EMPLOYING MODERN LC TECHNOLOGY TO SCALE A USP GRADIENT METHOD ON A SINGLE LIQUID CHROMATOGRAPHIC SYSTEM

Significance of the Topic

The continuous advancement of liquid chromatography in pharmaceutical laboratories is critical for ensuring high‐quality drug products while reducing operational costs. Modernizing gradient methods by transferring compendial protocols onto advanced LC systems with finer particle columns enables substantial savings in analysis time and solvent usage without compromising chromatographic integrity.

Objectives and Study Overview

This work focuses on scaling the USP monograph gradient procedure for quetiapine fumarate impurities to two progressively smaller column formats on a single ACQUITY Arc UHPLC system. By maintaining the original method’s key performance parameters, the study compares the standard HPLC method against scaled UHPLC (2.5 μm) and UPLC (1.7 μm) formats. The goal is to demonstrate equivalence in resolution, peak shape, and precision while achieving faster separations.

Methodology and Instrumentation

Instrumentation and setup

• ACQUITY Arc UHPLC System with active solvent preheating and 2998 PDA detector
• Column formats: 4.6×150 mm C8, 3.5 μm (HPLC); 3.0×100 mm C8, 2.5 μm (UHPLC); 2.1×75 mm C8, 1.7 μm (UPLC)
• Mobile phases: A – acetonitrile with 25% ammonium acetate buffer (75%); B – acetonitrile
• Buffer: 3.1 g/L ammonium acetate, adjusted to pH 9.2 with 25% ammonium hydroxide
• Detection: 250 nm, 4.8 nm bandwidth; column temperature 45 °C; sample temperature 4 °C

Method adaptation

• Particle size scaling and flow rate adjustments determined using Waters Columns Calculator
• Injection volumes adjusted to preserve sample loading across formats
• Gradient profile modified proportionally to column dimensions

Results and Discussion

Scaling to the 2.5 μm UHPLC format reduced run time by 51 % (from 70 to 34 min) and solvent consumption by 71 % compared to the original method. The 1.7 μm UPLC format further decreased runtime by 75 % (to 17 min) and solvent use by 89 %. All three methods met USP system suitability criteria for resolution (minimum 1.5), peak tailing, and retention time and area reproducibility. Slight resolution loss in the UPLC format remained within acceptable limits, demonstrating robust transferability.

Benefits and Practical Applications

• Significantly higher throughput for routine impurity analysis in quality control
• Reduced solvent costs and environmental footprint
• Preserved analytical performance ensures regulatory compliance
• Simplified method transfer using an online scaling tool

Future Trends and Potential Applications

Advances in column technology and automated method translation tools will continue to streamline assay development in pharmaceutical analytics. Wider adoption of sub-2 μm and core-shell particles, along with integrated system automation, will further enhance laboratory efficiency. Extending this approach to diverse compound classes and combining with mass spectrometric detection offer promising avenues.

Conclusion

This study confirms that established USP gradient methods can be effectively migrated to modern UHPLC and UPLC formats on a single system. The approach achieves faster separations and lower solvent consumption while maintaining or improving chromatographic quality, making it a valuable strategy for pharmaceutical quality control.

References

1. Fountain K. Transferring Compendial HPLC Methods to UPLC Technology for Routine Generic Drug Analysis Waters Application Note 720004251en 2012
2. United States Pharmacopeia. Official Monographs Quetiapine Fumarate USP 40 NF35 S1 2017
3. Neue U D, McCabe D, Vijaya R, Pappa H, DeMuth J. Transfer of HPLC Procedures to Suitable Columns of Reduced Dimensions Pharmacopeial Forum 2009 35(6):1622
4. Columns Calculator Online Help Waters Columns Calculator version 2.0
5. Hong P, McConville P. Dwell Volume and Extra-Column Volume Impact on Method Transfer Waters White Paper 720005723EN 2016