Nitrosamines Formation as Artefact... What are the Challenges?

Hi Everyone,

We have been seen a lot of discussions around nitrosamines formation as artefact during:
(1) Sample Preparation
(2) Sample Analysis

Here I have a case where we deal with a mixture of issues related to matrix effect and artefactual formation of Nitrosamines during SAMPLE PREPARATION:

:point_right: We know that Extended-Release (ER) formulations in general contain different types of polymer matrix systems within the formulation or use polymer coatings on solid dosage forms (e.g.: cellulose derivative HPMC in Metformin ER Tablets).

:point_right: CHALLENGES:
:white_check_mark: during sample preparation these high molecular weight polymers can swell and gelatinize in AQUEOUS solutions causing extraction efficiency issues: matrix effect causing artificial low quantification of NDMA in metformin ER Tablets
:white_check_mark: these high molecular weight polymers if extracted can contaminate the MS system causing ion suppression

Some preparation protocols for NDMA analysis in metformin ER Tablets consider the use of the following strategies to avoid this extraction efficiency issues and injection of these polymers into the MS system:
:white_check_mark: GC-MS/MS analysis: Extraction using Dichloromethane (DCM)
:white_check_mark: RPLC-MS/MS: Extraction using DCM, wash with water for the removal of residual HPMC, evaporation of DCM and resuspension in suitable solvent (in general a mixture of wa-ter/organic solvent)

Based on the amazing work published by Fritzsche, M et al in 2021 (Redirecting), there are evidences that the use of DCM as extraction solvent led to the extraction of dimethylamine (DMA)/nitrite (if present) and formation of NDMA in-situ in the DCM sample extract in the analysis of ER and Immediate Release Metformin Tablets

In this work they proposed two solutions for these cases:
:triangular_flag_on_post: Removal of DMA and nitrite by including an additional water washing step after extraction with DCM
:triangular_flag_on_post: addition of nitrite scavengers (4-methylbenzene-1,2-diamine and pyrrolidine) to consume nitrite

Did anyone here face the same issues for some other drug product? Which are the challenges and solutions you are using to avoid the overestimation of Nitrosamines due to their potential in-situ formation during sample prep?


@DAB Do you some experience/thoughts related to the issues highlighted in this post to share?

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Hi Amanda @AmandaGuiraldelli
This seems to be a very interesting & important case study for discussion. Thanks for tagging me on this discussion thread.
I was unable to access the ejps article from the link, however these points are exremely valid points from the Analytical Sample Prep perspective.
I was just thinking if SPE, Solid Phase Extraction can offer solutions for Sample Prep, extraction.
An optimised SPE procedure would make sample matrices more compatible with the target chromatographic method,concentrate analyte of interest - Nitrosamines in this case (trace enrichment) for increased sensitivity.remove interferences that cause background noise & ghost peaks. Once optimised the SPE process can be automated resulting in high throughput.
I have worked on this technique for sample prep & extraction of drugs & their metabolites from biological fluids, the Analytical challenges are quite similar to the one that are being discussed, analyte stability, trace analysis, potential risk of artefacts

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Dear Amanda, I would agree with you about SPE and go even further-it should allow sample concentration which would get us away from working at the low end of sensitivity, close always to the LOD. However for some reason it seems that this approach has not been applied to nitrosamine analysis. As it would seem a logical way to go I wonder if anyone has any reason why it is not used-does anyone have practical experience?

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Some colleagues in Brazil used SPE as a strategy to pre-concentrate the nitrosamines. Yesterday we had the last day of the Virtual Workshop on Nitrosamines organized by USP, Sindusfarma and ACNF, where Ache Labs discussed about using SPE.

@Diego, I know you have a lot of experience using SPE for Nitrosamines analysis. Would you have something to share about the experience you had with this protocol applied to nitrosamines quantification in pharmaceuticals?

Just listing here some challenges that we might face when we work with SPE for nitrosamines quantification:
:point_right: loss of nitrosamines during sample preparation (several nitrosamines are very volatile. Using internal stds may help to compensate that)
:point_right: matrix effect due to the concentration of interfering compounds. Do not select the best SPE packing material for the application only based on recovery. Use also a strategy to assess the presence of matrix effect and plan for minimizing that.

Does somebody else have more challenges and/or solutions to share regarding this topic?


I corrected the link. You should be able to access it now.

Good to know you have experience on that!

Are you used to assess the presence of matrix effect when selecting the ideal SPE cartridge for your applications? If yes, could you share your experience regarding selection of the ideal SPE packing material? If you can link that to applications for nitrosamines it would be amazing!

Thanks for your always valuable contributions here in the community!
Amanda Guiraldelli

Hi Amanda @AmandaGuiraldelli and all, thanks for raising the issue of gelling during sample preparation and it is something we have also observed in our method development at AZ. My colleague @giorgio blom will be able to offer further perspectives on this issue. Best Wishes. Tony

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Hi again, please take a look at the recent publication by Kausik Nanda and colleagues at Merck in US, which describes strategies to inhibit nitrosamine formation in solid dosage forms.
Inhibition of N-Nitrosamine Formation in Drug Products: A Model Study - ScienceDirect.
The approach is focussed on formulation optimisation to prevent nitrosamine formation, but has great potential to prevent artefactual formation during sample preparation for analysis.


Hello Amanda @AmandaGuiraldelli and all the community members who have been a part of this discussion thread

There are multiple challenges while estimating Nitrosamines from ER formulations as described by you.
Sharing my experiences here:

  1. ER formulations contain organo soluble polymers like HPMC that have a tendency to swell up on contact with aqueous media thereby providing extended release of drug over its Therapeutic window and this always poses a challenge for Analytical sample prep even while estimating actives from the ER dosage form, the sample prep in such cases normally involves multiple extraction steps followed by mechanical shaking for prolonged times using mechanical and orbital shakers and this shaking time needs to be optimised considering analyte recovery. At times use of Internal standard is required if labelled claim is low and recovery issues can pose a risk.

  2. SPE is employed for spl prep in the estimation of Nitrosamines in drinking water and in foods, there are couple of references available online. Sharing one of such references that also cites issues with usage of DCM as extraction solvent , Separations | Free Full-Text | Determination of N-Nitrosamines by Gas Chromatography Coupled to Quadrupole–Time-of-Flight Mass Spectrometry in Water Samples | HTML

  3. The SPE technique needs to be employed in such cases in conjunction with prior sample cleanups as is done with biological fluids otherwise the sample matrix can potentially clog the cartridges.

  4. Initial trials with spiked placebo can help in optimising the extraction procedure and evaluating Analyte recoveries. Once acceptable recoveries and LOD, LOQ are established the procedure can be extended/optimised with the ER formulation.

  5. Orthogonal screening methods also help in evaluating matrix effects and improvise signal to noise ratios for Analytes of interest.



Yes, we see a lot of protocols using SPE for nitrosamines analysis in water. The challenge it is not so high to use SPE to pre-concentrate Nitrosamines in Water but it canbe for nitrosamines in pharmaceuticals.

Yes, this is a great example of issues when using SPE. Another point is the concentration of an interfering compound of the matrix causing matrix effect. It’s important to assess the presence of matrix effect when selecting a suitable SPE cartridge.

Thanks for the discussion!

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Hi @AmandaGuiraldelli

Interesting discussion around the use of additional sample cleanup to minimise matrix effects and artefactual formation. SPE has proved to work in matrices such as water but as eluded earlier careful consideration needs to be made towards the entire matrix as for example you will always have approximately 1 billion times more API in comparison to nitrosamine by weight. This then has the potential to more preferentially adsorb to your SPE cartridge and potentially saturate the cartridge. Caus ing significant recovery issues.

The use of suitable stable labelled internal standards somewhat minimises the previous issues you highlighted but I would still recommend extensive method development to be performed to thoroughly understand method capability.

Another potential issue is could be the use of diluent required. In order to prevent the gelling, you will need to use less polar solvents (in comparison to water) such as DCM, IPA or acetone. This might then cause breakthrough of your nitrosamine, especially NDMA, when loading your sample on the SPE cartridge.

Kind regards,

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Amazing description about the challenges to work with SPE for pharmaceuticals compared to water matrix!

Great recommendations!