A Novel Approach for Post-Acquisition Mass Calibration in Metabolomic Profiling by High-Resolution Mass Spectrometry.

High-resolution mass spectrometry (HRMS) is essential for metabolomics due to its ability to deliver high mass accuracy for confident metabolite identification. However, maintaining this accuracy during large-scale studies is challenging because of instrumental drift, which can compromise data quality. Existing correction strategies, such as frequent recalibration, QC-based adjustments, or internal standard spiking, are often disruptive, costly, or limited in robustness. We present a novel post-acquisition mass calibration approach that uses endogenous metabolites as internal references, eliminating the need for additional experimental steps. Using hydrophilic interaction liquid chromatography coupled with HRMS (HILIC-HRMS) and a predefined metabolite panel, we demonstrate that a preliminary mass matching using a wide mass tolerance window provides sufficient reference points for estimating drift and correcting mass errors. The method effectively removes m/z-dependent deviations and restores accurate mass measurements across diverse sample types. This strategy is robust, easy to implement, and broadly applicable to targeted metabolomics workflows, offering a practical solution for improving mass accuracy in large-scale studies without added cost or complexity.