Anal. Chem. 1994,66,4202-4209
Liquid-Solid Extraction and Fast Atom Bombardment High-Resolution Mass Spectrometry for the Determination of Hydroxyatrazine in Water at Low-ppt Levels Zongwei Cai, V. M. Sadagopa Ramanujam, and M. L. Gross*,+ Midwest Center for Mass Spectrometry, Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304
S. J. Monson, D. A. Cassada, and R. F. Spalding Water Center, University of Nebraska, Lincoln, Nebraska 68583-0844
Parts-per-trillionlevels of hydroxyatrazine (HA) have been quantitatively extracted and eluted from water by liquidsolid extraction with a graphitized carbon-blackcartridge. Recoveries of HA (> 80%)for this procedure are superior to those obtained when a C-18 cartridge or liquid-liquid extractionwith dichloromethane are used. Quantification of HA at picogram levels was accomplished by using isotope dilution and fast atom bombardment high resolution mass spectrometry. No significant interfering spectral background occurs at the masses of interest, those of the [M HI+ ions of both the analyte and the internal standard, when the liquid matrix is 3:l dithiothreitoll dithioerythritol. Aqueous samples were forti6ed with l3C3-1abeled hydroxyatrazine ([13C31HA), and sample extracts were analyzed by recording the mass profiles of the target ions. Identification of HA is based on the exact mass of the [M HI+ ion, and confirmation is by tandem mass spectrometry (FAB-MS/MS) analysis. The method precision in relative standard deviation is lo%,and the accuracy in relative error is 99%purity) was obtained from Dr. James Carr of the Departcartridge are poor.38 Recoveries of over 85%for HA at ppb levels ment of Chemistry, University of Nebraska-Lincoln. Ring-labeled in fortified water have been reported by using a cyclohexyl silica [13C31HAwas synthesized via hydroxylation of [13C31ATRaccordcartridge3l and a propylbenzenesulfonic acid cation-exchange ing to the protocol provided by Dana Simmons (Ciba Geigy), for cartridge.32 Adoption of graphitized carbon-black cartridge extracsynthesizing native HA from ATR tion has provided good recoveries for atrazine and its dealkylation A mixture of 110 mg of [13C31ATR1.5 mL of concentrated HCl, metabolite~,3~-~~ as well as for other polar comp0unds,42~4~ but this and 1 mL of HzO was heated to reflux at 110 "C for 3 h. Upon approach has not bee applied to HA. cooling to room temperature, the reaction solution was diluted Fast atom bombardment (FAB), a soft ionization technique in with 20 mL of HzO, and 5 mL of aqueous NHIOH (4.5 M) was mass spectrometry, offers several advantages sufficient to meet added. A solid precipitate formed. The mixture was stirred for 45 min. After filtration, the solid [13C3]HAwas washed with 1 (31) Steinheimer,T. R; Ondrus, M. G. Water-Resour.Invest. Rep. (US.Geol. Sum.) 1990,89-4193. mL of water and 1mL of acetone and dried in a vacuum desiccator (32) Lerch, R N.; Donald, W. W. J. Affn'c. Food Chem. 1994,42,922. over PzOS. The chemical purity of the synthesized [13C31HAwas (33) Cochrane, W. P., J. Chromatogr. Sci. 1975,13, 246. approximately 95% according to a HPLC determination. An (34) Khan, S. U.; Greenhalgh, R; Cochrane, W. P. J. Agric. Food Chem. 1975, 23, 430. isotope purity of >99%for the synthesized [13C3]HAwas measured (35) Bushway, R J.; Perkins, B.; Savage, S. A.; Lekousi, S. J.; Ferguson, B. S. by both electron ionization (ED MS and FAB-MS analyses. Bull. Enuiron. Contam. Toxicol. 1988,40, 647. Stock standard solutions of native and 13C3-labeledHA were (36) Schlaeppi, J. M.; Werner, F.; Ramsteiner, K. 1.Agn'c. Food Chem. 1989, 37,1532. prepared separately in methanol. Solutes (2 mg) were weighed (37) Thomas, D. H., Beck-Westemeyer, H.; Hage, D. S., submitted for publication accurately and added to 50 mL of methanol. The solution was in Anal. Chem. sonicated and warmed to 50 "C to completely dissolve the (38) Cai, Z.; Spalding, R F.; Gross, M. L. GC/HRMS and FABHRMS Determinations of Atrazine and its Dealkylated and Hydroxylated Roducts in Ground compound. The stock solutions of HA and [13C31HAwere further Water. To be presented at the 42nd ASMS Conference on Mass Spectromdiluted with methanol to obtain the calibration solutions and a eby and Applied Topics, Chicago, IL, May 29, 1994. fortifying standard solution of 400 pg/pL. (39) Di Corcia, A; Marchetti, M.; Samperi, R 1,Chromatog, 1987,405, 357. (40) Di Corcia, A; Marchetti, M. Anal. Chem. 1991,63, 580. A fortifying standard solution of [l3C31ATR at 100 pg/pL was (41) Di Corcia, A; Samperi, R.; Marcomini, A; Stelluto, S. Anal. Chem. 1993, also prepared for the determination of ATR in water. The details 65, 907. for the calibration and determination of ATR by FAl3-HRMS were (42) Borra, C.; Di Corcia, A; Marchetti, M.; Samperi, R Anal. Chem. 1986,58, 2048. (43) Andriolini, F.; Borra, C.; Caccamo, F.; Di Corcia, A; Samperi, R Anal. Chem. 1987,59, 1720.
(4Caldwell, ) K. A; Sadagopa, R V. M.; Cai, Z.; Gross, M. L.Anal. Chem. 1993, 65. 2372.
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described elsewhere.u A standard solution containing 100 pg/ pL ATR and [13C3]ATR and 20 pg/pL HA and [W3]HA in methanol was prepared and used frequently to check the relative response factor (RRF). The matrix for the FAB-HRMS analysis was a mixture of dithiothreitol @'IT)and dithioerythritol @TE) (3:1, w/w), both of which were purchased from Aldrich Chemical Co. (Milwaukee, WI). The solid mixture was liquified by heating to 50 "C, and the mixture remained liquid upon cooling to room temperature. Water Samples. Standard water samples were prepared by fortifying puritied water with a native HA standard solution to obtain levels of 5, 100, and 500 ppt. Groundwater was pumped into amber bottles from multilevel samplers installed to depths of 60 ft in a sand and gravel aquifer located near Shelton, NE. The multilevel sampling system is capable of extracting groundwater from discrete levels throughout the 45ft saturation The samples were stored at 4 "C until analyzed. After addition of the internal standards, the water samples were extracted by the liquid-solid procedure using carbon-black cartridges. Liquid-Iiquid E.rrtraction Using Dichlommebne. Iiquidliquid extraction recoveries of HA with dichloromethane were determined from analyses of water samples fortified with 5 and 500 ppt of native HA. The fortified water samples (200 mL) were each extracted three times with dichloromethane (35 mL each). The extracts were combined and dried with anhydrous sodium sulfate. The [*3C3]HAinternal standard was added: 2 ng for the samples containing native HA at 5 ppt and 100 ng for those containing HA at 500 ppt. The solution was then concentrated by a slow stream of nitrogen to 20 pL for FAB-HRMS analysis.
Liquid-Solid Extraction Using the (2-18 Bonded-Silica Cartridge. The recovery of HA in the procedure using liquidsolid extraction via the C-18 bonded-silica cartridge was determined by analyzing standard water samples (each 200 mL) containing 5 and 500 ppt of HA. The detailed procedures of the C-18 cartridge extraction were reported elsewhere." Liquid-Solid Extraction Using the Graphitized CarbonBlack Cartridge. The liquid-solid extraction via a graphitized carbon-black cartridge was carried out for samples of standard water and groundwater. The carbon-black cartridge was prewashed sequentially with 2 mL each of dichloromethane, dichloromethane/methanol (80:20, v/v), methanol, and puritied water. A sample (200 mL, except as noted in the text) was fortilied with [13C31HAat levels of 10 or 500 ppt for standard water and 100 ppt for groundwater. The sample was drawn through the prewashed carbon-black cartridge at a flow rate of 15-20 mL/min by applying aspirator vacuum. The cartridge was first eluted with 0.5 mL of ethyl acetate to remove residual water. The analyte and the internal standard were then eluted by 5 mL of dichloromethane/ methanol (80:20, v/v). Residual water in the ethyl acetate eluant was removed by adding anhydrous sodium sulfate. Both ethyl acetate and dichloromethane/methanol eluants were combined and evaporated to 20-50 pL, depending on the analyte concentrations, by using a slow stream of nitrogen. For FAE&MS/MS analysis, 1L of a groundwater sample ( 1 M U 15) was extracted in order to obtain sufficient HA. The extraction procedure and volume of eluants were same as those described (45) Spdding, R F.; Exner, M. E. In Groundwater Residue SamplingDesign; Nash, R G., Leslie, A. R, Eds.: ACS Symposium Series 465; American Chemical Society: Washington, DC. 1991: Chapter 15, p 255.
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above. Breakthrough in the carbon-black cartridge extraction was determined with 2 L of standard water containing 100 ppt HA. Method blanks were obtained by passing 0.5 mL of ethyl acetate and 5 mL of dichloromethane/methanol (80:20, v/v) through prewashed carbon-black cartridges. The blank eluant was evaporated to approximately 20 pL and analyzed by FAB-HRMS to test for potential interfering compounds from the cartridge and the solvents. Safety: Because dichloromethane is a suspected carcinogen, all sample preparations were performed in a hood. Full Mass Spectra from EI-HRMS. The EI-HRMS analysis of native HA and [13C31HAstandards was conducted on a Kratos MS50 doublefocusing mass spectrometer. Instrument operating conditions were E1 at 70 eV, source temperature 250 "C, and resolution 10 000 (10%valley definition). The sample was introduced to the ion source via the direct insertion probe and heated to 150 "C. Spectra of 5-10 scans over the mass range m / z 50500 were acquired and averaged with a Kratos Mach-3 computer system. FABMS. Positive FAB-MS analyses of native and l3Cr1abeled HA were performed on a Kratos MS50 triple-analyzer @BE configuration) tandem mass spectrometer equipped with a Kratos FAB source, an Iontech FAI3 gun operated at 6 kV and 1-2 mA emission current, and a Kratos Mach-3 data system. The matrix was the D?T/DTE mixture. Quantitative determination was conducted on the Kratos M S 50 triple-analyzer instrument. The resolution was 8000-10 000 (10%valley definition). Data were acquired in the mass profile mode46by using the selected ion monitoring (SIM) program in the Mach-3 system. Narrow scans (300 ppm sweep width) over the following ions were obtained with the SIM program: [HA HI+ ( m / z 198.13551, [[13C31HA HI+ (m/z 201.1456), [ATR HI+ (m/z 216.1016), and [[13C31ATR + HI+ (m/z 219.1117). Typically, the first 10 scans were acquired and summed for each mass profile spectrum. One microliter of the sample extract was placed in 0.5 pL of D'IT/DTE matrix that had been loaded onto the FAB probe tip. The mass profile spectrum obtained from a sample analysis was subtracted from the matrix background of 0.5 pL of D'IT/DTE. The exact mass and peak intensity were used for qualitative and quantitative analysis, respectively, of ATR and HA. The quantitication of ATR was performed by comparing the peak areas of the [ATR HI+ and [[13C31ATR+ HI+ ions. The peak areas of [HA + HI and [ [13C31HA + HI ions were used for the determination of HA. The calibration solutions of HA/[13C3]HAwere prepared in methanol at levels of 10-1250 pg/pL HA and 100 pg/pL [W3]HA. The solutions were analyzed periodically throughout the method development and sample analysis to establish that data were within the calibration range. FABMS/MS. The collision activated dissociation (CAD) experiments were performed on the same mass spectrometer used for the quantitative FAB-HRMS analyses. The [HA HI+ ion ( m / z 198.1355) was selected and activated in the third field-free region by collisions with helium to give 50%beam reduction. Typically 10-15 scans were signal-averaged for each CAD spectrum.
+ +
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Ill
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142.'
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Figure 1. High-resolution El mass spectra of (A) native hydroxyatrazine and (B)[13C3]hydroxyatrazine. These spectra are low-resolution outputs of high-resolution spectra.
RESULTS AND DISCUSSION
Mass Spectrometry Analyses of HA and [13C31HAStandards. The E1 mass spectra of native HA and [13C31HAare shown in Figure 1. The fragmentation of HA occurs by elimination of CzH4, C3Hs, and CzH4 C3Hs from the molecular ion to give product ions of mlz 169, 155, and 127 via McLafferty rearrangements. The molecular ion also loses CH3 to give an abundant m/z 182 species that decomposes to mlz 139 by elimination of CzHsN. The abundant fragment of m/z 112 may form by loss of CHNO from the m/z 155 ion. This is confirmed by the spectrum of the [13C3]HA; the m / z 112 ion shifts to mlz 114 (the loss is now 13CHNO)and the m/z 155 ion shifts to m/z 158. We expect the homologous mlz 169 ion to give mlz 126 ion, and for the labeled compound, these ions should shift to m/z 172 and 128. This is observed. The above fragmentations, which are in accord with the mass shifts seen for the 13C-labeled compound, were
+
confirmed by measuring the exact masses of the ions to obtain the atom compositions; the mass deviations were less than 6 ppm. The mass spectra illustrate that the isotope purity of both native and labeled standards is high (299%). The mlz 214.1542 peak on the mass spectrum of [ 13C3]HA(Figure 1B) indicates that the labeled standard contains a chemical impurity. The scanning FAR MS spectrum of [13C31HA (not shown) further confirms an impurity exists in the standard. The impurity gives an ion of m/z 215 in the positive ion spectrum, indicating that the major contaminant has a molecular weight of 214 and is probably [13C31hydroxypropazine. The amount of the impurity, however, cannot be determined with high accuracy of either EI-HRMS or FAB-MS analysis because there are likely discrimination effects. The extent of this impurity, which is about 5%according to a HPLC determination, affects the RRF of HA/[13C3JHA but not the accuracy of the HA determination (see the following section). Analytical Chemistry, Vol. 66,No. 23, December 1, 1994
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Table I. Recoveriesmof Hydroxyatrazine in Fortified Water Samples at 5 and 500 ppt Levels
procedure
Q + 4 a
I
2i -
liquid-liquid extraction (dichloromethane) liquid-solid extraction (C-18 cartridge) liquid-solid extraction (carbon-black cartridge)
/
l
, 0
200
400
600
600
1000
1200
1400
Loaded Amount of Hydroxyatrazine (pg)
Figure 2. Dynamic ranges for the FAB-HRMS determination of hydroxyatrazine when 100 pg of [13C3jhydroxyatrazine was used as internal standard. Each data point is the average of two runs. The correlation coefficient for the calibration curve is 0.9994.
Calibration and Relative Response Factor of HA/t13CJHA. The RRF, the ratio of the intensity of native analyte signal to that of the corresponding internal standard, was obtained from the determination of the calibration standards. The average relative signal response (ratio of signals of native to those of labeled compound) vs concentration of native compound in standard solutions was plotted to obtain a calibration curve over the suitable concentration range. The plot, represented in Figure 2, shows that the method gives a linear dynamic range over 2 orders of magnitude (i.e., 10-1250 pg of native HA, with each solution containing 100 pg of [13C31HA). An average RRF of 1.32 was obtained from the analysis of seven standard solutions (RSD = 5.3%). The RRF of HA/[13C31HA did not vary by more than 10%at any time throughout the method development and sample analysis. Under ideal conditions, the RRF should be equal to 1. The deviation from unity may be due to the presence of the impurity in the internal standard, to errors introduced in the preparation of the standard solutions, and/or to the unresolved background from the matrix and solvent during the FAB-HRMS analysis. Because the chemical impurity in the internal standard does not interfere with the determination of the native HA (see under Potential Interferences), any errors caused by the impurity are nearly eliminated by the use of the relative response factor calibration. The RRF of ATRJ[W3]ATR obtained from the previous study is 1.07,44 which was used to measure ATR concentration in the simultaneous determination of HA and ATR The RRFs were checked periodically throughout the method development and sample analysis stages; the RRFs did not vary by more than 10%. Recoveries of Hydroxyatrazine with Different Extraction Methods. Three extraction procedures, liquid-liquid extraction with dichloromethane and liquid-solid extractions with either the C-18 bonded-silica cartridge or the graphitized carbon-black cartridge, were tested for the effectiveness in isolating HA from water. The standard water samples containing HA at levels of 5 and 500 ppt were used for determining recoveries (results are listed in Table 1). The data illustrate that HA can be isolated efficiently by the carbon-black extraction, whereas the other two procedures give poor extraction efficiencies. Two standard water samples (each 2 L) containing 100 ppt of native HA were extracted by using the carbon-black cartridge to test for breakthrough in the extraction. The recoveries of HA 4206
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rec, (5ppt RSD! soln) %
%
%
rec, (500ppt RSD,b soln)
