Stacked total ion chromatograms of purified α-CPS in MeOH/MilliQ (blue line) and a blank run (1:1 MeOH/MilliQ, red line) after ESI-MS in different ionization modes. Total ion counts are depicted. (A) negative ionization, (B) positive ionization. Retention times (RT) are depicted in the plots. The same chromatographic conditions as used for the chromatograms depicted in Figure 3 were applied. A reversed-phase column (100 × 2 mm Nucleosil C18, 100–3, Macherey Nagel, Düren, Germany) was used as stationary phase. After stacking of total ion chromatograms of α-CPS purified from carp bile and blank measurements of the same solvents used for α-CPS solvation, detected signals for both samples were almost identical throuoghut the chromatograms (indicating no contaminants) except for the time window between 1 and 3 min (SI Figure 4a). The extracted mass spectra corresponding to this time window indicated that the most prominent ion detected within these peaks derived from α-CPS (SI Figure 4a). Finally, the only differences between the purified α-CPS and the solvents in which it is diluted detected by LC-MS analyses, are signals derived from α-CPS, which indicates that the α-CPS enriched from carp bile is of great purity.