Gradient Method Transfer of the Iohexol USP Monograph HPLC Method for Related Compounds to Smaller Particle Size ZORBAX Columns

Importance of the Topic

In pharmaceutical and biopharmaceutical analysis, United States Pharmacopeia (USP) monograph methods often rely on older HPLC column technologies that lead to long analysis times and high solvent consumption. Modernizing these gradient methods to smaller particle size columns under the revised USP <621> guidelines enhances laboratory efficiency, reduces environmental impact, and maintains regulatory compliance.

Objectives and Study Overview

This application note describes the transfer of the USP monograph gradient HPLC method for related-compound analysis of iohexol from a conventional 4.6 × 250 mm, 5 µm column to smaller particle size columns (3.0 × 150 mm, 3.5 µm and 2.1 × 100 mm, 1.8 µm). The goal was to achieve significant reductions in run time and solvent usage without revalidation by adhering to the updated USP <621> criteria for gradient method modernization.

Methodology

A water-acetonitrile gradient (initial 1 % B rising to 13 % B) was employed across all column formats. To maintain consistent chromatographic performance, flow rates, gradient times, and injection volumes were scaled using USP‐recommended equations based on column dimensions and particle sizes. A system suitability solution, prepared per the iohexol monograph, ensured identity, selectivity, and resolution criteria were met for the exo- and endo-isomers and related impurities.

Instrumentation

• Agilent 1260 Infinity II LC system used for 5 µm and 3.5 µm columns, with 0.17 mm tubing, binary pump, multisampler, multicolumn thermostat, and diode array detector (254 nm).
• Agilent 1290 Infinity II LC system used for 1.8 µm column, with 0.12 mm tubing, high-speed pump, multisampler, multicolumn thermostat, and UV detector configured for 40 Hz sampling.

Main Results and Discussion

Transfer to the 3.5 µm, 3.0 × 150 mm column reduced analysis time from 60 to 25 minutes (58 % reduction) and solvent usage by 75 %. Use of the 1.8 µm, 2.1 × 100 mm column shortened run time to 8.6 minutes (86 % reduction) with a 92 % decrease in solvent consumption. All system suitability parameters—retention times, resolution between key impurities (>40), and relative peak areas—remained within USP specifications. L/dp ratios for the modified columns fell within the acceptable 37 500–75 000 range, confirming method equivalence without revalidation.

Benefits and Practical Applications

• Substantial improvements in sample throughput and laboratory productivity.
• Reduced operational costs and solvent waste, supporting greener analytical practices.
• Retention of monograph compliance and chromatographic selectivity without revalidation.
• Applicability to routine quality control of iohexol in pharmaceutical production.

Future Trends and Potential Applications

Ongoing adoption of superficially porous and sub‐2 µm particle columns will further accelerate gradient HPLC methods. Revised USP <621> guidelines pave the way for broader modernization of other monograph methods. Future integration with automated method transfer tools and AI-driven optimization will streamline cross-laboratory consistency and data analysis. Continued miniaturization and microflow LC may enhance sensitivity and further decrease solvent consumption.

Conclusion

The iohexol related-compound USP method was successfully transferred to smaller particle size columns under the revised USP <621> gradient method provisions. The optimized methods delivered substantial reductions in run time (up to 86 %) and solvent usage (up to 92 %) while preserving chromatographic performance and compliance, all without the need for revalidation.

References

1. USP Harmonized Standards Home Page, Supplement USP Stage 4 Harmonization, Official, December 1, 2022.
2. USP 35 Monographs. Iohexol, United States Pharmacopeia: 3534–3536.
3. Long W. J. A Simple Conversion of the USP Assay Method for Diphenhydramine HCl to the Agilent InfinityLab Poroshell 120 Column EC-C8. Agilent Technologies application note 5994-5400EN, 2022.