Analysis of four tobacco-specific nitrosamines in mainstream cigarette smoke of Virginia cigarettes by LC-MS/MS

An improved method was developed for the determination of the four major tobacco-specific nitrosamines（TSNAs） in mainstream cigarette smoke. The new method offers decreased sample preparation and analysis time as compared to traditional methods. This method uses isotope dilution liquid chromatography coupled with a tandem mass spectrometer with electrospray ionization and is significantly more sensitive than traditional methods. It also shows no evidence of artifactual formation of TSNAs. Sample concentrations were determined for four TSNAs in mainstream smoke using two isotopically labeled TSNAs analogues as internal standards. Mainstream smoke was collected on an industry standard 44-ram Cambridge filter pad, extracted with 0.1 mol/L ammonium acetate, purified by solid-phase extraction, and analyzed without further sample cleanup. The analytical column is a 3.9 mm×150 mm Waters Symmertry Shield RP18 column and volume fraction of the mobile phase is 50% methanol, 50% water containing 0.1% acetic acid. The results show that the linear range is 0.5-100.0 mg/L except for N-nitrosoanabasine （NAB） from 0.25 to 50.0 mg/L. The limits of detection are 0.1 mg/L for N-nitrosonornicotine （NNN）, 0.08 mg/L for 4-（methylnitrosamino）-1- （3-py-ridyl）-1-butanone （NNK）, 0.05 mg/L for N-nitrosoanatabine （NAT） and 0.06 mg/L for NAB. The recoveries of the four TSNAs are from 90.2% to 105.7%.

Hemoglobin Adducts as Biomarkers of Exposure to and metabolic Activation of Carcinogenic Tobacco-Specific Nitrosamines

The carcinogenic tobacco-specific nitrosamines N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK) form hemoglobin adducts in laboratory animals and humans. These adducts release 4-hydroxy-l-(3-pyridyl)-l-butanone (HPB) upon mild base hydrolysis. HPB released from human hemoglobin can be quantified by gas chromatography-mass spectrometry. It is the only available biochemical marker for determination of exposure to, and metabolic activation of, carcinogens present only in tobacco. Levels of HPB were highest in snuff-dippers, followed by smokers and nonsmokers. Large interindividual variations in adduct levels were observed. The relationship between HPB levels in globin and DNA of rats treated with NNK has been investigated in order to aid in interpretation of the data from humans. These studies have provided the initial database for understanding the metabolic activation of tobacco-specific nitrosamines in humans.

Association of a Tobacco-specific Nitrosamine Carcinogen with Urinary Cotinine,Urinary Sodium Excretion,and Total Energy Intake in Adolescents and Children

This study investigated the association of a tobacco-specific nitrosamine carcinogen,4-(methylnitrosamino)-1-(3-pyridy1)-I-butanol(NNAL)with urinary cotinine(uCot),urinary sodium(uNa)excretion,systolic blood pressure(sBP),and total energy intake in adolescents and children in relation to the subjects'age.A total of 790 subjects aged 6-19 years were evaluated.NNAL,uCot,corrected NNAL(cNNAL),the NNAL/uCot ratio,uNa,sBP,and nutrient intake were measured.A strong association between uCot and cNNAL was observed in children who were 11 years of age(r=0.881,P<0.001);however,no significant association was noted in adolescents who were 19 years of age.The uNa level was significantly higher(133.9 mmol/L vs.107.8 mmol/L,P<0.001)and sBP was significantly lower(105.3 mmHg vs.110.6 mmHg,P=0.012)in adolescents with elevated NNAL than in those without elevated NNAL.NNAL was significantly higher in subjects with increased uNa excretion than in those without increased uNa excretion.NNAL was positively correlated with uNa(r=0.183,P<0.001)and negatively correlated with sBP(r=0.142,P<0.001).Non-smokers with elevated NNAL/uCot ratios had significantly lower total energy intake than those without elevated NNAL/uCot ratios(1729.0 kcal/day vs.1911.0 kcal/day,P=0.008).The relationship between NNAL and uCot varied according to the subjects'age.NNAL seems to play a role in decreasing sBP by enhancing uNa excretion.Insufficient nutrient intake may contribute to endogenous formation of NNAL in non-smoking adolescents and children.

Nitrosamines crisis in pharmaceuticals-Insights on toxicological implications,root causes and risk assessment:A systematic review

The presence of N-nitroso compounds,particularly N-nitrosamines,in pharmaceutical products has raised global safety concerns due to their significant genotoxic and mutagenic effects.This systematic review investigates their toxicity in active pharmaceutical ingredients(APIs),drug products,and pharmaceutical excipients,along with novel analytical strategies for detection,root cause analysis,reformulation strategies,and regulatory guidelines for nitrosamines.This review emphasizes the molecular toxicity of N-nitroso compounds,focusing on genotoxic,mutagenic,carcinogenic,and other physiological effects.Additionally,it addresses the ongoing nitrosamine crisis,the development of nitrosamine-free products,and the importance of sensitive detection methods and precise risk evaluation.This comprehensive overview will aid molecular biologists,analytical scientists,formulation scientists in research and development sector,and researchers involved in management of nitrosamine-induced toxicity and promoting safer pharmaceutical products.

Hepatoprotective effect of Holothuria leucospilota methanolic extract on dimethyl nitrosamine-induced hepatotoxicity in rats

Background:Complementary medicine is an interesting field for extracting bio-active compounds from various plant and animal sources.The hepatoprotective effect of the methanolic extract of a species of sea cucumber called Holothuria leu-cospilota in an animal model of liver cancer caused by dimethyl nitrosamine(DMN)was studied.Methods:Wistar female rats were randomly divided into five groups(n=12):control(intact),positive control(received 1%DMN[10 mg/kg/week,intraperitoneally]for 12 weeks),and three treatment groups(received 50,100,and 200 mg/kg/day H.leu-cospilota extract orally for 12 weeks along with intraperitoneal administration of 1%DMN[10 mg/kg/week]).In all groups,ultrasound was performed on the liver every week to check its density.Blood sampling and liver isolation were performed on three occasions,at 4,8,and 12 weeks,to check liver enzymes and the histopathological condition of the liver tissue(every week,four animals from each group were randomly selected).Results:Liver density changes were evident from the eighth week onward in the positive control group.Histopathological results indicated pathologic changes in the positive control group after 4 weeks.The increase in liver enzymes in the posi-tive control group was significantly different from that in the treatment and control groups.Conclusions:We demonstrated the hepatoprotective effect of H.leucospilota on DMN-induced liver damage in rats using biochemical and histological parameters and ultrasonography.More additional research(in silico or in vitro)is needed to find the exact mechanism and the main biological compound in H.leucospilota.

Metabolism Study on Tobacco-specific N-Nitrosamines in Rabbit by Solid-phase Extraction and Liquid Chromatography-electrospray Ionization-mass Spectrometry Method

Metabolism of four tobacco-specific N-nitrosamines （TSNAs）, N＇-nitrosonornicotine （NNN）, N＇-nitrosoanatabine （NAT）, N＇-nitrosoanabasine （NAB） and 4-（methyl nitrosamino）-1- （3-pyridyl）-1-butanone （NNK） was studied by solid-phase extraction （SPE） and liquid chromato- graphy-electrospray ionization-mass spectrometry （LC-ESI-MS）. SPE and LC-ESI-MS method was evaluated to be rapid, simple, sensitive and selective for analysis of TSNAs in rabbit serum. The doses of TSNAs administrated were 4.666 μg/kg and 11.665 μg/kg according to different levels in cigarettes. Metabolic curves of four TSNAs and 4-（methyl nitrosamino）-1-（3-pyridyl）- 1-butanone （NNAL）, the metabolite of NNK, were obtained.

Intake of Volatile N-nitrosamines and Their Ability to Exogenously Synthesize in the Diet of Inhabitants from High-risk Area of Esophageal Cancer in Southern China

Objective Nan'ao County in Guandong Province is a high-risk area of esophageal cancer in Southern China. Of the suspected etiological factors in the environment, N-nitrosamines and their precursors have received the greatest attention. Methods Sixty samples of the diet ingested by the inhabitants were collected and detected for volatile N-nitrosamines and their precursors. Five N-nitrosamines detected by Gas Chromatography-Thermal Energy Analyzer were N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosopyrrolidine, N-nitrosopiperidine and N-nitrosomethyl-benzylamine. Results The average content of 5 volatile N-nitrosamines in the diet was 312.0 μg/kg (median). The daily intake of the nitrosamines was 286.5 μg/head/day. Only the ability to exogenously synthesize N-nitrosopiperidine was powerful among 5 volatile N-nitrosamines. By a computerized stepwise regression analysis and curve fitting, we studied the correlation among the nitrosamines, the precursors and the major food items in the samples. Conclusion It demonstrated that a relatively high content of volatile N-nitrosamines was present in the diet collected in the area.

Nitrate-nitrite-nitrosamines exposure and the risk of type 1 diabetes: A review of current data

The potential toxic effects of nitrate-nitrite-nitrosamine on pancreatic β cell have remained a controversial issue over the past two decades. In this study, we reviewed epidemiological studies investigated the associations between nitrate-nitrite-nitrosamines exposure, from both diet and drinking water to ascertain whether these compounds may contribute to development of type 1 diabetes. To identify relevant studies, a systematic search strategy of Pub Med, Scopus, and Science Direct was conducted using queries including the key words "nitrate", "nitrite", "nitrosamine" with "type 1 diabetes" or "insulin dependent diabetes mellitus". All searches were limited to studies published in English. Ecologic surveys, case-control and cohort studies have indicated conflicting results in relation to nitrate-nitrite exposure from drinking water and the risk of type 1 diabetes. A null, sometimes even negative association has been mainly reported in regions with a mean nitrate levels < 25 mg/L in drinking water, while increased risk of type 1 diabetes was observed in those with a maximum nitrate levels > 40-80 mg/L. Limited data are available regarding the potential diabetogenic effect of nitrite from drinking water, although there is evidence indicating dietary nitrite could be a risk factor for development of type 1 diabetes, an effect however that seems to be significant in a higher range of acceptable limit for nitrate/nitrite. Current data regarding dietary exposure of nitrosamine and development of type 1 diabetes is also inconsistent. Considering to an increasing trend of type 1 diabetes mellitus(T1DM) along with an elevated nitrate-nitrite exposure, additional research is critical to clarify potential harmful effects of nitrate-nitritenitrosamine exposure on β-cell autoimmunity and the risk of T1DM.

Ultra-Sensitive LC-MS/MS Method for the Trace Level Quantification of Six Potential Genotoxic Nitrosamine Impurities in Telmisartan

Nitrosamine impurities are potentially genotoxic which are considered under cohort of concern as per ICH M7 guidelines and need to be controlled at trace levels during quantification in drug substances and drug products for safe human consumption. Recent regulatory requirements also suggest the need to have highly sensitive analytical methods for the accurate quantification of Nitrosamine impurities. In this paper we have presented simple, rapid and ultra-sensitive LC-MS/MS method for six potential genotoxic nitrosamine impurities: N-Nitroso dimethyl amine (NDMA), N-Nitroso diethyl amine (NDEA), N-Nitroso Ethyl Iso propylamine (NEIPA), N-Nitroso-N-methyl-4-aminobutyric acid (NMBA) N-Nitroso diisopropylamino (NDIPA) and N-Nitroso dibutyl amine (NDBA) with a LOQ of 0.004 ppm. Chromatographic separation is achieved using Zorbax SB C18 150 × 3.0 mm, 3.5 μ column with 0.1% formic acid in water as mobile phase A and 0.1% formic acid in methanol as mobile phase B at a flow rate of 0.3 ml/min using gradient mode of elution at a total run time of 18 minutes. Six nitrosamine impurities are successfully ionized and quantified in positive mode of atmospheric pressure chemical ionization (APCI) using multiple reaction monitoring (MRM). Method validation is performed as per ICH guidelines evaluating the limit of quantification and detection and found to give good S/N ratios with good linearity range of 0.002 - 2 ppm with regression coefficient >0.99 for all the six nitrosamine impurities. Method recoveries are established using three-step sample preparation protocol and are found to be satisfactory within 80% - 120%. The method can be used routinely applied for the detection of Nitrosamines in Telmisartan at a concentration of 1.5 ng/ml (0.03 ppm with respect to telmisartan concentration of 50 mg/ml).

Modifying NaY Zeolite with Metal Oxide by Microwave Irradiation:Influence on the Adsorption and Decomposition of N-nitrosamines

Coating MgO.br ZrO2 on zeolite NaY accelerated decomposition of N-nitrosodimethylamine and N-nitrosopyrrolidine, resulting from variation of the surface state such as generation of basic sites.

A New Automated Method and Sample Data Flow for Analysis of Volatile Nitrosamines in Human Urine

Volatile nitrosamines (VNAs) are a group of compounds classified as probable (group 2A) and possible (group 2B) carcinogens in humans. Along with certain foods and contaminated drinking water, VNAs are detected at high levels in tobacco products and in both mainstream and side-stream smoke. Our laboratory monitors six urinary VNAs—N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine (NDEA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), and N-nitrosomorpholine (NMOR)—using isotope dilution GC-MS/ MS (QQQ) for large population studies such as the National Health and Nutrition Examination Survey (NHANES). In this paper, we report for the first time a new automated sample preparation method to more efficiently quantitate these VNAs. Automation is done using Hamilton STARTM and Caliper StaccatoTM workstations. This new automated method reduces sample preparation time from 4 hours to 2.5 hours while maintaining precision (inter-run CV < 10%) and accuracy (85% - 111%). More importantly this method increases sample throughput while maintaining a low limit of detection (<10 pg/mL) for all analytes. A streamlined sample data flow was created in parallel to the automated method, in which samples can be tracked from receiving to final LIMs output with minimal human intervention, further minimizing human error in the sample preparation process. This new automated method and the sample data flow are currently applied in bio-monitoring of VNAs in the US non-institutionalized population NHANES 2013-2014 cycle.

Rapid and Simple Extraction Method for Volatile N-Nitrosamines in Meat Products

A new methodology for extraction, pre-concentration and analysis of volatile nitrosamines in meat-derived products was developed and compared with conventional methods (Distillation and two-step solid-phase extraction). The samples (canned sausages, cured meat, luncheon and smoked meat) were treated with an aqueous sodium hydroxide (NaOH) by autoclaving at 121°C for 10 min and extracted by liquid-liquid extraction with dichloromethane, then the nitrosamines were pre-concentrated using activated silica. Then, gas chromatography coupled with flame ionization detector was used for the separation and determination of the different nitrosamines contained in a real sample and gas chromatography with mass spectrometry detection was used as the confirmation technique. The newly invented autoclaving method allowed the determination of nitrosamine compounds at trace levels with limit of detection ranged from 0.077 to 0.18 ppb and quantitation limits were from 0.26 to 0.6 ppb for all nitrosamines, and found to be superior to the conventional ones, yielding approximately about 10%-20% increasing in the recovery compared with the mean recovery obtained when applying conventional methods.

Analysis of <i>N</i>-Nitrosamine Migration from Rubber Teats and Soothers

A testing method for N-nitrosamines and N-nitrosatable substances in rubber teats and soothers was modified. N-Nitrosamines are generally analyzed using either a nitrogen chemiluminescence detector (NCD) or a thermal energy analyzer (TEA). However, because few testing laboratories are equipped with these devices, it is difficult to conduct these tests. Therefore, an analysis method for N-nitrosamines using the more widespread gas chromatography-mass spectrometry (GC-MS) method was improved. In addition, EN 12868 was used to prepare the test solutions because of its worldwide use and compliance with EU regulations. Using GC-MS, EN 12868 method targeting ten kinds of N-nitrosamines was modified. The determination limits of the method were 1.0 -1.5 μg/kg for N-nitrosamines and 4 -6 μg/kg for N-nitrosatable substances. Quantification was possible at 1/5 or less and 1/15 or less, respectively, of the regulation values listed in EU Directive 93/11/EEC. In terms of application, there were no problems with the selectivity of the detector. The recoveries were 58% -109% for N-nitrosamines and 59% -102% for N-nitrosatable substances. Screening and verification were possible by measuring the amount of secondary amines in the boiled solution and migration solution.

SYNTHESES OF N- METHYL-^(14)C BENZYL AMINE AND N- METHYL-^(14)C BENZYL NITROSAMINE

The syntheses of N-Methyl-14C benzyl amine and N-Methyl-14C benzyl nitrosamine are reported. Specific activities were approximately 920 MBq/mmol. Chemical and radiochemical purity checked by HPLC were more than 95%.

Analysis of Nitrosamines in Organic Foods

Nitrosamines are classified by IARC as Group 2B carcinogens. Usually they might be present in organic foods as products of reaction between secondary amines and nitrosation system. The aim of the study was to test the concentration of nitrosamines in Bulgarian products. High performance liquid chromatography with UV detector was used for identification and quantitation. A standard solution of N-nitrosodiethanolamine was used as a reference substance and in the validation procedure of samples. The limit of detection of the method was determined to 14× 10^-9 g/mL. The results of the testing showed that analyzed organic foods produced in Bulgaria did not contain nitrosamines above the limit of detection of the method.

MECHANISM OF PROMOTING EFFECTS OF RIBOFLAVIN DEFICIENCY ON CARCINOGENESIS OF NITROSAMINES (EFFECTS ON RAT LIVER GLUTATHIONE CONTENT)

The effects of riboflavin deficiency and simultaneously nitrosodimethylamine (NDMA) given by gastric intubation on the hepatic glutathione (GSH) content were examined in rats. On different days of the experiment, hepatic GSH content of the riboflavin deficient rats decreased to 55-61% of the control rats. When NDMA was given 6 mg kg by gastric intubation to riboflavin deficient rats, hepatic GSH content decreased markedly to 39-43% of the control rats. After supplying riboflavin, hepatie GSH content of the deficient rats recovered to the level of the control rats. These results suggest that alterations of rat hepatic GSH content during riboflavin deficiency may imply as one of the promoting effects of riboflavin deficiency on the carcinogenesis of nitrosamines.

IMMUNOCHEMICAL IDENTIFICATION AND LOCALIZATION OF CYTOCHROME P-450HSjISOZYME, AN ENZYME RELATED TO NITROSAMINE METABOLISM, IN HUMAN GASTRIC MUCOSA AND GASTRIC CARCINOMA

Monoclonal antibody (MAb) to rat liver cyto-chrome P-450j isozyme, an activating enzyme specific to nitrosamine metabolism, was used coupled with immunoblotting, densitometer scanning of SDS-PAGE gels and immunohistochemical technique. The trace P-450HSj isozyme (Mr. 51.5 Kd) was found in human gastric mucosa. It was similar to P-450j in molecular weight, catalytic and immunochemical properties. The concentrations of P-450HSj in mucosa of lesser curvature were higher than those in greater curvature. This might be one of the important reasons that lesser curvature is the commonest area for gastric carcinoma. But there was possibly less P-450HSj in gastric mucosa with cancer. Im-munohistochemically, P-450HSj was discovered in the cytoplasm of some glandular epithelial cells, especially in the glands with hyperplastic and intestinal metaplastic changes adjacent to carcinoma. It was also found in some normal glands and in tumor cells of high-differentiated adenocarcinoma, but not in those of low-differentiated ones. Following subjects are discussed: (1) the method of detecting trace P-450HSj, (2) the rule of distribution of P-450HSj, and (3) the relationship between the isozyme and the occurrence of gastric cancer caused by nitrosa-mines.

Carcinogenesis by Nitrosamines and Azoxyalkanes by Different Routes of Administration to Rats

The effects of administering similar doses of some simple alkylating agents to rats by different regimens have been compared.Two of the compounds were methylating agents,nitrosodimeth- ylamine and azoxymethane;two were ethylating agents,nitrosodiethylamine and azoxyethane and nitrosomethylethylamine was both a methylating and an ethylating agent.The treatments gave rise to tumors in almost all treated rats.The results indicate the importance ofpharmacoki- netics in determining which organs are the targets of the alkylating carcinogens.1989 Academic Press.Inc.

Application of Sargassum fusiforme(Harv.)Setch.Extract in Cigarettes

[Objectives]This study was conducted to investigate the application of Sargassum fusiforme(Harv.)Setch.in cigarettes.[Methods]Tobacco-specific nitrosamines in the smoke of cigarettes added with ethanol extract of S.fusiforme were determined,and the compounds related to the aroma of S.fusiforme were identified by flavor-smelling experiment.[Results]With the addition of the ethanol extract of S.fusiforme,the decrease in the total amount of four tobacco-specific nitrosamines in mainstream cigarette smoke reached 16.42%.The results of the flavor-smelling experiment showed that the aroma of S.fusiforme might be related to(R)-5,6,7,7A-tetrahydro-4,4,7A-trimethyl-2(4H)-benzofuranone,glycerol,ethyl palmitate,methyl palmitate,ethyl linoleate,methyl(Z,Z,Z)-9,12,15-octadecatrienoate,ethyl(Z,Z,Z)-9,12,15-octadecatrienoate,phytol,and tetradecanoic acid.[Conclusions]The ethanol extract of S.fusiforme has the potential function of reducing the content of tobacco-specific nitrosamines in smoke and improving the taste of cigarettes.

苦荞叶提取物清除亚硝酸钠及阻断亚硝胺合成作用研究

采用超声波辅助,响应面优化苦荞叶清除亚硝酸钠物质提取工艺,并以Vc和甘草提取物为对照,探究苦荞叶提取物对亚硝胺合成的阻断作用。结果表明,苦荞叶清除亚硝酸钠物质提取最佳工艺条件为:乙醇浓度44%,超声功率260 W,料液比21 mL/g,目数为80目,超声时间为30 min,提取温度50℃,此条件下苦荞叶提取物对亚硝酸钠的清除率为(83.97±0.12)%;在20~100 mg/L溶度范围内,Vc、苦荞叶提取物和甘草提取物对亚硝胺合成的阻断率线性关系良好,其对应的半阻断浓度IC50分别为61.26、80.09和90.20 mg/L。质量浓度继续增加时,苦荞叶提取物和甘草提取物对亚硝胺合成的阻断率随之下降,但同浓度情况下苦荞叶提取物对亚硝胺合成的阻断率高于甘草提取物,苦荞叶提取物对亚硝胺合成的阻断作用弱于Vc,但强于甘草提取物。