LC-MS/MS analysis of carcinogenic tobacco-specific nitrosamines in Spodoptera litura using the QuEChERS method

LC-MS/MS analysis of carcinogenic tobacco-specific nitrosamines in Spodoptera litura using the QuEChERS method | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article LC-MS/MS analysis of carcinogenic tobacco-specific nitrosamines in Spodoptera litura using the QuEChERS method Karthik Somala, Yashaswini Gummudala, Shankara Sai Reddy Morthala, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1707326/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Jul, 2023 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version 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 not a harmful substance except for the addiction it causes. 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, 5 th instar larvae of Spodoptera litura and their faeces were analyzed for the presence of NNN, NNK, and NNAL along with the stored tobacco leaves (PT-76) using an Agilent 6470B LC-MS/MS system following ISO/DIS 19290:2015 protocol. The LOD and LOQ were 0.001 mg/kg and 0.005 mg/kg for all the tested nitrosamines. NNN was found to be 0.365 mg/kg, 0.344 mg/kg, and 5.71 mg/kg in insect whole-body samples, faeces, and tobacco leaves, respectively. NNK was found to be 0.062 mg/kg, 0.036 mg/kg and 0.97 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 95-98% 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. Tobacco Tobacco-specific Nitrosamines Spodoptera litura Cancer Figures Figure 1 Figure 2 Figure 3 1. Introduction: Tobacco-specific nitrosamines (TSNAs) are a group of carcinogens found in tobacco and tobacco products (Sophia et al. 1989 ). They are formed during tobacco curing, storage, and fermentation by nitrosation of tobacco alkaloids such as nicotine, nornicotine, anabasine, and anatabine. TSNAs are not present in fresh leaves of tobacco and their formation starts a few days after harvesting (Hecht & Hoffmann 1988 ; Spiegelhalder & Fischer 1991 ; Sarlak et al. 2020 ). The TSNAs include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N’ -nitrosonornicotine (NNN), N’ -nitrosoanabasine (NAB) and N’ -nitrosoanatabine (NAT). The former two are potent carcinogens (Group 1) and are believed to be associated with the majority of lung cancers in smokers (“IARC” 2004; Hecht et al. 2016 ). Boyland et al. ( 1964 a & b) were the first to demonstrate the carcinogenic activity of NAB in rats and NNN in mice. NNN & NNK are reported to cause esophageal tumors, tumors of the olfactory epithelium, tracheal tumors, lung adenomas and adenocarcinomas in different animals as well as humans (Hecht et al. 1979 ; Hecht et al. 2016 ). 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a metabolite of NNK and is reported to cause lung cancers in humans. Patients having lung cancers had significantly higher levels of urinary NNAL (Yuan et al. 2009 ). Consuming tobacco in any form renders humans exposed to the risk of acquiring cancers. Insects feeding on tobacco and are not affected by the mutagenic compounds present in the tobacco leaves. Experiments on tobacco-feeding insects suggested that in a majority of insects either nicotine is not metabolized by the insects or nicotine and other likely harmful tobacco alkaloids are rapidly excreted by the insects before lethal concentrations are accumulated (Self et al. 1964 ; Farnham et al. 2007 ). Investigations to determine the nicotine metabolism in the tobacco hornworm, Manduca sexta revealed that nicotine is metabolized into cotinine-N-oxide and is rapidly excreted along with the free nicotine that is not metabolized (Synder et al . 1994). It is believed that midgut enzymes like the cytochrome P450 monooxygenases and Glutathione S-transferases are responsible for the rapid excretion of nicotine in Helicoverpa assulta (Lee and Boo 1993 ; Junfeng et al. 2002 ). The present investigation was aimed at the determination of TSNAs and their fate in a tobacco feeding insect, Spodoptera litura commonly referred to as tobacco cutworm. Tobacco leaves fermented in the gut of this insect should result in the formation of TSNAs as they are formed during the curing and fermentation of tobacco leaves. This is probably the first time TSNAs are being analyzed in insects. 2. Materials And Methods: 2.1 Reagents and Chemicals: Methanol, Ammonium acetate (E. Merck, India Ltd.), Agilent QuEChERS AOAC extraction kit (p/n 5982 − 5022) and Agilent QuEChERS dispersive SPE kit (p/n 5982 − 5022) was used for extracting the samples and the internal standards of NNN and NNK procured were mentioned below in the Supplementary material 1. Milli-Q water was used for the preparation of the samples. 2.2 Sample preparation: Spodoptera litura was reared on tobacco (PT-76) under controlled conditions (25°C, 70% RH). The insect was reared on tobacco for 5 generations and all the stages of its life cycle were healthy without any malformations. 5th instar larvae of Spodoptera litura from the 5th generation and their faeces were collected and stored at -20°C in a freezer. Leaves of the tobacco were collected and stored at -20°C in a freezer for 3 days to allow the formation of TSNAs. 2.2.1 Insect whole body and faeces The extraction method was followed as given by Saremba et al. ( 2018 ), which was adapted from a QuEChERS method (AOAC 2007.01, Agilent Inc. ®) as given by Chang ( 2013 ) for extracting nicotine and its metabolites in Trichoplusia ni. Insect whole body specimens and faeces were homogenized and extract ed in a buffered acetonitrile-water extraction and then centrifuged for 5 minutes at 5000 rpm. The supernatant from the extracts was collected and vortexed on an SPE-cleanup column (AOAC 2007.01) to remove pigments, lipids, and proteins then centrifuged for 5 min at 10000 rpm. The top supernatant layer was collected and filtered through 0.2 µm PTFE syringe filter to remove any particulates. 2.2.2 Tobacco leaves Tobacco leaves were extracted following the protocol used by Li et al. ( 2019 ) with slight modifications. 250 mg leaf sample was added with 50 µl internal standard solution, homogenized and filtered through a 425 µm sieve and extracted in ammonium acetate using the QuEChERS method (AOAC 2007.01, Agilent Inc. ®) and centrifuged at 5000 rpm for 5 minutes. The extractant is directly filtered into the injection vials using a 0.2 µm PTFE syringe filter. 2.3 Preparation of standard solutions 1mg/ml standard solutions were used to prepare stock solutions (10 µg/ml to 200 µg/ml) in LC-MS grade methanol through serial dilutions and were stored at -4°C until further usage. The calibration curves of NNN, NNK, and NNAL are given in Supplementary material 2 and chromatograms of standards are given in Supplementary material 3. 2.4 Instrumentation: The LC-MS/MS system (Agilent 6470B TQ LC/MS) consisted of a UHPLC device and an atmospheric pressure ionization triple quadrupole mass spectrometer (Agilent 6470 LC/TQ) equipped with electron spray ionization (ESI) Agilent Jetstream source. The UHPLC has a Zorbax XDB-C18 column (2.1 mm inner diameter x 100 mm long x 1.8 µm pore size). For quantification of TSNAs, LC-MS/MS was run with mobile phase A (5mM Ammonium acetate in 80:20 Water: Methanol with pH = 4) and mobile phase B (Methanol) at a flow rate of 0.25 ml/min coupled to a triple quadrupole Mass spectrometer with a capillary voltage of 3000V, Nozzle voltage of 500V, nebulizer pressure of 45 psi, the gas temperature of 200°C and sheath gas temperature of 350°C and gas flow and sheath gas flow of 8L/min and 11L/min, respectively. It was operated in electrospray positive mode and data collection was done in multiple reaction monitoring (MRM) mode. The LC-MS/MS system had an injection volume of 1µl and the total run time was 10 minutes. The column temperature was maintained at 35°C. ISO/DIS 19290:2015 method was used for the analysis of TSNAs. 2.5 Data Analysis: Data analysis was done using Lab Solutions software (Shimadzu). Ethical Statement: This article does not contain any studies involving humans/animals/plants that need approval from ethical committee. The plant material may be made available on request. 3. Results And Discussion: The standards are spiked at 0.001 and 0.005 mg/kg and recoveries obtained were 95-98% for all the three TSNAs being tested. The limit of detection and limit of quantification were worked out to be 0.001 and 0.005 mg/kg, respectively for all the TSNAs viz., NNN, NNK and NNAL. The RSD% was less than 2% for all the internal standards tested. The equations showing linearity of the standards is given in table-1. The retention time of the TSNAs and their levels in insect whole body and faeces are given below in table-2. The chromatogram of the samples analyzed is also given below in figure-1, 2 and 3. NNN and NNK are detected in both insect whole body and faeces. The data indicates the rapid excretion of the carcinogenic compounds just as soon as they are formed similar to the rapid excretion of nicotine and its metabolite cotinine-N-oxide as reported by Synder et al . (1994). NNAL, a metabolite of NNK is not detected in both the insect’s whole body and faeces indicating that NNK is not metabolized in S. litura , similar to the analysis of nicotine metabolism in cigarette beetle, Lasioderma serricorne conducted by Farnham et al. (2007) which revealed that nicotine is neither sequestered nor detoxified but is rather excreted. Pérez-Ortuño et al . (2016) reported that the mean NNN and NNK concentration in the oral fluid of people who smoked at least 1 cigarette per day was 118 and 6.6 pg/ml. Kavvadias et al . (2009) reported NNN mean concentration in the urine of smokers as 7.2 pg/ml. The NNN concentration we detected in S. litura larvae is 0.365 mg/kg on the whole body and 0.344 mg/kg in insect faeces which is approximately 4778 times more than what was reported in human urine by Kavvadias et al. (2009). The presence of TSNAs in the human urine or oral fluid is considered a biomarker for cancer risk (Pérez-Ortuño et al . 2016). More research is to be done to evaluate whether TSNAs possess a significant cancer risk in insects. The probability of insect getting is very low owing to several factors. First and foremost, the life span of a typical insect is rather short for any cancer to be developed at all, secondly, insects are popularly known for their xenobiotic detoxification potential and any carcinogenic agent (like pesticides for example) could be detoxified, moreover, the genome of the insect is of small size and therefore the chances for mutations are very less. Furthermore, insects undergo programmed cell death throughout their life cycle whenever they undergo a metamorphosis which will likely prevent any tumors (Battu et al. 2021). But again, insects can get cancer as well just like Peto’s paradox affirming that there is no correlation between the body size and cancer risk (Caulin and Maley 2011), but we are inconclusive as of now to answer this question. There had been reports of tumors in insects which likely explain this. Harker (1958) reported excessive endocrine secretions from sub-oesophageal ganglion-induced tumors in the midgut of Periplaneta americana . Federley (1936) reported male-killing in the species hybrids of the butterfly Pygaera pigra (Notodontitade: Lepidoptera). Male larvae of the species hybrids are killed due to cancer before they even reach pupation. Conclusion Tobacco-specific Nitrosamines are carcinogenic substances and are associated with various types of cancers. They are formed during the process of curing, storage and fermentation. TSNAs can also form in the midgut of tobacco-feeding insects, wherein ingested tobacco leaves are fermented. We have detected the presence of NNN and NNK in the insect’s whole body and faeces indicating their formation in tobacco-feeding insects. The genotoxic effects of TSNAs are widely reported in higher animals but there is no study conducted to investigate the effects of TSNAs on lower animals like invertebrates. This study prompts future investigations into how TSNAs are formed, metabolized, and detoxified (if any) in insects. This experiment should be conducted on all the tobacco-feeding insects to see how different insects have developed strategies to mitigate the genotoxic effects of TSNAs on them. Insects just like other living organisms are composed of living cells that undergo cell division and therefore hypothetically insects can get cancers as well. Cancer in insect for the first time has been reported in the larvae of the butterfly Pygaera pigra. It is easier to conduct cancer studies in vitro in an insect when compared to any other animal and the insect cell culturing makes it facile to maintain tumour cell lines in vitro and investigate cell biology. So, insects could be used as model organisms to study the effect of potential carcinogens and the potential of insects as models for cancer studies should also be evaluated given their xenobiotic detoxification potential as compared to the higher animals. Declarations: Acknowledgement: The authors are thankful to Departments of Entomology of Dr. Rajendra Prasad Central Agricultural University for unwavering support throughout the research. The authors would like to thank Dr Molamma P. Prabhakaran, National University of Singapore for reviewing this article. Authors Contribution: K.S.: Validation, Writing- Review& Editing. Y.G.: Writing- Review& Editing. S.R.M.S.: Resources, Supervision. H.T.: Writing- Review& Editing. K.A.: Writing- Review& Editing. S.K.M.P.: Writing- Review& Editing. J.R.B.: Conceptualization, Methodology, Writing - Original Draft. Data availability: The datasets generated during and/or analysed during this study are included in this published article (and its Supplementary Information files). Competing interests The authors declare no competing interests. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 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Tables: Table-1: Linearity of NNN, NNK and NNAL Standard R 2 Calibration curve LOD (mg/kg) LOQ (mg/kg) NNN 0.9523 5.819e3X-5.727e4 0.001 0.005 NNK 0.9994 5.494e4X-8.872e4 0.001 0.005 NNAL 0.9988 5.465e4X-1.09e5 0.001 0.005 Table-2: Retention time, Peak areas and concentration of NNN and NNK in the analyzed samples (average of three replicates) S.No Sample Analyte Retention time (min) Quantification (mg/kg) RSD% 1 S. litura whole body NNN 4.26 0.365 ∓ 0.014 1.78 NNK 6.35 0.062 ∓ 0.008 1.07 NNAL - Not Detected - 2 S. litura faeces NNN 4.26 0.344 ∓ 0.011 1.87 NNK 7.19 0.036 ∓ 0.009 1.23 NNAL - Not Detected - 3 Tobacco leaves (PT-76) NNN 6.35 5.71 ∓ 0.52 1.76 NNK 8.87 0.97 ∓ 0.064 1.13 Additional Declarations No competing interests reported. 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09:44:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1707326/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1707326/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-023-37656-2","type":"published","date":"2023-07-27T21:45:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":23234092,"identity":"85e09760-6994-4d8e-9a53-caa6bf2dc145","added_by":"auto","created_at":"2022-06-29 15:10:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":48171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromatograms of a) NNK, b) NNN in insect whole body\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1707326/v1/0570deb2705c9dc9e2ed703e.png"},{"id":23234091,"identity":"58de549a-0570-47e1-94f3-7fcda9b28c62","added_by":"auto","created_at":"2022-06-29 15:10:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":39515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromatograms of a) NNK, b) NNN in insect faeces\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1707326/v1/e24cd6bf1b95b37c92d6713b.png"},{"id":23234089,"identity":"1fe5fc24-c4a0-48fc-929f-437b32459a09","added_by":"auto","created_at":"2022-06-29 15:10:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":35321,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromatograms of a) NNK, b) NNN in tobacco leaves\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1707326/v1/af012aab350550e9160988fa.png"},{"id":44735622,"identity":"ec449fdc-07f1-4405-a02c-73f3e3174d2d","added_by":"auto","created_at":"2023-10-16 22:26:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":488131,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1707326/v1/84acaa8e-40c7-4b9e-b5d6-a6340bb6fdfb.pdf"},{"id":23234090,"identity":"a9497043-83d2-440b-9d98-eec0f803a03d","added_by":"auto","created_at":"2022-06-29 15:10:06","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":108544,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterials.doc","url":"https://assets-eu.researchsquare.com/files/rs-1707326/v1/c99da11614d20337013b3128.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"LC-MS/MS analysis of carcinogenic tobacco-specific nitrosamines in Spodoptera litura using the QuEChERS method","fulltext":[{"header":"1. Introduction:","content":"\u003cp\u003eTobacco-specific nitrosamines (TSNAs) are a group of carcinogens found in tobacco and tobacco products (Sophia et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). They are formed during tobacco curing, storage, and fermentation by nitrosation of tobacco alkaloids such as nicotine, nornicotine, anabasine, and anatabine. TSNAs are not present in fresh leaves of tobacco and their formation starts a few days after harvesting (Hecht \u0026amp; Hoffmann \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Spiegelhalder \u0026amp; Fischer \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Sarlak et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The TSNAs include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), \u003cem\u003eN\u0026rsquo;\u003c/em\u003e-nitrosonornicotine (NNN), \u003cem\u003eN\u0026rsquo;\u003c/em\u003e-nitrosoanabasine (NAB) and \u003cem\u003eN\u0026rsquo;\u003c/em\u003e-nitrosoanatabine (NAT). The former two are potent carcinogens (Group 1) and are believed to be associated with the majority of lung cancers in smokers (\u0026ldquo;IARC\u0026rdquo; 2004; Hecht et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Boyland et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1964\u003c/span\u003e a \u0026amp; b) were the first to demonstrate the carcinogenic activity of NAB in rats and NNN in mice. NNN \u0026amp; NNK are reported to cause esophageal tumors, tumors of the olfactory epithelium, tracheal tumors, lung adenomas and adenocarcinomas in different animals as well as humans (Hecht et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1979\u003c/span\u003e; Hecht et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a metabolite of NNK and is reported to cause lung cancers in humans. Patients having lung cancers had significantly higher levels of urinary NNAL (Yuan et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Consuming tobacco in any form renders humans exposed to the risk of acquiring cancers.\u003c/p\u003e \u003cp\u003eInsects feeding on tobacco and are not affected by the mutagenic compounds present in the tobacco leaves. Experiments on tobacco-feeding insects suggested that in a majority of insects either nicotine is not metabolized by the insects or nicotine and other likely harmful tobacco alkaloids are rapidly excreted by the insects before lethal concentrations are accumulated (Self et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1964\u003c/span\u003e; Farnham et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Investigations to determine the nicotine metabolism in the tobacco hornworm, \u003cem\u003eManduca sexta\u003c/em\u003e revealed that nicotine is metabolized into cotinine-N-oxide and is rapidly excreted along with the free nicotine that is not metabolized (Synder \u003cem\u003eet al\u003c/em\u003e. 1994). It is believed that midgut enzymes like the cytochrome P450 monooxygenases and Glutathione S-transferases are responsible for the rapid excretion of nicotine in \u003cem\u003eHelicoverpa assulta\u003c/em\u003e (Lee and Boo \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Junfeng et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The present investigation was aimed at the determination of TSNAs and their fate in a tobacco feeding insect, \u003cem\u003eSpodoptera litura\u003c/em\u003e commonly referred to as tobacco cutworm. Tobacco leaves fermented in the gut of this insect should result in the formation of TSNAs as they are formed during the curing and fermentation of tobacco leaves. This is probably the first time TSNAs are being analyzed in insects.\u003c/p\u003e"},{"header":"2. Materials And Methods:","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1 Reagents and Chemicals:\u003c/h2\u003e\n \u003cp\u003eMethanol, Ammonium acetate (E. Merck, India Ltd.), Agilent QuEChERS AOAC extraction kit (p/n 5982\u0026thinsp;\u0026minus;\u0026thinsp;5022) and Agilent QuEChERS dispersive SPE kit (p/n 5982\u0026thinsp;\u0026minus;\u0026thinsp;5022) was used for extracting the samples and the internal standards of NNN and NNK procured were mentioned below in the Supplementary material 1. Milli-Q water was used for the preparation of the samples.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.2 Sample preparation:\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eSpodoptera litura\u003c/em\u003e was reared on tobacco (PT-76) under controlled conditions (25\u0026deg;C, 70% RH). The insect was reared on tobacco for 5 generations and all the stages of its life cycle were healthy without any malformations. 5th instar larvae of \u003cem\u003eSpodoptera litura\u003c/em\u003e from the 5th generation and their faeces were collected and stored at -20\u0026deg;C in a freezer. Leaves of the tobacco were collected and stored at -20\u0026deg;C in a freezer for 3 days to allow the formation of TSNAs.\u003c/p\u003e\n \u003cdiv class=\"Section3\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.2.1 Insect whole body and faeces\u003c/h2\u003e\n \u003cp\u003eThe extraction method was followed as given by Saremba et al. (\u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e), which was adapted from a QuEChERS method (AOAC 2007.01, Agilent Inc. \u0026reg;) as given by Chang (\u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e) for extracting nicotine and its metabolites in \u003cem\u003eTrichoplusia ni.\u003c/em\u003e Insect whole body specimens and faeces were homogenized and extract ed in a buffered acetonitrile-water extraction and then centrifuged for 5 minutes at 5000 rpm. The supernatant from the extracts was collected and vortexed on an SPE-cleanup column (AOAC 2007.01) to remove pigments, lipids, and proteins then centrifuged for 5 min at 10000 rpm. The top supernatant layer was collected and filtered through 0.2 \u0026micro;m PTFE syringe filter to remove any particulates.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.2.2 Tobacco leaves\u003c/h2\u003e\n \u003cp\u003eTobacco leaves were extracted following the protocol used by Li et al. (\u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e) with slight modifications. 250 mg leaf sample was added with 50 \u0026micro;l internal standard solution, homogenized and filtered through a 425 \u0026micro;m sieve and extracted in ammonium acetate using the QuEChERS method (AOAC 2007.01, Agilent Inc. \u0026reg;) and centrifuged at 5000 rpm for 5 minutes. The extractant is directly filtered into the injection vials using a 0.2 \u0026micro;m PTFE syringe filter.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.3 Preparation of standard solutions\u003c/h2\u003e\n \u003cp\u003e1mg/ml standard solutions were used to prepare stock solutions (10 \u0026micro;g/ml to 200 \u0026micro;g/ml) in LC-MS grade methanol through serial dilutions and were stored at -4\u0026deg;C until further usage. The calibration curves of NNN, NNK, and NNAL are given in Supplementary material 2 and chromatograms of standards are given in Supplementary material 3.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003e2.4 Instrumentation:\u003c/h2\u003e\n \u003cp\u003eThe LC-MS/MS system (Agilent 6470B TQ LC/MS) consisted of a UHPLC device and an atmospheric pressure ionization triple quadrupole mass spectrometer (Agilent 6470 LC/TQ) equipped with electron spray ionization (ESI) Agilent Jetstream source. The UHPLC has a Zorbax XDB-C18 column (2.1 mm inner diameter x 100 mm long x 1.8 \u0026micro;m pore size). For quantification of TSNAs, LC-MS/MS was run with mobile phase A (5mM Ammonium acetate in 80:20 Water: Methanol with pH\u0026thinsp;=\u0026thinsp;4) and mobile phase B (Methanol) at a flow rate of 0.25 ml/min coupled to a triple quadrupole Mass spectrometer with a capillary voltage of 3000V, Nozzle voltage of 500V, nebulizer pressure of 45 psi, the gas temperature of 200\u0026deg;C and sheath gas temperature of 350\u0026deg;C and gas flow and sheath gas flow of 8L/min and 11L/min, respectively. It was operated in electrospray positive mode and data collection was done in multiple reaction monitoring (MRM) mode. The LC-MS/MS system had an injection volume of 1\u0026micro;l and the total run time was 10 minutes. The column temperature was maintained at 35\u0026deg;C. ISO/DIS 19290:2015 method was used for the analysis of TSNAs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003e2.5 Data Analysis:\u003c/h2\u003e\n \u003cp\u003eData analysis was done using Lab Solutions software (Shimadzu).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEthical Statement:\u0026nbsp;\u003c/strong\u003eThis article does not contain any studies involving humans/animals/plants that need approval from ethical committee. The plant material may be made available on request.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results And Discussion:","content":"\u003cp\u003eThe standards are spiked at 0.001 and 0.005 mg/kg and recoveries obtained were 95-98% for all the three TSNAs being tested. The limit of detection and limit of quantification were worked out to be 0.001 and 0.005 mg/kg, respectively for all the TSNAs \u003cem\u003eviz.,\u0026nbsp;\u003c/em\u003eNNN, NNK and NNAL. The RSD% was less than 2% for all the internal standards tested. The equations showing linearity of the standards is given in table-1. The retention time of the TSNAs and their levels in insect whole body and faeces are given below in table-2. The chromatogram of the samples analyzed is also given below in figure-1, 2 and 3.\u003c/p\u003e\n\u003cp\u003eNNN and NNK are detected in both insect whole body and faeces. The data indicates the rapid excretion of the carcinogenic compounds just as soon as they are formed similar to the rapid excretion of nicotine and its metabolite cotinine-N-oxide as reported by Synder \u003cem\u003eet al\u003c/em\u003e. (1994). NNAL, a metabolite of NNK is not detected in both the insect\u0026rsquo;s whole body and faeces indicating that NNK is not metabolized in \u003cem\u003eS. litura\u003c/em\u003e, similar to the analysis of nicotine metabolism in cigarette beetle, \u003cem\u003eLasioderma serricorne\u003c/em\u003e conducted by Farnham \u003cem\u003eet al.\u003c/em\u003e (2007) which revealed that nicotine is neither sequestered nor detoxified but is rather excreted. P\u0026eacute;rez-Ortu\u0026ntilde;o \u003cem\u003eet al\u003c/em\u003e. (2016) reported that the mean NNN and NNK concentration in the oral fluid of people who smoked at least 1 cigarette per day was 118 and 6.6 pg/ml. \u0026nbsp;Kavvadias \u003cem\u003eet al\u003c/em\u003e. (2009) reported NNN mean concentration in the urine of smokers as 7.2 pg/ml. The NNN concentration we detected in \u003cem\u003eS. litura\u003c/em\u003e larvae is 0.365 mg/kg on the whole body and 0.344 mg/kg in insect faeces which is approximately 4778 times more than what was reported in human urine by Kavvadias et al. (2009). The presence of TSNAs in the human urine or oral fluid is considered a biomarker for cancer risk (P\u0026eacute;rez-Ortu\u0026ntilde;o \u003cem\u003eet al\u003c/em\u003e. 2016). More research is to be done to evaluate whether TSNAs possess a significant cancer risk in insects. The probability of insect getting is very low owing to several factors. First and foremost, the life span of a typical insect is rather short for any cancer to be developed at all, secondly, insects are popularly known for their xenobiotic detoxification potential and any carcinogenic agent (like pesticides for example) could be detoxified, moreover, the genome of the insect is of small size and therefore the chances for mutations are very less. Furthermore, insects undergo programmed cell death throughout their life cycle whenever they undergo a metamorphosis which will likely prevent any tumors (Battu \u003cem\u003eet al.\u0026nbsp;\u003c/em\u003e2021). But again, insects can get cancer as well just like Peto\u0026rsquo;s paradox affirming that there is no correlation between the body size and cancer risk (Caulin and Maley 2011), but we are inconclusive as of now to answer this question. There had been reports of tumors in insects which likely explain this. Harker (1958) reported excessive endocrine secretions from sub-oesophageal ganglion-induced tumors in the midgut of \u003cem\u003ePeriplaneta americana\u003c/em\u003e. \u0026nbsp;Federley (1936) reported male-killing in the species hybrids of the butterfly \u003cem\u003ePygaera pigra\u003c/em\u003e (Notodontitade: Lepidoptera). Male larvae of the species hybrids are killed due to cancer before they even reach pupation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTobacco-specific Nitrosamines are carcinogenic substances and are associated with various types of cancers. They are formed during the process of curing, storage and fermentation. TSNAs can also form in the midgut of tobacco-feeding insects, wherein ingested tobacco leaves are fermented. We have detected the presence of NNN and NNK in the insect\u0026rsquo;s whole body and faeces indicating their formation in tobacco-feeding insects. The genotoxic effects of TSNAs are widely reported in higher animals but there is no study conducted to investigate the effects of TSNAs on lower animals like invertebrates. This study prompts future investigations into how TSNAs are formed, metabolized, and detoxified (if any) in insects. This experiment should be conducted on all the tobacco-feeding insects to see how different insects have developed strategies to mitigate the genotoxic effects of TSNAs on them. Insects just like other living organisms are composed of living cells that undergo cell division and therefore hypothetically insects can get cancers as well. Cancer in insect for the first time has been reported in the larvae of the butterfly\u003cem\u003e\u0026nbsp;Pygaera pigra.\u003c/em\u003e It is easier to conduct cancer studies \u003cem\u003ein vitro\u003c/em\u003e in an insect when compared to any other animal and the insect cell culturing makes it facile to maintain tumour cell lines \u003cem\u003ein vitro\u003c/em\u003e and investigate cell biology. So, insects could be used as model organisms to study the effect of potential carcinogens and the potential of insects as models for cancer studies should also be evaluated given their xenobiotic detoxification potential as compared to the higher animals.\u003c/p\u003e"},{"header":"Declarations:","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to Departments of Entomology of Dr. Rajendra Prasad Central Agricultural University for unwavering support throughout the research. The authors would like to thank Dr Molamma P. Prabhakaran, National University of Singapore for reviewing this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contribution:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.S.: Validation, Writing- Review\u0026amp; Editing. Y.G.: \u0026nbsp; Writing- Review\u0026amp; Editing. S.R.M.S.: Resources, Supervision. H.T.: \u0026nbsp;Writing- Review\u0026amp; Editing. K.A.: Writing- Review\u0026amp; Editing. S.K.M.P.: Writing- Review\u0026amp; Editing. J.R.B.:\u0026nbsp;Conceptualization, Methodology, Writing - Original Draft.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during this study are included in this published article (and its Supplementary Information files).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References:","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eBattu J.R., Karthik, S. \u0026amp; Anil, G. Programmed cell death: A destructive and constructive process in insects. 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Simultaneous determination of four tobacco-specific N-nitrosamines (TSNA) in human urine. J.Chromatogr.B. 877(11\u0026ndash;12), 1185\u0026ndash;1192. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jchromb.2009.03.009\u003c/span\u003e\u003c/span\u003e (2009).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLee, J.H. \u0026amp; Boo, K.S. Comparative effects of nicotine and diazinon on larval mortality and activity of cytochrome P-450 monooxygenases in \u003cem\u003eHelicoverpa assulta\u003c/em\u003e and \u003cem\u003eSpodoptera exigua\u003c/em\u003e. Korean J. Appl. Entomol. 32 (2), 225\u0026ndash;235. (1993).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLi, X., Liu, F., Wang, H., He, F., Yang, R. \u0026amp; Zhao, M. Gas chromatography-mass spectrometry method for simultaneous detection of nine alkaloids in tobacco and tobacco products by QuEChERS sample preparation. Anal. Sci. 35(8):849\u0026ndash;854. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2116/analsci.19P063\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eP\u0026eacute;rez-Ortu\u0026ntilde;o, R., Mart\u0026iacute;nez-S\u0026aacute;nchez, J.M., Fu, M., Ballb\u0026egrave;, M., Quir\u0026oacute;s, N., Fern\u0026aacute;ndez, E. \u0026amp; Pascual, J.A. Assessment of tobacco specific nitrosamines (TSNAs) in oral fluid as biomarkers of cancer risk: A population-based study. Environ. Res. 151, 635\u0026ndash;641. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envres.2016.08.036\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSaremba, B.M., Murch, S.J., Tymm, F.J. \u0026amp; Rheault, M.R. The metabolic fate of dietary nicotine in the cabbage looper, \u003cem\u003eTrichoplusia ni\u003c/em\u003e (H\u0026uuml;bner). J. Insect Physiol. 109, 1\u0026ndash;10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jinsphys.2018.05.010\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSarlak, S., Lalou, C., Amoedo, N.D. \u0026amp; Rossignol, R. Metabolic reprogramming by tobacco-specific nitrosamines (TSNAs) in cancer. Semin. Cell Dev. Biol. 98, 154\u0026ndash;166. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.semcdb.2019.09.001\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSelf, L.S., Guthrie, F.E. \u0026amp; Hodgson, E. Metabolism of nicotine by tobacco-feeding insects. Nature. 204(4955), 300\u0026ndash;301. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/204300a0\u003c/span\u003e\u003c/span\u003e (1964).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSnyder, M.J., Walding, J.K. \u0026amp; Feyereisen, R. Metabolic fate of the allelochemical nicotine in the tobacco hornworm, \u003cem\u003eManduca sexta\u003c/em\u003e. Insect Biochem. Mol. Biol. 24(8), 837\u0026ndash;846. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0965-1748(94)90112-0\u003c/span\u003e\u003c/span\u003e (1994).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSophia, F., Spiegelhalder, B. \u0026amp; Preussmann, R. Preformed tobacco-specific nitrosamines in tobacco\u0026mdash;the role of nitrate and influence of tobacco type. Carcinogenesis, 10(8), 1511\u0026ndash;1517. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/carcin/10.8.1511\u003c/span\u003e\u003c/span\u003e (1989).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSpiegelhalder, B. \u0026amp; Fischer, S. Formation of tobacco-specific nitrosamines. Crit. Rev. Toxicol. 21(4), 241. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3109/10408449109017911\u003c/span\u003e\u003c/span\u003e (1991).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eYuan, J.M., Koh, W.P., Murphy, S.E., Fan, Y., Wang, R., Carmella, S.G., Han, S., Wickham, K., Gao, Y.T., Yu, M.C. \u0026amp; Hecht, S.S. Urinary levels of tobacco-specific nitrosamine metabolites in relation to lung cancer development in two prospective cohorts of cigarette smokers. Cancer Res. \u003cstrong\u003e69\u003c/strong\u003e(7), 2990\u0026ndash;2995. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/0008-5472.can-08-4330\u003c/span\u003e\u003c/span\u003e (2009).\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables:","content":"\u003cp\u003e\u003cstrong\u003eTable-1: Linearity of NNN, NNK and NNAL\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"13.821138211382113%\"\u003e\n \u003cp\u003e\u003cstrong\u003eStandard\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.48780487804878%\"\u003e\n \u003cp\u003e\u003cstrong\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.691056910569106%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCalibration curve\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLOD (mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLOQ (mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"13.821138211382113%\"\u003e\n \u003cp\u003eNNN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.48780487804878%\"\u003e\n \u003cp\u003e0.9523\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.691056910569106%\"\u003e\n \u003cp\u003e5.819e3X-5.727e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20%\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20%\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"13.821138211382113%\"\u003e\n \u003cp\u003eNNK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.48780487804878%\"\u003e\n \u003cp\u003e0.9994\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.691056910569106%\"\u003e\n \u003cp\u003e5.494e4X-8.872e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20%\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20%\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"13.821138211382113%\"\u003e\n \u003cp\u003eNNAL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.48780487804878%\"\u003e\n \u003cp\u003e0.9988\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.691056910569106%\"\u003e\n \u003cp\u003e5.465e4X-1.09e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20%\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20%\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003cstrong\u003eTable-2: Retention time, Peak areas and concentration of NNN and NNK in the analyzed samples (average of three replicates)\u003c/strong\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"8.441558441558442%\"\u003e\n \u003cp\u003e\u003cstrong\u003eS.No\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.266233766233768%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.071428571428573%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnalyte\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.772727272727273%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRetention time (min)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.941558441558442%\"\u003e\n \u003cp\u003e\u003cstrong\u003eQuantification \u0026nbsp; \u0026nbsp; \u0026nbsp;(mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.506493506493506%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRSD%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"8.441558441558442%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"21.266233766233768%\"\u003e\n \u003cp\u003e\u003cem\u003eS. litura\u003c/em\u003e whole body\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.071428571428573%\"\u003e\n \u003cp\u003eNNN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.772727272727273%\"\u003e\n \u003cp\u003e4.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.941558441558442%\"\u003e\n \u003cp\u003e0.365\u0026nbsp;∓ 0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.506493506493506%\"\u003e\n \u003cp\u003e1.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.863741339491916%\"\u003e\n \u003cp\u003eNNK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.016166281755197%\"\u003e\n \u003cp\u003e6.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.792147806004618%\"\u003e\n \u003cp\u003e0.062\u0026nbsp;∓ 0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.32794457274827%\"\u003e\n \u003cp\u003e1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.863741339491916%\"\u003e\n \u003cp\u003eNNAL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.016166281755197%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.792147806004618%\"\u003e\n \u003cp\u003eNot Detected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.32794457274827%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"8.441558441558442%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"21.266233766233768%\"\u003e\n \u003cp\u003e\u003cem\u003eS. litura\u003c/em\u003e faeces\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.071428571428573%\"\u003e\n \u003cp\u003eNNN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.772727272727273%\"\u003e\n \u003cp\u003e4.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.941558441558442%\"\u003e\n \u003cp\u003e0.344\u0026nbsp;∓ 0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.506493506493506%\"\u003e\n \u003cp\u003e1.87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.863741339491916%\"\u003e\n \u003cp\u003eNNK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.016166281755197%\"\u003e\n \u003cp\u003e7.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.792147806004618%\"\u003e\n \u003cp\u003e0.036\u0026nbsp;∓ 0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.32794457274827%\"\u003e\n \u003cp\u003e1.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.863741339491916%\"\u003e\n \u003cp\u003eNNAL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.016166281755197%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.792147806004618%\"\u003e\n \u003cp\u003eNot Detected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.32794457274827%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"8.441558441558442%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"21.266233766233768%\"\u003e\n \u003cp\u003eTobacco leaves (PT-76)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.071428571428573%\"\u003e\n \u003cp\u003eNNN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.772727272727273%\"\u003e\n \u003cp\u003e6.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.941558441558442%\"\u003e\n \u003cp\u003e5.71\u0026nbsp;∓ 0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.506493506493506%\"\u003e\n \u003cp\u003e1.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.863741339491916%\"\u003e\n \u003cp\u003eNNK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.016166281755197%\"\u003e\n \u003cp\u003e8.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.792147806004618%\"\u003e\n \u003cp\u003e0.97\u0026nbsp;∓ 0.064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.32794457274827%\"\u003e\n \u003cp\u003e1.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Tobacco, Tobacco-specific Nitrosamines, Spodoptera litura, Cancer","lastPublishedDoi":"10.21203/rs.3.rs-1707326/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1707326/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNicotine 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 not a harmful substance except for the addiction it causes. 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, 5\u003csup\u003eth\u003c/sup\u003e instar larvae of \u003cem\u003eSpodoptera litura\u003c/em\u003e and their faeces were analyzed for the presence of NNN, NNK, and NNAL along with the stored tobacco leaves (PT-76) using an Agilent 6470B LC-MS/MS system following ISO/DIS 19290:2015 protocol. The LOD and LOQ were 0.001 mg/kg and 0.005 mg/kg for all the tested nitrosamines. NNN was found to be 0.365 mg/kg, 0.344 mg/kg, and 5.71 mg/kg in insect whole-body samples, faeces, and tobacco leaves, respectively. NNK was found to be 0.062 mg/kg, 0.036 mg/kg and 0.97 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 95-98% 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.\u003c/p\u003e","manuscriptTitle":"LC-MS/MS analysis of carcinogenic tobacco-specific nitrosamines in Spodoptera litura using the QuEChERS method","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-06-29 15:10:04","doi":"10.21203/rs.3.rs-1707326/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-08-24T04:12:02+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"72bc2776-5992-4602-b509-08089f081dbb","date":"2022-08-08T12:22:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-08-04T08:04:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-08-03T15:31:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2f276f04-84d2-4a20-a1a5-18d234000700","date":"2022-08-03T05:45:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"f97e1a05-fa9d-455f-9618-5544ff586c05","date":"2022-07-25T06:45:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"bb5c88a6-358f-421d-afd1-dd1ca9204a69","date":"2022-07-24T13:12:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-07-24T07:27:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-07-24T07:19:58+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-06-28T06:27:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-06-28T06:23:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-05-30T09:42:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9258f664-f72d-483f-8910-6861d61963cc","owner":[],"postedDate":"June 29th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T22:12:29+00:00","versionOfRecord":{"articleIdentity":"rs-1707326","link":"https://doi.org/10.1038/s41598-023-37656-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2023-07-27 21:45:04","publishedOnDateReadable":"July 27th, 2023"},"versionCreatedAt":"2022-06-29 15:10:04","video":"","vorDoi":"10.1038/s41598-023-37656-2","vorDoiUrl":"https://doi.org/10.1038/s41598-023-37656-2","workflowStages":[]},"version":"v1","identity":"rs-1707326","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1707326","identity":"rs-1707326","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}