*3.2. Extraction Solvent*

The first part of this investigation focused on determining the most suitable extraction solvent (ethanol or methanol) for the extraction of Blastocystis from cultures.

Two sets of triplicates of metabolite extractions from Blastocystis cells were trialled using ethanol or methanol as an extraction co-solvent with water. The efficacies of the extraction solvents were compared using C μMA/B and NA/B between the two samples calculating the ratio of ethanol/methanol. The ethanol extractions were labelled sample 1A– 1C and methanol extractions were labelled sample 2A–2C. The results of the extractions are shown in Figure 1a,b as C μME/M for a selected set of molecules and NE/M, respectively. The

triplicates shown in Figure 1a show that extraction from ethanol and water vs. extraction from methanol and water produced two consistent results. Four molecules from the 1A vs. 2A sample set were identified as outliers (Figure 1a). The 1A vs. 2A sample set was also identified as an outlier for the number of molecules extracted. The reproducibility of the triplicates was measured by the CV (Table S1, Supplementary Information) and the CV improved as the outliers were removed (Figure S1, Supplementary Information). All the reproducible results were below one, with the exception of formate and acetate in the sample set 1A vs. 2A and sample set 1A vs. 2A for the number of molecules extracted. The CV for the number of molecules extracted was 0.7, showing poor reproducibility. These results suggest that methanol worked better than ethanol. All six of the selected metabolites produced values below one in two of the three sample sets, and two of the three sample sets produced values below one for the number of metabolites extracted. Taken together, the results suggest that methanol was the better extraction solvent.

## *3.3. Lysis Method*

The lysis method for metabolite extraction was subsequently investigated as part of this experiment; here, samples which had been extracted with methanol (deemed the most suitable extraction solvent) were subjected to different lysis techniques.

Two sets of triplicates of metabolite extractions from Blastocystis cells were examined with either bead bashing or sonication as the differing lysis methods. The efficacies of the lysis methods were compared using C μMA/B and NA/B between the two samples calculating the ratio of sonication/bead bashing. The sonicated extractions were labelled sample 3A–3C and bead-bashed extractions were labelled sample 4A–4C. The results of the extractions are shown in Figure 2a,b as 'C μM S/B' for a selected set of molecules and 'N S/B', respectively.

The triplicates show that for lysis by bead bashing vs. lysis by sonication, bead bashing produced more consistent results for the number of metabolites extracted, with all three triplicates being below one (Figure 2b). For the metabolite concentrations extracted, two metabolites were noted as outliers: alanine and formate for the pair sample set 3C vs. 4C (Figure 2a) and were removed and the CVs dropped from 0.7 to 0.02 and 0.6 to 0.03, respectively (Figure S2 and Table S2, Supplementary Information). All other peaks yielded three reproducible triplicates (Figure 2a). Of the reproducible triplicates, seven gave C μMA/B ratios which were below 1.0 and five that were above; these produced no significant results on aggregate. The number of molecules extracted produced reproducible triplicates (Figure 2b) with no outliers and a CV of 0.27 (Figure 2b), all of which were below a ratio of 1.0. These results suggest that bead bashing was a more reliable method for lysis of Blastocystis when compared to sonication.

**Figure 1.** (**a**) Difference in metabolite concentrations between ethanol (1) and methanol (2) C μM E/M extractions for the triplicates A–C. (**b**) Difference in the number of different metabolites extracted between ethanol (1) and methanol (2) extractions NE/M for the triplicates. Numbers below 1.0 indicate an increased extraction in methanol, \* = outliers, numbers above the bars indicate measured ratios.

**Figure 2.** (**a**) Difference in concentrations between sonication (3) and bead bashing (4) C μMS/B lysis techniques for the triplicates A–C. (**b**) Difference in the number of different metabolites extracted between sonication (3) and bead bashing (4) lysis techniques NS/B for triplicates. Numbers below 1 indicate an increased extraction for bead bashing. \* = outliers, numbers above the bars indicate measured ratios.
