We also tested the quality of the new spectral library with spectra originating from the MCEBIO library, acquired in positive ionization mode. In this evaluation, all 48,000 MS 2 spectra were matched against open spectral databases, and the chemical space only of the acquired spectra was mapped by feature-based molecular networking (FBMN) using the GNPS web platform . Here, we limited the matching to open libraries, enabling the same spectral matching parameters and workflow. The results are visualized in Fig. . Here, each node represents one MS 2 spectrum from the MCEBIO positive data and the nodes are connected with an edge if the MS 2 spectra share a spectral similarity (cosine similarity ≥ 0.7 and at least four matched signals). Out of 48,000 spectra with multiple spectra acquired at different fragmentation energies per compound, only 20% showed a library match with a minimum cosine similarity of 0.6 and four matching fragment ions. This demonstrates the uniqueness of the acquired spectra and included compounds. To further evaluate the 20% matches, the same structure cleanup pipeline was used for the matched compounds (originating from the public spectral database) and we compared those structures with the actual structure. As a result, 68% showed a direct match based on the same InChIKey. As small structural changes result in different InChIKey strings, we added a comparison based on structural similarity assessed using the maximum common edge subgraph (MCES) algorithm () and Tanimoto similarity. Briefly, MCES counts the number of structural modifications resulting in MCES 0 for identical structures and MCES 2 if the position of a functional group changes, as highlighted in Fig. for acetophenazine and its library match. The structure stored in the open domain has the acetyl group at another position on the ring, but the spectra remain similar, as expected. Most likely, however, the structure in the open spectral library was wrong, as the declared name was acetophenazine. In the case of Tanimoto similarity, 88% of all matches showed high similarity values of ≥ 0.85. For MCES, 94% of all matches were identical or highly similar with MCES < 4. With this information, almost all annotations based on spectral library matching correspond to the same or very similar structure, showing the correct export and public library matching of the newly created spectral data. On the other hand, 80% of our spectral library entries remained new compared to the open libraries spectra. It is important to note that the same compound may be in the public database, but differences in instruments, fragmentation energies, or settings can affect the library match. We further evaluated the quality of the spectra based on the clustering of molecular families during FBMN (edges), where similar compound classes should fall into the same spectral subnetworks. This is the case for many clusters in the network and is highlighted in Fig. for the acetophenazine subnetwork, grouping compounds from the phenothiazine class. Here, acetophenazine connects two analog structures, namely piperacetazine and perphenazine, as they share the same core structure with only one modification. Both analogs remain unconnected, highlighting how modified cosine similarity primarily connects single modifications. Those networks demonstrate the spectral quality and correct exporting of the corresponding structures and names by the automatic library generation workflow. Finally, we emphasize that the libraries were acquired without chromatographic separation by flow injection-MS n , essential for obtaining deep and wide MS n trees in a high throughput workflow. The compound libraries, sourced from various companies, were not individually tested for quality. Consequently, structural isomers and other contaminants may be present during acquisition, annotation, and in the final MS n Lib. The top ten annotations for each scan were exported for structure cleanup and scoring of structural similarity with actual compounds analyzed. For this, the maximum common edge subgraph (MCES, ) and the Tanimoto similarity were calculated. MCES reflects the number of structural modifications, attaining a value of 0 for identical structures. MCES < 4 was chosen for similar structures, and MCES ≥ 4 was selected for lower or no similarity. Of all annotations, 94% were identical or similar. b, A subnetwork for acetophenazine and its neighbors for a distance of up to two edges. The node shapes reflect actual structures, and colors reflect MCES scores. Spectral similarity successfully clustered this molecular family of analog structures. The spectral library match of acetophenazine to an analog demonstrates how MCES = 2 often describes structural isomers differing in the position of one functional group. Piperacetazine and perphenazine yielded identical matches. Clidinium belongs to a different chemical class.