Detection and Quantitation of Nitrosamine Impurities in Drug Substances by LC-HRMS on LCMS-9030

Significance of the Topic

The presence of N-nitrosamine impurities in pharmaceutical products has become a major safety concern due to the probable carcinogenicity of compounds such as NDMA and NDEA. Regulatory agencies worldwide require highly sensitive and selective analytical methods to detect and quantify trace levels of these impurities in drug substances to ensure patient safety and compliance.

Objectives and Study Overview

This work describes the development and validation of a targeted high-resolution LC-HRMS (Q-TOF) method on the Shimadzu LCMS-9030 platform for simultaneous detection and quantitation of eight N-nitrosamines in drug substance matrices. The study aimed to optimize MS/MS acquisition parameters, establish calibration performance, and demonstrate applicability in metformin and angiotensin II receptor blocker (ARB) APIs.

Methodology

Standard Preparation:

• Individual nitrosamine stock solutions (100 µg/mL) in methanol.
• Mixed working solutions at 100 ng/mL for calibration in MeOH/H₂O (15:85 v/v) with 0.1% formic acid.

Sample Preparation:

• Metformin API: 150 mg dissolved in 1.5 mL diluent, vortexed, shaken and centrifuged; supernatant filtered (0.22 µm).
• ARB APIs (losartan, candesartan): 20 mg/mL extract prepared by FDA guidelines, flow diversion used to protect MS from high-concentration peaks.

Used Instrumentation

Shimadzu LCMS-9030 Q-TOF system with DUIS ESI source.
Shim-pack Solar C18 column (4.6 × 250 mm, 5 µm) at 0.8 mL/min, 45 °C.
Mobile phase A: water + 0.1% formic acid; B: methanol + 0.1% formic acid; gradient from 15% to 95% B.
MS/MS acquisition: quadrupole isolation ±2 m/z, collision energies 2–9 V, TOF mass range m/z 60–170, XIC tolerance ±15 ppm (±20 ppm for NDPA).

Main Results and Discussion

Calibration and Sensitivity:

• Linear dynamic range 0.5–100 ng/mL (NDBA: 0.25–10 ng/mL) with R² ≥ 0.994.
• LODs of 0.1–0.5 ng/mL and LOQs of 0.2–1 ng/mL in solvent.
• Accuracy at LOQ 92.3–116.5%; repeatability (RSD) 1.9–15.2% at 1 ng/mL.

Application in API Matrices:

• Metformin extract (100 mg/mL): LOQs correspond to 0.005–0.01 ppm; LODs 0.002–0.005 ppm.
• Losartan and candesartan extracts (20 mg/mL): LOQs 0.013–0.05 ppm; LODs 0.005–0.025 ppm.

The targeted MS/MS approach improved sensitivity over full-scan acquisition and effectively distinguished co-eluting interferences such as DMF isotopes from NDMA.

Benefits and Practical Applications

• Simultaneous quantitation of eight nitrosamines in a single run.
• High mass accuracy and resolution ensure selectivity in complex matrices.
• Compliance with FDA guidelines for nitrosamine testing in APIs.
• Robust calibration and low detection limits support routine QA/QC screening.

Future Trends and Opportunities

Further extension to additional nitrosamines and related impurities, automation of sample preparation, high-throughput screening workflows, integration with ion mobility, and adoption of ambient ionization techniques may enhance screening speed and applicability in continuous manufacturing settings.

Conclusion

A targeted LC-HRMS method on the LCMS-9030 Q-TOF was successfully developed and validated for eight N-nitrosamines in pharmaceutical substrates. The approach delivered excellent sensitivity, linearity, precision, and accuracy across solvent and API matrices, meeting regulatory requirements and enabling reliable routine monitoring.

References

1. S. Rane et al., Quantitation of 6 Nitrosamines in 5 Sartans by LC-MS/MS, USP <1469> Application Note.
2. US FDA, LC-HRMS Method for Six Nitrosamines in ARB Drugs, 05/21/2019.
3. US FDA, LC-ESI-HRMS Method for Nitrosamine Impurities in Metformin, 06/03/2020.
4. J. Yang et al., AAPS Journal 22:89 (2020).