Solving Matrix Suppression in Ultra-Trace NDSRI Analysis

The Recovery Experiment That Revealed Suppression

After initial method development to separate the API (the largest co-eluting interference) from the target nitrosamine, Tom performed a critical experiment to determine if the absence of analyte signal was genuine.

The approach: spike a known quantity of nitrosamine into samples where the API concentration varied at 0.5 mg/mL, 0.1 mg/mL, and 0.01 mg/mL.

Expected outcomes if suppression wasn’t occurring:

• If analyte present in product:5 mg/mL sample should show largest peak due to combined native + spiked analyte
• If analyte truly absent: All three concentrations should show equal intensity (only spiked amount)

Actual result: Neither pattern appeared.

This unexpected outcome revealed that suppression was present, masking the true analyte response. Without this experiment, the method would have reported results that didn’t reflect reality.

Identifying the Culprits: Polysorbate and Benzoates

Using full-scan mode on the mass spectrometer, Tom identified that polysorbate (a common pharmaceutical excipient) was co-eluting with the target analyte. The telltale chromatogram signature confirmed the interference.

Based on prior experience, Tom selected a pentafluorophenyl stationary phase, which provides alternative selectivity compared to standard C18 columns. This successfully separated the polysorbate from the analyte.

Problem solved? Not quite.

Further recovery experiments revealed that additional excipients were still co-eluting and causing suppression. Using the known excipient list, Tom determined that two benzoates had potential to interfere. Through additional mobile phase screening (column changes risked re-introducing polysorbate co-elution), one benzoate achieved baseline separation by 0.5 minutes.

The Systematic Approach to Matrix Complexity

Tom’s methodology exemplifies best practices for ultra-trace analysis in complex matrices:

1. Initial separation development – Distinguish API from analyte
2. Recovery experiments – Verify absence vs. suppression
3. Full-scan MS investigation – Identify specific interfering compounds
4. Strategic column selection – Exploit alternative selectivity mechanisms
5. Iterative screening – Address each interference source systematically
6. Validation beyond requirements – Build confidence through repeatability/precision

Each step builds upon previous findings, progressively eliminating sources of interference until the method delivers results you can trust.

The Performance Improvement

Following this tailored method development, the performance improvement was dramatic. By systematically eliminating excipient interferences, the method achieved:

• Confident determination that analyte levels were genuinely below detection limits
• Not artificially suppressed by matrix effects
• Validated repeatability and precision (exceeding ICH Q2 R1 requirements for limit test trace analysis)
• Increased confidence in reported results

While ICH Q2 R1 doesn’t require repeatability and precision validation for limit test methods, performing these experiments adds crucial confidence that results reflect product reality rather than analytical artifacts.

Why Tailored Method Development Matters

Formulated drug products contain diverse excipients, each with potential to interfere with ultra-trace analysis. Polysorbates, benzoates, surfactants, preservatives, and other formulation components can:

• Co-elute with target analytes
• Suppress ionization in the mass spectrometer
• Enhance signals through matrix effects
• Create interferences that vary with concentration

Standard method development approaches may work for simpler matrices, but complex formulations demand tailored strategies that:

• Systematically identify each interference source
• Test recovery across concentration ranges
• Verify that “no detection” means genuine absence
• Build confidence through rigorous validation

For nitrosamine analysis where patient safety depends on accurate results, this thoroughness isn’t optional.

Expert Method Development for Complex Matrices

Resolian’s analytical sciences team specializes in tailored method development for ultra-trace impurity analysis in complex formulated products. Our expertise includes:

• Systematic investigation of matrix suppression and enhancement effects
• Strategic selection of stationary phases for alternative selectivity
• Mobile phase optimization for challenging separations
• Recovery experiments that verify genuine results vs. matrix artifacts
• Validation approaches that exceed regulatory requirements
• Experience with diverse pharmaceutical excipients and their analytical impacts

When standard methods don’t deliver confident results, we bring the systematic problem-solving and chromatographic expertise that reveals the truth hidden in complex matrices.