The UPLC™-MS/MS Analysis of Free Thyroxine (FT4) and Free Triiodothyronine (FT3) With a Simplified Equilibrium Dialysis and SPE Workflow for Clinical Research

Importance of the Topic

The free fractions of thyroid hormones are biologically active and serve as critical biomarkers for thyroid disease diagnosis and research. While immunoassays are widespread, they can suffer from specificity issues. Equilibrium dialysis paired with liquid chromatography–tandem mass spectrometry (LC-MS/MS) is the recognized reference method but traditionally requires overnight processing.

Objectives and Study Overview

This work presents a complete clinical research workflow for FT4 and FT3 determination in human serum that integrates a simplified equilibrium dialysis device, microelution solid-phase extraction (SPE), and ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS), all achievable within a single working day.

Methodology and Instrumentation

Equilibrium dialysis was performed at 37 °C using a commercial device with HEPES buffer. Samples and calibrators underwent SPE on Oasis MAX µElution plates. Chromatography employed an ACQUITY UPLC I-Class FL system with an HSS T3 column (2.1×50 mm, 1.8 µm) at 45 °C using a 2.5 min gradient. Detection utilized a Xevo TQ Absolute mass spectrometer in positive electrospray ionization mode. Data processing was streamlined via waters_connect with QUAN Review Software.

Key Results and Discussion

The method achieved baseline separation of T3 and reverse T3 with limits of quantification of 0.97 pmol/L (FT4) and 1.15 pmol/L (FT3), precision ≤5% CV for spiked dialysate and ≤9.6% CV for serum QCs, and linear ranges of 1.11–177 pmol/L (FT4) and 1.32–211 pmol/L (FT3). Negligible carryover and minimal matrix effects were observed. Comparison with the CDC HoSt Phase 1 reference method yielded a Passing-Bablok regression of y=0.9198x+1.243 and Bland–Altman bias of 0.07% for FT4.

Benefits and Practical Applications

The protocol reduces total assay time to under one day, enhances throughput, and provides high specificity and sensitivity suitable for clinical research environments. Automated data review further accelerates result validation.

Future Trends and Potential Applications

Opportunities include further automation of dialysis and extraction steps, expansion to multiplexed free hormone panels, and potential adaptation for routine clinical diagnostics where rapid, accurate thyroid profiling is desired.

Conclusion

A robust, streamlined UPLC-MS/MS method for FT4 and FT3 analysis has been established, delivering high performance and same-day turnaround for clinical research needs.

References

• De Grande L et al. Standardization of Free Thyroxine Measurements Allows the Adoption of a More Uniform Reference Interval. Clinical Chemistry. 2017;63(10):1585–1593.