## 2 Results and Discussion
### Selenium Speciation in Yeast Using the UHPLC-ESI-QqQ MS and UHPLC-ESI-Orbitrap MS Methods
The HPLC-ESI-Orbitrap-MS chromatograms obtained were searched for the presence of a suitable isotope envelope characteristic of chemical compounds containing selenium in their composition. The element has 26 isotope forms, of which only six are durable [25]. The most significant ones have a mass from 74 to 82. The HPLC-ICP-MS method utilized allowed for the identification of five selenium compounds using commercial standards in extracts obtained from _C. utilis_ ATCC 9950 yeast biomass (Se-methyl-SeCys, SeMet, SeO\({}_{3}^{2-}\), \(\gamma\)-glutamyl-Se-methyl-SeCys, and SeO\({}_{4}^{2-}\)). The results are in line with the previously published results of [PERSON] et al. [20] and [PERSON] et al. [10]. In chromatograms obtained from yeast extracts, the presence of unidentified, overlapping signals could be discerned. In such a situation, the need to identify them arises, which requires additional analytical methods. Yeasts are microorganisms, which are capable of binding selenium ions from the extracellular environment and which accumulate them in their structures. They are undoubtedly among those microorganisms the biochemical activity of which is most frequently used for numerous biotechnological processes [26]. Yeast enriched in selenium contains over 60 unique compounds with this element [27]. The identification of individual metabolic pathways in which inorganic selenium transformation to organic forms occurs would contribute to broadening the understanding of selenometabolome in yeasts. The signal present on the chromatogram obtained from _C. utilis_ yeast extract using the HPLC-ICP-MS method (Figure 2B, fraction F1) and subsequently identified using the HPLC-ESI-Orbitrap-MS method consisted of dehydro-selenomethine-oxide (_m/z_ 195.9876). In fraction 2, the presence of SeMet-NH\({}_{3}\) (_m/z_ 180.9767) and SeMet (_m/z_ 198.0032) was determined. In the case of the F3 fraction, the presence of two different compounds was determined: an Se-S conjugate of selenoglutathione-cysteine (_m/z_ 475.0405), and methylthioiselenoglutathione (_m/z_ 402.0236). In the F4 and F5 fractions, the presence of only one compound was found: 2,3-dihydroxy-propionyl(DHP)-selenocysteine-cysteine with _m/z_ 376.9922. Probably due to their low concentrations, other selenium components present could not be identified. Identification analyses of _C. utilis_ yeast extract confirmed the presence of Se-methionine, which was identified using the HPLC-ICP-MS method. As an example, in an image of the fragmentation spectrum for an ion with _m/z_ 198.0032 (Figure 4) corresponding to selenomethine (Supplementary Materials, Figure S8), the presence of selected selenium components was found: C\({}_{2}\)H\({}_{5}\)Se\({}^{+}\) with a similar theoretical value _m/z_ 108.9698 (ethylselenurane), C\({}_{4}\)H\({}_{7}\)Se\({}^{+}\) with _m/z_ 134.9934 (selanylbut\(-\)1-ene), and C\({}_{5}\)H\({}_{9}\)O\({}_{2}\)Se\({}^{+}\) with _m/z_ 181.0291 ((4-methoxy-4-oxobutan-2-yl)selanyl) (Figure 4 \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Fraction** & **Exp. _m/z_** & **Theo. _m/z_** & **Appn** & **Formula** & **Name** & **RT/min** \\ \hline F1 & 195.9876 & 195.9870 & 3.06 & C\({}_{5}\)H\({}_{10}\)NO\({}_{5}\)Se\({}^{+}\) & Dehydro-Selenomethine-oxide & - \\ F2 & 198.0032 & 198.0028 & 2.02 & C\({}_{5}\)H\({}_{12}\)NO\({}_{5}\)Se\({}^{+}\) & Selenomethine & 6.59 \\ F2 & 180.9767 & 180.9875 & \(-\)59.67 & C\({}_{6}\)H\({}_{2}\)NO\({}_{5}\)Se\({}^{+}\) & Selenomethine–\(\rm NH_{3}\) & 6.59 \\ F3 & 475.0405 & 475.0396 & 1.89 & C\({}_{12}\)H\({}_{23}\)NO\({}_{5}\)Se\({}^{+}\) & Se-S conjugate of cysteine-eleagglut & 10.14 \\ F3 & 402.0236 & 402.0232 & 0.99 & C\({}_{11}\)H\({}_{23}\)NO\({}_{5}\)OS\({}^{+}\) & Methylthioiselenoglut & 13.88 \\ F4, F5 & 376.9922 & 376.9916 & 1.59 & C\({}_{9}\)H\({}_{12}\)N\({}_{2}\)O\({}_{5}\)Se\({}^{+}\) & 2,3-DHP-selenocysteine-cysteine & 12.08 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Selenium compounds identified in yeast extract using the UHPLC-(ESI)-Orbitrap-MS technique.