title: Development and validation of an ion-exchange chromatography method for heparin and its impurities in heparin products.
abstract: An anion-exchange liquid chromatography method for the determination of heparin and its impurities (dermatan sulfate and oversulfated chondroitin sulfate) was developed using chemometric-assisted optimization, including multivariate experimental design and response surface methodology. The separation of heparin, dermatan sulfate, and oversulfated chondroitin sulfate (Rs above 2.0) was achieved on a Dionex RF IC IonPac AS22 column with a gradient elution of 10-70% of 2.5 M sodium chloride and 20 mM Tris phosphate buffer (pH 2.1) at a flow rate of 0.6 mL/min and UV detection at 215 nm. Method validation shows good linearity (r > 0.99), acceptable precision (%relative standard deviations <11.4%) and trueness (%recovery of 92.3-103.9%) for all analytes. The limits of detection for dermatan sulfate and oversulfated chondroitin sulfate are equivalent to 0.11% w/w (10.5 μg/mL) and 0.07% w/w (7.2 μg/mL), while the limits of quantification are 0.32% w/w (31.5 μg/mL) and 0.22% w/w (22.0 μg/mL) relative to heparin, respectively. The method is specific for heparin, dermatan sulfate, and oversulfated chondroitin sulfate without interference from mobile phase and sample matrices and could be used for accurate quantitation the drug and its impurities in a single run. Applications of the method reveal contents of heparin between 90.3 and 97.8%. Dermatan sulfate and oversulfated chondroitin sulfate were not detected in any of the real-life samples.
authors.affiliation: Department of Pharmaceutical Chemistry, Mahidol University, Bangkok, Thailand; Bureau of Drug and Narcotic, Department of Medical Sciences, Ministry of Public Health, Nontaburi, Thailand.
authors.forename: Sumate
authors.initials: S
authors.lastname: Thiangthum
authors.forename: Yvan
authors.initials: Y
authors.lastname: Vander Heyden
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Buchberger
authors.forename: Johan
authors.initials: J
authors.lastname: Viaene
authors.forename: Brompoj
authors.initials: B
authors.lastname: Prutthiwanasan
authors.forename: Leena
authors.initials: L
authors.lastname: Suntornsuk
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000737:chemistry; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D002809:Chondroitin Sulfates / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003871:Dermatan Sulfate / Q000737:chemistry*; D004340:Drug Contamination; D006493:Heparin / Q000737:chemistry*; D007413:Intestinal Mucosa / Q000378:metabolism; D057230:Limit of Detection; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine; D014466:Ultraviolet Rays
chemical_list: D000925:Anticoagulants; D003871:Dermatan Sulfate; D006493:Heparin; D002809:Chondroitin Sulfates
keywords: Anion-exchange chromatography; Dermatan sulfate; Experimental design; Heparin; Oversulfated chondroitin sulfate