A Novel and Fast Online-SPE-LC-MS/MS Method to Quantify Thyroid Hormone Metabolites in Rat Plasma

Abstract

Since the focus in regulatory toxicology has drifted toward the identification of endocrine disruptors, the improvement in determination of alterations in the thyroid hormone system has become more important. THs are involved in several molecular processes important for a proper pre-and postnatal development so that disturbances can inter alia lead to incorrect brain maturation and/or disturbed metabolic processes (thermogenesis or lipolysis). In this publication, a new automated online solid-phase extraction (SPE)-liquid chromatography (LC)-tandem mass spectrometry (MS/MS, xLC-MS/MS) is introduced which simultaneously analyzes total T4, T3, rT3, T2, and T1. Method validation parameters are presented, and the method was positively verified by analyzing control and PTU-treated rat plasma samples (time points day 7, 14, and 28) for their total TH content. The obtained results were compared to published results by using a radioimmunoassay method. The automated SPE system ensures a consistent unified sample preparation, and this method overall showed sufficient specificity and accuracy to detect the given analytes in rat plasma. For the preparation of 50 μL of rat plasma, the following LOQs were established: 0.020 nM for T1, 0.029 nM for T2, 0.023 nM for rT3 and T3, and 3.22 nM for T4. This method is suitable to assess the identification of mechanisms leading to adverse effects, such as disturbed TH metabolism and regulation.

Thyroid Hormone Metabolites Quantified in Pup and Adult Rat Cerebellum, Cortex and Whole-Brain Samples Using an Automated Online SPE-LC-MS/MS Method

“…From the pulverized rat brain samples, a defined amount was weighed in depending on the age of the given rat samples. The following description of sample preparation is based on a previous method developed to measure THs in rat plasma, and this method was modified to analyze TH in rat brains [ 15 ]. From rat brains on PND21, 20 mg was taken; PND4 7 mg; and adult rats 40 mg; these samples were weighed in in a polypropylene Eppendorf tube (Safe-Lock Tubes, 2.0 mL, Eppendorf AG, Hamburg, Germany), 300 µL PBS was added and samples were sonicated for 5 min.…”