Abstract Background Measurement of free thyroxine (FT4) and free triiodothyronine (FT3) is typically performed by immunoassay, however these can be prone to suffer from interferences. Currently, the accepted gold standard for the measurement of FT4 and FT3 is using equilibrium dialysis (ED) combined with LC-MS/MS, which can overcome the issues with immunoassay in the measurement of FT4 and FT3. However, the ED procedure can be quite time consuming and often is performed overnight. Here, we describe a simple clinical research method to perform ED using a commercially available device followed by sample preparation using solid phase extraction (SPE), and UHPLC-MS/MS analysis using the Waters ACQUITY™ UPLC™ System and Xevo™ TQ Absolute Mass Spectrometer. This has allowed for the complete workflow including ED to be performed within a typical working day. Methods Only 200µL of serum sample was added to the sample chamber of the ED device and 400µL of dialysate buffer was added to the dialysate chamber. ED was performed at 37°C until equilibrium was reached and then samples were extracted using a simple SPE approach on an Oasis™ MAX µElution™ Plate. Calibrators were prepared directly into dialysate buffer across the ranges of 1. 3 – 129pmol/L for FT4 and 1. 55 – 154pmol/L for FT3 and were not subjected to the ED process, but extracted by SPE. Samples were injected onto an ACQUITY Premier HSS T3 Column with a VanGuard™ FIT Guard Column and FT4 and FT3 were chromatographically separated using a mobile phase gradient consisting of water, methanol, acetonitrile and formic acid with an injection to injection time of only 3. 5 minutes. Detection was performed using the Xevo TQ Absolute Mass Spectrometer and data analysis was simplified using watersconnect™ with QUAN Review Software. Results Within-run and total precision were =5. 0%CV for the dialysate QC samples and =9. 6%CV for the serum QC samples across all concentration levels for both FT4 and FT3. The LLoQ for FT4 and FT3 were determined to be 0. 97pmol/L and 1. 15pmol/L respectively. All calibration lines performed during testing were linear, having a coefficient of determination (r2) of 0. 995 and % deviations of within ±15% (±20% for Cal 1) and no significant carryover was observed. Method comparison to the CDC HoSt Phase 1 Program for FT4 gave a Passing-Bablok fit of y = 0. 9198x + 1. 243 and a Bland Altman bias of 0. 070% was obtained. Example chromatograms of FT4 and FT3 from human serum samples which have been dialyzed, extracted and analyzed, and then data analysis performed in QUAN Review are shown in Figure 1. Figure 1. Typical chromatograms of FT4 and FT3 from human serum samples which have been dialyzed, extracted and analyzed. Conclusion A clinical research method has been developed for the UHPLC-MS/MS analysis of free thyroxine (FT4) and free triiodothyronine (FT3) from human serum using a simple equilibrium dialysis and SPE procedure which can be completed within a typical working day. For Research Use Only. Not for use in diagnostic procedures ACQUITY, UPLC, Xevo, Oasis, µElution, VanGuard, watersconnect, are trademarks of Waters Technologies Corporation.
Wardle et al. (Wed,) studied this question.