For testing the potential of the optimized IP-HILIC method for impurity profiling, a mixture of the FLP and N-1, N+1, PO, DA, and NC was prepared with the impurities at 2% in weight relative to the FLP. The mixture was analyzed using IP-HILIC-UV-MS using an eluent comprising 25 mM TEAA (pH 6.3) and a column temperature of 80 °C. Fig. (top left) shows the obtained total-ion (TIC) and the extracted-ion chromatograms (EICs) for each ON species. The TIC is dominated by the FLP signal, but in the EICs the low-level impurities can be detected at suitable signal-to-noise ratios, confirming the good MS compatibility of the IP-HILIC method, despite the presence of TEAA as IPR. The detected impurities can now be selectively assigned according to their masses recorded with MS. The obtained impurity profile is corresponding to what could be expected from the exploring IP-HILIC-UV experiments (Fig. ): the NC, N-1 and DA impurities elute before the FLP, and the PO and N+1 are eluting after FLP, indicating satisfactory LC-MS interfacing. The DA and N-1 impurities are well separated from the FLP, co-elute together. Nevertheless, as their masses differ significantly, they can still be adequately differentiated by MS. Notably, the separation of the DA impurity from the FLP is essential, as these only differ less than 1 Da in mass, making their distinction based on MS only very challenging. The MS isotope patterns of DA and FLP strongly overlap, which hinders reliable assignment and quantification of DA in presence of excess FLP, as would be the case for IP-RPLC, in which FLP and DA are hardly separated (Figs. S7 and S8). Fig. (top right panel) provides the average mass spectra of the [M-4H] 4-ion recorded for the peaks at the retention times of FLP and DA (indicated as A and B, respectively). A clear shift in the isotopic pattern is observed, with the pattern of peak B being shifted one mass unit higher. This shift confirms the presence of the DA impurity in the highlighted B section. Further confirmation was obtained by charge deconvolution of the full mass spectra (Figs. and), which resulted in 6793 and 6794 Da for peaks A and B, respectively (i.e. the molecular masses of the FLP and DA impurity, respectively). To further showcase the obtained separation of DA from the FLP, EICs for the most abundant isotope of FLP (m/z 1697.32) and the first isotope observed for FLP (m/z 1696.32) was plotted (Fig. , red and teal traces, respectively). The most abundant isotope observed for FLP coincides with the second most abundant isotope observed for DA, and therefore, both FLP and DA will be visible in the EIC of m/z 1697.32. The first isotope of the FLP with m/z 1696.32 is lacking in the mass spectrum of the DA impurity. Indeed, by comparing the two constructed EICs, a small peak preceding the FLP peak can be observed in the m/z-1697.32 EIC, whilst this peak is not present in the m/z-1696.32 EIC. This again indicates that the small preceding peak corresponds to the DA impurity. In order to evaluate the separation space provided by IP-HILIC and probe its potential for broad impurity profiling, we analyzed the NC sample spiked with DA-NC by both IP-HILIC-MS and IP-RPLC-MS.