Determination of thyroid hormones in placenta using isotope-dilution liquid chromatography quadrupole time-of-flight mass spectrometry

The transplacental passage of thyroid hormones (THs) is of great significance since the maternal THs are vitally important in ensuring the normal fetal development. In this paper, we determined the concentrations of seven THs, viz. L-thyroxine (T₄), 3,3′,5-triiodo-L-thyronine (T₃), 3,3′,5′-triiodo-L-thyronine (rT₃), 3,3′-diiodo-L-thyronine (T₂), 3,5-diiodo-L-thyronine (rT₂), 3-iodo-L-thyronine (T₁) and 3-iodothyronamine (T₁AM), in placenta using isotope dilution liquid chromatography quadrupole time-of-flight mass spectrometry. We optimized the method using isotopically labeled quantification standards (¹³C₆-T₄, ¹³C₆-T₃, ¹³C₆-rT₃ and ¹³C₆-T₂) and recovery standard (¹³C₁₂-T₄) in combination with solid-liquid extraction, liquid–liquid extraction and solid phase extraction. The linearity was obtained in the range of 0.5–150 pg uL⁻¹ with R² values >0.99. The method detection limits (MDLs) ranged from 0.01 ng g⁻¹ to 0.2 ng g⁻¹, while the method quantification limits (MQLs) were between 0.04 ng g⁻¹ and 0.7 ng g⁻¹. The spike-recoveries for THs (except for T₁ and T₁AM) were in the range of 81.0%–112%, with a coefficient of variation (CV) of 0.5–6.2%. The intra-day CVs and inter-day CVs were 0.5%–10.3% and 1.19%–8.88%, respectively. Concentrations of the THs were 22.9–35.0 ng g⁻¹ T₄, 0.32–0.46 ng g⁻¹ T₃, 2.86–3.69 ng g⁻¹ rT₃, 0.16–0.26 ng g⁻¹ T₂, and < MDL for other THs in five human placentas, and 2.05–3.51 ng g⁻¹ T₄, 0.37–0.62 ng g⁻¹ T₃, 0.96–1.3 ng g⁻¹ rT₃, 0.07–0.13 ng g⁻¹ T₂ and < MDL for other THs in five mouse placentas. The presence of T₂ was tracked in placenta for the first time. This method with improved selectivity and sensitivity allows comprehensive evaluation of TH homeostasis in research of metabolism and effects of environmental contaminant exposures.