon. Recovery improvement for the other compounds is comparable. Since substituted groups on the structure (Table 3) influence pKa values of the pesticides, a relation between pKa and recoveries

obtained from SPE1 can be established for the s-triazines (Chart 1). Additional elution of the SPE material with methanol/dichloromethane after elution with acetonitrile eliminates this selective desorption of s-triazines obtained with elution by acetonitrile only. Recoveries are low for some phenoxy acids (2,4,5-T; 2,4-DB; 4-CPA; MCPA) if SPE1 is used. Recovery improves remarkably when SPE2 is applied. Increasing recovery reduces the limit of quantification (LOQ) for each compound (Table 2). For SPE2, the LOQ for 41 of 77 pesticides meets the EC limit of 0.1 µg/L, while only seven pesticides have quantification limits higher than 0.3 µg/L. In comparison, using SPE1, only 30 of 77 pesticides can be quantified in a concentration equal to or lower than 0.1 µg/L, while the concentration must be higher than 0.3 µg/L to determine 20 of the studied pesticides. Liquid-Liquid Microextraction. Especially for groundwater samples, matrix effects on the RP-HPLC chromatogram, after the described SPE procedure, are considerable, as a result of the generally elevated amounts of dissolved organic matter in natural groundwater. The effect of the matrix interferences due to humic acids on the RP-HPLC chromatogram is shown in Figure 3 for Analytical Chemistry, Vol. 72, No. 14, July 15, 2000

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Figure 3. Comparison of matrix interference in the RP-HPLC chromatograms between the injection of the extract of 20 mL of deionized water spiked with 10 mg/L of humic acids after solid-phase extraction (SPE2) (A) and 200 mL after solid-phase extraction (SPE2) and subsequent liquid-liquid microextraction (B). Sample volume was chosen 10× lower for (A) than for (B) because of important matrix interferences in (A).

Figure 4. RP-HPLC chromatogram of the extract of the groundwater sample (200 mL per HPLC injection; sample taken from a source from the Ledo-Paniselian aquifer in Flanders, Belgium) with identification and quantification of the detected pesticides (desethylatrazine, 0.38 µg/L; bentazone, 0.38 µg/L; atrazine, 0.29 µg/L). The other peaks appearing in the chromatogram could be not identified.

the extract of 20 mL of deionized water spiked with 10 mg/L of humic acids after acidification and SPE (line A). This humic acid concentration equals the concentrations observed in groundwater.38 Because of the matrix interferences after acidification of the sample, the extract of 20 mL was the maximum volume which could be analyzed. A major hump appears in the first 20 min of the chromatogram, rendering the identification of the pesticides insecure and quantification inaccurate.14,20,23 In comparison, line B (Figure 3) shows the chromatogram corresponding to the preconcentration of 200 mL of deionized water spiked with 10 (38) Matthess, G. Die Beschaffenheit des Grundwassers, Gebru ¨ der; Borntraeger: Berlin, Germany, 1994.

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mg/L of humic acids after acidification, SPE, and subsequent cleanup using micro-LLE with dichloromethane. The baseline is flat, and a lower attenuation range of the DAD can be used. This lowers the limit of detection and quantification. It is to be noted that the preconcentration volume leading to chromatogram B was 10 times larger than that for chromatogram A. In reality, the difference in matrix interference for the two chromatograms is therefore 10 times higher than shown in Figure 3. High recovery using dichloromethane for LLE was described for many different pesticide groups,9,24 such as phenylureas,13 triazines,13,17 and phenoxy acids.13 Micro-LLE with dichloromethane even has higher recoveries as indicated in Table 2, which

shows recoveries and relative standard deviations (RSD) of the analyzed pesticides. For 70% of the pesticides, recoveries are >90%, for 83%, recoveries exceed 80%. RSD values are small (70% of the pesticides have an RSD of 3% or less); only phenoxy acids (MCPA, MCPB, mecoprop) have somewhat higher RSD values. A high volume ratio (1/1) between both phases (aqueous phase/dichloromethane) in micro-LLE improves recoveries in comparison with those of classical LLE. Solvent volumes are reduced to a minimum (5 × 1.5 mL), thereby overcoming one of the main disadvantages of classical LLE. Analysis and Identification of a Groundwater Sample. The RP-HPLC chromatogram of the extract of a groundwater sample is indicated in Figure 4. It was taken from a source of the LedoPaniselian aquifer in Flanders, supplying drinking water in the area. Despite the relatively high amount of total dissolved solids in the sample (DOC, 7.8 mg/L), matrix effects on the chromatogram are low due to the cleanup by micro-LLE. Two pesticides and one s-triazine metabolite, in a concentration exceeding the EC-limit, could be identified on the basis of retention time and corresponding UV spectrum: desethylatrazine (0.38 µg/L), bentazone (0.38 µg/L), and atrazine (0.29 µg/L). For all three compounds, the wavelength used for quantification is 225 nm (cfr. Table 1). The other peaks appearing in the chromatogram (Figure 4) could not be identified. CONCLUSIONS A method for the quantification of 77 pesticides, extracted by SPE and subsequently cleaned up with micro-LLE, and analysis

using RP-HPLC with DAD is developed. An optimized choice of SPE extraction materials combines low costs and the extraction of more polar pesticides after acidification of the sample. By using different elution solvents to improve desorption of the pesticides from the SPE material, it is demonstrated that a low recovery is not always the result of a breakthrough of the pesticides during the adsorption on the SPE material, but can be caused by incomplete desorption during elution. Micro-LLE seems to be extremely useful as a cleanup after SPE on acidified samples due to its combination of high pesticide recoveries and selectivity with respect to humic acids. The applicability of the method was clearly illustrated on a real world groundwater sample, indicating that an extensive screening, in one run, of groundwater samples relatively highly loaded organically is possible. The potential of the method lies in the fact that, in one extraction and one RP-HPLC analysis, a large variety of basic/ neutral and acidic/phenolic pesticides can be determined and quantified with an acceptable limit of detection. ACKNOWLEDGMENT The authors wish to express their gratitude to Ghent University as well as to the Fund of Scientific Research (Flanders, Belgium) for providing the financial support necessary to complete this research. Received for review November 24, 1999. Accepted April 13, 2000. AC991359C

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