Journal of Chromatography A, 1101 (2006) 94–102 Selenium speciation in Agaricus bisporus and Lentinula edodes mushroom proteins using multi-dimensional chromatography coupled to inductively coupled plasma mass spectrometry Val´ eria Gergely a,1 , Kevin M. Kubachka b , Sandra Mounicou c , P´ eter Fodor a , Joseph A. Caruso b,∗ a Department of Applied Chemistry, Corvinus University of Budapest, Villanyi ´ ut 29-33, 1118 Budapest, Hungary b Department of Chemistry, University of Cincinnati, Cincinnati, P.O. Box 0172, OH 45221-0172, USA c CNRS EP132, H´ elioparc 2, av. du President Angot, 64000 Pau, France Received 4 April 2005; received in revised form 20 September 2005; accepted 26 September 2005 Available online 17 October 2005 Abstract In this study, selenium species from Se containing proteins in mushrooms (Agaricus bisporus and Lentinula edodes) were investigated with size-exclusion liquid chromatography coupled to UV and inductively coupled plasma mass spectrometry (ICP-MS). Different protein extraction protocols were investigated. Variability of the fractionation patterns with three extraction media (0.1M NaOH, 30mM Tris–HCl, and enzymatic digestions) was evaluated for both mushroom types. A 24 h Tris–HCl extraction followed by acetone addition was found to be optimal for protein precipitation. Presumably protein bound selenoamino acids were released using enzymes (proteinase K, protease XIV and trypsin). The selenium speciation of the proteolytic extract of the water soluble proteins fraction was carried out by using reversed-phase ion-pairing high performance liquid chromatography (RP-HPIPC) coupled on-line to ICP-MS for selenium specific detection. Selenocystine, selenomethionine, methylselenocysteine and inorganic selenium were established in both samples utilizing retention time standards and standard additions to the sample. © 2005 Elsevier B.V. All rights reserved. Keywords: Selenium speciation; Proteins; Mushroom; HPLC-ICP-MS 1. Introduction Selenium is an essential trace element for humans and ani- mals. Since the work of Clark et al. involving the role of selenium in cancer chemoprevention, potential “Se-delivery sys- tems” have been explored [1,2]. Research involving selenium has become an increasingly popular area in the research com- munity in the last 20 years. A lot is known about the health implications of selenium, but there are still numerous unexplored areas. One problem with selenium as a supplement is a narrow range between the selenium toxicity level and beneficial level [1]. Levels below the daily recommended amount (30 g per day) can lead to increased aging, increased risk of cardiovascular ∗ Corresponding author. Tel.: +1 513 556 9304; fax: +1 513 556 9239. E-mail address: joseph.caruso@uc.edu (J.A. Caruso). 1 VG studied as an exchange student at the University of Cincinnati, Autumn 2004. disease as well as other degenerative diseases [3]. Intake of sele- nium above the Tolerable Upper Intake Level (UL, 400 g per day) can lead to hair and nail brittleness and loss, gastrointesti- nal disturbances, skin rash, garlic breath, fatigue, irritability, and nervous system abnormalities [4]. Selenium is needed to pro- duce the thyroid hormone triiodothyronine (required for healthy brain and bone development, normal growth, and thermoreg- ulation) [2]. At the proper levels selenium has been shown necessary for proper immune system functioning [3]. Recent research has shown that selenium can inhibit the progression of human immunodeficiency virus to acquired immunodeficiency syndrome [5]. Also it has been shown to be important in cancer risk reduction [6]. Selenium exerts its biological effect through several seleno- proteins, of which there may be more than 30 in mammalian systems. It is incorporated by genetic coding to include selenocysteine in ribosome-mediated protein synthesis. These selenoproteins include a number of glutathione peroxidases (GPx) including cellular GPx (GPx1) and phospholipid 0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.09.061