Because of the low limit of quantification to attain, particular attention was given to extraction recovery and ion suppression. The overall recovery (including extraction and ionization) was superior to 90% for all compounds. Since 3,3′,5-triidothyronine (T3) and 3,3′,5′-triiodothyronine (rT3; reverse T3) are isobaric, complete chromatographic separation was required.
To improve assay ruggedness and accuracy, deuterated analogues were used as internal standards.
Mobile phase composition was optimized to generate the highest ionization efficiency. Both positive and negative ionization were investigated.
The limit of quantification was of 0.5 pg/mL and the calibration was linear up to 50 pg/mL for all compounds. These concentrations enable to monitor both normal and pathologic hormone levels.