# Commercial Hemp Seed Oils: A Multimethological Characterization
## 2 Materials and Methods
### NMR Analysis  
Each hemp seed oil sample (20 \(\upmu\)L) was dissolved in DMSO-d6 (20 \(\upmu\)L) and CDCl\({}_{3}\) (700 \(\upmu\)L) in a 5 mm NMR tube, which was flame-sealed. The NMR spectra were recorded at 27 \({}^{\circ}\)C on a Bruker AVANCE 600 spectrometer (Milan, Italy) operating at the proton frequency of 600.13 MHz and equipped with a Bruker multinuclear z-gradient 5 mm probe head. \({}^{1}\)H spectra were acquired using the following conditions: number of scans 1024, 90\({}^{\circ}\) pulse 9.5 \(\upmu\)s, time domain 64 K data points, relaxation delay plus acquisition time 3.5 s, and spectral width 10,802 Hz. \({}^{1}\)H NMR spectra were obtained by the FT (Fourier transform) of the free induction decay, applying an exponential multiplication with a line-broadening factor of 0.3 Hz and a zero filling (size 64 K) procedure. \({}^{1}\)H NMR spectra were manually phased. Chemical shifts were reported with respect to the residual CHCl\({}_{3}\) signal set at 7.26 ppm. The baseline was corrected using the Cubic Spline Baseline Correction routine in the Bruker Topspin software. The integrals of seven selected signals were measured. In particular, the selected proton resonances were due to hexanal (CH) at 9.699 ppm, _trans_-2-hexanal (CH) at 9.448 ppm, diallylic protons of tri-unsaturated fatty acids (CH\({}_{2}\)) at 2.778 ppm, diallylic protons of di-unsaturated fatty acids (CH\({}_{2}\)) at 2.729 ppm, \(\alpha\)-methylene protons of all fatty acids (CH\({}_{2}\)) at 2.251 ppm, allylic protons of all unsaturated fatty acids (CH\({}_{2}\)) at 1.999 ppm, and \(\beta\)-sitosterol (CH\({}_{3}\)) at 0.622 ppm. The integrals of the selected resonances were normalized with respect to the resonance at 2.251 ppm set to 100. \(\beta\)-Sitosterol, fatty acids, and aldehydes were expressed as molar %, following the Equations previously described [37], with some modifications:  
\[\%_{\beta\text{-SIT}}=100\times 0.66\text{I}_{\beta\text{-SIT}}/\text{I}_{ \text{tot}} \tag{5}\]  
\[\%_{\text{TRI}}=100\times 0.5\text{I}_{\text{TRI}}/\text{I}_{\text{tot}} \tag{6}\]  
\[\%_{\text{DI}}=100\times\text{I}_{\text{DI}}/\text{I}_{\text{tot}} \tag{7}\]  
\[\%_{\text{MONO}}=100\times 0.5\text{(I}_{\text{UNS}}-2\text{I}_{\text{DI}}- \text{I}_{\text{TRI}})/\text{I}_{\text{tot}} \tag{8}\]  
\[\%_{\text{SAT}}=100\times\text{(I}_{\text{FA}}-\text{I}_{\text{DI}}-0.5\text {I}_{\text{TRI}}-\text{\%}_{\text{MONO}})/\text{I}_{\text{tot}} \tag{9}\]  
\[\%_{\text{INS}}=\%_{\text{MONO}}\times\%_{\text{DI}}+\%_{\text{TRI}} \tag{10}\]  
\[\%_{\text{tot-2-HEX}}=100\times 2\text{I}_{\text{t-2-HEX}}/\text{I}_{\text{tot}} \tag{11}\]  
\[\%_{\text{HEX}}=100\times 2\text{I}_{\text{HEX}}/\text{I}_{\text{tot}} \tag{12}\]  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Sample** & **L*** & **a*** & **b*** & **  
where %\({}_{\rm{\beta-STI}}\), %TRI, %DL, %MONO, %SAT, %INS, %\({}_{\rm{\theta-2-HEX}}\), and %HEX are molar % of \(\beta\)-sitosterol, tri-unsaturated fatty acids, di-unsaturated fatty acids, mono-unsaturated fatty acids, saturated fatty acids, unsaturated fatty acids, _trans_-2-hexenal, and hexanal, respectively. I\({}_{\rm{\beta-STI}}\), I\({}_{\rm{TRI}}\), I\({}_{\rm{DI}}\), I\({}_{\rm{UNS}}\), I\({}_{\rm{FA}}\), I\({}_{\rm{\theta-2-HEX}}\), and I\({}_{\rm{HEX}}\) are integrals of \(\beta\)-sitosterol, tri-unsaturated fatty acids, di-unsaturated fatty acids, allylic protons of all unsaturated fatty acids, \(\alpha\)-methylene protons of all fatty acids, _trans_-2-hexenal, and hexanal, respectively. I\({}_{\rm{tot}}\) was calculated according to Equation (13).  
\[{\rm{I}_{tot}}={\rm{I}_{FA}}+0.66{\rm{I}_{\rm{\beta-STI}}}+2{\rm{I}_{\rm{t-2- HEX}}}+2{\rm{I}_{HEX}} \tag{13}\]