Development and validation of an LC–MS/MS Method for the quantitation of heparan sulfate in human urine

Abstract

Heparan sulfate is a linear polysaccharide and serves as an important biomarker to monitor patient response to therapies for MPS III disorder. It is challenging to analyze heparan sulfate intact owing to its complexity and heterogeneity. Therefore, a sensitive, robust and validated LC-MS/MS method is needed to support the clinical studies for the quantitation of heparan sulfate in biofluids under regulated settings. Presented in this work are the results of the development and validation of an LC-MS/MS method for the quantitation of heparan sulfate in human urine using selected high-abundant disaccharides as surrogates. During sample processing, a combination of analytical technologies have been employed, including rapid digestion, filtration, solid-phase extraction and chemical derivatization. The validated method is highly sensitive and is able to analyze heparan sulfate in urine samples from healthy donors. Disaccharide constitution analysis in urine samples from 25 healthy donors was performed using the assay and demonstrated the proof of concept of using selected disaccharides as a surrogate for validation and quantitation.

Characterization of Fluid Biomarkers Reveals Lysosome Dysfunction and Neurodegeneration in Neuronopathic MPS II Patients

“…Since HS is considered to be the primary driver of downstream pathophysiology in MPS (reviewed in Reference [ 34 ]), we evaluated HS and DS levels separately using an analytically qualified LC-MS/MS assay described in Wang et al IJMS co-submission ( Figure S1 ), as opposed to previously published total CSF GAG methods [ 29 ]. HS and DS levels were quantified in CSF and serum by measuring the most abundant HS derived disaccharides, D0A0 and D0S0, and DS derived disaccharide, D0a4 [ 35 , 36 ].…”

Quantitative analysis of heparan sulfate using isotopically labeled calibrants

“…Isotope-labeled Escherichia coli K5 polysaccharide by inserting 13 C/ 15 N into disaccharide units has been developed for investigations of protein/heparan sulfate interactions 9 and quantification analysis of heparan sulfate disaccharides 10 . Isotope dilution mass spectrometry technique has been applied for the quantification analysis of heparan sulfate disaccharides from biological samples 11 , 12 . Nevertheless, it is often quite challenging to perform quantitative analysis using the LC–MS or LC–MS/MS method due to the lack of appropriate internal reference standards.…”

High-Throughput Liquid Chromatography–Tandem Mass Spectrometry Quantification of Glycosaminoglycans as Biomarkers of Mucopolysaccharidosis II

“…This advancement has been directly enabled by the technological advances in liquid chromatography mass spectrometry (LC-MS/MS). Over the past decade, LC-MS/MS has become the preferred platform to analyze chemically or enzymatically degraded oligosaccharides from broad classes of GAGs [ 3 , 8 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. Acid hydrolysis of GAGs coupled with LC-MS/MS quantification is a highly sensitive method that has been successfully applied to biological samples from mouse models of MPS and MPS patient samples [ 19 ].…”