Use of frozen sera for FT4 standardization: investigation by equilibrium dialysis combined with isotope dilution-mass spectrometry and immunoassay.
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
After ED, we collected dialysate into tubes containing [13C6]-T4 for ID and [13C9]-T4 as carrier, purified the samples by solid-phase extraction, and analyzed them with LC/tandem 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.
Van Uytfanghe
Katleen
K
Laboratory for Analytical Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
Stöckl
Dietmar
D
Ross
H Alec
HA
Thienpont
Linda M
LM
D001798:Blood Proteins / Q000378:metabolism; D002247:Carbon Isotopes; D002853:Chromatography, Liquid; D015925:Cryopreservation; D003956:Dialysis; D006801:Humans; D007118:Immunoassay; D007201:Indicator Dilution Techniques; D013058:Mass Spectrometry; D011485:Protein Binding; D012015:Reference Standards; D013959:Thyroid Diseases / Q000175:diagnosis; D013974:Thyroxine / Q000097:blood / Q000592:standards*
D001798:Blood Proteins; D002247:Carbon Isotopes; D013974:Thyroxine