Analysis of Hydrocortisone - No. 582

Importance of the Topic

Purification of steroid drugs such as hydrocortisone is essential for ensuring pharmaceutical quality, safety and efficacy. Preparative reversed-phase LC coupled with MS detection offers high resolution and reliable identification of target compounds and related impurities, facilitating the production of high-purity APIs.

Study Objectives and Overview

This application note describes a preparative LC-MS method using the Shim-pack Scepter C18-120 column to separate hydrocortisone from two key impurities. The goal is to demonstrate efficient isolation, monitoring and collection of high-purity hydrocortisone fractions in a single workflow.

Methodology

The method employs a reversed-phase gradient on a preparative Shim-pack Scepter C18-120 (150 × 20 mm, 5 µm) column. Mobile phase A is 0.1 % formic acid in water and B is acetonitrile. The gradient runs from 25 % to 45 % B over 20 minutes, followed by a re-equilibration step. The preparative flow rate is 20 mL/min, with a 10 mL injection volume. A 2 mL sample loop and 5 mL syringe ensure reproducible injections. UV detection at 245 nm is combined with MS monitoring in positive and negative ESI/APCI modes (m/z 100–500).

Instrumentation

• LC system: Nexera X3
• Prep column: Shim-pack Scepter C18-120 (150 × 20 mm, 5 µm)
• MS detector: LCMS-2050 with dual-ionization ESI/APCI
• UV detector: PDA at 245 nm

Results and Discussion

The method achieved baseline separation of hydrocortisone and two impurities. Hydrocortisone was eluted within 20 minutes under the optimized gradient. MS detection confirmed target and impurity identities by characteristic m/z values. Fraction collection yielded hydrocortisone with purity exceeding 99 %. The use of a preparative flow rate and large injection volume demonstrated scalability and robustness.

Benefits and Practical Applications

• High-purity preparative isolation of steroids
• Online impurity monitoring via MS ensures identity confirmation
• Scalable protocol for pharmaceutical production
• Efficient solvent usage and rapid cycle times

Future Trends and Opportunities

Advances in preparative LC-MS integration may enable real-time fraction quality assessment. Adoption of greener solvents and automated fraction handling will improve sustainability and throughput. Emerging column materials and microfluidic preparative platforms may further enhance separation efficiency and reduce solvent consumption.

Conclusion

The described preparative LC-MS method using Shim-pack Scepter C18-120 provides a reliable, scalable solution for isolating high-purity hydrocortisone. Combining UV detection with MS confirmation ensures robust impurity control, supporting pharmaceutical development and production.

References

Application News 01-00651, Shimadzu Corporation (2024)