Analysis of Elemental Impurities in Synthetic Oligonucleotides by ICP-MS In compliance with USP <232>/<233> and ICH Q3D(R2)/Q2(R1)

Significance of the Topic

Synthetic oligonucleotides are essential in molecular biology and emerging therapeutic applications. Monitoring trace elemental impurities is critical to ensure product safety, regulatory compliance, and reliable performance in PCR, sequencing, and oligonucleotide-based drugs. Accurate impurity profiling guards against contamination introduced during synthesis, purification, and packaging processes.

Study Objectives and Overview

This study aimed to establish and validate an analytical workflow for quantifying 24 elemental impurities in a synthetic oligonucleotide matrix. The method aligns with USP<232>/<233> and ICH Q3D(R2)/Q2(R1) guidelines, evaluating system performance, accuracy, precision, specificity, and ruggedness using an Agilent 7850 ICP-MS.

Methodology and Instrumentation

Sample Preparation:

• Oligonucleotide stock at 10 mg/mL.
• Aliquot of 0.1 g diluted 500-fold in 3% HNO₃ and 1% HCl.

Instrumentation:

• Agilent 7850 ICP-MS with MassHunter software.
• Pre-set ICH/USP method automatically applied RF power, gas flows, cell mode (helium), and isotopic targets.

Analytical parameters were optimized for sensitivity and interference removal.

Main Results and Discussion

System suitability tests showed drift within ±20% and QC RSD below 2.6% over 7 hours. Method detectability and LOQs reached low ppt levels. Accuracy was confirmed by spike recoveries between 85% and 115% at 0.5J, 1J, and 1.5J levels. Precision for six replicates of the 1J spike exhibited RSDs under 2.5%, and intermediate precision across days and operators remained below 4%. Helium collision-cell mode effectively removed polyatomic interferences, and linearity achieved R²>0.999 for all elements.

Benefits and Practical Applications

The validated ICP-MS method provides a robust tool for routine monitoring of elemental impurities in oligonucleotide products. It ensures compliance with regulatory limits, supports quality control in manufacturing, and enhances confidence in therapeutic oligo safety.

Future Trends and Opportunities

Continued growth in oligonucleotide therapeutics will drive demand for high-throughput, automated impurity analysis. Advances may include coupling with chromatographic separations, miniaturized sampling workflows, and improved cell-gas technologies to further lower detection limits and enhance method sensitivity.

Conclusion

The Agilent 7850 ICP-MS method demonstrated full compliance with USP and ICH guidelines for elemental impurity analysis in synthetic oligonucleotides, offering reliable accuracy, precision, and specificity for routine quality assurance.

References

1. US FDA Guideline for Elemental Impurities Q3D(R2) Step 4, International Council for Harmonisation, April 2022
2. Elemental Impurities—Limits, Pharm. Forum, 2016, 42(2), Revision to Chapter <232>
3. US FDA Q2(R1) Validation of Analytical Procedures: Text and Methodology, September 2021
4. Elemental Impurities—Procedures, Pharm. Forum, 2014, 40(2), Revision to Chapter <233>
5. Octopole Collision/Reaction Cell and Helium Mode, Agilent Technologies publication, 5994-1172EN