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A rapid, sensitive and species preservative analytical method for the simultaneous determination of six selenium (Se) species has been developed. Enzymatic probe sonication (EPS) was investigated as a novel and alternative technology for the extraction of Se species from feed matrices and the results were compared with the conventional hot water extraction, enzymatic hydrolysis and sequential extraction. The critical parameters of EPS such as enzyme types, extraction time, temperature, ultrasonic power and sample/enzyme ratio were varied with control. The Se species were separated and quantitatively determined by ion chromatography-inductively coupled plasma mass spectrometry (IC-ICP-MS). Under current optimised conditions, six inorganic and organic Se species were completely separated within 15 min in a single chromatographic run. The spectral interferences from the argon plasma 40Ar2, 40Ar37Cl or 1H79Br were effectively removed by employing the kinetic energy discrimination (KED) mode. Quantitative extraction for total Se (>94.8%) and more than 89.0% for the sum of different Se chemical forms without species transformation were obtained in only 60 s by applying the EPS treatment using aqueous protease XIV. The limits of detection (LODs) and quantification (LOQs) for Se species were in the ranges of 0.21–0.56 µg kg–1 and 0.69–1.87 µg kg–1, respectively. The proposed method was successfully applied to the speciation of Se in several reference materials and feed samples collected from the markets and local farms. [ABSTRACT FROM AUTHOR]