Determination of endogenous glycosaminoglycans derived disaccharides in human plasma by HPLC: Validation and application in a clinical study

SB-424323 is a new, orally active anti-thrombotic agent presently in phase-II clinical development, with limited hemorrhagic risk and a unique mechanism of action involving the induction of glycosaminoglycans (GAGs) biosynthesis. The objective of the present study was to develop a simple and rapid high performance liquid chromatography (HPLC) method for determination of endogenous GAGs derived disaccharides in plasma samples from a phase-II clinical study of SB-424323. Sample preparation was a simple heat treatment of the diluted plasma followed by digestion of endogenous GAGs with chondroitinase ABC to yield unsaturated disaccharides, 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyluronic acid)-d-galactose (ΔDi-0S), 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose (ΔDi-4S), and 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose (ΔDi-6S). These disaccharides were recovered and purified using centrifugal filtration through a filter with 3000 molecular weight cut-off along with externally added internal standard 2-acetamido-2-deoxy-3-O-(2-O-sulfo-β-d-gluco-4-enepyranosyluronic acid)-d-galactose (ΔDi-UA2S). A gradient reverse phase HPLC separation was developed on a Waters Symmetry C18 column (4.6 mm × 150 mm, 5 μm) with a gradient mobile phase system consisting of 0.8 mM tetrabutylammonium hydrogen sulfate and 2 mM sodium chloride and acetonitrile at a flow rate of 1.0 mL/min. The eluate was monitored with an ultraviolet detector set at 230 nm. Plasma standard curves were linear (r2 ≥ 0.994) in the concentration range 1.0-20 μg/mL with a lower limit of quantification (LLOQ) of 1.0 μg/mL for each of the disaccharide. The mean measured quality control (QC) concentrations for the disaccharides deviated from the nominal concentrations in the range of −8.92 to 5.61% and −16.3 to 16.7%, for inter and intra-day, respectively. The inter and intra-day precision in the measurement of QC samples, were in the range of 3.21 to 18.2% relative standard deviation (R.S.D.) and 0.32 to 20.9% R.S.D., respectively. The inter and intra-day precision in the measurement of endogenous GAGs derived disaccharides in human control plasma, were in the range of 5.8 to 15.9% R.S.D. and 1.17 to 7.74% R.S.D., respectively. Stability of the processed samples was confirmed up to 48 h in the auto-sampler. The method is simple, reliable, and easily adaptable to analysis of large number of samples under logistics of a clinical study. The present method has been used to investigate the GAGs levels in the plasma of patients in a phase II clinical study of SB-424323.