Quantitation of 8 Nitrosamines in 12 different solvents by LC-MS/MS system

Significance of the Topic

Recent regulatory findings of nitrosamine impurities in pharmaceuticals have underscored the need for sensitive, reliable analytical methods to monitor N-nitroso compounds. Nitrosamines are potent genotoxic compounds that can form during chemical synthesis and solvent recovery. Ensuring solvent purity with low detection limits is essential for pharmaceutical quality control and patient safety.

Objectives and Study Overview

This study presents a unified LC-MS/MS protocol for the simultaneous quantification of eight nitrosamines—NDMA, NMBA, NDEA, NEIPA, NDPA, NDIPA, NMPA and NDBA—in twelve common organic solvents. The method aims to meet stringent regulatory thresholds (0.03 ppm) and to offer a flexible workflow adaptable to diverse solvent matrices.

Methodology and Used Instrumentation

Three sample-preparation strategies were optimized based on solvent properties:

• Direct injection for water, methanol, ethanol, acetonitrile, isopropanol and DMSO.
• Evaporation and reconstitution (40 °C, 40 min) for dichloromethane, acetone, chloroform and ethyl acetate.
• Dilution in methanol for toluene and dimethylformamide (DMF).

Quantitative analysis was performed using:

• UHPLC Nexera XS system with Shim-pack GIST C18-AQ column (100 × 4.6 mm, 3 µm) at 40 °C and a formic acid–methanol gradient.
• Shimadzu LCMSTM-8045 triple quadrupole mass spectrometer with APCI interface (350 °C), 3 L/min nebulizer and 5 L/min drying gas flows.
• Multiple reaction monitoring (MRM) transitions with four deuterated internal standards.

Main Results and Discussion

Calibration was linear from 1 to 100 ppb (R² > 0.993) for all analytes. Method limits of quantitation ranged from 0.001 ppm (water) to 0.010 ppm (various organics). Recoveries at LOQ levels fell between 90 % and 120 % for most compounds. Direct injection delivered rapid results for polar solvents, whereas evaporation improved sensitivity in mid‐boiling solvents. DMF and toluene required dilution to avoid matrix interferences. GC-MS remains a complementary option for NDMA in DMF due to coelution issues.

Benefits and Practical Applications

This single LC-MS/MS approach:

• Meets current EMA and FDA nitrosamine limits with LOQs well below 0.03 ppm.
• Reduces method development time by covering multiple solvent classes in one protocol.
• Utilizes stable isotope internal standards for robust quantitation across variable matrices.

The technique can be extended to additional solvents and to other nitrosamine analogues, supporting routine solvent screening in pharmaceutical and chemical industries.

Future Trends and Applications

Advances in high-throughput mass spectrometry and novel ionization techniques are expected to further lower detection limits. Automated sample preparation platforms may increase throughput and reduce operator variability. Expanding the nitrosamine panel to include emerging analogues and coupling with high-resolution mass spectrometry could enhance structural elucidation and regulatory compliance.

Conclusion

An efficient LC-MS/MS workflow was established for eight nitrosamines in twelve solvents, delivering high sensitivity, broad applicability and compliance with regulatory standards. This method enhances solvent safety assessment and streamlines quality control in pharmaceutical production.

Reference

Shailendra Rane, Devika Tupe, Deepti Bhandarkar, Jeetendra Kelkar and Pratap Rasam. Quantitation of Eight Nitrosamines in Twelve Different Solvents by LC-MS/MS, Shimadzu Analytical (India) Pvt. Ltd., May 2021.