Simplified Equilibrium Dialysis and SPE Method For The UHPLC-MS/MS Analysis of Free T4 and Free T3 For Clinical Research

Significance of Topic

Accurate measurement of free thyroxine (FT4) and free triiodothyronine (FT3) is essential for evaluating thyroid status in clinical research. Immunoassays may suffer from cross-reactivity and matrix effects, while equilibrium dialysis coupled with LC-MS/MS offers superior specificity but is traditionally time-consuming.

Objectives and Study Overview

This work presents a streamlined clinical research method that combines rapid equilibrium dialysis with solid phase extraction (SPE) and fast ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to quantify FT4 and FT3 in human serum within a standard workday.

Materials and Methodology

The method employs 13C6-labeled internal standards for T4 and T3, calibrators and tri-level quality controls prepared in HEPES buffer at pH 7.40, and unadulterated human serum samples.

• Equilibrium Dialysis: 200 µL serum against 400 µL buffer using a Rapid Equilibrium Dialysis (RED) insert at 37 °C for 5 h.
• SPE Cleanup: Post-dialysis samples processed on an Oasis MAX µElution plate with washing and elution steps; dilution directly to injection volume without evaporation.
• Chromatography: Waters ACQUITY PREMIER HSS T3 column, water/methanol/formic acid gradient, 2.5 min run time.
• Mass Spectrometry: Waters Xevo TQ Absolute in MRM mode monitoring transitions specific for FT4 and FT3 and their internal standards.

Used Instrumentation

• Rapid Equilibrium Dialysis device (Thermo Fisher Scientific RED system)
• Waters ACQUITY UPLC System
• Waters Xevo TQ Absolute Mass Spectrometer
• Oasis MAX µElution Plate
• ACQUITY PREMIER HSS T3 Column
• Orbital shaker with temperature control

Main Results and Discussion

The method demonstrated excellent analytical performance:

• Linearity: r2 > 0.995 over 1–100 pg/mL with deviations within ±15 % (±20 % at lowest level).
• Precision: ≤5.0 % CV for dialysate QC levels (3, 10, 85 pg/mL); ≤9.6 % CV for serum QC (FT4 25 pg/mL, FT3 6 pg/mL).
• Sensitivity: Lower Limit of Measuring Interval of 0.75 pg/mL for both analytes.
• Carryover: Negligible after 200 pg/mL injections.
• Matrix Effects: Normalized matrix factors 0.96–1.10 in stripped serum; endogenous interferents within 85–115 % recovery.
• Method Comparison: Strong agreement with the CDC HoSt Reference Procedure (Passing-Bablok y = 0.9198x + 1.243; Bland-Altman bias 0.07 %).

Benefits and Practical Applications

This approach uses only 200 µL of serum, achieves high sensitivity and reproducibility, and completes the entire workflow within a single working day. It provides a reliable alternative to traditional immunoassays for research laboratories and quality assurance settings.

Future Trends and Opportunities

Potential developments include automation of the dialysis and SPE steps, integration with high-resolution mass spectrometers for expanded analyte panels, miniaturization for reduced sample volume, and adaptation toward point-of-care thyroid testing platforms.

Conclusion

A rapid equilibrium dialysis and SPE-UHPLC-MS/MS protocol has been established for precise and efficient quantification of free T4 and T3. The method delivers robust analytical performance and aligns closely with reference measurement procedures, making it well suited for clinical research applications.

References

• Wardle R, Foley D, Hammond G, Kumar P (2025) Simplified equilibrium dialysis and SPE method for the UHPLC-MS/MS analysis of free T4 and free T3 for clinical research. Waters Corporation Application Note.