Abstract
Abstract Disclosure: L. Zhang: None. A. Ribera: None. Z. D'Zio: None. A. Doty: None. O. Sugahara: None. C. Tse: None. U. Danilenko: None. H.W. Vesper: None. A Reliable and accurate measurement of serum free thyroxine (FT4) is critical for diagnosis and treatment of thyroid disorders. Immunoassays (IAs) are most popularly used for FT4 measurements in-patient care. However, there are concerns about the accuracy of FT4 IAs. A reference measurement procedure (RMP) for FT4 based on well-defined equilibrium dialysis (ED) of serum has been established for standardization of routine FT4 IAs. FT4 RMP is highly accurate and precise, while it is labor-intensive and time-consuming which is not intended for routine clinical application. In the current study, we aim to develop a high-throughput FT4 method based on ED ID-LC/MS/MS that can be traceable to the RMP. FT4 in dialysate was isolated from protein-bound T4 in serum by using a commercially available micro-ED plate after 18-hours ED. The 13C6 labeled T4 was spiked into collected dialysate as an internal standard. FT4 in dialysate was extracted by using 96-well C18 SPE plate. FT4 in the samples was quantitated by using LC/MS/MS. The certified primary reference material was used to for assay calibration. T4, T3, reverse triiodothyronine (rT3), and other interferences in the dialysate were resolved on a C18 UPLC column within 4 min. The linear range of the routine FT4 assay covered 1-100 pg/mL. The assay sensitivity allowed detection of 0.3 pg/mL FT4 in serum, which is sufficient for FT4 measurement covering clinically relevant ranges including hypothyroid patient samples. Method comparison showed good agreement between our high-throughput method and RMP in Deming regression analysis with slope close to 1, while there was significant bias of IA from RMP. The intra and inter-day CV% of the assay was 6.96 and 3.38 %, respectively. The use of a micro-ED plate together with automation system could improve the throughput of the procedure. The accuracy, precision, and sensitivity of our method are suitable for the high-throughput FT4 measurements in clinical laboratories and large epidemiologic studies. The traceability of the described FT4 method to RMP can support the standardized FT4 measurements in patient care. Presentation: Saturday, June 17, 2023
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