Development of High-Throughput Measurement of Free Thyroxine in Serum Using Equilibrium Dialysis in Couple With Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Free thyroxine (FT4) measurements are critical in the diagnosis, classification, and treatment of thyroid diseases. It is estimated that about 18 million FT4 tests are requested in the USA per year annually. In clinical laboratories, most of FT4 assays are performed by using immunoassays (I As). However, the significant bias of IAs and large variation between laboratories have been reported. The reference measurement procedures (RMPs) of FT4based on equilibrium dialysis (ED) - liquid chromatography-tandem mass spectrometry (LC-MS/MS) have been established and recognized by the clinical chemistry community. However, the FT4 RMP is relatively low throughput and labor-intensive. Also, an aliquot of 1 mL sample is required for an RMP. A routine FT4 assay high-throughput procedure that is based on ED LC-MS/MS and utilized less sample volume will allow to conduct large biomonitoring studies and establish FT4 levels in US population. In the described method, FT4 in 150 uL of serum was separated from protein-bound T4 at 37.0 oC in 96-well Micro-ED Teflon devices from HTDialysis. The ED conditions suggested by CLSI C45-A guideline were followed. A volume of 150 uL dialysate samples with FT4 was obtained after the ED step. FT4 in the dialysate was purified by extractions before LC-MS/MS analysis. Chromatographic separation of T4 from the sample matrix is achieved on a C18 UPLC column with a gradient of methanol and water with 0.1% formic acid. Quantification of FT4 was performed by using selective reaction monitoring in positive electrospray ionization mode. The IRMM-468 certified primary reference material (JRC, Belgium) of T4 is used for the calibration curves. FT4 concentrations reached equilibrium after 4 hours under current dialysis conditions, which was also observed in the RMP setting. The developed routine FT4 assay 96-well ED system is within 5% bias from the FT4 RMP based on preliminary method comparison study using human serum. The studies to further characterize the FT4 routine method performance are ongoing. In summary, we are developing an analytical method based on a 96-well ED-LC-MS/MS system for the measurements of FT4 in serum. By comparison with RMP, the described method significantly improve throughput and reduce sample volume, which will fulfill the requirement of FT4 routine assay in clinical laboratories and allow for its use in the large biomonitoring studies and other activities in the research and public health settings.