Quantification of total thyroxine in plasma from Xenopus laevis

There is evidence that the endocrine systems of certain fish and wildlife can be affected by chemical contaminants, possibly resulting in developmental and reproductive problems. Perturbations in the hypothalamus-pituitary-thyroid (HPT) axis, in particular, can be detrimental during early development. Because the rate of amphibian metamorphosis is controlled by circulating thyroid hormones, tadpoles undergoing metamorphosis have been selected as relevant test organisms for evaluating the potential effects of a substance on the HPT axis in vertebrates. An indicative measure of HPT functioning in these assays is the concentration of the thyroid hormone, thyroxine (T4), in frog plasma. Therefore, there is a need for a validated method to measure T4 in plasma during amphibian metamorphosis. This study describes a method involving mixed-mode strong cation exchange solid-phase extraction (SPE) with ultrahigh-performance liquid chromatography and isotope dilution tandem mass spectrometry (UPLC-ID-MS-MS) to quantify total T4 in a small volume (10 µL) of plasma from Xenopus laevis (African clawed frog). The SPE procedure, together with MS detection, produced the required selectivity for the analysis of both T4 and the T4 internal standard. The limit of detection of the method was determined to be 0.2 ng/mL, whereas the lower limit of quantification was 0.5 ng/mL. The intra-day and inter-day precision values were less than ± 5 and ±10%, respectively. Concentrations of total T4 in the plasma of X. laevis tadpoles at metamorphic peak were calculated to be 10.7 ± 0.8 ng/mL, which is comparable to the results from radioimmunoassay. This validated UPLC-ID-MS-MS method for the determination of total T4 in plasma has demonstrated good accuracy and precision, with low susceptibility to interferences with the utilization of multiple reaction monitoring and ID.