Quantification of total thyroxine in plasma from Xenopus laevis

Area of Science:

• Environmental Toxicology
• Endocrinology
• Analytical Chemistry

Background:

• Chemical contaminants can disrupt wildlife endocrine systems, leading to developmental and reproductive issues.
• The hypothalamus-pituitary-thyroid (HPT) axis is vital for vertebrate development, with disruptions particularly harmful during early life stages.
• Amphibian metamorphosis, regulated by thyroid hormones, makes tadpoles suitable models for evaluating HPT axis effects of environmental substances.

Purpose of the Study:

• To develop and validate a precise method for quantifying total thyroxine (T4) in amphibian plasma.
• To establish a reliable assay for assessing HPT axis function in tadpoles exposed to potential endocrine disruptors.

Main Methods:

• Utilized mixed-mode strong cation exchange solid-phase extraction (SPE) for sample preparation.
• Employed ultra-high-performance liquid chromatography and isotope dilution tandem mass spectrometry (UPLC-ID-MS-MS) for quantification.
• Analyzed a small plasma volume (10 µL) from Xenopus laevis tadpoles.

Main Results:

• The UPLC-ID-MS-MS method demonstrated high selectivity for T4 and its internal standard.
• Achieved a lower limit of quantification of 0.5 ng/mL with excellent intra-day (< ±5%) and inter-day (< ±10%) precision.
• Measured total T4 concentrations in Xenopus laevis tadpoles at metamorphic peak as 10.7 ± 0.8 ng/mL, comparable to radioimmunoassay results.

Conclusions:

• The validated UPLC-ID-MS-MS method provides accurate and precise determination of total T4 in small amphibian plasma volumes.
• This method is suitable for assessing thyroid hormone levels in tadpoles, supporting research on endocrine disruption.
• The technique shows low susceptibility to interferences, ensuring reliable data for environmental risk assessment.