OBJECTIVE: A recent US Food and Drug Administration report highlighted concerns over nitrosamine (7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4] triazolo-[4,3-a]pyrazine [NTTP]) impurities in sitagliptin, prompting investigations into its safety profile. The present study aimed to determine if the use of NTTP-contaminated sitagliptin, in comparison with other dipeptidyl peptidase-4 (DPP-4) inhibitors, is associated with an increased cancer risk.
METHODS: This retrospective cohort study secondarily used the National Database of Health Insurance Claims and Specific Health Checkups of Japan, encompassing data on >120 million individuals. The study involved patients who initiated DPP-4 inhibitor therapy (sitagliptin or other DPP-4 inhibitors) and continued its exclusive use for 3 years. Sitagliptin users were compared with other DPP-4 inhibitor users for assessing the occurrence of cancers, as defined by diagnosis codes. Further analyses focused on specific types of cancer, using either diagnosis codes or a combination of diagnosis and procedure codes. We also carried out various sensitivity analyses, including those with different exposure periods.
RESULTS: Sitagliptin users (149,120 patients, 388,356 person-years) experienced 9,643 cancer incidences (2,483.0/100,000 person-years) versus 12,621 incidences (2,504.4/100,000 person-years) among other DPP-4 inhibitor users (199,860 patients, 503,952 person-years), yielding a minimal difference (incidence rate ratio 0.99, 95% confidence interval 0.97-1.02). A multiple Cox proportional hazards model showed no significant association between sitagliptin use and overall cancer incidence (hazard ratio 1.01, 95% confidence interval 0.98-1.04). Findings were also consistent across cancer types and sensitivity analyses.
CONCLUSIONS: We observed no evidence to suggest an increased cancer risk among patients prescribed NTTP-contaminated sitagliptin, although continued investigation is needed.

Nitrosamine compounds pose a significant concern as potential carcinogens, prompting heightened scrutiny from regulatory bodies, particularly regarding their presence in pharmaceuticals. The detection of unacceptable levels of N-nitrosodiethylamine (NDMA) in ranitidine has led to widespread recalls, driving interest in alternative medications such as nizatidine, which shares a similar pharmacological class and is used to treat various gastrointestinal conditions. Despite fewer reports on NDMA levels in nizatidine, its structural similarity to ranitidine, characterized by a tertiary amine, underscores the potential for NDMA formation. Addressing the analytical challenges associated with nitrosamine detection, this study focuses on developing and validating an ultra-high pressure liquid chromatography triple quadrupole mass spectrometry (UHPLC-MS/MS) method for quantifying NDMA in both nizatidine active pharmaceutical ingredients and tablet formulations. Method validation adheres to International Council for Harmonisation recommendations, with a demonstrated linear range of 0.25-100 ng/mL for NDMA, exhibiting excellent linearity (regression coefficient >0.999) and efficient recovery rates ranging from 95.98% to 109.57%. The method shows high sensitivity, with limits of detection and quantification of 0.25 and 0.5 ng/mL, respectively. The developed UHPLC-MS/MS method offers a simple, precise, accurate, and selective approach for monitoring NDMA levels in nizatidine formulations available in Australia, promising enhanced sensitivity and specificity with limits of quantification in the ppb and sub-ppb ranges.

Lung cancer remains the leading cause of cancer-related deaths, with cigarette smoking being the most critical factor, linked to nearly 90% of lung cancer cases. NNK, a highly carcinogenic nitrosamine found in tobacco, is implicated in the lung cancer-causing effects of cigarette smoke. Although NNK is known to mutate or activate certain oncogenes, its potential interaction with p27 in modulating these carcinogenic effects is currently unexplored. Recent studies have identified specific downregulation of p27 in human squamous cell carcinoma, in contrast to adenocarcinoma. Additionally, exposure to NNK significantly suppresses p27 expression in human bronchial epithelial cells. Subsequent studies indicates that the downregulation of p27 is pivotal in NNK-induced cell transformation. Mechanistic investigations have shown that reduced p27 expression leads to increased level of ITCH, which facilitates the degradation of Jun B protein. This degradation in turn, augments miR-494 expression and its direct regulation of JAK1 mRNA stability and protein expression, ultimately activating STAT3 and driving cell transformation. In summary, our findings reveal that: (1) the downregulation of p27 increases Jun B expression by upregulating Jun B E3 ligase ITCH, which then boosts miR-494 transcription; (2) Elevated miR-494 directly binds to 3\'-UTR of JAK1 mRNA, enhancing its stability and protein expression; and (3) The JAK1/STAT3 pathway is a downstream effector of p27, mediating the oncogenic effect of NNK in lung cancer. These findings provide significant insight into understanding the participation of mechanisms underlying p27 inhibition of NNK induced lung squamous cell carcinogenic effect.

This research summaries the development, optimization and validation of liquid chromatography tandem mass spectrometric (LC-MS/MS) method for concurrent measurement of seven nitrosamines viz; NDMA, NDEA, NDIPA, NDPA, NEIPA, NMPA & NMBA in Olmesartan tablet. Controlling these nitrosamines at trace levels is imperative for ensuring the safety of drug substances and products for consumption. Various regulatory authorities stress the significance of utilizing highly sensitive analytical methods to precisely measure nitrosamines at trace levels. The method applied effective chromatographic separation and optimized parameters for mass spectrometric detection. Detection was carried out using APCI positive ion mode. Chromatographic separation was achieved using a Thermo Accucore PFP column (150 mm x 4.6 mm, 2.6 µ), with a simple gradient elution of mobile phase consisting of 0.1 % formic acid in water (mobile phase A) and methanol (mobile phase B). The total run time was 20 min, with a flow rate of 0.800 mL/min. The method was validated according to the International Council on Harmonisation (ICH Q2 (R2)) guidelines. The established method demonstrated excellent linearity (R2> 0.99) and sensitivity for all the nitrosamines. Detection and quantification limits were sufficiently low for trace nitrosamine levels having good S/N ratio. The method showed good accuracy in Olmesartan tablet samples, with recoveries ranges between 80 % to 120 %. The new analytical approach has exceptional repeatability and reliability, making it possible to precisely quantify the levels of seven nitrosamines in Olmesartan medoxomil tablets in a single analytical run.

N-nitrosamines and nitrosamine drug substance related impurities (NDSRIs) became a critical topic for the development and safety of small molecule medicines following the withdrawal of various pharmaceutical products from the market. To assess the mutagenic and carcinogenic potential of different N-nitrosamines lacking robust carcinogenicity data, several approaches are in use including the published carcinogenic potency categorization approach (CPCA), the Enhanced Ames Test (EAT), in vivo mutagenicity studies as well as read-across to analogue molecules with robust carcinogenicity data. We employ quantum chemical calculations as a pivotal tool providing insights into the likelihood of reactive ion formation and subsequent DNA alkylation for a selection of molecules including e.g., carcinogenic N-nitrosopiperazine (NPZ), N-nitrosopiperidine (NPIP), together with N-nitrosodimethylamine (NDMA) as well as non-carcinogenic N-nitrosomethyl-tert-butylamine (NTBA) and bis (butan-2-yl) (nitros)amine (BBNA). In addition, a series of nitroso-methylaminopyridines is compared side-by-side. We draw comparisons between calculated reaction profiles for structures representing motifs common to NDSRIs and those of confirmed carcinogenic and non-carcinogenic molecules with in vivo data from cancer bioassays. Furthermore, our approach enables insights into reactivity and relative stability of intermediate species that can be formed upon activation of several nitrosamines. Most notably, we reveal consistent differences between the free energy profiles of carcinogenic and non-carcinogenic molecules. For the former, the intermediate diazonium ions mostly react, kinetically controlled, to the more stable DNA adducts and less to the water adducts via transition-states of similar heights. Non-carcinogenic molecules yield stable carbocations as intermediates that, thermodynamically controlled, more likely form the statistically preferred water adducts. In conclusion, our data confirm that quantum chemical calculations can contribute to a weight of evidence approach for the risk assessment of nitrosamines.

A quantitative testing method was developed for the analysis of low molecular weight (small molecules) nitrosamine impurities in cough syrups using solid phase extraction (SPE) on strong cation-exchange functionalized polymeric sorbent cartridges followed by gas chromatography-mass spectrometry. The matrix spike recoveries of the nitrosamine impurities from the cough syrup samples was observed to be within the range of 90 %-120 %. Limit of detection (LOD) achieved for NNitrosodimethylamine (NDMA) and NNitroso morpholine (NMOR) was about 0.1 ng/mL while the LOD for NNitrosodiethylamine (NDEA), NNitrosodiisopropylamine (NDIPA) and NNitrosoisopropylethylamine (NIPEA) impurities was about 0.02 ng/mL. The method was evaluated and found to meet the acceptable criteria as per the ICH Q2 guidelines for a working concentration range of 0.02 ng/mL to 1.2 ng/mL for the analyzed impurities. The selectivity of the nitrosamine impurities against the presence of drug product was established using multiple reaction monitoring (MRM) transitions during analysis.

Diet is one of the main exogenous sources of potentially carcinogenic nitrosamines (NAs) along with tobacco and cosmetics. Several factors can affect endogenous N-nitroso compounds (NOCs) formation and therefore the potential damage of the intestinal mucosa at initial colorectal cancer stages. To address this issue, 49 volunteers were recruited and classified according to histopathological analyses. Lifestyle and dietary information were registered after colonoscopy. The mutagenicity of fecal supernatants was assayed by a modified Ames test. Fecal heme-derived NOCs and total NOC concentrations were determined by selective denitrosation and chemiluminescence-based detection. Results revealed processed meats as the main source of dietary nitrites and NAs, identifying some of them as predictors of the fecal concentration of heme-derived and total NOCs. Furthermore, increased fecal NOC concentrations were found as the severity of colonic mucosal damage increased from the control to the adenocarcinoma group, these concentrations being strongly correlated with the intake of the NAs N-nitrosodimethylamine, N-nitrosopiperidine, and N-nitrosopyrrolidine. Higher fecal NOC concentrations were also noted in higher fecal mutagenicity samples. These results could contribute to a better understanding of the importance of modulating dietary derived xenobiotics as related with their impact on the intestinal environment and colonic mucosa damage.

N-nitrosamines are a type of nitrogen-containing organic pollutant with high carcinogenicity and mutagenicity. In the main drinking water sources of small and medium-sized towns in China, the contamination levels of N-nitrosamines remain unclear. In addition, there is still lack of research on the concentration of N-nitrosamines and their precursors in tributary rivers. In this study, eight N-nitrosamines and their formation potentials （FPs） were investigated in the Qingjiang River, which is a primary tributary of the Yangtze River. The sewage discharge sites were also monitored, and the environmental influencing factors, carcinogenic and ecological risks caused by N-nitrosamines, and their precursors were evaluated. The results showed that six N-nitrosamines were detected in water samples of the Qingjiang River, among which NDMA [（10 ±15） ng·L-1], NDEA [（9.3 ±9.3） ng·L-1], and NDBA [（14 ±7.8） ng·L-1] were the dominant N-nitrosamines, whereas seven N-nitrosamines were detected in chloraminated water samples, among which NDMA-FP [（46 ±21） ng·L-1], NDEA-FP [（26 ±8.3） ng·L-1], and NDBA-FP [（22 ±13） ng·L-1] were the dominant N-nitrosamine FPs. The concentrations of N-nitrosamines in the middle reaches of the Qingjiang River were higher than those in the upper and lower reaches. Furthermore, the concentrations of N-nitrosamines in the sample sites of sewage discharge and tributaries were significantly higher than those in other sampling sites. The monitoring results at the direct sewage discharge points indicated that the main source of N-nitrosamines in river water was the sewage carrying N-nitrosamines and their precursors. In addition, the concentrations of the three dominant N-nitrosamines including NDMA, NDBA, and NDEA were positively correlated with each other, mainly because of their similar sewage sources. The average carcinogenic risk to residents due to N-nitrosamine in drinking water sources was 2.4×10-5, indicating a potential carcinogenic risk. Moreover, due to the high concentrations of N-nitrosamine formation potentials in the Qingjiang River, the carcinogenic risk of drinking water may be even higher. The ecological risk assessment showed that the ecological risk quotient values of N-nitrosamines in the Qingjiang River watershed were lower than 0.002, which was negligible.

The Food and Drug Administration (FDA) recently reported a new nitrosamine impurity in sitagliptin that was named nitroso-STG-19 (NTTP), whose acceptable intake limit was extremely low at 37 ng per day. In addition, NTTP was found to be a degradation impurity in sitagliptin tablets, which was formed by the reaction of 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine hydrochloride and nitrite salts introduced via excipients. Consequently, the NTTP content in tablets was larger than that in active pharmaceutical ingredients (APIs). To control the impurity, an ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) procedure for the detection of NTTP in sitagliptin phosphate tablets and APIs was developed and validated. Furthermore, a derivatization method for the detection of nitrite salts at lower concentration was developed to select applicable excipients to decelerate the generation of NTTP. During validation of the analytical procedure for NTTP, the quantitation limit (LOQ) of NTTP was 56 ppb (0.056 ng mL-1), the linear correlation coefficient was 0.9998, and recoveries of NTTP in spiked samples ranged from 95.5% to 105.2%, indicating that the method is rapid, sensitive and accurate for an NTTP test. In the nitrite salt detection method, the LOQ was 0.21 ng mL-1, and recoveries of NTTP in spiked samples ranged from 87.6% to 107.8%, indicating a sensitive and accurate method, suitable for screening appropriate pharmaceutical excipients.

Due to their potential adverse health effects, some N-nitrosamines in drug products are strictly regulated with very low maximum daily intake limits. Nitrosamines can be formed from the reaction of nitrite and secondary or tertiary amines when both species co-exist in the drug synthesis or formulation process. One key strategy to mitigate nitrosamine risk in drugs is to select low-nitrite containing pharma excipients for formulation. It is necessary to develop a sensitive method for trace nitrite determination in pharma excipients as it enables drug producers to study nitrosamine formation kinetics and select excipient suppliers. This study details the development and validation of a two-dimensional ion chromatography mass spectrometry (2D-IC/MS) method for trace nitrite determination in hydroxypropyl methylcellulose (HPMC), one of the most important pharmaceutical excipients used in many drug formulations. The 2D-IC system was operated in heart-cutting mode with a concentrator column coupling the two dimensions. A standard bore anion-exchange column was used in the first dimension (1D) to enable a large volume injection for increased sensitivity and provide improved resolution between nitrite and the interfering chloride peak. A high efficiency microbore anion-exchange column with different selectivity was used in the second dimension (2D) to resolve nitrite from other interfering species. The use of 2D-IC resulted in significantly improved resolution, solving the sensitivity loss issue due to ion suppression from an otherwise 1D separation. MS detection with selective ion monitoring and isotope labeled nitrite internal standard further improve the method specificity, accuracy, and ruggedness, as compared with conductivity detection. For trace determination, it is also extremely important to have a clean blank. For this purpose, a novel cleaning procedure using a strong anion wash was developed to remove nitrite contamination from labware. The optimized method was validated with linearity of nitrite in the concentration range of 18.5-5005.8 ng/g having a regression coefficient of >0.9999, precision with RSD at 3.5-10.1 % and recovery of 90.5-102.4 %. The limit of detection and limit of quantitation were 8.9 and 29.6 ng/g relative to the HPMC sample, or equivalent to 89 and 296 pg/g in the sample solution, respectively.