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Technical Briefs

Use of Frozen Sera for FT4 Standardization: Investigation by Equilibrium Dialysis Combined with Isotope Dilution-Mass Spectrometry and Immunoassay

¹ Laboratory for Analytical Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium
² Department of Chemical Endocrinology, University Medical Centre, Nijmegen, The Netherlands

^* To whom correspondence should be addressed. E-mail: linda.thienpont{at}ugent.be.

Background: Serum-free thyroxine (FT₄) testing is recommended for diagnosis or monitoring of thyroid dysfunction, particularly in cases of hormone binding abnormalities. However, the poor intermethod agreement among commercial FT₄ assays suggests a need for standardization with a hierarchically higher measurement procedure. To that purpose, we applied equilibrium dialysis (ED) in combination with isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC-tandem MS).

Methods: We collected post-ED dialysate samples into tubes containing [¹³C₆]-T₄ for ID and [¹³C₉]-T₄ as carrier, purified the samples by solid-phase extraction, and analyzed them with LC/MS-MS. We evaluated the procedure's analytical performance and tested its suitability for measurement of hypo-, eu-, and hyperthyroid serum FT₄ concentrations. We conducted a pilot method comparison study with 3 commercial assays to investigate whether frozen sera could be used for the purpose of FT₄ standardization.

Results: The within-run, between-run, and total CVs (inclusive ED) were 3.7%, 4.2%, and 5.6%, respectively (17.7 pmol/L; n = 20). The mean accuracy, estimated from recovery experiments with dialysate and dialysis buffer supplemented at 8.7, 18.7, and 33.5 pmol/L, and from analysis of certified sera gravimetrically diluted to 9.8, 19.2, and 34.8 pmol/L, was 98.0% to 102.8%. The procedure's limit of detection and limit of quantification were 0.5 and 1.3 pmol/L, respectively. The method comparison demonstrated the suitability of the selected sera for standardization of FT₄ assays and confirmed the lack of assay comparability.

Conclusions: We demonstrated that the described ED-ID-LC/tandem MS procedure and the selected type of sera qualify for standardization of FT₄ measurements.