Pompe disease is a lysosomal disorder in which acid α-glucosidase (GAA) enzyme deficiency lead to intralysosomal accumulation of glycogen in different muscle tissues.1 Although it is a rare disease (1:40.000 live births in the world) this anomaly has a significant impact, which can lead to death in the first years of life.2 One of the ways to diagnose the disease is by enzymatic/molecular analysis and urine glucose tetrasaccharide (T4) screening by HPLC.2 However, the use of these approaches are time-consuming or expensive and the shipping of urine samples is a critical factor. In this sense, we have developed and validated a methodology for determination of T4 in dried urine on filter paper (DUFP) using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Then, this work has focused on 2 goals: (i) its applicability for screening, and (ii) to facilitate the collection and shipping of samples to reference centers as part of an early diagnostic investigation. Thus, control urine samples from health subjects were spiked with T4 standard solution, then mixed and added on 5 x 2.5 cm filter paper (Whatman 903). Then, it was dried for 24h on a plastic box at ambient temperature (22 ºC ± 0.3). Finally, the extraction was performed using a mixture of ACN:H2O 1:1 %v/v containing 0.1% v/v of NH4OH under constant agitation for 15 min. The extract was centrifuged and analyzed by LC-HRMS. To the evaluation of metabolites stability the DUFP cards were stored at different temperatures: 4, 22 and 50 °C and times: 1, 3, 7, 15, and 30 days. LC-HRMS data were processed by the MS Dial software and the aligned data matrix (retention time + m/z) vs detected peak area were submitted to statistical analysis using GraphPad Prism software for one-way analysis of variance (ANOVA) test followed by Tukey post-tests and Metaboanalyst for PCA. As result, the developed method presented a linear range between 1 and 20 μg/mL, repeatability of 3.8% (n = 9), and the limit of quantification of 1 μg/mL demonstrated the high sensitivity of the method., Extracts fortified with T4 at three concentration levels, led to an overall mean recovery of 93 ± 3.8% (n = 9). T4 and other metabolites in DUFP were stable up to 7 days at 4 and 22 °C, and up to 3 days at 50 °C. Wang et. al. has showed that metabolic stability for liquid urine is just 24h at 4 and 22 °C.3 The method was applied to analyze urines from 15 Pompe patients and 15 healthy individuals collected on papers. The PCA model was applied and the results demonstrated that there is a trend of separation between control samples and Pompe samples in the space of two principal components that explain 73% of the variability. Therefore, the collection of urine on filter paper can be a viable alternative to avoid its rapid metabolic degradation. This demonstrates that proposed methodology was robust and the use of dried urine samples rises as possible alternative for collection material and facilitate in shipping urine without refrigeration.

1Muraoka, T. et.al.; Internal Medicine, 2011, 50, 2987. 2Chien, Y.H. e Hwu, W.L.; Biologics, 2007, 1, 195. 3Wang, X. et al. Metabolites, 2019, 9, 203.