Quantitative Analysis of Nitrosamine Impurities in Synthetic Oligonucleotides

Significance of the Topic

Nitrosamines in synthetic oligonucleotides pose a high safety risk due to their genotoxic and carcinogenic nature. Regulatory bodies such as FDA, EMA and ICH require stringent assessment and control of these impurities in pharmaceutical products, including antisense oligonucleotides. A reliable analytical approach is essential to ensure patient safety and compliance.

Objectives and Study Overview

This work aims to develop a sensitive and robust LC/MS/MS method for simultaneous quantification of eight nitrosamine impurities in ASO matrices at ppt levels. Using the Agilent 6495D triple quadrupole LC/MS coupled with an atmospheric pressure chemical ionization source, the study evaluates calibration performance, sensitivity, accuracy, precision, recovery and reproducibility.

Methodology and Instrumentation

Sample Preparation:

• A 21-mer phosphorothioate ASO was prepared at 1 mg/mL in 10% methanol/water.
• Nitrosamine standards (0.05 to 25 ng/mL) were spiked for calibration; QC samples at low (0.2), mid (2) and high (20 ng/mL) levels were prepared in replicates.

Chromatographic and MS Conditions:

• Agilent 1290 Infinity II bio LC system with Poroshell 120 EC-C18 column (3.0×150 mm, 2.7 μm), 40 °C, 0.5 mL/min, gradient elution with 0.1% formic acid in water and methanol.
• Agilent 6495D LC/TQ with APCI source in positive mode; MRM transitions for eight nitrosamines optimized for sensitivity and selectivity.

Software:

• Agilent MassHunter Acquisition (v12.2)
• Agilent MassHunter Quantitative Analysis (v12.1)

Main Results and Discussion

Calibration and Sensitivity:

• Linear response (R²>0.99) across 0.05–25 ng/mL for most analytes; NPIP and NDBA from 0.25 and 0.1 ng/mL, respectively.
• Limits of detection and quantification were below 0.1 and 0.25 ng/mL, respectively, enabling ppt-level measurement.

Accuracy and Precision:

• Accuracy ranged 90–120% across all levels.
• Intra- and inter-day precision (RSD) remained below 4% and 8%, respectively.
• Retention time and peak area RSD in mid-QC samples did not exceed 0.17% and 3.71%.

Matrix Effects and Chromatography:

• Baseline interference observed for NPIP due to matrix coelution; use of divert valve recommended to improve specificity.
• Representative NDMA chromatograms confirm clear detection at 0.05 ng/mL with signal-to-noise >80.

Benefits and Practical Applications of the Method

The proposed LC/MS/MS workflow provides high-throughput screening of nitrosamines in oligonucleotide therapeutics, ensuring:

• Regulatory compliance through robust risk assessment.
• Dose-level safety verification with ppt sensitivity.
• Reliable quantification in complex biological matrices.

Future Trends and Potential Applications

Advances in high-resolution mass spectrometry and automated sample handling will further enhance sensitivity and throughput. Integration of online sample cleanup and real-time data processing could streamline routine QC in biopharma. Expansion to other genotoxic impurities and oligonucleotide formats is anticipated.

Conclusion

This study demonstrates a robust, sensitive LC/MS/MS method for quantifying eight nitrosamines at trace levels in ASO matrices. Excellent linearity, accuracy, precision, recovery and reproducibility support its routine use for impurity control in oligonucleotide-based drugs.

References

1. Borths C.J.; Burr T.; Figuccia A.; et al. Org. Process Res. Dev. 2023, 27, 1693–1702.
2. U.S. Food and Drug Administration. Control of Nitrosamine Impurities in Human Drugs: Guidance for Industry (Revision 2), 2024.
3. European Medicines Agency. Nitrosamine Impurities: Guidance for Marketing Authorisation Holders, 2025.
4. ICH. Assessment and Control of DNA Reactive (Mutagenic) Impurities: M7(R2), 2023.