Abstract
A new and improved method was developed for the determination and quantification of four “free” thyroid hormones (i.e. 3,5-diiodothyronine (T2), 3,3′,5-triiodothyronine (T3), 3,3′,5′-triiodothyrone (rT3) and 3,5,3′,5′-tetraiodothyronine (T4)) in human serum by low- and high-resolution liquid chromatography-mass spectrometry (LC-MS). Several sample preparation strategies were investigated to obtain matrix-independent results. These strategies included solid phase extraction and matrix dilution. The developed analytical methods were then directly compared, in a blind study using patient-derived human blood serum samples, to the current clinical routine testing methods, i.e. electrochemiluminescence immunoassay and enzyme-linked immunosorbent assay. Chromatographic separation was achieved on a pentafluorophenyl (F5) column with an isocratic method of 30% aqueous phase, 70% organic phase where mobile phase A is 0.1% formic acid in water (pH 4) and mobile phase B is 0.1% formic acid in methanol (pH 4) (v/v). The high-resolution LC-MS was able to give a significant improvement in sensitivity with limits of quantification of 0.002 to 0.008 pmol/L for all four “free” thyroid hormones, as well as reduced sample preparation, making this the preferred method. However, the increase in capital cost may be beyond the capabilities of some laboratories. The LC-MS methods allow for the analysis of “free” thyroid hormones to be carried out in a significantly reduced analysis time. Clinical sample analysis showed that there was no statistical difference between the results obtained by ECLIA/ELISA and both LC-MS methods.Graphical abstract
Highlights
Thyroid compounds are a group of hormones responsible for the regulation of a variety of biological functions, including basal metabolic rate, lipid, glucose and carbohydrate metabolism [1]
The ability to separate T3 and rT3 allows for reliable measurement of T3 in serum samples; in order to do this, liquid chromatography-mass spectrometry (LC-MS) parameters were optimised utilising both the internal tuning parameters and manual gas adjustment in order to achieve the optimum response for the thyroid hormone
Horse blood was chosen as it poses a reduction in the pathogenic risk while still containing the matrix material, of relevance for Bfree^ thyroid hormone determination, which could potentially interfere with the analysis
Summary
Thyroid compounds are a group of hormones responsible for the regulation of a variety of biological functions, including basal metabolic rate, lipid, glucose and carbohydrate metabolism [1]. This group of compounds contains tyrosine-based compounds including the physiologically active form triiodothyronine (T3). The majority of triiodothyronine is formed enzymatically by the deiodination of thyroxine (T4) [2, 3]. T4 can be deiodinated to form an inactive form of T3. Within the Newcastle upon Tyne NHS Trust, the reference ranges are 3.5–6.5 pmol/L and 9.5–21.5 pmol/L for T3 and T4, respectively [7, 8]
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