N-nitrosodimethylamine Research Articles

Development and Validation of a Rapid and Sensitive LC-APCI-MS/MS Method for the determination of three N-nitrosamine impurities in Varenicline tartrate drug substance and drug products

Nitrosamines impurities may be present in low levels in a variety of products that people are exposed to every day. After thorough investigations, regulatory agencies have outlined that the formation of nitrosamines is possible in the presence of secondary, tertiary, or quaternary amines and nitrite salts under acidic reaction conditions. For safety purpose, all API manufacturers must be developed methods to monitor the low levels of each nitrosamine impurity before the release the individual batches. In this article, we have developed novel, rapid and sensitive liquid chromatography Atmospiric pressure chemical ionization Mass spectroscopy (LC-APCI-MS/MS) method for the determination of N-Nitroso Dimethylamine (NDMA), N-Nitroso Diethylamine (NDEA) and N-Nitroso varenicline impurities in the Varenicline Tartrate drug substance and Drug products. Chromatographic separation is achieved using Inert sustain AQ C18 150 × 4.6mm, 3.0μ column with 0.1% formic acid in water as mobile phase A and 0.1% formic acid in methanol as mobile phase B at the 0.6 mL/min flow rate by using gradient mode of elution upto the 25 minutes total run time. The three impurities are successfully ionized and quantified in positive mode of atmospheric pressure chemical ionization (APCI) using multiple reaction monitoring (MRM). This method was validated according to ICH Q2 (R1) guidelines. The method provided excellent S/N ratios with a high linearity range of 0.66-19.88 ppm with respect to Varenicline Tartrate sample concentrations with a regression coefficient >0.995. The recovery for the method was established with a protocol of three-step sample preparation and was satisfactory within 85-115%. The determined LOQ (0.66 ppm) and LOD (0.22 ppm) values are very low with respect to sample concentration which shows the sensitivity Performance of the method. The method can be routinely applied for the detection of N-nitrosamine impurities (NDMA, NDEA and N-Nitroso varenicline) in Varenicline Tartrate drug substance and Drug products.

Development and validation of high-performance liquid chromatography-Orbitrap mass spectrometric method for quantification of NDMA in ranitidine drug products and evaluation of antioxidants as inhibitors of classical nitrosation reaction.

N-Nitroso dimethylamine (NDMA) is a mutagenic impurity detected in several ranitidine products. The amino functional group of ranitidine is a risk factor for classical nitrosation-induced NDMA formation in ranitidine drug products during storage conditions. The United States Food and Drug Administration (US FDA) recommended the use of antioxidants to control NDMA in drug products. Considering the need for sensitive analytics, a liquid chromatography/high-resolution mass spectrometry (LC-HRMS) method was developed and validated to detect NDMA in this pilot study to demonstrate the antioxidants as inhibitors of nitrosation reactions. The method, utilizing an EC-C18 column and tuned to atmospheric pressure chemical ionization/selected ion monitoring (APCI/SIM) mode, separated NDMA (m/z: 75.0553; tR: 3.71 min) and ranitidine (m/z: 315.1485; tR: 8.61 min). APCI mode exhibited four times higher sensitivity to NDMA than electrospray ionization (ESI) mode. Classical nitrosation of the dimethyl amino group of ranitidine was studied with sodium nitrite in solid pellets. Antioxidants (alpha-tocopherol, ascorbic acid, and trolox) were evaluated as NDMA attenuators in ranitidine pellets under vulnerable storage conditions. The developed method quantified NDMA levels in samples, extracted with methanol through vortex shaking for 45 min. The method achieved a limit of detection (LOD) and limit of quantitation (LOQ) of 0.01 and 0.05 ng/mL, respectively, with linearity within 1-5000 ng/mL (R1: 0.9995). It demonstrated good intra-day and inter-day precision (% RSD [relative standard deviation]: <2) and accuracy (96.83%-101.72%). Nitrosation of ranitidine induced by nitrite was significant (p < 0.001; R2 = 0.9579) at various sodium nitrite levels. All antioxidants efficiently attenuated NDMA formation during ranitidine nitrosation. Ascorbic acid exhibited the highest NDMA attenuation (96.98%), followed by trolox (90.58%). This study recommends 1% ascorbic acid and trolox as potent NDMA attenuators in ranitidine drug products. This study compared the effectiveness of antioxidants as NDMA attenuators in ranitidine under storage conditions susceptible to NDMA generation. The study concluded that ascorbic acid and trolox are potent inhibitors of NDMA formation and nitrosation attenuators in ranitidine drug products.

Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing

Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing.

N-nitrosamines in electroplating and printing/dyeing industrial wastewater treatment plants: Removal efficiency, environmental emission, and the influence on drinking water

The discharge of industrial wastewater containing high concentrations of N-nitrosamines to the aquatic environment can impair downstream source waters and pose potential risks to human health. However, the transport and fate of N-nitrosamines in typical industrial wastewater treatment plants (IWWTPs) and the influence of these effluents on source water and drinking water are still unclear. This study investigated nine N-nitrosamines in four full-scale electroplating (E-) and printing/dyeing (PD-) IWWTPs, two drinking water treatment plants (DWTPs) in the lower reaches of these IWWTPs, and the corresponding tap water in South China. The total concentrations of N-nitrosamines (∑NAs) were 382–10,600, 480–1920, 494–789, and 27.9–427 ng/L in influents, effluents, source water, and tap water, respectively. The compositions of N-nitrosamine species in different influents varied a lot, while N-nitrosodi-n-butylamine (NDBA) and N-nitrosodimethylamine (NDMA) dominated in most of the effluents, source water, and tap water. More than 70 % N-nitrosamines were removed by wastewater treatment processes used in E-IWWTPs such as ferric-carbon micro-electrolysis (Fe/C-ME), while only about 50 % of N-nitrosamines were removed in PD-IWWTPs due to the use of chlorine reagent or other inefficient conventional processes such as flocculation by cationic amine-based polymers or bio-contact oxidation. Therefore, the mass fluxes of N-nitrosamines discharged from these industrial wastewaters to the environment in the selected two industrial towns were up to 14,700 mg/day. The results based on correlation and principal component analysis significantly demonstrated correlations between E-and PD-effluents and source water and tap water, suggesting that these effluents can serve as sources of N-nitrosamines to local drinking water systems. This study suggests that N-nitrosamines are prevalent in typical IWWTPs, which may infect drinking water systems. The findings of this study provide a basis data for the scientific evaluation of environmental processes of N-nitrosamines.

A Novel Validated GC-MS/MS Method for the Estimation of N-Nitroso Dimethyl Amine and N-Nitroso Diethyl Amine in Zidovudine.

A novel method has been developed for the estimation of N-Nitroso dimethyl amine impurities (NDMA) and N-Nitroso diethyl amine (NDEA) in Zidovudine by using Gas chromatograph Triple Quadrupole Mass with Liquid autosampler (GC-MS/MS) and the method is validated as per International Conference on Harmonization recommendations. Sample analysis was executed for Zidovudine by developed method. Both NDMA and NDEA were detected in below quantitation limit for the Zidovudine batches. Efficient chromatographic separation was achieved on a DB-WAX 30m length × 0.25mm internal diameter, 0.5-μm film thickness, Triple quad-8040 GC-MS/MS. Quantification was carried out at Triple quad electron ionization source was at a column flow of 1.5mL/min at a column oven temperature 50°C. The precision was in the range of 0.9-2.5% for NDMA and 0.8-2.3% for NDEA, and regression analysis shows as r value (correlation coefficient) of is >0.99. This method is capable to detect the NDMA and NDEA impurities in Zidovudine at a level of 0.006ppm for limit of detection and 0.018ppm for limit of quantitation with respect to test concentration of 66.66mg/mL.

Isolation and purification of polysaccharides from Bupleurum marginatum Wall.ex DC and their anti-liver fibrosis activities.

Bupleurum marginatum Wall.ex DC [Apiaceae] (BM)is widely grown in southwestern China, and the whole plant is used as Traditional Chinese Medicine (TCM). Polysaccharides are main natural products in lots of TCM and have been studied for their effects of reducing oxidative stress, anti-inflammation and immune regulation. Herein, we investigated the extraction techniques of Bupleurum marginatum Wall.ex DC polysaccharides (BMP), the identification of their key components, and their ability to inhibit liver fibrosis in both cellular and animal models. Component identification indicated that monosaccharides in BMP mainly consisted of glucose, galactose, mannose, rhamnose, arabinose, and xylose. In vivo analysis revealed that BMP provided significant protective effects on N-Nitroso dimethylamine (NDMA)-induced liver fibrosis rats through reducing hepatomegaly, reducing tissue inflammation, and reducing collagen deposition. BMP also improved the hepatobiliary system and liver metabolism in accord to reduce the serum levels of ALT, AST, ALP, r-GT, and TBIL. In addition, BMP could reduce the level of inflammation and fibrosis through inhibition of IL-1β and TGF-β1. Cellular studies showed that the BMP could provide therapeutic effects on lipopolysaccharide (LPS)-induced cellular fibrosis model, and could reduce the level of inflammation and fibrosis by decreasing the level of TGF-β1, IL-1β, and TNF-α. Our study demonstrated that BMP may provide a new therapy strategy of liver injury and liver fibrosis.

Quantitative Analysis of NDMA in Drug Products: A Proposed High-Throughput Approach Using Headspace–SIFT-MS

Since the initial 2018 recall of angiotensin receptor blockers due to unacceptable levels of mutagenic N-nitrosodimethylamine (NDMA) impurity, numerous drug products delivering diverse active pharmaceutical ingredients (APIs) have been recalled. Regulators and the industry are working together to understand and address this widescale problem. Conventional analysis of NDMA utilizes liquid or gas chromatography-based procedures that can involve complicated sample preparation and slow sample analysis. Selected ion flow tube mass spectrometry (SIFT-MS) analyses NDMA directly in the gas phase using soft chemical ionization, with an LOQ of 2 ng g−1. Through the novel application of the multiple headspace extraction (MHE) technique, NDMA was quantified directly and rapidly from the drug product without dissolution, at levels well below the regulatory acceptable intake of 96 ng day−1. A comparative analysis of recalled metformin using MHE-SIFT-MS and a conventional liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) method showed good agreement. Use of the novel MHE-SIFT-MS approach may enable a wider screening of drug products to be conducted, since it provides around a three-fold increase in daily sample throughput.

Oxygen vacancy based WO3/SnO2-x promote electrochemical H2O2 accumulation by two-electron water oxidation reaction and toxic uniform dimethylhydrazine degradation

The key to constructing an anodic electro-Fenton system hinges on two pivotal criteria: enhancing the catalyst activity and selectivity in water oxidation reaction (WOR), while simultaneously inhibiting the decomposition of hydrogen peroxide (H2O2) which is on-site electrosynthesized at the anode. To address the issues, we synthesized novel WO3/SnO2-x electrocatalysts, enriched with oxygen vacancies, capitalize on the combined activity and selectivity advantages of both WO3 and SnO2-x for the two-electron pathway electrocatalytic production of H2O2. Moreover, the introduction of oxygen vacancies plays a critical role in impeding the decomposition of H2O2. This innovative design ensures that the Faraday efficiency and yield of H2O2 are maintained at over 80 %, with a noteworthy production rate of 0.2 mmol h−1 cm−2. We constructed a novel electro-Fenton system that operates using only H2O as its feedstock and applied it to treat highly toxic uniform dimethylhydrazine (UDMH) from rocket launch effluent. Our experiments revealed a substantial total organic carbon (TOC) removal, achieving approximately 90 % after 120 mins of treatment. Additionally, the toxicity of N-nitrosodimethylamine (NDMA), a byproduct of great concern, was shown to be effectively mitigated, as evidenced by acute toxicity evaluations using zebrafish embryos. The degradation mechanism of UDMH is predominantly characterized by the advanced oxidative action of H2O2 and hydroxyl radicals, as well as by complex electron transfer processes that warrant further investigation.

Nitrosamine Impurities in Pharmaceuticals: An Empirical Review of their Detection, Mechanisms, and Regulatory Approaches.

Since their discovery in valsartan-containing drugs, nitrosamine impurities have emerged as a significant safety problem in pharmaceutical products, prompting extensive recalls and suspensions. Valsartan, candesartan, irbesartan, olmesartan, and other sartans have been discovered to have additional nitrosamine impurities, such as N-nitroso-N-methyl-4-aminobutyric acid (NMBA), N-nitroso-Di-isopropyl amine (NDIPA), N-nitroso-Ethyl-Isopropyl amine (NEIPA), and N-nitroso-Diethyl amine (NDEA). Concerns about drug safety have grown in response to reports of nitrosamine contamination in pharmaceuticals, such as pioglitazone, rifampin, rifapentine, and varenicline. This review investigates the occurrence and impact of nitrosamine impurities in sartans and pharmaceutical goods, as well as their underlying causes. The discussion emphasizes the significance of comprehensive risk assessment and mitigation approaches at various phases of medication development and manufacturing. The link between amines and nitrosamine impurities is also investigated, with an emphasis on pH levels and the behaviour of primary, secondary, tertiary, and quaternary amines. Regulations defining standards for nitrosamine assessment and management, such as ICH Q3A-Q3E and ICH M7, are critical in resolving impurity issues. Furthermore, the Global Substance Registration System (GSRS) is underlined as being critical for information sharing and product safety in the pharmaceutical industry. The review specifically focuses on the relationship between ranitidine and N-nitroso dimethyl amine (NDMA) in the context of the implications of nitrosamine contamination on patient safety and medicine supply. The importance of regulatory authorities in discovering and correcting nitrosamine impurities is highlighted in order to improve patient safety, product quality, and life expectancy. Furthermore, the significance of ongoing study and attention to nitrosamine-related repercussions for increasing pharmaceutical safety and overall public health is emphasized.

Minimizing N-Nitrosodimethylamine Formation During Disinfection of Blended Seawater and Wastewater Effluent.

Augmenting seawater with wastewater has the potential to reduce the energy demand and environmental impacts associated with seawater desalination. Alternatively, as wastewater reuse becomes more widespread, augmenting wastewater with seawater can increase the available water supply. However, the chemistry of disinfecting a blended stream has not been explored. Toxic byproducts, including N-nitrosodimethylamine (NDMA), are expected to form during disinfection, and the extent of formation will likely be a function of which stream is chlorinated and whether disinfection happens before or after blending. In this work, three blending-disinfection scenarios were modeled and experimentally evaluated in bench-scale systems treating synthetic and authentic waters. Modeling results suggested that chlorinating preblended wastewater and seawater would produce the most NDMA because it yielded the highest concentrations of bromochloramine, which was previously found to promote NDMA formation. However, chlorinating wastewater prior to blending with seawater, which modeling indicated would form the most dichloramine, produced the most NDMA in experiments. When seawater was disinfected prior to blending with wastewater, bromide likely converted most chlorine to free bromine. Bromamines formed after blending, however, did not lead to an elevated level of NDMA formation. Therefore, to minimize NDMA formation when disinfecting blended wastewater-seawater, seawater should be disinfected prior to introducing wastewater.

Animal Feedlots and Domestic Wastewater Discharges are Likely Sources of N-Nitrosodimethylamine (NDMA) Precursors in Midwestern Watersheds.

N-nitrosodimethylamine (NDMA) precursor concentrations along four major rivers in Minnesota, USA were quantified and correlated with watershed land cover types, anthropogenic activity, and organic matter characteristics. River water samples (36 in total) were chloraminated under uniform formation conditions (UFC) before and after lime-softening treatment, and the resulting NDMA concentrations were quantified (NDMAUFC). Regarding land cover, NDMAUFC in raw river water exhibited weak positive correlations with urban land (ρ = 0.33, p = 0.05) and cropland coverage (ρ = 0.35, p = 0.04). For anthropogenic activity, NDMAUFC in raw river water positively correlated with the number of feedlots (ρ = 0.57), total weight of animals (ρ = 0.68), and total number of domestic wastewater treatment plants (WWTPs; ρ = 0.63) with p < 0.01. NDMAUFC positively correlated with region IV fluorescence intensity from fluorescence excitation-emission spectra (ρ = 0.70, p < 0.01). Lime softening of river water typically increased NDMAUFC and preferentially removed organic matter that fluoresces in region V, suggesting that the organic matter in this region decreases NDMAUFC by competing for available chloramines. Overall, animal feedlots, along with domestic WWTPs, are predominant sources of NDMA precursors in the studied watersheds, while croplands and urban runoff are of lesser importance.

Effects of transition-metal ions on the reduction of N-nitrosodimethylamine in water by zinc

The influencing factors, products and mechanism of N-nitrosodimethylamine (NDMA) reduction by zinc (Zn) in the presence of transition-metal ions (Mn2+, Co2+ and Ni2+), were investigated. The NDMA removal by Zn was significantly improved by them, and the promoting effect of Co2+ and Ni2+ were better than Mn2+. With the decrease of pH values and dissolved oxygen concentrations, the removals of NDMA were enhanced in all reaction systems. NDMA was mainly reduced to unsymmetrical dimethylhydrazine (UDMH) in Zn and Zn/Mn2+ systems. It was mainly reduced to dimethylamine (DMA) and ammonium (NH4+) in Zn/Co2+ and Zn/Ni2+ systems. There were undetected products in all reaction systems. Catalytic hydrogenation was the main mechanism, and active hydrogen atom was the main active species. The presence of Mn2+, Co2+ and Ni2+ promoted the corrosion of Zn and changed the surface morphology of Zn. The formation of type I film on Zn surface was promoted in the presence of Co2+ and Ni2+, which was conducive to the continuous corrosion of Zn and hydrogen generation. While type II film which would inhibit the corrosion of Zn formed on Zn surface in the presence of Mn2+. MnO2, Co3O4 and Ni(OH)2 were formed in Zn/Mn2+, Zn/Co2+ and Zn/Ni2+ systems, respectively. They would facilitate more generation of active hydrogen atoms.

Patient In-Use Stability Testing of FDA-Approved Metformin Combination Products for N-Nitrosamine Impurity.

Between February 2020 and January 2022, the Food and Drug Administration (FDA) recalled 281 metformin extended-release products due to the presence of N-nitrosodimethylamine (NDMA) above the acceptable daily intake (ADI, 96ng/day). Our previous studies indicated presence of NDMA levels above ADI in both metformin immediate and extended-release products. When metformin products have NDMA impurities, it is indispensable to check for the same impurities in metformin combination products. Therefore, the objective of the present study was to evaluate in-use stability of commercial metformin combination products for NDMA. For this purpose, metformin products in combination with glyburide (GB1-GB12), glipizide (GP1-GP8), pioglitazone (P1-P3), alogliptin (A1, A2), and linagliptin (L1, L2) were repacked in pharmacy vials, stored at 30°C/75% RH for 3months, and monitored for NDMA impurity. The NDMA level varied from 0 to 156.8 ± 32.8ng/tablet initially and increased to 25.4 ± 5.1 to 455.0 ± 28.4ng/tablet after 3months of exposure to in-use condition. Initially, 18 products have NDMA level below ADI limit before exposure which decreased to 7 products (GB5, GP3, GP5, A1, A2, L1, and L2) meeting specification. In conclusion, in-use stability study provides quality and safety risk assessment of drug products where nitroso impurities are detected in the probable condition of use.

Closing Dichloramine Decomposition Nitrogen and Oxygen Mass Balances: Relative Importance of End-Products from the Reactive Nitrogen Species Pathway.

In drinking water chloramination, monochloramine autodecomposition occurs in the presence of excess free ammonia through dichloramine, the decay of which was implicated in N-nitrosodimethylamine (NDMA) formation by (i) dichloramine hydrolysis to nitroxyl which reacts with itself to nitrous oxide (N2O), (ii) nitroxyl reaction with dissolved oxygen (DO) to peroxynitrite or mono/dichloramine to nitrogen gas (N2), and (iii) peroxynitrite reaction with total dimethylamine (TOTDMA) to NDMA or decomposition to nitrite/nitrate. Here, the yields of nitrogen and oxygen-containing end-products were quantified at pH 9 from NHCl2 decomposition at 200, 400, or 800 μeq Cl2·L-1 with and without 10 μM-N TOTDMA under ambient DO (∼500 μM-O) and, to limit peroxynitrite formation, low DO (≤40 μM-O). Without TOTDMA, the sum of free ammonia, monochloramine, dichloramine, N2, N2O, nitrite, and nitrate indicated nitrogen recoveries ±95% confidence intervals were not significantly different under ambient (90 ± 6%) and low (93 ± 7%) DO. With TOTDMA, nitrogen recoveries were less under ambient (82 ± 5%) than low (97 ± 7%) DO. Oxygen recoveries under ambient DO were 88-97%, and the so-called unidentified product of dichloramine decomposition formed at about three-fold greater concentration under ambient compared to low DO, like NDMA, consistent with a DO limitation. Unidentified product formation stemmed from peroxynitrite decomposition products reacting with mono/dichloramine. For a 2:2:1 nitrogen/oxygen/chlorine atom ratio and its estimated molar absorptivity, unidentified product inclusion with uncertainty may close oxygen recoveries and increase nitrogen recoveries to 98% (ambient DO) and 100% (low DO).

N-Nitrosodimethylamine investigations in Muta™Mouse define point-of-departure values and demonstrate less-than-additive somatic mutant frequency accumulations.

The N-nitrosamine, N-nitrosodimethylamine (NDMA), is an environmental mutagen and rodent carcinogen. Small levels of NDMA have been identified as an impurity in some commonly used drugs, resulting in several product recalls. In this study, NDMA was evaluated in an OECD TG-488 compliant Muta™Mouse gene mutation assay (28-day oral dosing across seven daily doses of 0.02-4 mg/kg/day) using an integrated design that assessed mutation at the transgenic lacZ locus in various tissues and at the endogenous Pig-a gene-locus, along with micronucleus frequencies in peripheral blood. Liver pathology was determined together with NDMA exposure in blood and liver. The additivity of mutation induction was assessed by including two acute single-dose treatment groups (i.e. 5 and 10 mg/kg dose on Day 1), which represented the same total dose as two of the repeat dose treatment groups. NDMA did not induce statistically significant increases in mean lacZ mutant frequency (MF) in bone marrow, spleen, bladder, or stomach, nor in peripheral blood (Pig-a mutation or micronucleus induction) when tested up to 4 mg/kg/day. There were dose-dependent increases in mean lacZ MF in the liver, lung, and kidney following 28-day repeat dosing or in the liver and kidney after a single dose (10 mg/kg). No observed genotoxic effect levels (NOGEL) were determined for the positive repeat dose-response relationships. Mutagenicity did not exhibit simple additivity in the liver since there was a reduction in MF following NDMA repeat dosing compared with acute dosing for the same total dose. Benchmark dose modelling was used to estimate point of departure doses for NDMA mutagenicity in Muta™Mouse and rank order target organ tissue sensitivity (liver > kidney or lung). The BMD50 value for liver was 0.32 mg/kg/day following repeat dosing (confidence interval 0.21-0.46 mg/kg/day). In addition, liver toxicity was observed at doses of ≥ 1.1 mg/kg/day NDMA and correlated with systemic and target organ exposure. The integration of these results and their implications for risk assessment are discussed.

Rapid analysis of N-nitrosamines in urine using ultra high-pressure liquid chromatography-mass spectrometry.

N-Nitrosamines, carcinogenic compounds present in dietary and environmental sources and formed endogenously, are believed to be linked with the presence of nitrate and nitrite, both within dietary sources and after intake. To fully evaluate this potential threat to human health, an accurate analytical method to measure N-nitrosamines in biological matrices is necessary. We report a simple, fast, selective mass spectrometry method to detect N-nitrosamines in human urine. Analysis of seven N-nitrosamines, N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine (NDEA), N-nitrosopiperdine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosodi-N-propylamine (NDPA) and N-nitrosodi-N-butylamine (NDBA) in urine was quantitated using Ultra High-Pressure Liquid Chromatography-tandem Mass spectrometry (UHPLC-MS/MS). A Sorbent supported Liquid Extraction (SLE) method was employed to extract N-nitrosamines from 24 hour collected human urine samples. The percent recovery varied between 74.3 to 110 and the limit of detection and limit of quantification ranged from 0.1 to 0.85 ng mL-1 and 0.22 to 2.06 ng mL-1 respectively. Precision for inter-day and intra-day assay yielded a % coefficient of variation between 4-10% for all measured compounds in urine. Linear regression analysis of calibration curves for N-nitrosamines measured in urine in the concentration range 0.4-12.8 ng mL-1 gave correlation coefficients, R2 0.9874-0.9962. Urinary excretion of N-nitrosamines measured in ten healthy subjects resulted in detection of most of the N-nitrosamines including NDMA, NDEA, NPYR, NDPA and NDBA by this method.

N-nitrosodimethylamine exposure to zebrafish embryos/larvae causes cardiac and spinal developmental toxicity

N-nitrosodimethylamine (NDMA), one of the new nitrogen-containing disinfection by-products, is potentially cytotoxic, genotoxic, and carcinogenic. Its potential toxicological effects have attracted a wide range of attention, but the mechanism is still not sufficiently understood. To better understand the toxicological mechanisms of NDMA, zebrafish embryos were exposed to NDMA from 3 h post-fertilization (hpf) to 120hpf. Mortality and malformation were significantly increased, and hatching rate, heart rate, and swimming behavior were decreased in the exposure groups. The result indicated that NDMA exposure causes cardiac and spinal developmental toxicity. mRNA levels of genes involved in the apoptotic pathway, including p53, bax, and bcl-2 were significantly affected by NDMA exposure. Moreover, the genes associated with spinal and cardiac development (myh6, myh7, nkx2.5, eph, bmp2b, bmp4, bmp9, run2a, and run2b) were significantly downregulated after treatment with NDMA. Wnt and TGF-β signaling pathways, crucial for the development of diverse tissues and organs in the embryo and the establishment of the larval spine, were also significantly disturbed by NDMA treatment. In summary, the disinfection by-product, NDMA, exhibits spinal and cardiac developmental toxicity in zebrafish embryos, providing helpful information for comprehensive analyses and a better understanding the mechanism of its toxicity.

Ames test study designs for nitrosamine mutagenicity testing: qualitative and quantitative analysis of key assay parameters.

The robust control of genotoxic N-nitrosamine (NA) impurities is an important safety consideration for the pharmaceutical industry, especially considering recent drug product withdrawals. NAs belong to the 'cohort of concern' list of genotoxic impurities (ICH M7) because of the mutagenic and carcinogenic potency of this chemical class. In addition, regulatory concerns exist regarding the capacity of the Ames test to predict the carcinogenic potential of NAs because of historically discordant results. The reasons postulated to explain these discordant data generally point to aspects of Ames test study design. These include vehicle solvent choice, liver S9 species, bacterial strain, compound concentration, and use of pre-incubation versus plate incorporation methods. Many of these concerns have their roots in historical data generated prior to the harmonization of Ames test guidelines. Therefore, we investigated various Ames test assay parameters and used qualitative analysis and quantitative benchmark dose modelling to identify which combinations provided the most sensitive conditions in terms of mutagenic potency. Two alkyl-nitrosamines, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) were studied. NDMA and NDEA mutagenicity was readily detected in the Ames test and key assay parameters were identified that contributed to assay sensitivity rankings. The pre-incubation method (30-min incubation), appropriate vehicle (water or methanol), and hamster-induced liver S9, alongside Salmonella typhimurium strains TA100 and TA1535 and Escherichia coli strain WP2uvrA(pKM101) provide the most sensitive combination of assay parameters in terms of NDMA and NDEA mutagenic potency in the Ames test. Using these parameters and further quantitative benchmark dose modelling, we show that N-nitrosomethylethylamine (NMEA) is positive in Ames test and therefore should no longer be considered a historically discordant NA. The results presented herein define a sensitive Ames test design that can be deployed for the assessment of NAs to support robust impurity qualifications.

Method Validation and Monitoring of N-nitrosodimethylamine in Metformin Hydrochloride Products in China by GC-MS/MS

Background: N-nitrosodimethylamine (NDMA) are a sort of genotoxic impurities (GTIs) having strong carcinogenic effects and obvious hepatotoxicity. To monitor the NDMA content of metformin hydrochloride sustained-release tablets and enteric capsules in China from 2018 to 2022, a GC-MS/MS method was established and validated. Materials and Methods: The chromatographic column was Agilent VF-WAXms capillary column (30 m×0.25 mm, 0.25 μm). The GC-MS/MS method was equipped with multiple reaction monitoring (MRM) modes. To assess the quantity of NDMA, the molecular ion at mass-tocharge (m/z) of 74-44 was monitored under the 6 V collision energy, and to assess the quality of NDMA monitoring, the molecular ions at m/z 74-42 were determined. A total of 143 batches of metformin hydrochloride-finished products from 35 enterprises were determined by this method Results: The linear range of the method was 0.25 ~ 50.00 ng/mL, r = 0.9998, S/N &gt; 10, and the limit of detection and quantitation were 0.06 ng/mL and 0.21 ng/mL, respectively. The average recovery was 98.62%, and the RSD was 4.31%. All batches of enteric capsules met the requirements; 38.21% of the 123 batches sustained-release tablets still exceeded the acceptable daily intake. Conclusion: The presented method is sensitive, accurate, precise, and available for both enteric capsules and sustained-release tablets of metformin hydrochloride, which can provide a reference for their quality control. The over-limit phenomenon of NDMA in metformin hydrochloride products poses new challenges and requirements for both the State Drug Administration and enterprises.

Preparation of MgIn2S4/g-C3N4 for enhanced ranitidine photocatalytic degradation activity via Z-scheme electron transfer

The evidence so far indicates that ranitidine (RAN) contains N-nitrosodimethylamine (NDMA), a strong carcinogen, which is extremely harmful to human health under long-term exposure. Hence, a well-design Z-scheme heterostructure composed of 3D MgIn2S4 micro-flower and 2D g-C3N4 nanosheets was fabricated and used to remove RAN. The optimal photocatalyst (MG-30) exhibited the fastest RAN degradation rate of 0.24685 min−1 within 30 min under visible light, which was 78.1 and 19.8 times higher than those of bare MgIn2S4 and g-C3N4, respectively. Impressively, MG-30 was exceedingly resistant to environmental variations including organic matter, inorganic salts and aqueous substrates. In the meantime, MG-30 boasted the merit such as wide pH range for RAN removal. The excellent results originated from the construction of Z-scheme heterojunctions, which improved the carrier separation efficiency and retained the strong redox ability of MG-30, thereby contributing to the creation of rich active species. Meanwhile, the 3D/2D structure of MgIn2S4/g-C3N4 overrode the limited reflection of visible light. The active species analyses revealed that superoxide radical (·O2−) and hydroxyl radical (·OH) mainly participated in RAN degradation. Furthermore, the possible degradation pathways of RAN and toxicity analysis of intermediates produced were presented. This work emphasizes practical application potential of Z-scheme heterostructure photocatalysts for emerging contaminants degradation in wastewater.