Simultaneous Determination of Eight Nitrosamine Impurities in Metformin Extended-Release Tablets Using the Agilent 6470 Triple Quadrupole LC/MS

Importance of the Topic

Trace levels of nitrosamine impurities in pharmaceuticals represent a critical safety concern due to their classification as probable human carcinogens. Recent regulatory recalls of metformin extended-release tablets worldwide highlight the need for highly sensitive, selective analytical methods to ensure product quality and patient safety.

Objectives and Study Overview

This work details the development and validation of two LC-MS/MS methods using the Agilent 6470 triple quadrupole system for simultaneous quantitation of eight nitrosamines (NDMA, NDEA, NEIPA, NMBA, NDIPA, NMPA, NDPA, NDBA) in metformin drug substance and extended-release tablet formulations. The goal was to achieve regulatory compliance with low ng/mL detection limits and robust performance across different sample matrices.

Methodology and Instrumental Setup

Sample Preparation

• Method 1 (Drug Substance and Product): Dissolution in methanol/water, filtration, dilution to 20 mg/mL metformin.
• Method 2 (Alternative Column Chemistry): Dissolution to 100 mg/mL metformin, extended extraction for dense formulations.

Chromatography and MS/MS

• UHPLC: Agilent 1290 Infinity II with Poroshell HPH-C18 or Poroshell 120 PFP columns (2.7 µm, 150 × 3–4.6 mm).
• Mobile phases: 0.1% formic acid in water (A) and methanol (B); flow rate 0.5 mL/min; 20–22 min gradients.
• Mass spectrometry: APCI positive mode, MRM transitions optimized for each analyte (precursor/product ion pairs, collision energy).

Key Results and Discussion

Method 1 achieved limits of detection (LOD) as low as 0.025 ng/mL and limits of quantitation (LOQ) down to 0.1 ng/mL for most analytes, with linearity R²>0.996 over 0.1–50 ng/mL. Recoveries in spiked metformin tablets ranged 93–116% with RSD<5%. Method 2, adapted for denser tablet extracts, yielded LODs of 0.05–0.25 ng/mL and LOQs of 0.1–0.5 ng/mL, with linearity R²>0.995 and recoveries between 91–109%. Chromatographic separation successfully resolved positional isomers NDIPA/NDPA and minimized matrix effects from metformin.

Benefits and Practical Applications

• Regulatory compliance: Sensitive detection of eight nitrosamines at sub-ppb levels.
• Versatility: Two sample preparation and column chemistries accommodate various tablet matrices.
• High throughput: UHPLC–MS/MS workflow suitable for routine quality control in pharmaceutical labs.

Future Trends and Applications

Continued method evolution may include coupling with high-resolution mass spectrometry for non-targeted screening, automation of sample preparation, integration into continuous manufacturing quality-by-design workflows, and extension to other drug classes susceptible to nitrosamine formation.

Conclusion

The Agilent 6470 triple quadrupole LC/MS platform delivers robust, ultra-sensitive analysis of eight critical nitrosamine impurities in metformin, meeting stringent regulatory demands and supporting pharmaceutical quality assurance.

References

1. FDA updates on ARB nitrosamine recalls and guidance for nitrosamine screening.
2. FDA guidance: RapidFire-MS/MS method development for nitrosamines.
3. FDA guidance: LC-HRMS method for six nitrosamine impurities in ARB drugs.
4. FDA guidance: LC-HRMS method for eight nitrosamines in metformin.
5. Mani C., Banerjee S., Application note: Nitrosamine determination using Ultivo triple quadrupole LC/MS, Agilent Technologies, 2019.
6. Agilent application note 5994-1383EN: Ultivo triple quadrupole LC/MS nitrosamine analysis.
7. FDA guidance on LC-ESI-HRMS methods for nitrosamines.
8. Chidella K.S., Mani C., Banerjee S., Vyas S., Agilent application note 5994-2286EN, 2020.