A UPLC-MS/MS method was developed to provide a reproducible, sensitive and quantitative assay to determine thyroid hormones in sea lamprey tissues and plasma. l-Thyroxine (T4) and its two triiodo-thyronine isomers have been simultaneously quantified and validated for plasma, gill, liver, and kidney matrices. Multiple sample preparation techniques were investigated to achieve optimal sample matrix digestion and clean-up. Enzymatic digestion followed by protein precipitation was selected to process the samples. The developed method exhibited excellent linearity for all analytes with regression coefficients higher than 0.99 for concentrations ranged from 10 to 50,000pg/mL. The limit of detection (LOD) was under 1pg/mL while the limit of quantification (LOQ) was estimated as 10pg/mL. This method was validated according to the FDA guidance and applied to determine thyroid hormone levels in plasma, gill and kidney of sea lamprey exposed to a sex pheromone. With appropriate implementation and further validation, this method could be applied to tissues in other species including humans.
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