Table 3.

Critical Features of Validation of the Analytical Method for Thyroid Hormone (TH) in Brain Tissue in Study 2.

1. MATRIX EFFECTS and CALIBRATION

TH are embedded in the rich phospholipid matrix of brain tissue. The degree to which this matrix can interfere with the accuracy of the measurement of TH needs to be minimized. Calibration curves were generated using 8 concentrations of each surrogate analyte with 5 replicates of solvent-based standards, matrix-matched standards, and blank standards. The solvent standards were spiked with unlabeled surrogates. Matrix and blank standards undergo the entire extraction procedure. After the extraction process, blank matrix standards were spiked with labeled surrogate TH analytes to generate the ‘spiked standards’. Each set of replicates was analyzed for T3, T4 and rT3 (Figure 3) and efficiencies of recovery, precision and matrix effects calculated as described below.

2. ACCURACY/RECOVERY

Accuracy of the analytical method for TH is reflected in the percent recovery of the T3, T4 and rT3 analyte from the matrix - greater recovery equating to better accuracy. Accuracy was determined by spiking 20 replicates of clean matrix with a concentration at or below the mid-point of the calibration curve and results are summarized in Table 4. The closer the measured analyte is to the spiked volume, the greater the accuracy of the method.

3. PRECISION

Precision of the analytical method refers to within- and between- day variability of the measurement of the TH analyte. Precision was determined by analyzing 5-replicate spiked samples at 3 different concentrations over 3 days. The replicate samples were prepared using blank matrix and spiked at concentrations that spanned the calibration range. The relative standard deviation (RSD) of the concentrations observed for the three sets of replicates provides the estimate of precision as reported in Tables 4 and 5.

4. SENSITIVITY

Sensitivity of the analytical method refers to the Method Detection Limit (MDL) and Method Quantitation Limit (MQL) for each TH analyte. The MDL is the lowest level that can be detected in the chromatogram, the MQL is the lowest level that can be accurately quantified in the chromatogram for each TH analyte. These values are reported in the text.