Analysis of Testosterone, Androstenedione, and Dehydroepiandrosterone Sulfate in Serum for Clinical Research

Importance of the Topic

Precise measurement of androgenic steroids in serum underpins research into endocrine disorders, contraceptive effects and metabolic regulation. High sensitivity and selectivity are essential to detect low physiological levels and to distinguish structurally similar compounds reliably.

Objectives and Overview

This study describes the development of a streamlined LC-MS/MS assay for simultaneous quantification of testosterone, androstenedione and DHEAS in human serum. Key goals included integrating selective sample preparation, ultraperformance liquid chromatography, tandem mass spectrometry and automated sample handling, and validating the method against external quality assessment schemes.

Methodology

• Sample Preparation: 100 µL serum spiked with isotope-labeled internal standards, proteins precipitated, then extracted on Oasis® PRiME HLB µElution plates using a Tecan Freedom Evo 100/4 liquid handler.
• Chromatography: ACQUITY UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) with water/methanol gradient containing 2 mM ammonium acetate and 0.1% formic acid, flow rate 0.6 mL/min, run time 4.7 min.
• Detection: Xevo TQD mass spectrometer in multiple reaction monitoring (MRM) mode; positive ESI for testosterone and androstenedione, negative ESI for DHEAS.
• Calibration and QC: Calibrators in stripped human serum covering 0.17–69 nmol/L (testosterone, androstenedione) and 0.14–54 µmol/L (DHEAS); QC at low, mid and high concentration levels.

Main Results and Discussion

• No detectable interference from eight related steroids; baseline resolution achieved for analytes and key epimers.
• Limits of quantification at 0.17 nmol/L for testosterone and androstenedione, and 0.14 µmol/L for DHEAS (signal-to-noise >10).
• Precision (total CV) ≤6.3% across QC levels and days; repeatability <4.7%.
• Excellent linearity (r² >0.998) over the calibration ranges.
• Minimal matrix effects (matrix factor 0.93–1.02; RSD ≤6.8%).
• Method comparison versus an independent LC-MS/MS assay showed biases of +5.0%, −3.3% and −6.3% for testosterone, androstenedione and DHEAS, respectively; EQA biases within ±6.9%.

Benefits and Practical Applications

The low sample volume requirement, automated SPE workflow and rapid UPLC-MS/MS analysis enable high throughput and reduce operator error. The assay’s sensitivity supports quantification at physiological levels, making it suitable for clinical research, pharmacokinetic studies and quality control laboratories.

Future Trends and Opportunities

Future enhancements may include expanding the steroid panel, adopting high-resolution mass spectrometry, integrating artificial intelligence for data processing, and translating the workflow to clinical diagnostic platforms for broader patient applications.

Conclusion

The validated method combining Oasis PRiME HLB µElution SPE, ACQUITY UPLC HSS T3 separation and Xevo TQD detection delivers a sensitive, selective and precise assay for testosterone, androstenedione and DHEAS in serum. Its automation and robust performance support reliable clinical research use.

Reference

Foley D and Calton L. A Clinical Research Method for the Analysis of Serum Testosterone and Androstenedione. Waters application note. 2015; P/N 720005274EN.