Analysis of Steroids by UltraPerformance Convergence Chromatography

Importance of the topic

Understanding and quantifying steroid profiles is critical in pharmaceutical development, clinical diagnostics, endocrine research, and anti‐doping analysis. Traditional gas chromatography methods require derivatization and extended run times, while conventional liquid chromatography approaches may lack sufficient speed or selectivity for closely related steroid isomers. The adoption of Supercritical Fluid Chromatography using CO2 as the primary mobile phase offers significant advantages in reducing analysis time, solvent consumption, and sample preparation steps.

Objectives and Study Overview

This study aimed to evaluate the performance of UltraPerformance Convergence Chromatography (UPC 2) for rapid separation of nine structurally similar steroids. The investigation compared three stationary phase chemistries to determine optimal selectivity and resolution under a fast screening gradient.

Methodology and Instrumentation

• Sample Preparation: A mixture of nine steroid standards (androstenedione, estrone, 17α-hydroxyprogesterone, testosterone, 11-deoxycortisol, estradiol, corticosterone, aldosterone, and cortisol) at 0.2 mg/mL in methanol/ethanol (88:12).
• Chromatographic System: ACQUITY UPC 2 with photodiode array detection set to 220 nm, data compensated between 380 and 480 nm.
• Columns Evaluated: BEH, BEH 2-EP, and CSH Fluoro-Phenyl, all 1.7 µm particle size, 3.0 × 50.0 mm.
• Mobile Phase: CO2 with methanol co-solvent; gradient from 2% to 17% methanol over 2 minutes at 3.65 mL/min.
• Operating Conditions: Column temperature 40 °C, ABPR at 1800 psi, 1 µL injection volume.

Key Results and Discussion

The three stationary phases exhibited distinct selectivity patterns, all achieving baseline resolution of the nine steroids within two minutes. BEH provided balanced retention for early‐eluting steroids, BEH 2-EP enhanced separation of mid-polarity analytes, and the CSH Fluoro-Phenyl phase excelled in resolving acidic and aromatic steroids. Rapid gradient and high flow rate reduced overall analysis time significantly compared to GC/MS and conventional LC/MS methods.

Benefits and Practical Applications

• Elimination of derivatization steps simplifies sample preparation and reduces potential artifacts.
• Direct analysis in organic extraction solvents without intermediate dilution or solvent exchange.
• Shorter run times increase sample throughput for clinical or high‐volume testing laboratories.
• Lower solvent consumption and waste generation decrease operational costs and environmental impact.

Future Trends and Potential Applications

Combining UPC 2 with high‐resolution mass spectrometry could further enhance sensitivity and structural elucidation of steroid metabolites. Automation of sample handling and data processing will streamline workflows in clinical and forensic settings. Expanding stationary phase chemistries may allow targeted profiling of larger steroid panels and emerging biomarkers.

Conclusion

UltraPerformance Convergence Chromatography demonstrates a rapid, selective, and efficient approach for steroid analysis, overcoming limitations of traditional methods. Its integration into analytical laboratories promises to accelerate drug development, improve diagnostic accuracy, and support regulatory testing with reduced cost and environmental footprint.

References

No external literature references were provided in the original text.