Envlron. Sci. Technol. W92, 26,1030-1035
(35) Eriksson, L. A strategy for ranking environmentally occurring chemicals; Thesis, University of Umea, Sweden, 1991. (36) Koester, C. J.; Hites, R. H. Chemosphere 1988, 17, 2355. (37) Sijm, D. T. H. M.; Wever, H.; de Vries, P. J.; Oppenhuizen, A. Chemosphere 1989,19, 263. (38) Tysklind, M.; Lundgren, K., Institute of Environmental Chemistry, University of Umeti, Sweden. Unpublished data. (39) Grainger, J.; Reddy, V. V.; Pattersson, D. J., Jr. Chemosphere 1989, 19, 249. (40) Nestrick, T. J.; Lamparski, L. L.; Townsend, D. I. Anal. Chem. 1980,52, 1865. (41) Dobbs, A. J.; Grant, C. Nature 1979, 278, 8.
(42) Tillitt, D. E.; Giesy, J. P.; Ankley, G. T. Environ. Sei. Technol. 1991, 25, 87. (43) Biological Basis for Risk Assessment of Dioxins and Related Compounds; Gallo, M. A., Scheuplien, R. J., Van der Heijden, K. A., Eds.; Banbury Report 35; Cold Spring Harbor Laboratory Press: New York, 1991. (44) Safe, S. Crit. Rev. Toxicol. 1990, 21, 51.
Received f o r review September 12, 1991. Revised manuscript received January 21,1992. Accepted January 22,1992. Financial support f r o m the Center for Environmental Research in Umea (CMF)is gratefully acknowledged.
High-Resolution Mass Spectrometry Method for the Analysis of 3-Chloro-4-( dichloromethyl)-5-hydroxy-2 (5H)-furanone in Waters M. Judlth Charles,* Gong Chen, Rohinl Kannigantl, and G. Dean Marbury Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7400
A method was developed for the of 3-ch10ro4-(dichlorometh~l)-5-h~drox~-2(5H)-furanone (MX) in drinking waters that is based on electron-ionization highresolution mass spectrometry and selected-ion monitoring of the m / z 199 and 201 ions of the methyl derivative of MX and the use of isotopically labeled benzoic acid as the internal standard. High-resolution mass spectrometry is needed to provide sensitivity, selectivity, and confidence in the identification of MX in water extracts. In the analysis of matrix spikes and replicate samples, the method provided recoveries of 102-108% and a precision of 8% RSD. Extrapolation of a signa1:noise-ratio that was measured on a chlorinated water sample provides a detection limit of 0.6 ng/L at a S:N of 3:1,thereby enabling the detection of pptr levels of MX in waters.
Introduction Environmental interest in the presence of the compound 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in drinking waters is growing for two reasons. One reason is that MX is a potent bacterial mutagen. The other reason is that MX has been shown to account for a significant (20-50%) portion of the mutagenic activity in chlorinated drinking waters (1-4). It appears that the mutagenic activity of MX is affected by the pH and temperature-sensitive equilibrium between the closed hydroxyfuranone form of MX and its tautomer, an open oxobutenoic acid (Figure 1). The mutagenic activity of the open form of MX parallels that of aflatoxin. The activity of MX in the Ames assay has been reported as 25.9 and 46.3 revertanta/ng ( 1 , 3 , 5 )and the activity of aflatoxin in the Ames assay has been reported as 22.62 revertants/ng (6).
Analytical methods that exist to measure the concentration of MX in waters rely on derivatization to methylate the hydroxyl group on the molecule followed by highresolution gas chromatography/low-resolution mass spectrometry (HRGC/LRMS) by selected-ion monitoring (SIM) of fragment ions at m/z 147, 199, 201, and 203. Quantification is accomplished by using either external standardization (7) or internal standardization (3). Internal standardization is a more accurate approach because any changes in the response of the analyte, due to the presence of other compounds or changes in the ion source conditions, are normalized to the response of the internal standard. In the method proposed by Hemming et al. (3), 1030
Environ. Scl. Technol., Vol. 26, No. 5, 1992
mucobromic acid was used as the internal standard and the response factorof MX to mucobromic acid was generated by measuring the response of 0.40p g / ~MX to 3.66 pg/L mucobromic acid. By definition, this method is semiquantitative because quantification is achieved on the basis of the response of a single standard of MX to mucobromic acid. A quantitative mass spectrometry method based on internal St-adardiZation is thus urgently n ~ d e d . In this study, we utilize information from previous work (8) to develop and validate a quantitative method for the analysis of MX in waters. The method described herein is based on electron-ionization high-resolution mass spectrometry and selected-ion monitoring of the m/z 199 and 201 ions of the methyl derivative of MX and the use of isotopically labeled benzoic acid as the internal standard.
Experimental Section Purity of MX Standard Material. Standard material of MX was obtained from the US.Environmental Protection Agency, Health Effects Research Laboratory. The purity of the material was determined to be 84%. We calculated this value in our laboratory by dividing the response (area) of MX by the total response (area) of all the peaks present in a chromatogram obtained by highresolution gas chromatography/ flame-ionization detection (GC/FID). In this analysis, the injector port of the gas chromatograph was 200 "C to prevent thermal degradation of MX. We and others (9) have observed the formation of 2-(dichloromethyl)-3-chloropropenalfrom the decarboxylation of MX at temperatures greater than 200 O C . Derivatization of Standard Materials and Sample Extracts. Solutions of the methyl derivatives of mucobromic acid (Aldrich Chemical Co., Inc., Milwaukee, WI), mucochloric acid (Aldrich Chemical Co., Inc.), MX (50-1000 pg/pL), benzoic acid, and [13C,]benzoic acid (MDS Isotopes, Merck Chemical Division, St. Louis, MO; 50 pg/pL) were prepared by reacting the standard materials independently or together, as indicated, with solutions of 14% (v/v) BF3 in methanol (Alltech Inc., Deerfield, IL) for 12 h. The resulting solutions were then neutralized with 3 mL or 500 pL of 2% (v/v) NaHC03, extracted twice with 250 pL of hexane, and concentrated to 100 pL under a stream of N2 gas. Preparation of Chlorination and Chloramination Extracts of Fulvic Acid. Monochloramine solutions were prepared by the method of Johnson and Overby (IO),
00113-936X/92/0926-1030$03.00/0
0 1992 American Chemical Society
CI
3-chloro-4-(dlchloromethyl)5-hydroxy-2(5H)furanone (Closed Form of MX)
(Z)-2-chloro-3-(dichioromethyi)4-oxobutenolc acid (Open Form of MX)
Flgure 1. Chemical equlllbrium of 3-chloro-4-(dichloromethyl)-5hydroxy-2(5H)-furanone (MX).
and chlorine and monochloramine concentrations were determined by the DPD ferrous titrimetric method. The fulvic acid used in this study was previously isolated from Lake Drummond, VA, waters by using the method of Thurman and Malcolm (11). Chlorination of the fulvic acid was performed by reacting NaOCl(300 mg/L as Clz) with 0.5-2 L of fulvic acid at pH 7.0 for 48 h at a 0.2 CkC molar ratio at room temperature with stirring. Monochloramine and fulvic acid were also at a 0.2 CkC molar ratio at room temperature with stirring. The monochloramine reaction mixture was maintained at pH 8.0 over a reaction period of 96 h to ensure that monochloramine was the only chloramine species in solution. The chlorination and chloramination reaction mixtures were acidified to pH 2.0 with HC1 in 250-mL aliquots, and MX was extracted with a total volume of 80 mL of diethyl ether. The extracts were then concentrated by using a KudernaDanish apparatus and nitrogen evaporation, and the residue was derivatized with 250 pL of 14% (v/v) BF3 in methanol. Preparation of Chlorinated and Ozonated Water Extracts. Water samples were collected from University Lake, Chapel Hill, NC. Samples (2 L) were chlorinated at a 1.0 CkC (w/w) ratio 60 h in the dark with stirring. The mixtures were maintained at pH 7.0 throughout the reaction period. At the end of the 60 h, the total chlorine residual in the samples was less than 0.1 mg/L. Other samples (2 L) were ozonated by bubbling ozone through the samples at a 1.0 03:C (w/w) ratio for 20 min. These samples were then chlorinated as previously described. The total chlorine residual at the end of the reaction period was less than 0.2 mg/L. The samples were adjusted to pH 2.0 by the addition of 4 N HC1, and the compound MX was extracted with EO-, 75-, and 75-mL volumes of diethyl ether. The three extracts obtained were combined and concentrated by using a Kuderna-Danish apparatus followed by nitrogen evaporation and then derivatized as previously described. Preparation of Samples Obtained from a Water Utility. Five samples of surface water that had been treated by perchlorination, coagulation, and filtration were collected by a water utility. Samples 1and 4 were chloraminated, and samples 2 and 3 were left with chlorine as a final disinfectant residual. These samples were acidified (pH 2) and then shipped on ice to the University of North Carolina by overnight express mail. A volume of 1.2 L for each sample was solvent extracted with 150,75, and 75 mL of diethyl ether. The extract was then treated as previously described. Preparation of Standard Solutions. Six standard calibration solutions that contained the methyl derivatives of MX and the methyl ester of isotopically labeled benzoic acid were prepared by mixing aliquots of solutions that contained these compounds. The final concentration of the methyl derivative of MX was either 50,100,250,500,
750, or 1000 pg/pL, and the concentration of the methyl ester of isotopically labeled benzoic acid was 50 pg/pL. Determination of Derivatization Yields. Standard material of mucobromic acid (41.7,39.5, 38.7 mg), mucochloric acid (40.7, 39.6, 38.1 mg), and benzoic acid (38.9, 26.7,41.9 mg) were derivatized with 14% BF, in methanol, and the resulting solutions were dried under nitrogen. The resulting residue was weighed, and the derivatization yield was calculated by the difference in weight before and after derivatization on a mole basis. Methylation of mucobromic acid and mucochloric acid produces two primary products. The percentage yield of these products was determined by analyzing the mixtures by high-resolution gas chromatography/flame-ionization detection and comparing the response (area) of the product to the total area of the methylation products. The identity of the products was verified by using high-resolution gas chromatography/mass spectrometry. Competitive Reaction Experiments. Replicates (n = 2) of standard solutions of MX (100 pg/pL) were derivatized with 14% BF, in methanol in the absence and presence of [13C6]benzoicacid (50 pg/pL) and mucochloric acid (250 pg/pL). Anthracene-d,, was added after methylation as the internal standard. The response of MX relative to the internal standard was then measured by using high-resolution gas chromatography/low-resolution mass spectrometry [resolving power (RP) 10001 in the selected-ion-monitoring mode. High-Resolution Gas Chromatography/Mass Spectrometry, High-resolution gas chromatography/ mass spectrometry (HRGC/MS) analyses were performed on a Hewlett-Packard 5890 gas chromatograph interfaced to a VG70-250SEQ mass spectrometer operating at a resolving power of 1000 or 10000 (10% valley definition). A 30-m, 0.25 mm i.d., 0.25-pm film, DB-5 fused capillary column was used in all experiments (J&W Scientific, Folsom, CA). The GC oven was held at an initial temperature of 50 "C for 1min and then programmed to rise at 2.5 "C/min to 150 "C and then at 5 "C/min to 300 "C. The mass spectra were acquired by using electron ionization at an electron energy of 70 eV, a 200-pA trap current, and a 250 "C source temperature. The methylation products of mucochloric acid, mucobromic acid, and MX were identified by using low-resolution (RP = 1000) gas chromatography/low-resolution mass spectrometry (HRGC/LRMS). In these analyses the magnet was scanned from 500 to 50 amu at 1 s/decade. In selected-ion-monitoring high-resolution gas chromatography/high-resolutionmass spectrometry (RP = 1000 and 10000) analyses, the M+ ion ( m / z 142.076) of the methyl ester of [13C6]benzoicacid and a PFK lock-mass (m/z 142.9920) were monitored from 3 to 11min, and the A and A + 2 ions ( m / z 198.9121, 200.9024), arising from the loss of M - OCH, and a PFK lock-mass (m/z 192.9888) were monitored from 11to 14 min. Each of these ions was monitored for 50 ms with a delay time of 10 ms. Quantitation of MX in Water Extracts. Quantitation of MX was achieved by performing a regression analysis of the data generated by the analysis of six calibration solutions (50,100,250,500,750, and 1000 pg/pL). The resulting equation was then used to determine the concentration of MX in sample extracts that were analyzed on the same day. The measured concentration of MX was corrected for the purity of the standard material by multiplying concentration by 0.84. Results and Discussion Justification for Electron-Ionization High-Resolution Mass Spectrometry, The mass spectrum of MX has Envlron. Sci. Technol., Vol. 26, No. 5, 1992
1031
1,000 Resolution
1,000 Resolution loo
loo]
A
1
A
S:N=1:1
50
c / /
100 1 %
c
.-2 Q, 5
0
1
c
n
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+
1 mlz 200.9
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o
-
l
l
0'
2 lool a
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I
11.24
11.28
10,000Resolution
c
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Y
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loo
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I
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I
n
mlz 198.9121
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0
I
11.32
mlz 198.9
501 0
11.48 11.52
A
mlz 200.9094
11.56 12.00
0
12.04
Time (minutes) Flgure 3. Mass chromatograms of a chlorinated extract of lake water obtained by using high-resolution gas chromatography/mass spectrometry (RP = 1000 and 10000).
11.8
12.0
Time (minutes) Flgure 2. Mass chromatograms of a chloramination extract of fulvic acid obtained by using high-resolution gas chromatography/mass spectrometry (RP = 1000 and 10000).
been described previously (8). In our former study, we determined that the chlorine isotope cluster observed at m/z 199 is composed of two fragment ions, each containing three chlorine atoms that overlap at mlz 201 and 203. The resulting doublet at mlz 201 requires a resolving power of 10 806, and thus the ratio between the ions at mlz 199 and 201 under low-resolution conditions deviates from the theoretical value for an isotope cluster that contains three chlorine atoms. Methods that are developed for the analysis of halogenated compounds often incorporate criteria for compound identification that set a limit on the deviation between the theoretical and the measured isotope ratios of the ions monitored. The use of high-resolution mass spectrometry thus needs to be considered because under low-resolution conditions the establishment of such criteria for compound identification would involve the comparison of ion signals that arise from at least two fragmentation pathways that are not mass resolved and that deviate from theoretical isotope ratios. By operating under high-resolution conditions, the ratio of the ions at mlz 199:201 changes from 62:lOO to 100:98. The latter ratio matches the theoretical ratio of the A and A + 2 ions in a threechlorine isotope cluster. Confirming criteria can therefore be established on the basis of the theoretical ratio of ions. Further evidence for high-resolution mass spectrometry was obtained by comparing the results of analyses per1032 Environ. Sci. Technol., Vol. 26, No. 5, 1992
formed under low-resolution and high-resolution conditions on extracts of samples that represent the type of waters that are analyzed for MX. These samples were a chloramination extract prepared by reacting monochloramine with fulvic acid, a water sample that was obtained from a local lake that is a source of drinking water and then chlorinated in our laboratory, and a sample from a water utility that employs chlorination for disinfection. In all these cases, identification and quantification of MX was impossible under low-resolution conditions due to the low signa1:noise values (Figures 2-4). The noise due to chemical interferences was eliminated by using high-resolution mass spectrometry and resulted in an improvement of signa1:noise values by 8-200X, thereby enabling the identification and quantification of MX. We thus chose electron-ionization high-resolution mass spectrometry because of its ability to provide the needed selectivity and because of our understanding of the electron-ionization mass spectrum of the methyl derivative of MX. Other techniques, such as electron-capture negative chemical ionization (ECNI), can be used to provide selectivity and sensitivity in the analysis of many halogenated compounds. However, the isotope cluster at mlz 199 in the electron-ionization mass spectrum is about 5X greater than either the (M - H)+ or the (M - C1)+isotope cluster in the ECNI mass spectrum (approximately 18% compared to 3%) (3). Furthermore, it is possible that other chlorinated compounds that are formed during chlorination of natural organics, known to exist at much higher concentrations than MX, could affect the relative response of MX to the internal standard. These considerations would not be problems if the sensitivity of ECNI compensates for the weak signal of the high mass ions or the relative intensity of the ions at high mass can be increased
1,000 Resolution
0 1
100
.-P>,
I
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1
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. I -
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o 1
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o 1 100
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I mlz 200.9094
50 0
( M HBri] 157
I
I
11.18
I
I
11.30 Time (minutes)
I
I
11.42
Flgure 4. Mass chromatograms of a chlorinated extract of a water utlllty water obtalned by using hlgh-resolutlon gas chromatography/ mass spectrometry (RP = 1000 and 10000).
by altering the operating parameters, and if a 13C isotopically labeled form of MX is commercially available. Choice of Internal Standard. Three compounds that were considered as candidates for an internal standard were mucochloric acid, mucobromic acid, and [13C6]benzoic acid. Mucochloric and mucobromic acid were chosen because they are structural analogues of MX and are likely to exhibit chemical and physical behavior similar to MX. The compound [13C6]benzoicacid was chosen because, to our knowledge, it is the only 13Cisotopically labeled aromatic compound with an acidic group (COOH) on the molecule that is commercially available. We tested the suitability of these compounds as internal standards by performing experiments to evaluate their methylation yields and artifacts arising from methylation of chlorinated waters. The methylation yields of mucobromic acid, mucochloric acid, and benzoic acid were determined in the first experiment. We could not determine the methylation yield of MX due to insufficient material [large quantities of material (mg) were required in these experiments to allow accurate gravimetric determinations of the product]. Interestingly, we observed the presence of two compounds produced by the methylation of mucobromic acid and mucochloric acid. The identification of these compounds was based on interpretation of the resulting mass spectrum. As an example, we present the mass spectra of the methylated producta of mucobromic acid in Figure 5. One of these compounds, the expected product, results from the methylation of the hydroxy group on the furanone ring (Figure 5a). The mass spectrum of the second compound suggests the presence of a compound containing three methoxy (OCH,) groups on the molecule (Figure 5b). Kronberg (12) previously reported that methylation of the compound EMX, the geometric isomer of the open form
50
75
100
125
150
mlz
Flgure 5. Mass spectrum of the closed hydroxyfuranone form of mucobromlc acld (a, top) and the open form of Its geometrlc Isomer (b, bottom).
of MX, results in the formation of a product that contains three methoxy groups due to methylation of the carboxyl and aldehyde groups on the molecule. Thus, we reasoned, on the basis of this information and the knowledge that, under the acidic conditions of methylation, mucobromic and mucochloric acid will exist as the closed forms, that the unexpected compound must be due to the presence of the geometric isomer of the closed forms of mucobromic acid and mucochloric acid in the standard material. The derivatization yield was therefore calculated by determining the formation of the desired methoxy product as a percentage of the total products formed (60.76% for mucobromic acid and 70.15% for mucochloric acid). This information was then used to calculate the methylation yield on a mole basis. Similar methylation yields were obtained for all the compounds (79 f 4 for mucobromic acid, 75 f 8 for mucochloric acid, and 77 f 7 for benzoic acid). In the second experiment, we discovered that mucochloric acid is formed during the chlorination and chloramination of fulvic acid, thereby suggesting the presence of this compound in treated waters. And the possible formation of mucobromic acid by an analogous mechanism may be inferred during disinfection of waters that contain high-levels of bromide. We chose [13C6]benzoicacid therefore as the internal standard because we were interested in developing a method applicable to all waters, independent of organic or chlorine content, and because the stability of the methyl derivative was shown to be about the same as the stability of the methyl derivative of MX. We are assuming that the methylation yield of [13C6]benzoicacid will be similar to that of MX since the results of the methylation yield exEnvlron. Scl. Technol., Vol. 26, No. 5, lQQ2 1033
periments show yields comparable to mucochloric acid and mucobromic acid, compounds that are structural analogues of MX. Linearity of the Response of the Methyl Derivative of MX to the Methyl Ester of [13C6]BenzoicAcid. In initial experiments, we observed large variabilities in the relative response factors of the methyl derivative of MX to isotopically labeled benzoic acid (e.g., RSD of 46%) that indicate a nonlinear response of the analyte to the internal standard. This nonlinear response was reduced when the standard materials of the analyte and the internal standard were derivatized separately and then mixed together to produce the standard solutions. We hypothesized that this occurrence was due either to competitive reactions during methylation or to a parameter that affects instrument performance. The possibility that a competitive reaction was occurring between MX and labeled benzoic acid seemed unlikely since the derivatizing agent is present in excess (>lo4mol) compared to the amounts of the analyte and the internal standard. Nonetheless, we performed an experiment in which the response of the methyl derivative of MX was compared when methylation was performed on solutions that contained MX in the absence and presence of other compounds (e.g., [13C6]benzoicacid and mucochloric acid). The results of these experiments indicate similar mean relative responses (area) of the methyl derivative of MX to anthracene (2.7 units) when it was derivatized independently as when it was derivatized in the presence of labeled benzoic acid (2.9 units), and labeled benzoic acid and mucochloric acid (2.7 units). A decrease in the response would be observed if competitive reactions were occurring. We therefore could not discern any differences in the response of MX in the absence or presence of other compounds, thereby eliminating the possibility of competitive reactions as a cause for the observed nonlinear responses. Regarding instrument performance, we became concerned about the ratio between the highest and lowest mass ions that were monitored. Difficulties are known to exist in voltages when the ratio of these masses exceed a value of 1.5. This is because the accelerating voltage is changed from 8 kV to a value that is inversely proportional to mass to transmit ions of different masses in the selected-ion-monitoring experiment. For example, the low mass in the one-window experiment is m/z 142.0776 and the high mass is m / z 200.9094, which means that the accelerating voltage changes from 8 to 5.7 kV. In the twowindow experiment, the greatest mass range occurs in the second window when monitoring a PFK lock-mass at m/z 192.9888 and the A + 2 ion of the (M - OCH,) fragment of the methyl derivative of MX at m / z 200.9024. In this case, the accelerating voltage changes from 8.0 to 7.7 kV. Changes in the accelerating voltage affect tuning of the source and the energy of the ions. The degree of the change should therefore be minimized to prevent a loss in sensitivity that occurs at either the low or, more typically, at the high mass end of the mass spectrum due to this change. We therefore compared the results of measurements made by selected-ion-monitoring ions for the analyte and the internal standard in one-mass group and two-mass groups to evaluate these effects. It was easy to make this modification because the retention time of the methyl derivative of MX (retention time is about 11.8 min) and that of labeled benzoic acid (retention time is about 7.15 min) differs by about 4 min. When this change was made we observed a difference in the setting of the ion energy 1034
Environ. Scl. Technol., Vol. 26, No. 5, 1992
and an improved response in the signal of the m/z 199 and 201 ions of the methyl derivative of MX. We analyzed three samples for MX-a chlorinated lake water extract, an extract of a water that was ozonated/chlorinated, and a lake water matrix spike. The concentration of MX in the chlorinated lake water was calculated to be 745 ng/L when the ions were monitored in one group and 33.7 ng/L when the ions were monitored in two groups. Concentrations of 433 and 18.9 ng/L of MX were determined when the ions were monitored in one and two groups, respectively. Thus, the analytically determined concentration of MX in these waters was found to differ by greater than 20X as an artifact of ion energy effects. In addition, the methyl derivative of MX was not detected when the ions were monitored in one window whereas 13.4 ng/L of MX was detected when the ions were monitored in two windows. Because we know that 12.6 ng/L of MX was added to the matrix spike prior and because of our observations about the effects on the ion energy, we believe the numbers derived from monitoring the ions in two windows are the correct values. Another experiment was performed to determine if this effect was the entire reason for nonlinear response of the analyte to the internal standard. In this experiment we prepared standard solutions either by mixing the internal standard and the analyte together prior to derivatization or by derivatizing standard solutions of the analyte and the internal standard independently and then preparing calibration solutions by mixing aliquots of these solutions together. The % RSD among the relative response factors, obtained from the analyses of a five-point calibration curve (the concentration of MX ranged from 50 to loo0 pg/pL), decreased from 40 to 30% when the standards were derivatized separately. It is ideal to selectively ion monitor the internal standard and the analyte ions in one window and to add the internal standard to a sample prior to extraction and derivatization. We found however that the accuracy and linearity of the method improved when the ions were monitored in two windows and when the internal standard was added to the extract postderivatization. The effect of these actions on quantitation was determined in validation of the method. Method Validation. The method was validated by the analysis of samples and matrix spikes. Recoveries of 106% and 108% were obtained in the analysis of lake water and chlorinated lake water samples that were spiked with 12.6 ng/L of MX. A 32% RSD was observed in the analysis of replicate sample extracts of the chlorinated water Samples. In the analysis of samples provided by the water utility, a 102% recovery of MX was obtained from the analysis of the matrix spike and an 8% RSD was observed from the repeated (three times) injection of a sample, thereby demonstrating instrument stability and reproducibility. Identification of the methyl derivative of MX in the sample extracts was based on the relative retention time of the analyte to the internal standard and the isotope ratio between the m/z 199 and 201 ions. In these analyses the relative retention time varied less than 1% and the ratio between the m/z 199 and 201 ions was within 10% of the theoretical ratio. The results are presented in Table I. The concentrations of MX reported in these samples are on the same order of those reported by Kronberg (2) for MX in drinking waters (13-67 ng/L) but less than those reported by Backlund (I) for MX in waters that were treated by chlorination (194 ng/L) and ozonation, followed by chlorination (128 ng/L). It is difficult to comment further on the differences observed among samples in this
Table I. Analysis of MX in Treated Waters by Using a High-Resolution Mass Spectrometry Method sample lake water matrix spikesb lake water lake water water utility 1 2
3a 3b 3c (matrix spike)b
treatment
measured concn MX, ng/L
none chlorination ozonation/chlorination
ND" 38.5, 29.0 18.9
none Chlorinated
13.4 13.7
chloramination chlorination chlorination chlorination chlorination chloramination
2.4 8.9 6.1, 7.1, 6.8 9.3 12.9 2.5
amount spiked
% recovery
12.6 12.6
106 108
12.6
102
"ND, not detected. bThe concentration of MX in the matrix spike was calculated by subtracting the sample concentration that was determined from the analvsis of unsDiked samDles from the measured concentration.
study or those observed by other investigators without more information about the waters (e.g., total organic carbon, pH, C1:C ratio). These data however do demonstrate that the method is suitable for the analysis of MX in treated waters. Method Detection Limits. A signaknoise ratio on the 42 pg/pL standard for 3 days of analysis was obtained to determine the detection sensitivity of the instrument. These values ranged from 5:l to 9:1, and extrapolation to a signa1:noise ratio of 3:l would provide an instrument detection limit of about 14-25 pg/pL. The signa1:noise ratio measured on a chlorinated water sample was 200:l for 38.6 ng/L of MX. This value extrapolates to a detection limit of 0.6 ng/L at a S:N of 3:l. Thus, it is reasonable to achieve method detection limits around l pptr by extracting a 2-L water sample and concentrating the extract to 100 FL.
Conclusions A high-resolution mass spectrometry method was developed for the analysis of the potent mutagen, 3-chloro4-(dichloromethyi)-5-hydroxy-2(5H)-furanone (MX) in waters in the low ppt range. This method is based on the use of [13C6]benzoicacid as the internal standard. Highresolution mass spectrometry was needed to increase confidence in compound identification and to remove chemical interferences present in sample extracts, thereby increasing the ability to identify and quantify MX in environmental matrices. Validation of the method involved repeated analysis of standard calibration solution to verify the linearity and reproducibility of the response of the methyl derivative of MX to the methyl ester of [l3C6]benzoic acid and analysis of treated waters and matrix spikes. Linear and reproducible responses of the methyl derivative of MX to the methyl ester of [13C6]benzoicacid were observed, although variability did exist in the absolute response factors on a daily basis. On the basis of the results of this study, we suggest the criteria for compound identification and quantitation of a 30% relative standard deviation among the relative response factors: a ratio of 1.02 f 20% for the measured isotope ratio between the m / z 199 and m / z 201 ions and relative retention time of f l s. The results of the analysis of a chlorinated water and the same water treated by ozonation followed by chlorination suggest that less MX is formed after ozonation of waters. This may be due to oxidation of the natural organic molecules that are precursors of MX. Future
studies are urgently needed on the toxicology of MX in mammalian systems and on the formation and environmental fate of MX. We have provided an accurate and reliable analytical method which previously did not exist that can be usedladapted to conduct such studies.
Acknowledgments We thank P. Ringhand, US. Environmental Protection Agency, for providing us with standard material of MX; B. Schwartz, University of North Carolina, for assisting in the mass spectrometry analyses; and J. Cavanaugh, University of North Carolina, for providing an ozonated sample of lake water. Registry No. MX, 77439-76-0; H,O, 7732-18-5.
Literature Cited (1) Backlund, P.; Kronberg, L.; Tikkanen, L. Chemosphere 1988, 1 7 (7), 1329-1336. (2) Kronberg, L.; Holmbom, B.; Reunanen, M.; Tikkanen, L. Environ. Sci. Technol. 1988, 22, 1097-1103. (3) Hemming, J.; Holmbom, B.; Reunanen, M.; Kronberg, L. Chemosphere 1986, 15 (5), 549-556. (4) Meier, J. R.; Knohl, R. B.; Coleman, W. E.; Ringhand, H. P.; Munch, J. W.; Kaylor, W. H.; Streicher, R. P.; Kopfler, F. C. Mutat. Res. 1987,189, 363-373. ( 5 ) Padmapriya, A. A.; Just, G.; Lewis, N. G. Can. J. Chem. 1985,63,828-832. (6) McCann, J.; Choi, E.; Yamaski, E.; Ames, B. N. h o c . Natl. Acad. Sci. U.S.A. 1975, 72, 5135-5139. (7) Munch, J. W.; Coleman, W. E.; Hodakievic, P. A.; Kaylor, W. H.; Meier, J. R. In Proceedings of the A WWA Water Technology Conference, Baltimore, MD, Nov 1987. (8) Charles, M. J.; Marbury, G. D.; Chen, G. Biol. Mass Spectrom. 1991,20, 529-531. (9) Colette, T. W.; Christman, R. F.; McGuire, J. M.; Trusty, C. Environmental Research Laboratory, Office of Research and Development: Athens, GA, Aug 1990. (10) Johnson, J. N., Overby, R. Anal. Chem. 1969, 41 (13), 1744-1 750. (11) Thurman, E. M.; Malcolm, R. L. Environ. Sci. Technol. 1981,15,463-466. (12) Kronberg, L. Ph.D. Dissertation, Abo Akademi, Turku, Finland, 1987. Received for review September 12, 1991. Revised manuscript received December 22,1991. Accepted December 30,1991. We thank the Metropolitan Water District of Southern California for funding this work and C. Hwang and S. Krasner of Metropolitan for their support.
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