Simultaneous derivatization of selenocysteine and selenomethionine in animal blood prior to their specific determination by 2D size-exclusion ion-pairing reversed-phase HPLC-ICP MS Katarzyna Bier1a, a Ve ´ronique Vacchina, b Joanna Szpunar, a Ge ´rard Bertin c and Ryszard qobi nski * ad Received 12th November 2007, Accepted 7th January 2008 First published as an Advance Article on the web 1st February 2008 DOI: 10.1039/b717448a A procedure for the simultaneous quantitative carbamidomethylation of selenocysteine (SeCys) and selenomethionine (SeMet) followed by their proteolytic release from blood proteins was developed. The fraction containing both derivatized selenoaminoacids was isolated by size-exclusion chromatography and submitted to ion-pairing HPLC-ICP MS analysis. The limit of detection was ca. 0.02 mgg 1 (dry mass) for either amino acid. The quantification of SeCys and SeMet was carried out by the method of standard additions. An internal standard of 77 Se-labelled SeMet was used to control the derivatization yield and chromatographic recovery. The determination of SeCys was validated by spiking with glutathione peroxidase. An additional proof of validity was achieved by monitoring the selenium mass balance (12 series of analysis over a period of 18 months; the Se amino acids accounted for 92  8% of the total Se). The method was applied to the monitoring of changes in SeCys and SeMet concentrations in lamb blood during supplementation studies (tolerance and dose effect) with selenium-rich yeast. Introduction Selenium is an essential element playing an important role in many metabolic functions. Supplementation of farm animals with selenium is stimulated by the beneficial effect of this element on animal health and, consequently, by the improved quality of foodstuffs of animal origin. Selenium status in animals is usually assessed via the determination of the total selenium in blood 1,2 or by the determination of the glutathione peroxidase activity 1,2 and does not take into account the selenium speciation. Most of the selenium present in blood is known to be protein-bound and to occur in the form of two seleno-amino acids. One of them, selenocysteine, considered to be biologically active, forms the active centre of selenoproteins, e.g. glutathione peroxidases (GPx), thioredoxin reductases (TrxR), and selenoprotein P (SeP). Selenomethionine is supposed to be incorporated randomly into methionine-containing proteins. The difference in function of these amino acids demands accurate methods for their routine determination. Literature reports on the determination of seleno-amino acids in blood samples have been scarce. To our knowledge, the only reference to selenomethionine determination in whole blood was made by Palacios et al. who observed a peak from selenomethio- nine (following those of Se-containing proteins) in size-exclusion chromatography (SEC)-ICP MS of blood from animals supple- mented with high doses of selenium. 3 Selenomethionine was quantified in serum 4,5 and tissue 6,7 samples by HPLC-ICP MS following enzymatic proteolysis. The only report referring to the selenocysteine determination concerned serum and was based on the isolation of this amino acid from a proteolytic extract after derivatization with iodoacetamide. 4 The objective of this research was to develop a method for the simultaneous determination of selenomethionine and selenocys- teine in animal blood. Emphasis was put on the quantification of selenocysteine and validation of its determination. Since attempts to selectively derivatize selenocysteine without affecting selenomethionine, as proposed elsewhere, 4 failed due to the matrix complexity, a different sample preparation strategy had to be developed. Experimental Instrumentation Chromatographic separations were carried out using a Model 1100 HPLC pump (Agilent, Wilmington, DE, USA) as the deliv- ery system. The exit of the column was directly connected to the Meinhard nebulizer (Glass Expansion, Romainmotier, Switzer- land) of the ICP MS equipped with a collision cell (Agilent 7500c, Tokyo, Japan) by means of PEEK tubing. Injections (both in size-exclusion and in reversed-phase LC) were performed using a Rheodyne valve with a 100 mL sample loop. Fractions from the preparative chromatography were lyophilized at 50  C using a Model LP3 lyophilizer (Jouan, France). Samples were shaken mechanically during extraction and derivatization (KS 250 basic, Kika Labortechnik, Staufen, Germany and OLS 200, GRANT, Chelmsford, UK) and then centrifuged (Force 6, Denver Instruments, Arvada, CO, USA). For the total selenium a Laboratoire de Chimie Analytique Bio-inorganique et Environnement, CNRS, UMR 5254, He´lioparc, 2Av. Angot, Pau, 64053, France. E-mail: ryszard.lobinski@univ-pau.fr b Ultratrace Analyses Aquitaine (UT2A), He´lioparc, 2Av. Angot, Pau, 64053, France c Alltech France, 14 Place Marie-Jeanne Bassot, 92593 Levallois-Perret, France d Department of Analytical Chemistry, Warsaw University of Technology, ul. Noakowskiego 3, Warszawa, 00-664, Poland 508 | J. Anal. At. Spectrom., 2008, 23, 508–513 This journal is ª The Royal Society of Chemistry 2008 PAPER www.rsc.org/jaas | Journal of Analytical Atomic Spectrometry