Using Gradient SmartStart Technology and an ACQUITY UPLC H-Class System to Emulate an Agilent 1100 Series LC System Separation for Impurity Testing

Significance of the Topic

Liquid chromatography method transfer across different instrument platforms is a common challenge in analytical laboratories. Differences in dwell volume and gradient delay between manufacturers can lead to shifts in retention times and relative retention times, jeopardizing system suitability criteria. Software tools that automate dwell volume compensation simplify method transfers, reduce manual adjustments, and increase confidence in reproducibility.

Objectives and Study Overview

The study aimed to replicate a validated clozapine impurity separation originally developed on an Agilent 1100 Series LC System using an ACQUITY UPLC H-Class System. The key objective was to assess whether the Gradient SmartStart feature could adjust for dwell volume differences and achieve comparable chromatographic performance, including retention time, relative retention time, resolution, and impurity quantitation.

Methodology and Instrumentation

The clozapine resolution standard from USP was prepared at 400 µg/mL in 80 percent methanol and 20 percent water. Three systems were evaluated:

• Agilent 1100 Series LC System with diode array detector
• Agilent 1290 Infinity Quaternary LC System with diode array detector
• ACQUITY UPLC H-Class System with PDA detector

All separations used a ZORBAX Eclipse XDB C18 column (4.6 × 150 mm, 3.5 µm) at 30 °C, a flow rate of 1.5 mL/min, and a gradient from 5 to 30 percent acetonitrile in 5 minutes, then to 95 percent in 4 minutes. Dwell volumes were measured using a propyl paraben gradient, yielding 1.290 mL for the 1100 system and 0.375 mL for the UPLC system. The difference (0.915 mL) was entered into the Gradient SmartStart feature as a post-injection delay on the UPLC system.

Main Results and Discussion

Initial transfers without dwell volume compensation showed retention time shifts of 0.45 to 0.62 minutes (4–9 percent), exceeding the 3 percent acceptance window, and relative retention time deviations for later eluting impurities. After applying a 915 µL post-injection delay on the UPLC system, retention time differences fell below 0.05 minutes (<1 percent), and relative retention times aligned with the 1100 system. Additionally, due to lower dispersion on the UPLC platform, USP resolutions improved by 4–8 percent for critical pairs. Percentage areas of clozapine and impurities remained unchanged, demonstrating quantitative equivalence.

Benefits and Practical Applications

• Automated dwell volume compensation without modifying gradient tables
• Faster method transfer and reduced risk of manual errors
• Enhanced resolution on UPLC improves impurity separation
• Consistent quantitative results facilitate regulatory compliance

Future Trends and Potential Applications

Future developments may include broader adoption of software-based method transfer tools across diverse LC platforms and integration with machine learning algorithms for automated method optimization. Increased connectivity between instruments and laboratory information management systems will support high-throughput impurity testing and real-time performance monitoring. Enhanced gradient compensation features could extend into two-dimensional and UHPLC applications.

Conclusion

Gradient SmartStart technology effectively compensates for dwell volume differences when transferring gradient methods from an Agilent 1100 Series LC System to an ACQUITY UPLC H-Class System. The approach achieved retention time and relative retention time matching, improved resolution, and maintained impurity quantitation, all without manual edits to the gradient program. This demonstrates a streamlined path for robust method transfer in pharmaceutical impurity analysis.

References

1. Protocol for Gradient Delay Measurement. Waters Corporation; 2013.
2. Snyder LR et al. Introduction to Modern Chromatography, Third Edition. John Wiley; 2009.
3. USP Chapter 621 Chromatography. United States Pharmacopeia; 2014.
4. Agilent Application Note on HPLC Method Transfer; 2006.
5. Official Monographs USP 37 NF32 S2. United States Pharmacopeia; 2014.
6. Snyder LR and Dolan JW. Gradient Equipment. John Wiley; 2009.