Enhanced Sensitivity in Free Thyroxine Measurement Using Equilibrium Dialysis and LC–MS/MS Coupled with Offline Anion-Exchange Solid-Phase Extraction

Abstract

INTRODUCTION: According to the free hormone hypothesis, only the unbound hormone fraction exerts biological effects. Diagnosis and monitoring of thyroid disorders often require the measurement of free thyroxine (fT4) by equilibrium dialysis, which is considered the gold-standard method for separating free hormones from biological samples. OBJECTIVES: Our objective was to reduce complexity and enhance the sensitivity of a liquid chromatography–tandem mass spectrometry method with online solid-phase extraction (SPE) for measuring fT4. METHODS: Serum aliquots of 200 µL were subjected to equilibrium dialysis. Post-dialysis, increases in analytical sensitivity and specificity were achieved using weak-anion-based SPE prior to liquid chromatographic separation with subsequent analyte detection by a high-sensitivity SCIEX Triple Quad^™ 7500 mass spectrometer. RESULTS: Lower and upper limits of quantitation (LLOQ and ULOQ) were 0.1 and 10 ng/dL, respectively. Intra- and inter-day precision evaluated using four matrix-matched quality control samples (0.15, 2.09, 5.10, and 7.94 ng/dL) indicated <10% imprecision, showing excellent agreement with the previous validated method (slope = 1.022, r = 0.9927, bias = 4.01%, n = 123). Chromatograms exhibited a substantial improvement in the signal-to-noise ratio at the LLOQ. CONCLUSION: A complex liquid chromatographic separation with online trapping-column method was successfully transitioned to a simpler off-line SPE liquid chromatographic separation method. Incorporating SPE desalting and using the improved instrument sensitivity of the SCIEX Triple Quad^™ 7500 mass spectrometer significantly improved the performance. A reduced injection volume allowed sample reanalysis, which is beneficial for pediatric samples. This method provides a superior analytical approach for clinical fT4 measurement.