Accelerated stability studies of heparin.
The objective of this study was to extend our understanding of the stability of heparin. Sodium heparin, derived from porcine intestinal mucosa, was first incubated in 0.1 N hydrochloric acid and 0.1 N sodium hydroxide at 30 and 60 degrees C and sampled at times ranging from 0 to 1000 h. The absorbance spectra of the products formed under basic conditions showed an ultraviolet maxima at 232 nm associated with chemically catalyzed beta-elimination at the uronic acid residues. The products formed under acidic conditions showed a decreased staining intensity consistent with desulfation and a decrease in molecular weight corresponding to hydrolysis of glycosidic linkages when analyzed by gradient polyacrylamide gel electrophoresis. Heparin samples were next prepared in 10 mM sodium phosphate buffer at pH 7.0 in sealed ampules that had been flushed with nitrogen and incubated at 100 degrees C. Samples taken at times ranging from 0 to 4000 h were then analyzed. Heparin was relatively stable over the first 500 h, after which it rapidly degraded. Heparin, assayed using both anti-factor Xa and anti-factor IIa amidolytic methods retained 80-90% of its activity over the first 500 h, but these activities dropped precipitously, to approximately 6% and approximately 0.5% of the initial activity at 1000 h and 2000 h, respectively. This rapid decomposition began only after the buffering capacity of the solution was overwhelmed by acidic degradants, which caused the pH to decrease. Decomposition processes observed under these conditions included the endolytic hydrolysis of glycosidic linkages and loss of sulfation, particularly N-sulfate groups, and were similar to the degradation processes observed in 0.1 N hydrochloric acid. This study provides initial observations on heparin degradation pathways. More complete, quantitative studies and studies leading to the isolation and characterization of specific degradants are still required.
Jandik
K A
KA
College of Pharmacy, University of Iowa, Iowa City 52242, USA.
Kruep
D
D
Cartier
M
M
Linhardt
R J
RJ
D000818:Animals; D000925:Anticoagulants / Q000737:chemistry*; D002240:Carbohydrate Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D012996:Solutions; D013552:Swine
D000925:Anticoagulants; D012996:Solutions; D006493:Heparin