# Combining NMR Spectroscopy and Chemometrics to Monitor Structural Features of Crude Hep-arin
## 2 Results
### Crude of Different Origin  
As porcine, bovine, and ovine API heparins differ in many aspects of their structure and activity [21; 22; 23; 24; 25], it is important to verify if their structural differences can be observed in the corresponding crude. Six heparin crude samples from bovine intestinal mucosa (BMHC), two from bovine lung (BLHC) and three from ovine intestinal mucosa (OMHC) were characterized by proton and HSQC analysis. The PCA of the GAGs signals region of proton spectra of these samples against the library of porcine crude heparin was not able to differentiate samples according their origin (Figure S7). In spite of the structural differences between the heparins in these crudes, the galactosaminoglycan content mostly contributes to the samples' separation in component 1 (Figure 3). By contrast, PCA performed exclusively on the anomeric region was able to group samples on the basis of their origin, separating them in function of their sulfation pattern in component 1 and of the 6-\(O\)-sulfation in component 3 (Figure 7 and Figure S8). Indeed, BMHC and PMHC are not fully separated in components 1 and 2 because of their similar degree of sulfation, while in component 3 BMHC are fully distinguished from other crude heparin (Figure 7).  
The monosaccharide composition of each crude family was determined by HSQC analysis (Tables 2 and 3). The bovine lung crude shows the highest degree of sulfation and lowest content of acetylation and nonsulfated uronic acid. The lower 6-O-sulfation of the bovine mucosa crude agree with those observed in the corresponding API [25], while the degree of sulfation and amount of \(N\)-acetylation measured for the ovine crude samples is intermediate to that of bovine lung and porcine mucosa heparin, as also observed by the integration of C13 spectra of the purified heparin samples [24]. The greater structural detail obtained applying the HSQC method allows the detection of minor residues in crude heparin, such as G2S, that is more abundant in BMHC compared to ovine or porcine heparins [25].  
\begin{table}
\begin{tabular}{c c c c c c c c c c c c c} \hline \hline
**BMHC** & **Hep** & **DeS** & **ChS** & **ANH26X** & **ANAc6X** & **ANS3S6X** & **A6S** & **G2 OH** & **G2S** & **I2 OH** & **I2S** & **SDEG** \\ \hline
**average** & 94.7 & 4.2 & 0.4 & 1.5 & 10.6 & 2.0 & 51.7 & 14.1 & 1.5 & 6.8 & 77.1 & 2.20 \\
**median** & 98.1 & 1.1 & 0.0 & 1.9 & 9.6 & 1.8 & 52.4 & 13.8 & 2.0 & 6.3 & 76.9 & 2.19 \\
**st.dev.** & 5.9 & 5.8 & 0.9 & 1.2 & 3.3 & 0.7 & 4.0 & 2.0 & 1.2 & 1.2 & 2.8 & 0.07 \\
**min** & 85.3 & 0.0 & 0.0 & 0.0 & 8.2 & 1.3 & 47.1 & 11.9 & 0.0 & 6.0 & 73.4 & 2.12 \\
**max** & 98.8 & 12.5 & 2.1 & 2.6 & 17.2 & 3.5 & 57.2 & 17.4 & 2.5 & 9.2 & 81.0 & 2.31 \\ \hline
**BLHC** & **Hep** & **DeS** & **ChS** & **ANH26X** & **ANAc6X** & **ANS3S6X** & **A6S** & **G2 OH** & **G2S** & **I2 OH** & **I2S** & **SDEG** \\
**Sample 1** & 94.7 & 4.2 & 0.4 & 1.9 & 6.6 & 2.8 & 85.8 & 7.8 & 0.0 & 4.6 & 87.6 & 2.68 \\
**Sample 2** & 98.1 & 1.1 & 0.0 & 0.0 & 1.6 & 2.5 & 90.2 & 2.3 & 0.5 & 2.8 & 93.4 & 2.85 \\ \hline
**OMHC** & **Hep** & **DeS** & **ChS** & **ANH26X** & **ANAc6X** & **ANS3S6X** & **A6S** & **G2 OH** & **G2S** & **I2 OH** & **I2S** & **SDEG** \\
**Sample 1** & 98.9 & 1.1 & 0.0 & 1.5 & 8.8 & 5.0 & 80.5 & 10.2 & 0.0 & 7.0 & 82.8 & 2.58 \\
**Sample 2** & 97.7 & 2.3 & 0.0 & 1.8 & 8.8 & 4.7 & 82.2 & 10.5 & 0.0 & 7.2 & 82.3 & 2.59 \\
**Sample 3** & 98.7 & 1.3 & 0.0 & 1.6 & 7.8 & 5.3 & 82.2 & 9.7 & 0.0 & 7.0 & 83.4 & 2.61 \\ \hline \hline \end{tabular}
\end{table}
Table 3: Molar average composition of crude heparin of different species/organ.  
Figure 7: Three-dimensional (3D) Score plot generated by PCA of part of the anomeric signals region. Sample clusters corresponding to the different families of crude heparin are defined. BMHC (above pointing triangles), OMHC (squares) and BLHC (below pointing triangles), against the library of PMHC (circles).