Heparin is a medical product, which is used as an anticoagulant to treat and prevent thrombotic and cardiovascular disorders. NMR spectroscopy is the standard method described in European and US Pharmacopoeias used for the identification and quality control of heparin sodium and calcium. Can we expand the ability of NMR to estimate other heparin characteristics? The answer is yes in case of application of multivariate methods for modelling of complex heparin profiles.


Qualitative analysis

NMR spectroscopy combined with chemometrics was used to distinguish heparin and low molecular weight heparin (LMWH) produced from porcine, bovine and ovine tissues as well as their blends. The models were validated for the modeling of spectral data of more than 200 authentic samples. Moreover, the intra-species differences within the each animal group according to the manufacturer were examined. Significant improvement of chemometric models was achieved by switching to 2D NMR experiments (heteronuclear multiple-quantum correlation (HMQC).

Moreover, retrospective multivariate analysis was performed on a big dataset of 990 NMR routine heparin spectra recorded over six years (2012-2017) in our laboratory. Several steps of statistical analysis of accumulated data were used to differentiate heparin samples according to animal origin (bovine, porcine and ovine), purity grade (crude and purified), distributing company as well as to estimate the closeness of their structure to the heparin reference sample provided by USP (Fig. 1).

Fig. 1.  Scatter plot of the PCA scores values for 870 heparin spectra of different origin (BOV – bovine; POR – porcine; OVI – ovine) and 111 crude porcine heparin (CH1 and CH2). Ellipses show 95% probability for each group.


Quantitative analysis

In addition to the above-mentioned qualitative characteristics, NMR also allows considerable amount of quantitative information to be obtained using the standard USP sample preparation. Our unique holistic approach includes the determination of molecular weight based on DOSY experiment as well as water content, inorganic ions (Na+, Ca2+, Cl, and CH3COO) based on the subsequent 1H, 2D, 23Na and 35Cl NMR runs with a total measurement time less than 20 minutes (Fig. 2). Validation results in terms of precision, reproducibility, limit of detection and recovery demonstrated that the developed method is fit-for-purpose for the authentic heparin samples. Semi-quantitative assessment of anticoagulant activity is also feasible.

Fig. 2 Water quantification in heparin by 1H NMR spectroscopy


Holistic control with only one sample preparation according to Pharmacopeia using just four sequential NMR experiments combined with chemometrics provides purity, assay and provenience of heparin samples as well as enables correlation with biological activities and macroscopic values.


For details please refer to our constantly growing list of scientific publications:

Monakhova YB, Diehl BWK, Do TX, Schulze M, Witzleben S. Novel method for the determination of average molecular weight of natural polymers based on 2D DOSY NMR and chemometrics: Example of heparin. J Pharm Biomed Anal. 2018; 149:128-132.


Monakhova YB, Diehl BWK, Fareed J. Authentication of animal origin of heparin and low molecular weight heparin including ovine, porcine and bovine species using 1D NMR spectroscopy and chemometric tools. J Pharm Biomed Anal. 2018; 149:114-119


Monakhova YB, Diehl BWK. Combining 1H NMR spectroscopy and multivariate regression techniques to quantitatively determine falsification of porcine heparin with bovine species. J Pharm Biomed Anal. 2015; 115:543-51


Monakhova YB, Diehl BWK. Nuclear magnetic resonance spectroscopy as a tool for the quantitative analysis of water and ions in pharmaceuticals: example of heparin J Pharm Biomed Anal. 2018, accepted


Monakhova YB, Fareed J., Yao Y., Diehl BWK, Improving reliability of chemometric models for authentication of species origin of heparin by switching from 1D to 2D NMR experiments J Pharm Biomed Anal. 2018, accepted