Publication: " Modified NAP Test: A Simple and Responsive Nitrosating Methodology for Risk Evaluation of NDSRIs" by Sharma et al.
Nitrosamines have been recognized as one of the cohorts of concern as per ICH M7. In recent years, the regulatory focus has shifted from common nitrosamines to nitroso-impurities of drug products. Thus, the detection and quantification of unacceptable levels of nitrosamine drug substance-related impurities are of great concern for analytical scientists during drug development. Moreover, risk assessment of nitrosamines is also an essential part of the regulatory filling. For risk assessment, the Nitrosation Assay Procedure suggested by WHO expert group in 1978 is being followed. However, it could not be adopted by the pharmaceutical industries due to the limitation of drug solubility and artefact formation in the test conditions. In this work, we have optimized an alternative nitrosation test to investigate the likelihood of direct nitrosation. The technique is simple, where the drug solubilized in an organic solvent is incubated at 37°C with a nitrosating agent named tertiary butyl nitrite in a 1:10 molar ratio. LC-UV/MS-based chromatographic method was developed to separate drug substances and respective nitrosamine impurities using the C18 analytical column. The methodology was successfully tested on five drugs with varying structural chemistry. The procedure is straightforward, effective, and quick for the nitrosation of secondary amines. This modified nitrosation test and WHO prescribed nitrosation test have been compared and found that the modified methodology is more effective and time-saving.
Thank you Naiffer for bringing this up.
I cannot access the paper at the moment but from the abstract I wanted to raise one question.
This procedure has been made in organic solvents with an organic nitrite source in order to overcome the poor solubility of some drugs.
Is this really going to provide valid results? I mean, normally, for example in drug product formulations where most of the NDSRIs are being formed and where the largest concern is at the moment, you don’t have an organic solvent being used, and nitrites are normally from inorganic origin.
So how would this translate into a risk assessment? A positive finding in this modified assay may not be representative for the drug product, were the solvent is usually water.
Yes you are right @JavierFernandez,
This approach seems to be of more of an academic research interest.
It is important to take note of the IQ consortium’s following set of three conditions to investigate the potential formation of a N-nitrosamine from an vulnerable amine.
Amine, AcOH (~ a third of overall reaction volume), NaNO2 solution in water (1.5 eq), overall reaction media concentration 0.1 M (add water if required to reach this concentration), 20-25⁰C temperature with a cap on to avoid NOx depletion. No co-solvents to be added - reaction could be a slurry.
Amine, dilute HCl* so that pH is between 3 and 4, NaNO2 solution in water (1.5 eq), overall reaction media concentration 0.1 M (add water if required to reach this concentration), 20-25⁰C temperature with a cap on to avoid NOx depletion. Monitor pH at each time points and adjust if necessary. No co-solvents to be added - reaction could be a slurry.
*if the API is a salt of a strong acid, use the API salt in water instead of dilute HCl and do not adjust the pH
Amine free base, organic solvent that solubilises the amine (e.g. acetonitrile, tetrahydrofuran, or other aprotic solvent), tert-butyl nitrite (1.5 eq), overall reaction media concentration 0.1 M, 20-25⁰C temperature with a cap on to avoid NOx depletion. Reaction could be performed with deuterated solvent in an NMR tube if desired. Note that this anhydrous method can lead to the formation of nitrosamines that are unstable under aqueous conditions. Such instability can be used as part of a rationale to justify the absence of N-nitrosamine risk in the drug substance and/or product.
do we have a link to the full paper?
The manuscript is talking about risk assessment pertaining to possibility of N-Nitroso formation of API. There are almost 12000 molecules [including Drug and Impurities], which can theoretically make NDSRI. However, many of NDSRIs are not feasible chemically due to substitutes near to -NH, steric hinderance, extended resonance etc. This paper is talking about checking chemical possibility of NDSRI formation based on structure of API/impurity. WHO had published NAP test more than a decade ago for checking possibility of N-Nitroso formation in food and other substances including drug. This manuscript is talking about modification/improvement of that test.
Your interpretation is correct that there is no organic solvent in actual condition. Thus, this manuscript can not simulate or predict formation of NDSRIs. However, this manuscript is only talking about/predict “Is there any possibility for the API structure to produce NDSRI in presence of favourable -NO source?” Incase, API is not capable to make NDSRI, scientific justification along with this data may suffice the regulatory need.