Monitoring of Trace Impurities Using the Agilent 1260 Infinity II Prime Online LC System with High Dynamic Range-DAD

Importance of the Topic

The precise monitoring of trace impurities alongside highly concentrated reagents and products is critical in small-molecule active pharmaceutical ingredient (API) manufacturing. Impurities formed under reaction conditions can impact safety, efficacy, and regulatory compliance. Traditional approaches often require separate analyses for major components and low-level impurities, increasing cycle times and resource consumption.

Objectives and Study Overview

This application note evaluates an online process analytical technology (PAT) solution that combines the Agilent 1260 Infinity II Prime Online LC system with a High Dynamic Range Diode Array Detector (HDR-DAD). The aim is to demonstrate simultaneous quantification of high-abundance reactants/products and trace byproducts in a single chromatographic run, using an aldol condensation of p-anisaldehyde and acetone as a model reaction.

Methodology and Instrumentation

Reagents and reaction conditions:

• Reactants: p-anisaldehyde and acetone in acetone:water (2:1, v/v) with NaOH catalyst at room temperature.
• Sampling: Automated 500 µL draws every 3 minutes into deep-well plates, 1:10 dilution with water/acetonitrile + 0.1% formic acid.

Chromatographic setup:

• Column: Agilent InfinityLab Poroshell 120 EC-C18, 2.1 × 30 mm, 1.9 µm.
• Mobile phase: Water + 0.1% formic acid (A) and Acetonitrile + 0.1% formic acid (B); gradient 40→90% B in 0.85 min, flow 1.3 mL/min.
• Column temperature: 45 °C; injection volume: 1 µL.

Detector configuration:

• Two diode array detectors with 60 mm and 3.7 mm Max-Light flow cells.
• Electronic combination into a normalized 10 mm equivalent HDR signal, dynamic range ~6500 mAU.

Main Results and Discussion

The HDR-DAD approach captured the declining educt peak at 0.26 min, the growing main product (E-anisylidene acetone) at 0.36 min, and a low-level byproduct at 0.84 min in a single run. Individual detectors alone either overloaded on major peaks or lacked sensitivity for impurities. The combined HDR response extended the linear range, enabling quantification of both high and low concentration species without detector saturation. Real-time trending of area percentages illustrated reaction progress: educt consumption from 100% to <5% over 90 minutes, product formation to >96%, and byproduct detection down to 0.02% area.

Benefits and Practical Applications

• Single-injection analysis reduces cycle time and solvent consumption.
• Simultaneous quantification of major and trace components enhances process understanding.
• Automated sampling and online trending support real-time decision making in API production.

Future Trends and Opportunities

The HDR-DAD methodology can be extended to other reaction types and inline process monitoring applications. Combining HDR detection with mass spectrometry could further improve impurity identification. Integration into continuous flow manufacturing and advanced data analytics platforms will drive smarter process control and regulatory compliance.

Conclusion

This study demonstrates that the Agilent 1260 Infinity II Prime Online LC coupled with HDR-DAD delivers a robust, high-throughput solution for simultaneous monitoring of high-level reactants, products, and low-level impurities. The enhanced dynamic range simplifies workflows, accelerates analysis, and supports real-time process control.

References

1. Agilent Technologies. Impurity Testing of Fixed-Dose Combination Drugs Using the Agilent 1290 Infinity II HDR-DAD Impurity Analyzer Solution. Application note 5991-5743EN, 2015.
2. Viviano M. et al. A Scalable Two-Step Continuous Flow Synthesis of Nabumetone and Related 4-Aryl-2-butanones. Org. Process Res. Dev. 2011, 15, 858–870.
3. Agilent Technologies. Automated Reaction Monitoring by the Agilent 1260 Infinity II Prime Online LC System. Application note 5994-3980EN, 2021.
4. Agilent Technologies. Sensitive Detection of Low-Level By-Products in a Small Molecule Reaction by the Agilent 1260 Infinity II Prime Online LC/MS System. Application note 5994-3981EN, 2021.