biostudies-literatureDeng ZNational Natural Science Foundation of ChinaNational Key Research and Development ProgramChongqing Science and Technology Bureau8105https://www.ebi.ac.uk/biostudies/studies/S-EPMC11890610biostudies-literatureUnknown15(1)Understanding DOM composition is essential for investigating the biogeochemical carbon cycle and related elements. However, methods for assigning molecular formulas (MF) in high-resolution mass spectrometry remain poorly defined, which often resulting in the misidentification of DOM components. In this study, we established a metrics evaluation framework for assessing the assignment results of each method based on similarity, accuracy and correctness. We selected six methods and then evaluated them using different settings of elemental limits, filter rules and selection rules. Our findings reveal that Formularity and TRFU are the most suitable methods for MF assignment in DOM. These two methods show high similarity ratios (93-99%) and low Bray-Curtis distances (0.13-0.14), indicating more Scientific reportsEvaluation of the current methods for assigning molecular formulas to dissolved organic matter using high resolution mass spectrometry.PMC11890610U23402222022YFC3203504CSTB2022NSCQ-MSX0599Li ZOuyang WLi CTang QHe XWang DDeng ZChen YYing HfalseEvaluation of the current methods for assigning molecular formulas to dissolved organic matter using high resolution mass spectrometry.Understanding DOM composition is essential for investigating the biogeochemical carbon cycle and related elements. However, methods for assigning molecular formulas (MF) in high-resolution mass spectrometry remain poorly defined, which often resulting in the misidentification of DOM components. In this study, we established a metrics evaluation framework for assessing the assignment results of each method based on similarity, accuracy and correctness. We selected six methods and then evaluated them using different settings of elemental limits, filter rules and selection rules. Our findings reveal that Formularity and TRFU are the most suitable methods for MF assignment in DOM. These two methods show high similarity ratios (93-99%) and low Bray-Curtis distances (0.13-0.14), indicating more 2025-01-01T00:00:00Z2025 Mar2025-04-04T08:23:18.47Z2025-04-04T08:23:18.47ZS-EPMC118906104005751910.1038/s41598-025-87539-x