Analytical Performance of a Standardized Kit for Mass Spectrometry-based Measurements of Human Glycosaminoglycans

Abstract

Glycosaminoglycans (GAGs) are long linear sulfated polysaccharides implicated in processes linked to disease development such as mucopolysaccharidosis, respiratory failure, cancer, and viral infections, thereby serving as potential biomarkers. A successful clinical translation of GAGs as biomarkers depends on the availability of standardized GAG measurements. However, owing to the analytical complexity associated with the quantification of GAG concentration and structural composition, a standardized method to simultaneously measure multiple GAGs is missing. In this study, we sought to characterize the analytical performance of a liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (LC-MS/MS)-based kit for the quantification of 17 GAG disaccharides. The kit showed acceptable linearity, selectivity and specificity, accuracy and precision, and analyte stability in the absolute quantification of 15 GAG disaccharides. In native human samples, here using urine as a reference matrix, the analytical performance of the kit was acceptable for the quantification of CS disaccharides. Intra- and inter-laboratory tests performed in an external laboratory demonstrated robust reproducibility of GAG measurements showing that the kit was acceptably standardized. In conclusion, these results indicated that the LC-MS/MS kit was standardized for the simultaneous measurement of GAG disaccharides allowing for comparability of measurements and enabling translational research.

Analysis of normal levels of free glycosaminoglycans in urine and plasma in adults

“…In this study, we took advantage of a standardized analytical method using ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-MS/MS) ( 15 ) to analyze the urine and plasma protein-free fraction of GAGomes (or free GAGomes, in short). We analyzed the free GAGomes in a good laboratory practice (GLP)-compliant blinded central laboratory in two prospectively enrolled independent cohorts of 308 self-reported healthy subjects with comprehensive demographic and blood chemistry data.…”

Analysis of Normal Levels of Urine and Plasma Free Glycosaminoglycans in Adults

“…In this study, we established reference intervals for the urine and plasma free GAGomes in a large adult healthy population by taking advantage of standardized high throughput UHPLC-MS/MS method ( 15 ). In addition, the extensive demographic and biochemical characterization of subjects allowed us to assess novel correlations with the free GAGome.…”

“…Sample preparation was performed in a single blinded GLP-compliant laboratory using the MIRAM™ Free Glycosaminoglycan Kit (Elypta AB, Sweden) to extract glycosaminoglycans (GAGs) from urine samples. GAG detection and quantification was obtained through ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (Waters® Acquity I-class Plus Xevo TQ-S micro) in accordance with the instruction for use in of the kit ( 15 ).…”

“…Specifically, we quantified eight CS disaccharides (0s CS, 2s CS, 6s CS, 4s CS, 2s6s CS, 2s4s CS, 4s6s CS, Tris CS) and eight HS disaccharides (0s HS, 2s HS, 6s HS, Ns HS, Ns6s HS, Ns2s HS, 2s6s HS, Tris HS) – corresponding to different sulfation patterns of CS and HS – and one HA disaccharide. A measured GAGome feature was considered detectable in a fluid (plasma or urine) if its mean concentration across all samples was > 0.1 ug mL -1 ( 15 ).…”

“…In this study, we took advantage of a standardized analytical method using ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-MS/MS) ( 15 ) to analyze the urine and plasma free GAGomes in a Good Laboratory Practice (GLP)-compliant blinded central laboratory in two prospectively enrolled independent cohorts of 308 self-reported healthy subjects with comprehensive demographic and blood chemistry data. We first determined the correlation of free GAGomes with demographic and blood chemistry variables.…”