Anal Bioanal Chem (2006) 385: 1504–1512 DOI 10.1007/s00216-006-0550-y ORIGINAL PAPER L. Ayouni . F. Barbier . J. L. Imbert . J. Y. Gauvrit . P. Lantéri . M. F. Grenier-Loustalot New separation method for organic and inorganic selenium compounds based on anion exchange chromatography followed by inductively coupled plasma mass spectrometry Received: 5 January 2006 / Revised: 24 April 2006 / Accepted: 12 May 2006 / Published online: 21 July 2006 # Springer-Verlag 2006 Abstract We describe a new method for separating the organic and inorganic selenocompounds methaneseleninic acid, selenite, selenate, methylselenocysteine, selenocys- tine as well as both selenomethionine and its oxidized form. The separation is performed on a Hamilton PRP- X100 column. According to the literature, the oxidized form of selenomethionine—which is easily formed—is eluted close to the dead volume when this column is used. The choice of parahydroxybenzoic acid as mobile phase enabled us to elute all of these species after this oxidized form, resulting in better identification and quantification. The factors determining separation (eluent concentration, pH, gradient) were optimized via an experimental design. Application of the method to yeast and commercial tablets showed that the principal Se compound present was selenomethionine, which was also present in its oxidized form. Keywords Selenium . Speciation . Anion exchange chromatography . Experimental design Introduction Selenium was considered to be toxic until the 1950s, when the essential role played by it in organisms was first observed [1]. Its principal property is its antioxidant activity [2]. It has been shown that its presence is indispensable to the formation of the active site of glutathione peroxidase, an enzyme that combats the peroxidation of lipid chains by free radicals [3]. The latter entities are highly reactive and are responsible for aging and certain diseases. Clinical studies conducted over the past decade have thus shown that selenium supplementa- tion can help prevent certain cancers [4]. Selenium is used as a dietary supplement in the form of enriched yeasts in which a number of selenocompounds are present. The bioavailability and toxicity of elemental selenium depend on its concentration and chemical form. This is why considerable research has been conducted recently [5–9] into the speciation of all forms of selenium. Although the techniques used enable the detection of a large number of species, their physicochemical identifica- tion is far from complete. This lack of information caused the European Parliament to establish a “positive list” (in Directive 2002/46EC [10]) of dietary supplements, which states that the only authorized forms of selenium are selenite, selenate and sodium hydrogen selenite; any other chemical form of selenium was prohibited as of 1st August 2005. Selenite, selenate, selenocystine and selenomethionine are the principal compounds that are separated and detected in dietary supplements [11–15] by liquid chromatography followed by inductively coupled plasma mass spectrometry (ICP–MS). The dual organic/inorganic aspect of these organometallic species, however, poses difficulties when analyzing the four products simultaneously. Reversed- phase liquid chromatography is often used for the speci- ation of selenium, but the inorganic compounds are eluted in the dead volume [13, 16]. Potin-Gautier et al. [17] separated selenomethionine and selenocystine by reversed- phase ion pair chromatography, but selenite and selenate were not well-separated [11]. The ion exchange technique appears to be the most suitable separation method for these compounds. The four products are also often analyzed by anion exchange chromatography [5, 11, 14, 18, 19], but the conditions described by these authors involve systematic elution of one of the compounds around the dead volume characteristic of unretained species. Selenoamino acids, in particular selenomethionine, however, are easily oxidized L. Ayouni . F. Barbier . J. L. Imbert . M. F. Grenier-Loustalot (*) CNRS USR 59, Service Central d’Analyse, Chemin du Canal, BP 22, 69390 Vernaison, France e-mail: mf.grenier-loustalot@sca.cnrs.fr L. Ayouni . J. Y. Gauvrit . P. Lantéri UMR 5180, Laboratoire de Chimiométrie, Université Claude Bernard Lyon1, 43, Bd du 11 novembre 1918, Bâtiment 308D, 69622 Villeurbanne, France