Analysis of Estrogens Using a Solid Core HPLC Column

Significance of the Topic

Aromatic steroid hormones such as estrogens play critical roles in human health, being central to female reproductive biology and widely used in hormone therapies and contraceptives. Reliable separation and quantitation of these structurally related compounds are essential for pharmaceutical quality control, environmental monitoring and clinical analysis. Advanced chromatographic phases that enhance selectivity and speed can significantly improve laboratory efficiency and analytical accuracy.

Objectives and Study Overview

This application study aims to evaluate the performance of a solid-core Accucore Phenyl-X HPLC column for the isocratic separation of four key estrogens: estriol, estradiol, estrone and ethynylestradiol. Performance metrics are benchmarked against a conventional fused-core C18 column to demonstrate improvements in selectivity, retention and resolution.

Methodology and Instrumentation

A mixed standard of the four estrogens (100 µg/mL each) was prepared in a mobile phase of acetonitrile : methanol : water (15 : 40 : 45, v/v). Separations were conducted at 40 °C with a 0.40 mL/min flow rate and UV detection at 220 nm. Key chromatographic conditions were kept identical on both columns to directly compare selectivity and retention behaviour.

Instrumentation Used

• HPLC system: Thermo Scientific
• Column: Accucore Phenyl-X, 2.6 µm, 100 × 2.1 mm
• Autosampler vial kit: Premium Autosampler Vial Convenience Kit
• Solvents: HPLC-grade water, acetonitrile, methanol

Main Results and Discussion

The Accucore Phenyl-X column delivered sharper peaks, increased hydrophobic retention and enhanced aromatic selectivity compared to the C18 phase. Notably, estrone and ethynylestradiol exhibited an inversion in elution order, highlighting complementary selectivity. The solid-core particles provided efficient mass transfer at reduced backpressure, enabling faster runs.

• Retention times for all four estrogens were reduced by approximately 10–20 % on Phenyl-X compared to C18.
• Resolution between key peak pairs increased (resolution factor RS ~2.0 vs. 1.25 on C18).
• Unique π–π interactions with the phenyl bonded phase improved separation of closely related aromatic steroids.

Benefits and Practical Applications

The Phenyl-X phase offers several practical advantages for routine estrogen analysis:

• Enhanced selectivity for aromatic steroid compounds, enabling clear baseline separation.
• Reduced analysis time and lower backpressure, extending column lifetime and reducing solvent consumption.
• Compatibility with aqueous-rich mobile phases, supporting bioanalytical workflows.

Future Trends and Possibilities for Application

Advances in superficially porous particle technology will continue to push speed and resolution boundaries. Future work may include:

• Integration with mass spectrometry for enhanced sensitivity in trace-level estrogen detection.
• Expansion to other hormone classes and polar metabolite profiling.
• Automation and high-throughput platforms for clinical diagnostics and environmental screening.

Conclusion

The Accucore Phenyl-X HPLC column demonstrated superior performance over a conventional C18 column for the isocratic separation of estrogens. Its unique phenyl selectivity, efficient solid-core design and robustness to aqueous conditions make it a valuable tool for pharmaceutical, clinical and environmental steroid analyses.

References

1. Women-Health-Info.com (accessed 2023)
2. Dionex.com (accessed 2023)