Anal. Chem. IOSZ, 64, 1985-1990
1985
Mixed-Mode Isolation of Triazine Metabolites from Soil and Aquifer Sediments Using Automated Solid-Phase Extraction M. 5.Mills' and E. M. Thurman
US.Geological Survey, Water Resources Division, 4821 Quail Crest Place, Lawrence, Kansas 66049
Revemed-phau lrolationand lon-exchange purlflcatlonwere combinedin the automatedrolld-phase extractbnof two polar etrlazlne metabolltes, t-amlno-4-chloro-&( Iropropytamlno)strlazine (deethylatrazlne) and t-amlno-4-chloro-&( ethylamln0)-etrlazlne (delropropylatrazlne) from clay-loam and dlt-loam rdk and sandy aqulfer wdlments. Flrst, methanol/ water (4/1, v/v) rdl extractswere transferredto an automated workstatton following evaporatlon of the methanol phase for the rapid revemd-phau kolatlon of the metabolltes on an 0ctad.Cyiredn(C18). The retmtbnof the trlazlne metablltes on C18 decreased rubstantlally when trace methanol concentratlons (1%) remalned. Furthermore, the retentlon on C18Increasedwlth decrecrdng aqwousso4Mlty and Increadng alkyl-chain kngth of the metabolltes and parent herbkldes, lndlcatlng a reversedphaw Interactlon. Tho analytes were eluted wlth ethyl acetate, whlch iefl much of the roll organlcmatter Impurltles on the redn. Second, the small-volume organic eluate was purlfled on an anlon-exchange redn (0.5 mUmin) to extract the remalning roll plgmentsthat could foul the ion source of the GWMS system. Recoveries of the analytes were 75%, udng deuterated atrazlne as a surrogate, and were comparable to recoverles by roxhlet extraction. The detectbnlhnnwas 0.1 pg/kg wlth a coeffkkntof varlatton of 15%. The ease and efflclency of thls automated method makes lt viable, practlcal technlque for studying trlazlno metabolltes in the envlronment.
INTRODUCT10N Solid-phase extraction (SPE) is a technique used extensively in environmental chemistry to isolate organic compounds from aqueous solutions.1-7 In fact, the U.S. Environmental Protection Agency (EPA) recently added solidphase extraction to their methods of analysis for organic contaminants in water (EPA Method 525).8 Furthermore, the ease of automation of SPE aids development of rapid, inexpensive methods for the isolation and purification of analytes, with reproducible results."l However, there have been only a few applications of SPE to s0il'~-14and none to analysis of aquifer sediments. Recently, innovative new (1)Wolkoff, A. W.; Creed, C. J. Liq. Chromatogr. 1981,4,1459-1472. (2)Andrewa, J. S.;Good, T. J. Am. Lab. 1982,14,70-75. (3)Chladek, E.; Marano, R. S. J. Chromatogr. Sci. 1984,22,313-320. (4)Junk, G. A,; Avery, M. J.; Richard, J. J. Anal. Chem. 1988,60, 1347-1350. (5)Junk, G. A,; Avery, M. J.; Richard, J. J. Anal. Chem. 1988,60, 451-454. (6)McDonald, P. D. Waters Sep-Pak Cartridge Applications Bibliography; Millipore Corp.: Milford, MA, 1991. (7)Applications Bibliography Sample PreparationProducts; Varian: Harbor City, CA 90710; 1990. (8)Eichelberger, J. W.; Behymer, T. D.; Budde, W. L. Determination of Organic Compounds in Drinking Water by Liquid-Solid Extraction and Capillary Column Gas ChromatographylMass Spectrometry Methods for the Determination of Organic Compounds in Drinking Water, Environmental Protection Agency, . Method 525;U S . GPO: Washinaton, DC, 1988; pp 325-356. (9) Castellani, W. J.; Lente, F. V.; Chou, D. J.Autom. Chem. 1990.12, 141-144.
methods in SPE such as mixed-mode resins are being developedfor environmental chemistry, where reversed-phase and ion-exchange mechanisms of isolation are combined in a single resin or used ~equentially.'~J~ These mixed-mode resins allow sorption of the analyte whilst removing less strongly sorbed interferents with solvent washes. In this paper, we examine a mixed-mode application of SPE applied in tandem, with the automated sequential use of two resins, in order to isolate triazine metabolites from soil and aquifer sediments. The triazine herbicides are some of the most extensively used pesticides in the central United States11 and are frequently detected in surface water.16 Furthermore, the degradation products of the triazine herbicides are known to persist in soil and exhibit herbicidal effects that prevent crop rotation from corn (Zea mays L.) to soybeans (Glycine mar L.) in successive year^.^^^^^ Unlike their parent compounds, these soluble triazine degradation products have not been analyzed by solid-phase extraction from soil due to the difficulty of isolation and detection. Thus, the polar dealkylated metabolites of triazine herbicides provide a challenging target for automated solid-phase extraction by application of mixed-mode resins. The objectives of the study reported herein were (1) to examine the effects of aqueous solubility and the structure of dealkylated triazine metabolites on their retention on a solid-phase Cl8 resin, (2) to measure the metabolite capacity of the resin in the presence of trace concentrations of methanol (1-575 ), (3) to examine the tandem use of two resins for the mixed-mode isolation and purification of the triazine metabolites from soil extracts, and (4)to determine if solidphase extraction and gas chromatography/massspectrometry (GC/MS) with selected ion monitoring (SIM) could be combined into a rapid, automated procedure for the analysis of triazine metabolites from soil and aquifer sediments.
EXPERIMENTAL SECTION Reagents. Methanol (Burdick and Jackson, Muskegon, MI), ethyl acetate, and isooctane (Fisher Scientific, Springfield, NJ) (10)Thurman, E.M.; Meyer, M.; Pomes, M.; Perry, C. A,; Schwab, A. P. Anal. Chem. 1990,62,2043-2048. (11)Marvin, C. H.; Brindle, I. D.; Hall, C. D.; Chiba, M. Anal. Chem. 1990,62,1495-1498. (12)Wachob, G. D. J. Liq. Chromatogr. 1984,10, 756-759. (13)Huang, L.Q.J. Assoc. Off. Anal. Chem. 1989,72, 344-354. (14)Huang, L. Q.;Frink, C. R. Bull. Enuiron. Contam. Toxicol. 1989, 4.7. - -, 151)-164. __ - - - -. (15)Patel, R.;Jagodzinski, J. J.; Benson, J. R.; Hometchko, D. LC-CC, 1990,8(ll),874-878. (16)Patel, R.; Benson, J. R.; Hometchko, D.; Marshall, G. Am. Lab. 1990,Feb, 92-99. (17)Gianessi, L. P.; Puffer, C. M. Use of selected pesticides for agricultural crop production in the United States; NTIS: Springfield, VA, 1982-1985;Vol. 490. (18)Thurman, E. M.; Goolsby, D. A.; Meyer, M. T.; Kolpin, D. W. Enuzron. Scz. Technol. 1991,25 (lo),1794-1796. (19)Sirons, G. J.; Frank, R.; Sawyer, T. J. Agric. Food Chem. 1973, 21, 1016-1020. (20)Muir, D. C. G.; Baker, B. E. Weed. Res. 1978, 18 (2),111-120.
This article not subject to U.S.Copyright. Published 1992 by the American Chemical Society
1986
ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992
were pesticide-grade solvents. Deionized water was charcoalfiltered and glass-distilled prior to use. Simazine, atrazine, and propazine were obtained from Supelco (Bellefonte,PA), and the triazine metabolites, 2-amino-4-chloro-6-(ethylamino)-s-triazine and 2-amino-4-chloro-6-(isopropylamino)-s-triazine were from Ciba Geigy (Greensboro, NC). The CIScartridges (SEP-PAK from Waters,Milford,MA) contained 360 mg of 40-pm CIS-bonded silica. Standard solutions were prepared in methanol, and deuterated phenanthrene (phenanthrene&) (EPA,Cincinnati, OH) was used as an internal standard for GC/MSquantitation. Deuterated atrazine (New England Nuclear, Boston, MA) was used as a surrogate for soil extraction. Soil Extraction. A 20-g sample of soil (or its wet-weight equivalent) was weighed into a Teflon-lined screwcap test tube. Then 5 mL of distilled water was added, and the surrogate standard (deuterated atrazine) was spiked into the soil. The samplewas equilibrated for 1h by mixing on a mechanicalshaker. Methanol (15 mL) was added, and the mixture was vortexed to a slurry. The slurry was heated to 75 "C for 30 min, with periodic vortexing. Next, the sample was mixed for 15 min on a mechanical shaker to allow cooling and then was centrifuged. The clear supernatant was poured directly into a 40-mL tube for evaporation. This extraction procedure was repeated on the soil sample and the second supernatant combined with the first. The combined extract was evaporated using a Turbovap (Zymark, Palo Alto, CA) until only 10 mL of water remained. This was transferred to a test tube for automated solid-phase extraction. Automated Solid-Phase Extraction. A Waters Millilab workstation (Milford, MA) was used for solid-phase extraction of the analytes. Cls Sep-Pak cartridges were preconditioned sequentiallywith 2 mL each of methanol, ethyl acetate, methanol, and distilled water. Solventswere drawn up from reagent stations by a robotic probe and aspirated through the cartridge under positive pressure. Each 10-mL soil extract was spiked by the robotic probe with 100pL of surrogate standard, terbuthylazine (2.4 ng/pL), drawn up by the probe and pumped through the cartridge at a rate of 20 mL/min. The exterior of the probe was washed of adhering sampleby immersingin a test tube containing 4 mL of ethyl acetate and agitating the solvent by bubbling air through the probe. Analytes were eluted with 4 mL of ethyl acetate into a centrifuge tube, and following successive preparation of all water samples, each eluate was spiked with 500 pL of internal standard phenanthrene-& (0.2ng/pL) by the robotic probe. Approximately 100 pL of water, trapped in the cartridge, was coeluted with the ethyl acetate. The ethyl acetate and water layers were mixed after addition of the internal spike, and after settling, 3.5 mL of the homogenized ethyl acetate layer was drawn off the denser water layer by robotic probe and aspirated through an anion-exchangecartridge (QMA Accell resin, Waters) into a second centrifuge tube. The ethyl acetate was passed through the anion-exchange cartridge at a rate of 0.5 mL/min, for the sequential removalof coeluted humic substances by ion exchange. The anion-exchangecartridge had previously been sequentially conditioned by the probe with 2 mL of distilled water, methanol, and ethyl acetate. The exterior of the probe was immersed in the ethyl acetate wash tube prior to transferring successive eluates. Finally, the colorlessethyl acetate extract was evaporated automatically to 100 r L by a Turbovap (Zymark,Palo Alto, CA) at 45 "C under a nitrogen stream and transferred to a glasslined vial for GC/MS analysis. GC/MS Analysis. Automated GC/MS analyses of the eluates were performed on a Hewlett-Packard Model 5890 gas chromatograph (Palo Alto, CA) and a 5970A mass-selectivedetector (MSD). Operating conditionswere as follows: ionizationvoltage, 70 eV; ion source temperature, 250 "C; electron multiplier, 2200 V; direct capillary interface at 280 "C, tuned daily with perfluorotributylamine; 50-ms dwell period. Separation of the herbicides was accomplishedwith a fused-silica capillary column of methylsilicone (HP-1) with a film thickness of 0.33 pm, 12-m x 0.2-mm i.d. (Hewlett Packard, Palo Alto, CA). Helium was used as the carrier gas at a flow rate of 1mL/min and a head pressure of 35 kPa. The column temperature was held at 50 "C for 1min and then ramped at 6 "C/min to 250 "C where it was held for 10 min. The injector temperature was 280 "C. The filament and multiplier were not turned on until 5 min into the analysis.
Table I. Selectively Monitored Ions Used To Identify Metabolite and Parent Herbicides atomic mass unit retention molecular base compd time (rnin) ion peak ion 1 ion2 173 173 158 145 deisopropylatrazine 17.98 18.32 187 172 145 deethylatrazine 19.71 201 201 186 176 simazine 20.02 215 200 173 atrazine 20.25 229 214 propazine 172 100
A
90 BO
i B e
100
0 100r
40
,
,
200 ,
.
:,
300
,;
,
500
400
,
,
,
.
,*
c
0
o
1,000
1 2.000
3.000
4,000
5.000
6.000
VOLUME. IN MILLILITERS
Flgurr 1. Volume required for breakthrough of triazine metabolltes (A) and parent herbicides (B) on ClS resin in pure water.
Quantification of the base peak of each compound was based on the response of the 188( m u )ion of the internal standard, phenanthrene-&. Confirmation of the compound was based on the presence of the molecular ion and two confirming ions with a retention-time match of f0.2 % relative to phenanthrene-dlo and correct area ratios for confirming ions. Compounds selectively monitored included deisopropylatrazine (2-amino-4-chloro-6(ethy1amino)-s-triazine),deethylatrazine (2-amino-4-chloro-6(isopropy1amino)-s-triazine), simazine, atrazine, and propazine (Table I).
RESULTS AND DISCUSSION Solid-Phase Extraction. The capacity of the CIS cartridges for the s-triazine metabolites and their parent herbicides was determined from pure-water solutions to 80% methanol/water. Figure 1A shows the relative retention of each of the metabolites in pure water, and Figure 1B shows the retention of parent herbicides. The C18 resin had the least capacity for deisopropylatrazine, with approximately 80% retention from a 100-mLsample volume that commonly is used in environmental analyais.l0 Deethylatrazine had 100% retention from a 100-mL sample, but breakthrough began at approximately 200 mL. The parent herbicides, simazine, atrazine, and propazine, had 100% retention from the same volume with initial breakthrough beginning a t 750 mL for simazine, 1250 mL for atrazine, and 2500 mL for propazine. The retention of the metabolites and parent herbicides on Cle resin from aqueous solution increases with increasing alkylchain length, in the order deisopropylatrazine, deethylatrazine, simazine, atrazine, and propazine. This order of increasing retention on CISclearly follows structural changes,
ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992
1987
DEETHYUTRUINE
60 50 -
-
::I 30
,
,
,
,
,
,
,
1
2
3
4
5
6
7
,J-j
0
0
6
9
10
100 90 80
0
70
0
DEISOPROPYLATRUINE DEETHYUTRUINE SIMAZINE
60
A
PROPUINE
0
1'
2
I
1
3
I
I 4
Log of Breakthrough Volume (mL)
50 40
Figuro 2. Llnear correiatlonof the number of methylene groups in the slde chains of triazines versus log of 100% breakthrough volume milliliter from pure water on Cle resin.
30 20
10
with capacity on C18 (measured by volume for 100% breakthrough of analyte) increasing in a linear logarithmic fashion with the addition of a methylene group to the triazine alkyl side chain (Figure 2). It has been noted previously that addition of a methylene group to a homologous series of compounds linearly increases the free energy of reversedphase sorption by a consistent amount for each methylene group.21 Furthermore, the order of retention of the metabolites and parent herbicides on C18 resin nearly follows the order of decreasing aqueous solubility, which is deisopropylatrazine > deethylatrazine > atrazine > simazine > propazine. The exception is simazine, which has a lower solubility (3.5 mg/L) than atrazine (33 mg/L) but less retention on C18 than atrazine. Chiou and other^^^,^^ have shown that solubility is inversely proportional to sorption capacity. Furthermore, they have shown that improved correlations of the solubility relationship were obtained when the aqueous solubilities of a solid analyte were converted to corresponding aqueous solubilities of the supercooled liquid, thereby eliminating the energy of crystallization term that must be overcome during solvation. The lower solubility of simazine probably is due to a larger energy of crystallization than either atrazine or propazine, which is expressed as a much higher melting point for simazine (225 OC) than for either atrazine (175 OC) or propazine (212 "C).This larger energy of crystallization for simazine lessens the aqueous solubility. Thus, the order of sorption onto Cl8 follows structural changes but not exact solubility changes. Because methanol is used as an effective solvent for soil extraction, the effect of percent methanol on CMrecovery was examined. Figure 3A shows the retention on the C18 resin for the triazine metabolites, and Figure 3B shows the retention on CISfor parent herbicides in the presence of 080% methanol from 100-mL water samples. Initial losses in retention on Cl8 began in the presence of only 1%methanol for deisopropylatrazine and deethylatrazine,5 % for simazine, 10% for atrazine, and 20% for propazine (Figure 3A,B). In the presence of 10% methanol, the retention of deisopropylatrazine is reduced to between 18% and 20%. At 30% methanol, the retention of deethylatrazine was zero. The order in which these compounds are affected is a function of (21) Thurman, E. M.; Malcolm, R. L.; Aiken, G. R. Anal. Chem. 1978,
50 (6), 775-779.
(22) Chiou, C. T.; Peters, L. J.; Freed, V. H. Science 1979,206, 831832. (23) Chiou, C. T.; Schmedding, D. W.; Manes, M. Enuiron. Sci. Technol. 1982,16, 4-10.
0 0
10
20
30
40
50
60
70
BO
PERCENT METHANOL. IN MOBILE PHASE
Flgure 3. Percentretentkmof trlazlne metabolitesand parent herbicides on Cle resin as a function of percent methanol (based on a 100-mL volume and 360-mg of CIe resin).
their alkyl-chain length (or decreasing polarity). The compound with the shortest alkyl-chain length, deisopropylatrazine, was affected most by trace quantities of methanol, and the compound with the longest alkyl-chain length, propazine, was affected the least. The parent herbicides lose their retention on C18 in the order simazine, atrazine, and propazine at between 40 and 80% methanol. From these differences among polar metabolites and more hydrophobic parents, it is postulated that the presence of methanol affects the sorption mechanisms of the metabolites more so than the parent compounds, which may be the result of both primary and secondary sorption me~hanisms.2~ The percent methanol remaining after evaporation of the methanol/water soil extract to 4 mL was approximately 0.06 % , as determined by dissolved organic carbon measurements. All traces of methanol must be evaporated from soil extracts prior to SPE to ensure full recoveryof the triazine metabolites. Recognitionof this effect has implicationsfor soil-extraction techniques. For example, in the analysis of the three parent herbicides from soils, the step of methanol evaporation from soil extracts could be eliminated. Thus, the methanol/water (80/20, v/v) extract could be diluted with water and applied directly to the CU resin. Sample-preparation time would be decreased by elimination of this evaporation step. In fact, this procedure has been reported recently,'3J4>25but no consideration was given to possible reductions in retention in accordance with the quantity of methanol present. Furthermore, it has been hypothesized that methanol could be added to the mobile phase to increase C18 capacity for the triazines and other more hydrophobic pesticides, becauselarge volumes of sample wash away the conditioning methanol, reducing the contact that the water sample may have with the C18 chains.2~25Results from the study described herein go against this concept for more-polar metabolites. For example, Figure 1B shows that as much as 750 mL of water may be passed through a C18 cartridge before the first triazine, simazine, begins to break through, and 1250 mL for (24) Van Horne, K. C. Sorbent Extraction Technology;Analytichem Internationak Varian, Harbor City, CA 90710, 1985; 16 pp. (25) Brooks, M. W.; Jenkins, J.; Jimenez, M.; Quinn, T.; Clark, J. M. Analyst 1989,114, 405-406.
1968
ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992
Table 11. Color Removal from CISCartridges by Various Eluting Solvents. eluting solvent methanol acetonitrile methylene chloride/ 2-propanol 80/20% v/v ethyl acetate
% color in solvent extract
Table 111. Percent Recovery of Metabolites and Parent Herbicide from a Field-Contaminated Eudora Silt-Loam Soil (63% Silt, 26% Sand, 11% Clay, 0.99% Organic Carbon), Using Methanol and Acetonitrile as the Extracting Solvents.
20 10
8 2
Color was measured as the optical density at 400 nm.
atrazine. Addition of methanol is not required for increased capacity. On the other hand, a trace of methanol (1%) will decrease the capacity of Cle for more-polar degradation products, such as deisopropylatrazine (Figure 3A). Thus, evaporation of all methanol is essential in order to isolate metabolites for G U M S analysis. The efficiency of four solvent systems was tested for the elution of the C18cartridge. The solvents included methanol, acetonitrile, methylene chloride/2-propano1(80/20% v/v), and ethyl acetate. All four solvents eluted the metabolites and parent herbicides from the resin with 100% removal. However, because solid-phase extraction isolates natural aquatic humic substances,26 as well as the triazines, elution solvents were evaluated for the efficiency of desorption of these compounds from CISresin. Adsorption of polar and nonpolar natural organic components of soil commonly seen as a discoloration a t the head of the C18 cartridge. Table I1 shows the color removed by the elution solvents methanol, acetonitrile, methylene chloride/ 2-propanol, and ethyl acetate from CIScartridges treated with 10 mL of approximately 1 mg/mL solutions of humic and fulvic acids isolated from water.26 Color was measured as the optical density of the solution at 400 nm.26327Methanol and acetonitrile desorbed 20 and 10% of the original color, respectively; methylene chloride/%-propanoldesorbed 8% ; the ethyl acetate eluate desorbed only 2%, which left the eluate nearly clear. Finally, the ethyl acetate is immiscible with water. This is an advantage in that the extract is free of water, which simplifies the evaporation step. Ethyl acetate appears to be the best solvent for elution of the solid-phase extraction cartridge. A small percentage of colored humic substances from soil are still eluted with the ethyl acetate solvent, because soil extracts contain various polar organic acids. Because the organic acids are negatively charged (pK, = 4.5), they can be selectively removed with the sequential use of an anionexchange cartridge. The reversed-phase and ion-exchange resins were used sequentially instead of combining the two mechanisms into one resin for the following reasons: (1)A slower flow rate is needed for effective ion exchange to take place. Processing a 3-mL organic eluate instead of a 10-mL aqueous extract greatly speeds analysis time, (2) inorganic ions present in an aqueous soil are lost from the sample during reversed-phase isolation of the triazines and cannot interfere with the anion-exchange isolation of the organic acids. Soil Extraction. Table I11 shows a comparison of methanol and acetonitrile used in the extraction and recovery of deethylatrazine, deisopropylatrazine, simazine, atrazine, and propazine (performed in triplicate) from a fieldcontaminated wet Eudora silt-loam soil. Deethylatrazine extraction from the silt-loam soil increased by nearly 50% (26) Thurman, E. M. Organic Geochemistry of Natural Waters; Dordrecht, Martinus Nijhoff: The Netherlands, 1985; Chapter 10. (27) American Public Health Association; American Water Works Association; Water Pollution Control Federation. Standard Methods for the Examination of Water and Wastewater;American Public Health Association: Washington, DC, 1980.
compd
methanol
acetonitrile
% recovery D5
deisopropylatrazine deethylatrazine simazine atrazine propazine
75 f 15 75f15 75f15 7 5 f 15 7 5 f 15
30 f 15 48f15 75f15 7 5 f 15 7 5 f 15
75 f 15 75 f 15 75 f 15 75 f 15 75 f 15
a Recoveries of analytes are based on the percent recovery of the surrogate spike, deuterated atrazine.
Table IV. Comparison of the Percent Recovery (f15%) of Deisopropylatrazine and Deethylatrazine (Present at Approximately 6.0 rg/kg) and Atrazine (Present at Approximately 150 pg/kg) from a Field-Contaminated Eudora Silt-Loam Soil (63% Silt, 26% Sand, 11% Clay, 0.99% Organic Carbon) by the Automated Solid-Phase Extraction Method and Soxhlet Extraction Method. compd
SPE
soxhlet extraction
% recovery D5
deisopropylatrazine deethylatrazine atrazine
75 75 75
0 0 70
75 75 75
automated
Recoveries are based on the percent recovery of the surrogate spike, deuterated atrazine.
using methanol as the extracting solvent as compared to acetonitrile. However, the less-polar parent compounds, simazine, atrazine, and propazine, were extracted efficiently with either methanol or acetonitrile. Apparently, the morepolar methanol desorbs the polar degradation products more efficiently, whereas the more-hydrophobic atrazine is desorbed almost equally efficiently by either methanol or acetonitrile. The equality of extraction of the parent herbicide, atrazine, using either solvent has been noted previously.28-z9 When the silt-loam soil was spiked with herbicides and metabolites, both solvents recovered with equal efficiency, which reflects the lack of soil binding of spiked herbicide. Table IV shows the extraction recovery of deisopropylatrazine, deethylatrazine, and atrazine from a field-contaminated Eudora silt-loam soil performed in triplicate, by conventional soxhlet extraction and the automated soil extraction method. Soxhlet extraction was carried out by refluxing a 20-g silt-loam soil with 400 mL of methanol for 24 h, rotary evaporating the solvent to 10 mL, and then nitrogen evaporating the sample to 100 pL for analysis by GC/MS. Recoveries of atrazine were comparable between the two methods, but deisopropylatrazine and deethylatrazine present a t approximately 6.0 Hg/kg were recovered only by the automated procedure and not by soxhlet extraction. One reason for this may be the use of a watedmethanol mix in the automated procedure, increasing the wetting ability of methanol to extract the more-polar analytes from soil. Previously, it has been reported that the presence of 20% water can improve the efficiency of extraction of herbicides from a variety of soil t y p e ~ . ~Another ~ J ~ reason may be the increased temperature of extraction in the automated extraction procedure (Table V). A field-contaminated Kim0 clay-loam soil was heated during the methanol/water extraction in an attempt to increase recovery of the triazine metabolites (Table V). Extractions were performed in triplicate, and the recovery of ~
~
~~
(28) Cotterill. E. G. Pestic. Sci. 1980. 1 1 . 23-28.
(29) Huang, L. Q.;Pignatello, J. J. J.’Assoc. Off. Anal. Chem. 1990,
73 (3), 443-446.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992
Table V. Percent Herbicide Recovery (h15%) from Field-Contaminated Kim0 Clay-Loam Soil (60% Silt, 17% Sand, 23% Clay, 0.63% Organic Carbon Content) a# a Function of Temperature of Solvent Extractions temp of solvent extraction (OC) compd
45
55
65
75
85
deisopropylatrazine deethylatrazine atrazine simazine
60 49 53 50 55
55 66 68 67 70
68 69 68 68 70
75 75 75 75 75
75 75 75 75 75
propazine
manual
the metabolites, deisopropylatrazine and deethylatrazine,and parent herbicides from the clay-loam soil increased from between 49 and 60% at 45 "C to 75% at 75 "C, at which point the methanol/water mixture was almost boiling. The 75 "C extraction temperature was previously reported as an optimum temperature for extraction of atrazine and metolachlor from a fine sandy-loam ~0i1.29 Uncontaminated aquifer sediments (3% coarse sand and pebbles, 96% sand, 1% silt, approximately 0.1% organic carbon content) and an uncontaminated Eudora silt-loam field soil (0.99% organic carbon content) were spiked in triplicate with the triazine metabolites and parent herbicides, to determine the efficiency of the automated method to extract the triazines from soils and sediments containing different amounts of soil organic matter. Recoveries of all parent triazines and metabolites were between 70 and 75% for both soil and aquifer sediment, indicating that more organic-rich soils can be extracted equally as efficiently as sediments with low organic-matter content. Furthermore, the silt-loam field soil was spiked with both triazine metabolites and parent herbicides and mixed at room temperature for 24,144, and 240 h. Recovery of the analytes ranged between 70 and 75% from the silt-loam soil of all equilibration times, indicating that the recovery of spiked analytes is not proportional to the equilibration period. Because the automated method has recoveries of the triazines comparable to soxhlet extraction procedures in the part-per-billion range and has greater sensitivity at the sub-part-per-billion range, determining the recovery of surrogate deuterated compounds is necessary in order to determine the recovery of nonspiked analytes from soils. It is important that the method has consistent recoveries of spiked materials, regardless of soil type and equilibration period, in order to accurately determine method recoveries. Robotics and G U M S Analysis. Figure 4 shows the sample flow path for the method described and how a methanol/water soil extract is linked into a solid-phase extraction, GC/MS with SIM procedure for water samples. A 10-mL water sample is the end product of the methanol/ water soil extraction,and a small-volumeCl8 isolation method was developed on a Waters Millilab workstation. Because the robotic probe was used to transfer these small volumes, it was important to ensure no carryover of the previous sample via adhesion of contaminated water or solvent to the exterior of the probe. The test tube of ethyl acetate used to wash the exterior of the probe contained up to 1% of the previous sample, or approximately 50 p L of liquid had adhered to the probe exterior. Contamination was most extensive during transferal of the ethyl acetate eluate through the anionexchange resin, due to rapid evaporation of ethyl acetate on the probe exterior. Therefore washing the exterior of the probe is an important step in the method to ensure accurate and precise results. The precision of robotic spiking of both the surrogate and internal standard was checked by measuring the delivered
r l Soil extraction procedure
Soil
Water sample
extract
a Recoveries of herbicides are based on recovery of surrogatespike, deuterated atrazine.
1989
automated
Robotic solid-phase extraction
automated
Evaporation
Flgure 4. Flow diagram of the automated soll-extractlon method, using solid-phase extraction and W / M S SIM.
volume of the spiking syringe. The 1.00-mLsyringe delivered the 500- and 100-pLspikes with a precision of 1.3% relative standard deviation. This result was determined by measuring the volume for 12repetitions of the spike. The robotic method required 33 min for completion of each water sample, and 21-30 samples could be prepared consecutively, depending on the size of the test-tube rack. Because the millilab delivers exactly the same volume of eluate to each test tube, a precisely timed evaporation can be operated on the turbovap to achieve 100-pL volumes uniformly throughout the whole sample set. Precision data for the dealkylated metabolites and their parent triazine herbicides, simazine, atrazine, and propazine, using wet 20-guncontaminated Eudora silt-loam soil samples (performed in triplicate), spiked with the analytes, were examined. The coefficients of variation for deisopropylatrazine, deethylatrazine, simazine, atrazine, and propazine were greater than 15% at 0.1 pglkg and less than 15% at 0.2 pg/kg. From these variances, a quantitation limit for all compounds of 0.2 pg/kg was established, with the detection limit set at 0.1 pg/kg. Variations with time in the recovery of deisopropylatrazine and deethylatrazinemay occur. These variations were traced to losses of these compounds in the inlet of the injector port in the gas chromatograph. These compounds are susceptible to adsorption onto any nonvolatile organic matter on the inlet sleeve, which must be changed weekly according to the responses of metabolite standards. Losses of up to 40% for deisopropylatrazine and 20% for deethylatrazine are possible if the inlet is dirty.
CONCLUSIONS The retention of the s-triazine metabolites and parent herbicides on C18is related to the structure of the compound, with increasing retention as alkyl-side-chain length of the compound increases. This effect is demonstrated by the more rapid breakthrough of the less-hydrophobic dealkylated triazine metabolites on C18 compared to more-hydrophobic parent triazines. Trace concentrations of methanol in the aqueous mobile phase cause desorption of the dealkylated
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triazine metabolites from Cl8 (1% methanol) and the parent herbicides ( 5 % methanol and more). All traces of methanol must be evaporated from soil extracts prior to SPE to ensure full recovery of the triazine metabolites. Ethyl acetate is the best eluting solvent to desorb the analytes and leave the majority of soil organic-matter impurities a t the head of the Cla column. Any remaining soil organic-matter impurities present in the eluate are removed by the sequential use of an anion-exchange resin. The automated soil-extraction procedure has several positive aspects compared to previous soil methods. First, it is capable of efficiently recovering the two triazine metabolites, deisopropylatrazine and deethylatrazine. Second, it is labor conservative, time and cost effective, and uses small soil samples (20 g) and minimal solvent volumes (30 mL of methanol in total). The method uses nonchlorinated solvents and is reliable and safe. Third, this method can achieve an environmentally significant quantitation limit (0.2rg/kg) for all compounds, which is not possible by soxhlet extraction techniques. Fourth, SPE and GC/MS SIM analysis in
conjunction with soil extraction allows automation of nearly the entire method, with only a single manual transfer of the solvent extract. The ease and efficiency of this automated method make it viable for the analysis of large numbers of soil samples and aquifer sediments for the triazine metabolites and parent herbicides.
ACKNOWLEDGMENT We thank Gail Mallard, Surface and Ground Water Toxics Program, US.Geological Survey, for financial support of this research. Brand names are for identification purposes only and do not imply endorsement by the US.Geological Survey.
RECEIVED for review February 6, 1992. Accepted May 18, 1992. Registry No. Deisopropylatrazine, 1007-28-9; deethylatrasimazine, 122-34-9; atrazine, 1912-24-9; propazine, zine, 6190-65-4; 139-40-2.
