(M1230-09-56) Trace Level Quantification of Nitrosamine Impurities in Tuberculosis Medicines by LC-MS/MS

Purpose: Since 2018, the presence of mutagenic nitrosamine impurities in drugs has attracted international attention. In 2020, the FDA became aware of the nitrosamines, 1-cyclopentyl-4-nitrosopiperazine (CPNP) and 1-methyl-4-nitrosopiperazine (MNP), in rifapentine and rifampin, essential medicines for the treatment of tuberculosis. Highly sensitive analytical methods are desired due to suggested limits being as low as 0.1 ppm for CPNP in rifapentine and 0.16 ppm for MNP in rifampin. An LC-HRMS/MS method using a hybrid quadrupole-orbitrap mass spectrometer was reported previously for quantification of CPNP and MNP in those drugs. To increase the applicability of the method, our laboratory adapted the reported method to LC-QqQ MS (LC-MS/MS).

Methods: When adapting the LC-HRMS method to LC-QqQ MS, recovery of CPNP from spiked rifapentine samples was low, due to ionization suppression from matrix interferences (e.g., impurities, excipients) that coeluted with CPNP. To reduce matrix effects, we modified the LC gradient, reduced sample concentration, and used stable isotope-labeled internal standard. We also quantified the samples against linearity curve rather than against a standard solution at a single level as in the reported method. For the quantification of MNP in rifampin, a stable isotope-labeled internal standard and quantification against linearity curve were also necessary for method accuracy. No further modification of the reported method was required. Finally, two LC-MS/MS methods using multiple reaction monitoring were validated for quantification of CPNP and MNP.

Results: The two LC-MS/MS methods were validated in the range of 0.05 – 10 ppm for CPNP in rifapentine drug substance and products and 0.05 – 5 ppm for MNP in rifampin drug substance and products. The methods were determined to be specific and linear. Accuracy was within 70% – 130% and reproducibility was below 20% RSD. The levels of CPNP found in the tested rifapentine samples ranged from 8 to 17 ppm, and the levels of MNP found in the tested rifampin samples ranged from 2 to 9 ppm. These levels far exceed the suggested limits and highlight the urgency for nitrosamine control in pharmaceuticals. The two methods presented may be used by rifapentine and rifampin drug manufacturers to monitor and investigate ways to reduce the levels of mutagenic nitrosamines in their products. Matrix effects were shown to be critical considerations and should be effectively mitigated in trace level quantification of nitrosamine impurities in pharmaceuticals by LC-MS/MS.

Conclusion: Two LC-MS/MS methods for nitrosamine quantification were developed, validated, and may be applied to help ensure quality of tuberculosis medicines.

Acknowledgements: Certain commercial equipment, instruments, or materials may be identified in this poster to specify adequately the experimental procedure. Such identification does not imply approval, endorsement, or certification by USP of a particular brand or product, nor does it imply that the equipment, instrument, or material is necessarily the best available for the purpose.

Method details

Representative calibration curve for CPNP

Representative chromatograms