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

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: 543-551. 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): 13-19. More than 100 citations!!! Alban S, et al. Anal Bioanal Chem. 2011; 399(2): 605-620.

1H NMR porcine bovine ovine

Animal origin: PCA of 1H NMR Heparin Enoxaparin

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

HMQC: Spectral region selection

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

Individual heparin + mixtures: PCA Enoxaparin Heparin

Heparin: DA LDA 100% (calibration) FDA 100% (LOOCV) PLS-DA 92-100% (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)

Retrospective PCA of 859 spectra: animal origin and purity

Crude heparin

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

Routine analysis of heparin

DOSY NMR Heparin Enoxaparin PPG 16000 D 2000 D 4000 D

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

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

Validation Parameter Heparin LMWH Reference range [Da] 12130 - 15090 3858 - 4778 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

MW vs. Activity

NMR: a tool for structure - activity relationship

NMR: a tool for structure - activity relationship

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-, …)

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.

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