Evaluation of detection levels (sensitivity) One of the main parameters that allows for a sensitivity comparison between two methods is signal-to-noise ratio (S/N). Nevertheless, comparing S/N between different levels of MS data, i.e., MS 1 for SWATH-only and MS 2 for MRM-HR+SWATH would not provide a meaningful comparison. One of the main reasons for this is the expected increase in specificity when dealing with MS 2 data, resulting, frequently in a flat (non-existent) baseline which, consequently, makes noise estimation impractical. In addition, S/N estimation algorithms included into SCIEX OS (namely Standard Deviation and Peak to Peak) require a noise region to be defined. Nevertheless, due to the use of very narrow RT windows for MRM experiments, selecting this noise region becomes unrealistic and, as a consequence, estimated S/N values turn out to be not representative. For these reasons, we have opted to report, for each analyte, the lowest concentration level that could be detected in the spiked matrix (and not detected in the non-spiked matrix) as a sensitivity comparison basis. Two criteria were considered to define if a peak was detected or not: a minimum intensity threshold of 100 counts and the visual inspection of the baseline (when possible) to estimate S/N. For a few borderline cases, we opted for a more conservative approach to avoid overestimation of the method's performance. This approach was taken for all compounds that were not detected in non-spiked matrix (for compounds detected in non-spiked matrix, see next section). For SWATH-only data, MS 1 level is reported since compounds could be detected at lower levels when compared to MS 2 level (which may be explained for the nonspecific CE chosen for each compound). For MRM-HR +SWATH data, on the other hand, MS 2 level data showed larger values for peak areas when compared to MS 1 data, as also expected due to the optimized CE and the use of the Zeno. In summary, mean detected concentrations in MRM-HR+SWATH were 2.1 and 2.3 times lower for SRM 1950 in ESI-and ESI+ mode, respectively. For SRM 1958, a four times higher mean detected concentration was found for ESI-, while ESI+ presented a two-fold increase. Even for compounds in which the lowest detected level was the same between MRM-HR + SWATH and SWATHonly, there were prevalent larger peak areas and calibration curve slopes for the first, as depicted in Figure for four exemplary compounds. The complete description of the lowest detected levels for all compounds using both methods is reported in Table .