Differentiation of industrial hemp strains by their cannabinoid and phenolic compounds using LC × LC-HRMS

Abstract

Cannabis is an ancient plant that has been used for therapeutic and recreational purposes. Nowadays, industrial hemp, a variety with low concentration of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) and high concentration of non-psychoactive cannabinoids, is getting more and more interest in the food, pharmaceutical, and cosmetic industry. However, cannabis not only contains cannabinoids as bioactive components but also other metabolites like terpenes and phenolic compounds, and the content of these interesting secondary metabolites greatly differs with the genetic variety of the plant. Due to the huge complexity of composition of the cannabis matrix, in this work, a comprehensive two-dimensional liquid chromatography (LC × LC) method has been developed as a very power separation technique coupling a pentafluorophenyl (PFP) and a C18 in the first and second dimensions. Two industrial hemp strains (cookie and gelato) were analyzed to determine the difference in their content of cannabinoids and phenolic compounds. To do this, a new demodulation process was applied for the first time to transform 2D raw data into 1D data which allowed carrying out the chemometric analysis needed to determine the statistical differences between the hemp strains. The cookie strain presented a total of 41 cannabinoid markers, while the gelato strain presented more representative phenolic compounds, in total 24 phenolic compounds were detected as potential markers of this sample. These differences in the chemical composition could determine the industrial destiny of the different hemp strains. Graphical abstract

Multi-2D LC × LC as a Novel and Powerful Implement for the Maximum Separation of Complex Samples

“…For the optimization of the multi- 2 D LC × LC, PFP was selected as 1 D column due to its good efficiency for the separation of compounds with aromatic rings like phenolic compounds. This separation is enhanced when using methanol as an organic solvent in the mobile phase since this protic solvent increases the π–π selectivity between the phenyl stationary phase group and the aromatic rings. , A second important reason for the selection of PFP as 1 D separation is the good orthogonality achieved between PFP × HILIC as well as when this stationary phase is coupled in an RP × RP method using C18 as 2 D . The optimized 1 D separation of the vermouth samples carried out in the PFP column can be observed in Figure .…”

“…Moreover, when two RP separations are coupled in LC × LC, the most useful alternative to achieve higher orthogonality values is the use of shifting gradients . Therefore, LC × LC analysis using PFP in the 1 D and C18 in the 2 D have already shown very high orthogonal separations …”

“…This separation is enhanced when using methanol as an organic solvent in the mobile phase since this protic solvent increases the π−π selectivity between the phenyl stationary phase group and the aromatic rings. 12,13 A second important reason for the selection of PFP as 1 D separation is the good orthogonality achieved between PFP × HILIC as well as when this stationary phase is coupled in an RP × RP method using C18 as 2 D. 4 The optimized 1 D separation of the vermouth samples carried out in the PFP column can be observed in Figure 2. In this 1D chromatogram, it is important to highlight the complexity of the vermouth sample regarding the variety of compounds that could not be completely resolved in 120 min as well as the large difference in the concentration of the compounds present in the sample.…”

“…21 Therefore, LC × LC analysis using PFP in the 1 D and C18 in the 2 D have already shown very high orthogonal separations. 4 The optimization of the PFP × C18 analysis of vermouth consisted of the selection of the most suitable 2 D-C18 columns, modulation times (0.65, 1, and 2 min), make-up flow (0.02 and 0.03 mL/min), and the shift gradient program (shifting and multi-segment gradients).…”

“…The large interest in LC × LC is the increase of peak capacity that is possible to achieve in comparison with one-dimensional liquid chromatography (1DLC) methods. With highly orthogonal LC × LC separations, peak capacity values of several thousand can be obtained for very complex samples, , while, usually, 1DLC analysis provides a value not higher than a few hundred.…”