Untargeted Metabolomics of Dried Urine Spot Samples from Pompe Disease Patients

Dried blood samples on filter paper have been used in newborn screening programs for decades and have recently gained attention as a practical alternative to traditional liquid matrices for untargeted metabolomics. This material provides benefits, such as long-term stability and cost-effective transportation, which are particularly valuable for rare disease studies. While dried blood spots are well-established in clinical and research settings, dried urine spots (DUS) remain largely unexplored for untargeted metabolomics, despite their potential as a noninvasive matrix for biomarker discovery. In this study, it was conducted an exploratory untargeted metabolomics investigation using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) to analyze DUS samples from individuals with Pompe disease (PD). Using an integrated data analysis strategy that combined univariate testing with multivariate and machine learning methods, 29 metabolites that differed significantly (p < 0.05) between Pompe disease patients and controls were annotated. Of these, six have been previously reported in liquid urine, confirming that DUS is a reliable sampling method for biomarker detection and discovery. In conclusion, our results demonstrate that untargeted LC-HRMS analysis of DUS offers a reliable alternative for urine studies, highlighting specific metabolic changes in Pompe disease and supporting its application in research on rare disorders.