Amlund, Heidi

Sele, Veronika

Sloth, Jens Jørgen

Ørnsrud, Robin

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: Dec 18, 2017There is a need for speciation analysis of selenium in fish feed and fish tissueSele, Veronika; Sloth, Jens Jørgen; Ørnsrud, Robin; Amlund, HeidiPublication date:2016Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Sele, V., Sloth, J. J., Ørnsrud, R., & Amlund, H. (2016). There is a need for speciation analysis of selenium infish feed and fish tissue. Poster session presented at 8th Nordic Conference on Plasma Spectrochemistry, Loen,Norway.BackgroundSelenium (Se) is an element that is both essential and toxic for living organisms. The element can be present in organic and inorganic forms, and the bioavailability and toxicity of Se are affectedby its chemical form, or Se species. Seafood and marine raw materials have natural high levels of Se. Hence, in traditional salmon feeds where there is a high inclusion of marine raw materials,such as fish meal, the Se levels are high. The Se levels typically range from 0.2 to 9.0 mg Se kg-1 feed [1, 2]. The Se levels in fish feeds are dependent on the Se levels in the feed ingredients, andreplacement of marine-based ingredients with plant-based ingredients lowers the Se levels in the final feeds. In turn, this may cause a need for supplementation of Se to assure a robust farmedsalmon. Hence, the substitution of feed ingredients and supplementation of feeds with e.g. Se-containing yeast or inorganic Se salts also affect the Se species pattern in the final feeds andpossible in tissues of farmed Atlantic salmon. To study the bioavailability and toxicity of different Se feed additives, there is a need for speciationanalysis of both feeds and of fish.IntroductionFor Se speciation analysis High Pressure Liquid Chromatography (HPLC) coupled to Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is commonlyused. A range of natural occurring Se species exist in environmental samples, both inorganic Se species (selenite and selenate) and organic Se species(e.g. selenomethionine; SeMet), Selenomethylselenocysteine; SeMetSeCys and selenocysteine; SeCys). In fish tissue SeMet is normally the major Se speciesdetected [3, 4]. Since SeMet and SeCys are bound to proteins, Se was extracted from sample matrix using enzymatic hydrolysis using a non-specificprotease. The extraction recovery (%) of Se in the soluble extracts was determined by measurement of total Se in the extracts by ICP-MS, and comparing the results with total Se concentrations in the samples(Fig 1). For the Se speciation analysis two chromatographic separation modes were used to achieve the optimal chromatographic separation of the Se species; an anion-exchange HPLC-ICP-MS method wasapplied for the determination of the inorganic Se species, whereas a cation-exchange HPLC-ICPMS method was applied for determination of organic Se species. Fig 2 shows fish feed supplemented with a highlevel of inorganic Se analysed by anion-exchange HPLC-ICPMS, and a feed supplemented with high level organic Se-containing yeast analysed by cation-exchange HPLC-ICPMS.THERE IS A NEED FOR SPECIATION ANALYSIS OF SELENIUM IN FISH FEED AND FISH TISSUE  Veronika Sele 1*, Jens J. Sloth 1,2, Robin Ørnsrud 1 & Heidi Amlund11 ) National Institute of Nutrition and Seafood Research (NIFES), Bergen , Norway. 2) National Food Institute, Technical University of Denmark, Copenhagen, Denmark*) Contact mail address: veronika.sele@nifes.noConclusion & future work• The extraction efficiency of Se seems to depend on the type of sample and supplementation source. The extraction recovery is lower for thefeeds supplemented with inorganic Se compared to feeds supplemented with organic Se yeast. The extraction recovery is 99 ± 12% (n = 17) forsalmon muscle tissue.• Two separate chromatographic methods have been developed for speciation analysis of inorganic and organic Se species in fish feeds andsalmon muscle tissue. The methods needs yet to be fully validated.• Speciation analysis of supplemented feeds shows that the feed supplemented with high inorganic Se contains several unknown Se species.Selenomethionine is the major Se species in the organic supplemented feed. Preliminary results shows that SeMet is the major Se species in thesalmon tissues.References[1] Sanden, M., Hemre, G.-I., Måge, A., Lunestad, B.T., Espe, M. , Lundebye, A.-K. & Ørnsrud, R. (2014). . Program for overvåking av fiskefôr. Årsrapport 2013. Nasjonalt institutt for ernærings-og sjømatforskning, Bergen. [2] Sanden, M., Hemre, G.-I., Måge, A., Lunestad, B.T., Espe, M. , Lundebye, A.-K., Amlund, H., Torstensen, B. & Ørnsrud, R. (2015). Program for overvåking av fiskefôr. Årsrapport for prøver innsamlet i 2014. Nasjonalt institutt for ernærings-og sjømatforskning, Bergen.[3] Diaz Huerta V, Fernàndez Sànches ML, Sanz-Medel, A. Quantitative selenium speciation in cod muscle by isotope dilution ICP-MS with a reaction cell: comparison of different reported extraction procedures. J Anal At Spectrom 2004; 19:644-648.[4] Malgorzata AB, Måge A. Determination of selenium and its compounds in marine organisms. J Trace Elem Med Biol 2015; 29:91-98.  This work is funded by FHF, project nr 900871. Table 1. Instrumental settings for ICP-MS and HPLC (1260, both Agilent Technologies) in Se speciation analysis.Aim of this study was develop analytical method for determination of Se species in feed ingredients, fish feeds and in fish tissue.Results & DiscussionFig 1. The extraction recovery (%) of Se in the protease extractscompared to total Se in samples (n=3), determined by ICP-MS.The extraction procedure was carried out three repetitive timesfor selected samples .Outline of Se speciation analysesApproximately 0.2 g of freeze dried sample was mixed with 2.5mL of1mM ammonium phosphate, pH 7, containing 20 mg protease type XIVfrom Streptomyces griseus (Sigma Aldrich). The samples were left onwater bath at 37°C and shaking at 100/min for 24h. The samples werecooled, centrifuged (3500rpm, 10min) and filtered with 0.45µm,membrane filters (Meck Millipore) and Amicon (0.5mL, 10kDa cut-off,Merck Life Science AS) prior to Se speciation analysis with HPLC-ICPMS.Fig 2. Anion-exchange HPLC-ICPMS of inorganic Se species of a standard mixture (100µg L-1; A) and diet 3 (B). Cation-exchange HPLC-ICPMS analysis of Se species in astandard mixture (C), fish feed diet 3 (E) and diet 5 (x4 dilution). The assigned SeMetpeak in diet 5 was determined by spiking the sample with a standard mixtture.Table 2. Se levels in experimental diets; mean ± SD, n = 3.A BECSeMet04000800012000160000 5 10 15 20Se signal (78Se)Retention time (min)SeleniteSelenateSeMetOrganic Se02000400060008000100000 5 10 15 20Se signal (78SeRetention time (min)01002003004005000 5 10 15Se signal (78Se)Retention time (min)D02000040000600000 5 10 15Se signal (78Se)Retenton time (min)SeCys2SeMetSeCysSeMetinorgSe02000400060000 5 10 15Se signal (78Se)Retention time (min)020406080100120140160% Recoveryextraction 3extraction 2extraction 1Diet Information on feed dietsSe concentration(mg kg-1 dw)Diet 1 Plant-based 0.35 ± 0.02Diet 2 Supplemented- low inorganic Se 1.10 ± 0.03Diet 3 Supplemented- low inorganic Se 15.0 ± 0.5Diet 4 Supplemented- low organic Se 2.10 ± 0.05Diet 5 Supplemented- low organic Se 15.0 ± 0.24Diet 6 Marine-based 0.89 ± 0.03RF power 1520 WCarrier gas flow 0.9 L min-1Plasma gas flow 0.20 L min-1H2 gas flow (collision-reaction cell) 4 mL min-1Spray chamber temperature 2 °CInterface cones NickelNebulizer BabingtonIntegration time 0.1 secIsotope monitored 78, 80, 82HPLC settings – Anion-exchange HPLCColumn Hamilton PRP-X100  (4.6 x 150mm, 5µm)Injection volume 25µLFlow rate 1 mL min-1Mobile phase A: 0.5mM ammonium acetate, pH 5.2.B: 100mM ammonium acetate, pH 5.2Gradient 0-3min: 95% A, 5% B, 3-5min: 5-100% B, 5-13min: 100% B, 13.1 – 20min: 95% A, 5% B.HPLC settings – Cation-exchange HPLCColumn Ionosphere 5C (3 x 100mm, 5µm) Injection volume 25 µLFlow rate 1 mL min-1Mobile phase A: 0.5mM Pyridine, pH 3.B: 10mM Pyridine, pH 3Separation mode 0-2min: 90% A, 10% B, 2-4min: 10-100% B, 4-10min: 100% B, 10.1-15min: 90% A, 10% B.

There is a need for speciation analysis of selenium in fish feed and fish tissue

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There is a need for speciation analysis of selenium in fish feed and fish tissue

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