Article pubs.acs.org/est
Identification of Precursors and Mechanisms of Tobacco-Specific Nitrosamine Formation in Water during Chloramination Beibei Chen,†,‡ Yichao Qian,† Minghuo Wu,† Lifang Zhu,†,§ Bin Hu,‡ and Xing-Fang Li*,† †
Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2G3, Canada ‡ Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, Hubei 430072, People’s Republic of China § Zhejiang University of Water Resources and Electric Power, 583 Xuelin Street, Xiasha Higher Education District, Hangzhou, Zhejiang 310018, People’s Republic of China S Supporting Information *
ABSTRACT: We report here that tobacco-specific nitrosamines (TSNAs) are produced from specific tobacco alkaloids during water chloramination. To identify the specific precursors for the formation of specific TSNAs in drinking water, we have developed a solid-phase extraction−liquid chromatography−tandem mass spectrometry (SPE−LC−MS/MS) method for simultaneous determination of five TSNAs and three tobacco alkaloids. Using this method, we detected nicotine (NIC) at 15.1 ng/L in a source water. Chloramination of this source water resulted in the formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK) (0.05 ng/L) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) (0.2 ng/L) along with the reduction of NIC to 1.1 ng/L, suggesting that NNK and NNAL were formed from NIC. To confirm that tobacco alkaloids are the precursors of TSNAs, we chloraminated water-leaching samples of tobacco from three brands of cigarettes and found that the formation of TSNAs coincides with the reduction of the alkaloids. Chloramination of individual alkaloids confirms that NNK and NNAL are produced from NIC, N-nitrosonornicotine (NNN) from nornicotine (NOR), and N-nitrosoanabasine (NAB) from anabasine (ANA). Furthermore, we have identified specific intermediates of these reactions and proposed potential pathways of formation of TSNAs from specific alkaloids. These results confirm that NNK and NNAL are the disinfection byproducts (DBPs) resulting from NIC in raw water.
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detected N-nitrosamine in drinking water systems.9 Consequently, a number of studies have focused on identification of its precursors and elucidation of the formation mechanisms during drinking water treatment.10−14 However, NDMA only accounts for ∼5% of the estimated total nitrosamines,15 suggesting that the majority of nitrosamines in drinking water are still unknown. Therefore, it is important to identify other Nnitrosamine DBPs and to understand their formation
INTRODUCTION Water disinfection kills pathogens to prevent waterborne disease1 but will inevitably cause the generation of unintended disinfection byproducts (DBPs) from the reaction of disinfectants (e.g., chlorine and chloramines) with natural organic matter (NOM) and organic compounds in water.2,3 Epidemiological studies have found a potential association of exposure to DBPs with adverse health effects, such as bladder cancer.4,5 However, animal bioassays indicate that the regulated DBPs, such as trihalomethanes and haloacetic acids, do not account for the increased risk of bladder cancer.6 N-Nitrosamines are a group of non-halogenated DBPs associated with chloramination classified as probable bladder carcinogens.7,8 NNitrosodimethylamine (NDMA) is the most commonly © 2014 American Chemical Society
Received: Revised: Accepted: Published: 459
October 17, 2014 November 28, 2014 December 3, 2014 December 3, 2014 dx.doi.org/10.1021/es505057h | Environ. Sci. Technol. 2015, 49, 459−466
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Table 1. Structures and Molecular Weights of the Five TSNAs and Three Tobacco Alkaloids
of NNK increased in the formation potential (FP) tests of the source water after chloramination. These results raise several questions: (1) what their precursors are, (2) why the other TSNAs are absent, and (3) how they are produced during water treatment. TSNAs can be formed from nicotine (NIC) and related compounds by a nitrosation reaction.26 Because of the worldwide use of tobacco products, NIC exists at several to hundreds of nanograms per liter in surface water or groundwater.27−31 Its concentration could be higher at the microgram per liter level in wastewater.31,32 On the basis of these facts, we hypothesized that tobacco alkaloids, including NIC and its related compounds, are the precursors of TSNAs and that individual TSNAs are produced from specific precursors. The objectives of this study are to confirm specific tobacco alkaloids as the precursors of certain TSNAs in drinking water and to elucidate the formation pathways of TSNAs during chloramination. To achieve these goals, we have developed a new solid-phase extraction liquid chromatography−tandem mass spectrometry (SPE−LC−MS/MS) method to simultaneously determine both TSNAs and tobacco alkaloids in source water and treated water. The new SPE−LC−MS/MS method can identify and quantify five TSNAs (NNN, NNK, NNAL,
mechanisms during water treatment to limit the formation of N-nitrosamines in drinking water. Several toxicology studies have shown that tobacco-specific nitrosamines (TSNAs) are carcinogenic.16,17 According to the International Agency for Research on Cancer (IARC) monograph,18 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) are carcinogenic to humans (group 1), with higher carcinogenicity than NDMA, a probable human carcinogen (group 2A). N-Nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT) are not classifiable regarding their carcinogenicity to humans (group 3). NNN, NNK, or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) can induce oral, lung, pancreatic, and esophageal cancer in animals.16,19 An increased number of oral tumors was observed in rats given drinking water containing NNK and NNN.20,21 NNK is a more potent mutagen than NDMA in the F344 rat.22 α-Hydroxylation of NNK and NNAL can lead to the formation of DNA adducts, single-strand breaks, and other macromolecular damage.19,23 TSNAs may exist in source water because of contamination by wastewater.24 Our previous results25 confirmed the existence of NNK in source water impacted by wastewater and the occurrence of both NNK and NNAL in the disinfected water. In addition, the concentrations 460
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transferred into the vials, to which 10.7 mL of 140 mM monochloamine solution was added. The pH of the mixture was adjusted to 8 and maintained with the addition of 1.5 mL of 200 mM ammonium acetate buffer. Then, the mixture was diluted with Optima water to 30 mL (10 μg/L NIC, NOR, or ANA, 50 mM monochloramine, and 10 mM buffer), and the chloramination reactions were conducted at 25 °C. At given time points, the reaction solution (50 μL) was sampled and 20 μL was directly injected into the LC−MS/MS (MRM) for the determination of residual tobacco alkaloids and formed TSNAs. Duplicate analyses were performed for each time point. FP control experiments were also performed using Optima water. All FP experiments were performed in triplicate, and the average values along with standard deviations were reported. To elucidate the formation mechanisms of TSNAs from NIC, NOR, and ANA, we used LC−high-resolution Q-TOF− MS/MS to identify the intermediates and TSNAs in the reaction mixtures, when NIC, NOR, and ANA (1 mg/L) were reacted with monochloramine (100 mM). The other FP conditions were the same as described above. Solid-Phase Extraction (SPE). To preconcentrate tobacco alkaloids and TSNAs in drinking water samples collected from a treatment plant, we developed and validated a SPE method using Oasis hydrophilic−lipophilic balanced (HLB) cartridges (6 mL, 200 mg per cartridge, Waters, Milford, MA). The HLB cartridges on a Visiprep SPE manifold (Supelco, Bellefonte, PA) were connected to a vacuum. Each HLB cartridge was initially rinsed with 10 mL of methanol and equilibrated with 10 mL of water. A 500 mL water sample passed through the HLB cartridge at a flow rate of ∼3 mL/min. After the cartridge was washed with 5 mL of 10% methanol, the analytes adsorbed on the cartridge were eluted with 5 mL of methanol. The eluate was collected and condensed to approximately 0.2 mL with a high-purity nitrogen stream in a 40 °C water bath and reconstituted with Optima water to 1 mL for subsequent LC− MS/MS (MRM) analysis. For each set of SPE experiments, a blank control of 500 mL of Optima water and a positive control of 500 mL of Optima water containing 4 ng/L each of the analytes were included. Water Sample Collection and Preparation. Water samples including the source water and the treated water from a drinking water treatment plant were collected in 500 mL amber glass bottles. Ascorbic acid (0.5 g/L) was immediately added to the samples after collection to quench the free chlorine and the samples were stored at 4 °C. The source water samples were filtered with glass microfiber filters (47 mm × 1.5 μm, Waterman) and nylon membrane disc filters (47 mm × 0.45 μm, Pall Corporation) prior to SPE. The SPE combined with the LC−MS/MS (MRM) method was used to determine the concentrations of tobacco alkaloids and TSNAs in the source water and the treated water samples. The source water samples were also used for FP tests of TSNAs. We randomly selected three brands of cigarettes (C1, C2, and C3) that are most commonly available in grocery stores in Edmonton. All three are domestic brands in Canada. Tobacco in each cigarette was removed, weighed, and then soaked in 100 mL of Optima water in a 250 mL beaker. After this suspension was stirred for 24 h at room temperature, the water leaching sample of the cigarette was collected through filtration with a glass microfiber filter (47 mm × 1.5 μm, Waterman) and a nylon membrane disc filter (47 mm × 0.45 μm, Pall Corporation) and stored at 4 °C. The cigarette-leaching samples were diluted with water 20 000 times for analysis of
NAB, and NAT) and three tobacco alkaloids [NIC, nornicotine (NOR), and anabasine (ANA)] at sub-nanogram per liter levels. To confirm that tobacco alkaloids are the precursors of various TSNAs, we further studied the formation of TSNAs from the chloramination of water-leaching samples of tobacco in three brands of cigarettes. In addition, chloramination of individual NIC, NOR, and ANA standard solutions was conducted to verify that NIC, NOR, and ANA are the precursors of specific TSNAs. Finally, we used a high resolution triple quadrupole time-of-flight (TOF) mass spectrometer to identify the intermediates formed from the reactions of tobacco alkaloids with monochloramine to elucidate possible reaction pathways.
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EXPERIMENTAL SECTION Chemicals. TSNA standards of NNK, NNAL, NNN, NAB, and NAT were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). The standards of tobacco alkaloids NIC, NOR, and ANA were obtained from SigmaAldrich (Milwaukee, WI). The structures and molecular weights of the five TSNAs and three alkaloids are described in Table 1. NNK and NNAL are the nitroso derivatives of NIC; NNN is the nitroso derivative of NOR; NAB is the nitroso derivative of ANA; and NAT is the nitroso derivative of anatabine. Methanol and water of Optima LC−MS grade were provided by Fisher Scientific (Fair Lawn, NJ). All other chemicals used were of analytical grade. Stock solutions (1 g/L) of the five TSNAs and three tobacco alkaloids were prepared in methanol and stored at −20 °C. Working solutions were prepared daily by diluting the corresponding stock solution in water. LC−MS/MS Analysis. An Agilent 1290 LC system (Santa Clara, CA) with a Kinetex C18 column (100 mm × 3 mm × 2.6 μm, Phenomenex, Torrance, CA) was used for the separation of three alkaloids and five TSNAs. The mobile phase consisted of solvent A (10 mmol/L ammonium acetate and 0.01% acetic acid in water) and solvent B (100% methanol), and the flow rate was 0.4 mL/min. A gradient program was performed as follows: linearly increased B from 35 to 80% over 3.5 min, changed B to 90% in 0.1 min and kept B at 90% from 3.6 to 4.6 min, returned to 35% B for column equilibration from 4.7 to 6 min. The sample injection volume was 20 μL, and the column temperature was 25 °C. Multiple-reaction monitoring (MRM) methods were performed using a triple quadrupole ion-trap tandem mass spectrometer (QTRAP 5500, AB Sciex, Concord, Ontario, Canada) for the quantification of the eight analytes. A highresolution quadrupole time-of-flight tandem mass spectrometer (Q-TOF−MS/MS, TripleTOF 5600, AB Sciex) was used to identify the intermediate products during the chloramination process to elucidate the formation pathways of TSNAs. The conditions of the QTRAP−MS/MS and Q-TOF−MS/MS experiments are described in the Supporting Information, including Table S1 of the Supporting Information. Formation Potential (FP) of TSNAs from Chloramination. Stock solution of monochloramine (NH2Cl) solution (140 mM) was prepared according to the procedures previously reported.33 To study the formation of TSNAs during water chloramination, we conducted sets of reactions in batches of 40 mL Supelco amber EPA vials with caps (SigmaAldrich, Oakville, Ontario, Canada). The volume of each reaction solution was kept at 30 mL. For FP tests, an aliquot of 6 mL of 50 μg/L NIC, NOR, or ANA was separately 461
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Figure 1. MRM chromatograms of five TSNAs and three tobacco alkaloids by LC−MS/MS. Concentration = 1 μg/L.
9.2%; NNAL, 74.0 ± 8.3%; NIC, 64.7 ± 11.7%; NOR, 56.0 ± 2.7%; and ANA, 61.6 ± 4.7%). In comparison to other reported methods,34−39 this method is more sensitive and faster and can simultaneously determine TSNAs and tobacco alkaloids, enabling the identification of the precursors of the TSNAs in the following studies. Identification of Tobacco Alkaloids as the Precursors of TSNAs. To confirm the hypothesis that TSNAs are formed from specific tobacco alkaloids, we analyzed drinking water samples for the tobacco alkaloids and TSNAs using the developed SPE−LC−MS/MS (MRM) method. NIC was detected in the source water at 15.1 and 1.1 ng/L in the treated water of a treatment plant. None of the other alkaloids and TSNAs were detected in the source water samples. NNK and NNAL were determined to be 0.05 and 0.25 ng/L in the treated water samples, respectively. NNK and NNAL are carcinogens,16−19 and NNK is more carcinogenic to humans than NDMA, according to the IARC monograph.18 Although the concentrations of NNK and NNAL are lower than that of NDMA commonly detected in treated water (several dozen nanograms per liter),25 it is reported that the concentration of NNK corresponding to a 10−6 cancer risk is 0.8 ng/L,24 suggesting that long-term exposure to drinking water containing TSNAs at a sub-nanogram per liter level is a health risk. To confirm that NNK and NNAL are produced from NIC in the water, we performed the chloramination of the same source water samples. We found that chloramination of the source water samples resulted in the reduction of NIC from 15.1 to 1.2 ng/L and the formation of NNK (0.2 ng/L) and NNAL (0.6 ng/L). The results suggest that NIC is the precursor of NNK and NNAL formation. To further demonstrate that tobacco alkaloids can be the precursors of the TSNAs, we performed chloramination of water-leaching samples of cigarettes. Using LC−MS/MS (MRM), we first analyzed the 20 000-fold diluted leaching samples for the three tobacco alkaloids and the five TSNAs.
tobacco alkaloids and TSNAs in cigarettes. The diluted leaching samples were reacted with monochloramine for the FP test described above. After the FP reactions, the samples were directly analyzed using LC−MS/MS (MRM) without SPE.
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RESULTS AND DISCUSSION Determination of TSNAs and Tobacco Alkaloids in Water. To confirm the precursors and TSNAs in water samples, we have developed a LC−MS/MS (MRM) method to determine the five TSNAs and three tobacco alkaloids. Figure 1 shows a typical chromatogram of the TSNAs and tobacco alkaloids. The confirmation of the compounds is based on the mass/charge ratio (m/z) and the retention time. This analysis takes 3.5 min for the separation of the compounds and 2.5 min for the column cleaning and equilibration. Table S2 of the Supporting Information summarizes the analytical performance of the developed LC−MS/MS method. The limits of detection (LODs, 3σ) were 0.4−4.7 ng/L for the five TSNAs and 2.7− 14.4 ng/L for the three tobacco alkaloids. The linear response range was 0.01−10 μg/L for NAB, NNN, NAT, and NNK, 0.02−10 μg/L for NNAL, NOR, and ANA, and 0.05−10 μg/L for NIC, with R2 greater than 0.99. The method provides reproducible retention times and peak areas. The accuracy of the method is 85−114%, with the relative standard deviations (RSDs; n = 7) of 5.1−9.7%, when three concentration levels of 0.050, 0.20, and 2.00 μg/L were examined. To enable the determination of these compounds in drinking water samples from a water treatment plant, we developed and evaluated a SPE method using Oasis HLB cartridges prior to the LC−MS/MS (MRM) analysis. In tap water samples, the LODs of the SPE−LC−MS/MS method were 0.005, 0.002, 0.009, 0.010, 0.043, 0.136, 0.020, and 0.068 ng/L for NAB, NNN, NAT, NNK, NNAL, NIC, NOR, and ANA, respectively. The SPE recoveries of the eight compounds (1 ng/L each) in tap water were obtained in a range of 56.0−81.5% (NAB, 73.5 ± 3.9%; NNN, 62.9 ± 2.7%; NAT, 64.4 ± 5.5%; NNK, 81.5 ± 462
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Figure 2. Time course of the formation of TSNAs and reduction of tobacco alkaloids from the reactions of cigarette-leaching samples of C1, C2, and C3 with monochloramine (50 mM) (error bars represent the standard deviation value for triplicate analysis).
Figure 2 shows that the concentrations of NNK and NNAL are significantly higher than the other three TSNAs, because their specific precursor NIC is 40 times higher than other tobacco alkaloids (NOR and ANA) in cigarettes. The higher NIC also results in higher yields for NNK and NNAL than other TSNAs during chloramination. The molar yields (Ym) of TSNAs generated from specific tobacco alkaloids in the diluted leaching samples were calculated on the basis of eq 1
The mean concentrations were determined to be 8.0 ng/L for NIC, 0.15 ng/L for NOR, and 0.21 ng/L for ANA in the diluted leaching samples. No TSNAs were detected in the diluted leaching samples. Table S3 of the Supporting Information presents the concentrations of the detected alkaloids in diluted leaching samples and in one cigarette, providing that the leaching of alkaloids was complete. On the basis of the concentrations of alkaloid precursors, the diluted cigarette-leaching samples were chloraminated with ca. 25 000fold molar excess of monochloramine. During chloramination, the concentrations of TSNAs and tobacco alkaloids were analyzed every 1−2 h for up to 20 h. Figure 2 clearly shows the time course of the formation of TSNAs along with the reduction of the precursor alkaloids. The reaction kinetics of NIC, NOR, and ANA in FP tests was similar, although the concentrations of NOR and ANA were significantly lower than NIC. With an increase in reaction time, the concentrations of NIC, NOR, and ANA decreased, while the concentrations of the five TSNAs increased. After a reaction time of about 10 h, all three tobacco alkaloids were consumed, while the TSNAs reached the maximum concentrations. The reduction of tobacco alkaloids coincides with the formation of TSNAs, supporting that tobacco alkaloids can be the precursors of TSNAs during water chloramination.
Ym = C Tt /(CA0 − CAt ) × 100%
(1)
where CTt and CAt are the concentrations (ng/L) of TSNAs and corresponding tobacco alkaloids at a specified reaction time (t), respectively, and CA0 is the initial concentration (ng/L) of the tobacco alkaloid. At a given time of 18 h when the reaction reached equilibrium, the Ym for NNK, NNAL, NNN, and NAB is 4.9, 41.8, 0.4, and 2.7%, respectively, in C1 diluted leaching sample. The yield (4.9%) of NNK is 8 times lower than NNAL (41.8%), supporting that NNAL is more stable than NNK under chloramination conditions. Similar results were also found in C2 and C3 diluted leaching samples (see Table S4 of the Supporting Information). Different molar yields were obtained for different TSNAs probably because of structuraldependent reactivity. Figure 2 also shows that the formation of NNK and NNAL reaches a plateau, suggesting that they are relatively stable in 463
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Figure 3. XICs of (a) NIC standard solution and (b) NIC FP test solution, isotope pattern and MS/MS spectra of the intermediates (c) A and (d) B produced in the NIC FP test system, and (e) possible formation pathway. (NIC, 1 mg/L; monochloramine, 100 mM).
explains why NNK and NNAL are detected in the drinking water distribution system but not other TSNAs. The higher concentration of NIC in water kinetically favors the formation
the reaction system. The high content of NIC in cigarettes leads to its higher concentration in wastewater. The chloramination of source water impacted by wastewater 464
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oamino)-2-(pyridine-3-yl)piperidinium ion (CAPPI), with mass accuracy of −2.6 ppm. A formation pathway of NAB from ANA is also proposed (see Figure S2d of the Supporting Information), which is similar to that of NNN from NOR. It consists of the (i) formation of the CAPPI intermediate from the reaction of ANA with monochloramine and (ii) oxidation of CAPPI by monochloramine to NAB.
of NNK and NNAL over other TSNAs. The low concentrations of NOR and ANA lead to a low yield of other TSNAs. 3.3. Mechanisms of the Formation of TSNAs during Chloramination. To further confirm the formation of the TSNAs from specific tobacco alkaloids, we performed the reactions of monochloramine with the standard solutions of NIC, NOR, and ANA. The reaction mixtures were analyzed using the LC−Q-TOF−MS/MS method. Figure 3 shows the extracted ion chromatograms (XICs) of the (a) NIC standard solution and (b) reaction mixture of NIC with monochloramine solution. Figure 3a shows that only NIC is detected without monochloramine. After the reaction of NIC with monochloramine, Figure 3b shows that the peaks of NNK and NNAL along with two new peaks A and B are detected, while NIC is reduced to be barely detectable. To identify the two new peaks, we carefully examined the accurate mass and isotope patterns of the parent ions and the MS/MS spectra of peaks A and B (panels c and d of Figure 3). The results are presented in Table S5 of the Supporting Information. Peaks A and B have accurate masses of m/z 178.1337 and 192.1130, respectively. PeakView analysis of the isotope patterns identified peak A corresponding to [C10H16N3]+ and peak B corresponding to [C10H14N3O]+, with the mass accuracy of peak A of −1.0 ppm and peak B of −1.8 ppm. On the basis of the accurate masses and MS/MS spectra and with a pyridine group in the composition of the compound taken into consideration, the PeakView search matches peak A with 1amino-1-methyl-2-(pyridine-3-yl)pyrrolidinium ion (AMPP) and peak B with 1-methyl-1-nitroso-2-(pyridine-3-yl)pyrrolidinium ion (MNPP). On the basis of these results described above, we proposed a possible formation pathway of NNK and NNAL from NIC, as shown in Figure 3e. It consists of the (i) formation of the AMPP intermediate from the reaction of NIC and monochloramine, (ii) oxidation of AMPP by monochloramine to MNPP, (iii) oxidation of MNPP by monochloramine to open the ring to form NNK, and (iv) transformation of NNK to NNAL. Similarly, we identified the intermediates of the reaction of NOR with monochloramine. Figure S1 of the Supporting Information presents the XIC chromatograms of the (a) NOR standard solution and (b) reaction mixture of NOR with monochloramine solution. As NOR is consumed, the formation of NNN and a new compound is detected in the reaction mixture. Figure S1c of the Supporting Information presents the new compound of m/z 198.0785, its isotope pattern, and MS/ MS spectrum (details summarized in Table S5 of the Supporting Information). The PeakView search identifies this compound as 1-(chloroamino)-2-(pyridine-3-yl)pyrrolidinium ion (CAPP). The mass accuracy for CAPP was −2.3 ppm. The results suggest a possible formation pathway of NNN from NOR, as shown in Figure S1d of the Supporting Information. It consists of the (i) nucleophilic attack of the amine group on monochloramine to form CAPP, followed by (ii) oxidation of CAPP to NNN. Figure S2 of the Supporting Information presents the results obtained from the reaction of ANA with monochloramine solution. In comparison to control ANA (see Figure S2a of the Supporting Information), an intermediate (see Figure S2b of the Supporting Information) was detected during the formation of NAB from ANA. Figure S2c of the Supporting Information shows the isotope pattern and MS/MS spectrum of the intermediate (more details presented in Table S5 of the Supporting Information), which was identified as 1-(chlor-
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ASSOCIATED CONTENT
S Supporting Information *
Additional information on LC−MS/MS analysis, MRM conditions of the five TSNAs and three tobacco alkaloids (Table S1), analytical performance of the developed LC−MS/ MS method (Table S2), concentrations of the tobacco alkaloids in cigarettes (Table S3), molar yields of TSNAs at 18 h from the reaction in the diluted leaching samples (Table S4), theoretical and measured isotope patterns of new intermediates produced in reactions of NIC, NOR, or ANA with monochloramine (Table S5), XICs of (a) NOR standard solution and (b) NOR FP test solution, (c) isotope pattern and MS/MS spectra of the intermediate produced in the NOR FP test system, and (d) possible formation pathway (Figure S1), and XICs of (a) ANA standard solution and (b) ANA FP test solution, (c) isotope pattern and MS/MS spectra of the intermediate produced in the ANA FP test system, and (d) possible formation pathway (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Telephone: 780-492-5094. Fax: 780-492-7800. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The project is supported by grants from the Natural Sciences and Engineering Research Council of Canada, Alberta Health, and Alberta Innovates−Energy and Environmental Solutions. Beibei Chen and Lifang Zhu acknowledge the visiting scholarship from the China Scholarship Council.
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REFERENCES
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(7) Schreiber, I. M.; Mitch, W. A. Nitrosamine formation pathway revisited: The importance of dichloramine and dissolved oxygen. Environ. Sci. Technol. 2006, 40, 6007−6014. (8) Mitch, W. A.; Gerecke, A. C.; Sedlak, D. L. N-Nitrosodimethylamine (NDMA) precursor analysis for chlorination of water and wastewater. Water Res. 2003, 37, 3733−3741. (9) Hrudey, S. E.; Bull, R. J.; Cotruvo, J. A.; Paoli, G.; Wilson, M. Drinking water as a proportion of total human exposure to volatile Nnitrosamines. Risk Anal. 2013, 33, 2179−2208. (10) Shah, A. D.; Mitch, W. A. Halonitroalkanes, halonitriles, haloamides, and N-nitrosamines: A critical review of nitrogenous disinfection byproduct formation pathways. Environ. Sci. Technol. 2012, 46, 119−131. (11) Zhao, Y.-Y.; Boyd, J.; Woodbeck, M.; Andrews, R. C.; Qin, F.; Hrudey, S. E.; Li, X.-F. Formation of N-nitrosamines from eleven disinfection treatments of seven different surface waters. Environ. Sci. Technol. 2008, 42, 4857−4862. (12) Choi, J. H.; Valentine, R. L. Formation of N-nitrosodimethylamine (NDMA) from reaction of monochloramine: A new disinfection by-product. Water Res. 2002, 36, 817−824. (13) Mitch, W. A.; Sedlak, D. L. Formation of N-nitrosodimethylamine (NDMA) from dimethylamine during chlorination. Environ. Sci. Technol. 2002, 36, 588−595. (14) Shen, R.; Andrews, S. A. Formation of NDMA from ranitidine and sumatriptan: The role of pH. Water Res. 2013, 47, 802−810. (15) Dai, N.; Mitch, W. A. Relative importance of N-nitrosodimethylamine compared to total N-nitrosamines in drinking waters. Environ. Sci. Technol. 2013, 47, 3648−3656. (16) Hecht, S. S. Biochemistry, biology, and carcinogenicity of tobacco-specific N-nitrosamines. Chem. Res. Toxicol. 1998, 11, 559− 603. (17) Schuller, H. M. Mechanisms of smoking-related lung and pancreatic adenocarcinoma development. Nat. Rev. Cancer 2002, 2, 455−463. (18) International Agency for Research on Cancer (IARC). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans; IARC: Lyon, France, 2014; http://monographs.iarc.fr/ENG/Classification/. (19) International Agency for Research on Cancer (IARC). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans; IARC: Lyon, France, 2007; Vol. 89: Smokeless Tobacco and Some TobaccoSpecific N-Nitrosamines. (20) Hecht, S. S.; Hoffmann, D. The relevance of tobacco-specific nitrosamines to human cancer. Cancer Surv. 1989, 8, 273−294. (21) Hecht, S. S.; Hoffmann, D. Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco-smoke. Carcinogenesis 1988, 9, 875−884. (22) Hecht, S. S.; Trushin, N.; Castonguay, A.; Rivenson, A. Comparative tumorigenicity and DNA methylation in F344 rats by 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosodimethylamine. Cancer Res. 1986, 46, 498−502. (23) Bodhicharla, R.; Ryde, I. T.; Prasad, G. L.; Meyer, J. N. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK) induces mitochondrial and nuclear DNA damage in Caenorhabditis elegans. Environ. Mol. Mutagen. 2014, 55, 43−50. (24) Andra, S. S.; Makris, K. C. Tobacco-specific nitrosamines in water: An unexplored environmental health risk. Environ. Int. 2011, 37, 412−417. (25) Wu, M.; Qian, Y.; Boyd, J. M.; Leavey, S.; Hrudey, S. E.; Krasner, S. W.; Li, X.-F. Identification of tobacco-specific nitrosamines as disinfection byproducts in chloraminated water. Environ. Sci. Technol. 2014, 48, 1828−1834. (26) Brunnemann, K. D.; Prokopczyk, B.; Djordjevic, M. V.; Hoffmann, D. Formation and analysis of tobacco-specific N-nitrosamines. Crit. Rev. Toxicol. 1996, 26, 121−137. (27) Boleda, M. R.; Galceran, M. T.; Ventura, F. Behavior of pharmaceuticals and drugs of abuse in a drinking water treatment plant (DWTP) using combined conventional and ultrafiltration and reverse osmosis (UF/RO) treatments. Environ. Pollut. 2011, 159, 1584−1591.
(28) Bureau of Water, Illinois Environmental Protection Agency (EPA). Report on Pharmaceuticals and Personal Care Products in Illinois Drinking Water; Bureau of Water, Illinois EPA: Springfield, IL, 2008; http://www.epa.state.il.us/water/pharmaceuticals-in-drinking-water. pdf. (29) Robles-Molina, J.; Gilbert-López, B.; García-Reyes, J. F.; MolinaDíaz, A. Monitoring of selected priority and emerging contaminants in the Guadalquivir River and other related surface waters in the province of Jaén, South East Spain. Sci. Total Environ. 2014, 479−480, 247− 257. (30) Estévez, E.; Cabrera, M. D. C.; Molina-Díaz, A.; Robles-Molina, J.; Palacios-Díaz, M. D. P. Screening of emerging contaminants and priority substances (2008/105/EC) in reclaimed water for irrigation and groundwater in a volcanic aquifer (Gran Canaria, Canary Islands, Spain). Sci. Total Environ. 2012, 433, 538−546. (31) Huerta-Fontela, M.; Galceran, M. T.; Ventura, F. Ultra performance liquid chromatography−tandem mass spectrometry analysis of stimulatory drugs of abuse in wastewater and surface waters. Anal. Chem. 2007, 79, 3821−3829. (32) Subedi, B.; Kannan, K. Mass loading and removal of select illicit drugs in two wastewater treatment plants in New York state and estimation of illicit drug usage in communities through wastewater analysis. Environ. Sci. Technol. 2014, 48, 6661−6670. (33) Zhou, W. J.; Boyd, J.; Qin, F.; Hrudey, S.; Li, X.-F. Formation of N-nitrosodiphenylamine and two new N-containing disinfection byproducts from chloramination of water containing diphenylamine. Environ. Sci. Technol. 2009, 43, 8443−8448. (34) Sleiman, M.; Maddalena, R. L.; Gundel, L. A.; Destaillats, H. Rapid and sensitive gas chromatography−ion-trap tandem mass spectrometry method for the determination of tobacco-specific Nnitrosamines in secondhand smoke. J. Chromatogr. A 2009, 1216, 7899−7905. (35) Xiong, W.; Hou, H. W.; Jiang, X. Y.; Tang, G. L.; Hu, Q. Y. Simultaneous determination of four tobacco-specific N-nitrosamines in mainstream smoke for Chinese Virginia cigarettes by liquid chromatography tandem mass spectrometry and validation under ISO and “Canadian intense” machine smoking regimes. Anal. Chim. Acta 2010, 674, 71−78. (36) Wu, W. J.; Ashley, D. L.; Watson, C. H. Simultaneous determination of five tobacco-specific nitrosamines in mainstream cigarette smoke by isotope dilution liquid chromatography/electrospray ionization tandem mass spectrometry. Anal. Chem. 2003, 75, 4827−4832. (37) Xia, B.; Xia, Y.; Wong, J.; Nicodemus, K. J.; Xu, M.; Lee, J.; Guillot, T.; Li, J. Quantitative analysis of five tobacco-specific Nnitrosamines in urine by liquid chromatography−atmospheric pressure ionization tandem mass spectrometry. Biomed. Chromatogr. 2014, 28, 375−384. (38) Pedrouzo, M.; Borrul, F.; Pocurull, E.; Marce, R. M. Drugs of abuse and their metabolites in waste and surface waters by liquid chromatography−tandem mass spectrometry. J. Sep. Sci. 2011, 34, 1091−1101. (39) Huerta-Fontela, M.; Galceran, M. T.; Martin-Alonso, J.; Ventura, F. Occurrence of psychoactive stimulatory drugs in wastewaters in north-eastern Spain. Sci. Total Environ. 2008, 397, 31−40.
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Identification of Precursors and Mechanisms of Tobacco-Specific Nitrosamine Formation in Water during Chloramination Beibei Chen,†,‡ Yichao Qian,† Minghuo Wu,† Lifang Zhu,†,§ Bin Hu,‡ and Xing-Fang Li*,† †
Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2G3, Canada ‡ Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, Hubei 430072, People’s Republic of China § Zhejiang University of Water Resources and Electric Power, 583 Xuelin Street, Xiasha Higher Education District, Hangzhou, Zhejiang 310018, People’s Republic of China S Supporting Information *
ABSTRACT: We report here that tobacco-specific nitrosamines (TSNAs) are produced from specific tobacco alkaloids during water chloramination. To identify the specific precursors for the formation of specific TSNAs in drinking water, we have developed a solid-phase extraction−liquid chromatography−tandem mass spectrometry (SPE−LC−MS/MS) method for simultaneous determination of five TSNAs and three tobacco alkaloids. Using this method, we detected nicotine (NIC) at 15.1 ng/L in a source water. Chloramination of this source water resulted in the formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK) (0.05 ng/L) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) (0.2 ng/L) along with the reduction of NIC to 1.1 ng/L, suggesting that NNK and NNAL were formed from NIC. To confirm that tobacco alkaloids are the precursors of TSNAs, we chloraminated water-leaching samples of tobacco from three brands of cigarettes and found that the formation of TSNAs coincides with the reduction of the alkaloids. Chloramination of individual alkaloids confirms that NNK and NNAL are produced from NIC, N-nitrosonornicotine (NNN) from nornicotine (NOR), and N-nitrosoanabasine (NAB) from anabasine (ANA). Furthermore, we have identified specific intermediates of these reactions and proposed potential pathways of formation of TSNAs from specific alkaloids. These results confirm that NNK and NNAL are the disinfection byproducts (DBPs) resulting from NIC in raw water.
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detected N-nitrosamine in drinking water systems.9 Consequently, a number of studies have focused on identification of its precursors and elucidation of the formation mechanisms during drinking water treatment.10−14 However, NDMA only accounts for ∼5% of the estimated total nitrosamines,15 suggesting that the majority of nitrosamines in drinking water are still unknown. Therefore, it is important to identify other Nnitrosamine DBPs and to understand their formation
INTRODUCTION Water disinfection kills pathogens to prevent waterborne disease1 but will inevitably cause the generation of unintended disinfection byproducts (DBPs) from the reaction of disinfectants (e.g., chlorine and chloramines) with natural organic matter (NOM) and organic compounds in water.2,3 Epidemiological studies have found a potential association of exposure to DBPs with adverse health effects, such as bladder cancer.4,5 However, animal bioassays indicate that the regulated DBPs, such as trihalomethanes and haloacetic acids, do not account for the increased risk of bladder cancer.6 N-Nitrosamines are a group of non-halogenated DBPs associated with chloramination classified as probable bladder carcinogens.7,8 NNitrosodimethylamine (NDMA) is the most commonly © 2014 American Chemical Society
Received: Revised: Accepted: Published: 459
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Table 1. Structures and Molecular Weights of the Five TSNAs and Three Tobacco Alkaloids
of NNK increased in the formation potential (FP) tests of the source water after chloramination. These results raise several questions: (1) what their precursors are, (2) why the other TSNAs are absent, and (3) how they are produced during water treatment. TSNAs can be formed from nicotine (NIC) and related compounds by a nitrosation reaction.26 Because of the worldwide use of tobacco products, NIC exists at several to hundreds of nanograms per liter in surface water or groundwater.27−31 Its concentration could be higher at the microgram per liter level in wastewater.31,32 On the basis of these facts, we hypothesized that tobacco alkaloids, including NIC and its related compounds, are the precursors of TSNAs and that individual TSNAs are produced from specific precursors. The objectives of this study are to confirm specific tobacco alkaloids as the precursors of certain TSNAs in drinking water and to elucidate the formation pathways of TSNAs during chloramination. To achieve these goals, we have developed a new solid-phase extraction liquid chromatography−tandem mass spectrometry (SPE−LC−MS/MS) method to simultaneously determine both TSNAs and tobacco alkaloids in source water and treated water. The new SPE−LC−MS/MS method can identify and quantify five TSNAs (NNN, NNK, NNAL,
mechanisms during water treatment to limit the formation of N-nitrosamines in drinking water. Several toxicology studies have shown that tobacco-specific nitrosamines (TSNAs) are carcinogenic.16,17 According to the International Agency for Research on Cancer (IARC) monograph,18 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) are carcinogenic to humans (group 1), with higher carcinogenicity than NDMA, a probable human carcinogen (group 2A). N-Nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT) are not classifiable regarding their carcinogenicity to humans (group 3). NNN, NNK, or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) can induce oral, lung, pancreatic, and esophageal cancer in animals.16,19 An increased number of oral tumors was observed in rats given drinking water containing NNK and NNN.20,21 NNK is a more potent mutagen than NDMA in the F344 rat.22 α-Hydroxylation of NNK and NNAL can lead to the formation of DNA adducts, single-strand breaks, and other macromolecular damage.19,23 TSNAs may exist in source water because of contamination by wastewater.24 Our previous results25 confirmed the existence of NNK in source water impacted by wastewater and the occurrence of both NNK and NNAL in the disinfected water. In addition, the concentrations 460
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transferred into the vials, to which 10.7 mL of 140 mM monochloamine solution was added. The pH of the mixture was adjusted to 8 and maintained with the addition of 1.5 mL of 200 mM ammonium acetate buffer. Then, the mixture was diluted with Optima water to 30 mL (10 μg/L NIC, NOR, or ANA, 50 mM monochloramine, and 10 mM buffer), and the chloramination reactions were conducted at 25 °C. At given time points, the reaction solution (50 μL) was sampled and 20 μL was directly injected into the LC−MS/MS (MRM) for the determination of residual tobacco alkaloids and formed TSNAs. Duplicate analyses were performed for each time point. FP control experiments were also performed using Optima water. All FP experiments were performed in triplicate, and the average values along with standard deviations were reported. To elucidate the formation mechanisms of TSNAs from NIC, NOR, and ANA, we used LC−high-resolution Q-TOF− MS/MS to identify the intermediates and TSNAs in the reaction mixtures, when NIC, NOR, and ANA (1 mg/L) were reacted with monochloramine (100 mM). The other FP conditions were the same as described above. Solid-Phase Extraction (SPE). To preconcentrate tobacco alkaloids and TSNAs in drinking water samples collected from a treatment plant, we developed and validated a SPE method using Oasis hydrophilic−lipophilic balanced (HLB) cartridges (6 mL, 200 mg per cartridge, Waters, Milford, MA). The HLB cartridges on a Visiprep SPE manifold (Supelco, Bellefonte, PA) were connected to a vacuum. Each HLB cartridge was initially rinsed with 10 mL of methanol and equilibrated with 10 mL of water. A 500 mL water sample passed through the HLB cartridge at a flow rate of ∼3 mL/min. After the cartridge was washed with 5 mL of 10% methanol, the analytes adsorbed on the cartridge were eluted with 5 mL of methanol. The eluate was collected and condensed to approximately 0.2 mL with a high-purity nitrogen stream in a 40 °C water bath and reconstituted with Optima water to 1 mL for subsequent LC− MS/MS (MRM) analysis. For each set of SPE experiments, a blank control of 500 mL of Optima water and a positive control of 500 mL of Optima water containing 4 ng/L each of the analytes were included. Water Sample Collection and Preparation. Water samples including the source water and the treated water from a drinking water treatment plant were collected in 500 mL amber glass bottles. Ascorbic acid (0.5 g/L) was immediately added to the samples after collection to quench the free chlorine and the samples were stored at 4 °C. The source water samples were filtered with glass microfiber filters (47 mm × 1.5 μm, Waterman) and nylon membrane disc filters (47 mm × 0.45 μm, Pall Corporation) prior to SPE. The SPE combined with the LC−MS/MS (MRM) method was used to determine the concentrations of tobacco alkaloids and TSNAs in the source water and the treated water samples. The source water samples were also used for FP tests of TSNAs. We randomly selected three brands of cigarettes (C1, C2, and C3) that are most commonly available in grocery stores in Edmonton. All three are domestic brands in Canada. Tobacco in each cigarette was removed, weighed, and then soaked in 100 mL of Optima water in a 250 mL beaker. After this suspension was stirred for 24 h at room temperature, the water leaching sample of the cigarette was collected through filtration with a glass microfiber filter (47 mm × 1.5 μm, Waterman) and a nylon membrane disc filter (47 mm × 0.45 μm, Pall Corporation) and stored at 4 °C. The cigarette-leaching samples were diluted with water 20 000 times for analysis of
NAB, and NAT) and three tobacco alkaloids [NIC, nornicotine (NOR), and anabasine (ANA)] at sub-nanogram per liter levels. To confirm that tobacco alkaloids are the precursors of various TSNAs, we further studied the formation of TSNAs from the chloramination of water-leaching samples of tobacco in three brands of cigarettes. In addition, chloramination of individual NIC, NOR, and ANA standard solutions was conducted to verify that NIC, NOR, and ANA are the precursors of specific TSNAs. Finally, we used a high resolution triple quadrupole time-of-flight (TOF) mass spectrometer to identify the intermediates formed from the reactions of tobacco alkaloids with monochloramine to elucidate possible reaction pathways.
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EXPERIMENTAL SECTION Chemicals. TSNA standards of NNK, NNAL, NNN, NAB, and NAT were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). The standards of tobacco alkaloids NIC, NOR, and ANA were obtained from SigmaAldrich (Milwaukee, WI). The structures and molecular weights of the five TSNAs and three alkaloids are described in Table 1. NNK and NNAL are the nitroso derivatives of NIC; NNN is the nitroso derivative of NOR; NAB is the nitroso derivative of ANA; and NAT is the nitroso derivative of anatabine. Methanol and water of Optima LC−MS grade were provided by Fisher Scientific (Fair Lawn, NJ). All other chemicals used were of analytical grade. Stock solutions (1 g/L) of the five TSNAs and three tobacco alkaloids were prepared in methanol and stored at −20 °C. Working solutions were prepared daily by diluting the corresponding stock solution in water. LC−MS/MS Analysis. An Agilent 1290 LC system (Santa Clara, CA) with a Kinetex C18 column (100 mm × 3 mm × 2.6 μm, Phenomenex, Torrance, CA) was used for the separation of three alkaloids and five TSNAs. The mobile phase consisted of solvent A (10 mmol/L ammonium acetate and 0.01% acetic acid in water) and solvent B (100% methanol), and the flow rate was 0.4 mL/min. A gradient program was performed as follows: linearly increased B from 35 to 80% over 3.5 min, changed B to 90% in 0.1 min and kept B at 90% from 3.6 to 4.6 min, returned to 35% B for column equilibration from 4.7 to 6 min. The sample injection volume was 20 μL, and the column temperature was 25 °C. Multiple-reaction monitoring (MRM) methods were performed using a triple quadrupole ion-trap tandem mass spectrometer (QTRAP 5500, AB Sciex, Concord, Ontario, Canada) for the quantification of the eight analytes. A highresolution quadrupole time-of-flight tandem mass spectrometer (Q-TOF−MS/MS, TripleTOF 5600, AB Sciex) was used to identify the intermediate products during the chloramination process to elucidate the formation pathways of TSNAs. The conditions of the QTRAP−MS/MS and Q-TOF−MS/MS experiments are described in the Supporting Information, including Table S1 of the Supporting Information. Formation Potential (FP) of TSNAs from Chloramination. Stock solution of monochloramine (NH2Cl) solution (140 mM) was prepared according to the procedures previously reported.33 To study the formation of TSNAs during water chloramination, we conducted sets of reactions in batches of 40 mL Supelco amber EPA vials with caps (SigmaAldrich, Oakville, Ontario, Canada). The volume of each reaction solution was kept at 30 mL. For FP tests, an aliquot of 6 mL of 50 μg/L NIC, NOR, or ANA was separately 461
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Figure 1. MRM chromatograms of five TSNAs and three tobacco alkaloids by LC−MS/MS. Concentration = 1 μg/L.
9.2%; NNAL, 74.0 ± 8.3%; NIC, 64.7 ± 11.7%; NOR, 56.0 ± 2.7%; and ANA, 61.6 ± 4.7%). In comparison to other reported methods,34−39 this method is more sensitive and faster and can simultaneously determine TSNAs and tobacco alkaloids, enabling the identification of the precursors of the TSNAs in the following studies. Identification of Tobacco Alkaloids as the Precursors of TSNAs. To confirm the hypothesis that TSNAs are formed from specific tobacco alkaloids, we analyzed drinking water samples for the tobacco alkaloids and TSNAs using the developed SPE−LC−MS/MS (MRM) method. NIC was detected in the source water at 15.1 and 1.1 ng/L in the treated water of a treatment plant. None of the other alkaloids and TSNAs were detected in the source water samples. NNK and NNAL were determined to be 0.05 and 0.25 ng/L in the treated water samples, respectively. NNK and NNAL are carcinogens,16−19 and NNK is more carcinogenic to humans than NDMA, according to the IARC monograph.18 Although the concentrations of NNK and NNAL are lower than that of NDMA commonly detected in treated water (several dozen nanograms per liter),25 it is reported that the concentration of NNK corresponding to a 10−6 cancer risk is 0.8 ng/L,24 suggesting that long-term exposure to drinking water containing TSNAs at a sub-nanogram per liter level is a health risk. To confirm that NNK and NNAL are produced from NIC in the water, we performed the chloramination of the same source water samples. We found that chloramination of the source water samples resulted in the reduction of NIC from 15.1 to 1.2 ng/L and the formation of NNK (0.2 ng/L) and NNAL (0.6 ng/L). The results suggest that NIC is the precursor of NNK and NNAL formation. To further demonstrate that tobacco alkaloids can be the precursors of the TSNAs, we performed chloramination of water-leaching samples of cigarettes. Using LC−MS/MS (MRM), we first analyzed the 20 000-fold diluted leaching samples for the three tobacco alkaloids and the five TSNAs.
tobacco alkaloids and TSNAs in cigarettes. The diluted leaching samples were reacted with monochloramine for the FP test described above. After the FP reactions, the samples were directly analyzed using LC−MS/MS (MRM) without SPE.
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RESULTS AND DISCUSSION Determination of TSNAs and Tobacco Alkaloids in Water. To confirm the precursors and TSNAs in water samples, we have developed a LC−MS/MS (MRM) method to determine the five TSNAs and three tobacco alkaloids. Figure 1 shows a typical chromatogram of the TSNAs and tobacco alkaloids. The confirmation of the compounds is based on the mass/charge ratio (m/z) and the retention time. This analysis takes 3.5 min for the separation of the compounds and 2.5 min for the column cleaning and equilibration. Table S2 of the Supporting Information summarizes the analytical performance of the developed LC−MS/MS method. The limits of detection (LODs, 3σ) were 0.4−4.7 ng/L for the five TSNAs and 2.7− 14.4 ng/L for the three tobacco alkaloids. The linear response range was 0.01−10 μg/L for NAB, NNN, NAT, and NNK, 0.02−10 μg/L for NNAL, NOR, and ANA, and 0.05−10 μg/L for NIC, with R2 greater than 0.99. The method provides reproducible retention times and peak areas. The accuracy of the method is 85−114%, with the relative standard deviations (RSDs; n = 7) of 5.1−9.7%, when three concentration levels of 0.050, 0.20, and 2.00 μg/L were examined. To enable the determination of these compounds in drinking water samples from a water treatment plant, we developed and evaluated a SPE method using Oasis HLB cartridges prior to the LC−MS/MS (MRM) analysis. In tap water samples, the LODs of the SPE−LC−MS/MS method were 0.005, 0.002, 0.009, 0.010, 0.043, 0.136, 0.020, and 0.068 ng/L for NAB, NNN, NAT, NNK, NNAL, NIC, NOR, and ANA, respectively. The SPE recoveries of the eight compounds (1 ng/L each) in tap water were obtained in a range of 56.0−81.5% (NAB, 73.5 ± 3.9%; NNN, 62.9 ± 2.7%; NAT, 64.4 ± 5.5%; NNK, 81.5 ± 462
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Figure 2. Time course of the formation of TSNAs and reduction of tobacco alkaloids from the reactions of cigarette-leaching samples of C1, C2, and C3 with monochloramine (50 mM) (error bars represent the standard deviation value for triplicate analysis).
Figure 2 shows that the concentrations of NNK and NNAL are significantly higher than the other three TSNAs, because their specific precursor NIC is 40 times higher than other tobacco alkaloids (NOR and ANA) in cigarettes. The higher NIC also results in higher yields for NNK and NNAL than other TSNAs during chloramination. The molar yields (Ym) of TSNAs generated from specific tobacco alkaloids in the diluted leaching samples were calculated on the basis of eq 1
The mean concentrations were determined to be 8.0 ng/L for NIC, 0.15 ng/L for NOR, and 0.21 ng/L for ANA in the diluted leaching samples. No TSNAs were detected in the diluted leaching samples. Table S3 of the Supporting Information presents the concentrations of the detected alkaloids in diluted leaching samples and in one cigarette, providing that the leaching of alkaloids was complete. On the basis of the concentrations of alkaloid precursors, the diluted cigarette-leaching samples were chloraminated with ca. 25 000fold molar excess of monochloramine. During chloramination, the concentrations of TSNAs and tobacco alkaloids were analyzed every 1−2 h for up to 20 h. Figure 2 clearly shows the time course of the formation of TSNAs along with the reduction of the precursor alkaloids. The reaction kinetics of NIC, NOR, and ANA in FP tests was similar, although the concentrations of NOR and ANA were significantly lower than NIC. With an increase in reaction time, the concentrations of NIC, NOR, and ANA decreased, while the concentrations of the five TSNAs increased. After a reaction time of about 10 h, all three tobacco alkaloids were consumed, while the TSNAs reached the maximum concentrations. The reduction of tobacco alkaloids coincides with the formation of TSNAs, supporting that tobacco alkaloids can be the precursors of TSNAs during water chloramination.
Ym = C Tt /(CA0 − CAt ) × 100%
(1)
where CTt and CAt are the concentrations (ng/L) of TSNAs and corresponding tobacco alkaloids at a specified reaction time (t), respectively, and CA0 is the initial concentration (ng/L) of the tobacco alkaloid. At a given time of 18 h when the reaction reached equilibrium, the Ym for NNK, NNAL, NNN, and NAB is 4.9, 41.8, 0.4, and 2.7%, respectively, in C1 diluted leaching sample. The yield (4.9%) of NNK is 8 times lower than NNAL (41.8%), supporting that NNAL is more stable than NNK under chloramination conditions. Similar results were also found in C2 and C3 diluted leaching samples (see Table S4 of the Supporting Information). Different molar yields were obtained for different TSNAs probably because of structuraldependent reactivity. Figure 2 also shows that the formation of NNK and NNAL reaches a plateau, suggesting that they are relatively stable in 463
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Figure 3. XICs of (a) NIC standard solution and (b) NIC FP test solution, isotope pattern and MS/MS spectra of the intermediates (c) A and (d) B produced in the NIC FP test system, and (e) possible formation pathway. (NIC, 1 mg/L; monochloramine, 100 mM).
explains why NNK and NNAL are detected in the drinking water distribution system but not other TSNAs. The higher concentration of NIC in water kinetically favors the formation
the reaction system. The high content of NIC in cigarettes leads to its higher concentration in wastewater. The chloramination of source water impacted by wastewater 464
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oamino)-2-(pyridine-3-yl)piperidinium ion (CAPPI), with mass accuracy of −2.6 ppm. A formation pathway of NAB from ANA is also proposed (see Figure S2d of the Supporting Information), which is similar to that of NNN from NOR. It consists of the (i) formation of the CAPPI intermediate from the reaction of ANA with monochloramine and (ii) oxidation of CAPPI by monochloramine to NAB.
of NNK and NNAL over other TSNAs. The low concentrations of NOR and ANA lead to a low yield of other TSNAs. 3.3. Mechanisms of the Formation of TSNAs during Chloramination. To further confirm the formation of the TSNAs from specific tobacco alkaloids, we performed the reactions of monochloramine with the standard solutions of NIC, NOR, and ANA. The reaction mixtures were analyzed using the LC−Q-TOF−MS/MS method. Figure 3 shows the extracted ion chromatograms (XICs) of the (a) NIC standard solution and (b) reaction mixture of NIC with monochloramine solution. Figure 3a shows that only NIC is detected without monochloramine. After the reaction of NIC with monochloramine, Figure 3b shows that the peaks of NNK and NNAL along with two new peaks A and B are detected, while NIC is reduced to be barely detectable. To identify the two new peaks, we carefully examined the accurate mass and isotope patterns of the parent ions and the MS/MS spectra of peaks A and B (panels c and d of Figure 3). The results are presented in Table S5 of the Supporting Information. Peaks A and B have accurate masses of m/z 178.1337 and 192.1130, respectively. PeakView analysis of the isotope patterns identified peak A corresponding to [C10H16N3]+ and peak B corresponding to [C10H14N3O]+, with the mass accuracy of peak A of −1.0 ppm and peak B of −1.8 ppm. On the basis of the accurate masses and MS/MS spectra and with a pyridine group in the composition of the compound taken into consideration, the PeakView search matches peak A with 1amino-1-methyl-2-(pyridine-3-yl)pyrrolidinium ion (AMPP) and peak B with 1-methyl-1-nitroso-2-(pyridine-3-yl)pyrrolidinium ion (MNPP). On the basis of these results described above, we proposed a possible formation pathway of NNK and NNAL from NIC, as shown in Figure 3e. It consists of the (i) formation of the AMPP intermediate from the reaction of NIC and monochloramine, (ii) oxidation of AMPP by monochloramine to MNPP, (iii) oxidation of MNPP by monochloramine to open the ring to form NNK, and (iv) transformation of NNK to NNAL. Similarly, we identified the intermediates of the reaction of NOR with monochloramine. Figure S1 of the Supporting Information presents the XIC chromatograms of the (a) NOR standard solution and (b) reaction mixture of NOR with monochloramine solution. As NOR is consumed, the formation of NNN and a new compound is detected in the reaction mixture. Figure S1c of the Supporting Information presents the new compound of m/z 198.0785, its isotope pattern, and MS/ MS spectrum (details summarized in Table S5 of the Supporting Information). The PeakView search identifies this compound as 1-(chloroamino)-2-(pyridine-3-yl)pyrrolidinium ion (CAPP). The mass accuracy for CAPP was −2.3 ppm. The results suggest a possible formation pathway of NNN from NOR, as shown in Figure S1d of the Supporting Information. It consists of the (i) nucleophilic attack of the amine group on monochloramine to form CAPP, followed by (ii) oxidation of CAPP to NNN. Figure S2 of the Supporting Information presents the results obtained from the reaction of ANA with monochloramine solution. In comparison to control ANA (see Figure S2a of the Supporting Information), an intermediate (see Figure S2b of the Supporting Information) was detected during the formation of NAB from ANA. Figure S2c of the Supporting Information shows the isotope pattern and MS/MS spectrum of the intermediate (more details presented in Table S5 of the Supporting Information), which was identified as 1-(chlor-
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ASSOCIATED CONTENT
S Supporting Information *
Additional information on LC−MS/MS analysis, MRM conditions of the five TSNAs and three tobacco alkaloids (Table S1), analytical performance of the developed LC−MS/ MS method (Table S2), concentrations of the tobacco alkaloids in cigarettes (Table S3), molar yields of TSNAs at 18 h from the reaction in the diluted leaching samples (Table S4), theoretical and measured isotope patterns of new intermediates produced in reactions of NIC, NOR, or ANA with monochloramine (Table S5), XICs of (a) NOR standard solution and (b) NOR FP test solution, (c) isotope pattern and MS/MS spectra of the intermediate produced in the NOR FP test system, and (d) possible formation pathway (Figure S1), and XICs of (a) ANA standard solution and (b) ANA FP test solution, (c) isotope pattern and MS/MS spectra of the intermediate produced in the ANA FP test system, and (d) possible formation pathway (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Telephone: 780-492-5094. Fax: 780-492-7800. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The project is supported by grants from the Natural Sciences and Engineering Research Council of Canada, Alberta Health, and Alberta Innovates−Energy and Environmental Solutions. Beibei Chen and Lifang Zhu acknowledge the visiting scholarship from the China Scholarship Council.
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REFERENCES
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