ABSTRACT Serum total thyroxine (TT4) serves as a clinically essential biomarker for thyroid disorder diagnosis and therapeutic monitoring. The aim of this study was to establish and validate a sensitive and reliable liquid chromatography‐tandem mass spectrometry (LC–MS/MS) method for the quantification of serum TT4, and to compare its performance with that of the Abbott automated immunoassay platform. Serum samples underwent protein precipitation using acetonitrile prior to analysis. Chromatographic separation was achieved on an Agilent Poroshell 120 SB‐C18 column (4.6 × 50 mm 2 , 2.7 µm) with a gradient elution system comprising solvent A (0.1% aqueous formic acid) and solvent B (0.1% formic acid in acetonitrile) at a 0.8 mL/min flow rate, enabling efficient analysis within a 7‐min runtime. The method demonstrated excellent linearity ( r 2 > 0.995) across the clinically relevant range of 15–500 ng/mL, with a lower limit of quantification (LLOQ) at 15 ng/mL. Precision studies revealed intra‐day and inter‐day variations below 7.4%, accompanied by accuracies ranging from 91.7% to 111.4% across quality control levels. Stability assessments confirmed TT4 integrity under routine storage conditions: 7 days at 2°C–8°C and 30 days at −20°C. Comparative analysis with immunoassay results showed strong correlation ( r = 0.93), though absolute concentrations exhibited expected methodological differences due to distinct detection principles. The developed LC–MS/MS method demonstrates superior analytical performance through its combination of high sensitivity, simplified sample preparation, and enhanced specificity compared to antibody‐based methods. This validated methodology has been successfully implemented in clinical thyroid diagnostics, providing a reliable reference system for resolving discordant immunoassay results and improving test standardization.
Sun et al. (Wed,) studied this question.