Simplified, high-throughput separation of glucocorticoids

Significance of the Topic

This application note addresses the analytical challenge of separating structurally similar glucocorticoids in pharmaceutical quality control and drug development. Rapid, reproducible assays for hormones such as prednisone, cortisone, prednisolone, hydrocortisone, corticosterone, betamethasone, and dexamethasone are critical for high‐throughput screening and regulatory compliance.

Goals and Study Overview

The primary goal was to demonstrate that alternative UHPLC stationary phase selectivities, specifically a solid‐core C30 phase, can simplify the isocratic separation of seven glucocorticoids and reduce analysis time to two minutes or less. Key performance indicators included resolution of critical pairs, retention time precision, and solvent consumption.

Methodology and Instrumentation

Sample preparation involved dissolving each steroid in water/acetonitrile mixtures to obtain 1 mg/mL primary solutions and 0.1 mg/mL mixed working standards. Separations were performed on a Thermo Scientific Vanquish Flex Quaternary UHPLC system with SmartInject and LightPipe DAD technology. The column was Accucore C30 (150 × 2.1 mm, 2.6 µm). Mobile phase composition was isocratic 73% water/8% methanol/19% tetrahydrofuran at 0.6 mL/min, 60 °C, detection at 240 nm.

Main Results and Discussion

— All seven glucocorticoids eluted within two minutes with baseline resolution (minimum 1.5).
— Temperature increase from 50 °C to 60 °C reduced retention times and peak widths without compromising selectivity.
— Flow rate variation from 0.4 to 0.7 mL/min showed ≥80% of optimal efficiency at 0.6 mL/min.
— Twenty‐four replicate injections at 0.6 mL/min and 60 °C yielded retention time RSDs of 0.10–0.14%, greatly surpassing typical USP 2% criteria.
— Compared to legacy 20-minute methods (250 × 4.6 mm columns), the new method achieved a ten-fold throughput increase and twenty-fold reduction in solvent use.

Benefits and Practical Applications

• High‐throughput screening: sub-2-minute analysis enables rapid sample turnover.
• Cost and waste reduction: Lower mobile phase consumption and THF usage.
• Robust performance: Solid-core C30 column delivers consistent retention and reproducibility.
• Regulatory alignment: Resolution meets or exceeds USP monograph requirements.

Future Trends and Potential Applications

Emerging UHPLC phases and multi-omics integrations may further accelerate steroid profiling. Automation and AI-driven method optimization could refine selectivity and solvent usage. Adaptation to LC-MS platforms would enhance sensitivity for trace analysis. Expanding to other small molecule classes can leverage solid-core advantages for high‐throughput pharmaceutical and clinical testing.

Conclusion

The use of a solid-core C30 column in a simple isocratic UHPLC method achieves rapid, reproducible separation of seven glucocorticoids in under two minutes. This approach offers significant improvements in throughput, solvent economy, and regulatory compliance over conventional methods, making it well suited for pharmaceutical R&D and QC environments.

References

1. Thermo Fisher Scientific. Rapid analysis of nine corticosteroids using an Acclaim RSLC C18 column. Application Note AN205. 2016.
2. Thermo Fisher Scientific. Accucore HPLC Columns Technical Guide. Brochure TG20666. 2017.