1. Breakthrough in heparin analysis: holistic control by NMR spectrometry and chemometrics
Yulia Monakhova, Bernd W.K. Diehl
XI Winter Symposium on Chemometrics, February 2018

2. Inspiration Holistic NMR control of heparin 2017 2015 2013 2011 2008
Monakhova YB, Diehl BWK et al. J Pharm Biomed Anal. 2018
2011
New crisis in Germany
NMR method to differentiate bovine and porcine heparin
2008
USP Monograph revisions
Monakhova YB, Diehl BWK J Pharm Biomed Anal. 2015; 115:
Renewed interest to alternative heparin sources
NMR control of DS and OSCS
Heparin crisis in China
B.W.K Diehl, et al. J Pharm Biomed Anal. 2008; 48(1):
More than 100 citations!!!
Alban S, et al. Anal Bioanal Chem. 2011; 399(2):

3. 1H NMR
porcine
bovine
ovine

4. Animal origin: PCA of 1H NMR
Heparin
Enoxaparin

5. 2D NMR heparin profiling: HMQC (Heteronuclear Multiple Bond Correlation)
Ovine: olive
Porcine: blue
Bovine: green
N-acetyl group
Sugar
ring signals
Anomeric signals

6. HMQC: Spectral region selection

7. Alignment Original data 4096 x 2048 Bucketing 1H direction 80 x 2048
Bucketing 13C direction 80 x 150
Unfolding 1 x 12000
1H direction: 0.01 ppm bucket width
13C direction
0.1 ppm bucket width

8. Individual heparin + mixtures: PCA
Enoxaparin
Heparin

9. Heparin: DA LDA 100% (calibration) FDA 100% (LOOCV)
PLS-DA % (independent set validation)
Individual + mixtures (Calibration/validation set):
7/2 Ovine
22/8 Bovine
13/5 Porcine
4/2 Mixture1 (ovine-porcine)
4/2 Mixture2 (bovine-porcine)

10. Retrospective PCA of 859 spectra: animal origin and purity

11. Crude heparin

12. Retrospective PCA of 859 spectra: producer and the closeness to the reference USP standard

13. Routine analysis of heparin

14. DOSY NMR
Heparin
Enoxaparin
PPG
16000 D
2000 D
4000 D

15. Preprocessing Original data Diffusion fit function + Lb=1 Hz
Exclusion of water and impurities signals
TSP as calibrant (0; -9.3 m2/s)
8192 x 64
Bucketing (1H axis)
Exclusion of zero values (D axis)
440 x 30
Unfolding
1 x 13200
Normalization to the total intensity
1H direction: 0.01 ppm bucket width

16. MW of LMWH: PLS based on DOSY and GPC
7 Ovine
19 Porcine
GPC reference data
2 minutes measurement

17. Validation Parameter Heparin LMWH Reference range [Da] 12130 - 15090
Number of PLS factors 5 6
RMSECV [Da]
443
159
RMSEP [Da]
498
179
Repeatability
(CV [%], n=5)
Porcine
2.1
1.8
Ovine
2.0
1.3
Bovine
1.2 -
Precision [%]
0.26
1.9
1.4
2.5
1.6

18. MW vs. Activity

19. NMR: a tool for structure - activity relationship

20. NMR: a tool for structure - activity relationship

21. Holistic control of heparin by NMR
Evaluation based on 1D and 2D NMR and chemometrics Qualitative Analysis - Animal source of the raw material - Producer - Closeness to the reference USP material Quantitative Analysis - molecular weight based on 2D DOSY experiments - semiquantification of anticoagulant activity based Also possible - organic components - impurities (DS, OSCS) - water content - absolute quantification of heparin - inorganic components - counter ions (Na+, Ca2+, Mg2+, Li+, …) - anions (Cl-, CH3COO-, F-, PO43-, …)

22. Holistic control with only one simple sample preparation according the pharmacopeia using NMR provides purity, assay and provenience of the heparin samples and enables a successful correlation by multivariate analysis with biological activities.

23. Thank you for your attention!!! monakhova@spectralservice.de