Quantitation of 7 N-nitrosamines in Monoclonal Antibody (mAb) Formulations Using LC-MS/MS

Significance of the Topic

Trace levels of N-nitrosamines in pharmaceutical products present significant mutagenic and carcinogenic risks. Regulatory agencies such as ICH M7 and EMA have extended their oversight to include biological medicines, including monoclonal antibody formulations. Reliable detection and quantitation of these impurities are essential for patient safety and compliance with evolving global guidelines.

Objectives and Study Overview

This study aimed to develop and validate a robust LC-MS/MS method for the quantitation of seven structurally diverse N-nitrosamines in monoclonal antibody (mAb) formulations. The workflow leverages advanced sample preparation and MRM-based detection to achieve low limits of quantitation in complex biological matrices.

Methodology and Instrumentation

The analytical protocol involved:

• Sample preparation: mAb formulation samples incubated at 70 ºC for 30 minutes, centrifugation at 12 000 rpm at 5 ºC, and direct injection of the supernatant.
• Chromatography: Shimadzu Shim-pack Scepter C8 column (150 mm × 4.6 mm, 5 μm) at 40 ºC with a water/formic acid and methanol gradient (0.5 mL/min).
• Mass spectrometry: Shimadzu LCMS-8060NX with an APCI source; MRM transitions optimized via LabSolutions auto-MRM for collision energy and voltage settings.
• Calibration: Eight-point matrix-matched curves (50–2000 ppb) spiked with four isotope-labeled internal standards; LOQ established at 50 ppb (S/N > 18, RSD < 20%).

Used Instrumentation

• UHPLC System: Shimadzu Nexera X3
• Analytical Column: Shim-pack Scepter C8-120 (150 mm × 4.6 mm, 5 μm)
• Mass Spectrometer: Shimadzu LCMS-8060NX Triple Quadrupole
• Ion Source: Atmospheric Pressure Chemical Ionization (APCI)

Main Results and Discussion

Calibration curves for all seven N-nitrosamines demonstrated excellent linearity (r2 ≥ 0.996). Repeatability at the LOQ level yielded RSD values below 20%. Recovery experiments in a representative mAb sample spiked at 200 ppb produced recoveries between 70 % and 130 %, confirming method accuracy. Analysis of six commercial mAb formulations showed that most N-nitrosamines were below the LOQ, underscoring the method’s suitability for routine quality control.

Benefits and Practical Applications

This validated approach offers:

• High sensitivity for trace-level N-nitrosamine detection in proteinaceous matrices.
• Rapid sample throughput with minimal column fouling.
• Regulatory compliance support for biologics in global markets.

Future Trends and Potential Applications

As regulatory scrutiny intensifies, laboratories will adopt higher-throughput LC-MS platforms and enhanced ion optics for greater robustness. Automation of sample preparation and integration with data-management systems will accelerate release testing for a wider range of biological therapeutics. Emerging soft ionization techniques may further improve sensitivity for novel nitrosamine analogs.

Conclusion

The reported LC-MS/MS method on the Shimadzu LCMS-8060NX achieved precise quantitation of seven N-nitrosamines in mAb formulations, with low LOQs, strong linearity, and acceptable recoveries. The instrument’s advanced ion-lens design enhanced robustness, making this workflow well suited for routine monitoring of critical impurities in biopharmaceutical quality control.

References

• Shimadzu Analytical (India) Pvt. Ltd. Application note: Quantitation of 7 N-nitrosamines in mAb formulations using LCMS-8060NX, First Edition: Aug. 2025.