# Commercial Hemp Seed Oils: A Multimethological Characterization
## 3 Results and Discussion
### NMR Analysis  
In Figure 2, the \({}^{1}\)H spectrum of a hemp seed oil is reported. Hemp seed oils spectra, similarly to other vegetable oils, were characterized by strong signals, due to the fatty chains on the glycerol moiety and minor compounds. The amount of \(\beta\)-sitosterol, hexanal, _trans_-2-hexenal, saturated fatty chains, and unsaturated fatty chains (including mono-unsaturated, di-unsaturated, and tri-unsaturated fatty chains) are reported as molar percentages in Figure 3.  
Figure 1: Spectral reflectance curves obtained by CIEL*a*b* analyses applied to 1–9 hemp seed oils. Samples 6 and 7 were measured after filtration.  
Figure 2: 600.13 MHz \({}^{1}\)H NMR spectrum of a hemp seed oil. Quantified selected NMR signals are reported in expanded regions. (**A**) \(\beta\)-sitosterol (0.622 ppm); (**B**): allyic protons of all fatty chains (1.999 ppm); (**C**): \(\alpha\)-methylene protons of all acyl chains (2.251 ppm); (**D**): diallyic protons of linoleic fatty chains (2.729 ppm); (**E**): diallyic protons of linolenic fatty chains (2.778 ppm); (**F**): _trans_-2-hexenal (9.448 ppm); (**G**): hexanal (9.699 ppm).  
As expected, fatty acid chain profiles of all nine hemp seed oils were characterized by a higher content of unsaturated fatty acid chains with respect to saturated ones, and, among them, di-unsaturated fatty chains resulted in more abundance (35-48%), compared to mono- (15-28%) and tri-unsaturated ones (8-20%). In particular, sample 1 was characterized by the lowest tri-unsaturated fatty chain content.  
Hemp seed oils are renowned for their optimal 3:1 ratio in \(\omega\)-6:\(\omega\)-3. However, on the bottle labels of samples 2, 6, and 9 (see Table 4), a ratio lower than 3:1 was reported, and, in some cases, the \(\omega\)-6:\(\omega\)-3 ratio measured by NMR was different from the ratio reported on the labels. For instance, 2, 3, and 4 hemp seed oil samples showed an \(\omega\)-6:\(\omega\)-3 ratio lower than that declared in the labels. Only 7, 8, 5, and 1 hemp seed oil samples, with values of 3.6, 3.6, 3.3, and 4.7, respectively, turned out to have an optimal \(\omega\)-6:\(\omega\)-3 ratio. In particular, sample 1 presented a particularly high ratio compared to other ones. According to NMR data, samples 4, 2, 9, and 6 did not reach the 3:1 \(\omega\)-6:\(\omega\)-3 ratio value, with sample 3 having a particularly low value.  
It is interesting to note that sample 9, with an \(\omega\)-6:\(\omega\)-3 ratio of 2.86, was sold at 80 \(\xi\)/L, whereas sample 5, with a 3.36 ratio value, was sold at 26 \(\xi\)/L, a much more affordable price.  
Regarding the content of minor compounds, the \(\beta\)-sitosterol amount ranged from 0.15% (sample 8) to 0.33% and 0.45% (samples 6 and 5, respectively).  
\begin{table}
\begin{tabular}{c c c} \hline \hline  & \multicolumn{2}{c}{\(\omega\)-6:\(\omega\)-3 Ratio} \\ \hline
**Samples** & **Bottle Label** & **NMR Results** \\ \hline
1 & 4.20 & 4.71 \\
2 & 2.80 & 2.40 \\
3 & 3.37 & 1.99 \\
4 & 3.17 & 2.30 \\
5 & 3.21 & 3.36 \\
6 & 2.39 & 2.27 \\
7 & 3.11 & 3.64 \\
8 & 3.37 & 3.61 \\
9 & 2.57 & 2.86 \\ \hline \hline \end{tabular}
\end{table}
Table 4: Comparison between \(\omega\)-6:\(\omega\)-3 ratios reported on the bottle labels and the ones measured by NMR analysis. Integrals of signals D (diallylic protons of linoleic fatty chains at 2.729 ppm) and E (diallylic protons of linoleic fatty chains at 2.778 ppm) of the nine commercial hemp seed oils analyzed were measured.  
Figure 3: Histograms resulting from the quantitative NMR analysis of (**A**) \(\beta\)-sitosterol and aldehydes, and (**B**) fatty acids. The relative molecular abundances (molar percentage) are reported.  
The content of _trans_ 2-hexanal and hexanal was generally low in all the samples (between 0 and 0.5%). However, sample 1 showed the highest content of hexanal (0.34%) and _trans_-2-hexanal (0.17%).