Regulatory authorities in various countries have successively issued notices and guidance to pharmaceutical manufacturers and distributors to evaluate the risk of contamination of nitrosamines in pharmaceutical products and to take appropriate measures. Analysis of nitrosamines in pharmaceutical products is not easy due to the large number of foreign substances, and the risk of contamination is determined by first conducting a desk investigation of the manufacturing process of the APIs or pharmaceutical products. However, a desk investigation may miss the risk since this method is not based on actual measurements. Therefore, in addition to conventional desk-based investigation, a new method is required to pick up risks that cannot be covered by a desk investigation. Nitrosamines are known to be formed by the reaction of amines with nitrosating agents such as nitrite. In the case of small alkyl nitrosamines such as NDMA and NDEA, the origin of the amines is mostly residual amines in the APIs. Residual amines in the APIs are a potential nitrosamine contamination risk, although the extent of that risk has rarely been reported. In this study, we developed and validated a simultaneous analytical method for amines corresponding to 10 typical small alkyl nitrosamines. Good linearity was obtained in the range of 0.003 to 10 μg/mL for MPA, 0.003 to 2 μg/mL for DIPA and DBA, 0.003 to 1 μg/mL for MeP, DEA, EIPA, and DPA, and 0.003 to 0.2 μg/mL for DMA, MOR, and MBA. The limits of quantitation and detection were 0.003 and 0.001-0.003 μg/mL, respectively. The recovery rates ranged from 70 to 130% for 121 APIs and were more than 40% for 83 APIs. Repeatability was also good, with %RSD < 15%. Although the correlation between the amount of amines detected in the APIs and the nitrosamines in the pharmaceutical products is under investigation, we expect that this analytical method will be used to determine the residual amine contents in APIs and contribute to the risk assessment of the nitrosamine contamination.

The high-sensitivity analytical method for the determination of N-nitroso duloxetine (NDXT), which can be carcinogenic and harmful in duloxetine drug products, was successfully developed utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). Tandem mass spectrometric detection at positive electrospray ionization in multiple reaction monitoring (MRM) mode was then employed for the determination of NDXT. The quantitative range for NDXT was found in 0.075-3.75 ng/mL in terms of concentration in the dilution solvent for duloxetine active pharmaceutical ingredient (API) and capsules and 0.075-1.875 ng/mL for duloxetine tablets, and the recovery rates were in the range of 82.5-91.6% for the API, 91.0-113.4% for capsules, and 70.6-109.1% for tablets, respectively. The repeatability was 6.9% with a %RSD of n = 9 for the API, 10.9% with a %RSD of n = 9 for capsules, and 21.6% with a %RSD of n = 9 for tablets, respectively. For reproducibility, the %RSD of the n = 6 measurements between the two sites was 3.5%. The calibration curve of NDXT in the concentration range of 0.075-3.75 ng/mL was carried out, and the correlation coefficient (R) was found to be 1.000. The sample solution was stable for 7 days. The applicability of the determination of the content of NDXT in a variety of duloxetine drug products was demonstrated. This manuscript seeks to aid the risk assessment process of NDXT in duloxetine drug products through providing a fast and reliable quantitative LC-MS/MS analytical method.

N- Nitrosodimethyl amine, the simplest member of the N-Nitrosamine family, is a carcinogenic and mutagenic agent that has gained considerable research interest owing to its toxic nature. Ozonation of industrially important hydrazines, such as unsymmetrical dimethylhydrazine (UDMH) or monomethylhydrazine (MMH), has been associated with NDMA formation and accumulation in the environment. UDMH/MMH - ozonation also leads to several other transformation products such as acetaldehyde dimethyl hydrazine (ADMH), tetramethyl tetra azene (TMT), diazomethane, methyl diazene, etc, which can be either precursors or competitors for NDMA formation. However, the relevant chemistry detailing the formation of these transformation products from UDMH/MMH -ozone reaction and their subsequent conversion to NDMA is not well understood. In this work, we explored the formation mechanism of ADMH and TMT from UDMH-ozonation and their further oxidation to NDMA using the second-order Moller Plesset perturbation theory employing the 6-311G(d) basis set. We have also investigated how MMH selectively forms methyl diazene and diazomethane under normal conditions and NDMA in the presence of excess ozone. Our calculations indicate that the reactions proceed via an initial H abstraction from the hydrazine -NH2 group, followed by the oxidation of the generated N-radical species. The formation of ADMH from the UDMH-ozone reaction involves an acetaldehyde intermediate, which then reacts with a second UDMH molecule to generate ADMH. The preferable attack of ozone molecule on N=C bond of ADMH generates DMAN intermediate, which subsequently undergoes oxidation to form NDMA. Unlike other transformation products, TMT formation occurs via the dimerization of DMAN. 1Though there exists an N=N bond in the TMT, which are preferable attacking sites for ozone, experimental studies show the lower yields of NDMA formation, which corroborates with the high activation barrier required for the process (42 kcal/mol). Overall, our calculated results agree well with the experimental observations and rate constants. Computational calculations bring new insights into the electronic nature and kinetics of the elementary reactions of this pathway, enabled by computed energies of structures that are not possible to access experimentally.

Over the past few years, an increasing number of commercially available drugs have been reported to contain N-nitrosamine impurities above acceptable intake limits. Consequent interruption or discontinuation of the manufacturing and distribution of several marketed drugs has culminated into shortages of marketed drugs, including the antidiabetic drug metformin and the potentially life-saving drug rifampin for the treatment of tuberculosis. Alarmingly, the clinical development of new investigational products has been complicated as well by the presence of N-nitrosamine impurities in batches of marketed drug. In particular, rifampin is a key clinical index drug employed in drug-drug interaction (DDI) studies, and as a result of nitrosamine impurities regulatory bodies no longer accept the administration of rifampin in DDI studies involving healthy subjects. Drug developers are now forced to look at alternative approaches for commonly employed perpetrators, which will be discussed in this review.