A Gradient USP Method Modernization Using Agilent InfinityLab Poroshell 120 Aq-C18

Importance of the Topic

The modernization of USP monograph methods for pharmaceutical quality control enhances laboratory productivity and resource efficiency. Shorter analysis times and reduced solvent consumption are essential for high-throughput environments and greener chromatographic practices.

Objectives and Study Overview

This study aimed to transfer the gradient USP method for iohexol related compounds from a conventional 5 µm column to a superficially porous 2.7 µm column under the revised USP <621> guidelines. Key goals included significant reductions in analysis time and solvent usage without requiring method revalidation.

Methodology and Instrumentation

Gradient HPLC methods were adapted as follows:

• Original column: Agilent ZORBAX SB-C18, 4.6×250 mm, 5 µm
• Modernized column: Agilent InfinityLab Poroshell 120 Aq-C18, 4.6×100 mm, 2.7 µm
• LC system: Agilent 1260 Infinity II with binary pump, multisampler, column thermostat, and DAD detector
• Mobile phase: water (A) and acetonitrile (B) gradient
• System suitability based on tR/Wh2 ratios and resolution for iohexol impurities

Flow rate, gradient time, and injection volume were adjusted using USP-specified equations to preserve chromatographic performance.

Main Results and Discussion

Adoption of the 2.7 µm superficially porous column achieved:

• Analysis time reduction from 60 to 13 minutes (78% decrease)
• Solvent consumption reduction by 78%
• System suitability metrics (tR/Wh2 ratios and resolution) within USP limits
• Retention and selectivity maintained via gradient volume scaling

This approach enhances sample throughput while maintaining impurity separation and sensitivity.

Benefits and Practical Applications

• Increased laboratory productivity through faster run times
• Lower operational costs and environmental impact via reduced solvent use
• Direct method transfer allowed under revised USP <621> without revalidation
• Applicable to routine impurity profiling in generic pharmaceutical QC

Future Trends and Opportunities

Advancements in superficially porous particle design and higher-pressure UHPLC systems will further decrease analysis times and enhance resolution. Integration of green solvents and automated method transfer tools can expand application to diverse APIs and complex formulations.

Conclusion

This work demonstrates successful modernization of a USP gradient HPLC method for iohexol using a superficially porous 2.7 µm column. Significant time and solvent savings were realized without compromising analytical performance, in full compliance with regulatory and sustainability objectives.

Instrumentation Used

• Agilent 1260 Infinity II Binary Pump (G7112B)
• Agilent 1260 Infinity II Multisampler (G7167A)
• Agilent InfinityLab Multicolumn Thermostat (MCT)
• Agilent 1260 Diode Array Detector WR (G7115A)
• Agilent OpenLab CDS Version C.01.07

References

1. USP <621> Chromatography, Supplement, Official December 2022.
2. USP Monograph: Iohexol, USP 35, 3534–3536.
3. Agilent Technologies Application Note 5994-6544EN, 2023.
4. Agilent Technologies Application Note 5994-5400EN, 2022.