Abstract In the present work, a comparative study of analytical methods for the simultaneous and quantitative determination of trivalent and hexavalent chromium is presented. For the analysis by ion chromatography-inductively coupled plasma-mass spectrometry, two different columns were tested, as well as different mobile phases and different pH of the samples. The optimized analytical method permitted the separation of Cr(III) and Cr(VI) using 75 mmol/L NH 4 NO 3 pH 3 as chromatographic eluent. The method was validated and applied to real samples, allowing the determination of both species simultaneously, even when there is a huge difference of concentration between Cr(III) and Cr(VI). Limit of detection and limit of quantification for Cr(III) were found to be 0.016 and 0.054 $$\upmu$$ μ g/L (0.3 and 1.1 $$\upmu$$ μ g/kg), respectively, and for Cr(VI) 0.13 and 0.43 $$\upmu$$ μ g/L (7 and 22 $$\upmu$$ μ g/kg), respectively. Possible species interconversions were monitored through the use of chromium isotopic standards, which confirmed that the optimized methodology preserves chromium speciation during extraction and analysis. Fourier-transform ion cyclotron resonance-mass spectrometry permitted the structure elucidation of the complex formed during ethylenediaminetetraacetic acid extraction. Graphical Abstract