A LOW-LEVEL ANALYTICAL METHOD FOR THE QUANTIFICATION OF SERUM FREE TESTOSTERONE USING LC-MS/MS FOR CLINICAL RESEARCH

Significance of the Topic

Accurate determination of free testosterone in human serum underpins clinical research into hormone balance and related disorders. Given that free testosterone constitutes less than 3 % of total circulating testosterone, highly sensitive and reproducible analytical methods are essential to support studies in endocrinology, pharmacology and diagnostic assay development.

Objectives and Study Overview

This work presents a streamlined LC-MS/MS protocol designed for clinical research applications. It aims to combine rapid equilibrium dialysis with robust liquid-liquid extraction to achieve low‐level quantification of free testosterone in serum, while controlling factors such as temperature, pH and equilibrium conditions.

Methodology

The method employs a 2-hour equilibrium dialysis step at 37 °C using a Rapid Equilibrium Dialysis insert and 52.75 mM HEPES buffer at pH 7.40. Following dialysis, the serum diasylate is spiked with a testosterone-13C3 internal standard and extracted with methyl tert-butyl ether. Samples are dried under nitrogen at 40 °C and reconstituted in a 50:50 mixture of aqueous and organic mobile phases.

Used Instrumentation

• Rapid Equilibrium Dialysis (RED) insert and reusable base plate
• Orbital shaker with temperature control
• Waters ACQUITY UPLC I-Class FL System
• ACQUITY BEH C18 analytical column
• Waters Xevo TQ Absolute triple-quadrupole mass spectrometer operating in MRM with positive electrospray ionization
• Nitrogen evaporator setup for sample drying

Main Results and Discussion

• Linearity was proven from 0.5 to 650 pg/mL with correlation coefficients r2 ≥ 0.995 over the 1–500 pg/mL measuring range.
• The lower limit of the measuring interval (LLMI) was established at 0.5 pg/mL (≤20 % CV and ≤15 % bias).
• No significant carryover was detected following 500 pg/mL injections.
• Matrix effect evaluation in stripped serum showed normalized matrix factors between 1.03 and 1.07, indicating negligible ion suppression or enhancement.
• Precision studies at low, mid and high QC levels (2.79, 8.80 and 134 pg/mL) demonstrated repeatability and total precision ≤ 8.4 % CV.
• Accuracy assessment against UK NEQAS external quality assurance samples (n=45) yielded a Deming regression slope of 0.947 and a mean bias of –7.6 %, reflecting strong concordance with the all-laboratory trimmed mean.

Benefits and Practical Applications

The method requires only 200 µL of serum and completes equilibrium dialysis in 2 hours without sacrificing sensitivity or precision. Its high analytical sensitivity (0.5 pg/mL), robust reproducibility and agreement with external quality schemes make it suitable for research studies on androgen physiology and for validation of clinical assays.

Future Trends and Opportunities

Advances may include automation of equilibrium dialysis and extraction workflows, integration of microfluidic devices for faster sample processing, and coupling with high-resolution mass spectrometry for multiplexed free steroid profiling. Such enhancements could broaden applications to therapeutic monitoring and deeper investigation of hormonal regulation.

Conclusion

This LC-MS/MS method offers a sensitive, precise and efficient approach for quantifying serum free testosterone in clinical research settings. By combining rapid equilibrium dialysis with targeted mass spectrometric detection, it addresses previous challenges in low-level hormone analysis and aligns well with external quality standards.