To test the isotopologue classification model on real samples, the isotopologue detection performance was evaluated for the feature lists obtained from forty-four wastewater influent samples and three quality control samples. Additionally, a reference compound list comprised of forty-five chemicals was used, containing the monoisotopic masses (i.e., protonated molecular mass), retention times, and parent isotopologue distributions (Table ). The isotopologue distributions for these chemicals were obtained from the isotope pattern preview tool in MZmine2 (v2.53), using the protonated molecular formula, a minimum intensity of 0.01%, a merge width of 0.0001 Da, and a charge of 1,which showed to cover an isotopologue mass depth of six. The presence of a reference compound was confirmed based on the reference retention time ± 0.1 minutes and the monoisotopic parent mass with a mass tolerance of 0.01 Da.