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.

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.

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.

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.

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.

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.

Intake of tobacco nitrosamines of smokers in various provinces of China and their cancer risk:A meta-analysis

Nitrosamines are a class of carcinogens which have been detected widely in food,water,some pharmaceuticals as well as tobacco.The objectives of this paper include reviewing the basic information on tobacco consumption and nitrosamine contents,and assessing the health risks of tobacco nitrosamines exposure to Chinese smokers.We searched the publications in English from“Web of Science”and those in Chinese from the“China National Knowledge Infrastructure”in 2022 and collected 151 literatures with valid information.The content of main nitrosamines in tobacco,including 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone(NNK),N-nitrosonornicotine(NNN),N-nitrosoanatabine(NAT),N-nitrosoanabasine(NAB),total tobacco-specific nitrosamines(TSNA),and N-nitrosodimethylamine(NDMA)were summarized.The information of daily tobacco consumption of smokers in 30 provinces of China was also collected.Then,the intakes of NNN,NNK,NAT,NAB,TSNAs,and NDMA via tobacco smoke were estimated as 1534ng/day,591 ng/day,685 ng/day,81 ng/day,2543 ng/day,and 484 ng/day by adult smokers in30 provinces,respectively.The cancer risk(CR)values for NNN and NNK inhalation intake were further calculated as 1.44×10^(-5)and 1.95×10^(-4).The CR value for NDMA intake via tobacco smoke(inhalation:1.66×10^(-4))indicates that NDMA is similarly dangerous in tobacco smoke when compared with the TSNAs.In China,the CR values caused by average nitrosamines intake via various exposures and their order can be estimated as the following:smoke(3.75×10^(-4))>food(1.74×10^(-4))>drinking water(1.38×10^(-5)).Smokers in China averagely suffer 200%of extra cancer risk caused by nitrosamines in tobacco when compared with non-smokers.

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.

Occurrence and formation potential of nitrosamines in river water and ground water along the Songhua River, China

The presence of mutagenic and carcinogenic nitrosamines in water is of great concern. In this study, seven nitrosamines including N-nitrosodimethylamine （NDMA）, N-nitrosodiethylamine （NDEA）, N-nitrosomethylethylamine （NMEA）, N-nitrosopyrrolidine （NPyr）, N-nitrosopiperidine （NPip）, N-nitrosodi-n-propylamine （NDPA）, and N-nitrosodi-n-butyl-amine （NDBA） were investigated in river water and ground water samples collected from 5 representative cities （Jilin, Songyuan, Harbin, Jiamusi and Tongjiang） along the Songhua River. The total concentrations of nitrosamines in ground water were n.d. （not detected） to 60.8 ng/L, NDMA was the most frequently detected nitrosamines in ground water, followed by NDEA and NPip. Relatively high detected frequency and concentrations of NDMA were also observed in river water samples, and the total nitrosamines＇ concentration at midstream is always higher than that at upstream and downstream. After 24 hr chlorination, concentration of NDMA, NDBA was obviously increased but NDEA was reduced. Furthermore, UV2s4 showed a better relationship with NDMA-FP rather than dissolved organic carbon （DOC）. NI-h-N. and TDN.

Effect of activated sludge treatment on the formation of N–nitrosamines under different chloramination conditions

Municipal wastewater discharge is considered as one of the main sources of N-nitrosamine precursors which can impact the qualities of downstream source waters and reclaimed wastewaters for potable reuse. N –Nitrosamine precursors can be removed to various degrees during biological wastewater treatment(e.g., the activated sludge(AS) process). So far, little is known about the impact of the AS process on N-nitrosamine formation under practical disinfection condition(e.g., uniform formation condition(UFC)). In this study, N-nitrosamine UFC from selected model compounds, sewage components(i.e., blackwaters and greywaters) and sewage samples were comprehensively investigated during batch AS treatment tests. N –Nitrosodimethylamine(NDMA) formation from the tested precursor compounds(i.e., trimethylamine(TMA) and sumatriptan(SMTR)) under UFC chloramination decreased mostly after 6 or 24 hr treatment with different types of AS(i.e., domestic rural AS, domestic urban AS, and textile AS), and the reductions in NDMA UFC were comparable to their NDMA formation potential(FP) reductions. In urine and feces blackwaters, NDMA UFC increased after 6 or 24 hr treatment with the domestic(i.e., rural and urban) AS, while NDMA FP decreased substantially. The increases in NDMA UFC after AS treatment was presumably attributed to the removal of bulk organic matters(e.g., dissolved organic carbon(DOC)) which favored NDMA formation under UFC. On the other hand, in laundry greywaters having relatively abundant DOC, N-nitrosamine UFC was less affected by DOC removal before or after AS treatment, but decreased to similar degrees with N-nitrosamine FP. In sewage samples collected from wastewater treatment plants, N-nitrosamines UFC tended to increase or remain constant during AS treatment, despite the decreases in their FPs. These results suggest that biological wastewater treatment(e.g., the AS process) may not effectively reduce N-nitrosamine formation(e.g., measured under UFC) partially because the concurrent removal of bulk organic matters(e.g., DOC) favored N-nitrosamine formation in s econdary effluents.

Study on environmental etiology of high incidence areas of liver cancer in China

INTRODUCTIONChina is a country with a high incidence of liver cancer insome areas.Liver cancer has a wide distribution andthreatens human health seriously.A rough estimationshows that out of a population of 1.2×10~8 in liver

Risk assessment of chemical substances of safety concern generated in processed meats

This manuscript is reviewing the presence of substances of safety concern that may be generated in different types of meat products.Such substances include N-nitrosamines that can be generated under certain conditions when using nitrite as preservative,the polycyclic aromatic hydrocarbons generated as a consequence of particular smoking processes,heterocyclic aromatic amines generated under particular cooking conditions,compounds released from the oxidation of lipids and proteins,Maillard reaction products like acrylamide and carboxymethyl lysine,and amines that can be generated and accumulated in fermented meats.These hazardous compounds,their mechanisms of generation,risks for health and ways of preventing its presence in meat products are briefly described in this review.

Role of CYP2E1 gene polymorphisms association with hepatitis risk in Northeast India

AIM:To investigate hepatitis virus, genetic and environmental factors, and their interactions in predisposing patients to liver diseases in Northeast India. METHODS:A total of 104 jaundice patients and 124 community controls were included. Serological analysis was performed by routine enzyme-linked immunosorbent assay, and nucleic acid testing for hepatitis viruses was done by polymerase chain reaction (PCR), followed by PCR direct sequencing for viral genotyping. Cytochrome P450 2E1 (CYP2E1) polymorphism was studied by PCR-restriction fragment length polymorphism. Nitrite and volatile nitrosamines in indigenous foods consumed routinely by the Northeast Indian ethnic population were estimated by Griess’s reagent and GC-MS, respectively.RESULTS: Hepatitis A virus (HAV) infection was predominantly prevalent (36.5%) in our cohort, followed by hepatitis B virus (HBV), hepatitis E virus (HEV) andhepatitis C virus. HBV genotype D and HEV genotype 1 were the most dominant. CYP2E1 c1/c2 genotype frequency was comparatively higher in alcoholic (P<0.0001,OR =30.5) and cryptogenic (P=0.014, OR=8.714) patients, and was associated with significantly higher hepatitis risk (P=0.0.007,OR=6.489). Mutant C allele of Cyp2E1 DraⅠ frequency was comparatively higher in HAV (P=0.006), alcoholic (P =0.003) and cryptogenic (P=0.014) cases, and was associated with overall hepatitis risk (P=0.026, OR=5.083). Indigenous foods, Gundruk, Kharoli, betel leaf and nuts were found to have the highest nitrite content. CONCLUSION: Apart from viral factors, CYP2E1 polymorphism might be associated with increased risk of liver diseases in Northeast India. Indigenous foods that contain nitrite and nitrosamine might be an associated risk factor.

Comparison of Mechanisms of N-Nitrosation and N-Nitration of Ammonia and Dimethylamine by Reactive Nitrogen Oxygen Species:A Theoretical Study

Reactive nitrogen oxygen species（RNOS） implicate damage in biological systems,especially leading to inflammation,neurodegenerative and cardiovascular diseases,and cancer by altering the functions of biomolecules through the N-nitrosation and N-nitration reactions.The mechanisms of N-nitrosation and N-nitration reactions of ammonia and dimethylamine by RNOS,i.e.,N2O3,N2O4,N2O5 and ONOOH,were investigated at the CBS-QB3 level of theory.The computational results indicate that the N-nitrosation reaction prefers a concerted mechanism,in which a H-abstraction and ON-addition occur simultaneously,whereas a stepwise mechanism（also called a free radical mechanism） is more favorable for most nitrating agents in the N-nitration reaction,where NO2 first abstracts a hydrogen atom from the nitrogen of amines and then the induced intermediate reacts with NO2 once more to form the nitration products.However,the concerted pathway is still a feasible process for some nitrating agents such as N2O5.In addition,the relationship between the structures of different RNOS and their nitrosating or nitrating abilities was also investigated.

Inhibition of EGFR Suppresses Ethyl Alcohol and Tobacco Cell Effects on Growth of Human Oral Keratinocytes and Human Papillomavirus 16 Entry as a Function of Furin

Background: Reported are increased risks for malignant transformation in human oral keratinocytes (HOK) from ethyl alcohol (ETOH), tobacco products or human papilloma virus oncogenic subtype 16 (HPV 16) infections. We examined various HOK cell responses to these factors to show inhibitors of epidermal growth factor receptor (EGFR) also inhibits furin;proprotein convertase (FC) and HPV 16 entry in HOK. Methods: Immortalized HOK by HPV 16 (HPV 16B) or human telomerase (hTERT);primary foreskin keratinocytes (NHFK), primary HOK, buccal keratinocytes (NHBK) and oral SCC-25 were treated with dibenz[a,l]pyrene (DBP), anthraquinone;nitrosamine (NNAL) or ethyl alcohol (ETOH) and acetaldehyde (AA). ETOH was tested for synthesis of malondialdehyde (MDA) and alcohol dehydrogenase expression (ADH). ETOH, and PAH were evaluated by Western immunoblot for oncogene changes, and phosphorylated EGFR expression. Inhibition of EGFR by WZ4002 and Erlotinib and/or carcinogens effect on HPV 16 entry were studied. A green fluorescent pseudovirus (PsV);chloromethylketone (CMK) an inhibitor of furin activity and Western immunoblot of furin cell distribution further characterized HPV 16 entry. Results: ETOH (10 μM) increased expression of phosphorylated EGFR and HPV 16 entry through furin activity, and membrane, nuclear and cytoskeletal accumulations. CMK suppressed HPV 16 entry and blockage of ADH while aldehyde dehydrogenase (ALDH) enhanced HPV 16 entry. Similarly PAH, DBP (4-8 nM), anthraquinone (98 nM) and NNAL (6.9 μM) enhanced HPV 16 entry through furin activity and membrane, nuclear and cytoskeletal accumulations. Furthermore, WZ4002 and Erlotinib suppressed expressions of phosphorylated EGFR, FC activity, and HPV 16 entry. ETOH and DBP treatments also enhanced expressions of protease activated receptor-1 (PAR-1), and p21waf1 while depressed p16 and p27KIP1 expressions in HOK/HPV 16B cells. Conclusion: EGFR inhibitors are candidates for suppression of alcohol and tobacco effects on EGFR phosphorylated expression;keratinocyte growth, and HPV 16 entry and prevention treatment for HPV related diseases.

A Participant-assigned Interventional Research of Precesarean Internal Iliac Artery Balloon Catheterization for Managing Intraoperative Hemorrhage of Placenta Previa and Placenta Accreta Spectrum Disorders After Cesarean Section

Placenta accreta spectrum disorder (PASD) and placenta previa (PP) are two of the mosthideous obstetric complications which are usually associated with a history of cesarean section(CS). Moreover, women with PASD, PP and/or a cesarean scarred uterus are more likely to haveadverse pregnancy outcomes, including blood transfusion, hysterectomy, pelvic organs damage,postpartum hemorhage, disseminated intravascular coagulation, multi-organ dysfunction syndromeand even maternal or fetal death. This study aimed to investigate the efficacy of precesareaninternal iliac artery balloon catheterization (BC) for managing severe hemorhage caused by PASDand PP with a history of CS. This participant-assigned interventional study was conducted inTongji Hospital. We recruited 128 women with suspected PASD, PP and a history of CS. Womenin the BC group accepted precesarean BC of bilateral internal iliac arteries before the scheduledcesarean delivery. Women in the control group underwent a conventional cesarean delivery.Intraoperative hemorrhage, transfusion volume, radiation dose, exposure time, complicationsand neonatal outcomes were discussed. There were significant differences in calculated bloodloss (CBL) between BC group and control group (1015.0±144.9 vs. 1467.0±171.0 mL, P=0.04).Precesarean BC could reduce intraoperative red blood cell (RBC) transfusion as compared withcontrol group (799.5±136.1 vs. 1286.0±161.6 mL, P=0.02) and lessen the rate of using bloodproducts (57.1% vs. 76.4%, P=0.02). The incidence of hysterectomy was also lower in BC groupthan in control group. Postpartum outcomes showed no significant differences between the twogroups, except that postoperation hospitalization was longer in BC group than in control group(6.7±0.4 vs. 5.8±0.2 days, P=0.03). Precesarean BC of internal iliac artery is an effective methodfor managing severe hemorrhage caused by PASD and PP with a cesarean scarred uterus, as it couldreduce intraoperative blood loss, lessen intraoperative RBC transfusions and potentially decreasehysterectomies.

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%.

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.

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.

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).