Hi Dear All members,
Need guidance on below topic,
We have recently received the regulatory query for our one of the product regarding Specificity parameter on LCMS method.
“Conduct specificity studies using samples spiked with known drug substance-related impurities. These studies should demonstrate that the presence of other impurities does not interfere with the accurate quantification of peak of interest.”
To support you with a practical and well-structured reference, I’d recommend taking a look at the following paper:
A multianalyte LC-MS/MS method for accurate quantification of Nitrosamines in Olmesartan tablets
This article describes how specificity was assessed, including testing with blank and unspiked matrix samples, and comparing them with samples spiked with impurities. It’s an approach that could serve as a useful starting point for your case.
Let me know how things progress — I’d be interested to hear how you end up approaching this.
Lucas Maciel
Thanks for your guidance,
However as per ICH Q2(R2)
In case of LCMS method,
The specificity or selectivity of an analytical procedure can be demonstrated through absence of interference
LCMS methods are inherent Technology and as per guidance below guidance is provided:
In some cases where the specificity of the analytical technology can be ensured and predicted by technical parameters (e.g., resolution of isotopes in mass spectrometry, chemical shifts of NMR signals), no experimental study may be required.
Technology inherent justification: Inferred through use of specific and selective MS detection (e.g., MRM transition with specified quantitative to qualitative ion ratio, accurate m/z value) in combination with retention time, consider potential for isotopes Absence of interference: from other components in sample matrix.
In case of LCMS methods we are providing required things such as accurate m/z value, retention time, quantitative to qualitative ion ratio
From this I can interpreted that for specificity parameter in case of LCMS method spiking of other impurities is not essential and only absence interference study e.g. blank and sample matrix (Placebo)
Is anybody having different opinion than this, your valuable guidance is always welcome.
Dear Yuvraj,
Technology inherent justification may not suffice as there is a probability of an impurity having same or similar m/z values as your nitrosamine and may be co eluting with your nitrosamine of interest and causing a specificity issue. This may be more relevant if you are using a triple quadrapole mass spectrometry and not a HRMS. MRM transitions by itself do not guarantee that the method is specific.
There is ample evidence of this for nitrosamines.
Thanks Amit for your valuable guidance.
As per USP general chapter <736> Mass Spectrometry
Specificity:
The purpose of a specificity test is to demonstrate that measurements of the intended analyte signals are free of interference from components and impurities in the test material. Specificity tests can be conducted to compare spectra of components and impurities that are known from synthetic processes, formulations, and test preparations. Specificity is also to be demonstrated for any materials added as part of the procedure (e.g., specificity versus isotope-labeled internal standards).
For an identification MS analytical procedure (Category I and II), validation experiments may include multidimensional MS experiments to validate correct assignments of an ion’s structure or origin.
From this I could understand that, during validation specificity of method demonstrated by checking interference from components and impurities that are known from synthetic processes, formulations, and test preparations.
So if we consider product which is having 4 specified degradation product under organic impurities, 2 genotoxic impurities, 2 Nitrosamines impurities
We have to demonstrate specificity by injecting individual identification solution of each of impurities, spiked sample with all impurities, as well as blank and placebo interference to be checked.
Is my understanding is correct or wrong?
If this is correct, then we have to perform the specificity parameter for nitrosamine impurity method by including all possible small nitrosamines as well as including all possible NDSRI’s.
I think your query itself states that samples spiked drug substance related impurities to be injected to demonstrate specificity.
Hence in my interpretation all specified impurities need to be evaluated.
I cannot help sensing a constant intent to fit the nitrosamines impurities into a compendial compliance basket. I highly suggest to see nitrosamines impurities as a parenthesis in compendial compliance. None of the validation, specifications or compendial requirements were developed considering the challenges of Nitrosamines. That’s exactly why regulators conducted and published the round robin study reference by Lucas to understand the depth of the analytical performance complications.
excellent help Lucas,
thanx a lot!!
Christos
You’re right that LC-MS/MS brings intrinsic specificity through selectivity of MRM transitions, accurate mass, and retention time — and that for many routine applications, absence of interference in blanks and placebo matrices may be considered sufficient. That’s particularly true when the goal is qualitative confirmation or quantitative analysis in relatively simple matrices.
However, the nuance lies in the regulatory expectation behind the query you received, especially when it comes to nitrosamines, genotoxic impurities, or complex degradation profiles. These are not standard cases — and regulators are aware of it.
Naiffer made an excellent point: nitrosamines shouldn’t be forced into legacy validation frameworks without acknowledging their unique analytical challenges. A good example of this is discussed in the recent publication:
Cross-Signal Contribution as a Challenge in LC-MS/MS Bioanalysis
This article explores a key issue that often flies under the radar:
“Furthermore, the guidelines address the method’s specificity, but a cross-signal contribution experiment between monitored compounds has not been suggested directly, which may leave some researchers unaware of the problem.”
This highlights why regulators may ask for spiking studies with known impurities even when LC-MS/MS selectivity is well justified. It’s not just about resolving the analyte from matrix components — it’s about ruling out cross-talk, in-source fragmentation, isobaric interferences, and other subtle signal contributions that can impact accuracy at ultra-trace levels.
The request to assess specificity by injecting each individually and spiking them together is aligned with the understanding that specificity is not only about selectivity, but also about signal integrity.
The requirement may not be about checking the box of ICH Q2(R2) — it’s about demonstrating fitness-for-purpose in a risk-driven, impurity-specific context.
For nitrosamines and NDSRIs, this often includes:
Hope this helps. It’s an evolving space, and we’re all learning as the science and regulatory landscape mature.
Lucas Maciel
Thanks for your excellent guidance and help.