Double-Disk Solid-Phase Extraction: Simultaneous Cleanup and

Cleanup and Trace Enrichment of Herbicides and. Metabolites from ... extraction disks, a method called double-disk solid-phase extraction. The first d...
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Anal. Chem. 1999, 71, 1009-1015

Double-Disk Solid-Phase Extraction: Simultaneous Cleanup and Trace Enrichment of Herbicides and Metabolites from Environmental Samples Imma Ferrer,† Damia` Barcelo´,*,† and E. M. Thurman‡

Department of Environmental Chemistry, CID-CSIC c/ Jordi Girona, 18-26, 08034 Barcelona, Spain, and U.S. Geological Survey, 4821 Quail Crest Place, Lawrence, Kansas 66049

In 1990, the Empore disk was introduced as a new method in sample preparation for isolation of organic contaminants from water and other aqueous samples.1-3 Empore disks consist of 8-10-µm octyldecyl silica particles embedded in a matrix of Teflon, where approximately 10% of the disk by weight is Teflon. The remainder of the disk is the C18 particle. Initially, the disk was 47 mm in diameter and contained 500 mg of packing material. The Empore disk was used for the rapid isolation of organic contami-

nants from a 1-L water sample because 1 L of water can be passed through the disk in less than 15 min with effective recovery of many pesticides.1 In 1994, other chemistries of the solid phase were incorporated into disks including styrene divinylbenzene (SDB), cation exchange, and anion exchange, thus permitting the effective isolation of phenolic and other polar analytes.4 C18 cartridges have been used extensively over the past decade for the isolation of many types of organic contaminants from water.5,6 However, there have been few applications of anionexchange solid-phase extraction (SPE) cartridges for the isolation of organic compounds. A major reason is that anion-exchange SPE isolates organic compounds by the anion-exchange mechanism; therefore, the molecule must contain an anion-exchange site. Second, inorganic substances present in the water samples also compete for the anion-exchange site and will lower the recovery of organic substances from the water sample. Third, the rate of anion exchange on 40-60-µm particles is considerably lower than the rate of adsorption of organic molecules onto a C18 adsorbent, suggesting that the analytes could be inefficiently extracted. For these reasons, there have been limited applications of the use of anion-exchange cartridges for the isolation of organic substances from water. Since the introduction of the Empore strong anion-exchange (SAX) disks in 1994, there have been several applications for the isolation of negatively charged pesticide degradation products on the disks.7-9 In these studies, the capacity of the SAX disk was sufficient to isolate the negatively charged metabolites of the pesticide dacthal from groundwater. The disk was found to have a high capacity for the doubly charged dacthal metabolites and a rapid rate of adsorption with the 8-10-µm particle size. Apparently, the SAX disk had a much lower selectivity for inorganic ions in the groundwater sample; thus, the Empore SAX disk was an effective solid-phase adsorbent for negatively charged species that contained carboxylic groups, which showed that the SAX disk could be an effective sorbent for the isolation of traces of anionic organic contaminants in water samples.

* To whom correspondence should be addressed (e-mail dbcqam@ cid.csic.es). (1) Hagen, D. F.; Markell, C. G.; Schmitt, G. A.; Blevins, D. Anal. Chim. Acta 1990, 236, 157-164. (2) Barcelo´, D.; Durand, G.; Bouvot, V.; Nielen, M. Environ. Sci. Technol. 1993, 27, 271-277. (3) Barcelo´, D.; Hennion, M.-C. Trace Determination of Pesticides and their Degradation products in Water; Elsevier: Amsterdam, 1997; Vol. 19.

(4) Puig, D.; Barcelo´, D.; Silgoner, I.; Grasserbauer, M. J. Mass Spectrom. 1996, 31, 1297-1307. (5) Thurman, E. M.; Mills, M. S. Solid-Phase Extraction: Principles and Practice; John Wiley & Sons: New York, 1998. (6) Ferrer, I.; Thurman, E. M.; Barcelo´, D. Anal. Chem. 1997, 69, 4547-4553. (7) Field, J. A.; Monohan, J. J. Chromatogr. A 1996, 741, 85-90. (8) Field, J. A.; Monohan, J. Anal. Chem. 1995, 67, 3357-3362. (9) Field, J. A.; Monohan, J.; Reed, R. Anal. Chem. 1998, 70, 1956-1962.

Phenylurea and triazine herbicides, including some metabolites, were isolated from water and soil extracts by solid-phase extraction using a layered system of two extraction disks, a method called double-disk solid-phase extraction. The first disk consisted of strong anion exchange (SAX) of 10-µm styrene divinylbenzene (SDB) particles embedded in Teflon, and the second disk was a C18 disk of 10-µm particles also embedded in Teflon. A volume of 500 mL of water or aqueous soil extract is passed through the layered system with the SAX disk first. The purpose of the SAX disk is to remove the humic and fulvic acids from the water or aqueous soil extract by ion exchange through their carboxyl groups. Even during methanol elution of herbicides, the humic substances remain bound to the SAX disk with >85% retention. Elution with methanol results in more than 90% recovery of the herbicides from the layered extraction disks. Removal of the humic and fulvic acids results in greater sensitivity for diode array detection quantitation (0.05 µg/L for herbicides) by substantially reducing the absorbance of the humic peak on the LC chromatogram. The herbicides adsorb to the SAX disk either through hydrogen bonding to the anion-exchange sites or by hydrophobic interaction with the SDB surface of the anion-exchange disk. The method was tested for the analysis of natural water samples from the Mississippi Embayment, a cottongrowing area of the southeastern United States.

10.1021/ac980975q CCC: $18.00 Published on Web 01/27/1999

© 1999 American Chemical Society

Analytical Chemistry, Vol. 71, No. 5, March 1, 1999 1009

From the work of Field and Monohan,8 we realized that the rapid and effective anion-exchange capacity of the SAX disk could also be used to remove humic substances, which are present in surface and groundwater samples and soil extracts. In this paper, advantage is taken of the high selectivity of the SAX disk for humic substances; thus, these substances can be effectively removed from water samples during trace enrichment of herbicides from water or soil extracts. The concept of layering the disk is used to first remove the humic impurities on the SAX disk with simultaneous isolation of herbicides onto the lower C18 disk. The concept of stacking adsorbents for trace enrichment was first introduced in the early 1980s with XAD adsorbents. In these earlier examples, both anion-exchange and reversed-phase methods were used to isolate both natural and contaminant organic compounds from water.10 More recently, SPE cartridges have been introduced with layered adsorbents.11 Nevertheless, these adsorbents consist of 40-60-µm particles, which may have a lower kinetic adsorption rate and hence lower effective recoveries of pesticides. In this paper, it is shown for the first time that the Empore disks may be stacked for the effective removal of trace humic substances with simultaneous isolation of herbicides. The purpose of this paper is first to demonstrate that humic substances are effectively removed by the SAX disk and are not then eluted with organic solvents used for isolation of herbicides. The second purpose is to determine whether herbicides are isolated not only on the C18 disk (lower adsorbent) but also on the SAX disk. The third purpose is to discuss the various mechanisms of sorption of herbicides on the SAX disk, including strong hydrogen-bonding sites that effectively isolate some herbicides from water samples, and hydrophobic interactions on the styrene divinylbenzene matrix of the ion-exchange sorbent. Finally, this paper shows the first application of reversed-phase isolation of herbicides from water using the double-disk methodology, and the method was tested on natural water samples from the Mississippi Embayment that were thought to contain phenylurea herbicides. EXPERIMENTAL SECTION Chemicals. HPLC grade solvents acetonitrile, methanol, and water were purchased from Merck (Darmstadt, Germany). Pesticide standards [didemethylfluometuron, demethylfluometuron, demethyldiuron, chlorotoluron, fluometuron, isoproturon, diuron, linuron, diflubenzuron, deisopropylatrazine, deethylatrazine, simazine, atrazine, propazine terbuthylazine, and 2,4-dichlorophenoxyacetic acid (2,4-D)] were obtained from ChemService (Westchester, PA). Stock standard solutions of 1000 µg/mL were prepared by weighing the solutes and dissolving them in methanol. A stock solution of 10 µg/mL was used to spike tap water at the 1 µg/L level for preconcentration through the disks and further determination of recoveries. The final standard solutions did not contain more than 0.5% methanol. Chromatographic Conditions. The HPLC analyses of the methanol extracts for phenylurea and triazine herbicides were performed on a Hewlett-Packard model 1090, series II, liquid chromatograph with a photodiode-array (PDA) detector (Hewlett(10) Leenheer, J. L. Environ. Sci. Technol. 1981, 15, 578-587. (11) Raisglid, M.; Burke, M. F. Abstracts of the Pittsburgh Conference and Exposition on Analytical Chemistry and Applied Spectroscopy, Atlanta, GA, March 1621, 1997; Abstract 653.

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Figure 1. Double-disk solid-phase extraction (DD-SPE) apparatus.

Packard). The HPLC was equipped with a 25-cm × 4.6-mm-i.d. column packed with 3 µm of C18 (Hewlett-Packard). The analytical column temperature was set at 60 °C in order to achieve a better separation between the compounds analyzed. The mobile phase consisted of three different solvents: A (HPLC-grade water), B (methanol), and C (60/35/5 HPLC-grade water at pH ) 7/MeOH/ ACN). The gradient elution was performed as follows: from 25% A, 25% B, and 50% C, conditions kept for 5 min, to 5% A, 45% B, and 50% C in 30 min. The flow rate of the mobile phase was 0.6 mL/min, and the sample injection volume was 50 µL. The phenylurea and the triazine herbicides were monitored at 245 and 220 nm, respectively. Sample Preparation. (a) Water Samples. Tap water samples from Lawrence, KS, spiked at 1 µg/L, were extracted onto SAX and C18 disks using an Empore extraction system (see Figure 1). The first step of the SPE consisted of conditioning the disks with 20 mL of methanol and 20 mL of HPLC-grade water. Afterward, 500 mL of the sample was percolated through the disks. The compounds trapped on the double disk were eluted, either together or separately, with 4 mL × 2 of methanol. The extracts were spiked with 100 µL of a solution of 10 µg/L 2,4-D as an internal standard for the quantification of the compounds. Afterward, the extracts were evaporated to a volume of 100 µL under nitrogen at 45 °C using a Turbovap (Zymarck, Palo Alto, CA). Finally, a volume of 50 µL of the sample extract was injected into the HPLC system. The methodology was tested on 500 mL of a

Table 1. Recoveries of Extraction (%) of Herbicides from a 500-mL Tap Water Sample Spiked at 1 µg/L Using the DD-SPE Described in Figure 1 and a Single C18a

compound

% recovery onto the DD-SPE disk (SAX + C18)

% recovery onto a single C18 disk

80 85 75 101 95 99 90 90 85

50 97 79 99 97 98 108 92 82

25 85 110 109 101 104

23 83 115 110 107 102

phenylureas di-demethylfluometuron demethylfluometuron demethyldiuron chlorotoluron fluometuron isoproturon diuron linuron diflubenzuron triazines deisopropylatrazine deethylatrazine simazine atrazine propazine terbuthylazine

a The relative standard deviation (RSD) varied between 3 and 12% (n ) 3).

natural water sample from the Steele Bayou River (Mississippi), located in a cotton-growing area of the southeastern United States. (b) Soil Extracts. Soil samples were collected from an experimental corn field near Topeka, KS. Ten grams of soil was extracted twice with a methanol (15 mL)/water (5 mL) mixture at 75 °C for 30 min. The soil extracts were combined and evaporated to 10 mL under nitrogen, removing most, if not all, of the methanol. The extracts obtained were brought up to a volume of 500 mL with distilled water and then spiked with a mixture of four phenylurea herbicides. Afterward, the aqueous samples were extracted onto the layered disks, according to the same procedure as described before for water samples, to determine the recoveries of extraction onto the double disk.

RESULTS AND DISCUSSION Pesticide Sorption onto Disks. Figure 1 illustrates the concept of double-disk solid-phase extraction (DD-SPE). Because the disks are only 0.5 mm in thickness, they may be easily stacked for sample application and even used separately for sample elution. Figure 1 shows how the anion-exchange disk is placed above the reversed-phase C18 disk. Because of the convenience of the disks, they may be readily stacked and eluted either together or separately as needed. The same vacuum apparatus that is used for a single disk may also be used for the double disk without leaking or handling problems. The two disks may be conditioned at the same time and eluted simultaneously in order to use a minimum amount of solvent. Table 1 shows the recovery of the phenylurea and triazine herbicides from a 500-mL water sample using the DD-SPE system described in Figure 1. In this first experiment both disks were eluted together. The phenylurea herbicides were spiked at 1 µg/L and were recovered at an average of 89 ( 9%. These data indicate that the combination of the two disks results in efficient sorption and recovery of the phenylurea herbicides at the trace levels that occur in natural surface and groundwater samples. Furthermore, with the exception of deisopropylatrazine, all the triazines studied presented high recoveries, indicating good performance of the double-disk methodology for this class of compounds too. Deisopropylatrazine was poorly recovered due to its breakthrough as a consequence of its high polarity. For comparison, Table 1 also shows the recovery of the same compounds on a single C18 disk only, using a 500-mL water sample spiked at a concentration of 1 µg/L, too. The average recovery was 97 ( 8%, with the exception of the most polar herbicides, didemethylfluometuron and deisopropylatrazine. Again, these data suggest that the C18 disk is effective at both the sorption and recovery of the herbicides at trace levels of concentration. Table 2 shows the recovery of the same herbicides from the strong anion-exchange disk. In this experiment, both disks were stacked together as before for the extraction of the herbicides, but in this case they were eluted separately. Surprisingly, the SAX disk has some adsorption capacity for the herbicides on the basis

Table 2. Individual Disk Recoveries of Extraction (%) of Herbicides from a 500-mL Tap Water Sample Spiked at 1 µg/L Using the DD-SPE Disk (SAX + C18), Where Both Disks Were Eluted Separatelya compound phenylureas di-demethylfluometuron demethylfluometuron demethyldiuron chlorotoluron fluometuron isoproturon diuron linuron diflubenzuron triazines deisopropylatrazine deethylatrazine simazine atrazine propazine terbuthylazine a

% recovery onto the SAX disk

% recovery of the eluate from the SAX disk onto the C18 disk

total recovery (SAX + C18)

65 53 70 57 19 54 48 68 25

12 33 20 41 86 41 52 35 65

77 86 90 98 105 95 100 103 90

4 10 62 90 97 96

27 86 55 22 12 6

31 96 117 113 109 102

The relative standard deviation (RSD) varied between 2 and 10% (n ) 3).

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of a 500-mL water sample spiked at 1 µg/L. The average recovery was 51 ( 15% for the phenylurea herbicides. It is important to note that, for didemethylfluometuron, the recovery onto the SAX disk was higher than that obtained on the C18 disk (see Table 1 for C18 recoveries). On the other hand, for the triazine metabolites deisopropylatrazine and deethylatrazine, the recoveries obtained on the SAX disk were poor as compared with those obtained on the C18 disk. As can be observed in this table, the retention of the triazines onto the SAX disk is related to the hydrophobicity of the molecule, indicating a strong affinity of the disk for the less polar triazine compounds, as in a C18 disk. Table 2 also shows the recoveries of the herbicides on the second disk, the C18 disk, after its elution separately. Excluding deisopropylatrazine, the average of the total recovery of each compound adds up to 98 ( 4%. This result confirms the recovery of the SAX disk for each of the herbicides under study. Initially, the recovery of the SAX disk was quite surprising because the herbicides studied are not negatively charged species; thus, their removal by the SAX disk occurs as a result of another mechanism. Several possibilities exist. The first possibility is the sorption of the herbicides onto hydrophobic zones of the SAX disk. The disk consists of a polymeric network of styrene and divinylbenzene that contains quaternary anion-exchange sites. These positively charged sites are present at 0.2 mequiv/disk (manufacturer’s datum), and the disk contains 0.5 g of sorbent with a surface area of approximately 500 m2/disk (manufacturer’s datum based on nitrogen sorption). If one considers the surface area as a flat square surface of 500 m2 and that the anion-exchange sites are equally distributed across the surface, then the distance between sites may be calculated according to the following approximations:

500 m2/disk ) a square 22.35 m in length or 22.35 × 1010 Å in length (1) 0.2 mequiv/disk ) 2 × 10-4 equiv/disk

(2)

2 × 10-4 equiv × 6 × 1023 sites/equiv ) 1.2 × 1020 sites/disk (3) From eq 3, a square can be imagined that has the square root of 1.2 × 1020 sites per side, which would be the number of sites per side of the square, which is 1.1 × 1010 sites per side of the square. Thus, we have a square that is 22.35 × 1010 Å long with 1.1 × 1010 sites per length, which gives an approximate distance of 20 Å between sites. Consequently, the surface of the SAX disk is quite hydrophilic because of the sorbed water layers around each of the positively charged sites and the ordered water that exists around each of those sites. This simple model suggests that there are equal distances among sites, which of course may not be entirely accurate, but it does give us an idea of the approximate distances between sites and the potential for hydrophobic zones on the styrene divinylbenzene matrix of the SAX disk. The next possibility one must consider is the polar interactions that are occurring on the surface of the SAX disk at the anionexchange sites. The sites consist of amine groups of both strong (continually positively charged) and weak sites (may be charged 1012 Analytical Chemistry, Vol. 71, No. 5, March 1, 1999

Table 3. Recoveries of Extraction (%) of Phenylureas from a 500-mL Tap Water Sample Spiked at 1 µg/L Using the DD-SPE Described in Figure 1 with Two SAX Disksa compound

% recovery onto the DD-SPE disk (SAX + SAX)

demethylfluometuron chlorotoluron fluometuron isoproturon diuron linuron diflubenzuron

105 103 49 103 106 117 51

a The relative standard deviation (RSD) varied between 4 and 14% (n ) 3).

at acid pH). The strong sites are the fixed anion-exchange sites of the disk. The weak sites may share hydrogen atoms, and these hydrogen atoms may bond to the nitrogen atoms of the phenylurea herbicides (see Figure 2). This is another possible mechanism of sorption that may occur on the SAX disk. A comparison of the percent recoveries of each of the herbicides may give more insight into which of the two mechanisms is occurring on the SAX disk. For example, Table 3 shows the recovery of a double SAX disk, instead of the SAX and C18 disk for some of the phenylurea herbicides. Only fluometuron and diflubenzuron had poor recoveries on the double SAX disk; presumably, this is caused by a much weaker hydrogen-bonding interaction with the polar sites. This conclusion is reached from the fact that the demethyl metabolite of fluometuron has a greater bonding capacity than the parent compound, which is the most hydrophobic of the two compounds. The demethylfluometuron has an extra hydrogen atom present on the amide nitrogen (see Figure 2) and is more susceptible to the hydrogen-bonding mechanism. Furthermore, diuron and linuron also have a greater bonding capacity than fluometuron. One reason for this may be that the CF3 group of fluometuron is less electron withdrawing than the chlorine substituents of diuron and linuron. The effect of the electron withdrawing is to increase the acidity of the amide proton of the phenylurea, which increases its ability to hydrogen bond to the polar sites. More compounds of different pesticide classes are being examined in order to better understand the nature of the hydrogen-bonding mechanism and the nature of the polar interaction and will be the topic of a future paper. Removal of Humic Substances by SAX. Surface water samples commonly contain 1-5 mg/L of dissolved humic substances.12 Humic substances are naturally occurring colored organic acids of 500-2000 molecular weight that are readily leached from soils and plant materials into water.12 They comprise a majority of the natural dissolved organic carbon (DOC) in water samples, and they are commonly the major interferences that occur in high-performance liquid chromatography (HPLC) analysis of surface and estuarine water samples. Humic substances are somewhat soluble in methanol and may elute with the herbicides and interfere in HPLC analysis. Therefore, removal of these compounds during sample preparation is important for the most (12) Thurman, E. M. Organic Geochemistry of Natural Waters; MartinusNijhoff: Dordrecht, The Netherlands, 1985.

Figure 2. Chemical structures of the herbicides studied.

reliable pesticide analysis by HPLC with diode array detection (DAD). The amount of natural humic material that may occur in a chromatogram is shown in Figure 3, which is the HPLC/DAD analysis of a sample of tap water from Lawrence, KS, spiked with phenylureas. Figure 3a shows that, when only a C18 disk is used, the large peak eluting at the beginning of the chromatogram increases to over 500 mAU with a bimodal peak. This peak is associated with natural DOC and is considered to be a major fraction of the DOC.12-14 When the combination of SAX and C18 disks is used to remove the phenylurea herbicides, the early-eluting peak (the DOC peak) is decreased to a full scale of approximately 120 mAU, a decrease of approximately 4 times. This dramatic decrease in the “humic peak” presumably is due to the ion-exchange removal of the natural organic matter. The “humic peak” in Figure 3b can be further reduced if a double SAX disk is used (Figure 3c). All of the phenylurea herbicides are extracted with >95% recovery except fluometuron and diflubenzuron, as discussed in the preceding section. The “humic peak” has a full scale of ap(13) Walton, H. F. Ion Exchange Chromatography; Dowden, Hutchninoon & Rox: Stroudsburg, PA, 1976. (14) Thurman, E. M.; Malcolm, R. L.; Aiken, G. R. Anal. Chem. 1978, 50, 775779.

Table 4. Recoveries of Fulvic Acid from Suwannee River onto a SAX Disk Using a 500-mL Sample with a DOC Concentration of 20 mg/La sample treatment

% of recovery

% retained on the disk % passing the disk % eluted with methanol % eluted with acetonitrile % eluted with acidic methanol % eluted with basic methanol

95 5 10 5 10 15

a The relative standard deviation (RSD) varied between 5 and 10% (n ) 3).

proximately 100 mAU and a decreased area. This is especially useful when analyzing polar metabolites by HPLC, which often coelute with the matrix peak and are difficult to identify and quantify. The ability of the SAX disk to sorb humic substances was tested directly with fulvic acid from the Suwannee River in Mississippi. Table 4 shows the recoveries of fulvic acid from a 500-mL sample at pH 7. Nearly all of the fulvic material was removed from the sample (95% removal being determined by UV Analytical Chemistry, Vol. 71, No. 5, March 1, 1999

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Table 5. Recoveries of Extraction (%) of Phenylurea Herbicides from a 500-mL Distilled Water Sample Containing a Soil Extract and Spiked with a Mixture of Phenylureas Using the DD-SPE Described in Figure 1a

compound

% recovery onto a single C18 disk

% recovery onto the DD-SPE disk (SAX + C18)

% recovery onto the DD-SPE disk (SAX + SAX)

demethylfluometuron fluometuron diuron linuron

110 112 110 114

107 107 99 90

108 56 110 108

a The relative standard deviation (RSD) varied between 2 and 11% (n ) 3).

Figure 3. HPLC/DAD chromatograms of a tap water sample spiked at 1 µg/L with phenylurea herbicides and processed with a single C18 disk (a), with DD-SPE using SAX and C18 disks (b), and with DD-SPE using two SAX disks (c). Peak numbers: 1, 2,4-D; 2, demethylfluometuron; 3, fluometuron; 4, diuron; 5, linuron.

absorbance). Furthermore, the fulvic material could not be eluted effectively from the disk with methanol, acetonitrile, or water mixtures. Also acid and organic solvents were tried. Only trace levels of fulvic material were removed with either methanol/base or methanol/acid (Table 4). These data suggest that the SAX disk binds the humic material quite strongly by anion exchange of the carboxyl groups. The fulvic acid from the Suwannee River has approximately 6 mequiv/g of carboxyl group,15 which amounts to 5-10 carboxyl groups per molecule on the basis of a molecular weight of 2000.12 Thus, the fulvic material is tightly bound through interaction with multiple ion-exchange sites. (15) Oliver, B. G.; Thurman, E. M.; Malcolm, R. L. Geochim. Cosmochim. Acta 1983, 47, 2031-2035.

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Figure 4. HPLC/DAD chromatograms of a surface water sample from the Mississippi Embayment (stream called Steele Bayou) with a single C18 disk (a), with DD-SPE using two SAX disks (b), and with DD-SPE using two SAX disks at a wavelength of 245 nm, which enhances phenylurea peaks. Peak numbers: 1, 2,4-D; 4, diuron.

Table 6. Identification of Herbicides by GC/MS in a Surface Water Sample (Steele Bayou) from the Mississippi Embayment Compared to HPLC/DADa compound

detected by GC/MS

detected by HPLC/DAD

atrazine cyanazine cyanazine amide deethylatrazine deisopropylatrazine demethyldiuron demethylfluometuron demethylnorfluorazon 3,4-dichloroaniline didemethylfluometuron diuron fluometuron linuron metolachlor metribuzin molinate norflurazon prometryn simazine trifluoromethylaniline

yes yes yes yes yes no, not volatile for GC yes yes yes no no, not volatile for GC yes no, not volatile for GC yes yes yes yes yes yes yes

yes yes yes yes no, below detection limit yes yes yes yes no yes yes no yes no, below detection limit yes yes yes no, below detection limit no, below detection limit

a GC/MS detection limit for all compounds was 0.05 µg/L, and the detection limit by HPLC/DAD was 0.1 µg/L for all compounds.

Soil Samples. Table 5 shows the spike recoveries of extraction of four phenylureas in soil extracts from Topeka, KS. The same recovery behavior as that observed for water samples was noticed for the soil samples. All the phenylureas were recovered onto the DD-SPE disk, with the exception of fluometuron with a recovery of extraction of 56%, showing that the disks are effective at the sorption of herbicides from environmental samples. Moreover, the chromatographic behavior observed in the analysis of the soil extracts was the same as that encountered for water samples, being that the DOC peak decreased when compared with a single C18 extraction. Water Samples from Mississippi. Figure 4 shows the chromatograms for phenylureas isolated by a single C18 (Figure 4a) and the DD-SPE using two SAX disks (Figure 4b,c). The upper chromatogram shows a rather complex chromatogram because of the many herbicides present in the sample. However, when using the DD-SPE with two SAX disks, a chromatogram showing a cleaner baseline is obtained (see Figure 4b). Table 6 shows the identification of 20 herbicides and metabolites by gas chromatography/mass spectrometry (GC/MS) analysis of this sample. The phenylurea herbicide fluometuron is identified by GC/MS as well as its metabolites, didemethylfluometuron and demethylfluometuron. Atrazine is present at more than 1 µg/L. The

analysis by DD-SPE followed by HPLC/DAD shows that diuron is present at 0.5 µg/L. The method detection limit for diuron was 0.05 µg/L, calculated using a signal-to-noise ratio of 3 (the ratio between the peak intensity and the noise), using the double-disk approach, as compared to 0.1 µg/L using a single C18 disk. Thus, it is demonstrated that detection limits can be decreased by using the DD-SPE approach. Figure 4c shows that, for some compounds, the HPLC chromatogram may be further simplified by DAD detection at 245nm wavelength. The phenylureas sorb at this wavelength, whereas many other herbicides do not. Quantitation by this method increases the reliability of detection by DAD, and Figure 4 shows the DAD confirmation of diuron in this most complex water sample, since the UV peak purity is enhanced due to a cleaner baseline in the chromatogram. CONCLUSIONS In conclusion, the concept of DD-SPE has been demonstrated for herbicides and their metabolites from complex surface water samples. Humic and fulvic acids are effectively removed by SAX disks. Moreover, a combination of SAX and C18 is an effective method for herbicide isolation and cleanup when HPLC/DAD is used for detection. The concept of DD-SPE may have much broader application than demonstrated here because of the removal of anionic interferences by the SAX disk followed by the hydrophobic removal of organic compounds by the C18 disk. Examples could include drugs from urine and serum, pesticides from food extracts, and insecticides from soil and sediment samples. These examples will be examined in future work. ACKNOWLEDGMENT This work was partly supported by the Commission for Cultural, Educational and Scientific Exchange between the USA and Spain (Contract No. HNCCT 98148), the Commission of the European Communities, Environment and Climate Program (Contract ENV4-CT95-0066), and PLANCICYT (Contract AMB962808-CE). This work was carried out at the US Geological Survey organic research laboratory in Lawrence, KS; special thanks are given to the members of this group for their kindness and valuable help. The use of trade, firm, or brand names in this paper is for identification purposes only and does not constitute endorsement by the U.S. Government.

Received for review August 31, 1998. Accepted December 2, 1998. AC980975Q

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