Assay of endogenous 3,5-diiodo-L-thyronine (3,5-T2) and 3,3’-diiodo-L-thyronine (3,3’-T2) in human serum.

Background: 3,5-diiodo-L-thyronine (3.5-T2) is an endogenous derivative of thyroid hormone with potential metabolic effects. It has been occasionally detect in human blood using High Performance Liquid Chromatography- tandem Mass Spectrometry (HPLC-MS/MS), but the results have been quite variable and quality control data were often missing. In this study we used an ad-hoc optimized Solid Phase Extraction (SPE)-based extraction method which allowed us to quantitate 3,5-T2 and its isomer 3,3’-T2 in human serum. Methods: Serum samples were obtained from 28 patients (8 healthy volunteers and 20 women undergoing endocrinological screening and found to be euthyroid). Two ml of serum were deproteinized with acetonitrile and then extracted using an SPE based process. To lower background noise, after extraction the samples were furtherly cleaned by hexane washing and acetonitrile precipitation of residual proteins. 3,5-T2 and 3,3’-T2 were analyzed by a Sciex API4000 mass spectrometer coupled to an Agilent Infinity 1290 LC system using isotope dilution method. In 20 patients total T3 and T4 were also assayed by HPLC-MS/MS. Results: Accuracy and precision for 3,5-T2 assay were 88-104% and 95-97%, respectively. Recovery, matrix effect and process efficiency averaged 78%, 108%, and 84% respectively. 3,5-T2 was detected in all samples and its concentration averaged (mean±SEM) 41±5 pg/ml, i.e. 78±9 pmol/l. In the same samples the concentration of 3,3’-T2 averaged 133±15 pg/ml, i.e. 253±29 pmol/l. 3,5-T2 concentration was significantly related to 3,3’-T2 concentration (r=0.540, P