When Multiple Analytical Challenges Converge
Your pharmaceutical sponsor needs ultra-trace quantification of an N-nitrosamine drug substance-related impurity in an allergy relief product. Sub-1 ng/mL detection limits are required. The sample matrix is complex. And there’s no commercially available deuterated internal standard to compensate for matrix effects.
Challenging, but manageable with the right approach.
Then you discover that croscarmellose sodium in the formulation swells to 4-8 times its original volume when it contacts water, rendering your proven offline solid-phase extraction cleanup process completely impossible to perform.
Now you have compounding analytical challenges with no obvious path forward. This is where standard method development approaches reach their limits.
Watch: Advanced Separation Strategy for Impossible Analytics
In this video, Resolian analytical scientist Tom Kershaw demonstrates how online SPE with mixed-mode column switching overcomes multiple simultaneous obstacles to achieve GMP-validated ultra-trace nitrosamine quantification.
N-nitrosamines represent one of the pharmaceutical industry’s most significant impurity challenges. These carcinogenic compounds form readily from nitrosatable amine groups present in many active pharmaceutical ingredients, creating mutagenic impurities that pose serious patient health risks. Regulatory authorities worldwide require manufacturers to risk-assess all products for nitrosamine presence and develop validated analytical methods where risks are identified.
Tom’s project involved developing and validating a quantitative method for NDSRI analysis in a complex drug product formulation where standard approaches simply wouldn’t work.
What You’ll Learn in This Video:
- The regulatory landscape requiring ultra-sensitive nitrosamine analysis
- Why this particular allergy relief formulation presented unique challenges
- How croscarmellose sodium’s swelling properties blocked offline SPE approaches
- The limitations of adding pre-extraction steps to remove problematic excipients
- Why lack of deuterated internal standard compounds analytical difficulties
- Online SPE theory and mixed-mode separation strategy
- Column switching approach: C18 for API/excipient retention, HILIC for salt management
- How the method prevents matrix effects from enhancing or suppressing analyte signal
- Comparative performance: offline SPE versus online SPE
- GMP validation according to ICH Q2 R1 guidelines despite analytical obstacles
Tom’s approach demonstrates that when conventional techniques fail, advanced separation science provides alternative pathways to validated methods.
Understanding the Croscarmellose Sodium Problem
Croscarmellose sodium is a common pharmaceutical excipient used as a superdisintegrant in tablets. Its purpose is to rapidly absorb water and swell, facilitating tablet disintegration and drug release. This same property that makes it valuable in formulations creates severe analytical challenges.
When sample solutions contact water (the typical diluent for offline SPE cleanup), the croscarmellose rapidly expands to 4-8 times its original volume. This:
- Blocks SPE cartridges, preventing flow
- Renders extraction impossible to complete
- Eliminates the proven cleanup approach used successfully for other formulations
Attempting to remove croscarmellose through additional pre-extraction steps introduces new problems:
- Dramatically increased procedure complexity
- Extended preparation time
- Additional opportunities for analyte loss
- Multiple extraction steps without internal standard compensation
Without a deuterated internal standard to account for recovery variations, these additional manipulations severely impact method reproducibility and sensitivity.
The Online SPE Advantage
Online solid-phase extraction fundamentally changes the analytical approach by integrating cleanup directly into the chromatographic separation. Rather than performing offline extractions and cleanups before injection, the method uses column switching to achieve separation and cleanup simultaneously.
Tom’s mixed-mode approach uses intelligent column switching:
- First dimension (C18 reverse-phase column):
- Retains the API and excipients
- Allows poorly retained analyte to pass through
- Provides initial separation and cleanup
- Second dimension (HILIC normal-phase column):
- Retains salts and matrix components
- Allows analyte to elute cleanly
- Prevents matrix effects from suppressing or enhancing signal
This two-dimensional separation addresses multiple challenges simultaneously. The API doesn’t interfere with the analyte. Salts don’t cause matrix effects. The problematic excipient doesn’t block the process. And the entire approach is automated, eliminating manual extraction steps that would amplify the impact of having no internal standard.
Achieving Validation Without Internal Standard
Deuterated internal standards are standard practice in LC-MS/MS quantification because they compensate for:
- Matrix effects during ionization
- Recovery variations during extraction
- Instrument response variability
- Sample preparation inconsistencies
When no appropriate internal standard exists commercially, method development becomes significantly more challenging. Every source of variability that an internal standard would normally correct must be controlled through method design instead.
Online SPE provides this control through:
- Automated, reproducible injection and switching
- Consistent separation eliminating matrix effects
- No manual extraction steps that introduce variability
- Superior cleanup reducing ion suppression/enhancement
The result: effective and reproducible quantification despite the lack of internal standard, achieving GMP validation according to ICH Q2 R1 guidelines.
When Standard Approaches Aren’t Enough
This project exemplifies the reality of pharmaceutical method development: regulatory requirements don’t adjust for analytical difficulty. When ultra-trace quantification is required for patient safety, the method must deliver regardless of matrix complexity, excipient interference, or internal standard availability.
The difference between successful and failed method development often lies in recognizing when to abandon conventional approaches and explore advanced separation strategies.
Expert Problem-Solving for Challenging Analyses
Resolian’s analytical sciences team specializes in ultra-trace analysis and complex method development for regulatory compliance. Our expertise includes:
- NDSRI and nitrosamine impurity analysis at sub-ng/mL levels
- Advanced separation techniques including online SPE and multi-dimensional chromatography
- Method development for challenging matrices and formulations
- Strategies for quantification when internal standards aren’t available
- GMP validation for regulatory submission
When analytical challenges seem insurmountable, we bring the separation science expertise and creative problem-solving that transforms obstacles into validated methods.
Ready to discuss your challenging nitrosamine analysis or ultra-trace quantification needs?
Contact our analytical sciences team to explore how advanced separation strategies can address your specific requirements.
