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.

Recent evidence suggests that vitamin D insufficiency is associated with an increased risk of stress fracture, total body inflammation, infectious illness and impaired muscle function (1,2) and is thus likely to impair the training ability of athletes in addition to affecting their overall health. Though not widely studied, vitamin D deficiency and insufficiency have been reported among athletes, irrespective of geographic location (3) . The aim of the current study was to assess vitamin D status in two groups of elite Irish athletes sampled in different months of the year.Non-fasting blood samples were taken from consenting volunteers (n 66) and total serum 25-hydroxyvitamin D (25(OH)D) was quantified by enzymeimmunoassay (IDS Limited, Boldon, UK). Serum calcium and plasma parathyroid hormone (PTH) concentration were also assessed (using the I-lab 650 Clinical Chemistry analyser and ARCHITECT i1000SR integrated system, respectively). *Significant difference between athletic groups (P < 0.05, independent t-test). Significance remained after controlling for age and gender (P < 0.001, ANCOVA); †mean daily intakes estimated from a validated food frequency questionnaire . Among the paralympians, the serum 25(OH)D and calcium concentrations of wheelchair-bound athletes were significantly lower compared to that of their able-bodied counterparts (P = 0.013 and P = 0.001, respectively), without significantly affecting PTH concentrations (P = 0.676). There were no significant differences in mean daily vitamin D (P = 0.903) or calcium (P = 0.210) intakes between the two groups of paralympians. Mean daily vitamin D and calcium intake were significantly correlated after controlling for age and gender (r = 0.464, P = 0.030), however were not associated with circulating 25(OH)D, PTH or calcium concentrations.This study highlights a high prevalence of vitamin D deficiency among elite Irish athletes, particularly at the end of winter. Inadequate sun exposure, rather than diet, is the likely cause of lower 25(OH)D concentrations observed in the wheelchair athletes. A serum 25(OH)D concentration ‡ 80 nmol/L has recently been postulated as desirable for athletes (2) however, the majority of athletes sampled in this study failed to reach this level. Vitamin D supplementation should therefore be considered for these athletic groups.

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