Analytical method for the determination of disaccharides derived from keratan, heparan, and dermatan sulfates in human serum and plasma by high-performance liquid chromatography/turbo ionspray ionization tandem mass spectrometry.

We established a highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method to analyze the disaccharides produced from keratan sulfate (KS), heparan sulfate (HS), and dermatan sulfate (DS). It was revealed that KS, HS, and DS in human serum and plasma were digested to each disaccharide by keratanase II, heparitinase, and chondroitinase B, respectively. Analysis of disaccharides 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.x150 mm, 5 microm) with a gradient elution of acetonitrile-0.01M ammonium bicarbonate (pH 10). The mobile phase flow rate was 0.2ml/min. An API-4000 mass spectrometer equipped with a turbo ionspray was used to determine each glycosaminoglycan (GAG) in the serum of control subjects and plasma of mucopolysaccharidose (MPS) patients. The intraday precision expressed as a coefficient of variation was within 15.8% for five replicate analyses with three human control samples. The interday (overall, n=15) precision was within 14.8% for 3 days. This method is sensitive and reproducible, and it would be useful for clinical diagnosis.