LC-MS analysis of thyroid hormones in human serum

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. 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.

Liquid chromatography mass spectrometry (LC-MS) has been reported 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. The present work demonstrates successful online SPE with LC-MS for rapid determination of T4, T3, and 3,3',5'-triiodo-Lthyronine (rT3) from biological matrices.

An online SPE-LC-MS method has been developed for the rapid detection of thyroid hormones in human serum with minimal hands-on effort and time-consuming steps. Both C8 and RP-Amide online cartridges were shown to be capable of capturing a trace amount of analyte from protein precipitated human serum samples. All three analytes, T3, rT3 and T4 were resolved on a Biphenyl column, with sharp and symmetric peak shapes. In addition, reproducibility (%RSD) of the retention times of the thyroid hormones from 120 consecutive injections is between 0.1% and 0.2%, with either C8 or RPA online cartridges, while the peak area reproducibility (%RSD) is between 5.1% and 7.7%. These RSD’s indicate great ruggedness of the online SPE-LC-MS system.