Online Reaction Monitoring of In-Process Manufacturing Samples by UPLC

Significance of the Topic

The ability to monitor chemical reactions in real time is critical for modern pharmaceutical and biopharmaceutical manufacturing. UltraPerformance liquid chromatography (UPLC) applied as a process analytical technology (PAT) tool delivers precise, quantitative insights into the concentration of active pharmaceutical ingredients (APIs) and trace impurities during synthesis. This capability supports improved yield, reduced waste, and tighter process control compared to traditional spectroscopic PAT sensors.

Objectives and Study Overview

This application note describes the deployment of the Waters PATROL UPLC Process Analysis System for online monitoring of the conversion of acetylsalicylic acid (ASA) to salicylic acid. The goals were to demonstrate system repeatability, chromatographic resolution of multiple components, and automated reaction endpoint detection under typical manufacturing conditions.

Methodology and Instrumentation

The reaction was carried out by heating an aqueous ASA solution (0.3 g/L) at 75 °C and initiating hydrolysis with nitric acid. Aliquots were taken automatically and transferred to the UPLC system at 4-minute intervals. Chromatographic separation employed a Waters ACQUITY UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) with a 5–80 % acetonitrile gradient in 2 minutes, 0.8 mL/min flow, and UV detection at 243 nm. System suitability was confirmed by assessing repeatability prior to reaction initiation, yielding %RSD < 0.2 % for retention time and peak area.

Used Instrumentation

• PATROL UPLC Process Analysis System for automated online/at-line sampling
• ACQUITY UPLC HSS T3 column, 2.1 × 50 mm, 1.8 µm
• Empower Software with 21 CFR Part 11 compliance options
• Connections INSIGHT and NuGenesis SDMS for data management
• UV detector operating at 243 nm with 40 Hz sampling frequency

Main Results and Discussion

• Repeatability: six replicate injections of the starting material showed %RSD ≤ 0.19 % for peak areas.
• Chromatographic resolution allowed baseline separation of API and four process impurities in under 2.5 minutes.
• Large dynamic range permitted quantification of major and minor species, including impurities below 0.05 % of the main peak.
• Real-time summary plots of peak area percentages enabled clear mapping of reaction progress and precise determination of optimal quench time.
• Automated alerts flagged when impurities exceeded critical thresholds or when the API reached target concentration.

Benefits and Practical Applications

• Enhanced process understanding through quantitative, component-specific data in real time.
• Improved throughput and yield by pinpointing reaction endpoints and minimizing over-reaction or side-product formation.
• Reduced reliance on offline QC laboratories and spectroscopic sensors requiring frequent calibration.
• Streamlined workflows with minimal user intervention and secure data handling.

Future Trends and Opportunities

• Integration of UPLC-based PAT with advanced control systems for closed-loop process optimization.
• Expansion to monitor multistep syntheses and biologics production in real time.
• Application of machine learning to chromatographic PAT data for predictive modeling and anomaly detection.
• Development of miniaturized, portable UPLC modules for on-site manufacturing and continuous processing.

Conclusion

Implementing UPLC as an online PAT sensor delivers unparalleled selectivity and sensitivity for in-process reaction monitoring. The Waters PATROL UPLC System demonstrated reproducible performance, rapid analysis times, and automated endpoint detection, enabling higher yields, consistent quality, and efficient scale-up from development to commercial production.