Analysis of Free Testosterone in Serum using the Xevo TQ Absolute for Clinical Research

Significance of the topic

The accurate measurement of free testosterone in serum is critical for clinical research into endocrine disorders and hormonal therapies. Free testosterone constitutes less than 3% of total testosterone in serum, demanding highly sensitive and selective analytical methods. Rapid and reliable quantification supports studies of hormone metabolism, pharmacokinetics, and patient stratification in translational research.

Objectives and Study Overview

This study aimed to develop and validate a robust LC-MS/MS method to quantify free testosterone in human serum at femtogram levels. Key objectives included achieving low limits of quantitation, ensuring precision and accuracy across a broad concentration range, minimizing sample volume, and demonstrating agreement with external quality assurance materials.

Methodology

The method combines equilibrium dialysis to separate free from protein-bound testosterone with liquid-liquid extraction (LLE) for sample cleanup and concentration. Rapid Equilibrium Dialysis (RED) inserts and plates were incubated with 200 µL of serum and HEPES buffer (pH 7.4) at 37 °C, 800 rpm for two hours. The dialysate was spiked with a 13C-labeled internal standard, extracted with methyl tert-butyl ether, evaporated under nitrogen at 40 °C, and reconstituted in mobile phase.

Instrumental Setup

• Chromatography: ACQUITY UPLC I-Class PLUS FL System with BEH C18 column (2.1 mm × 100 mm, 1.7 µm) at 50 °C and a 4.5-minute run time.
• Mobile phases: 0.2 mM ammonium fluoride in water (A) and in methanol (B); gradient elution.
• Mass spectrometry: Xevo TQ Absolute triple quadrupole with ESI+; capillary 1.2 kV; desolvation 650 °C; source 150 °C; MRM detection with unit resolution.
• Data processing: MassLynx v4.2 and TargetLynx XS v4.2.

Main Results and Discussion

The method demonstrated linearity from 0.5 to 650 pg/mL (r2 ≥ 0.995), with a lower limit of measuring interval (LLMI) of 0.5 pg/mL defined by precision ≤20% and bias ≤15%. Precision across low (2.8 pg/mL), medium (8.8 pg/mL), and high (134 pg/mL) QC levels was ≤8.4% RSD. Matrix effects ranged from 96% to 102%, indicating negligible ion suppression or enhancement. Endogenous interferents (albumin, bilirubin, lipids, etc.) showed recoveries within 87–115%. External comparison with 45 UK NEQAS male serum samples yielded a Passing-Bablok slope of 0.947 and mean bias of –7.6% in Bland-Altman analysis.

Benefits and Practical Applications

• High sensitivity (LLMI 0.5 pg/mL) using only 200 µL of serum.
• Short two-hour equilibrium dialysis reduces turnaround time.
• Capability for sample re-analysis from the prepared extract.
• Excellent reproducibility and agreement with external quality schemes.

Future Trends and Opportunities

Further developments may include automation of the dialysis and extraction workflow, miniaturization to reduce volumes and increase throughput, integration with online sample preparation, and application to other low-abundance steroids. Emerging high-resolution instruments and ion mobility separation may enhance specificity for complex matrices.

Conclusion

A sensitive and precise LC-MS/MS method for free testosterone quantification in human serum has been established using equilibrium dialysis and the Xevo TQ Absolute. The approach meets stringent criteria for sensitivity, accuracy, and throughput, supporting advanced clinical research into hormonal status and therapy monitoring.

References

No external literature references were provided in the original text.