## 4 Discussion
As the value of the quadratic coefficient was small, if the calibration range was reduced by removing the two highest concentrations (50 and 20 mg/L points), the calibration would become linear. However, based on the experiments, it was decided to keep the quadratic calibrations in order to enable the quantification of a wider range of concentrations [55; 57]. Dilution of the sample is another practical approach worth considering when the expected concentrations are approximatively known and/or when they are presumed to be high. However, there is a disadvantage to the dilution approach. As the analysis of other yeast strains demonstrated, there were additional compounds possibly of relevance to the study of yeast metabolism. Some of these compounds were at high enough concentration to be included in the further testing of the method (i.e., palmitoleic acid and squalene), but others were present at levels that were too low to be included in the quantitation. By diluting the samples, the chance of detecting and possibly identifying and quantifying these low concentration compounds would be an issue. The natural variability of the matrix should already be considered at method development stages, especially in the case of biological samples and when there is not much information available in the literature with regard to the composition and/or concentrations. The application of a screening method first is advisable, but not always possible. However, some targeted methods can also be used for screening purposes first when a new matrix is analyzed (in this study new yeast strains, but it can also be applied to grape juice or wine); retention indices from the literature would then have to be used for tentative identification before proceeding further. In case the concentration ranges for compounds of interest varies greatly, and taking into account the dynamic range of the instrument, a combination of internal and standard addition calibration could be considered. A mixture of IS with the standards for the low concentration compounds would be added to the sample to fit into the calibration range with or without further dilution, based on the high level compounds already present. Even though not very practical for big sample sets, this approach might allow the quantification of minor lipids that are potentially of relevance for the yeast metabolism. Even though the application included here was only a proof of concept, there were some interesting findings. _S. cerevisiae_ is known to only produce monounsaturated fatty acids (oleic and palmitoleic acid) due to the absence of specific desaturases responsible for the biosynthesis of polyunsaturated acids, as confirmed in this study [58]. On the other hand, _K. marxianus_[31] and _T. delbruvecki_[59] are known to produce linoleic and linoleic acid with functions related to stress resistance, and this study confirmed the presence of linoleic acid and linoleic acid in all non-_Saccharomyces_ yeasts even in SGM without the supplementation of linoleic acid (Figure 6). Moreover, it was observed that all yeasts were capable of taking up linoleic acid from the exogenous environment. However, lipid uptake varied between strains, as the highest amount of linoleic acid was observed in _M. pulcherrina_, whereas _S. cerevisiae_ incorporated this fatty acid into its membranes the least highlighting differences in the metabolism and transport of lipids in yeasts. In a previous study, a high amount of squalene in the cell pellet was previously attributed to high ergosterol biosynthetic activity [59]. Thus, given the divergent fermentative performances of the yeasts in this study, it was assumed that these yeasts were at different physiological stages when samples were harvested for lipid analyses with _T. debruvecki_ and _S. cerevisiae_ being at a later stage of fermentation. From this perspective, further studies are necessary to evaluate the impact of lipids at defined physiological growth stages. Conceptualization, B.D. and A.B. ; methodology, A.B., L.L.M., C.W. ; validation, C.W., A.B., L.L.M.