Anal, Chem. 1993, 85, 2372-2379
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Herbicide Trace Analysis by High-Resolution Fast Atom Bombardment Mass Spectrometry: Quantification of Low Parts per Trillion Levels of Atrazine in Water Kenneth A. Caldwell, V. M. Sadagopa Ramanujam, Zongwei Cai, and Michael L. Gross. Midwest Center for Mass Spectrometry, Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304 Roy F. Spalding
Water Center, University of Nebraska, Lincoln, Nebraska 68588-0844
A method for determining trace levels of atrazine in groundwater and tap water by using fast atom bombardment mass spectrometry is reported. Low-picogram detection limits were achieved by considering explicitly the chemistry leading to the production of background ions from the liquid matrix ( 3 1dithiothreitoVdithioerythrito1)and by using the minimum volume of matrix required to achieve ionization. Protonated atrazine was produced abundantly in the gas phase with relatively low abundance background ions at the atrazine ion mass region. The method, which uses a highresolution magnetic sector mass spectrometer, is sensitive, fast, and simple. The response curve for atrazine is linear from 1 to 1000 pg. Appropriate procedures for isolating atrazine from water were developed. The analytical results compared well with those obtained by using the capillary gas chromatography/high-resolution mass spectrometry method developed in this laboratory. The accuracy and precision of the method were assessed by analyzing six standard water samples containing 1 part-per-trillion atrazine. Both the precision and accuracy of the method were better than 10% (relative standard deviation) for standard water samples. After the sample was fortified with the [W~]atrazineinternal standard, the native atrazine in local tap water samples could be determined directly or after a single-step extraction with dichloromethane. The method has also been successfully used for analysis of atrazine at 6-94 parts-per-trillion levels in several wellwater samples. INTRODUCTION Atrazine (structure I) is the most widely used herbicide in the Midwest. It is applied to corn, soybean, wheat, barley, (CHJ2CHNHyNyCI
NYN HNCH,CH, I
and sorghum crops for broadleaf weed control. Residues of atrazine and related triazines (cyanazine,propazine, simazine, etc.) have been detected in soils and surface waters in several 0003-2700/93/0385-2372sO4.0010
states of the United States, owing to their persistence and relative high water solubility.132 Determination of triazines present in natural waters at parts-per-billion (ppb) levels is now routine. Quantification can be achieved by means of a variety of methods including capillary gas chromatography (GC),S1OGC combinedwith low-resolution mass spectrometry (GC/MS)11-21 or ion trap mass spectrometry,= high-performance liquid chromatography,- liquid chromatography/ low-resolution mass spectrometry (LC/MS),31e32and enzyme(1)Patrick, R.; Ford, E.; Quarles, J. Ground Water Contamination in the United States, 2nd ed.; University of Pennsylvania Press: Philadelphia, PA, 1987. (2)Ragsdale,N. N., Kuhr,R. J., Eds. Pesticides: Minimizing the Risks; ACS Symposium Series 336;American Chemical Society Washington, DC. 1987. (3)Graves, R. L. Determination of Nitrogen- and PhosphorusContaining Pesticidesin Water by Gas Chromatography with a NitrogenPhosphorus Detector. Revision 2.0; Environmental Protection Agency, Method 507;U.S.G P O Washington, DC, 1989; pp 143-170. (4)Lee,H. B.; Stokker, Y. D. J.-Assoc. Off. Anal. Chem. 1986,69, 568-572. (5)Popl, M.; Voznakova, Z.; Tatar, V.; Stmadova, J. J. Chromatogr. Sci. 1983.21. 39-42. (6)W d o w , J. E.; Majewski, M. S.; Sieber, J. N. J. Enuiron. Sci. Health 1986, B21,143-164. (7) Aaromon,M. J.;Kitby, K. W.;Tessari, J. D.Bul1. Enuiron. Contam. Toricol. 1980,25,492-497. (8) Brooke, M. W.; Jenkins, J.: Jimenez, M.: Quinn, T.: Clark, J. M. Analyst 1989,114,405-406. (9)Grob, K.; Li, Z.J. Chromatogr. 1989,473,423-430. (10)Edgell, K. W.; Jenkins, E. L.; Lopez-Avila, V.; Longbottom, J. E. J.-Assoc. Off. Anal. Chem. 1991,74,295-309. (11)Mangani, F.; Bruner, F. Chromatographica 1983,17, 377-380. (12)Lopez-Avila, V.;Hirata, P.; Kraska, S.; Flanagan, M.; Taylor, J. H., Jr. Anal. Chem. 1986,57,2797-2801. (13)Barcelo, D. Chromatographia 1988,2S,295-299. (14)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. In Methods for the Determination of Organic Compounds in Drinking Water; Environmental Protection Agency, Method 525; U.S. G P O Washington, DC, 1988; pp 325-356. (15)Bagnati, R.; Benfenati, E.; Davoli, E.; Fanelli, R. Chemosphere 1988,17, 59-65. (16)Rustad, C. E.; Pereira, W. E.; Leiker,T. F.Biomed. Enuiron. Mass. Spectrom. 1989,18,820-827. (17)Huang, L. Q. J.-Assoc. Off. Anal. Chem. 1989, 72,349-354. (18)Benfenati, E.;Tremolada, P.; Chiappetta, L.; Frassanito, R.; Bassi, G.; Di Toro, N.; Fanelli, R.; Stella, G. Chemosphere 1990,21,1411-1422. (19)Schuette, S.A.; Smith, R. G.; Holden, L. R.; Graham, J. A. Anal. Chim. Acta 1990,236,141-144. (20)Pereira, W. E.; Rostad, C. E.; Leiker, T. J. Anal. Chim. Acta 1990, 22469-75. (21)Kraut-Vase, A.; Thoma, J. J. Chromatogr. 1991, 538, 233-240. (22)Periera, W. E.; Rostad, C. E.; Leiker, T. J. Anal. Chim. Acta 1990, 22469-75. (23)Brown, D. F.; McDonough, L. M.; McCool, D. K.; Papendick, R. I. J . Agric. Food Chem. 1984,32,195-200. (24)Owens, D. S.;Sturrock, P. E. Anal. Chim. Acta 1986,188,269274. (25)Reupert, R.; Ploeger, E. Fresenius Z . Anal. Chem. 1988,331,503509. 0 1993 Amerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, 1993
linked immunosorbent assays.33 The detection limits range from 60 to lo00 pg and are limited by the instrumental methods, but not by the isolation procedures. Parte-per-trillion (ppt) and sub-ppt-level detection limits for herbicides and pesticides are necessary for providing benchmark data, for studying the kinetics of degradation in the environment, and for assessing contamination of pristine waters, such as those in confined or semiconfined aquifers. Greater specificity can be gained by using magnetic sector mass spectrometers because their resolving power is greater than that of quadrupole spectrometers. Recently Cai et al.34 reported the use of a magnetic sector mass spectrometer to determine sub-ppt levels of atrazine. The herbicide was isolated by solid-phase extraction and quantified by using GC combined with high-resolution mass spectrometry (GC/ HRMS). The detection limit for the analysis of 500-mL groundwater samples was approximately 300 parta-perquadrillion (ppq), 104 times lower than the maximum concentration (3 ppb) allowed by the United States Environmental Protection Agency for potable water. Although GC/MS is appropriate for many environmental analyses, a major challenge is quantifying pollutants, such as hydroxy-containing degradates of parent pesticides. It was estimated recently6 that, among the various chemical pollutants, only 10% are amenable to traditional GC/MS methods because both the GC and the electron ionization (EI) source of the mass spectrometer require that the analyte be in the gas phase. Another challenge is decreasing the analysis time. One approach to overcoming these challenges is to use fast atom bombardment (FAB) MS, a soft ionization technique in which the analyte is dissolved in a vacuum-compatible liquid matrix and the resultant solution is bombarded by kiloelectronvolt-energy atoms or ions.mQ Bombardment leads to the formation of sample-specific ions such as the protonated molecule (positive ion mode) and characteristic fragments. Quantitative applications of FAB ionization for determining nonvolatile molecules have been limited because the absolute abundances of sample ions are not very reproducible from loading to loading. The low reproducibility can be overcome, however, by using an internal standard, either an isotopically labeled molecule or a surrogate. Quantitative applications using an internal standard include the determination of (1) biomolecules,(2) environmental pollutants, such as cationic surfactants," and (3) paint stabilizers.48 The detection limits for these applications range from nanograms (26) Froehlich, D.; Meier, W. J. High Resolut. Chromatogr. 1989,12, 340-342. (27) Stahl, M.; Luehrmann,M.; Kicinski, H. G.;Kettrup, A. 2.Wasser Abwasser Forsch. 1989,22,124-127. (28) Battista, M.; Di Corcia, A.; Marchetti, M. Anal. Chem. 1989,61, 935-939. (29) Kicinski, H. G.Lebemm.-Biotechnol. 1990, 7,159-164. (30) Di Corcia, A,; Marchetti, M. Anal. Chem. 1991,63, 580-585. (31) Barcelo, D. Chromatographia 1988,25,928-936. (32) Durand, G.;Barcelo, D. J. Chromatogr. 1990,243, 275-286. (33) Thurman, E. M.; Meyer, M.; Pomes, M.; Perry, C. A.; Schwab, A. P. Anal. Chem. 1990,62, 2043-2048, and references therein. (34) Cai, Z.; Sadagopa Ramanujam,V. M.; Giblin, D. E.; Gross, M. L. Anal. Chem. 1993,65,21-26. (35) DePauw, E.; Agnello, A.; Derva, E. Mass Spectrom. Rev. 1991,10, 283-301. (36) Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N. J. Chem. Soc.. Chem. Commun. 1981.325-327. (37) Barber, M.; Bordoli,R. S.; Sedgwick, R. D.; Tyler, A. N. Nature 1981,293, 270-275. (38) Barber, M.; Bordoli, M.; Sedwick, R. D. In Biological Mass Spectrometry; Morris, H. R., Ed.; Heyden & Sons: London, 1981; pp
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to micrograms and are higher than those that can be obtained by GC/MS. This is because the abundance of the samplespecific ion of many analytes at low-nanogram levels is not significantly greater than that of the matrix-derived background ion of the same nominal mass. If the interference from matrix-derived background ions can be eliminated, then FAB ionization offers several advantages for the trace analysis of environmental pollutants. First, fragmentation is less extensive than that observed upon electron ionization, especially for amine-containing compounds. These latter compounds, when converted to radical cations by El, fragment extensively as a result of cleavages directed by the nitrogen heteroatom. Second, the analyte does not have to be volatile, only soluble in the liquid matrix. Third, the run time is short compared with GC/MS analyses. In this paper, we report a method that combines the advantages of high-resolution mass spectrometry and FAB ionization (HRFAB-MS) to determine atrazine in groundwater and tap water a t part-per-trillion levels. The method was designed to achieve low detection limits by taking measures to reduce interference from matrix-derived background ions. Specificity was sought by using high mass resolving power and a selective ionization method (i.e., FAB) in place of chromatography interfaced to mass spectrometry. We hoped the design would result in a fast analysis. The results from the HRFAB-MS method are compared to those from the recently reported34 capillary GC/HRMS method.
EXPERIMENTAL SECTION Solvents and Materials. Methanol (HPLC grade), ethyl acetate (pesticidegrade),and dichloromethane (pesticidegrade) were purchased from Fisher Scientific(Pittaburgh,PA). Purified water was obtained by passing tap water through a Fisher Barnstead 4-Module Nanopure cartridge system located at the Water Center of the University of Nebraska at Lincoln (UNL). Anhydroussodium sulfate (analyticalgrade) was purchased from Fisher Scientific, purified by soxhlet extraction with methanol for 48 h, and then heated overnight at 450 O C . Bottled nitrogen gas (dry grade), used for evaporating solvents, was purchased from Air Products & Chemicals, Inc. (Allentown, PA) and transferred through precleaned copper tubing. Matrices for FAB-MS. Solid dithiothreitol (DTT) and dithioerythritol (DTE) were purchased from Aldrich Chemical Co. (Milwaukee, WI), mixed in the ratio 3:l (DTT/DTE, w/w), and heated to 50 "C to form a mixture which, upon cooling to room temperature, remained liquid. Hydroxyethyl disulfide (HEDS),3-nitrobenzyl alcohol (3-NI3A),thioglycerol, and glycerol matrices as well as all other chemicals were reagent grade and used as received. Standard Solutions. Native atrazine was purchased from AccuStandard Inc. (New Haven, CT). Ring-labeled [WaIatrazine, used as an internal standard was specially synthesized for the Water Center at UNL by Merck Frost. Deethylatrazine was purchased from Crescent Chemical Co. (Hauppauge, NY). Synthesized hydroxyatrazine* was obtained from Dr. J. Carr of the Department of Chemistry, UNL.
(42) Beckner, C. F.; Caprioli, R. M. Biomed. Moss Spectrom. 1984,11, 60-62. (43) Lehmann, W. D.; Kessler, M.; Konig, W. A. Biomed. Mass Spectrom. 1984,11, 217-222. (44) Millington, D. S.; Roe, C. R.; Maltby, D. A. Biomed. Mass SDectrom. 1984.11.236-241. Clav. K. L.; Stone, D. 0.:Muruhv, _ - R. C. B'iomed. Mass Spectrom. 1984,1f,.47-49.(45) Chilton, F. H.; Murphy, R. C. Biomed. Mass Spectrom. 1986,13, 71-76. (46) Kusmierz, J. J.; Sumrada,R.; Desiderio, D. M. Anal. Chem. 1990, 62,2395-2400. 137-152. (47) Simms, J.; Keough, T.;Ward, S. R.; Moore, B.; Bandurraga, M. __ . __ _. M. Anal. Chem. 1988,60,2613-2620. (39) Barber, M.; Bordoli, R. S.; Elliot, G.J.; Sedgwick, R. D.; Tyler, (48) Riley, T.L.; Prater, T.J.; Gerlock,J. L.; de Vrries,J. E.;Schuetzle, A. N. Anal. Chem. 1982,54,645A-657A. D. Anal. Chem. 1984,56, 2145-2147. (40)Gaskell,S.J.;Browneey,B.G.;Brooke,P.W.;Green,B.N.Biomed. (49) Skipper, H. D.; Volk, V. V.; Frech, R. J. Agric. Food Chem. 1976, Mass Spectrom. 1983, 10, 215-219. 24,126-129. (41) Gaskell, S. J. Biomed. Enuiron. Mass Spectrom. 1988,15,99-104.
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Stock solutions of native and ring-labeled atrazine were prepared separately in methanol. Standard solutions used for the linear response curve were prepared by mixing different amounts of the native atrazine stock solution with a measured amount of the [13C3]atrazinestock solutionand dilutingto volume so that the concentration of native atrazine ranged from 1 to 1000 pg/pL. For the solutions that contained 1, 5, 10, and 25 pg/pL native atrazine, the internal standard concentration was 50 pg/pL. For the solutions that contained 40,100,250,500, and 1000pg/pL native atrazine, the internal standard concentration was 200 pg/pL. For the 50-pgdata point, solutionswere prepared that contained both 50 and 200 pg/pL internal standard. Standard Water Samples. Standard water samples (500 mL) that were used to determine accuracy and precision were prepared from purified water by adding known amounts of native and [W3latrazine whereas those used to assess the recovery of C-18cartridges were prepared by using only native atrazine; the recovery was determined by HRFAB-MS(videinfra) after adding, just prior to analysis, a known amount of ['3C3]atrazine to the eluants. Isolation of Atrazine from GroundwaterSamples. Atrazine was removed from groundwater by using C-18 bonded-silica solid-phase extraction cartridges, which were obtained from Waters Chromatography. (Millipore, MA; Environmental, lo00 mg), Supelco (Bellefonte, PA; Supraclean LC-18, 200 mg), and Baxter Diagnostics,Inc. (Muskegon,MI; glass cartridge for water matrices, 500 mg). Except where noted in the text, the cartridge from Waters Chromatography was used. Each cartridge was prewashed sequentially with 5 mL each of methanol, dichloromethane, ethyl acetate,methanol, and purifiedwater by passing them through the cartricige at a flow rate of approximately 2 mL/min. Cartridge blanks were obtained by passing 2 mL of ethyl acetate through prewashed cartridges, evaporating the eluant to approximately 20 pL, and analyzing 3 pL of the concentrated solution in 0.2 p L of the DTT/DTE matrix by FAB desorption. Atrazine was isolated from the standard and groundwater samples (500mL) by pulling,by means of water aspirator vacuum, the sample through a prewashed cartridge at a flow rate of approximately 10 mL/min. Prior to aspiration, the internal standard was added so that the final concentration was 5 ppt for low-level samples and 50 ppt for high-level samples. Retained native and [lY!s]atrazine were eluted by passing 2 mL of ethyl acetate through the cartridge at a flow rate of approximately 2 mL/min. Although it was not necessary to remove completely the residual water from the eluant for HRFAB-MS analysis, a water-freesampleis needed for the capillaryGC/HRMSmethod.% Therefore, the eluant was dried over 0.5 g of anhydrous sodium sulfate and then removed from the drying agent and placed into a reaction vial. Preconcentration. The volume of the water-free eluant was reduced to 20-50 pL by using a gentle stream of dry nitrogen obtained from a dedicated cylinder. Samplingand Storageof GroundwaterSamples. Samples of groundwater from a sand and gravel aquifer located southeast of Grand Island, NE,W were collected into precleaned brown bottles by staff of the UNL Water Center. The samples were obtained from a specially installed multilevel sampling system capable of extracting groundwater from discrete 5-ft levels throughout the 100-ft saturation zone;s1 the depth in feet is indicated by the sample identification number. After collection, the bottles were stored in a cold room. Samples of tap water from the Lincoln municipal water supply, which originates as groundwater, were collected at the Department of Chemistry just prior to analysis. Isolation of Atrazine from Tap Water. Tap water samples were collected from a tap in the Chemistry Department, UNL. Water (5 mL) was collected into a glass centrifuge tube and fortifiedwith 5 ng of [W3]atrazine (finalconcentration of internal (50) Spalding,R. F.; Exner, M. E.; Martin, G. E.; Snow, D. D. J.Hydrol., in press. (51)Spalding, R. F.;Exner, M. E.; Burbach, M. E. In Groundwater Residue Samplkg Design; Nash, R. G., Leslie, A. R., Eds.; ACS Symposium Series 465; American Chemical Society: Washington, DC, 1991;Chapter 15,pp 255-261.
standard, lo00 ppt). Five microliters of the fortified water was analyzed directly, without extraction,but the signal-to-noiseratio (S/N) was low. Higher S/N Was obtained by concentrating the atrazine by performing a simple liquid-liquid extraction. Dichle romethane (300 pL) was added to the sample, and the mixture was then vortexed for approximately 2 min. Two microliters of the dichloromethane phase w a ~removed by syringe from the centrifuge tube and analyzed directly. Becausedichloromethane is a suspected carcinogen,the sample loading and the isolation step should be performed in a hood. Mass Spectrometry. A Kratos MS-50 triple analyzef12 equipped with a standard Kratos FAB source and an Iontech FAB gun operated at 6 kV and 1-2-mA discharge current was tuned to a resolution of 10 000 (10% valley definition). The data shown in scans of Figures 1and 2 were acquired by using a Nicolet 1170 signal averager. Data were then transferred to the DS-55 data system (Kratos) and processed with software written in this laboratory. The data shown in the scans of Figures 3, 5, and 6 and the data for Figure 4 were acquired by using the selected ion monitoring program of a Kratos Mach-3 data system. Atrazine standards (1 pL) that were used to determine detection limits and obtain the calibration curve, concentrated eluants (1-3 pL) from solid-phase extractions, liquid-liquid extracts (2 pL) of tap water, and unextracted fortified tap water (5 pL) were analyzed by adding the liquids to the DTT/DTE matrix that had been applied to a small (d = 3.5 mm) copper probe tip. To obtain the data in Figures 1and 2, approximately 1pL of matrix was used, but for all other analyses, the volume of matrix deposited onto the probe tip was 0.2 pL, as measured by dispensing from a 1-pL syringe. The atrazine-containing liquids were mixed with the matrix to form a homogeneous solution. To distribute the matrix uniformly over the probe tip and to avoid loss of the liquid mixture as a result of bumping, any residual volatile solvent was allowed to evaporate for 20-30 s at room temperature before inserting the probe into the vacuum lock of the spectrometer. After evacuation, a thin, shiny layer of matrix containing the analyte remained. Best results were obtained when the copper probe tip was clean so that the matrix could wet the surface. The probe was rinsed with water and acetone between sample runs;the tip was polished flat and cleaned by immersion in a nitric acid solution prepared by diluting the concentrated acid by 50 % . Quantification. Mass profiles for the ions of [atrazine + HI+ (m/z 216.1016), [[S'ClIatrazine + HI+ (m/z 218.0988, confirmation ion), and [[Wslatrazine + HI+ (m/z 219.1117, internal standard) were collected by using a Kratos Mach-3 data system. The width of the mass window used to monitor each ion was 300 ppm (0.06 u). Either the peak areas or the peak heights for the [atrazine + HI+and [ [W~latrazine + HI+ions were used to quantify atrazine. Peak areas were obtained from raw, unsmoothed data by summing the intensity of the whole peaks, and the peak heights were determined by summing the intensity of only the data points at the top of the peak. The exact locations of the peak positions were determined at the beginning and the end of each set of samples by analyzing standards that contained 200 pg each of native and labeled atrazine; the same locations were used to process all data within arun. Specifically, for Figure 3 peak areas were measured, and for Figure 5 peak heights were measured. A relative response factor (RRF)was used for the quantitative analysis to improve the accuracy of the method RRF = (A,C,)/(A,C,) (1) where A, and A, are the peak areas of the [atrazine + HI+ and [[W3]atrazine + HI+ ions, respectively, and C, and C, are the concentrations of atrazine and [Wslatrazine, respectively, in the standard solutions. The RRF was obtained by analysisof the calibration solutions. The relative standard deviation (RSD) for the RRF of 1.07 is 8.3% for nine determinations. The RRF was checked periodically throughout the development of the method and did not vary by more than 10% from 1.07 at any time. (52)Gross, M.L.; Chess, E. K.; Lyon, P. A.; Crow, F. W.; Evans,S.; Tudge, H. Int. J. Mass Spectrom. Ion Phys. 1982,42, 243-254.
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The concentration of atrazine in the samples was calculated by the formula C = [CL(A/A,)I/RRF
(2) where C is the concentration of atrazine in the sample and A is the peak area for the [atrazine + HI+ ion. For sample in which peak heights were used to determine the abundance of the ions, the peakareas in the equationswere replaced by the peak heights.
RESULTS AND DISCUSSION The specific objective of this research was to develop a rapid, selective,and sensitiveHRFAB-MS method to quantify ppt levels of atrazine and its degradates, which are found in groundwater. High-resolution fast atom bombardment mass spectrometry would be a powerful addition to the repertoire of analytical methods, especiallyfor the polar degradates (e.g., hydroxyatrazine). The use of an ionization technique that produces a background and does not incorporate on-line chromatographic separation, however, requires that certain problems be addressed; examples are the interferences from matrix-derived background ions and from the solid-phase extraction procedure. Background Ions and Choice of Matrix. The bombardment-induced chemistryleadingto background ions bears on the choice of a matrix for trace level detection of analytes. The development of the method reported here builds on the results of fundamental studies of the origin of background ions from glycerol.5~7 The matrix chemistry leading to background ions determines their elemental composition and hence their exact mass. If a matrix can be chosen in which the masses of the background ions differ significantly from those of the analyte ions, then analyte ions can be resolved from those of the matrix by using a practical resolving power. The exact mass of an isotope of an element can be compared to the rounded, integer mass to assess the mass defect.% Isotopes of elements with atomic numbers lower than or equal to seven (i.e., nitrogen) have a positive mass defect, and those of elements higher in atomic number have a negative mass defect-the exact mass is less than the integer value-and the magnitude of the defect tends to increase up through the fifth row of the periodic table. Most analytes and FAB matrices are composed of C, H, N, and 0. Therefore, background ions are typically not resolved from those of the analyte by using low to moderate resolving power, and it is often difficult to resolve them by using high resolving power (210 OOO), which accounts for the relatively high detection limits reported previously. For atrazine, which has the empirical formula CeH14N5C1, background ions from glycerol and 3-nitrobenzylalcohol matrices overlap in mass completely with that of the protonated molecule (Figure 1);the negative mass defect of the C1 atom is offset by the positive mass defect of the N and H atoms so the analyte ion is not shifted significantly to lower mass. Because third-row elements such as sulfur have negative mass defects, matrices containing them are good candidates for producing background ions with masses that are lower (53) Caldwell, K. A,; Gross, M. L. Identificationand Characterization of Matrix Background Ions in FAB. Proceedinge of the 38th ASMS
Conference on Mass Spectrometry and Allied Topics, Tucson, AZ,June 3-8,1990. (54) Caldwell, K. A.; Groes, M. L. Matrix Chemistry and Background Ions in FAB and Liquid SIMS. Proceedii of the 39thASMS Conference on MW Spectrometry and Allied Topics, Nashville, TN, May 19-24, 1991. (55) Caldwell, K. A.; Gross, M. L. 3. Am. SOC. Mass Spectrom., in press. (56)Field, F. H. 3. Phys. Chem. 1982,86,5115-5123. (57) Keough, T.; Ezra, F. S.; Russell, A. F.; Pryne, J. D. Org. Mass Spectrom. 1987,22,241-247. (58)Williams, W. S. C. Nuclear and Particle Physics; Clarendon Press: London, 1991; p 106.
I
3-NBA mb 216
m/z Flgure 1. Narrow scans (1000 ppm sweep width) of the background ions of nominal mass 216 u from (A) glycerol and (B) 3-NBA. The volume of matrix was approximately 1 pL. Tic marks denote the exact mass of Iatrazine H]+ (m/z 216.1016).
+
than those of most analyte ions. Moreover, for sulfurcontaining matrices such as thioglycerol and DTT/DTE, HzO loss is more facile than H2S 10ss,5~?~9 and the background ions tend to retain the sulfur atoms. For thioglycerol, 2-hydroxyethyl disulfide, and DTT/DTE matrices, the background ions of m/z 216 are shifted to lower mass than those of the same nominal mass fromglyceroland 3-NBA (Figure2A-C). Thus, the absolute abundance of the matrix background ions at the [atrazine + HI+ ion region is low, and low-picogram levels of atrazine can be detected (Figure 2D). The DTT/DTE and HEDS matrices contain two sulfur atoms, and as a result, the background ions from these matrices are shifted to lower mass than those from the thioglycerol matrix, which contains only one sulfur atom per molecule. The amount and abundance of the background ions from HEDS at the [atrazine HI+ ion region are lower than those from DTT/DTE. A possible reason is that DTT/DTE-derived background ions can lose HoS and produce background ions that contain only C, H, and 0, like those from glycerol. The HEDS-derived background ions, however, are likely to contain sulfur atoms as disulfide or thioether functionalities, and losses of sulfurcontaining neutrals are not facile. HEDS was not selected as matrix because of the ionization efficiency of the analyte is lower than that for DTT/DTE (seesection on Samplingand IonizationEfficiencyfor details). Because the same number of sweeps comprised each scan in Figures 1 and 2 and because each was normalized to the most abundant data point, the noise level indicates the magnitude of the ion abundances. Although there were background ions from DTT/DTE at the [atrazine + HI+ ion region, they were much less abundant than those from either the glycerol or 3-NBA matrices. If the scan in Figure 2C were normalized to that in Figure lA, then the peaks from the background ions of DTT/DTE would be just above the baseline. Effect of Volume of Matrix. Analytes dissolved in the FAB matrix can be viewed as being partitioned between the surface region sampled by the fast atom beam and the underlying bulk liquid. As the bulk concentration of the analyte is increased, more sample will be distributed to the surface region, regardless of whether or not the analyte is hydrophobic, and the abundance of the sample-specificions
+
(59) Caldwell, K. A,; Gross, M. L., unpublished resulta.
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7 Thioglycerolm k 216
atrazine is partitioned more into the bulk of the HEDS matrix). Second, the ability of the HEDS matrix to promote protonation during bombardment is lower than that of the DTT/DTE matrix. The DTT/DTE matrix provided the lowest detection limit for atrazine, even though the mass regions were almost completely free of background ions for the HEDS matrix. If ultimate detection limits are not required, then the HEDS matrix can also be used because it provides cleaner mass windows. Detection Limits and Linear Response. The lowest amount of atrazine detectable and the linear response were determined from standard solutions that were loaded directly onto the FAB probe. Using either peak areas (Figure 4) or peak heights (not shown), we found the response was linear from 1pg to nearly lo00 pg. Because the contribution of the unresolved background ion to the signal became significant at very low levels, the data points less than or equal to 50 pg were obtained after subtracting a matrix blank. The background ion signal was not subtracted from the data points for the higher levels because its contribution to the [atrazine HI+signal was low. Two calibration curves were obtained approximately 1month apart, and the slopes of the curves are not significantly different. The detection limit of 1pg should not be considered routinely achievable. It was necessary to take great care in loading the sample, to have source slits in good condition, and to have a well-aligned and relatively clean source. Nevertheless,the ultimate detection limit for the standards translates ideally-for complete transfer of the analyte in the final eluant and for 100% recoveries-to a detection limit of 1ppq for the analysis of 1L of water. For real samples, however, interferences and incomplete recoveries will raise the detection limit. Analysis of water more pristine than that analyzed here will require low-ppt to high-ppq detection limits. Our experience thus far with water collected from a confined aquifer indicates that, fortuitously, the level of interferences is much lower than that from surface waters, which allows the instrument detection limit to be achived. Isolation of Part-per-TrillionLevels of Atrazine from Water. Three types of water samples were analyzed for atrazine: standard water, groundwater from Grand Island, NE, and tap water from Lincoln municipal supply line. Atrazine from the water samples was isolated by using C-18 solid-phase extraction. Because the sensitivity of the HRFAB-MS detection scheme was high and because there was no additional separation step (e.g., GC or LC), cartridges for removingthe atrazine had to be evaluated to determine which cartridge gave rise to the lowest abundance of interfering ions at the [atrazine + HI+, [[37C11atrazine+ HI+,and [['w31atrazine + HI+ ion regions. Of the three commercially available cartridges tested, the one from Waters Chromatography produced lower abundance interferences than those from Baxter Diagnostics, Inc. and from Supelco. The recoveries for the Waters cartridge ranged from 60 to 80% for standard water samples containing 1 ppt atrazine. Both dichloromethane and ethyl acetate can be used to elute efficiently the atrazine from the C-18 cartridges?@but the interferences were less when ethyl acetate was used. For both solvents, the blanks obtained by reducing the volume of the pure solvents 1000-fold-10 times more than the volume reduction of the atrazine-containingeluants-were relatively free of interferences. Quantification. Recently, Tong et alP demonstratedthat mass profile monitoring mode is superior to peak top monitoring mode for quantitative mass spectrometry. The
+
I
4 2
Flgure 2. Narrow scans (1000 ppm sweep wldth) of the background ions of nominal mass 216 p from (A) thioglycerol, (6)hydroxyethyl disulfkle,and (C)D?l/DTE matrlces. The spectrum of 20 pg of atrazine in DTTlDTE is shown in D. The volume of matrix for each scan was approximately 1 pL. Tic marks denote the exact mass of [atrazine H]+ (mlz 216.1016) (Le., where the peak center should appear).
+
should increase, whereas the abundance of the matrix-derived background ions should remain approximately constant up to the point at which the surface is saturated and then decrease. The abundances of the background ions within the mass windows monitored in this high-resolution method were approximatelyconstant for 0.1-l.0pL of matrix (Figure 3; the scansfor 0.1 pL of matrix are not shown). For a constant amount of analyte, however, the absolute abundance of the sample-specific ions increased as the volume of the matrix was reduced from 1.0 to 0.2 pL,in proportion to the increasing concentration of the atrazine in the matrix. Below 0.2 pL, however, the abundance of the protonated atrazine ions decreased, apparently because there was not enough matrix to promote efficient ionization. Sampling and Ionization Efficiency. Although DTT/ DTE and HEDS are preferred over glycerol or 3-NBA for trace-level detection of atrazine, the absolute abundance of the [atrazine + HI+ion was approximately a factor of 2 lower for the HEDS matrix than for the DTT/DTE matrix for the same volume of matrix (0.2 pL) and for the same amount of atrazine. This is likely due to two factors. First, atrazine is more surface active in the polyhydroxy DTT/DTE matrix than in the less polar, more hydrophobic HEDS matrix (Le.,
(60) Shepherd, T. R., Can,J. D.J. AOAC Int., in preaa. (61) Tong, H.Y.; Giblin, D.E.;Lapp, R. L.; Monaon, S. J.; Groae, M. L.Anal. Chem. 1991,63,1772-1780.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, 1993
loo
1
DTTDTE (0.2 uL)
216
218
I
219
216
218
219
216
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219
m/z
m/z
m/z 100 3
2977
I
216
218
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216
218 m/z
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m/z
Flgure 3. Narrow scans of the background ions from the DTTIDTE matrix that appear wlthin the mass windows monltoredto detect the protonated molecules of native atrazine and [13C3]atrazine(top scans) and the spectra obtained after adding 1 ML of a standard solution (100 pglpL native atrazine and 200 pg/pL [ 13C3]atrazine)to the same volumes of matrix (bottom scans). Each spectrum Is a composite of three 300 ppm narrow scans that were centered at mlz 216.1016, 218.0988, and 219.1 117, respectively. The line with darkened circles on the ends indicatesthe mass range over which the intensity was summed to obtain the peak area.
p
15
5
-C p.-
+I
v
10
+I
m
z $ 5
0 200 400 600 800 10001200
Native Atrazine (pg) Figure 4. Response curve for atrazine obtained from the peak areas of the [atrazine i- H]+ and [[I3C3]atrazine H]+ ions detected by using HRFAB-MS. The inset is the region from 1 to 100 pg. Standard solutions ( 1 pL) contalnlng varying amounts of native atrazine and either 200 (open circles) or 50 pg (black circles) of [ I3C3]atrazinewere added to 0.2 pL of a DTTIDTE matrix. Each data point is the average of two runs, and the data points below 50 pg were obtained by subtracting the matrix blank before measuring the peak areas. The correlation coefficient for this response cure was 0.9980.
+
contribution of unresolved ions to the signal can be assessed directly by the former, but not by the latter. Mass profile monitoring mode provides the additional benefit that either
peak areas or peak heights can be used to determine the abundance of the monitored ions. For interferencefree peaks, peak areas provide greater precision because a greater fraction of the peak is used to determine the magnitude of the signal. If there is an incompletely resolved interference, however, using the heights of the centers of the peaks reduces the contribution of the interference, providing more accurate but slightly less precise data. Because interferences for the standard water and tap water samples were low, either peak areas or heights can be used, whereasfor groundwatersamples, the interferences were more abundant, and peak heights were used exclusively. The accuracy and precision of the overall method for pptlevel analyses were determined by using six 500-mL standard water samples containing 0.5 ng of native atrazine (1.0 ppt) and 2.5 ng of ['3C3]atrazine (5.0 ppt). The atrazine was isolated from each sample by using solid-phase extraction, and the concentrated eluant was analyzeddirectly by HRFABMS (see Table I for results). The precision of the method is good. When peak areas are used, all the results are within 10% of each other. When peak heights are used, the precision is worse, which ia expected because less scans are used for quantification. The accuracy of the method is also good. The relative percent error is better than 10% for standard water samples. Comparison of FAB and Capillary G-C/HRMSResults. Groundwater samples were obtained from eight wells of different depths. Someof the atrazine levels were determined previously to be below the detection limits of quadrupolebased GC/MS methods. By using HRFAB-MS,however, atrazine could be detected. The levels range from 6 to 94 ppt
Table I. Results for the Analysis of Standard Water Samples Containing 1 ppt Native Atrazine fortified found' foundb (PPt) AIAi (PPt) HIHi (ppt) 1.0 1.0 1.0 1.0 1.0 1.0
0.22 0.23 0.24 0.24 0.22 0.24
1.0 1.1 1.1 1.1 1.0 1.1
*
0.25 0.25 0.25 0.23 0.20 0.26
1
1.2 1.2 1.2 1.1
0.9 1.2
1.1 & 0.06 5
mean SDC RSD,d (% )
loo 80
1.1
0.1 10
0 From peak areas. b From peak heighta (see text for discussion). SD, standard deviation. d RSD, relative standard deviation.
216
218
219
216
m/z
Table 11. Average Levels of Atrazine (ppt) in Groundwater Determined by Using HRFAB-MS and the Capillary GC/HRMS Method. capillary capillary GC/ sample HRFABGCI sample HRFABID MS HRMS ID MS HRMS MLE75 MLESO
mEs5 MLESO a
94 71 21 13
132 107 22 13
MLE95 MLElOO MLE105 MLEllO
40 15 6 11
219
Figure 5. Trace-level detectlon of atrazine in groundwater by HRFABMS. Peak helghts were used for the detectlon of Intensities. The mass range over whlch the Intensity was summed was reduced to Includeonly the center of the peak, as indlcated by the almost complete overlap of the darkened circles that occurs at the ends of the line that Indicates summation range.
n j.
3
22 16 3 12
Method reported recently by Cai et al." See text for discussion.
(Table 11). Because the run time for the FAB-MS method was short, all determinations were readily made in duplicate, and the two values agree within 20%. The precision for the real samplesis worse than that for the standard water samples because some interferenceswere not completely resolved from either the [atrazine HI+ or the [[Wslatrazine + HI+ ions. For most samples, it was only necessary to load 1p L of the concentrated eluant onto the probe, but for low-levelsamples, the S/N could be improved by simply loading more of the eluant. To check the accuracy of the HRFAB-MS method, the concentrated eluants were also analyzed by using capillary GC/HRMS (see Table I1for results). The HRFAB-MS results comparewell with those from the more conventionalmethod. For samples relatively free of interferences, the agreement is within a few percent. For samples that produce incompletely resolved interferences, either at the [atrazine HI+ or the [[Ws]atrazine+ HI+ionregions (seeFigure5),thedifferences are larger, but do not exceed a factor of 2. NonvolatileDegradates. Unlike electron ionization used in most GUMS applications,FAB ionization is readily applied to polar, nonvolatile analytes and potentially allows for simultaneous detection whereas a GUMS method may require changing columns and derivatizing analytes such as atrazine and ita degradates. The degradation products of atrazine, which may also be metabolized by microorganisms, include hydroxyatrazine,'32*athe principal species, and deethylatrazinea (structures I1and 111). Determinationof these
218 m/z
2
3
1(
+
+
(cHJ,c"HyNyoH
NYN HNCH,CH,
(CH3)2C"HyNyC1
NYN "2
I1
188
198
m/z
216
188
198
216
m/z
Figure 8. Narrow scans (300 ppm sweep width) of protonated (1) deethylatrazine (mlz 188.0703), (2) hydroxyatrazine(mlz 198.1355), and (3)natlve atrazlne (mlz 2 18.10 18) detectedafter loadlnga solution that contained (A) 1 and (B) 10 pmol of each component.
than the parent compound, and hydroxyatrazine chromatographs poorly.22 Two standard mixtures containing equimolar amounts (1 and 10pmol each) of atrazine, hydroxyatrazine,and deethylatrazine were analyzed to demonstrate the feasibility of detecting atrazine and its less volatile degradates in one analysis (see Figure 6). Although all species were not represented equally, each was readily detected above the background. Discrimination of sample components in the analysis of mixtures of FAB-MS was reported previously for peptideand fatty acid mixtures,'37and the discrimination that occurred for the atrazine mixture parallels that for the peptides and fatty acids; that is, at low concentrations of the analytes in the matrix, less surface-active, more hydrophilic components were discriminated against. It is possible that solubility differences also contribute to the discrimination,
111
degradationproducts is difficult because both are less volatile (62) Dao, T. H.; Lavy, T. L.; Sorensen,R. C. Soil Sci. SOC.Am. 1979, 43,1129-1134. (63) Skipper, H. D.; Volk,V. V.; Frech, R. J. Agric. Food Chem. 1976, 24,126-129.
(64)Clench, M. R.; Gamer, G. V.; Gordon, D. B.; Barber, M. Biomed. Mass Spectrom. 1985,12,355-357. (65) Naylor, 5.;Findeis, F. A.; Gibson, B. A,; Williams, D. H. J. Am. Chem. SOC.1986,108,6359-6362. (66)Caprioli, R. M.; Morre, W. T.; Fan, T. Rapid Commun. Mass Spectrom. 1987, I, 15-18. (67) Caldwell, K. A.; Grow, M. L. Anal. Chem. 1989,61, 496-499.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, 1993
but these can be reduced by acidifying the matrix. Regardless, the detection limits for the more polar components of the mixture are higher than that for native atrazine. Analysis of Tap Water. The analysis of Lincoln tap water by using the FAB-MS method is very simple owing to the sensitivity of the method. Solid-phase extraction and preconcentration were used to prepare one sample so that the results can be compared with those from the capillary GC/ HRMS method. For both methods, the sample signals were high and the interferences were relatively low, and the agreement for data obtained by the two methods is very good (493 ppt by HRFAB-MS compared to 463 ppt using GC/ HRMS) Subsequently, two simpler and faster methods of sample preparation were used (1)fortification of [lsCdatrazine (5 ng) into the tap water (5 mL) and analysis of the sample directly (loading 5 pL to the probe tip) without extraction or (2) simple vortex extraction of the fortified tap water ( 5 mL) with dichloromethane (300 pL) and analysis of the dichloromethane phase (2 pL onto the probe tip). The levels of native atrazine (method 1,446 ppt; method 2,501 ppt) in the tap water sample were found to be in agreement with the value obtained by using (2-18cartridge extract (493ppt). Even though the quantified atrazine level was comparable in these two sample preparation methods, it should be emphasized, however, that the signal-to-noiseratio was worse in the sample prepared by method 1. For a fast and accurate analysis, the sample preparation method 2 is highly recommended. Although the volume of dichloromethane was approximately 17 times lower than the volume of water, analysis of the aqueous phase indicated that most of the atrazine was transferred to the organiclayer. Becausean internal standard was used, the recovery does not have to be 100% to assure accuracy.
.
2379
poratingelectron ionization: (a) lower detection limits (1pg), (b) faster throughput (no chromatographic separation step), (c) no apparent effect from traces of water, acids, and polar solvents, (d) no derivatization needed for the polar analytes investigated here, (e) less fragmentation than from EI, and (f) no significant memory effects. The principal disadvantage of the HRFAB-MS method is that it is more susceptible to interferences. In groundwater and tap water, the interferences should be small. For the most contaminated samples encountered here, the error resulting from incompletely resolved interferences is not significantly greater than the normal error associated with a trace analysis. The specificity afforded by a gas chromatographic separation step is compensated for, in part, by the specificity inherent in the FAB ionization method. Analytes with basic sites are more readily ionized (by protonation) than are ethers, esters, acids, and, especially, nonpolar interferences such as hydrocarbonsand halogenatedmaterials. Moreover, there is a reduction in fragment ion interferences when the analyte ions are detected at high mass resolving power. If additional specificity beyond that provided by highmass resolution is needed, one could turn to MS/MS and selected reaction monitoring. Preliminary results for the analysis of a sample containing a mixture of atrazine, hydroxyatrazine, and deethylatrazine suggest that the FAB-MS method can be applied to simultaneous determinations of the products of atrazine degradation in the environment.
ACKNOWLEDGMENT We thank Professor J. D. Carr, Department of Chemistry, UNL, for the sample of hydroxyatrazine standard. The research was supported by the UNL Water Center and by the National Science Foundation (DIR 9017262).
CONCLUSIONS The HRFAB-MS method for trace analysis of atrazine has several advantages over traditional GUMS methods incor-
RECEIVEDfor review November 20, 1992. Accepted May 17, 1993.
