Highly sensitive LC-MS/MS method for the determination of NDMA, NDEA, NDIPA, NMBA, NEIPA and NDBA in Metformin drug substance

Importance of the Topic

Nitrosamine impurities such as NDMA, NDEA, NDIPA, NMBA, NEIPA and NDBA are classified as probable human carcinogens and have triggered global regulatory recalls of metformin products when detected above acceptable limits. A unified, high-sensitivity analytical method is essential for routine quality control and risk mitigation in pharmaceutical manufacturing and regulatory compliance.

Objectives and Study Overview

This study aimed to develop and validate a single ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method capable of simultaneously quantifying six nitrosamines in metformin drug substance. The method was evaluated for sensitivity, linearity, accuracy and the ability to eliminate false positives, with confirmation by high-resolution mass spectrometry.

Methodology and Instrumentation

Sample Preparation

• Standard solutions of mixed nitrosamines (0.25–100 ng/mL) spiked with internal standards NDMA-D6 and NDEA-D10 at 10 ng/mL.
• Metformin samples: 100 mg dissolved in 1 mL water, filtered through 0.2 µm syringe filter.

Chromatographic and Mass Spectrometric Conditions

• UHPLC system: Shimadzu Nexera X2 with Shim-pack GIST C18 column (150 × 4.6 mm, 5 µm), column temperature 30 °C.
• Mobile phase: A = 0.1% formic acid in water, B = 0.1% formic acid in methanol; flow rate 0.7 mL/min; gradient from 5% to 90% B over 12 minutes.
• Injection volume: 30 µL.
• MS detector: Shimadzu LCMS-8045 triple quadrupole with APCI positive mode; desolvation line and heating block at 200 °C; interface at 350 °C; nebulizing gas 3 L/min; drying gas 5 L/min.
• Confirmation: Shimadzu LCMS-9030 Q-TOF for high-resolution verification of suspect peaks.

Main Results and Discussion

All six nitrosamines were well resolved from the metformin matrix, with metformin diverted to waste via a switching valve. Calibration curves exhibited linearity (r² > 0.99) over 0.25–100 ng/mL. Limits of detection ranged from 0.10 to 0.25 ng/mL (0.0010–0.0025 ppm), and limits of quantitation from 0.25 to 0.50 ng/mL (0.0025–0.0050 ppm). Recovery tests at 0.005 ppm spiking levels met acceptance criteria. A case study revealed a false positive NDMA signal on the triple quadrupole system, which was resolved and ruled out by Q-TOF high-resolution mass accuracy.

Benefits and Practical Applications of the Method

• Single-run analysis of six nitrosamines streamlines QC workflows in pharmaceutical laboratories.
• High sensitivity and selectivity facilitate compliance with stringent regulatory limits.
• Integration of high-resolution MS confirmation reduces the risk of false positives and supports investigative follow-up.

Future Trends and Potential Applications

Advances may include expansion to additional nitrosamine chemotypes and other drug matrices, automation of sample preparation, and wider adoption of high-resolution MS platforms for routine confirmation. Machine learning algorithms could further optimize chromatographic separation and peak identification.

Conclusion

A robust UHPLC-MS/MS method was established for simultaneous quantitation of six nitrosamines in metformin with excellent sensitivity, linearity and accuracy. The use of high-resolution Q-TOF mass spectrometry proved critical for confirming and eliminating false positives, reinforcing the method’s suitability for comprehensive QC screening.

Reference

• [1] HSA Press Release – HSA Recalls Three out of 46 Metformin Medicines, December 2019.
• [2] Tabrez et al Journal of Advances in Pharmacy Practices Volume 2 Issue 1 48-57 2020.