Stable Isotopic Analysis of Porcine, Bovine, and Ovine Heparins

Abstract

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 (δ18O), 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 (i) stable-isotope measurements on these highly-sulfated polysaccharide (MW ~15 kDa) natural products (“biologics”) were feasible; (ii) in bivariate plots, the δ13C versus δ18O plot reveals a well-defined relationship for source differentiation of hogs raised in the US from hogs raised in Europe and China; (iii) 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 (iv) the δ15N versus δ18O and δ34S versus δ18O relationships are both very similar, possibly reflecting the food sources used by the different heparin producers.

Pharmacology of Heparin and Related Drugs

“…MS can also play a different part in the stable isotope analysis of heparin from different species and areas of origin (Jasper et al, 2015).…”

Recent innovations in the structural analysis of heparin

“…These findings resonate with a recent theoretical report stating that positively charged amino acids (Arg and Lys) are poor predictors of heparin binding sites on protein surfaces, while the uncharged side chains of Gln and Asn were posited as important for their ability to hydrogen bond [65]. The geographical origin of heparin, which is of particular concern in light of problems with provenance, has been examined through analysis of stable isotopes, revealing that the content of 13 C and 18 O isotopes provided good overall ability to differentiate its origin [66]. The possibility of de-N-sulfation in heparin samples, arising from heating and/or exposure to acidic conditions or solvents has also been assessed, employing labelling of the resultant free amine groups and disaccharide analysis employing HILIC MS [21].…”

Combining 1H NMR spectroscopy and multivariate regression techniques to quantitatively determine falsification of porcine heparin with bovine species

“…However, this method is characterized by time-consuming sample preparation. Stable isotope analysis (δ 13 C, δ 15 N, δ 18 O, δ 34 S, and δD) is another feasible method for assessing of geographical and animal origin of heparin, which, unfortunately, was not applied to heparin blends of different animal origin [14].…”