Solid-phase immunoextraction followed by liquid chromatography-tandem mass spectrometry for the selective determination of thyroxine in human serum.

In this work, an analytical method based on solid-phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) has been developed for the selective determination of thyroxine (T4) in human serum. For this purpose, two immunosorbents (ISs) specific to T4 were synthesized by grafting two different T4-specific monoclonal antibodies on a cyanogen bromide (CNBr)-activated-Sepharose® 4B solid support. The grafting yields obtained from the immobilization of each antibody on the CNBr-activated-Sepharose® 4B were over 90%, demonstrating that most of the antibodies were covalently bound to the solid support. The SPE procedure was optimized by studying the retention capability and selectivity of the two ISs in pure media fortified with T4. Under the optimized conditions, high elution efficiencies were achieved in the elution fraction for both specific ISs (i.e., 85%), whereas low ones were obtained in the control ISs (ca. 2%), showing the selectivity of the specific ISs. The ISs were also characterized by studying extraction and synthesis repeatability (RSD <8%), and capacity (104 ng of T4 per 35 mg of ISs, i.e., 3 μg g

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Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.