Comprehensive Profiling of Free and Conjugated Estrogens by Capillary Electrophoresis-Time of Flight-Mass Spectrometry

Importance of the Topic

The bioavailability and metabolism of estrogens are critical to human health, and comprehensive profiling of both free and conjugated forms is essential for clinical diagnostics and research. Conventional assays often lack the specificity, sensitivity, or dynamic range needed to quantify diverse estrogen metabolites in complex matrices such as urine. This study addresses these challenges by presenting a streamlined analytical method that combines separation and high-resolution mass spectrometry. The ability to distinguish positional isomers and accurately predict migration behavior further strengthens the relevance of the approach for endocrine studies and environmental monitoring.

Goals and Study Overview

The primary objective was to establish a robust capillary electrophoresis–time-of-flight mass spectrometry (CE-TOF-MS) workflow for simultaneous analysis of free estrogens and their phase II conjugates. A central composite experimental design was applied to optimize ionization parameters and sheath liquid composition. Predictive modeling of relative migration times was incorporated to facilitate identification of known and unknown metabolites. The method was validated using standard mixtures, simulated urine, and authentic human urine samples.

Methodology and Instrumentation

The analytical protocol included the following steps:

• Capillary electrophoresis separation in a 50 µm i.d., 85 cm fused silica capillary at 20 °C with 50 mM ammonium bicarbonate buffer (pH 9.5)
• Coaxial sheath liquid electrospray interface using 5 mM ammonium bicarbonate in 80:20 methanol:water at 14 µL/min
• Time-of-flight mass spectrometry in negative-ion mode, scanning m/z 50–1100 with real-time mass correction (<2 ppm)
• Design of experiments for optimization of ESI voltage, methanol fraction, and sheath liquid flow rate via a 23 central composite design
• Off-line sample preparation for human urine involving 10-fold dilution and optional solid-phase extraction (Oasis HLB cartridges) for 125-fold preconcentration

Applied Instrumentation

• Agilent G7100A capillary electrophoresis system
• Agilent 6224 orthogonal-axis TOF LC/MS spectrometer
• 3D-CE ChemStation and Agilent MassHunter Workstation for control and data acquisition
• Igor Pro 5.0 software for data processing, electrophoregram analysis, and response surface modeling

Key Results and Discussion

Baseline resolution was achieved for 12 estrogen targets, including free steroids, glucuronides, and sulfates, with clear separation of positional isomers. Limits of detection ranged from 0.07 to 0.23 µM, and calibration exhibited excellent linearity (R2 > 0.99). Precision was demonstrated with relative peak area CVs below 14% and migration time CVs under 0.2%. Predicted relative migration times based on molecular volume and effective charge correlated strongly with measured values (R2 = 0.975). Matrix effects in diluted urine were negligible, and additional steroid conjugates were tentatively identified from authentic samples.

Benefits and Practical Applications

• Simultaneous profiling of free and conjugated estrogens with minimal sample handling
• Naturally efficient desalting and reduced ion suppression through CE separation
• Accurate isomer discrimination and structural assignment using combined migration prediction and high-resolution MS
• Applicability to clinical research, endocrine monitoring, environmental screening, and food safety analysis

Future Trends and Potential Applications

• Integration with microfluidic CE platforms for higher throughput and reduced sample volume
• Expansion to broader steroid and metabolite panels using advanced predictive modeling
• Automation of sample preparation and on-line coupling with solid-phase extraction
• Application in longitudinal patient monitoring, exposome studies, and personalized medicine

Conclusion

The developed CE-ESI-TOF-MS method offers a versatile and sensitive approach for comprehensive estrogen profiling, combining high selectivity, predictive migration modeling, and minimal matrix interference. This platform can support diverse applications in analytical chemistry and biomedicine, enabling reliable detection and identification of hormone metabolites in complex biological fluids.

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