# Combining NMR Spectroscopy and Chemometrics to Monitor Structural Features of Crude Hep-arin
## 2 Results
### HSQC Analysis
\begin{table}
\begin{tabular}{l c c c c c c c c c c c c} \hline
**PMHC** & **Hep** & **DeS** & **ChS** & **ANH26X** & **ANAc6X** & **ANS356X** & **A6S** & **G2 OH** & **G2S** & **I2 OH** & **I2S** & **SDEG** \\ \hline
**average** & 89.3 & 8.6 & 2.4 & 1.8 & 16.3 & 4.5 & 73.3 & 18.0 & 0.01 & 10.5 & 71.5 & 2.31 \\
**median** & 89.2 & 8.7 & 2.1 & 1.9 & 16.3 & 4.4 & 73.7 & 17.9 & 0.00 & 10.6 & 71.4 & 2.32 \\
**stdev** & 4.1 & 3.1 & 1.9 & 0.7 & 2.0 & 0.7 & 3.4 & 1.9 & 0.09 & 0.9 & 25.0 & 0.08 \\
**min** & 79.9 & 0.0 & 0.0 & 0.0 & 11.6 & 3.1 & 63.3 & 14.0 & 0.00 & 8.2 & 65.4 & 2.13 \\
**max** & 100.0 & 16.6 & 7.7 & 3.0 & 21.1 & 5.9 & 79.8 & 22.4 & 0.75 & 12.4 & 76.8 & 2.46 \\ \hline \end{tabular}
\end{table} 
Table 2: Molar average composition of 88 crude heparin samples. PMHC = pig mucosa heparin crude;  
Hep = Heparin; DeS = Dermatan sulfate; ChS = Chondroitin sulfate; A = Glucosamine; I = Iduronic acid; G = Glucuronic acid; X = SO3 – or H; SDEG = Heparin disaccharide degree of sulfation. Figure 6: Anomeric region of a crude heparin HSQC spectrum. Signal assignments of heparin and DeS/ChS components are in black and red, respectively. above 3%, compared to 2% for API heparin with 2 h at 600 MHz and 3\(\frac{1}{2}\) h at 500 MHz [10]. This was considered a good result also in view of the fact that the concentration in the NMR tube was limited to 20 mg/0.6 mL compared to 35 mg/0.6 mL for API heparin. Clearly, the limit of quantification (LOQ) of heparin fragments might change according to the dermatan and chondro content. It was verified that a reliable estimation of the LOQ is given by the formula  
\[\text{LOQ}=2000\times\text{VOL}/(\text{VOL\_SUM}\times\text{SNR}) \tag{1}\]  
where VOL_SUM is the sum of all heparinomeric signals, while VOL and SNR are the volume and signal to noise ratio of the anomeric signal of I-(A\({}_{\text{NY}^{\prime},68}\)) or I-(A\({}_{\text{NY}^{\prime},60\text{H}}\)) (Figure 6; both residues give comparable values for LOQ). If the LOQ is too high, the spectrum can be acquired again with more scans (NS) for better SNR, as the SNR term is proportional to the square root of NS. The robustness of the method was also evaluated by identifying the most critical parameters of the analytical method: pH of buffer, sample concentration, acquisition temperature, and spectrum phasing. These four parameters where studied at two levels in a Plucket-Burman design (Tables S4-S6). The heparin fragment quantification showed good robustness with respect to all of the parameters. The GAGs content was sensitive to variations of temperature, probably due to the different mobility of the heparin, DeS, and ChS chains. A summary and the details of the obtained composition of a series of 75 out of 88 crude heparin samples are shown in Table 2 and Tables S10-S13, respectively. As already observed in the PCA, crude heparin samples are characterized by a large variation in galactosaminoglycan content (0-20%), while the heparin component varies in \(N\)-acetylation, 6-\(O\)-sulfation, and nonsulfated uronic acid content (11-21%, 63-79%, and 23-35% respectively). Notably, the serine of the linkage region is almost exclusively present in its intact form (CH\(\alpha\) at 4.00/57.4 ppm; Figure S5). However, the oxidized form of serine [20] was detected in the samples of group A by the reduction or absence of the CH\(\alpha\) signal and the presence of the anomeric signal of xylose linked to oxidized serine residue (4.48/105.5 ppm; Figure S6). The presence of the oxidized forms in the linkage region suggests that crude heparin has been subject to an oxidizing process; usually, such a step is applied during the purification of crude to API heparin. In fact, the monosaccharide composition of the samples present in group A of the PCA results is compatible with a semi purified product. In addition to showing serine in the oxidized form, their spectra do not contain signals belonging to galactosaminoglycan or DNA/RNA impurities, making the whole spectral profile similar to that of an API product (Figure S6).