Method Development for the Analysis of Endogenous Steroids Using Convergence Chromatography with Mass Spectrometric Detection

Importance of the Topic

The precise measurement of endogenous steroids is crucial for clinical research, drug development, and diagnostic applications. Structural similarities among steroid hormones demand high-resolution chromatographic separation prior to detection. Traditional GC/MS requires derivatization and lengthy run times (~40 minutes), while typical LC/MS methods range from 4 to 12 minutes. Convergence chromatography (UPC2) coupled with mass spectrometry offers a rapid, efficient alternative that can accelerate steroid profiling and reduce solvent consumption.

Objectives and Study Overview

This study aimed to develop and validate a two-minute chromatographic method for nine endogenous steroids using UPC2 with tandem quadrupole mass spectrometric detection. Key goals included:

• Screening multiple UPC2 stationary phases for optimal selectivity.
• Establishing MRM transitions and ionization conditions for each steroid.
• Assessing method reproducibility, linearity, and sensitivity in a post-spiked human plasma matrix.

Methodology and Instrumentation

Sample Preparation:
A 3:1 acetonitrile protein crash of human plasma was performed, supernatant collected, and post-spiked with nine steroid standards (0.98–500 ng/mL).

Chromatographic Conditions:

• System: Waters ACQUITY UPC2 with photodiode array detector.
• Column screening: BEH, BEH 2-EP, CSH Fluoro-Phenyl, HSS C18 SB (3.0×50 mm, 1.7–1.8 µm).
• Mobile Phase A: CO2; Modifier B: methanol. Gradient 2→17% B in 2 min; flow rate 3.65 mL/min; column temp. 40 °C; ABPR 1800 psi.
• Injection volume: 1 µL (UV screening), 5 µL (MS quantification).

Mass Spectrometric Conditions:

• Instrument: Waters Xevo TQ with ESI source (positive or negative mode for estradiol).
• MRM transitions optimized by direct infusion (IntelliStart).
• Make-up flow: methanol with 2.5% water and 0.1% ammonium hydroxide at 0.4 mL/min.
• Capillary voltage 1.0 kV; desolvation temperature 500 °C; gas flow 750 L/h.

Instrumentation Used:

• Waters ACQUITY UPC2 System (BSM, SM, CCM, CM, PDA detector).
• Waters Xevo TQ Mass Spectrometer.
• Empower 3 and MassLynx software.

Main Results and Discussion

Stationary Phase Selection:
All four UPC2 columns achieved baseline separation of nine steroids within two minutes, with BEH providing the best selectivity.

Ionization and MRM Optimization:
Ammonium hydroxide in the make-up flow enhanced MS signal for eight steroids. Optimal MRM transitions and source parameters yielded robust detection.

Reproducibility:
One-microliter injections of 50 ng/mL spiked plasma over 100 injections showed peak area RSDs of 5.6–13.7%.

Linearity:
Five-microliter injections across 0.98–500 ng/mL produced correlation coefficients r² ≥0.9926 for all compounds. Deviations were generally within ±20%.

Sensitivity and Throughput:
Analysis time reduced by 50–95% compared to GC/MS and LC/MS, with less solvent consumption and waste generation.

Benefits and Practical Applications

• High-throughput steroid profiling in under two minutes.
• Minimal sample preparation without derivatization.
• Compatibility with UV and MS detection modes.
• Lower operating costs and reduced environmental impact due to CO2 mobile phase.
• Potential for routine QA/QC, clinical research, and pharmaceutical bioanalysis.

Future Trends and Applications

Continued method refinement could include:

• Derivatization strategies to further improve ionization efficiency and sensitivity.
• Integration with high-resolution MS for broader steroid panels.
• Miniaturized sample preparation or on-line extraction to enhance throughput.
• Application in clinical diagnostics for low-abundance hormone quantification.
• Expansion to metabolomic profiling and green analytical protocols.

Conclusion

The developed UPC2-MS workflow delivers rapid, reproducible analysis of nine endogenous steroids with excellent linearity and acceptable precision. By leveraging convergence chromatography and optimized MS settings, the method represents a significant improvement over conventional GC/MS or LC/MS approaches in speed, solvent use, and operational cost. Further enhancements in sensitivity will broaden its application in clinical and research environments.

Reference

1. Sellers K. Rapid analysis of steroid hormones by GC/MS using the new Rxi-1ms column. Restek Application Note.
2. Licea-Perez H, Wang S, Szapacs ME, Yang E. Development of a highly sensitive and selective UPLC/MS/MS method for the simultaneous determination of testosterone and 5α-dihydrotestosterone in human serum to support testosterone replacement therapy for hypogonadism. Steroids. 2008;73:601–610.
3. Schwarz E, Liu A, Randall H, Haslip C, Keune F, Murray M, Longo N, Pasquali M. Use of steroid profiling by UPLC-MS/MS as a second tier test in newborn screening for congenital adrenal hyperplasia: The Utah experience. Pediatric Research. 2009;66:230–235.