16 February / March 2020
Online Solid Phase Extraction and LC/MS Analysis of Thyroid Hormones in Human Serum
by Olga I. Shimelis, Candace Price, MilliporeSigma, Bellefonte, PA , USA
olga.shimelis@
milliporesigma.com;
candace.price@
milliporesigma.com
Thyroid hormones play critical roles in the regulation of biological processes, such as growth, metabolism, protein synthesis, and brain development. Specifically, both 3,3’,5,5’-tetraiodo-L-thyronine (thyroxine or T4) and 3,3’,5-triiodo-L-thyronine (T3), are essential for development and maintenance of normal physiological functions [1]. For a clinical laboratory, measurements of total T4 and total T3, along with estimates of free T4 (FT4) and free T3 (FT3), are important for the diagnosis and monitoring of thyroid diseases. Most clinical laboratories measure thyroid hormones using immunoassays. The immunoassay-based methods offer a relatively rapid, high patient sample throughput that lends itself to automation, but are significantly compromised by problems with assay interference and are complicated by changes in protein levels that alter the free hormone availability [1,2]. These drawbacks lead to inaccuracies of immunoassays and can lead to false high or low results [3].
Liquid chromatography mass spectrometry (LC/MS) has been reported [1-3] to offer superior specificity and speed over the immunoassays for determination of thyroid hormones in biological matrices such as serum and tissues. Nevertheless, the reported sample preparation procedures, typically liquid-liquid extraction followed by solid phase extraction (SPE), involve multiple time-consuming steps, and are less compatible with automation [3,4]. The
Chemical structures of the thyroid analytes
present work demonstrates successful on- line SPE with LC/MS for rapid determination of T4, T3, and 3,3′,5′-triiodo-L-thyronine (rT3) from biological matrices. The method development process included the use of 2 on-line SPE cartridge chemistries: C8 and RP-Amide. The serum samples underwent protein precipitation procedure to release the protein-bound thyroid hormones. The capture of analytes on the on-line SPE cartridges was confirmed by washing the
cartridges directly into analytical HPLC column using higher concentrations of organic solvent and tandem mass spectrometry detection.
Experimental:
Materials: SupelTMGenie C8 and RP-Amide (RPA) on-line cartridges (2 cm length x 4.0 mm i.d.), human serum (MilliporeSigma Cat. H-1388), protein crashing solvent: methanol with 1% (w/v) ammonium formate.
Figure 1: The chemical structures of the thyroid analytes. Note T3 and rT3 are isobaric.
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