Human serum albumin (HSA) is known as a low affinity iron binding protein and it has been proposed as a ligand for the non-transferrin-bound iron (NTBI) pool existing in the sera of iron-overload patients, but definitive evidence is still lacking. In this study, gel filtration linked to inductively coupled plasma mass spectrometry (GF-ICP-MS) was employed to assess iron speciation in solutions containing physiological concentrations of citrate and HSA at physiological values of pH and ionic strength. The influence of common non-enzymatic post-translation modifications on the ability of HSA to bind iron was also studied. HSA was found to bind between 60 and 20% of the available iron when iron concentration was varied over the common clinical range of NTBI concentrations (1 to 10 microM), thus proving to be a relevant ligand for NTBI speciation. Analysis of modified albumins showed that both glycation and oxidation increase the albumin iron binding capacity. The results presented indicate that non-enzymatic modifications of albumin, that are prevalent in diabetes and increased oxidative stress, may have a fundamental role in NTBI speciation. Experiments with the nitrilotriacetic acid (NTA) based assay for NTBI determination demonstrate the inability of NTA to quantitatively mobilize albumin-bound iron, especially after the albumin had undergone oxidation or glycation. This result would indicate that the low NTBI levels so far measured in diabetic sera may be understated and that different analytical techniques are required to determine NTBI levels in diabetic patients.