A Rapid and Analytically Sensitive LC-MS Method for the Simultaneous Analysis of a Panel of Steroid Hormones for Clinical Research

Importance of the Topic

Efficient and sensitive quantification of steroid hormones in serum is essential for clinical research into endocrine disorders, metabolic syndromes, and cancer. Traditional assays can be time-consuming, labor-intensive, and prone to variability, driving the need for harmonized LC-MS-based workflows that can handle multiple analytes simultaneously from small sample volumes.

Objectives and Study Overview

The primary goal was to establish a rapid, reproducible LC-MS method using an online solid-phase extraction (SPE) manager to measure testosterone, androstenedione, 17-hydroxyprogesterone, and dehydroepiandrosterone sulfate in a single run. The method targets clinical research applications requiring high throughput and limited sample availability.

Methodology and Instrumentation

• Sample Preparation
  50 μL serum was mixed with ZnSO₄ solution and acetonitrile containing isotopic internal standards, followed by centrifugation.
• Online SPE
  Waters ACQUITY UPLC Online SPE Manager with MassTrak C18 cartridges was used for automated conditioning, equilibration, loading, washing, and elution.
• Chromatography and MS
  ACQUITY UPLC HSS SB C18 column (2.1×50 mm, 1.8 μm) with a water/methanol gradient (both containing 2 mM ammonium acetate and 0.1% formic acid). Waters Xevo TQ-S tandem quadrupole MS operated in negative mode for DHEAS and positive mode for other steroids using MRM transitions.

Main Results and Discussion

• Separation and Run Time
  All four steroids were baseline separated within a 5.5-minute run.
• Analytical Sensitivity
  Lower limits of quantitation ranged from 0.12 to 0.27 nmol/L for the steroid panel and 0.17 μmol/L for DHEAS, with imprecision <8% at LLOQ levels.
• Matrix Effects
  Online SPE virtually eliminated ion suppression, as confirmed by comparison of blank serum extracts to solvent blanks.
• Method Comparison
  Passing-Bablok regression showed excellent agreement with liquid-liquid extraction and protein precipitation methods, demonstrating accuracy and reproducibility.

Benefits and Practical Applications

• Simultaneous quantification of four key steroid hormones reduces instrument time and labor.
• Minimal sample volume (50 μL) is advantageous for pediatric or limited-volume studies.
• Automated SPE integration allows high throughput and consistent sample cleanup.
• Avoids use of large volumes of flammable solvents typical for liquid-liquid extraction.

Future Trends and Potential Applications

Advancements may include expanded steroid panels, incorporation of additional internal standards, and further miniaturization for microliter-scale assays. Automated online SPE-LC-MS workflows could be adapted for hormone profiling in multiplexed clinical studies and for biomarker discovery in precision medicine.

Conclusion

The developed online SPE-LC-MS method enables rapid, sensitive, and reproducible simultaneous measurement of testosterone, androstenedione, 17-hydroxyprogesterone, and DHEAS from micro-volume serum samples. This approach streamlines clinical research workflows while maintaining analytical robustness.

Used Instrumentation

• Waters ACQUITY UPLC System
• ACQUITY UPLC HSS SB C18 Column, 2.1×50 mm, 1.8 μm
• ACQUITY UPLC Online SPE Manager with MassTrak C18 cartridges
• Waters Xevo TQ-S Tandem Quadrupole MS

Reference

Owen L., Keevil B. A Rapid and Analytically Sensitive LC-MS Method for the Simultaneous Analysis of a Panel of Steroid Hormones for Clinical Research. University Hospital South Manchester, UK, 2014.