A Clinical Research Method for the Analysis of Serum Testosterone and Androstenedione

Importance of the Topic

Accurate measurement of serum testosterone and androstenedione is essential for clinical research into endocrine function and pathology. These androgenic steroids play key roles in sexual development and metabolic regulation, and their low physiological concentrations demand highly sensitive and selective analytical approaches.

Objectives and Study Overview

This study aimed to develop a robust, automated UPLC-MS/MS method for quantifying testosterone and androstenedione in human serum. Emphasis was placed on harmonization through the CDC HoSt program and reduction of operator variability via a plate based extraction workflow.

Methodology

Serum samples were spiked with 13C3 internal standards and processed using Oasis MAX microelution SPE plates. Extraction was automated on a Tecan Freedom Evo 100/4 liquid handler. Chromatographic separation employed an ACQUITY UPLC I-Class system with an HSS SB C18 column and a rapid gradient. Detection used a Xevo TQD tandem quadrupole mass spectrometer in positive ESI mode with MRM transitions for quantifier and qualifier ions. Data acquisition and processing were performed with MassLynx v4.1 and TargetLynx.

Used Instrumentation

• Tecan Freedom Evo 100/4 Liquid Handler
• Waters ACQUITY UPLC I-Class System
• ACQUITY UPLC HSS SB C18 Column (2.1×50 mm, 1.8 μm)
• Waters Xevo TQD Mass Spectrometer
• MassLynx Software v4.1 with TargetLynx Application Manager

Key Results and Discussion

No interferences were detected for eight related steroids and epimers, and baseline separation of testosterone and epitestosterone was achieved. The method exhibited linearity from 0.17 to 52 nmol/L (r2>0.994) and a lower limit of quantification of 0.17 nmol/L (S/N>10). Intra and interday precision were ≤4.0% RSD for testosterone and ≤4.7% for androstenedione. Matrix effects were effectively compensated by internal standards, yielding net effects near unity. Accuracy assessment against CDC HoSt samples showed r2=0.999 and mean bias within ±3.3%. Comparison with independent LC-MS/MS methods and manual SPE demonstrated equivalent performance.

Benefits and Practical Applications

The automated workflow reduces sample handling time, minimizes operator variability, and supports high throughput. Its analytical sensitivity and selectivity ensure reliable quantification at low physiological levels, aiding clinical research and laboratory harmonization efforts.

Future Trends and Potential Applications

Further development may extend this approach to additional steroid panels and integrate with laboratory information systems. Advances in microelution SPE and ultra high sensitivity mass spectrometry could enhance throughput and detection limits. Continued standardization initiatives will support cross-laboratory comparability.

Conclusion

A fully automated UPLC-MS/MS method for serum testosterone and androstenedione provides excellent selectivity, sensitivity, precision, and accuracy. Its compatibility with CDC HoSt standards and flexible extraction options make it a valuable tool for clinical research laboratories.

Reference

• Foley D and Calton L. A Clinical Research Method for the Analysis of Serum Testosterone and Androstenedione. Waters Corporation; 2015.
• CDC Hormone Standardization Program (HoSt).