To evaluate the model performance for real samples, isotopologue detection was performed for forty-four wastewater influent and three quality control samples. A total of 391 features were evaluated as potential isotopologues from the forty-fivev reference compounds in question. Overall, 212 TP cases, one FN case, and one FP case were found for the classification model, Resulting in an average TP r of 99.8% and an FD r of 0.5%. The FN case was caused by an 0.011 Da mass error between the monoisotopic and isotopologue mass, which is larger than the minimum mass error (i.e., 0.01 Da) assumed for the true negative cases that are used for training the model. As for the FP case, the detected isotopologue mass was 155.068 m/z and the monoisotopic parent ion mass was 152.072 m/z. If the decreasing intensity for less likely isotopologues would have been taken into account, this ion would not have been included due to the absence of the isotopologues with a higher probability (e.g., 153.068 and 154,075 m/z, Figure ). From this, it can be concluded that the classification model can also be used for real data.