title: Determination of endogenous glycosaminoglycans derived disaccharides in human plasma by HPLC: validation and application in a clinical study.
abstract: 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-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (DeltaDi-0S), 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (DeltaDi-4S), and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (DeltaDi-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-beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (DeltaDi-UA2S). A gradient reverse phase HPLC separation was developed on a Waters Symmetry C(18) column (4.6 mm x 150 mm, 5 microm) with a gradient mobile phase system consisting of 0.8 mM tetrabutylammonium hydrogen sulfate and 2mM 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 (r(2)> or =0.994) in the concentration range 1.0-20 microg/mL with a lower limit of quantification (LLOQ) of 1.0 microg/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.
authors.affiliation: Pharmacokinetics and Biopharmaceutics Laboratory, Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, 20 Penn Street, Baltimore, MD 21201, USA.
authors.forename: Vijay V
authors.initials: VV
authors.lastname: Upreti
authors.forename: Manoj
authors.initials: M
authors.lastname: Khurana
authors.forename: Donna S
authors.initials: DS
authors.lastname: Cox
authors.forename: Natalie D
authors.initials: ND
authors.lastname: Eddington
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017322:Clinical Trials, Phase II as Topic; D004187:Disaccharides / Q000097:blood*; D005260:Female; D006025:Glycosaminoglycans / Q000097:blood*; D006027:Glycosides / Q000494:pharmacology; D006801:Humans; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004187:Disaccharides; D006025:Glycosaminoglycans; D006027:Glycosides