Qualitative Analysis of 15 Traditional Nitrosamines in Thalassemia drug at Ultra-trace Levels Using LC/TQ

Significance of the Topic

Nitrosamine impurities are potent mutagens that can form during manufacturing or storage of active pharmaceutical ingredients. Even ultra-trace levels pose significant health concerns and are tightly regulated by health authorities. A reliable analytical method for detecting and quantifying multiple nitrosamines in critical drugs such as deferiprone ensures patient safety and supports compliance with stringent impurity guidelines.

Objectives and Study Overview

This study aimed to develop and validate a targeted LC–TQ method for simultaneous analysis of fifteen traditional nitrosamines in deferiprone formulations at sub-ppb levels. The workflow included spiking known amounts of each impurity into tablet matrices, optimizing chromatography and mass spectrometry parameters, and assessing method performance metrics such as linearity, precision, recovery, and limit of quantitation.

Methodology and Instrumentation

Sample Preparation
• 100 mg deferiprone powder dissolved in 1 mL ultrapure water
• Spike: 1 ng NDELA and 0.1 ng for each remaining nitrosamine per 100 mg sample
• Vortex 5 min, centrifuge at 10000 g for 5 min, filter supernatant through 0.2 µm membrane

Chromatography and Detection
• Agilent 1290 UHPLC with built-in flow diverter valve to divert deferiprone (retention at 280 nm) to waste
• Poroshell 120 EC-C18 column at 40 °C, gradient from 100 % aqueous to organic over 20 min
• Agilent 6495D triple quadrupole MS with APCI positive mode, dynamic MRM transitions

Mass Spectrometry Parameters
• Multisampler 15 °C, injection 20 µL
• Drying gas 290 °C at 11 L/min, nebulizer 55 psi, corona current 4 µA, vaporizer 350 °C

Main Results and Discussion

Chromatographic Separation
• Baseline resolution of all fifteen nitrosamines, with effective deferiprone diversion preventing source contamination

Calibration and Linearity
• Concentration range 0.05–5 ng/mL, correlation coefficients ≥0.998 for each analyte

Precision and Recovery
• RSD for six replicate injections at 0.05 ng/mL ranged from 2.5 % to 9 %
• Recovery at LOQ levels between 81 % and 118 %, within ±20 % acceptance criteria

Limits of Quantitation
• Obtained LOQs were at least three-fold lower than regulatory impurity limits, in some cases over 8000-fold better

Benefits and Practical Applications

• High sensitivity enables reliable detection of nitrosamines at ultra-trace levels
• Robust chromatography with dynamic MRM ensures selectivity and minimizes matrix effects
• The method supports routine quality control in pharmaceutical laboratories, ensuring compliance with global regulatory requirements

Future Trends and Applications

• Extension to other drug substances and formulations with minimal method adaptation
• Integration with high-resolution MS for non-targeted impurity screening
• Automation of sample preparation and data analysis to increase throughput
• Application of machine learning for predictive impurity profiling and risk assessment

Conclusion

The developed LC–TQ method provides a sensitive, precise, and reproducible approach for quantifying fifteen nitrosamine impurities in deferiprone at ultra-trace levels. Performance metrics meet or exceed regulatory criteria, supporting its implementation in pharmaceutical QC to safeguard drug safety.

Reference

1. FDA Updates and Press Announcements on ARB Recalls: control of nitrosamine impurities in valsartan and losartan products
2. FDA Guidance for Industry: Control of Nitrosamine Impurities in Human Drugs