Rapid UPLC-MS/MS Dried Blood Spot Analysis of Steroid Hormones for Clinical Research

Importance of the Topic

Steroid hormone profiling in dried blood spots (DBS) provides a minimally invasive, low-cost sampling approach that is highly valuable for clinical research, epidemiology and biomarker discovery. By combining DBS with advanced UPLC-MS/MS, researchers gain high analytical specificity and sensitivity, overcoming limitations of traditional ligand binding assays.

Aims and Study Overview

• Develop a rapid, high-throughput UPLC-MS/MS method for simultaneous analysis of key steroid hormones in DBS.
• Demonstrate chromatographic separation of isobaric steroids within a short cycle time.
• Establish analytical sensitivity, linearity and reproducibility suitable for clinical research concentrations (0.5–500 ng/mL).

Used Instrumentation

• Waters ACQUITY UPLC I-Class System
• Xevo TQ-S micro Mass Spectrometer
• CORTECS C18 2.7 micrometer, 2.1 × 50 mm Column with VanGuard Pre-Column
• Tecan Freedom Evo 100 liquid handling platform
• Oasis MAX microElution SPE 96-well plate

Methodology

Two 3 mm DBS punches were spiked with internal standard, extracted in aqueous buffer and processed via automated Oasis MAX microElution SPE. Eluates were diluted and injected (25 µL) onto the UPLC system using a 0.05 mM ammonium fluoride/methanol gradient. Separation of cortisol, 17-OHP, androstenedione, 11-deoxycortisol and 21-deoxycortisol from endogenous interferences was achieved in a 2.3-minute injection-to-injection cycle. Detection employed multiple reaction monitoring on the Xevo TQ-S micro.

Main Results and Discussion

• Calibration curves were linear from 0.5–500 ng/mL (r² > 0.99) across five occasions.
• Lower limits of quantification (LLOQ) demonstrated signal-to-noise ratios ≥12 for 0.5–1 ng/mL levels.
• Total precision and repeatability were ≤9.3% CV at QC levels (2–400 ng/mL).
• Baseline separation of critical steroid isobars achieved within 1.4 minutes of elution window, ensuring high specificity.

Benefits and Practical Applications

• High throughput: extraction and analysis of 384 DBS samples in under 18 hours.
• Reduced manual handling and improved reproducibility through automated SPE workflow.
• Enhanced analytical specificity compared to ligand binding assays, enabling multiplex steroid panels.
• Minimal sample volume requirement supports pediatric and field studies.

Future Trends and Potential Applications

Expansion of this workflow to broader steroid panels and other small-molecule biomarkers, integration into large-scale population studies and newborn screening programs, and coupling with emerging point-of-care sampling devices may further enhance clinical and translational research. Advances in high-resolution chromatography and ion mobility could allow even faster separations and multiplexing.

Conclusion

The described UPLC-MS/MS protocol delivers rapid, sensitive and reproducible analysis of key steroid hormones in dried blood spots, addressing critical needs in clinical research and biomarker validation with high throughput and minimal sample handling.

Reference

Foley D, Brown H, Calton L. Rapid UPLC-MS/MS dried blood spot analysis of steroid hormones for clinical research. Waters Corporation; 2019.