ADLM: A FAST, NOVEL AND ANALYTICALLY SENSITIVE CLINICAL RESEARCH METHOD FOR THE ANALYSIS OF FREE TESTOSTERONE USING UPLC-MS/MS

Importance of the Topic

A precise measurement of free testosterone in serum is essential for clinical research into endocrine disorders, hormone replacement therapies and metabolic diseases. Because free testosterone constitutes less than 3% of total testosterone, its accurate quantification demands highly sensitive and reproducible analytical methods.

Study Objectives and Overview

This work presents a fast, novel and analytically sensitive UPLC-MS/MS method to quantify free testosterone in human serum. The aim was to overcome limitations of traditional equilibrium dialysis, achieve sub-picogram sensitivity, minimize carryover and demonstrate strong agreement with external quality assurance schemes.

Methodology and Instrumentation

The procedure uses 200 μL of serum subjected to a two-hour rapid equilibrium dialysis at 37 °C with 52.75 mM HEPES buffer (pH 7.40). After dialysis, 30 μL of testosterone-13C3 internal standard are added and free testosterone is extracted by liquid-liquid partitioning into methyl tert-butyl ether. Following nitrogen drying, samples are reconstituted in a 50:50 mixture of mobile phases.

Instrumentation:

• Waters ACQUITY UPLC I-Class FL system with BEH C18 column
• Waters Xevo TQ Absolute mass spectrometer in MRM mode (ESI+)

Main Results and Discussion

Linearity was demonstrated from 0.5 to 650 pg/mL with correlation coefficients ≥0.995. Analytical sensitivity (LLMI) of 0.5 pg/mL was established at ≤20% CV and ≤15% bias. No significant carryover was observed from high-level injections. Normalized matrix factor assessments indicated absence of ion suppression or enhancement (mean factors 1.03–1.07). Repeatability and total precision across low, mid and high QC pools (2.79, 8.80, 134 pg/mL) were ≤8.4% CV. Method accuracy was confirmed by analysis of 45 NEQAS external quality samples (50.1–268.6 pg/mL), yielding a Deming regression of y=0.947x–2.869 and Bland-Altman bias of –7.62%.

Practical Benefits and Applications

This streamlined protocol delivers rapid equilibrium dialysis (2 hours) without sacrificing analytical performance. Its high sensitivity and precision support clinical research applications in endocrinology, pharmaceutical development and quality control laboratories.

Future Trends and Potential Applications

Advances may include automated sample preparation platforms for higher throughput, expansion of the approach to other low-abundance steroids, development of ultra-sensitive assays for pediatric and veterinary endocrinology, and integration of MS/MS data into personalized medicine workflows.

Conclusion

A novel UPLC-MS/MS assay for free testosterone achieves sub-picogram sensitivity, excellent precision and strong external accuracy. The method’s speed, robustness and minimal matrix interference make it a valuable tool for clinical research settings.