Research Article Received: 7 January 2014
Revised: 24 March 2014
Accepted: 5 May 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 1658–1664 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6943
Characteristic fragmentation behavior of tobacco-specific Nnitrosamines using electrospray ionization multistage tandem mass spectrometry incorporating deuterium labeling An-Fu Hu, Jian Jiang, Guo-Jun Zhou*, Jun Yang, Wei-Qiang Xiao and Jian Xu Technology Center, China Tobacco Zhejiang Industrial Co., Ltd, Hangzhou 310024, P.R. China RATIONALE: Tobacco-specific N-nitrosamines (TSNAs) mainly include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK), N-nitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT) that are formed from tobacco alkaloids during the curing process and contained in tobacco and tobacco smoke. They are linked with carcinogenesis. Analytical methods for quality control of products and determination of their metabolites are therefore of great importance. METHODS: The characteristic fragmentation behaviors of tobacco-specific TSNAs have been studied by electrospray ionization multistage tandem mass spectrometry. The deutero-labeled TSNA compounds have also been employed to clarify the fragmentation mechanism, which further confirms the proposed fragmentation patterns. RESULTS: Detailed analysis of the resultant fragments shows there are two different kinds of fragmentation patterns with the general fragment backbone of pyrrolidine or piperidine rings. In one route, pyrrolidine or piperidine rings undergo direct fragmentation and form some stable intermediates without affecting the parent rings. The other, however, involves ring opening and then ring closure at the pyridine-2 carbon atom to form multi-membered rings. CONCLUSIONS: This characteristic fragmentation behavior therefore provides useful information on identification of TSNAs that may be used to monitor such kinds of compound in the biological metabolism. Copyright © 2014 John Wiley & Sons, Ltd.
Tobacco-specific N-nitrosamines (TSNAs) mainly include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), Nnitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT) that are formed from tobacco alkaloids during the curing process and contained in tobacco and tobacco smoke.[1–3] The TSNAs are of interest because of their link with carcinogenesis. Various studies have implicated that TSNAs are the potential sources of carcinogenesis in animal models,[4] and Hoffmann and co-workers have identified that TSNAs are potentially associated with increased risk of illness.[5,6] The International Agency for Research on Cancer has classified NNK and NNN as group 1 carcinogens – "carcinogenic to humans".[7] NAB is classified as a weak esophageal carcinogen in rats, whereas NAT is a group 3 carcinogen – "not classifiable for human carcinogenicity", showing no carcinogenicity in animal studies featuring rats.[8] In view of the health implications, analytical methods for quality control of products and determination of their metabolites are therefore of significant importance. The most commonly used detection technologies so far are mainly based on gas chromatography/mass spectrometry (GC/MS),[9,10] gas chromatography/thermal energy analyzer (GC/TEA),[11,12] and liquid chromatography/ tandem mass spectrometry (LC/MS/MS).[13–16]
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* Correspondence to: G.-J. Zhou, Technology Center, China Tobacco Zhejiang Industrial Co., Ltd, Hangzhou 310024, P.R. China. E-mail: [email protected]
Rapid Commun. Mass Spectrom. 2014, 28, 1658–1664
Electrospray ionization tandem mass spectrometry (ESI-MSn) is a very powerful tool for structural determination and has been widely used in chemistry, biochemistry and pharmaceutical research.[17] Although a few pioneering works have been carried out to gain insights into the fragmentation patterns of TSNAs by utilizing ESI-MSn, none of them give a detailed discussion about particular fragmentation mechanisms.[18,19] Herein we present the ESI-MSn behavior of tobacco-specific TSNAs and try to establish fragmentation patterns for these compounds that can be used for their characterization. Furthermore, the ESI-MSn of deutero-labeled TSNA compounds is used to support the proposed identities of the product ions and therefore the mechanism. Identification of the ways in which such kinds of compounds reorganize into small fragments may provide some useful information and guidance to monitor their metabolisms in the biological system.[20,21]
EXPERIMENTAL MSn characteristic fragmentation behavior of tobacco-specific N-nitrosamines (and TSNA-d4) was achieved using an LCQ™ Deca XP Plus ion trap mass spectrometer (Thermo, USA) utilizing electrospray ionization (ESI), which is capable of analyzing ions up to m/z 6000. The scan range was generally from m/z 50 to 250. The compounds, dissolved in methanol at a concentration of 1.0 × 10–5 mol L–1, were infused into
Copyright © 2014 John Wiley & Sons, Ltd.
Fragmentation behavior of tobacco-specific N-nitrosamines Table 1. Chemical structures of the four TSNAs and deutero-labeled TSNAs NNK/NNK-d4
NAB/NAB-d4
the ESI probe at a rate of 3 μL min–1. The nebulizer pressure was 7 psi and the capillary was typically held at 4 kV. The capillary temperature was maintained at 250 °C. Nitrogen was used as drying gas at a flow rate of 4 L min–1. The TSNA compounds were purchased from Sigma-Aldrich. The chemical structures of the four TSNAs and deutero-labeled TSNAs are shown in Table 1. The selected ion was first isolated and then fragmented through collisions with helium to yield tandem mass spectra. The fragmentation amplitude values were 0.5–1.0 V and the fragmentation time was 40 ms. For each of the compounds the [M + H]+ ion was selected for MS2 analysis and the resulting most abundant product ion was selected for MS3, and so on. An isolation width of 1 m/z unit was used during each MSn stage.
NAT/NAT-d4
178 ion. For this to happen the nitrogen atom has to have a hydrogen atom donated from the 2-position of the pyridine ring and another hydrogen from 4-cleavage of butan-1-one. In ESI-MS4 (Fig. 1(D1)), the fragment ion at m/z 119 corresponds to loss of ethane, which could be the radical that directly attacks the carbonyl to form a four-membered ring.
Table 2. MS/MS spectral data of significant ions of compound 1–8 [m/z with relative abundance (%) in parentheses]
Compound NNK (1)
RESULTS AND DISCUSSION The ESI-MSn analyses of the TSNAs and TSNA-d4 are summarized in Table 2.
NAB (2) NAT (3)
ESI-MSn fragmentation pathways of [M + H]+ for NNK (1)
Rapid Commun. Mass Spectrom. 2014, 28, 1658–1664
NNN (4)
NNK-d4 (5) NAB-d4 (6)
NAT-d4 (7)
NNN-d4 (8)
Precursor ions, m/z
Fragment ions, m/z (relative abundance, %)
208[M + H] 178 147 122 192[M + H] 162 133 190[M + H] 160
178 (75) 147 (8) 122 (100) 147 (100) 119 (20) 132 (20) 119 (90) 93 (10) 80 (15) 162 (100) 147 (8) 133 (100) 106 (8) 118 (100) 160 (100) 143 (25) 133 (85) 131 (80) 118 (90) 106 (100) 93 (50) 79 (8) 148 (100) 120 (100) 105 (30) 93 (35) 70 (15) 93 (100) 79 (30) 182 (65) 126 (100) 150 (100) 122 (40) 84 (25) 166 (100) 151 (20) 136 (100) 110 (25) 84 (8) 121 (25) 164 (100) 146 (15) 137 (85) 134 (40) 121 (40) 110 (100) 97 (40) 83 (8) 152 (100) 124 (100) 109 (20) 97 (30) 70 (20) 97 (100) 82 (15)
178[M + H] 148 120 212[M + H] 182 126 196[M + H] 166 136 194[M + H] 164 182[M + H] 152
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The corresponding ESI-MSn mass spectrum of NNK (1) is summarized in Fig. 1. Compound 1 (Fig. 1(A1)) gave the protonated molecule ion peak [M + H]+ at m/z 208, which fragmented at MS2 (Fig. 1(B1)) to give two major product ions at m/z 122 and 178, and less abundant ions at m/z 147 and 148. The fragment ion at m/z 178 is the result of α-cleavage, producing the indicated ion and the NO · radical. There were, however, another three ions which were not easily explained. In order to clarify their structures, ESI-MS3(Fig. 1(C1, E1)) was performed. Two major ions at m/z 147 and 119 were detected in the ESI-MS3 spectrum of the ion at m/z 178. It did not show ions at m/z 122 and 148. The plausible fragmentation pattern is shown in Scheme 1. The product ions at m/z 122 and 148 could derive from the direct loss of N-methyl-N-nitrosoethenamine and N-nitrosomethanamine from NNK (1), respectively. In addition, the ions at m/z 93 and 80 correspond to the 3-methylpyridine radical ion and the pyridine ion, respectively. Another ion at m/z 147 rather than 148 is attributed to the loss of CH3NH2 from the m/z
NNN/NNN-d4
A.-F. Hu et al.
A1
B1
C1
D1
E1
Figure 1. ESI-MSn spectra of compound NNK (1): (A1) all MS; (B1) ESI-MS2 of m/z 208; (C1) ESI-MS3 of m/z 178; (D1) ESI-MS4 of m/z 147; (E1) ESI-MS3 of m/z 122.
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Scheme 1. ESI-MSn fragmentation pathway of NNK (1) (corresponding to Fig. 1).
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Rapid Commun. Mass Spectrom. 2014, 28, 1658–1664
Fragmentation behavior of tobacco-specific N-nitrosamines In another way, the ion at m/z 132 is due to the radical attack of an α-carbon to form a five-membered cyclic ring followed by loss of the methyl radical, and double-bond formation. The ESI-MSn spectrum of NNK-d4 (Fig. 2) showed major ions at m/z 212, 182, 150, 122, 126 and 84. The ion at m/z 212 of NNK-d4 (5) displayed [M + H]+ ions at m/z values 4 Th higher than the native analytes. The ion at m/z 182 corresponded to the indicated ion. As to m/z 126 this indicates the pathway of the direct fragment. The ESI-MS3 spectrum (Fig. 1(C2)) reveals ions at m/z 150, 122, 3 Th higher than in the native analytes, respectively, supporting the cyclization mechanism. Thus, the characteristic fragmentation behavior in the ESI-MS process may be used to monitor this compound in the biological metabolism.
ESI-MSn fragmentation pathways of [M + H]+ for NAB (2) NAB (2), which has a molecular mass of 191, showed a protonated molecular ion, [M + H]+, at m/z 192 in ESI-MS. On fragmentation at the MS2 level, as with NNK, an initial loss of 30 Da was observed to give a major product ion at m/z 162, with less abundant ions at m/z 147, 106 and 118. The plausible fragmentation pattern is shown in Scheme 2. There were two possible pathways. In one route, the ion at m/z 133 corresponds to the loss of methanimine from the opening of the piperidine ring, causing rearrangement to form a six-membered radical ring. This ion further fragmented to give m/z 118 due to the removal of the methyl radical. In the other, the product ion at m/z 147 is suggested to
A2
B2
C2
D2
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Figure 2. ESI-MSn spectra of compound NNK-d4 (5): (A2) all MS; (B2) ESI-MS2 of m/z 212; (C2) ESI-MS3 of m/z 182; (D2) ESI-MS3 of m/z 126.
A.-F. Hu et al. originate from the loss of the methyl radical and formation of a double bond in the heterocyclic ring. As to m/z 106 this is due to the direct loss of the prop-2-en-1-amine radical from the piperidine ring. This fragmentation is similar to nicotine, as has been previously reported.[22] The ESI-MSn spectrum of NAB-d4 showed major ions at m/z 166, 136, 121, 151, 110 and 84. The ions at m/z values 3 Th higher than the native analytes support the first pathway, and ions 4 Th higher support the second pathway. ESI-MS displays a different pattern with breakage of the bond between the pyridine and piperidine entities. ESI-MSn fragmentation pathways of [M + H]+ for NAT (3) NAT (3) gave an [M + H]+ ion at m/z 190 by ESI-MS, which under MS2 conditions showed an initial loss of 30 Da to give an ion at m/z 160. The ESI-MS3 spectrum exhibited richer fragment ions. This is illustrated in Scheme 3.The ion at m/z 143 corresponds to the loss of NH3 from the opening of the tetrahydropyridine ring, causing ring expansion and
rearrangement to form a seven-membered ring. The major ion at m/z 133 presumably corresponds to loss of the ethane radical. As to the fragment ions at m/z 131 and 106, these are similar to NAB (2), that has been shown above. ESI-MSn analysis of NATd4 gave ions at m/z 164, 137, 110 and 97, at m/z values 4 Th higher than the native analytes, and m/z 146, 134 and 121, at m/z values 3 Th higher than the native analytes, which supports the postulated ESI-MSn fragmentation. ESI-MSn fragmentation pathways of [M + H]+ for NNN (4) NNN (4) showed a [M + H]+ ion at m/z 178 in ESI-MS. The MS2 characteristic ion spectrum was an ion at m/z 148. This ion further fragmented to give m/z 120, 105, 93, 79 and 70. According to a previous report, the ion at m/z 120 was thought to have two structures. One is loss of the ethane radical from the opening of the pyrrolidine ring and rearrangement to form a five-membered ring to the 2-position of the pyridine ring.[18] Another is α-cleavage and the resulting ring opening of the pyrrolidyl group in m/z 148,
Scheme 2. ESI-MSn fragmentation pathway of NAB (2).
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Scheme 3. ESI-MSn fragmentation pathway of NAT (3).
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Rapid Commun. Mass Spectrom. 2014, 28, 1658–1664
Fragmentation behavior of tobacco-specific N-nitrosamines
Scheme 4. ESI-MSn fragmentation pathway of NNN (4).
followed by pseudo-α-cleavage resulting in the loss of ethane.[19] In ESI-MSn of NAB-d4, a group of characteristic ions at m/z 152, 124, 109, 97 and 70 were observed. The ions at m/z 152, 124, 109 and 97 are at m/z values 4 Th higher than the native analytes, which indicates the second structure. Furthermore, it is interesting that the ion at m/z 105 is directly fragmented from the ion at m/z 148 rather than 120, corresponding to loss of the propane radical. The ions at m/z 70 represent cleavage of the bond between the pyridine and pyrrolidine subunits. This is illustrated in Scheme 4. Spectra of other TSNAs not shown here are given in the Supporting Information.
CONCLUSIONS The ESI-MSn fragmentation patterns of tobacco-specific Nnitrosamines have been studied. The structures of product ions proposed for ESI-MSn have been supported by deutero-labeled TSNAs. Detailed analysis of the resultant fragments shows there are two types of fragmentation patterns in relation to the backbone of pyrrolidine or piperidine rings. In one route, pyrrolidine or piperidine rings undergo direct fragmentation and form some stable intermediates without affecting the parent rings. The other, however, involves ring opening and then ring closure at the pyridine-2 carbon atom to form multi-membered rings. The deutero-labeled TSNAs have also been employed to clarify the fragmentation patterns and further confirm the proposed fragmentation patterns. This characteristic fragmentation behavior therefore provides useful information on identification of TSNAs that may be used to monitor such kinds of compound in the biological metabolism.
Acknowledgements The research was supported by the China National Tobacco Corporation, and the Major Programme (ZJZY2010C00201).
REFERENCES
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[1] K. D. Brunnemann, D. Hoffmann. Analytical studies on tobacco-specific N-nitrosamines in tobacco and tobacco smoke. Crit. Rev. Toxicol. 1991, 21, 235.
[2] J. D. Adams, S. J. Lee, N. Vinchkoski, A. Castonguay, D. Hoffmann. On the formation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone during smoking. Cancer Lett. 1983, 17, 339. [3] A. Wiernik, A. Christakopoulos, L. I. Johansson. Effect of air curing on the chemical composition of to-bacco. Recent Adv. Tobacco Sci. 1995, 21, 39. [4] S. S. Hecht, D. Hoffmann. The relevance of tobaccospecific nitrosamines to human cancer. Cancer Surveys 1989, 8, 273. [5] K. D. Brunnemann, B. Prokopczyk, M. V. Djordjevic, D. Hoffmann. Formation and analysis of tobacco-specific N-nitrosamines. Crit. Rev. Toxicol. 1996, 26, 121. [6] B. Prokopczyk, J. E. Cox, D. Hoffmann, S. E. Waggoner. Identification of tobacco- specific carcinogen in the cervical mucus of smokers and nonsmokers. J. Nat. Cancer Inst. 1997, 89, 868. [7] International Agency for Research on Cancer, IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans – Tobacco habits other than smoking; betel-quid and areca-nut chewing; and some related nitrosamines, IARC Press, Lyon, 1985, p. 37. [8] S. S. Hecht. Biochemistry, biology, and carcinogenicity of tobacco-specific N-nitrosamines. Chem. Res. Toxicol. 1998, 11, 559. [9] J. Zhou, R.-S. Bai, Y.-F. Zhu. Determination of four tobaccospecific nitrosamines in mainstream cigarette smoke by gas chromatography/ion trap mass spectrometry. Rapid Commun. Mass Spectrom. 2007, 21, 4086. [10] S. C. Moldoveanu, M. Borgerding. Formation of tobacco specific nitrosamines in mainstream cigarette smoke. Beitr. Tabakforsch. Int. 2008, 23, 19. [11] J. Adams, K. Brunnemann, D. Hoffmann. Chemical studies on tobacco smoke LXXV. Rapid method for the analysis of tobacco-specific N-nitrosamines by gas– liquid chromatography with a thermal energy analyser. J. Chromatogr. A 1983, 256, 347. [12] CORESTA Recommended Method No. 63. Determination of tobacco specific nitrosamines in cigarette mainstream smoke GC-tea method. CORESTA 2005. [13] M.-J. Wu, Y.-H. Dai, S.-X. Tuo, N.-N. Hu, Y. Li, X.-M. Chen. Analysis of four tobacco-specific nitrosamines in mainstream cigarette smoke of Virginia cigarettes by LCMS/MS. J. Cent. South Univ. Technol. 2008, 15, 627. [14] C. Jansson, A. Paccou, B. G. Osterdahl. Analysis of tobaccospecific N-nitrosamines in snuff by ethyl acetate extraction and liquid chromatography-tandem mass spectrometry. J. Chromatogr. A 2003, 1008, 135.
A.-F. Hu et al. [15] W.-J. Wu, D. L. Ashley, C. H. Watson. 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. [16] M.-J. Wu, N.-N. Hu, Y. Li, X.-J. Sun, X.-M. Chen. Analysis of tobacco-specific nitrosamines in mainstream cigarette smoke with HPLC-MS-MS. Tobacco Chem. 2007, 243, 41. [17] Z. Wang, C. E. C. A. Hop, M.-S. Kim, S.-E. W. Huskey, T. A. Baillie, Z.-Q. Guan. The unanticipated loss of SO2 from sulfonamides in collision-induced dissociation. Rapid Commun. Mass Spectrom. 2003, 17, 81. [18] D. Kavvadias, G. Scherer, M. Urban, F. Cheung, G. Errington, J. Shepperd, M. McEwan. Simultaneous determination of four tobacco-specific N-nitrosamines (TSNA) in human urine. J. Chromatogr. B 2009, 877, 1185. [19] P. M. Clayton, A. Cunningham, J. D. H van Heemst. Quantification of four tobacco-specific nitrosamines in cigarette filter tips using liquid chromatography-tandem mass spectrometry. Anal. Methods 2010, 2, 1085.
[20] Q. Xiao, Y. Ju, X.-P. Yang, Y.-F. Zhao. Electrospray ionization mass spectrometry of AZT H-phosphonates conjugated with steroids. Rapid Commun. Mass Spectrom. 2003, 17, 1405. [21] P.-X. Xu, A.-F. Hu, D. Hu, X. Gao, Y.-F. Zhao. Negative ion electrospray ionization mass spectrometry of nucleoside phosphoramidate monoesters: elucidation of novel rearrangement mechanisms by multistage mass spectrometry incorporating in-source deuterium labeling. Rapid Commun. Mass Spectrom. 2008, 22, 2977. [22] J. Thomas, V. N. Smyth, R. Alex McGuigan, J. Hopps, W. Franklin Smyth. Characterisation of nicotine and related compounds using electrospray ionisation with ion trap mass spectrometry and with quadrupole time-of-flight mass spectrometry and their detection by liquid chromatography/ electrospray ionisation mass spectrometry. Rapid Commun. Mass Spectrom. 2007, 21, 557.
SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s website.
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Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 1658–1664
Revised: 24 March 2014
Accepted: 5 May 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 1658–1664 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6943
Characteristic fragmentation behavior of tobacco-specific Nnitrosamines using electrospray ionization multistage tandem mass spectrometry incorporating deuterium labeling An-Fu Hu, Jian Jiang, Guo-Jun Zhou*, Jun Yang, Wei-Qiang Xiao and Jian Xu Technology Center, China Tobacco Zhejiang Industrial Co., Ltd, Hangzhou 310024, P.R. China RATIONALE: Tobacco-specific N-nitrosamines (TSNAs) mainly include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK), N-nitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT) that are formed from tobacco alkaloids during the curing process and contained in tobacco and tobacco smoke. They are linked with carcinogenesis. Analytical methods for quality control of products and determination of their metabolites are therefore of great importance. METHODS: The characteristic fragmentation behaviors of tobacco-specific TSNAs have been studied by electrospray ionization multistage tandem mass spectrometry. The deutero-labeled TSNA compounds have also been employed to clarify the fragmentation mechanism, which further confirms the proposed fragmentation patterns. RESULTS: Detailed analysis of the resultant fragments shows there are two different kinds of fragmentation patterns with the general fragment backbone of pyrrolidine or piperidine rings. In one route, pyrrolidine or piperidine rings undergo direct fragmentation and form some stable intermediates without affecting the parent rings. The other, however, involves ring opening and then ring closure at the pyridine-2 carbon atom to form multi-membered rings. CONCLUSIONS: This characteristic fragmentation behavior therefore provides useful information on identification of TSNAs that may be used to monitor such kinds of compound in the biological metabolism. Copyright © 2014 John Wiley & Sons, Ltd.
Tobacco-specific N-nitrosamines (TSNAs) mainly include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), Nnitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT) that are formed from tobacco alkaloids during the curing process and contained in tobacco and tobacco smoke.[1–3] The TSNAs are of interest because of their link with carcinogenesis. Various studies have implicated that TSNAs are the potential sources of carcinogenesis in animal models,[4] and Hoffmann and co-workers have identified that TSNAs are potentially associated with increased risk of illness.[5,6] The International Agency for Research on Cancer has classified NNK and NNN as group 1 carcinogens – "carcinogenic to humans".[7] NAB is classified as a weak esophageal carcinogen in rats, whereas NAT is a group 3 carcinogen – "not classifiable for human carcinogenicity", showing no carcinogenicity in animal studies featuring rats.[8] In view of the health implications, analytical methods for quality control of products and determination of their metabolites are therefore of significant importance. The most commonly used detection technologies so far are mainly based on gas chromatography/mass spectrometry (GC/MS),[9,10] gas chromatography/thermal energy analyzer (GC/TEA),[11,12] and liquid chromatography/ tandem mass spectrometry (LC/MS/MS).[13–16]
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* Correspondence to: G.-J. Zhou, Technology Center, China Tobacco Zhejiang Industrial Co., Ltd, Hangzhou 310024, P.R. China. E-mail: [email protected]
Rapid Commun. Mass Spectrom. 2014, 28, 1658–1664
Electrospray ionization tandem mass spectrometry (ESI-MSn) is a very powerful tool for structural determination and has been widely used in chemistry, biochemistry and pharmaceutical research.[17] Although a few pioneering works have been carried out to gain insights into the fragmentation patterns of TSNAs by utilizing ESI-MSn, none of them give a detailed discussion about particular fragmentation mechanisms.[18,19] Herein we present the ESI-MSn behavior of tobacco-specific TSNAs and try to establish fragmentation patterns for these compounds that can be used for their characterization. Furthermore, the ESI-MSn of deutero-labeled TSNA compounds is used to support the proposed identities of the product ions and therefore the mechanism. Identification of the ways in which such kinds of compounds reorganize into small fragments may provide some useful information and guidance to monitor their metabolisms in the biological system.[20,21]
EXPERIMENTAL MSn characteristic fragmentation behavior of tobacco-specific N-nitrosamines (and TSNA-d4) was achieved using an LCQ™ Deca XP Plus ion trap mass spectrometer (Thermo, USA) utilizing electrospray ionization (ESI), which is capable of analyzing ions up to m/z 6000. The scan range was generally from m/z 50 to 250. The compounds, dissolved in methanol at a concentration of 1.0 × 10–5 mol L–1, were infused into
Copyright © 2014 John Wiley & Sons, Ltd.
Fragmentation behavior of tobacco-specific N-nitrosamines Table 1. Chemical structures of the four TSNAs and deutero-labeled TSNAs NNK/NNK-d4
NAB/NAB-d4
the ESI probe at a rate of 3 μL min–1. The nebulizer pressure was 7 psi and the capillary was typically held at 4 kV. The capillary temperature was maintained at 250 °C. Nitrogen was used as drying gas at a flow rate of 4 L min–1. The TSNA compounds were purchased from Sigma-Aldrich. The chemical structures of the four TSNAs and deutero-labeled TSNAs are shown in Table 1. The selected ion was first isolated and then fragmented through collisions with helium to yield tandem mass spectra. The fragmentation amplitude values were 0.5–1.0 V and the fragmentation time was 40 ms. For each of the compounds the [M + H]+ ion was selected for MS2 analysis and the resulting most abundant product ion was selected for MS3, and so on. An isolation width of 1 m/z unit was used during each MSn stage.
NAT/NAT-d4
178 ion. For this to happen the nitrogen atom has to have a hydrogen atom donated from the 2-position of the pyridine ring and another hydrogen from 4-cleavage of butan-1-one. In ESI-MS4 (Fig. 1(D1)), the fragment ion at m/z 119 corresponds to loss of ethane, which could be the radical that directly attacks the carbonyl to form a four-membered ring.
Table 2. MS/MS spectral data of significant ions of compound 1–8 [m/z with relative abundance (%) in parentheses]
Compound NNK (1)
RESULTS AND DISCUSSION The ESI-MSn analyses of the TSNAs and TSNA-d4 are summarized in Table 2.
NAB (2) NAT (3)
ESI-MSn fragmentation pathways of [M + H]+ for NNK (1)
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NNN (4)
NNK-d4 (5) NAB-d4 (6)
NAT-d4 (7)
NNN-d4 (8)
Precursor ions, m/z
Fragment ions, m/z (relative abundance, %)
208[M + H] 178 147 122 192[M + H] 162 133 190[M + H] 160
178 (75) 147 (8) 122 (100) 147 (100) 119 (20) 132 (20) 119 (90) 93 (10) 80 (15) 162 (100) 147 (8) 133 (100) 106 (8) 118 (100) 160 (100) 143 (25) 133 (85) 131 (80) 118 (90) 106 (100) 93 (50) 79 (8) 148 (100) 120 (100) 105 (30) 93 (35) 70 (15) 93 (100) 79 (30) 182 (65) 126 (100) 150 (100) 122 (40) 84 (25) 166 (100) 151 (20) 136 (100) 110 (25) 84 (8) 121 (25) 164 (100) 146 (15) 137 (85) 134 (40) 121 (40) 110 (100) 97 (40) 83 (8) 152 (100) 124 (100) 109 (20) 97 (30) 70 (20) 97 (100) 82 (15)
178[M + H] 148 120 212[M + H] 182 126 196[M + H] 166 136 194[M + H] 164 182[M + H] 152
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The corresponding ESI-MSn mass spectrum of NNK (1) is summarized in Fig. 1. Compound 1 (Fig. 1(A1)) gave the protonated molecule ion peak [M + H]+ at m/z 208, which fragmented at MS2 (Fig. 1(B1)) to give two major product ions at m/z 122 and 178, and less abundant ions at m/z 147 and 148. The fragment ion at m/z 178 is the result of α-cleavage, producing the indicated ion and the NO · radical. There were, however, another three ions which were not easily explained. In order to clarify their structures, ESI-MS3(Fig. 1(C1, E1)) was performed. Two major ions at m/z 147 and 119 were detected in the ESI-MS3 spectrum of the ion at m/z 178. It did not show ions at m/z 122 and 148. The plausible fragmentation pattern is shown in Scheme 1. The product ions at m/z 122 and 148 could derive from the direct loss of N-methyl-N-nitrosoethenamine and N-nitrosomethanamine from NNK (1), respectively. In addition, the ions at m/z 93 and 80 correspond to the 3-methylpyridine radical ion and the pyridine ion, respectively. Another ion at m/z 147 rather than 148 is attributed to the loss of CH3NH2 from the m/z
NNN/NNN-d4
A.-F. Hu et al.
A1
B1
C1
D1
E1
Figure 1. ESI-MSn spectra of compound NNK (1): (A1) all MS; (B1) ESI-MS2 of m/z 208; (C1) ESI-MS3 of m/z 178; (D1) ESI-MS4 of m/z 147; (E1) ESI-MS3 of m/z 122.
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Scheme 1. ESI-MSn fragmentation pathway of NNK (1) (corresponding to Fig. 1).
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Fragmentation behavior of tobacco-specific N-nitrosamines In another way, the ion at m/z 132 is due to the radical attack of an α-carbon to form a five-membered cyclic ring followed by loss of the methyl radical, and double-bond formation. The ESI-MSn spectrum of NNK-d4 (Fig. 2) showed major ions at m/z 212, 182, 150, 122, 126 and 84. The ion at m/z 212 of NNK-d4 (5) displayed [M + H]+ ions at m/z values 4 Th higher than the native analytes. The ion at m/z 182 corresponded to the indicated ion. As to m/z 126 this indicates the pathway of the direct fragment. The ESI-MS3 spectrum (Fig. 1(C2)) reveals ions at m/z 150, 122, 3 Th higher than in the native analytes, respectively, supporting the cyclization mechanism. Thus, the characteristic fragmentation behavior in the ESI-MS process may be used to monitor this compound in the biological metabolism.
ESI-MSn fragmentation pathways of [M + H]+ for NAB (2) NAB (2), which has a molecular mass of 191, showed a protonated molecular ion, [M + H]+, at m/z 192 in ESI-MS. On fragmentation at the MS2 level, as with NNK, an initial loss of 30 Da was observed to give a major product ion at m/z 162, with less abundant ions at m/z 147, 106 and 118. The plausible fragmentation pattern is shown in Scheme 2. There were two possible pathways. In one route, the ion at m/z 133 corresponds to the loss of methanimine from the opening of the piperidine ring, causing rearrangement to form a six-membered radical ring. This ion further fragmented to give m/z 118 due to the removal of the methyl radical. In the other, the product ion at m/z 147 is suggested to
A2
B2
C2
D2
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Figure 2. ESI-MSn spectra of compound NNK-d4 (5): (A2) all MS; (B2) ESI-MS2 of m/z 212; (C2) ESI-MS3 of m/z 182; (D2) ESI-MS3 of m/z 126.
A.-F. Hu et al. originate from the loss of the methyl radical and formation of a double bond in the heterocyclic ring. As to m/z 106 this is due to the direct loss of the prop-2-en-1-amine radical from the piperidine ring. This fragmentation is similar to nicotine, as has been previously reported.[22] The ESI-MSn spectrum of NAB-d4 showed major ions at m/z 166, 136, 121, 151, 110 and 84. The ions at m/z values 3 Th higher than the native analytes support the first pathway, and ions 4 Th higher support the second pathway. ESI-MS displays a different pattern with breakage of the bond between the pyridine and piperidine entities. ESI-MSn fragmentation pathways of [M + H]+ for NAT (3) NAT (3) gave an [M + H]+ ion at m/z 190 by ESI-MS, which under MS2 conditions showed an initial loss of 30 Da to give an ion at m/z 160. The ESI-MS3 spectrum exhibited richer fragment ions. This is illustrated in Scheme 3.The ion at m/z 143 corresponds to the loss of NH3 from the opening of the tetrahydropyridine ring, causing ring expansion and
rearrangement to form a seven-membered ring. The major ion at m/z 133 presumably corresponds to loss of the ethane radical. As to the fragment ions at m/z 131 and 106, these are similar to NAB (2), that has been shown above. ESI-MSn analysis of NATd4 gave ions at m/z 164, 137, 110 and 97, at m/z values 4 Th higher than the native analytes, and m/z 146, 134 and 121, at m/z values 3 Th higher than the native analytes, which supports the postulated ESI-MSn fragmentation. ESI-MSn fragmentation pathways of [M + H]+ for NNN (4) NNN (4) showed a [M + H]+ ion at m/z 178 in ESI-MS. The MS2 characteristic ion spectrum was an ion at m/z 148. This ion further fragmented to give m/z 120, 105, 93, 79 and 70. According to a previous report, the ion at m/z 120 was thought to have two structures. One is loss of the ethane radical from the opening of the pyrrolidine ring and rearrangement to form a five-membered ring to the 2-position of the pyridine ring.[18] Another is α-cleavage and the resulting ring opening of the pyrrolidyl group in m/z 148,
Scheme 2. ESI-MSn fragmentation pathway of NAB (2).
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Scheme 3. ESI-MSn fragmentation pathway of NAT (3).
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Fragmentation behavior of tobacco-specific N-nitrosamines
Scheme 4. ESI-MSn fragmentation pathway of NNN (4).
followed by pseudo-α-cleavage resulting in the loss of ethane.[19] In ESI-MSn of NAB-d4, a group of characteristic ions at m/z 152, 124, 109, 97 and 70 were observed. The ions at m/z 152, 124, 109 and 97 are at m/z values 4 Th higher than the native analytes, which indicates the second structure. Furthermore, it is interesting that the ion at m/z 105 is directly fragmented from the ion at m/z 148 rather than 120, corresponding to loss of the propane radical. The ions at m/z 70 represent cleavage of the bond between the pyridine and pyrrolidine subunits. This is illustrated in Scheme 4. Spectra of other TSNAs not shown here are given in the Supporting Information.
CONCLUSIONS The ESI-MSn fragmentation patterns of tobacco-specific Nnitrosamines have been studied. The structures of product ions proposed for ESI-MSn have been supported by deutero-labeled TSNAs. Detailed analysis of the resultant fragments shows there are two types of fragmentation patterns in relation to the backbone of pyrrolidine or piperidine rings. In one route, pyrrolidine or piperidine rings undergo direct fragmentation and form some stable intermediates without affecting the parent rings. The other, however, involves ring opening and then ring closure at the pyridine-2 carbon atom to form multi-membered rings. The deutero-labeled TSNAs have also been employed to clarify the fragmentation patterns and further confirm the proposed fragmentation patterns. This characteristic fragmentation behavior therefore provides useful information on identification of TSNAs that may be used to monitor such kinds of compound in the biological metabolism.
Acknowledgements The research was supported by the China National Tobacco Corporation, and the Major Programme (ZJZY2010C00201).
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SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s website.
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