Validated ultra high performance liquid chromatography-tandem mass spectrometry method for quantitative analysis of total and free thyroid hormones in bovine serum

Thyroid hormones are essential hormones for regulating growth and development. Methods to accurately monitor low-levels (ppb-ppt) of these hormones in serum are needed to assess overall health, both from a clinical perspective as from environmental contaminant or drug exposures. In general, the separation of the free thyroid hormone fraction from animal sera is performed through labour intensive equilibrium dialysis, while detection of total and free thyroid hormone fractions in animals is done with commercially available radioimmunoassays (RIAs). This study reports newly developed analysis methods for both the total and free fractions of triiodothyronine (T3), reverse-triiodothyronine (rT3) and thyroxin (T4) from bovine serum, with a much higher specificity and selectivity than commercially available RIAs. The bovine serum extraction procedures of total and free T3, rT3, T4 were optimised with fractional factorial designs and consisted of, respectively, deproteinisation followed by liquid-liquid extraction, 30 kDA ultracentrifugation and solid phase extraction. Both free and total thyroid hormone UHPLC-HESI-MS/MS based analysis methods were successfully validated. The limits of quantification for T4, rT3 and T3 amounted respectively 0.04 ng mL(-1), 0.05 ng mL(-1), 0.03 ng mL(-1) for the total fraction, and 6.6 pg mL(-1), 2.6 pg mL(-1) and 2.7 pg mL(-1) for the free fraction. Individual recoveries of total and free thyroid hormone fractions ranged between 95.6 and 106.3% and 92.1 and 106.5%. Good results for repeatability and intra-laboratory reproducibility (RSD%) were observed, i.e. respectively ≤8.0% and ≤7.3% for the total and free fractions. Excellent linearity (R(2)≥0.99) and lack-of-fit was proven for both fractions. In conclusion, these methods show excellent in-house performance and possibilities for elaboration to application in other animal sera (e.g. feline, canine, equine).