Determination of total thyroxine in human serum by hollow fiber liquid-phase microextraction and liquid chromatography–tandem mass spectrometry

• Hollow fiber liquid-phase microextraction for total thyroxine in human serum.
• Extracts had comparable cleanness with those obtained using solid-phase extraction.
• Serum samples were spiked with 13C6- thyroxine and analyzed by LC–MS/MS.
• Replicates of 6 gave RSD of <1.3%. The difference from the SPE method was <1.2%.
• Two reference materials of human serum were analyzed with good precision and accuracy.

Determination of total thyroxine in human serum using hollow fiber liquid-phase microextraction (HF-LPME) has been accomplished for the first time. HF-LPME serves as an inexpensive sample pretreatment and the cleanup method that is nearly solvent-free. Thyroxine was extracted through a water immiscible organic solvent immobilized in the wall pores of a polypropylene hollow fiber into 20 μl of an aqueous acceptor phase inside the lumen of the hollow fiber. This technique produced extracts that had comparable cleanness with those obtained using solid-phase extraction (SPE). Serum samples with endogenous thyroxine were spiked with isotopically-labeled thyroxine and analyzed by liquid chromatography–tandem mass spectrometry after HF-LPME extraction. Extraction parameters including the organic phase, acid/base concentration of acceptor phase, stirring speed and extraction time were optimized. The calibration range was found to be linear over 1–1000 ng/g with the limit of detection (LOD) of 0.3  ng/g. For quantification of total thyroxine in human serum, 6 subsamples were prepared and the results indicated very good precision with a relative standard deviation of <1.3%. The difference from the SPE method was less than 1.2%, with independent t-test showing insignificant bias. Two reference materials of human serum were analyzed, and our obtained values were compared with the reference values. The results showed very good precision with RSD around 0.2% and the deviation from the reference values were −3.1% and −2.1%. The newly developed method is precise, accurate, inexpensive, and environmentally friendly.

HF-LPME configurations. (A) 3 cm hollow fiber (straight) and (B) 7 cm hollow fiber (U-shape with extension).Figure optionsDownload as PowerPoint slide