Development of analytical method for thyroid hormones (T3 and T4) in blood using LC-MS/MS

抄録

The Organisation for Economic Co-operation and Development (OECD) guidelines for toxicity testing methods was updated in 2018 to add endocrine disruptor relevant endpoints to the repeated dose 90-day oral toxicity study in rodents (TG408), requiring measurement of thyroid-related hormones (T3, T4, and TSH).

The enzyme-linked immunosorbent assay (ELISA), which has been the mainstream method for measuring T3 and T4 in blood since its inception, is an immunoreaction-based assay that enables highly sensitive measurement by specifically recognizing the target molecule. However, this assay has problems including the presence of many variations due to differences in test kit lots, analysts, etc., and requiring a lot of time because T3 and T4 are measured in separate kits.

With the aim of solving these problems, we investigated a method to simultaneously measure T3 and T4 using a mass spectrometer (LC-MS/MS) as an alternative to the ELISA method.

T3 and T4 labeled with 13C were used as internal standards. Rat serum was purified by solid-phase extraction using Oasis HLB. Water-acetonitrile containing 0.1% formic acid was used as the mobile phase, and the concentration gradient was controlled by changing the mixing ratio. MS/MS detection was performed in positive electrospray ionization mode to obtain calibration curves for T3 and T4 in the concentration ranges of 0.05 to 25 ng/mL and 0.5 to 250 ng/mL, respectively. Using this method, we investigated the linearity, repeatability, etc. of the resulting calibration curves.