Analytical Methods Evaluation of selenium species in selenium-enriched pakchoi (Brassica chinensis Jusl var parachinensis (Bailey) Tsen & Lee) using mixed ion-pair reversed phase HPLC–ICP-MS Witphon Thosaikham a , Kritsana Jitmanee b , Rossukon Sittipout a , Sarunya Maneetong a , Anut Chantiratikul c , Piyanete Chantiratikul a,⇑ a Department of Chemistry and Center of Excellence for Innovation in Chemistry (PERCH-CIC), Faculty of Science, Mahasarakham University, Kantarawichai, Maha Sarakham 44150, Thailand b Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand c Department of Agricultural Technology, Faculty of Technology, Mahasarakham University, Kantarawichai, Maha Sarakham 44150, Thailand article info Article history: Received 30 September 2011 Received in revised form 4 October 2012 Accepted 28 August 2013 Available online 5 September 2013 Keywords: Brassicaceae plants Ion-paired reversed phase HPLC–ICP-MS Pakchoi (Brassica chinensis Jusl var parachinensis (Bailey) Tsen & Lee) Selenium speciation Se-enriched pakchoi abstract HPLC–ICP-MS based on ion-paired reversed phase chromatography for the selenium speciation using the mixture of 1-butanesulfonic acid (BA) and trifluoroacetic acid (TFA) as the mixed ion-pairing reagents was developed and applied to selenium-enriched pakchoi (Brassica chinensis Jusl var parachinensis (Bailey) Tsen & Lee). Several conditions of ion-paired reversed phase HPLC–ICP-MS, such as pH of the mobile phase, concentration of ion pairing reagents, types and length of analytical column, and flow rate of the mobile phase, were optimised for five selenium species; selenate (Se(VI)), Selenite (se(IV)), selenocys- teine (SeC), Se-methylselenocysteine (SeMC) and selenomethionine (SeM). The results showed that the optimum conditions for pH, BA and TFA condition, type of separating column and flow rate, were 4.5, 8 mM, 4 mM, C18 (250 mm length  4.6 mm I.D) and 1.2 mL min 1 , respectively. These conditions archived separation of the organic selenium species. The limits of detection (LOD) and quantitation (LOQ) of each selenium species were lower than 5 and 16 ng Se mL 1 , respectively. Furthermore, the recoveries of most selenium species were good, except for SeC. In this research, selenium-enriched pak- choi was cultivated by supplementing inorganic selenium from selenate into sand. The result showed that inorganic selenium, SeMC, SeM and several unknown species were found in selenium-enriched pak- choi sprouts by using the proposed method. Thereby, the biotransformation of selenate in pakchoi was similar to other Brassicaceae plants such as kale and broccoli. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Selenium (Se) is a trace element and recognised as an essential nutrient and toxic reagent for animals including humans (Burke & Opeskin, 2002; Klein, 2004). However, the toxicity or benefit of selenium depends on the chemical form and concentration of sele- nium in environmental and biological systems (Brown & Arthur, 2001; Li et al. 2010). Generally, the inorganic species of selenium such as selenate (SeVI) and selenite (SeIV) have high toxicity at large doses (Mazan, Gilon, Crètier, Rocca, & Mermet, 2002; Orero, Roig-Navarro & Hermanández, 2004). Meanwhile, the organic spe- cies of selenium, selenocysteine (SeC), Se-methylselenocysteine (SeMC) and selenomethionine (SeM) are amino acid forms of sele- nium in organisms and have several benefits for health (Bock et al., 1991; Hartikainen, 2005). Thus, the production of Se-enriched food by supplementing inorganic selenium into organisms such as yeast, mushroom and bacteria for providing an organic selenium source for humans has received wide interest around the world (Alzate et al., 2007; Casiot et al., 1999; Larsen et al., 2006). The Brassicaceae is one of the most popular vegetables that can be produced as a Se-enriched food. Several Brassicaceae species accumulate selenium in high concentration (Montes-Bayón et al., 2002). As such, these species could provide cost-effective technol- ogy for removing selenium from contaminated soils and waters. Furthermore, they can transform selenium into bioactive com- pounds that have important implications for human nutrition and health (Kahakachchi, Boakye, Uden, & Tyson, 2004). Broccoli is a species in Brassicaceae used for Se-enriched plant production because it can reduce the toxicity of inorganic selenium and transform to several organic species of selenium such as Se-meth- ylselenocysteine, Se-methylselenomethionine, and gamma-glut- amyl-Se-methyl selenocysteine (Terry, Zayed, De Souza, & Tarun, 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.08.116 ⇑ Corresponding author. Tel.: +66 871735444; fax: +66 43754246. E-mail addresses: piyanete_s@yahoo.com, piyanete.c@msu.ac.th (P. Chantiratikul). Food Chemistry 145 (2014) 736–742 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem