Method of Analysis for N nitroso Diclofenac and Quetiapine

Dear Team,

I just want to know if i can find method of analysis for N nitroso diclofenac and N nitroso Quetiapine.

Defiantly methods are available, but I am not Shure anyone will share on common forum.

I’m afraid I have to disagree with that statement. This community comes together to learn and support on a daily basis. As you spend time in our community, I hope you change your perception.

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Last year, i joined this public event:


Pharma Conclave 2023 by Sciex - Mérieux NutriSciences (merieuxnutrisciences.com)
Pharma conclave 2023 (sciex.com)

Presentation: Nitrosamine impurities in dermal products: from APIs and their salts (secondary amines) evaluations through analytical chemistry and mass spectrometry

Here are my personal notes from this event:

The lab has screened a topical gel containing diclofenac diethylammonium API for nitrosamines:

  • N -nitroso-diclofenac (possible root cause nitrosation of diclofenac diethylammonium salt API)
  • NDEA (possible root cause: link with the diethylamine base used to make the diclofenac diethylammonium salt API).

4 samples of drug product were screened for NDEA and N-nitroso-diclofenac. In 3 out of 4 samples no NDEA or N-nitroso-diclofenac could be detected. A limit of detection was not reported. In one sample however 300 ppb of NDEA and 500 ppb of N-nitroso-diclofenac was found.

Subsequently, nitrite testing was performed on the 4 samples. The 3 samples containing no NDEA or N-nitroso-diclofenac contained no detectable nitrite, but the one sample with nitrosamine positivity, did contain 5 ppm of nitrite (LOD of nitrite testing method not shared (method: UPLC-MS with precolumn nitrite derivatization, most probably using 2,3-diaminonaphthalene)).

As such the high nitrosamine detection outlier was rationalized to be linked to an equal outlier in nitrite contamination.

This led to further investigation to explore whether:

  • The two nitrosamines were formed from their precursors in situ during the sample preparation and are artefacts (the high nitrite content in the sample could not be tolerated).
  • The high nitrite contamination during manufacturing and shelf life of the gel led to increased nitrosamine formation.

Due to proprietary reasons it was not explicitly confirmed which one of the two options applied. The presenter did confirm in general recommendations and with other examples that the first cause (analytical root cause – artefacts) is more typical.

Nonetheless, it can be concluded that in diclofenac gels with a more typical nitrite contamination profile for gels, the detection of N-nitroso-diclofenac is atypical (and including the outlier result based on a 5 ppm nitrite samples not significantly above 10% of the anticipated real AI of 78000 ng/day).

This is aligned with the published NAP test on diclofenac, showing low susceptibility of diclofenac to nitrosation (cf. reduced nucleophilicity of the nitrogen in the diclofenac). Comparison of published NAP tests further stressed the likelihood of an analytical root cause for the OOT result. (In general a 5 ppm nitrite contamination for diclofenac gels is possibly atypical).

Schmidtsdorff, S., Neumann, J., Schmidt, A. H., & Parr, M. K. (2022). Risk assessment for nitrosated pharmaceuticals: A future perspective in drug development. Archiv Der Pharmazie (Weinheim) , 355(4), e2100435–n/a. https://doi.org/10.1002/ardp.202100435.

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Can you provide details of the exact problem you are facing

Hi Sbasaleh,

Diclofenac NDSRI- MW of NDSRI is 325.1 Da & Diclofenac monoisotopic mass is 295.1 Da. This NDSRI is very very unstable under thermal treatment of ESI & undergoes the loss of NO atoms to show 294.1 as Q1 mass. Further Q3 fragments are 242.1 & 214.05 as Quantifier & Qualifier respectively.

The behavior of impurity cross checked with LC method where there is proved that Diclofenac & NDSRI have different RT on chromatograms. Based on this theorem we have proceeded and executed the activity.

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Dear C. Wybon
I’m the Business Director of MXNS Italy.

I would like to comment your phrase “The presenter did confirm in general recommendations and with other examples that the first cause (analytical root cause – artefacts) is more typical” in order to explain better our position.

Artefacts formation during the analytical phase should be always taken into consideration expecially when precursors sensitive to nitrosation (e.g. API with secondary amine groups, DEA, etc) are present in matrices (e.g. finished drug products) which could likely contain trace amounts of nitrite.

This is why we use scavengers during sample preparation and systematically verify the robustness of our methods respect to any eventual in situ nitrosation.

When significant amount of NDRSIs are found, the application of robust methods can guarantee that the results are related only to the native presence of the target nitrosamine(s) in the tested product without other contribution (i.e. in situ generation).

Of course, when the tested product contains high native concentration of NDSRI it means that, coherently, the risk or artefacts formation in presence of nitrite residues could be very high, as well, and should be investigated properly during the analytical phase.

In conclusion, the analytical phase could be a supplementary root cause only if non-robust analytical method are applied, which is not the case reported in our presentation.
The applied method has been in fact verified in advance to avoid artefacts formation by means of the APNS verification procedure before presenting the results of the cited case study.

Best regards.

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Hi Sergio,

Explicit proof that (and how) the robustness of the method was verified for the relevant amine and nitrosating agent and/or catalyst content of worst case sample(s) preparation(s) I could understandably not derive from your presentation. At the same time I would also have too little information on the product composition, nitrosamine risk assessment and sample preparation to judge a possible design of robustness studies, as this requires knowing if alternative nitrosation risks apply. NDSRIs like nitrosodiclofenac have an increased theoretic risk for participating in transnitrosation (as flagged by the EMA), formaldehyde contamination catalysis can be more important for slightly higher pH formulas like expected for such a gel, the NAP for nitrosodiclofenac should be interpreted in the context of the API and DP manufacturing vs. sample preparation conditions and how the vulnerable amines interact with the often polymeric gel structure and (other) acids/bases in the formula can be important for availability for nitrosation and thus robustness.
Higher detection with higher nitrite is in theory both compatible with analytical and manufacturing/shelf life root causes so I’m not drawing conclusions based on this alone to conclude whether or not the developed method is robust in > 5 ppm nitrite (while understanding you must have the proprietary data at hand). The combined expression of a NDEA and nitrosodiclofenac risk on its own doesn’t tell much.
If an analytical root cause would apply, the for example non-repeatability of sample 2 approximate results on sample 1 + > 5 ppm nitrite spike are on its own not all-telling without further understanding the possible risks and no scavenging impact on sample 2 depends on the mechanism of nitrosation and the scavenger used, hence I’m careful (especially because the original results were reported without scavenging studies).

So I’m was in no liberty to record it my notes and rather valued the presentation very much for linking nitrite and nitrosamine testing in one reported project and creating continued awareness on robustness risks, also for APIs with low NAP outcome (regards being made to the literature NAP being MS-based only and competitive C-nitrosation risks that surround APIs like diclofenac).
As a company always bringing up the robustness topic in public presentations, surely you have the bases covered.

Of note, in general, the real NDSRI risk profile for the product and the NDSRI risk profile for the analytical testing can be significantly different as well.

Yes…that’s correct…You can also try using the ammonium adduct at 342 for quantification