Analysis of genotoxic impurities in pharmaceuticals

Importance of the Topic

Genotoxic impurities (GTIs) are trace-level contaminants capable of inducing DNA damage and driving mutagenesis and carcinogenesis even at nanogram concentrations. In recent years, recalls of common pharmaceuticals—including ranitidine, metformin, and sartan-class drugs—have underscored the urgent need for highly sensitive, selective, and reliable analytical methods to monitor GTIs throughout drug development and manufacturing.

Objectives and Study Overview

This work presents an integrated, single-vendor solution for the detection and quantification of GTIs, including nitrosamines, azido impurities (AZBT), and nitroso drug substance related impurities (NDSRIs), in active pharmaceutical ingredients (APIs) and finished dosage forms. The study benchmarks method performance across multiple sample matrices and impurity classes to demonstrate compliance with ICH S2(R1) regulatory thresholds.

Methodology and Instrumentation

Analyses were performed using Thermo Scientific Vanquish Flex UHPLC systems coupled to an Orbitrap Exploris 120 high-resolution mass spectrometer, managed by Chromeleon chromatography data system (CDS). Key features include:

• SmartFlow pumping technology for precise gradient control and Viper fingertight fittings for near-zero dead volume.
• Segmented quadrupole for isolation widths down to 0.4 Da and an ion routing multipole with HCD fragmentation.
• High-field Orbitrap mass analyzer achieving up to 120,000 resolution (FWHM at m/z 200) and mass accuracy below 1 ppm with EASY-IC internal calibration.
• Chromeleon CDS for full 21 CFR Part 11 compliance, audit trails, and electronic signatures.
• Diverse column chemistries—C18, biphenyl, mixed mode WCX—to optimize separation and enable divert-valve protection of the MS from high-concentration APIs.
• Optima LC/MS-grade solvents, PVDF filters, and dedicated vials to minimize chemical noise and ensure reproducible sample preparation.

Main Results and Discussion

Across nine sample types (tablets, APIs, and formulations), limits of detection ranged from 0.025 to 0.3 ng/mL and limits of quantitation from 0.25 to 1 ng/mL, with regulatory specification limits up to 13.7 ng/mL. The method demonstrated:

• Excellent linearity across multiple concentration ranges (e.g., 0.5–30 ng/mL for eight nitrosamines).
• Robustness over 40 hours of continuous injection with consistent peak areas and retention times.
• Chromatographic resolution sufficient to separate seven nitrosamines from a dydrogesterone peak and to isolate AZBT in sartan mixtures using a biphenyl column.
• Mass accuracy stability: <3 ppm drift over 24 h (external calibration), <1 ppm with EASY-IC internal lock mass, and <3 ppm over 4 weeks with one-point calibration.

Benefits and Practical Applications of the Method

This fully integrated UHPLC–HRMS platform streamlines method development, sample throughput, and data compliance for quality control laboratories. High sensitivity and selectivity enable simultaneous screening of multiple GTIs at levels well below regulatory thresholds. Automated calibration and robust column performance reduce downtime and support rapid analytical throughput for both API and final product testing.

Future Trends and Potential Applications

Advancements may include further automation of sample preparation, AI-driven method optimization for broader impurity classes, and miniaturized flow systems to reduce solvent consumption. Integration with laboratory information management systems (LIMS) and expansion into nontraditional drug modalities will extend the applicability of high-resolution MS workflows.

Conclusion

The combination of Vanquish Flex UHPLC, Orbitrap Exploris 120 MS, and Chromeleon CDS provides a compliant, sensitive, and high-throughput solution for quantifying genotoxic impurities. This approach ensures confidence in data integrity and supports regulatory compliance for modern pharmaceutical quality control.

Reference

1. Genotoxic impurities in pharmaceutical products. European Pharmaceutical Review, 2020. Available at https://www.europeanpharmaceuticalreview.com/article/108031/genotoxic-impurities-in-pharmaceutical-products/
2. “10 Prominent Drugs Recalled Over Nitrosamines,” Pharmaceutical Processing World. Available at https://www.pharmaceuticalprocessingworld.com/10-prominent-drugs-recalled-over-nitrosamines/