Non-target screening using fast comprehensive two-dimensional liquid chromatography-high-resolution mass spectrometry II: the mosaic workflow - development of a scalable and open-source non-target screening data processing workflow for LC×LC-HRMS and its application to wastewater effluent.

Comprehensive two-dimensional liquid chromatography-high-resolution mass spectrometry (LC × LC-HRMS) offers improved group-type separation and mass spectral quality than one-dimensional LC-HRMS, but its widespread adaption remains limited, partly, due to labor-intensive and non-scalable data-processing workflows. Here, we present Mosaic, an open-source NTS workflow for LC × LC-HRMS that groups ions originating from a single compound, including adducts, isotopes, and in-source fragments, into single components within and across samples. Mosaic was evaluated using three componentization strategies: mass filtering (MF), non-negative matrix factorization (NMF), and their combination (MF+NMF). The workflow was validated using 78 LC × LC-HRMS chromatograms originating from wastewater effluents samples from six wastewater treatment plants, quality control samples, field blanks and three instrument blanks. Fourteen internal standards (IS), present in 75 chromatograms, were used to quantify detection performance, across-sample grouping efficiency, and the accurracy of automated peak volume extraction. Within-sample true positive rates of detection (TPRs) were 52% (NMF), 91% (MF), and 99% (MF+NMF), respectively. All detected IS were componentized correctly for the MF+NMF. Only the combined MF+NMF componentization approach could extract peak volumes comparable to those obtained by manual integration (p = 0.99). This performance relied on the generation of higher purity mass spectral loadings, achieved through correlation-based removal of interfering ions in both chromatographic dimensions as done in MF, and NMF's ability to resolve peaks and corresponding ions with varying relative abundances across samples. The Mosaic workflow provides an open-source, transparent, and scalable NTS solution for complex samples analysed by LC × LC-HRMS, enabling reliable detection and peak volume extraction for trace-level contaminants at ng/mL concentrations.