Assay of possible economically motivated additives or native impurities levels in heparin by 1H NMR, SAX-HPLC, and anticoagulation time approaches.
New assays have been developed to detect the presence of economically motivated additives (EMAs) and poor manufacturing processes in heparin. Here, selected oversulfated glycosaminoglycans that are possible EMAs to heparin were synthesized and the utility of current (1)H NMR, SAX-HPLC or anticoagulation time protocols were evaluated for the detection of native impurities (chondroitin sulfate A or B (CSA or CSB), or heparan sulfate (HS)), or synthetic contaminants (oversulfated-(OS)-CSA, OS-CSB, OS-HS or OS-Heparin) spiked into heparin sodium active pharmaceutical ingredients (APIs). The range of w/w percent LOD values from the SAX-HPLC analysis for heparin spiked with CSA, CSB, HS, OS-CSA, OS-CSB, OS-HS, OS-Heparin and two partially oversulfated CSA analogs was 0.05-0.12%. The 500 MHz 1D-(1)H NMR spectra of heparin spiked with 1.0-10% CSA, CSB, OS-CSA, or OS-CSB showed unique signal pattern changes while the samples spiked with HS, OS-HS, OS-Heparin or partially sulfated CSA were more difficult to identify using NMR data. The ratio of anticoagulation time values obtained with factor Xa and factor IIa were found to remain within USP specifications in the presence of 10% amounts of these impurities or contaminants. In a separate test, using OS-CSA spiked API heparin samples, the factor Xa or factor IIa to USP standard ratio were found to fall below the USP 0.9 specification value in samples spiked at ca. weight percent of 15% or greater of OSCS. We conclude that the SAX-HPLC assay is the most sensitive and robust assay to identify and quantitate possible GAG-based EMAs in heparin.
Keire
David A
DA
Food and Drug Administration, Division of Pharmaceutical Analysis, 1114 Market St., St Louis, MO 63101, USA. david.keire@fda.hhs.gov
Mans
Daniel J
DJ
Ye
Hongping
H
Kolinski
Richard E
RE
Buhse
Lucinda F
LF
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006493:Heparin / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D011522:Protons
D004364:Pharmaceutical Preparations; D011522:Protons; D006493:Heparin