Clinical applications of antimonials in the treatment of leishmaniasis are a unique opportunity to study the metabolism of antimony and its species in the human body. As little is known about antimony metabolism, studies are required to improve the knowledge about the excretion mechanism of this toxic element; in particular, those which deal with antimony metabolites and the action of antimonials for the eradication of leishmaniasis causing parasites. In contrast with its chemical analogue arsenic, only a few methods concerning antimony speciation in clinical samples have been described in the literature. The aim of the present work was to improve a method previously developed to separate and quantify N-methyl meglumine antimoniate, Sb(V) and Sb(III) in blood plasma and urine samples by using anion exchange high performance liquid chromatography (IC-HPLC) coupled with inductively-coupled plasma mass spectrometry (ICPMS). Results on the application of an ion chromatography column (Metrosep - A Supp5; 250 mm x 4.0 mm; Metrohm, Swiss) with better resolution for the separation of TMSb, Sb(V) and Sb(III) are presented. The optimum conditions for the separation of the three species were established with 0.006 mol L-1 EDTA at pH 4.7 as the mobile phase. The retention times for TMSb, Sb(V) and Sb(III) were 44,2 s, 151,6 s and 548,4 s, respectively. A robust linear relationship between concentration and peak area was obtained for the three species, both, in aqueous standard solutions and spiked urine samples, with an associated correlation factor higher than 0,999. Relative standard deviations below 4 percent have been obtained in the peak area quantification mode. When using the ELAN 5000 (PerkinElmer-Sciex, EUA) equipped with a concentric Meinhard nebulizer in combination with a cyclonic spray chamber as detector, and employing a 100-uL injection loop in the HPLC system, typical absolute limits of quantification were 0.02 ng of Sb for each of the three antimony species studied. Stability tests showed that Sb(V) suffers chemical alterations within a short time period (>8h in aqueous solutions and >4h in spiked urine), whereas TMSb and Sb(III) presented better stability conditions in both solutions.