Non-target screening using fast comprehensive two-dimensional liquid chromatography-high-resolution mass spectrometry I: investigating stability across a set of wastewater samples.

Comprehensive two-dimensional liquid chromatography-high-resolution mass spectrometry (LC × LCHRMS) provides high separation capacity, but its routine use in environmental non-target screening (NTS) remains limited. A key unresolved question is whether LC × LCHRMS methods can maintain retention-time precision, peak width, and signal stability over large analytical sequences. In this study, the long-term analytical stabilility of a LC × LCHRMS method applied to effluent wastewater and point source samples collected upstream of wastewater treatmeant plant, quality control and blank samples. The method provided a corrected two-dimensional peak capacity of 1.63 × 103 (σ: 112) and a peak production rate of approximately 40 min⁻¹. Stability was assessed for a chromatographic sequence with 96 injections, corresponding to >106 h of continuous run time, using 14 isotope-labelled internal standards spiked into 75 samples. For the 12 internal standards retained in both dimensions, retention-time precision was high, with mean relative standard deviation (RSD) of 0.7% in the first dimension and 0.6% in the second dimension. Second-dimension peak broadening was minor: the median increase during the sequence was 4.4 × 10⁻² s for the 14 internal standards, corresponding to less than one HRMS scan point and approximately 6% of the median second-dimension peak width. Signal intensities for retained internal standards were also stable, with an average RSD of 6.8%. The results demonstrates that LC × LCHRMS can maintain high chromatographic efficiency and satisfactory analytical stability over extended wastewater NTS sequences. Overall, the study supports LC × LCHRMS as a platform for detailed characterization and comparison of complex environmental samples.