How to circumvent matrix effect in confirmatory testing

Dear all,

We are facing some analytical challenges with the CRO performing confirmatory testing on a drug product and I wanted to seek advice on how it would be best to move forward.

The drug product is a polypeptide API with a simple formulation made of mannitol and lactose. Method development worked swiftly with the API, being able to have a proper method to detect the nitrosamine.
Problem arises when trying to test the drug product, since there is a strong matrix effect and it is not possible to recover any of the standard, even in spiked samples.

I know different equipment have been tested with similar outcomes and we are a bit at a loss on how to move forward at this point. We believe that the risk is most likely going to be low in the drug product, but authorities are requiring confirmatory testing data in order to move forward

Thanks for your help in advance!

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Just an opinion:
I don’t know the particularities of the used method or about the drug product but did you tried alternative extraction or even filtration methods in order to see which one is more sensitive? What about the stationary phase in order to obtain the separation of different eluting components.
Maybe will work.
Good luck with it!
Thank you, Cristina

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Hi Javier! Some common tricks can be applied like decrease the injection volume is one of them (may be the easiest primary test to perform if your method is by LC-MS, for example).

We are facing a pronounced matrix effect in metformin analysis, because in our case, the direct extraction described in FDA or HSA method`s has not worked in our API or DP. So, we are trying to develop an extraction protocol using SPE.

The paper “A robust analytical method for simultaneous quantification of 13 low-molecular-weight N-Nitrosamines in various pharmaceuticals based on solid phase extraction and liquid chromatography coupled to high-resolution mass spectrometry” can be useful for you.


Including @lucas10mauriz @AmandaGuiraldelli

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@JavierFernandez ,

In this case, I can contribute by brainstorming some topics for discussion:

  1. Extraction methods,
  2. Chromatographic conditions,
  3. Calibration approach (matrix matched calibration curve, standard addition, internal standard), and
  4. Dilution of samples or
  5. Reduction of injection volume.

Based on the context you provided, I believe we can delve deeper into two points: 1) Sample Preparation Optimization (as suggested by my colleague @CesarJr) and 2) chromatographic conditions.

Regarding sample preparation, consider evaluating various techniques such as solid-phase extraction (SPE) or liquid-liquid extraction (LLE) to mitigate matrix interference and enhance analyte recovery. For more details, you can refer to the article: “Overview, consequences, and strategies for overcoming matrix effects in LC-MS analysis: a critical review.”

On the chromatographic aspect, explore the discussion presented by the authors in the article: “An improved analytical method for quantitation of nitrosamine impurities in ophthalmic solutions using liquid chromatography with tandem mass spectrometry.” The authors examine how their method effectively retains polar nitrosamine impurities. They achieve this by employing an enhanced pentafluorophenyl column, which proves beneficial when handling complex matrices. Additionally, the method’s ability to withstand organic solvents in mobile phases and diluents is highlighted as a significant advantage.

These are my suggestions. Feel free to continue the discussion.

I kindly request that you keep us updated on the progress of your challenge and whether any of the proposals presented here were satisfactory.

Lucas Maciel


I’m considering the context of an LC-MS methodology.

Thank you very much for your suggestions Lucas! I will discuss them with the team and surely will get back to you

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