Introduction 

The US-based pharmaceutical giant Merck's blockbuster diabetes medication, sitagliptin (marketed under the trade name Januvia), has undoubtedly reemerged as a topic of significant interest recently, particularly in light of the detected nitrosamine impurities affecting the compound. Nitrosamines represent a class of chemical carcinogens, categorizing them among the four primary food contaminants. Not only may chronic exposure to low doses increase the risk of cancer in both animals and humans, but substantial high-dose "shocks" can also initiate the development of cancerous cells. The food and pharmaceutical sectors maintain an uncompromising stance towards nitrosamines. In this edition, we will delve into the unique nuclear magnetic resonance (NMR) data characteristics of nitrosamine-like impurities, using the NTTP as a case study to assist analysts in comprehending the structure of such compounds.

As the world's first marketed DPP-4 inhibitor, the sales data of Sitagliptin is undoubtedly very eye-catching, with annual sales of more than $3 billion between 2017 and 2020. With the expiration of compound patents in 2024, there are more than 30 domestic enterprises applying for single and compound formulations of Sitagliptin tablets in China. If a similar "valsartan incident" is caused by the influence of nitrosamine impurities in the raw material of Sitagliptin, domestic companies will undoubtedly suffer a“blow”; As a widely used prescription drug for the treatment of type 2 diabetes, the shortage of Sitagliptin may cause harm to patients. What is the future of Sitagliptin? What will Merck do? How do domestic enterprises follow up? Many questions are waiting for us to see.

As a member of the "notorious" nitrosamine impurities, Nitroso-STG-19 (NTTP) , a substance related to Sitagliptin, has become "black hand" that affects the safety of sitalliptin related drugs. Concontaminated with NTTP impurities, NTTP content in specific batches of sitalliptin-related products exceeded the lifetime daily exposure requirement required by FDA. Although there are no detailed carcinogenesis data on NTTP, based on the existing data on nitrosamine compounds with similar structures, the FDA has sets the lifetime daily exposure limit for NTTP at 37 ng, making NTTP content control an important task for drug quality control.

To study NTTP, it is necessary to understand the structure of the related substance. NTTP chemical is 7-nitroso-3- (trifluoromethyl) -5,6,7,8-tetrahydro- [1,2,4] triazole [4,3-A] pyrazine, which is an N-nitrosamine compound (N-nitrosamines, abbreviated NAs). The special chemical structures of N-nitrosamines may exhibit different properties and results in NMR, and the particularity of this structure can sometimes cause analysts to question the identification and use of this compound. For nitrosamines compounds, we are often more familiar with the drawing method of the nitrosamine structure in Figure A below, where the nitrogen-oxygen double bond is connected to one side of the N-N single bond. However, it should be noted that, in fact, the resonance structure B of the compound can often better reflect the chemical characteristics of the compound. In resonance structure due to the N-N bond obvious double bond properties, for nitrosamine structure, due to the space of oxygen atoms towards the different, specific nitrosamine structure showed two different configurations: structure B1 and B2, and two structures can mutually converted (The editor actually wants to tell you again about the effects of bond length, bond angle, and rotational energy barrier on the structure of N-nitrosamine. However, it is beyond the scope of the article. Welcome to contact the editor in chief in the background to obtain detailed literature materials). Thus the asymmetrically substituted N-nitrosamine compounds will generate two sets of signals under special conditions, corresponding to two different configurations. For example, the common nitrosamine compounds NMPA and NEPA have been well known, and a set of signals produced in the NMR map make people helpless, even nicknamed "map clown-NXPA brothers" by teachers.

For the protagonist NTTP structure in this study, due to significant differences in amino substitutions, the differences in graph data for different configurations are more obvious. Due to the influence of environmental variables such as temperature and medium on the conversion between configurations, we used different solvents for NMR hydrogen spectrum testing in the spectral study of this product, in order to provide a more intuitive view of the mutual conversion between the two configurations of NTTP in different solvents. The data shown in the following figure is the NMR spectrum results of NTTP product (QCS CAT NO.: RM-S200807, LOT NO.: 24852) in deuterated chloroform and deuterated dimethyl sulfoxide. It can be clearly seen that under the condition of using deuterated chloroform as the solvent, the two configurations of NTTP coexist in a 1:3 ratio; Under the condition of using deuterated dimethyl sulfoxide, the ratio is 1:4, and a similar phenomenon can also be seen in the carbon spectrum results.This is the result of the changes in molecular configuration ratios of nitrosamines under different solvent conditions, influenced by environmental variables such as temperature and medium, rather than the "quality" issue that some teachers mistakenly believe.

Note: The experimental data was collected from samples of batch 24852 from the QCS Standard Product Development Center, with a sample size of 20mg, collection instrument: Bruker AVANCE 400.

Taking the sample test data of batch 24852 from QCS standard product development center as an example, it can be seen that the product meets the usage requirements in terms of high-performance liquid chromatography, mass spectrometry and TGA data results. The following is the graph data.

The use of high-quality impurity reference materials is the primary condition for obtaining reliable results. QCS provides elaborately developed NTTP standard products to meet your needs for NTTP content detection (if customers need sample products, click on our official account below and send "sample" to the background to get them).