Agilent 1200 Infinity Series HDR‑DAD Impurity Analyzer System for the Quantification of Trace Level of Genotoxic Impurity

Significance of the Topic

Trace level genotoxic impurities may form during drug degradation and must be controlled according to global regulatory guidelines to ensure patient safety.
The quantification of these low concentration species while preserving the main drug peak requires advanced detection strategies.

Study Objectives and Overview

The study aimed to demonstrate the use of an Agilent 1200 Infinity Series HDR-DAD Impurity Analyzer System for accurate quantification of an aromatic N-oxide genotoxic impurity generated from peroxide-degraded omeprazole.
Peroxide treatment at varying hydrogen peroxide concentrations (1–4 %) induced degradation, and spiked standards spanning 0.1–2 ppm were used to establish linearity.

Methodology and Instrumentation

• Sample preparation: Omeprazole capsules diluted to 1 mg/mL; degradation by H2O2, speed-vacuum, dilution, vortex, centrifugation.
• Standards: Stock solutions of omeprazole (1250 ppm) and N-oxide impurity (1000 ppm); spiked API for linearity levels.
• Chromatography: Agilent 1290 Infinity Binary LC with ZORBAX Eclipse Plus C8 column (4.6×150 mm, 3.5 µm); phosphate buffer/acetonitrile (3:1); 0.8 mL/min; 25 °C; 30 min run.
• Detection: HDR-DAD combining two DADs with 60-mm and 3.7-mm flow cells, normalizing to 10-mm path length to extend dynamic range.

Main Results and Discussion

• HDR-DAD enabled a 20 µL injection without main peak saturation, revealing a 0.009 % impurity peak automatically integrated.
• Noise reduction up to 88 % improved signal-to-noise ratios for impurity detection at trace levels.
• Linearity across 0.1–2 ppm showed R2 > 0.9989 with consistent retention times, and peak overlay confirmed reproducibility.
• Degradation samples yielded impurity levels proportional to H2O2 concentration: 0.43–1.32 ppm for 1–4 % treatments.

Benefits and Practical Applications

• Extended dynamic range allows higher injection volumes for enhanced impurity visibility while preventing main peak overload.
• Automated integration with HDR improves precision and reduces manual intervention.
• Reduced baseline noise supports reliable quantitation at regulatory trace limits.
• The workflow is compatible with pharmaceutical QA/QC settings for monitoring genotoxic impurities.

Future Trends and Opportunities

• Application of HDR-DAD to a broader range of genotoxic and degradation impurities.
• Integration with mass spectrometry for structural confirmation at trace levels.
• Automation and AI-driven data analysis to streamline impurity profiling.
• Miniaturized and high-throughput formats to meet evolving regulatory demands.

Conclusion

The Agilent 1200 Infinity Series HDR-DAD Impurity Analyzer System offers a robust solution for trace genotoxic impurity quantitation, delivering high sensitivity, expanded dynamic range, and automated data processing that align with pharmaceutical quality standards.

References

1. Seshadri RK, Raghavaraju TV, Chakravarthy IE. A Single Gradient Stability-indicating Reversed-Phase LC Method for the Estimation of Impurities in Omeprazole and Domperidone Capsules. Scientia Pharmaceutica. 2013;81:437-458.
2. Benigni R, Bossa C. Structural Alerts of Mutagens and Carcinogens. Current Computer-Aided Drug Design. 2006;2(2):169-176.
3. Huesgen AG. Reliable and Automatic Integration of Trace Compounds Using the Agilent 1200 Infinity Series High Dynamic Range Diode Array Detector Solution. Agilent Technologies Technical Overview. 2013;5991-1409EN.
4. United States Pharmacopeia. USP29-NF24. Omeprazole Monograph. 2007; p.1577.
5. Agilent Technologies. Agilent 1200 Infinity Series High Dynamic Range Diode Array Detector Solution User Manual. 2014; G2199-90000.