An improved method for glycosaminoglycan analysis by LC–MS/MS of urine samples collected on filter paper

BackgroundMucopolysaccharidoses are complex lysosomal storage disorders caused by any of eleven different enzyme deficiencies resulting in the accumulation of substrates, mainly glycosaminoglycans (GAGs), in various tissues and biological fluids.MethodWe developed and validated a urine filter paper methodology for the analysis of GAGs using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for mucopolysaccharidoses type I, type II and type VI patients. We focused on 2 objectives: first, its applicability to high-risk screening, and secondly, to facilitate the collection and shipping of samples to reference centers as part of diagnostic investigation, as well as from treated patients needing to be monitored for assessment of the efficacy of treatment. GAGs in urine dried onto filter paper were extracted and subjected to methanolysis to obtain the repeating disaccharides of the molecules. We devised a multiple reaction monitoring method in positive electrospray ionization mode.ResultsThe use of deuterated internal standards for dermatan sulfate (DS) and heparan sulfate (HS) reduced a troubling matrix effect. The resulting CVs were < 14%. Linearity assessment showed Pearson correlation coefficients of 0.999 and 0.997, for DS and HS, respectively. The stability on filter paper was good for DS and HS for up to 6 weeks at various temperatures.ConclusionWe devised a robust and efficient LC–MS/MS methodology for GAGS quantification in urine dried on filter paper and subjected to environmental conditions likely to be encountered during collection, storage and shipping of specimens from referring physicians to medical centers.

► This LC–MS/MS technique allows quantification of GAGS for mucopolysaccharidoses.
► A methanolysis procedure is performed for dermatan and heparan sulfate analysis.
► Urine sample collected on filter paper facilitates collection, storage and shipment.
► This method allows adequate differentiation of MPS patients from healthy controls.
► It is a useful method for diagnosis, monitoring, and evaluation of efficacy of treatment.