Development of a ClinicalResearch Method for theMeasurement of Testosteroneand Dihydrotestosterone

Importance of the Topic

Clinical measurement of testosterone and dihydrotestosterone (DHT) at physiological concentrations is essential for endocrinology research and patient monitoring, yet it remains challenging due to low analyte levels in pediatrics and females and the limited specificity of immunoassays.

Objectives and Study Overview

The study aimed to establish a rapid, high-throughput LC-MS/MS method with automated online solid-phase extraction (SPE) to directly quantify testosterone and DHT from plasma without derivatization, streamlining sample preparation while maintaining sensitivity.

Methodology and Instrumentation

• Instrumentation: ACQUITY UPLC system coupled to a Xevo TQ-S triple quadrupole mass spectrometer.
• Column: ACQUITY UPLC BEH C18, particle size 1.7 µm, dimensions 2.1 × 50 mm.
• Online SPE: ACQUITY UPLC Online SPE Manager using MassTrak™ C8 cartridges.
• Sample preparation: 200 µL plasma, 20 µL isotopically labeled internal standards (d3-testosterone, 13C3-DHT), protein precipitation with zinc sulfate–methanol, centrifugation, water dilution to 1 mL, injection of 25 µL directly onto the SPE trap.
• Chromatography: gradient from 60% to 0% aqueous (10% methanol/0.05% formic acid) to 100% organic (methanol/0.05% formic acid) over 3 minutes at 0.4 mL/min; total run time <5 minutes.
• Online SPE conditions: multi-step conditioning, equilibration, loading, wash, elution and clamp-flush steps optimized via Design of Experiments and Advanced Method Development to ensure analyte retention and matrix removal.

Main Results and Discussion

• Optimized SPE parameters provided efficient isolation of testosterone and DHT, with retention times of approximately 1.96 min and 2.32 min, respectively, achieving baseline separation.
• Male plasma samples produced strong signals for both analytes, while female samples yielded quantifiable testosterone at picomolar levels and DHT below the limit of quantification, demonstrating method sensitivity.
• Integration of online SPE with UPLC-MS/MS significantly reduced matrix effects and improved reproducibility compared to traditional offline workflows.

Benefits and Practical Applications

• Simultaneous analysis of testosterone and DHT in a single, fast assay without derivatization.
• Minimal sample preparation and automated extraction reduce hands-on time and variability.
• High throughput (<5 min per sample) is suitable for large clinical research studies and routine hormone profiling.
• Applicable across diverse populations including male, female, and pediatric cohorts.

Future Trends and Opportunities

Advances in mass spectrometry instrumentation are expected to drive further automation of sample handling, miniaturized SPE formats for lower sample volume, expanded steroid panels for comprehensive hormone profiling, and integration with high-resolution analyzers to push detection limits even lower. Coupling these methods with data-driven biomarker discovery could open new clinical applications.

Conclusion

The developed online SPE–LC-MS/MS workflow enables robust, sensitive, and rapid quantification of testosterone and DHT from plasma without derivatization. This integrated method addresses key analytical challenges and is well suited for clinical research and high-throughput hormone monitoring.

Reference

• van Faassen M., Kema I. Development of a Clinical Research Method for the Measurement of Testosterone and Dihydrotestosterone. University Medical Center Groningen, 2014.