2.3. Glycosaminoglycan assay

We developed a highly accurate, sensitive, and cost-effective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure the four disaccharides produced from GAGs; CS, DS, HS and KS [[15], [16], [17]]. Briefly, DS, HS, and KS in human serum and plasma were digested to disaccharides by chondroitinase B, heparitinase, and keratanase II, respectively. Analysis of disaccharides [DiHS0S (HS0S), DiHSNS (HSNS), DiHS6S (DS), mono-sulfated KS, di-sulfated KS] was performed by LC-MS/MS with multiple reactions monitoring in the negative ion mode. Separation of LC was performed on a Hypercarb (2.0 mm i.d. × 150 mm, 5 μm) with a gradient elution of acetonitrile–0.01 M ammonium bicarbonate (pH 10). The mobile phase flow rate was 0.2 ml/min. The 6460 triple quad mass spectrometer with a 1290 Infinity LC system (Agilent Technologies, Palo Alto, CA) was used to determine each GAG in the serum or plasma of MPS patients and controls.