Highly Sensitive Quantitation of N-Nitroso Timolol Impurity in Timolol API using the Agilent 6495D LC/TQ

Importance of the Topic

Nitrosamine impurities in pharmaceuticals are potent mutagens and potential carcinogens.
Regulatory agencies have imposed strict limits on nitrosamine drug substance-related impurities after several product recalls to safeguard patients.
Screening APIs such as timolol for N-nitroso derivatives is essential for quality control and compliance.

Objectives and Study Overview

• Develop a sensitive and specific LC–TQ method for quantifying N-nitroso timolol in timolol API.
• Achieve detection limits below 10% of the specified threshold.
• Validate the method in accordance with ICH guidelines.

Methodology and Instrumentation

The analysis employed reversed-phase chromatography on an Agilent InfinityLab Poroshell Phenyl-Hexyl column (150×3.0 mm, 2.7 μm) coupled to an Agilent 6495D triple quadrupole mass spectrometer and 1290 Infinity II LC.

• Mobile Phase A: 1 mM ammonium trifluoroacetate with 0.05% formic acid in water.
• Mobile Phase B: 0.1% formic acid in methanol.
• Gradient elution over 20 minutes at 0.4 mL/min and 40 °C column temperature.
• MRM transitions for N-nitroso timolol: m/z 346.2→128, 346.2→144, 346.2→130 with optimized collision energies.

Source settings included positive AJS-ESI, 3000 V capillary voltage, 12 L/min gas flows, and ionization temperatures of 250–350 °C.

Main Results and Discussion

• Linearity: 5–1500 pg/mL (0.5–150 ppb) with R² > 0.9990 using 1/x² weighting.
• LOD: 2 pg/mL (0.2 ppb) with S/N > 118:1; LOQ: 5 pg/mL (0.5 ppb) with S/N > 1680:1.
• Precision: %RSD below 5% across six replicate injections at LOD, LOQ, and mid-range levels.
• Recovery: 98.5% average at 50 pg/mL spike (5 ppb) in API matrix, within 90–110% criteria.
• Chromatographic separation achieved baseline resolution at retention time ~8.96 min, confirmed by UV and MRM traces.

Benefits and Practical Applications

• Enables reliable monitoring of trace-level nitrosamines in timolol API.
• Supports compliance with global regulatory requirements for nitrosamine limits.
• Methodology adaptable to other pharmaceutical nitrosamine analyses.

Future Trends and Opportunities

• Expansion of MRM workflows to cover multiple nitrosamine impurities.
• Automation of sample preparation for higher throughput.
• Integration of high-resolution mass spectrometry for confirmation and structure elucidation.

Conclusion

The developed LC/TQ method delivers high sensitivity, precision, and accuracy for quantifying N-nitroso timolol at trace levels, ensuring robust quality control and regulatory compliance in pharmaceutical production.

Reference

1. FDA Guidance: Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities, CDER, August 2023.
2. Agilent Application Note 5994-7066E: Low-Level Quantitation of N-Nitroso Dabigatran Etexilate Impurity Using the Agilent 6495C LC/TQ.