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

Víctor Vállez-Gomis; Juan L Benedé; Audrey Combès; Alberto Chisvert; Valérie Pichon

doi:10.1016/j.talanta.2023.124864

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

Talanta. 2023 Dec 1:265:124864. doi: 10.1016/j.talanta.2023.124864. Epub 2023 Jun 22.

Authors

Víctor Vállez-Gomis¹, Juan L Benedé², Audrey Combès³, Alberto Chisvert², Valérie Pichon⁴

Affiliations

¹ Department of Analytical, Bioanalytical Sciences, and Miniaturization, UMR 8231 Chemistry, Biology and Innovation, ESPCI Paris, PSL University, CNRS, 10 rue Vauquelin, Paris 75005, France; GICAPC Research Group, Department of Analytical Chemistry, University of Valencia, Burjassot, Valencia 46100, Spain.
² GICAPC Research Group, Department of Analytical Chemistry, University of Valencia, Burjassot, Valencia 46100, Spain.
³ Department of Analytical, Bioanalytical Sciences, and Miniaturization, UMR 8231 Chemistry, Biology and Innovation, ESPCI Paris, PSL University, CNRS, 10 rue Vauquelin, Paris 75005, France.
⁴ Department of Analytical, Bioanalytical Sciences, and Miniaturization, UMR 8231 Chemistry, Biology and Innovation, ESPCI Paris, PSL University, CNRS, 10 rue Vauquelin, Paris 75005, France; Sorbonne Université, Paris 75005, France. Electronic address: valerie.pichon@espci.fr.

PMID: 37379751
DOI: 10.1016/j.talanta.2023.124864

Abstract

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^-1). Finally, the methodology was applied to a pooled human serum sample in order to study its analytical utility and accuracy. Relative recovery (RR) values between 81 and 107% were obtained, showing no matrix effects during the global methodology. Furthermore, the need to perform the immunoextraction was evidenced by comparing the LC-MS scan chromatograms and RR values with and without applying the immunoextraction procedure on a serum sample submitted to protein precipitation. This works exploits, for the first time, the use of an IS on the selective determination of T4 in human serum samples.

Keywords: Human serum; Immunosorbent; Liquid chromatography-tandem mass spectrometry; Solid-phase extraction; Thyroxine.