## 2 Results and Discussion
### Selenium Speciation in Yeast Using the UHPLC-ESI-QqQ MS and UHPLC-ESI-Orbitrap MS Methods  
The UHPLC-QqQ-MS method was used for the quantitative analysis of selenium compounds present in yeast extracts. By using the UHPLC-ESI-QqQ-MS method, the presence of Se-methionine (Figure 3a) was confirmed in the sample, which was performed by registering fragmentation passages characteristic of the compound. Figure 3b presents a chromatogram for SELM-1 certified material. The sample of _C. utilis_ ATCC 9950 was not found to have passages typical of Se-methyl-SeCys. This led to the conclusion that the yeast sample did not contain Se-methyl-SeCys. In the case of selenomethionine in _C. utilis_ ATCC 9950 yeasts, the content of this amino acid was at about the level of 31.57 \(\upmu\)g\(\cdot\)g\({}^{-1}\). The selenomethionine level obtained in the reference material-SELM\(-\)1 (selenium-enriched yeast certified reference material) was 2480.37 \(\upmu\)g\(\cdot\)g\({}^{-1}\) (77%) The recovery of SeMet from products introduced on the market depends on the physical and chemical properties of a sample, which may stem from the drying process conducted [9]. Experiments carried out by [PERSON] et al. [22] showed that commercial yeast (_Saccharomyces cerevisiae_) may contain approximately 1563 \(\upmu\)g\(\cdot\)g\({}^{-1}\) of selenomethionine. This amino acid (selenomethionine) is the main selenium-containing organic compound found in yeasts. It is considered to be the best selenium form intended for humans and animals [23]. It may constitute from 60 to 80% of the total selenium content in a biomass [21]. However, in accordance with EU regulations [24], the production of dietary supplements containing selenium may only utilize dietary supplements in the form of Se-methionine and selenium-enriched yeasts. Also, yeasts should not contain more than 2500 \(\upmu\)g Se\(\cdot\)g\({}^{-1}\) in their biomass. The study conducted by [PERSON] et al. [9] demonstrated that _Torula_ (syn. _Candidia_) yeasts are capable of metabolizing selenium in a different manner from _Saccharomyces cerevisiae_, leading to elevated biosynthesis of selenohomolanthionine as the primary selenium metabolite. In Figure 2A,B, apart from the signals confirmed via the availability of standards, the presence of unidentified selenium forms was observed. Thus, in the subsequent part of the study, the UHPLC-ESI-Orbitrap MS method was used, primarily to identify the unknown selenium compounds as well as to confirm the presence of those forms identified with the HPLC-ICP-MS coupled technique. The absence of standards for selenium compounds meant that mass spectrometry methods with electrospray were indispensable for the identification of selenium compounds [9]. In the  
Figure 3: Chromatogram registered for extract of yeast _C. utilis_ ATCC 9950 (transitions precursor ion \(\rightarrow\) product ion) (**a**) and SELM\(-\)1 (selenium-enriched yeast certified reference material) (**b**) by UHPLC-ESI-QqQ-MS (the colors correspond to the intensity of the SeMet compounds, Supplementary Materials, Figure S8). method presented, ions are transferred from the liquid to the gaseous phase. The analyzed compounds in the liquid phase must be ionized to a minor degree. The efficiency of the electrospray method (ESI) improves the more polar the compound is. For the analysis of selenium compounds using the HPLC-ESI-Orbitrap-MS method, yeast extracts were analyzed. As a result of the analysis, the presence of Se-methionine and its metabolites, previously identified via the HPLC-ICP-MS method, was confirmed. In addition, other selenium compounds were identified using the HPLC-ESI-Orbitrap-MS method. The identified compounds found in the collected fractions are presented in Table 1.  
The search for and identification of unknown selenium compounds is presented on the basis of the example of selenometabolite precursor identification: Se-S conjugate of selenoglutathione -cysteine, selenomethionine, methylthioiselenoglutathione, and 2,3-DHP-selenocysteine-cysteine.