Preventing False Positives During the Development of Targeted Analytical Methodologies

3^rd annual genotoxic impurities summit: Beyond nitrosamines

Nathanael Page

1^st October 2025

1. Brief Introduction to Resolian
2. Identifying Sources of False Positives
3. Mitigating Risk During Method Development
4. Considerations for High Resolution Accurate Mass Spectrometry (HRAMS) Analysis

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AGENDA

1. Brief Introduction to Resolian
2. Identifying Sources of False Positives
3. Mitigating Risk During Method Development
4. Considerations for High Resolution Accurate Mass Spectrometry (HRAMS) Analysis

AGENDA

LIAN means integrity

Resolvers

Resolute

Five state-of-the-art labs�Four Continents. One Partner.

USA�FOUNDED 2007

UK

FOUNDED 1997

CHINA

FOUNDED 2017

AUSTRALIA

FOUNDED 2023

Fordham

UNITED KINGDOM | ANALYTICAL SCIENCES | FOUNDED IN 1997

Analytical Sciences Fordham and its teams are divided between two laboratory areas, Separation Sciences and Elemental Analysis.

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Both areas are purpose-built, state-of-the-art analytical laboratories �ideal for our scientists to deliver for you.

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SERVICES PROVIDED

• Separation Sciences: Is subdivided into discrete, Sample Management, �Wet Chemistry, and Instrument areas, to enable efficient working.

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• Elemental Analysis: Is access controlled and under positive pressure�to reduce possible contamination issues.

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Sandwich

UNITED KINGDOM | ANALYTICAL SCIENCES | FOUNDED IN 2012

Based at Discovery Park in Sandwich, Kent, our Sandwich facility is a dedicated materials science and analytical development lab, offering you tailored services and partnership to navigate your pharmaceutical development journey from discovery to commercial manufacture..

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CORE SERVICES

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• Materials Science Solutions: Dedicated characterization services to enable �better understanding of the solid state and physical properties of �your materials.

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• Analytical Solutions: Bespoke, phase-appropriate analytical methods for �identification, characterization and testing of your material to underpin �chemistry, manufacturing and controls (CMC).

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• Foreign Matter Analysis: Forensic analysis of contamination to determine its identity and source, and to support root cause investigations and remediation for more robust processes.

1. Brief Introduction to Resolian
2. Identifying Sources of False Positives
3. Mitigating Risk During Method Development
4. Considerations for High Resolution Accurate Mass Spectrometry (HRAMS) Analysis

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AGENDA

False Positives: An Introduction

Measurement of a component at a level higher than its original level in the received sample.

Due to the ubiquity of nitrosation sources, false positive results have been commonplace throughout the history of generic nitrosamine testing.

These false positive results have consequently led to unnecessary financial implications.

At Resolian we specialise in the efficient development of highly selective, tailored methods which differentiate between nitrosamines and structurally similar impurities that would otherwise lead to a false positive.

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Identifying Sources of False Positives

The initial aim is to consider the aspects of the sample and analytical approach which, under suitable conditions, could potentially result in a false positive:

• Excipients
• Process Impurities
• Packaging Leachables
• Sample Storage
• Sample Handling
• Sample Preparation
• Sample Introduction Technique
• Isobaric Interferences

Fire/Combustion Triangle

A fire triangle can help identify potential ways to prevent a fire.

• Oxygen <- Smothering
• Heat <- Cooling
• Fuel <- Starving

“Nitrosamine Triangle”

An N-nitrosamine triangle �can help identify potential ways to prevent generation of your target N-nitrosamine.

• Nitrite <- Scavenging
• Heat/Acid <- Cooling/Solvent
• 2° or 3° Amine <- Removal

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1. Brief Introduction to Resolian
2. Identifying Sources of False Positives
3. Mitigating Risk During Method Development
4. Considerations for High Resolution Accurate Mass Spectrometry (HRAMS) Analysis

​

AGENDA

Sample Handling

Nitrile and rubber gloves are a well-known source of nitrite in analytical laboratories.

Multiple studies have reported that nitrosamines can be detected when analysing solutions that have contacted these gloves.

Solutions:

• Minimise contact between gloves and samples
• Automation

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Sample Preparation

Many common extraction procedures can induce the formation of nitrosamines.

Approaches validated for assay/related substances should not be blindly trusted.

Solutions:

• Utilise a suitable scavenger
• Utilise solvents and buffers that do not encourage formation or require heat
• Utilise a highly selective extraction to remove amines related to your target prior to heating

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Sample introduction technique

Caution needs to be taken when considering gas chromatography for the measurement of nitrosamines.

Regardless of system, there is a requirement to use significant heat to evaporate your nitrosamine.

Solutions:

• Utilise a highly selective extraction to remove amines related to your target prior to heating
• Use liquid chromatography

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Evaporation

Degradation

Formation

Isobaric Interferences

Isobaric interferences are a potential risk for all mass spectrometry-based methods.

Single Quadrupole (SIM/SRM):

NDEA by has multiple potential Isobaric interferences from common solvents: Triethylamine, Diethylformamide, DMSO+Na.

Triple Quadrupole (MRM):

Many nitrosamines have a structurally similar “Formamide” impurity. The A+1 isotope produces the same fragments in MRM experiments, leading to a potential interference.

Solutions:

• Utilise selective chromatography.

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1. Brief Introduction to Resolian
2. Identifying Sources of False Positives
3. Mitigating Risk During Method Development
4. Considerations for High Resolution Accurate Mass Spectrometry (HRAMS) Analysis

​

AGENDA

Considerations for HRAMS Analysis

HRAMS is a very powerful technique that can maximise your confidence in the identity of a trace-level component.

This is achieved by deriving elemental composition information for the intact component and its fragments.

However as with many other analytical techniques, you need to have an intricate understanding of your system and how its parameters can impact your results.

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The Benefits of HRAMS

NDEA 102.07931 Da

N-Nitroso Folic Acid 470.12984 Da

Irganox 1010 1176.78408 Da

HRAMS

Multi Reflecting Time-of-Flight MS (MRT)

Poster available upon request

N-Nitrosatable APIs By LC-MRT-MS

Note: An electron is 0.55 mDa

Poster available upon request

Structural Elucidation of NDSRIs

When identifying nitrosamines in pharmaceuticals and medical devices, generation of component specific spectra for interpretation is key to maximising confidence in the assignment.

The use of the latest software tools, with modern LC-HRAMS instrumentation, enables the confident identification of trace-level components.

A data independent acquisition (DIA) approach utilising selective chromatography enables the confident elucidation of targeted and untargeted components.

How Much MS Resolution do you need?

MS resolution alone is not enough to confidently identify a nitrosamine in the presence of interferences.

High chromatographic selectivity is still critical to achieving accurate identification.

While >200,000 mass resolution is nice to have, it is not essential. The combination of a modern LC-HRAMS system with the latest software tools is equally suitable for the confident elucidation of nitrosamines.

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Maximising Sensitivity For Your Target

The default StepWave settings are optimised to cover a wide range of small molecules and typically do not need to be adjusted.

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However, the StepWave RF, StepWave 2 Wave Height and StepWave DC StepWave 2 Offset can be reduced for more efficient Transport of lower m/z and more labile species

Information adapted from Xevo G2-XS ToF training document

NDMA Sensitivity Optimisation

Default Settings

> 5-Fold Improvement

Results processed in MODDE 13 Pro Design of Experiments (DoE) Software

Maximising Sensitivity For Your Targets

While the specific optimal parameters shown are system dependent, it highlights the benefits of a tailored analytical development approach.

StepWave system-level optimisation is a small example of how we go beyond the routine and demonstrates the benefit a tailored method development approach on both our research and GMP systems.

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Conclusions

• Thorough evaluation of the method development process is necessary to identify and mitigate any risks of false positives.
• Generic nitrosamine methods should be avoided.
• A tailored method development approach is recommended to avoid false positives and maximise sensitivity.
• An in-depth understanding of your instrumentation is required to maximise the sensitivity of your methods.
• LC-HRAMS can maximise confidence in the identity of nitrosamine and the accuracy of a positive result.

Acknowledgements

Resolian Analytical Sciences

• Matthew Knox, Samantha Marshall

Resolian Bioanalytics

• Simon Noble, Szabolcs Szarka

Waters

• Martin Palmer, Emma Marsden-Edwards, Dale Cooper- Shepherd, James I. Langridge, David Eatough, Michael McCullagh

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Thank you for your Attention

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