Rapid quantification of 12 nitrosamines in metformin using triple quadrupole GC-MS/MS with Advanced Electron Ionization (AEI)

Significance of the topic

Nitrosamine impurities have emerged as critical safety concerns in pharmaceutical products since the discovery of N-nitrosodimethylamine (NDMA) in valsartan in 2018. Classified as probable or known human carcinogens by ICH M7 and IARC, nitrosamines can form during drug synthesis, storage, or due to degraded API. Regulatory agencies worldwide have established stringent limits (e.g., 30 ng/g) to protect patient safety. Accurate, rapid, and reliable methods for detecting multiple nitrosamines at trace levels in active pharmaceutical ingredients (APIs) such as metformin are therefore essential for quality control and regulatory compliance.

Objectives and scope of the study

This application note demonstrates a high-throughput GC-MS/MS approach using Thermo Scientific TSQ 9000 and TSQ 9610 triple quadrupole mass spectrometers with Advanced Electron Ionization (AEI) and the XLXR detector to quantify 12 nitrosamines in metformin. Key performance characteristics—including chromatographic separation, calibration linearity, sensitivity (MDL/IDL), precision, accuracy, and dynamic range—were assessed. The work also compares standard ion source operation on TSQ 9000 to extended linearity capabilities provided by the TSQ 9610 NeverVent AEI configuration.

Used instrumentation

• Thermo Scientific TRACE 1310 Gas Chromatograph
• Thermo Scientific TSQ 9000 and TSQ 9610 triple quadrupole MS with Advanced Electron Ionization (AEI)
• XLXR electron multiplier detector (TSQ 9610)
• Chromeleon 7.3 Chromatography Data System
• TraceGOLD TG-1701 MS column (30 m × 0.25 mm × 0.50 μm)
• TriPlus RSH autosampler with robotic liquid-liquid extraction capability

Methodology

Samples of metformin (100 mg) were spiked with internal standards (13C2D6-NDMA, D14-NDPA) and subjected to automated liquid-liquid extraction in amber vials: addition of water/internal standard, vortex mixing, dichloromethane extraction, centrifugation, and 100 µL DCM injection. GC conditions employed splitless injection (2 µL), helium carrier at 1.3 mL/min, and a temperature program from 40 °C to 270 °C. MS operated in SRM (timed-SRM) with optimized precursor/product ­ion transitions for each analyte. Calibration standards covered 0.025–2,000 ng/mL (depending on analyte), matrix-matched spikes at 0.25–2.0 ng/g for MDL/LOQ assessment, and extended range up to 4,000 ng/mL on the TSQ 9610 with XLXR.

Main results and discussion

• Chromatographic separation of 12 nitrosamines achieved in <11 min with peak asymmetry <1.5.
• Excellent calibration linearity over >4 orders of magnitude (R² > 0.995 for all analytes; AvCF %RSD < 14%).
• Method detection limits ranged from 0.05 to 0.28 ng/g (MDL), significantly below the 30 ng/g regulatory threshold. Instrument detection limits (IDL) were 11–56 fg on-column.
• Precision and accuracy met ICH Q2(R1) criteria: recovery 70–130% and RSD < 20% in triplicate spiked metformin samples.
• TSQ 9610 AEI with XLXR extended dynamic range to 4,000 ng/mL (>5.5 orders of magnitude) while maintaining R² > 0.998 and AvCF %RSD < 9%.
• Ion ratio monitoring prevented false positives and ensured data quality at trace levels.

Benefits and practical applications of the method

• High throughput analysis with automated sample preparation and rapid GC cycle time.
• Sensitivity and selectivity of triple quadrupole MS/MS reduce background noise and false positives.
• Broad dynamic range accommodates both trace-level monitoring and higher concentration testing in a single assay.
• Ease of method deployment via Chromeleon eWorkflows and SmartTune for robust performance and regulatory compliance (FDA 21 CFR Part 11, EU Annex 11).
• Applicability to quality control laboratories for routine nitrosamine surveillance in APIs and final drug products.

Future trends and potential applications

Advances in GC-MS/MS ion source designs and detector technologies will further improve sensitivity and dynamic range, enabling detection of emerging nitrosamine species. Integration with automated headspace sampling or solid-phase microextraction can expand application to diverse dosage forms. Coupling with high-resolution MS and data-mining algorithms may facilitate non-targeted screening for unknown nitrosamine analogs. Enhanced laboratory automation and LIMS/CDS interoperability will drive higher throughput while ensuring data integrity and regulatory traceability.

Conclusion

This work validates a robust, sensitive, and rapid GC-MS/MS method for simultaneous quantification of 12 nitrosamines in metformin. Using Thermo Scientific TRACE 1310 GC and TSQ 9000/9610 triple quadrupole MS with AEI, the approach delivers trace-level detection, wide dynamic range, and reliable quantitation—fully compliant with regulatory guidelines and suitable for routine pharmaceutical quality control.

References

1. World Health Organization. Information Note: Nitrosamine Impurities – Update on Nitrosamine Impurities. 2019.
2. International Council for Harmonisation. ICH M7(R1): Assessment and Control of DNA-Reactive (Mutagenic) Impurities in Pharmaceuticals. March 31, 2017.
3. International Agency for Research on Cancer. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 89: Smokeless Tobacco and Some Tobacco-Specific N-Nitrosamines. Lyon; 2007.
4. Thermo Fisher Scientific. Technical Note 10499: Practical Determination and Validation of Instrument Detection Limit of Thermo Scientific ISQ LT GC-MS. 2018.