An automated sample preparation workflow for the analysis of nitrosamines in metformin

Significance of the topic

Nitrosamines are classified as probable carcinogens and have been detected as impurities in pharmaceutical products such as valsartan and metformin, triggering recalls and regulatory scrutiny. Accurate, sensitive, and selective detection of these compounds is essential to ensure patient safety and compliance with global guidelines. Traditional manual sample preparation for nitrosamine analysis is labor-intensive, time-consuming, and prone to contamination and human error. Automating this process can improve throughput, data quality, and analyst safety.

Objectives and overview

This study evaluates an automated workflow using the Thermo Scientific TriPlus RSH SMART autosampler for extraction of nitrosamines from metformin drug substance. Key goals include demonstrating long-term extraction repeatability, recovery accuracy, and compliance with ICH and U.S. FDA requirements for nitrosamine impurity assays in active pharmaceutical ingredients.

Methodology and instrumentation

The automated procedure employs liquid–liquid extraction of 100 mg metformin with water and dichloromethane (DCM), followed by vortex mixing, centrifugation, and transfer of the organic phase into amber GC vials. A pre-compiled autosampler sequence handles reagent dispensing, tool changes, vial mixing, centrifugation, and sample transfers. Optional modules enable headspace or multiple headspace extraction for LC analysis. Extracts are analyzed by gas chromatography coupled to high-resolution accurate-mass Orbitrap Exploris GC-MS, with sequence control and data processing managed in Chromeleon 7.3 CDS.

• TriPlus RSH SMART autosampler with Automatic Tool Change, vortexer, and centrifuge combi
• Orbitrap Exploris GC-MS
• Chromeleon 7.3 Chromatography Data System

Main results and discussion

Long-term repeatability was assessed by monitoring the 13C-NDMA-d6 internal standard peak area over 100 injections, achieving 7.7 % RSD. Recovery experiments at spiking levels of 1–10 ng/g in metformin yielded recoveries between 77 % and 111 % with precision (RSD) below 13 %, satisfying ICH Q2(R1) criteria (70–130 % recovery, bias within 20 %). The automated protocol reduced solvent use to 0.3 mL DCM per sample compared to 2 mL in manual methods and minimized the risk of cross-contamination and carryover.

Benefits and practical applications

• Unattended operation with a throughput of six minutes per sample
• Enhanced reproducibility and compliance with regulatory standards
• Reduced solvent consumption and cost savings
• Lower risk of contamination and human error
• Improved analyst safety by limiting exposure to toxic reagents
• Full data traceability and adherence to 21 CFR Part 11 and EU Annex 11

Future trends and opportunities

Automation of sample preparation is poised to expand across pharmaceutical QC labs for a wider range of analytes and matrices. Integration with liquid chromatography and mass spectrometry platforms will enable multi-residue workflows. Advances in robotics, smart syringe tracking, and digital connectivity will further enhance throughput, predictive maintenance, and data integrity. Ongoing regulatory focus on nitrosamine impurities will drive continued method optimization and adoption of high-throughput automated solutions.

Conclusion

The automated TriPlus RSH SMART workflow delivers reliable, precise, and compliant extraction of nitrosamines from metformin, substantially improving throughput, reducing solvent use, and enhancing data confidence. This approach offers a robust solution for pharmaceutical QC laboratories to meet stringent regulatory requirements while ensuring analyst safety and operational efficiency.

Reference

• Thermo Fisher Scientific. TriPlus RSH SMART robotic sampling system brochure, BR52235-EN 0921C.
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