Simultaneous Analysis of 10 Nitrosamines in an Active Pharmaceutical Ingredient Using a Triple Quadrupole Mass Spectrometer

Importance of the Topic

Trace levels of nitrosamines in pharmaceutical ingredients pose significant carcinogenic and genotoxic risks. Regulatory bodies including IARC, FDA, EMA and regional pharmacopeias require stringent control and limit levels of these mutagenic impurities to ensure drug safety.

Objectives and Study Overview

This study aimed to develop and validate a robust LC/MS/MS method for simultaneous quantification of ten nitrosamines in an active pharmaceutical ingredient (API), meeting Japanese, U.S. and European regulatory standards. The analytical workflow covers calibration, sample preparation, spike recovery and repeatability evaluations.

Methodology and Instrumentation

Sample Preparation:

• Mixed nitrosamine standard prepared in methanol and diluted in water/methanol (9:1) to 0.1–25 ng/mL.
• API sample extracted at 50 mg/mL following a modified TFDA protocol with external standard approach.

Chromatography:

• Shim-pack Velox Biphenyl column (100 mm × 2.1 mm, 2.7 µm) on Nexera X3.
• Mobile phases: water/formic acid (1000:0.5) and methanol/formic acid (1000:0.5).
• Gradient elution over 14 min, flow rate 0.6 mL/min, column temperature 45 °C, injection volume 20 µL.

Mass Spectrometry:

• LCMS-8060NX triple quadrupole with APCI in positive MRM mode.
• Probe voltage 4.0 kV, nebulizing gas 4 L/min, drying gas 3 L/min.
• DL 150 °C, heat block 200 °C, interface 300 °C.

Main Results and Discussion

• Calibration linearity 0.1–25 ng/mL for all ten nitrosamines, correlation coefficients R > 0.99.
• Accuracy ranged 80–120% and repeatability <10% RSD at 0.1 ng/mL standard level.
• Spike recovery in API at 0.01–0.1 ppm yielded 70–105% for most compounds; NDBA required further optimization.
• API matrix separation was achieved: no coelution of API components with target analytes.
• Repeatability in API spike tests remained <10% RSD across all concentrations.

Benefits and Practical Applications

• Enables compliance with ICH M7, FDA, EMA and Ph. Eur. nitrosamine limits.
• Delivers reliable quantification of mutagenic impurities at sub-ppm to ppb levels in drug substances.
• Supports QA/QC laboratories with a rapid, high-throughput analytical protocol.

Future Trends and Potential Applications

Expansion of this method could include additional nitrosamine analogs and diverse pharmaceutical matrices. Integration with high-resolution MS, automation for impurity profiling, and adoption of greener sample preparation solvents will enhance throughput and sustainability. Anticipated regulatory updates may drive further method refinement for ultra-trace controls.

Conclusion

The LCMS-8060NX triple quadrupole platform enabled a validated method for ten nitrosamines in an API with excellent linearity, sensitivity, accuracy and precision. This workflow fulfills global regulatory requirements and is suitable for routine monitoring of genotoxic impurities.

References

1. International Council for Harmonisation M7 (R1), Addendum: Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk.
2. Method of Test for Nitrosamines in Medicines – Multiple Analysis (LC-MS/MS Method), TFDA RA01I004.002, accessed May 15, 2022.