Development of an equilibrium dialysis ID-UPLC-MS/MS candidate reference measurement procedure for free thyroxine in human serum

Practical considerations for accurate determination of free thyroxine by equilibrium dialysis

“…All solutions and sample extracts were dried under nitrogen flow at room temperature and reconstituted in 200 µL 10% acetonitrile in water with 0.1% formic acid. All samples were analyzed by LC-MS/MS according to the reference method procedure at CDC [16] . The percent of T4 recovered after exposure to labware was determined according to the formula , where A is the mean T4/ 13 C 6 -T4 peak area ratios of set 1 and B is the peak area ratios of set 2.…”

“…The study was conducted at two locations operating independent FT4 RMPs: CDC Hormone Reference Laboratory and at the Reference Laboratory at Ghent University (Ref4U). Previously completed and ongoing studies have demonstrated excellent comparability between these two RMPs [15] , [16] .…”

Analysis of free, unbound thyroid hormones by liquid chromatography‐tandem mass spectrometry: A mini‐review of the medical rationale and analytical methods

“… 17 LC‐MS/MS‐based methods are by many considered the “gold standard” for clinical measurement of free TH and form the basis of the proposed reference methods for free TH measurement. 14 , 18 , 19 , 20 However, most clinical laboratories consider LC‐MS/MS‐based methods to be too laborious, technically difficult and too expensive for routine analysis of free THs, and the method is not widely used. Recently, the application of 96‐well plate‐based microdialysis 21 has greatly enhanced the throughput and some specialized clinical labs (e.g.…”

“… 20 , 69 Several different methods and validation results have been reported in recent times (Table 1 ). 18 , 19 , 20 , 21 , 35 , 70 Equilibrium dialysis of serum (plasma also appears acceptable) should be performed at 37°C and involve a (maximum of) 1:1 volume ratio of serum to a physiologically relevant dialysis buffer. Furthermore, pH must be maintained at 7.4 (at 37°C) to ensure the equilibrium between free and bound THs is not disturbed.…”

“…regular) extraction is possible, and both SPE, liquid‐liquid extraction and supported liquid extraction have been used (e.g. comparison in 19 ). Because THs have amine and carboxyl groups, they can be utilized in ion exchange‐sample preparations.…”