# Combining NMR Spectroscopy and Chemometrics to Monitor Structural Features of Crude Hep-arin
## 2 Results
### HSQC Analysis  
Quantitative HSQC was recently applied to heparin and low molecular weight heparins (LMWHs). The \({}^{1}\)H,\({}^{13}\)C-HSQC data acquired on heparin APIs were used for calculating the percentage of mono- and disaccharides by normalizing volumes with reference to the sum of volumes of signals corresponding to each monosaccharide type (glucosamines or uronic acids) and the same carbon-proton pair type (e.g., anomeric or C2 position pairs) [10; 15; 16; 17]. In the HSQC spectra collected here, signals belonging to high molecular weight species, mainly DNA and RNA, were not detected, presumably due to the increased linewidths associated with these slower tumbling larger molecules. The HSQC spectrum of crude heparin also contains signals belonging to galactosaminoglycan components (DS and CSA), which partially overlap with those of the heparin/heparan sulfate (HS) components. By contrast, in the H2/C2 region, signals belonging to \(N\)-acetyl-glucosamine were completely separated from those of the \(N\)-acetyl-galactosamine residues (3.90 ppm/56.0-56.6 ppm and 4.02 ppm/53.8-54.6 ppm, respectively; Figure S5). Thus, the percentage of glucosamine and galactosamino residues could be calculated by the integration of the corresponding residues (Table 2). In addition, the uronic acid components of the galactosaminoglycans were well separated in the anomeric region of the crude heparins (Figure 6). Beside the major IdoA-GalNAc4S sequences, DeS  
Figure 5: The 5 min to 25 min portion of the strong anion exchange HPLC (SAX-HPLC) chromatograms with UV detection at 215 nm of crudes differentiated into groups A, B, and C by NMR data as shown in Figure 2. Generally, the lower resolution HPLC method shows these groups have increasing levels of different impurities, going from group A to B to C, respectively. contains about 10% of disulfated disaccharide sequences, mostly consisting of IdoA2S-GalNAc_4S_[18]. The anomeric signals of IdoA and IdoA2S of dermatan sulfate, at 4.88/106.1 ppm and 5.18/103.4 ppm, respectively, could be integrated to determine the IdoA2S/IdoA ratio. Similarly, the anomeric signal of the glucuronic acid of the chondro presents distinct chemical shift values on the basis of the position of the sulfate group of the following \(N\)-Acetyl-galactosamine residue (Figure 6) (4.47/106.6 ppm and 4.50/107.1 ppm for the ChS4S or ChS6S, respectively) [19], allowing the evaluation of the GalNAc_6S/GalNAc_4S_ ratio. Unfortunately, the anomeric signals of the DeS and ChS \(N\)-acetyl-galactosamine residues partially overlap with the anomeric signal of heparin glucuronic acid. In particular, if chondro is present, the amount of glucuronic acid linked to trisulfated glucosamine (G-\(\mathrm{A}_{\mathrm{NS},\mathrm{3S},\mathrm{6X}}\)) could be overestimated. Finally, the composition of the heparin fraction was obtained as for the API product [10]. Tables S2 and S3 show the signals assignment and the detailed formulas for the calculations. The precision of the method was evaluated using a crude sample positioned close to the origin (0) of the PCA scores plot of Figure 2, therefore having an average composition in DeS/ChS comparable with the whole library of 88 samples. The intermediate precision was determined combining two operators and two spectrometers operating at 500 and 600 MHz (Tables S7-S9). Using a reasonable experimental time (6 h at 600 MHz and 8 h at 500 MHz) it was possible to quantify with sufficient precision all of the fragments