Quantification of Total Thyroxine in Plasma from Xenopus laevis

Abstract

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.

Quantification of 11 thyroid hormones and associated metabolites in blood using isotope-dilution liquid chromatography tandem mass spectrometry

“…All analytes yielded two SRM-ion transitions; however, it was only possible to obtain a single high abundance ion-transition for T 0 and T 1 AM. Our MS/MSfragmentation is in agreement with other studies that also found similar ion fragments caused by loss of carboxylic acid for T 4 , T 3 , rT 3 [8,[21][22][23], 3,5-T 2 , and 3,3′-T 2 [16,24]. For T 1 AM, we observed a MS/MS-fragment at m/z 212, corresponding to loss of aromatic hydroxyl and iodine, which agrees with other findings [25,26].…”

“…The average surrogate recoveries were 70.7 % for cT 4 , 89.4 % for cT 3 , and 83.9 % for cT 2 , and we did not detect any other analyte in these samples. These plasma levels for T 4 are in accordance with another X. laevis study where NF stage 62 [40] tadpoles yielded 10.7 ± 0.8 ng mL -1 at this metamorphic climax stage [23]. Metamorphic climax is the period in which maximal levels of T 4 and T 3 are typically reached in the frog.…”

“…Accordingly, we focused on denaturing the proteins, thereby unfolding and bringing the macromolecules into solution. Typically, other researchers have done this by adding strong acids, such as trichloroacetic acid and hydrochloric acid [8,23,29]. As per our stability study ( Fig.…”

“…Compared to other methodologies for analyzing only T 4 , the developed methodology is at least twice as sensitive [23,39]. Compared to methods analyzing multiple thyroid hormones, the developed method is 5-to 20-fold more sensitive [16,17].…”

Data collection in support of the Endocrine Disruption (ED) assessment for non‐target vertebrates

“…The method by Simon et al (2002) (reference 8692 in Annex B -) differed from the other two both with regards to the sampling matrix (whole body homogenates of tadpoles of African Clawed frogs) and therefore sample preparation, and the instrumentation (use of the LC-ICP-MS as opposed to the more selective LC-MS/MS methods). Luna et al (2013) used ultrahighperformance liquid chromatography and isotope dilution tandem mass spectrometry (UPLC-ID-MS-MS) to quantify total T4 in a small volume (10 μL) of plasma also from tadpoles and adult Xenopus laevis (reference 3463 in Annex B -). Hansen et al (2016) validated a LC-MS/MS method to quantify absolute and total concentrations of eleven thyroid hormones and associated metabolites in both 50 μL plasma of adult Xenopus or 50 μL serum of tadpoles (American bullfrog, Rana (Lithobates) catesbeiana) (reference 8142 in Annex B -).…”