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

Importance of the Topic

The accurate measurement of androgenic steroids such as testosterone, androstenedione and dehydroepiandrosterone sulfate (DHEAS) in human serum is critical for clinical research into hormone-related disorders and metabolic studies. High selectivity and sensitivity are required to quantify low physiological concentrations and to distinguish structurally similar steroids in complex biological matrices.

Objectives and Study Overview

This article presents the development and validation of an automated LC-MS/MS method for the simultaneous quantification of testosterone, androstenedione and DHEAS in serum. The goal is to enhance sample throughput, minimize interferences and generate reliable data for clinical research applications.

Methodology

Sample preparation was performed on 100 µL of serum using solid-phase extraction with Oasis PRiME HLB µElution plates, fully automated on a Tecan Freedom Evo 100/4 liquid handler. Chromatographic separation was carried out on an ACQUITY UPLC HSS T3 column with a water–methanol gradient containing 2 mM ammonium acetate and 0.1% formic acid. Detection was achieved on a Xevo TQD mass spectrometer in multiple reaction monitoring mode, using positive ionization for testosterone and androstenedione and negative ionization for DHEAS. Calibration curves were constructed in stripped human serum over physiological ranges, with isotopically labeled internal standards for each analyte.

Used Instrumentation

• Oasis PRiME HLB µElution Plate
• Tecan Freedom Evo 100/4 Liquid Handler
• ACQUITY UPLC I-Class System with HSS T3 VanGuard Pre-column and HSS T3 Column
• Xevo TQD Mass Spectrometer
• MassLynx v4.1 Software with TargetLynx Application Manager

Main Results and Discussion

The method achieved limits of quantification of 0.17 nmol/L for testosterone and androstenedione and 0.14 µmol/L for DHEAS, with signal-to-noise ratios above 10 at the lowest calibrators. Total precision and repeatability were below 6.3% across low, mid and high quality control levels. Linearity was demonstrated over 0.15–76 nmol/L for testosterone, 0.15–74 nmol/L for androstenedione and 0.13–58 µmol/L for DHEAS, with correlation coefficients (r²) exceeding 0.998 on multiple occasions. Matrix effects were minimal (matrix factors between 0.93 and 1.09) with relative standard deviations under 7%. Method comparison with an independent LC-MS/MS procedure and external quality assessment samples yielded mean biases of +5.0% for testosterone, −3.3% for androstenedione and −6.3% for DHEAS, indicating strong agreement.

Benefits and Practical Application of the Method

• High sensitivity enables quantification at low physiological concentrations
• Enhanced selectivity via optimized SPE, UPLC separation and MRM detection
• Automated sample preparation improves throughput and reduces operator error
• Robust validation covering linearity, precision and matrix effects
• Alignment with external quality schemes ensures method reliability

Future Trends and Applications

Further integration with laboratory information management systems and expanded automation can boost throughput in clinical laboratories. Extending the steroid panel to include additional metabolites will support more comprehensive endocrine profiling. Emerging high-resolution mass spectrometry platforms and novel microextraction techniques may further enhance sensitivity and selectivity for trace-level analytes.

Conclusion

An automated LC-MS/MS workflow combining Oasis PRiME HLB µElution SPE and UPLC-MS/MS detection offers a sensitive, selective and high-throughput solution for simultaneous quantification of testosterone, androstenedione and DHEAS in serum. The method exhibits excellent analytical performance and strong concordance with external quality assessment, supporting its use in clinical research.

References

• Foley D. and Calton L. A Clinical Research Method for the Analysis of Serum Testosterone and Androstenedione. Waters application note, 2015. p/n 720005274EN.