HRMS QTOF-PRM (Parallel reaction Monitoring) for ultra-trace level quantification of N-Nitroso Moxifloxacin in API and Formulations.

Significance of the topic

Accurate quantification of nitrosamine impurities in pharmaceuticals is critical due to their potential genotoxicity and carcinogenicity. N-Nitrosomoxifloxacin (N-MOX), a nitrosamine related to the widely used antibiotic moxifloxacin, is subject to strict regulatory intake limits (CPCA Category 4: 1500 ng/day). Sensitive and selective analytical workflows are therefore required to detect and quantify N-MOX at concentrations well below these regulatory thresholds in API, placebo, and finished dosage forms to ensure patient safety and compliance.

Objectives and overview of the study

The study aimed to develop and validate a high-resolution mass spectrometry (HRMS) workflow using Parallel Reaction Monitoring (PRM) on an Agilent Revident LC/Q-TOF for ultra-trace quantification of N-MOX in API and tablet formulations. Key goals were method transfer from triple-quadrupole MRM to Q-TOF PRM, attainment of regulatory-relevant sensitivity, avoidance of false positives via accurate-mass MS/MS, and demonstration of robustness across matrices (API, placebo, tablets).

Methodology

Sample preparation:

• Tablet samples: Five 400 mg moxifloxacin tablets were weighed, crushed, homogenized; an aliquot corresponding to a 1.5 mg/mL API test solution was prepared (2.74 mg tablet powder per mL).
• API & placebo: Spiked solutions were prepared to simulate finished product matrices.
• Regulatory threshold: CPCA Cat 4 (1500 ng/day per 400 mg tablet) corresponds to ~5.63 ng/mL in the 1.5 mg/mL test solution and served as the decision concentration for method performance.

Analytical approach:

• Initial optimization of MRM transitions, collision energies (CE) and fragmentor voltages (FragV) was performed on a triple quadrupole instrument.
• Optimized parameters were transferred to the Agilent Revident LC/Q-TOF PRM workflow by averaging CE and FragV across transitions to promote comparable fragmentation on Q-TOF.
• High-resolution full MS/MS spectra were acquired for targeted precursors to enable accurate-mass quantitation and fragment confirmation, improving selectivity and eliminating false positives.

Used instrumentation

• Liquid chromatography: Agilent 1290 Infinity III (LC).
• Mass spectrometry: Agilent Revident LC/Q-TOF operated in PRM (high-resolution full MS/MS) mode.

Main results and discussion

Analytical performance:

• Sensitivity: The method achieved a lower limit of quantitation (LLOQ) of 12.2 pg/mL and an upper limit of quantitation (ULOQ) of 25 ng/mL — well below the regulatory decision threshold (~5.63 ng/mL).
• Linearity: Calibration across the validated range produced excellent linearity with R² = 0.9997 (weighted 1/x linear regression).
• Selectivity: The monitored PRM transition m/z 431.1725 → 301.1 delivered high selectivity with no detected interference from moxifloxacin API or excipient matrix components. Chromatographic separation confirmed lack of crosstalk between analyte and API peaks.
• Precision and recovery: Quality control repeatability showed low variability (example CV 1.17% at QC concentration), and recovery and accuracy metrics met expectations across API, placebo and tablet matrices.
• Signal quality: High signal-to-noise ratios were reported for both standard LLOQ injections and tablet extracts, supporting confident peak detection at ultra-trace levels.

Transfer strategy and benefits of PRM on Q-TOF:

• Transferring MRM-optimized parameters to PRM allowed retention of targeted-fragment specificity while adding accurate-mass confirmation from HRMS full MS/MS data.
• Accurate-mass fragment verification minimizes false positives, a key benefit when screening complex pharmaceutical matrices for nitrosamines.

Benefits and practical applications of the method

• Regulatory relevance: Sensitivity and accuracy meet or exceed CPCA category limits, supporting impurity control and release testing of APIs and finished products.
• Matrix robustness: Demonstrated performance in API, placebo and tablet matrices indicates suitability for routine QC and stability testing.
• High confidence reporting: Accurate-mass PRM enables simultaneous quantitation and structural confirmation, reducing risk of misidentification common with nominal-mass methods.
• Method transferability: The described parameter-averaging approach smooths migration from triple-quadrupole MRM workflows to HRMS PRM implementations.

Future trends and potential applications

• Extension to other nitrosamines: The PRM/HRMS workflow is broadly adaptable to additional NDSRIs (nitrosamine drug substance related impurities) after suitable optimization.
• Use of isotopically labelled internal standards: Incorporating labeled surrogates would further improve accuracy and compensate for matrix effects in complex formulations.
• Automation and high-throughput screening: Integration with automated sample prep and batch-processing workflows can enable routine screening of large sample sets in QC environments.
• Orthogonal confirmation: Combining HRMS PRM with orthogonal techniques (GC-HRMS for volatile nitrosamines or orthogonal fragmentation methods) will strengthen regulatory submissions.
• Regulatory harmonization and adoption: As regulatory expectations for nitrosamine control evolve, HRMS-based PRM methods are likely to gain wider acceptance for their selectivity and confirmatory power.

Conclusions

The study demonstrates that PRM on an Agilent Revident LC/Q-TOF provides a regulatory-ready, high-confidence platform for ultra-trace quantification of N-nitrosomoxifloxacin in API and formulated products. With an LLOQ of 12.2 pg/mL, excellent linearity, strong matrix selectivity and robust precision, accurate-mass PRM eliminates false positives and meets the sensitivity required by CPCA Category 4 limits. The approach is extensible to other nitrosamine impurities and supports implementation in QC laboratories concerned with nitrosamine risk management.

References

1. Balfour JA, Wiseman LR. Moxifloxacin. Drugs. 1999 Mar;57(3):363-373; discussion 374. doi:10.2165/00003495-199957030-00007.
2. Nakka S, et al. A facile and eco-friendly simultaneous quantification LC-TQMS/MS approach for N-Nitroso Moxifloxacin and di-nitroso pyrrolopiperidine in Moxifloxacin tablets and eye drops. Green Analytical Chemistry. 2025;12:100188.
3. U.S. Food and Drug Administration. Guidance for Industry: Recommended Acceptable Intake Limits for NDSRIs. August 2023.
4. European Medicines Agency. Nitrosamine impurities: guidance for marketing-authorisation holders. European regulatory guidance on nitrosamine control.