Method Development for the Separation of Sulfated Estrogens by UltraPerformance Convergence Chromatography (UPC2)

Significance of the Topic

Conjugated sulfated estrogens are key therapeutic agents in hormone replacement therapy and present a significant analytical challenge due to their structural similarity and isobaric nature. Traditional gas chromatography methods require lengthy enzymatic cleavage and chemical derivatization steps, leading to analysis times exceeding three hours and incomplete resolution of critical compound pairs. UltraPerformance Convergence Chromatography (UPC2) offers a high-efficiency, rapid alternative that can analyze these compounds in their native sulfated form without hazardous reagents.

Objectives and Study Overview

This study aimed to develop a robust UPC2 method for baseline separation (resolution >1.5) of ten structurally similar sulfated estrogens using UV detection. The method development followed a systematic approach: initial screening of stationary phases and mobile-phase modifiers, evaluation of additive effects, optimization of pressure and temperature, exploration of flow and gradient programs, and final refinement to meet resolution criteria.

Methodology and Instrumentation

Samples were prepared by simple dilution of individual estrogen sulfates in organic solvents, eliminating enzymatic cleavage and derivatization. An ACQUITY UPC2 system equipped with a PDA detector was used, employing a 3.0×100 mm, 1.7 µm BEH 2-Ethylpyridine column. The mobile phase consisted of CO2 (A) and methanol with additives (B). Key parameters optimized included CO2 backpressure (up to 4100 psi), column temperature (20 °C to 5 °C), flow gradients (1.4 mL/min to 0.9 mL/min), and modifier gradients (8 % to 27 % MeOH with 0.3 % isopropyl amine). Instrument washes and injection volumes were standard for UPC2 operation.

Main Results and Discussion

• Initial column and modifier screening identified BEH 2-EP chemistry with methanol as optimal for selectivity.
• Additive evaluation showed that 0.3 % isopropyl amine facilitated elution and gave superior peak shapes under UV detection.
• Increasing backpressure from 1700 to 4100 psi improved resolution of critical pairs (peaks 6 and 7), reaching R S = 1.00 at constant flow and further improvements with a flow gradient.
• Lowering the column temperature from 50 °C to 20 °C progressively enhanced resolution of all isobaric pairs, achieving R S > 1.5 at 20 °C.
• Reverse flow gradients maintained system pressure near specification limits, maximizing peak capacity and reducing analysis time.
• Final gradient optimization (8 % to 27 % modifier over 12 min with a 1.4 → 0.9 mL/min flow gradient) delivered baseline resolution of all ten analytes.

Benefits and Practical Applications

• Over 90 % reduction in total analysis time compared to GC methods.
• Elimination of enzymatic and derivatization steps simplifies workflow and reduces error opportunities.
• Use of benign solvents (CO2 and methanol) enhances safety and sustainability.
• High resolution of isobaric estrogens supports accurate quantitation in pharmaceutical QC and research laboratories.

Future Trends and Potential Applications

• Coupling UPC2 with mass spectrometry for enhanced structural confirmation and trace quantitation.
• Application of UPC2 methods to glucuronide conjugates and other steroid derivatives.
• Exploration of alternative stationary phases and green modifiers to further reduce analysis time and solvent consumption.
• Customization of UPC2 workflows for clinical and environmental steroid monitoring.

Conclusion

A fully optimized UPC2 method was established for rapid, high-resolution separation of ten sulfated estrogens in their native form. The approach eliminates complex sample preparation steps, achieves baseline resolution (R S > 1.5) in under 15 minutes, and significantly improves laboratory efficiency for steroid analysis.

Instrumentation

• Acquity UPC2 system with photodiode-array detector
• BEH 2-Ethylpyridine 1.7 µm, 3.0×100 mm column
• CO2 mobile phase supply (medical grade)
• Modifier: methanol with isopropyl amine additive
• ABPR capable of up to 4100 psi

References

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