Nitrosamines Analysis in Pharmaceuticals - Consumables workflow ordering guide

Significance of the Topic

Mutagenic nitrosamines in pharmaceutical substances and finished drug products represent a serious health hazard due to their DNA-damaging and potentially carcinogenic nature. Regulatory bodies worldwide, including the US FDA, EDQM and Health Canada, have tightened guidelines and initiated recalls of drugs such as metformin and ranitidine after detecting traces of N-nitrosodimethylamine and related impurities. Robust analytical methods capable of detecting nitrosamines at low parts-per-billion levels are essential to ensure compliance, protect patient safety and maintain product quality.

Objectives and Study Overview

This report outlines the development and application of liquid chromatography–tandem mass spectrometry techniques for simultaneous detection and quantification of eight nitrosamine impurities in active pharmaceutical ingredients and finished products. Key goals include:

• Establishing sensitive, selective LC/MS/MS and LC/Q-TOF workflows
• Comparing two chromatographic methods for separation of positional isomers NDIPA and NDPA
• Demonstrating method performance at sub-nanogram per milliliter levels
• Providing a consumables ordering guide to streamline laboratory setup

Methodology and Instrumentation

Sample preparation employs liquid extraction and filtration using certified LC/MS solvents, PVDF or PES syringe filters and deactivated vials. Two UHPLC methods were developed:

• Method 1 on a hybrid-particle HPH-C18 column, requiring careful pH and gradient optimization to resolve isomers.
• Method 2 on a pentafluorophenyl Poroshell 120 PFP column, offering orthogonal selectivity and straightforward separation of NDIPA and NDPA.

Both methods use 0.1% formic acid in water and methanol mobile phases at 0.5 mL/min with reversed-phase gradients. Triple quadrupole MS in multiple reaction monitoring mode provides specificity, while quadrupole time-of-flight MS offers high-resolution confirmation when required.

Applied Instrumentation

• Agilent 1290 Infinity II and 1260 Infinity UHPLC systems
• Agilent 6470B Triple Quadrupole LC/MS for high-sensitivity MRM analysis
• Agilent 6550 iFunnel Q-TOF LC/MS for high-resolution accurate-mass detection
• Agilent Ultivo Triple Quadrupole LC/MS for compact footprint applications
• InfinityLab Poroshell 120 PFP and HPH-C18 analytical columns

Main Results and Discussion

Representative chromatograms demonstrate baseline separation of eight nitrosamines at 0.5 ng/mL using Method 1 and at 3 ng/mL with Method 2. Key performance metrics include low limits of detection and quantification, excellent linearity across trace-level concentrations, and reliable resolution of positional isomers. Method 2 showed superior selectivity for halogenated and polar analytes, while Method 1 provided robust performance across a wider pH range.

Benefits and Practical Applications

The described workflows deliver:

• High sensitivity and specificity to meet stringent regulatory thresholds
• Flexible column choices for complex sample matrices
• Streamlined consumables ordering and laboratory implementation
• Compatibility with routine QA/QC and stability testing in pharmaceutical development

Future Trends and Applications

Advances in mass spectrometry are expected to further lower detection limits and improve structural confirmation of novel nitrosamine analogues. Emerging directions include:

• Integration of automated sample preparation and online SPE
• Expansion of high-resolution MS libraries for unknown impurity screening
• Miniaturized LC/MS configurations for field or point-of-use testing
• Machine-learning algorithms to predict formation pathways and prioritize targets

Conclusion

The implementation of triple quadrupole and quadrupole time-of-flight LC/MS methods provides a powerful and flexible platform for the trace-level analysis of nitrosamine impurities in pharmaceuticals. Through careful column selection, optimized gradients, and targeted MS detection, laboratories can achieve reliable compliance with global regulatory requirements and safeguard drug quality.

References

1. Nitrosamines analysis in pharmaceuticals using single quadrupole GC/MS and triple quadrupole GC/MS/MS: Consumables workflow ordering guide, Agilent publication 5994-2979EN.
2. Nitrosamine Impurities Application Guide – Confidently detect and quantify mutagenic impurities in APIs and Drug Products, Agilent 5994-2393EN.
3. Determination of a Genotoxic NDMA Impurity Using the High-Resolution Agilent 6546 LC/Q-TOF in ranitidine, Agilent 5994-1626EN.
4. Simultaneous Determination of Eight Nitrosamine Impurities in Metformin Using Agilent 6470 Triple Quadrupole LC/MS, 5994-2286EN.
5. Determination of Nitrosamine Impurities Using the Ultivo Triple Quadrupole LC/MS, Agilent 5994-1383EN.
6. Determination of NDMA Impurity in Ranitidine Using the Agilent 6470 Triple Quadrupole LC/MS, 5994-1668EN.
7. Determination of Nitrosamine Impurities Using the High-Resolution Agilent 6546 LC/Q-TOF, 5994-1372EN.
8. Simultaneous Determination of Eight Nitrosamine Impurities in Metformin Extended-Release Tablets Using the Agilent 6470 Triple Quadrupole LC/MS, 5994-2533EN.