Determination of free thyroid hormones in animal serum/plasma using ultrafiltration in combination with ultra-fast liquid chromatography-tandem mass spectrometry

Thyroid hormones (THs), which mainly consist of 3, 3′, 5-triiodo-l-thyronine (T3) and L-thyroxine (T4), play a critical role in regulating biological processes such as growth and metabolism in various animal species. Thus, accurate measurement of T3 and T4, especially physiologically active free (protein-unbound) forms, in serum/plasma is needed for the evaluation of TH homeostasis. However, such high-precision determination of free THs is lacking for non-human species. The present study aimed to develop a highly sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of six free THs in serum/plasma, which is applicable to not only humans but also non-human species. Two different physical separation steps, ultrafiltration (UF) and equilibrium dialysis (ED), were examined to obtain the free TH fraction. Several experimental conditions were carefully optimized and validated for UF or ED using the commercially available bovine serum. As a result, UF at 1100 × g and 37 °C for 30 min with a 30 kDa ultrafiltration device (Centrifree YM-30, Millipore) yielded excellent precision (CV: <10%). The optimized ED step also yielded high precision (CV: <10%) and the measurement values were approximately equal to those of UF, but at least 16 h were required to reach equilibrium. Thus, UF combined with LC-MS/MS was finally chosen, in terms of the time needed for the measurement. Acceptable accuracy (recovery: 70%-110%) and intra- and inter-day precision (CV: <10% and <12%, respectively) were obtained, when triplicate analyses in three different days were conducted using the bovine serum. The developed analytical method was successfully applied to the determination of free THs in serum/plasma samples of humans, cats, and dogs. Furthermore, comparison with free T4 concentrations measured by a common immunoassay method evidently indicated that the ultrafiltration-LC-MS/MS method developed in this study can increase the specificity and accuracy of TH measurement.