Stable Isotopic Analysis of Porcine, Bovine, and Ovine Heparins

The assessment of provenance of heparin is becoming a major concern for the pharmaceutical industry and its regulatory bodies. Batch-specific [carbon (δ13C), nitrogen (δ15N), oxygen (S18O), sulfur (δ34S), and hydrogen (δD)] stable isotopic compositions of five different animal-derived heparins were performed. Measurements readily allowed their differentiation into groups and/or subgroups based on their isotopic provenance. Principle component analysis showed that a bivariate plot of δ13C and δ18O is the best single, bivariate plot that results in the maximum discrimination ability when only two stable isotopes are used to describe the variation in the data set. Stable isotopic analyses revealed that (1) stable isotope measurements on these highly sulfated polysaccharide (molecular weight ~ 15 kDa) natural products (“biologics”) were feasible; (2) in bivariate plots, the δ13C versus δ18O plot reveals a well-defined relationship for source differentiation of hogs raised in the United States from hogs raised in Europe and China; (3) the δD versus δ18O plot revealed the most well-defined relationship for source differentiation based on the hydrologic environmental isotopes of water (D/H and 18O/16O); and (4) the δ15N versus δ18O and δ34S versus δ18O relationships are both very similar, possibly reflecting the food sources used by the different heparin producers.