Analysis of Carcinogenic Nitrosamines at Ultra-trace Levels Among Terbinafine Using LC/TQ

Significance of the Topic

The emergence of genotoxic N-nitroso impurities in pharmaceutical products has prompted regulatory recalls by the FDA, underscoring the need for ultra-trace analysis of nitrosamine drug substance-related impurities (NDSRIs). Terbinafine, an amino-containing antifungal agent, may harbor N-nitroso derivatives that pose health risks even at sub-ppm levels. Reliable detection and quantitation of these impurities ensure product safety and compliance with stringent regulatory limits.

Objectives and Study Overview

This study aimed to develop and validate a highly sensitive LC-MS/MS method for the simultaneous determination of three NDSRIs in terbinafine: N-nitroso-desmethyl terbinafine (NDT), N-nitroso terbinafine impurity A (NTA), and N-nitroso terbinafine degradant (NTD). Key goals included achieving a limit of quantitation (LOQ) below 0.007 ppm, demonstrating linearity, precision, and recovery in both API and tablet formulations.

Methodology and Instrumentation

Sample Preparation
A 37 mg terbinafine powder sample was dissolved in 1 mL ultrapure water, vortex-mixed for 10 minutes, and centrifuged at 10 000 rpm. The clear supernatant was injected directly.

Chromatographic Conditions

• Instrument: Agilent 1290 Infinity II UHPLC
• Column: HPH-C18, 3 × 150 mm, 2.7 µm, 30 °C
• Mobile Phase: (A) 5 mM ammonium acetate in water; (B) methanol
• Gradient: 2% B (0–5 min), 50% B (5–10 min), 75% B (10–25 min), 90% B (25–38 min), re-equilibration to 2% B
• Flow Diversion: Terbinafine diverted to waste via an integrated valve to protect the mass spectrometer

Mass Spectrometry

• Instrument: Agilent 6495C Triple Quadrupole MS
• Ionization: ESI in positive mode
• Drying Gas: 250 °C, 15 L/min; Sheath Gas: 350 °C, 9 L/min; Nebulizer: 45 psi
• Capillary/Nozzle Voltage: 2000 V; Dynamic MRM with narrow resolution
• UV Detection: 220 nm (for monitoring terbinafine diversion)

Results and Discussion

Chromatographic Separation
Efficient baseline separation of NDT, NTA, and NTD was achieved, while the high-concentration terbinafine peak was diverted to waste, preventing contamination of the MS source.

Sensitivity and Linearity
Calibration was linear over 0.125–2 ng/mL with correlation coefficients (r²) ≥ 0.997 for all analytes. Signal-to-noise ratios at the LOQ (0.25 ng/mL) exceeded 15:1 for all NDSRIs.

Precision and Recovery

• Precision (RSD) at LOQ: NTD (6.8%), NTA (6.6%), NDT (5.1%)
• Recovery in API: 96.1–99.2%; in tablet: 93.3–108.1%

The results confirm robustness, reproducibility, and accuracy at the target sub-ppb concentration (0.007 ppm).

Benefits and Practical Applications

• Regulatory Compliance: Meets stringent guidelines for nitrosamine impurities.
• High Sensitivity: Enables detection at sub-ppb levels.
• Operational Robustness: Efficient diversion of matrix-rich drug peak protects MS system.
• Versatility: Applicable to both API and finished dosage forms.

Future Trends and Opportunities

Advancements may include:

• Automation of sample preparation and data processing.
• Integration with high-resolution MS for broader impurity profiling.
• Adaptation to other nitrogen-containing APIs prone to nitrosamine formation.
• Real-time monitoring in continuous manufacturing environments.

Conclusion

• A robust LC-MS/MS method for three terbinafine NDSRIs was established.
• LOQ of 0.25 ng/mL (0.007 ppm) with excellent linearity (r² ≥ 0.997).
• Precision at LOQ: RSD < 7%; recovery within 90–110% in API and tablets.
• Method supports stringent safety testing in pharmaceutical quality control.

Reference

• Low-Level Quantitation of N-Nitroso Dabigatran Etexilate Impurity in Dabigatran Etexilate Mesylate API Using the Agilent 6495C. Agilent Application Note, 5994-7066EN.
• Nitrosamine Impurities Application Guide. Agilent Application Guide, 5994-2393EN.