Degradation of the Neonicotinoid Pesticides in the Atmospheric Pressure Ionization Source
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During the analysis of neonicotinoid pesticide standards (thiamethoxam, clothianidin, imidacloprid, acetamiprid, and thiacloprid) by mass spectrometry, the degradation of these pesticides (M-C=N-R is degraded into M-C=O, M is the skeleton moiety, and R is NO2 or CN) was observed in the atmospheric pressure ionization interfaces (ESI and APCI). In APCI, the degradation of all the five neonicotinoid pesticides studied took place, and the primary mechanism was in-source ion/molecule reaction, in which a molecule of water (confirmed by use of H2 18O) attacked the carbon of the imine group accompanying with loss of NH2R (R=NO2, CN). For the nitroguanidine neonicotinoid pesticides (R=NO2, including thiamethoxam, clothianidin, and imidacloprid), higher auxiliary gas heater temperature also contributed to their degradation in APCI due to in-source pyrolysis. The degradation of the five neonicotinoid pesticides studied in ESI was not significant. In ESI, only the nitroguanidine neonicotinoid pesticides could generate the degradation products through in-source fragmentation mechanism. The degradation of cyanoamidine neonicotinoid pesticides (R=CN, including acetamiprid and thiacloprid) in ESI was not observed. The degradation of neonicotinoid pesticides in the ion source of mass spectrometer renders some adverse consequences, such as difficulty interpreting the full-scan mass spectrum, reducing the sensitivity and accuracy of quantitative analysis, and misleading whether these pesticides have degraded in the real samples. Therefore, a clear understanding of these unusual degradation reactions should facilitate the analysis of neonicotinoid pesticides by atmospheric pressure ionization mass spectrometry.
KeywordsNeonicotinoid pesticide Degradation Atmospheric pressure ionization In-source ion/molecule reaction
High performance liquid chromatography-mass spectrometry (HPLC-MS) is acknowledged to be an extremely versatile and indispensable instrumental technique for determination of pesticide residues as well as other toxic chemicals [1, 2]. In this instrumentation, the HPLC is attached to the MS via an ionization interface. The most commonly used interface for HPLC-MS is atmospheric pressure ionization (API), which mainly includes electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). ESI and APCI are well-known as soft ionization, since there is very little fragmentation. The analytes are converted into quasi-molecular ions through protonation, deprotonation, or formation of other ionic adducts ([M+H]+, [M–H]–, [M+Na]+, [M+AcO]–, etc.) in the API ion source, which is advantageous in the sense that the molecular weight of the analyte can be easily obtained. However, ESI and APCI do not always produce these conventional precursor ions for all the compounds. The exceptions are often reported. One of our authors had reported the formation of unexpected [M–H]+ ions of 1,4-diphenyl-3-benzoyl-1,4-dihydropyridines in the ESI positive mode . The mechanism was attributed to an electrochemical oxidation reaction. Owing to the high voltage in the ESI interface, the 1,4-dihydropyridine was oxidized to the corresponding aromatic pyridine. More examples about electrochemical redox reactions leading to unexpected ions in the ESI and APCI interfaces had also been observed by other mass spectrometrists [4, 5, 6, 7]. Pan et al. had reported the formation of unexpected [M+15]+ ions during the analysis of aromatic aldehydes in ESI positive mode . They explained that a gas-phase aldol reaction between the aromatic aldehydes and methanol (the solvent) occurred in the ion source to produce the [M+15]+ ions. A similar result was also presented by Jiang et al. in the analysis of heteroaromatic aldehydes using ESI-MS . They observed the unusual [M+15]+, [M+33]+, and [M+47]+ ions in the ESI-MS spectra, which were the intermediates of in-source aldolization reactions between the heteroaromatic aldehydes and the solvent methanol. In addition, the formation of other kinds of unusual ions in the ESI or APCI interfaces, such as open-shell radical molecular ions [10, 11, 12, 13], covalently bound dimers [14, 15], oxygen addition species , and so on, had been recognized and reported in literature. These side reactions in the ion source are mechanistically interesting that can help us to more deeply understand the ionization mechanism, and are potentially useful to design novel ion sources, such as active capillary dielectric barrier discharge ionization that is significantly softer than APCI [17, 18, 19]. However, theses anomalies during ionization processes must draw attention of analytical chemists. The side reactions may interfere with obtaining reliable molecular mass of the analyte, generating false information about the composition of the sample, and reducing the sensitivity and accuracy of quantitative analysis.
Chemical Structures of the Five Neonicotinoid Insecticides and their Degradation Products Studied
Thiamethoxam (98.5%), clothianidin (99.0%), imidacloprid (98.0%), acetamiprid (98.1%), and thiacloprid (98.0%) standards used in this study were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany) and used as received. Water-18O (97% atom, 18O) was purchased from Beijing InnoChem Science and Technology Co., Ltd. (Beijing, China). Methanol (HPLC grade) was purchased from Tedia (Fairfield, OH, USA). Deionized water was purified with a Mill Q-Plus system (Millipore, Billerica, MA, USA).
The thiamethoxam urea was synthesized according to previous reported procedures . The structure was confirmed by NMR spectroscopy and mass spectrometry. 1H NMR (400 MHz, CDCl3) δ 7.38 (s, 1H), 4.79 (s, 2H), 4.74 (s, 2H), 4.55 (d, J=0.6 Hz, 2H), 2.90 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 153.94, 152.99, 139.62, 137.40, 80.02, 78.38, 41.03, 31.36.
The neonicotinoid insecticide standards were prepared in methanol at a final concentration of 1 μg mL–1. After preparation, the samples were analyzed by MS immediately.
The HPLC-MS analysis was performed on an UltiMate 3000 system (ThermoFisher Scientific, Bremen, Germany) coupled with a hybrid quadrupole-Orbitrap mass spectrometer, Q-Exactive (ThermoFisher Scientific, Bremen, Germany). A ThermoFisher Hypersil GOLD column (C18, 100 × 2.1 mm, 3 μm) was used for chromatographic separation. The column temperature was set at 30 °C and the autosampler temperature was set at 10 °C. The isocratic mobile phase consisted of methanol/water (30/70, v/v) at a flow rate of 0.2 mL min–1. The injection volume was 3 μL. Ionization in APCI positive mode was performed using the following settings: spray current 5 μA, sheath gas (N2) flow rate 35 arb, auxiliary gas (N2) flow rate 10 arb, sweep gas (N2) flow rate 3 arb, heated capillary temperature 320 °C, auxiliary gas heater temperature 320 °C, S-lens radio frequency (rf) level 50. Ionization in ESI positive mode was performed using the following settings: spray voltage 3.5 kV, sheath gas (N2) flow rate 35 arb, auxiliary gas (N2) flow rate 10 arb, heated capillary temperature 320 °C, S-lens rf level 50. Orbitrap resolution was set to 70,000 full-width at half-maximum (FWHM) at m/z=200. The MS/MS spectra were obtained with nitrogen (99.99%) as the collision gas after isolation of the desired precursor ion. The normalized collision energy (NCE) was varied in a range of 10%–50% to provide suitable energy for the fragmentation of the [M+H]+ ions.
The samples were also analyzed by using flow inject analysis (FIA) method without chromatographic separation. Unless otherwise indicated, the HPLC and MS conditions were the same as those in HPLC-MS analysis. The mobile phase was methanol/water (90/10, v/v) and methanol/water (50/50, v/v) in APCI and ESI, respectively. Different auxiliary gas heater temperatures (220 °C, 320 °C, and 420 °C) in APCI were investigated.
18O Labeling Experiment
Thiamethoxam and acetamiprid were dissolved in methanol/water-18O (50/50, v/v) to a final concentration of 1 μg mL–1. Solutions were infused to the mass spectrometer with a syringe pump at a flow rate of 20 μL min–1. The MS conditions were the same as those in FIA-MS analysis.
Results and Discussion
The mass spectrometric results of the five neonicotinoid insecticides studied using HPLC-MS method a
Protonated neonicotinoid insecticide
Protonated degradation product
Protonated neonicotinoid insecticide
Protonated degradation product
292.0261 (94.1) b
292.0264 (64.6) c
250.0163 (50.1) c
256.0594 (75.3) c
Pesticide degradation or metabolism is an important research topic and the mass spectrometer has become an indispensable tool to detect the structure and concentration of the degradation products and metabolites. In the degradation or metabolism of neonicotinoid pesticides, one of the pathways is that their pharmacophore (C=N-R, R is a substituent) is converted into a carbonyl group (C=O). In this study, the degradation of neonicotinoid pesticides (thiamethoxam, clothianidin, imidacloprid, acetamiprid, and thiacloprid) inside the APCI and ESI ion sources was revealed and investigated based on various experimental methods. Their degradation in the APCI was clearly faster than that at normal conditions in the environment because of some in-source ion/molecule reactions with water molecules (based on 18O-labeling experiments). In addition, intramolecular fragmentation (the oxygen of the nitro group attacked the carbon of the imine group accompanying with loss of N2O) in the ion source also contributed to the degradation of nitroguanidine neonicotinoid pesticides (R=NO2) in APCI and ESI. The degradation of neonicotinoid pesticides inside the ion source may interfere with the qualitative and quantitative analysis of these compounds, including their degradation products and metabolites. This work permits us to highlight the importance of understanding unexpected reactions in mass spectrometry and improves our knowledge on the ionization process of atmospheric pressure ionization.
This work was supported by the Public Welfare Technology Application Research Project of Zhejiang Province (2016C32025), the Innovative Research Team in Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2017-TRICAAS-7), the National Natural Science Foundation of China (21775164), the National Agricultural Product Quality and Safety Risk Assessment Project (GJFP2018005), and the Central Institute Basic Scientific Research Expenses Foundation (1610212016005).
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