Thyroid hormones (TH) are known to play an important role in the growth and development of vertebrates. In fish species, TH regulates the larval-juvenile metamorphosis, and is crucial for development during early life stages. Monitoring the variations in TH levels at different life stages can provide insights into the regulation of metamorphosis and fish development. In this study, we developed an extremely sensitive method for the quantification of thyroxine (T4), triiodothyronine (T3), and reverse-triiodothyronine (rT3), in lake sturgeon (Acipenser fulvescens) tissues from eggs, free embryos, larvae, and juveniles. The target compounds were extracted by an enzymatic digestion method, followed by protein precipitation. Further cleanup was performed by liquid-liquid extraction (LLE) and solid phase extraction (SPE) using SampliQ OPT cartridges. The liquid-chromatography tandem mass spectrometry (LC-MS/MS) method used to quantify TH compounds showed remarkably high sensitivity with the limit of detection (LOD) and the limit of quantification (LOQ) ranging from < 1 pg/mL to 10 pg/mL and linearity in the range of 10-50,000 pg/mL. This method was validated for tissue samples across several early developmental stages and was checked for intra- and inter-day accuracy (78.3-111.2 %) and precision (0.1-4.9 %), matrix effect (75.4-134.1 %), and recovery (41.2-69.0 %). The method was successfully applied for the quantification and comparison of T4, T3 and rT3 in hatchery raised lake sturgeon samples collected at unique time points (i.e., days post fertilization dpf) including fertilized eggs (11 dpf), free embryos (14 dpf), larvae (22 dpf), juveniles (40 dpf) and older juveniles (74 dpf). With modifications, this method could be applied to other species important for agriculture or conservation.
Keywords: Developmental stages; Lake sturgeon; Method validation; T3; T4; rT3.
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