Development of a kit for urine collection on filter paper as an alternative for Pompe disease screening and monitoring by LC-HRMS

Pompe disease (PD) is an inborn error of metabolism caused by α-glucosidase acid enzyme deficiency. It significantly impacts patients' health and life quality and may lead to death in the first few years of life. Among the well-established diagnostic methods, urinary glucose tetrasaccharide (Glc4) screening by high performance-liquid chromatography has been helpful in monitoring Glc4 levels in patients on enzyme replacement therapy, demonstrating therapy efficacy. However, the specimen shipping process from a sample collecting location to a specialized laboratory for monitoring the Glc4 is costly and presents preanalytical challenges. In this work, we developed a filter paper based-urine collection kit to facilitate specimen shipment, and liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) analysis to determine Glc4 and creatinine in dried urine on filter paper. The LC-HRMS was based on a combination of targeted and untargeted screening on the same specimen injection and was successfully developed and validated. Bland-Altman statistics revealed a good relationship between dried and liquid urine samples and Glc4 and creatinine. Glc4 and other metabolites in dried urine showed stability for at least 7 days at 4 and 22 °C, and 3 days at 50 °C. The stability of the analytes and the efficiency of the kit were tested simulating real conditions by sending it by post. After two days in transit without refrigeration, the stability of compounds was maintained, showing the reliability of the urine collection kit and analysis method to determine the PD biomarker Glc4.