Selenium speciation and total Se analyses

The Se speciation analysis (organic and inorganic Se) of the soluble and protease extracts from agretti is described in detail by Bañuelos et al. (2012). Selenium speciation analyses used an Agilent 1200 HPLC equipped with a Hamilton PRP-X100 strong anion exchange column (10 μm particle size 250 mm length and 4.1 mm internal diameter) coupled to the Agilent 7500 CX ICP-MS (SAX-HPLC-ICP-MS). The ICP-MS was equipped with a quadrupole detector and an Octopole Reaction System (ORS) utilizing hydrogen as a cell gas (5.5 mL/min) to minimize Se polyatomic interferences. Dried ground agretti was analyzed for total Se concentrations by Agilent 7500 CX ICP-MS (Agilent Technologies Santa Clara, USA) and other elements with the inductively coupled plasma optical emission spectrometry (ICP-OES) (Varian Vista-Pro Santa Clara, CA, USA) after wet-acid digestion with HNO₃, and H₂O₂, and HCl. Generally, for Se speciation a single analysis (30 μL injection) was conducted for each of the broths, soluble, and protease extract replicates (n = 3). Chromatographic separation of Se was achieved with an isocratic mobile phase of 5 mM ammonium citrate buffer (pH 5.2) with 2% methanol at flow rate of 1 mL/min. The two instruments (Agilent 1200 HPLC and Agilent 7500 CX ICP-MS) were integrated through Agilent Chemstation software with chromatographic data analysis. The retention times of Se-78 containing peaks were monitored using the ICP-MS and directly compared to the authentic standard (listed below), retention times, and secondary confirmation by spiking samples with standards to account for any matrix induced changes to the chromatographic analysis, as described by Bañuelos et al. (2012). The SAX-HPLC-ICP-MS standards utilized included sodium selenate (Na₂SeO₄), sodium selenite (Na₂SeO₃), SeMet, and SeCys₂ (all purchased from Sigma-Aldrich, St. Louis, MO). Additionally, methyl-selenocysteine (MeSeCys), selenocystathionine (SeCyst), and γ-glutamyl-methyl-selenocysteine (γ-gluMeSeCys) were all purchased from Pharma Se.