Because iron in the calibrator solution was bound to HEDTA used as a calibrator, ferrozine did not form a complex with iron in this elimination step.
HEDTA + iron calibrator [right arrow] HEDTA-Fe (1st step)
The iron calibrator that was bound to HEDTA was dissociated from HEDTA under the acidic condition and formed a colored complex with ferrozine.
HEDTA-Fe + ferrozine [right arrow] HEDTA + ferrozine-Fe
We also examined the effect of the HEDTA concentration used as a calibrator, which behaved in the same manner as transferrin.