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:
- Evaluation of signal suppression/enhancement from co-eluting analytes,
- Cross-signal contribution experiments,
- Demonstration that impurities do not affect quantification of each other — even indirectly.
Hope this helps. It’s an evolving space, and we’re all learning as the science and regulatory landscape mature.
Lucas Maciel