LC–MS/MS analysis of carcinogenic tobacco-specific nitrosamines in Spodoptera litura using the QuEChERS method
Abstract Nicotine is a highly addictive alkaloid and a neurostimulator found in tobacco that causes addiction in humans and makes tobacco a high-demand commercial product. It is popularly used for recreational purposes and is a harmful substance (Oral LD50 value for rat is 50 mg/kg) and causes addic...
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| Format: | Article |
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Nature Portfolio
2023-07-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-37656-2 |
| _version_ | 1797769490679726080 |
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| author | Jabez Raju Battu Somala Karthik Gummudala Yashaswini Himanshu Thakur Alagesan Keerthana M. P. Shireesh Kumar Morthala Shankara Sai Reddy |
| author_facet | Jabez Raju Battu Somala Karthik Gummudala Yashaswini Himanshu Thakur Alagesan Keerthana M. P. Shireesh Kumar Morthala Shankara Sai Reddy |
| author_sort | Jabez Raju Battu |
| collection | DOAJ |
| description | Abstract Nicotine is a highly addictive alkaloid and a neurostimulator found in tobacco that causes addiction in humans and makes tobacco a high-demand commercial product. It is popularly used for recreational purposes and is a harmful substance (Oral LD50 value for rat is 50 mg/kg) and causes addiction. The metabolites of nicotine such as the Tobacco-specific Nitrosamines (TSNAs) are hazardous substances whose metabolites are highly electrophilic and form DNA adducts, which will initiate the process of carcinogenesis. TSNAs are formed during curing, storage and fermentation due to the nitrosation of nicotine and other tobacco alkaloids. TSNAs are used as biomarkers for cancer risk assessment in humans exposed to tobacco and its products. To determine the occasional formation of TSNAs in tobacco-feeding insects, 5th instar larvae of Spodoptera litura and their faeces were analyzed for the presence of N′-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) along with the stored tobacco leaves (PT-76) using an Agilent 6470B LC–MS/MS system following ISO/DIS 19290:2015 protocol. The larvae are extracted in a buffered acetonitrile–water extraction and the amount of TSNAs are quantified in multiple reaction monitoring (MRM) mode. 20 $$\upmu$$ μ l of each extracted and cleaned up sample was injected into the LC–MS/MS system for quantification. The Limit of Detection (LOD) and Limit of Quantification (LOQ) were 0.001 mg/kg and 0.005 mg/kg for all the tested nitrosamines. NNN was found to be 0.361 mg/kg, 0.340 mg/kg, and 5.66 mg/kg in insect whole-body samples, faeces, and tobacco leaves, respectively. NNK was found to be 0.060 mg/kg, 0.035 mg/kg and 0.93 mg/kg in insect whole body samples, faeces and tobacco leaves, respectively. However, NNAL was not detected in both the insect’s whole body and faeces. Recoveries ranged between 95 and 98% for all compounds when spiked at LOD and LOQ. The presence of TSNAs is a biomarker for cancer risk and their presence in insects would point to cancer risk assessment in tobacco feeding insects and any possible TSNA-detoxifying pathways in insects that might prevent mutagenesis caused these compounds. |
| first_indexed | 2024-03-12T21:08:48Z |
| format | Article |
| id | doaj.art-344b8cd7e49f4b25993366902d8380dd |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-12T21:08:48Z |
| publishDate | 2023-07-01 |
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| spelling | doaj.art-344b8cd7e49f4b25993366902d8380dd2023-07-30T11:15:58ZengNature PortfolioScientific Reports2045-23222023-07-011311710.1038/s41598-023-37656-2LC–MS/MS analysis of carcinogenic tobacco-specific nitrosamines in Spodoptera litura using the QuEChERS methodJabez Raju Battu0Somala Karthik1Gummudala Yashaswini2Himanshu Thakur3Alagesan Keerthana4M. P. Shireesh Kumar5Morthala Shankara Sai Reddy6Department of Entomology, CSK Himachal Pradesh Agricultural UniversityDepartment of Entomology, PG College of Agriculture, Dr. Rajendra Prasad Central Agricultural UniversityDepartment of Entomology, PG College of Agriculture, Dr. Rajendra Prasad Central Agricultural UniversityDepartment of Entomology, CSK Himachal Pradesh Agricultural UniversityDepartment of Entomology, PG College of Agriculture, Dr. Rajendra Prasad Central Agricultural UniversityDepartment of Entomology, PG College of Agriculture, Dr. Rajendra Prasad Central Agricultural UniversityDepartment of Entomology, PG College of Agriculture, Dr. Rajendra Prasad Central Agricultural UniversityAbstract Nicotine is a highly addictive alkaloid and a neurostimulator found in tobacco that causes addiction in humans and makes tobacco a high-demand commercial product. It is popularly used for recreational purposes and is a harmful substance (Oral LD50 value for rat is 50 mg/kg) and causes addiction. The metabolites of nicotine such as the Tobacco-specific Nitrosamines (TSNAs) are hazardous substances whose metabolites are highly electrophilic and form DNA adducts, which will initiate the process of carcinogenesis. TSNAs are formed during curing, storage and fermentation due to the nitrosation of nicotine and other tobacco alkaloids. TSNAs are used as biomarkers for cancer risk assessment in humans exposed to tobacco and its products. To determine the occasional formation of TSNAs in tobacco-feeding insects, 5th instar larvae of Spodoptera litura and their faeces were analyzed for the presence of N′-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) along with the stored tobacco leaves (PT-76) using an Agilent 6470B LC–MS/MS system following ISO/DIS 19290:2015 protocol. The larvae are extracted in a buffered acetonitrile–water extraction and the amount of TSNAs are quantified in multiple reaction monitoring (MRM) mode. 20 $$\upmu$$ μ l of each extracted and cleaned up sample was injected into the LC–MS/MS system for quantification. The Limit of Detection (LOD) and Limit of Quantification (LOQ) were 0.001 mg/kg and 0.005 mg/kg for all the tested nitrosamines. NNN was found to be 0.361 mg/kg, 0.340 mg/kg, and 5.66 mg/kg in insect whole-body samples, faeces, and tobacco leaves, respectively. NNK was found to be 0.060 mg/kg, 0.035 mg/kg and 0.93 mg/kg in insect whole body samples, faeces and tobacco leaves, respectively. However, NNAL was not detected in both the insect’s whole body and faeces. Recoveries ranged between 95 and 98% for all compounds when spiked at LOD and LOQ. The presence of TSNAs is a biomarker for cancer risk and their presence in insects would point to cancer risk assessment in tobacco feeding insects and any possible TSNA-detoxifying pathways in insects that might prevent mutagenesis caused these compounds.https://doi.org/10.1038/s41598-023-37656-2 |
| spellingShingle | Jabez Raju Battu Somala Karthik Gummudala Yashaswini Himanshu Thakur Alagesan Keerthana M. P. Shireesh Kumar Morthala Shankara Sai Reddy LC–MS/MS analysis of carcinogenic tobacco-specific nitrosamines in Spodoptera litura using the QuEChERS method Scientific Reports |
| title | LC–MS/MS analysis of carcinogenic tobacco-specific nitrosamines in Spodoptera litura using the QuEChERS method |
| title_full | LC–MS/MS analysis of carcinogenic tobacco-specific nitrosamines in Spodoptera litura using the QuEChERS method |
| title_fullStr | LC–MS/MS analysis of carcinogenic tobacco-specific nitrosamines in Spodoptera litura using the QuEChERS method |
| title_full_unstemmed | LC–MS/MS analysis of carcinogenic tobacco-specific nitrosamines in Spodoptera litura using the QuEChERS method |
| title_short | LC–MS/MS analysis of carcinogenic tobacco-specific nitrosamines in Spodoptera litura using the QuEChERS method |
| title_sort | lc ms ms analysis of carcinogenic tobacco specific nitrosamines in spodoptera litura using the quechers method |
| url | https://doi.org/10.1038/s41598-023-37656-2 |
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