UHPLC Separation of Nine Corticosteroids in Under Four Minutes

Importance of the Topic

Rapid and reliable analysis of corticosteroids is essential for pharmaceutical quality control, clinical diagnostics, and regulatory compliance. High-throughput methods save time and resources while ensuring accurate quantitation of multiple steroid hormones in complex matrices.

Objectives and Study Overview

This study demonstrates an ultra-high-performance liquid chromatography (UHPLC) method capable of separating nine commonly used corticosteroids in under four minutes. The goal was to evaluate different column formats and mobile phase compositions to optimize resolution, analysis time, and solvent consumption.

Methodology and Applied Instrumentation

Methodological Approach:

• Mobile phase: water, tetrahydrofuran (THF), and methanol in isocratic or gradient modes.
• Columns tested: 5 µm C18 (4.6 × 250 mm), 3 µm C18 (4.6 × 150 mm), and 2.2 µm RSLC C18 (2.1 × 100 mm).
• Temperatures: 40 °C and 50 °C, with a 2 µL precolumn heater where needed.
• Flow rates from 0.75 to 1.5 mL/min, injection volumes from 5 to 50 µL.

Applied Instrumentation:

• Dionex UltiMate 3000 RSLC system with SRD-3600 degasser, DGP-3600RS pump, WPS-3000RS sampler, and TCC-3000RS compartment.
• Diode array detector (DAD-3000RS) with semi-analytical and semi-micro flow cells.

Main Results and Discussion

Condition 1 (5 µm, 4.6×250 mm, gradient): resolved seven steroids in 30 min; prednisolone and cortisone coeluted. Condition 2 (5 µm, 4.6×250 mm, isocratic at 50 °C): complete separation in 25 min with satisfactory resolution and asymmetry. Condition 3 (3 µm, 4.6×150 mm): reduced run time and solvent use, maintaining resolution; precolumn heating required to prevent thermal mismatch. Condition 4 (2.2 µm, 2.1×100 mm RSLC): full separation in under 4 min at 586 bar without precolumn heater. Performance metrics (USP resolution and asymmetry) showed no sacrifice in chromatographic quality across formats.

Benefits and Practical Applications of the Method

• High throughput: sub-4-minute analysis per sample.
• Lower solvent consumption: miniaturized column formats.
• Flexibility: scalable to different laboratory requirements; compatible with routine QC and clinical workflows.
• Robustness: consistent resolution and peak shape across column types.

Future Trends and Potential Applications

Advances may include sub-2 µm core–shell columns for even faster separations, integration with mass spectrometry for enhanced sensitivity, adoption of greener solvents, and automated online sample preparation. Emerging high-pressure UHPLC systems will further push speed and resolution boundaries.

Conclusion

The described UHPLC method provides a rapid, solvent-efficient, and reliable separation of nine corticosteroids. The approach offers laboratories a practical solution for high-throughput steroid analysis without compromising chromatographic performance.

Reference

No additional literature references were provided in the original text.