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here, the gradient method may permit some flexibility to build in a weaker solvent hold prior to the 75-90% MPB gradient in order to clear matrix interferences. Analysts must also assess whether other endogenous cannabis compounds, such as terpenes and terpenoids, potentially interfere with identification of cannabinoids.
In some assays, analysts are concerned with improving the resolution of certain critical pairs. This may be especially true in cases where one component is far more abundant than the other. In the gradient separations shown in Figure 2, the resolutions between Δ9-THC and Δ8-THC are approximately 1.50. These isomers are neutral, and their retentions are largely unaffected by changes in mobile phase pH or ionic strength. Often, it is possible to improve resolution by running an isocratic analysis and by reducing eluent strength. In the case of Δ9-THC and Δ8-THC, the greatest effect is observed by changing the composition of mobile phase B with various ratios of acetonitrile and methanol.
Acetonitrile and methanol are two of the most common organic modifiers used in reversed-phase HPLC, and many studies have detailed the differing and often complementary selectivities that they provide. Fundamental understandings of the solute-mobile phase, solute-stationary phase, and stationary phase-mobile phase molecular interactions can inform the strategies used in HPLC method development [8]. For instance, it has been noted that, depending on the modifier used and how it has partitioned or adsorbed into the stationary phase, differences in hydrophobicity, hydrogen-bonding, and dipole-type interactions can be observed [9,10]. When developing methods and selecting appropriate mobile phases, it can be useful to consult Snyder’s solvent selectivity triangle, which plots solvents according to their acidic, basic, and dipolar properties [11,12,13]. Solvents that feature one of those properties more prominently than the other two can be readily identified from the plot. For example, methanol has acidic properties, and acetonitrile has dipole properties. Since they are miscible, they can be mixed in any ratio to achieve intermediate or new solvent properties.
Figure 3 plots the effect of varying the percentage and composition of mobile phase B (MPB) on the isocratic resolution of 1:2 Δ9-THC:Δ8-THC using the same Evoke C18, 15 cm x 4.6 mm column. Consider the analysis when performed with H2
O/
MPB = 10/90. The resolution of Δ9-THC and Δ8-THC is 1.06 when MPB = 100%
acetonitrile. When MPB = 100% methanol, the resolution is 2.84. Maximum resolution (Rs = 3.12) is observed when MPB = 15:85 acetonitrile:methanol. That relatively minor improvement in resolution afforded by the blended MPB might suggest pure methanol to be the preferred organic modifier for this analysis, especially given the convenience of using a single solvent over pre-mixing a blend of acetonitrile:methanol or investing in alternative pumping instrumentation (e.g. quaternary pumps). With complex samples, though, care must be taken to observe how a desired change in selectivity can affect other analytes in the separation.
A brief example serves to illustrate that several parameters should be considered when developing a chromatographic method for the resolution
Figure 4: Separation of 1:2:3 Δ9-THC:Δ8-THC:CBL. An organic modifier of pure methanol results in the coelution of Δ8-THC and CBL while pure acetonitrile results in incomplete resolution of the THC isomers. A 50:50 blend of acetonitrile:methanol resolves all three analytes. Evoke C18, 15 cm x 4.6 mm, 3 µm, 1.5 mL/min, H2
O/MPB = 10/90.
of complex samples involving key critical pairs. Consider again the separation of 1:2 Δ9-THC:Δ8-THC in the presence of cannabicyclol (CBL). In Figure 3 it can be seen that the resolution of the THC isomers is superior with pure methanol than with pure acetonitrile as the organic modifier. As shown in Figure 4, though, if CBL is present, it coelutes with Δ8-THC in H2
O/methanol =
10/90. CBL elutes well away from the critical pair if pure acetonitrile is used, but the THC isomers are insufficiently resolved (Rs = 1.06). A 50:50 blend of acetonitrile:methanol provides good resolution, with Rs > 2.5 for both pairs. So, while binary mobile phase systems are very common in reversed-phase HPLC separations, ternary mobile phases can provide access to unique selectivities based on the combination of acidic, basic, and dipolar properties of the mobile phases used.
To recap, we developed an HPLC method that fully resolves 17 cannabinoids by using screening runs that altered concentrations of organic and acid modifiers and provided the foundation for further development. The addition of ammonium formate to mobile phase A gave a means to shift the retentions of the carboxylated species relative to the neutral ones, and an optimised concentration allowed for the baseline resolution of all cannabinoids in the test mixture. In addition, the use of a ternary mobile phase system (water, methanol, acetonitrile) was shown to improve the resolution of THC isomers while permitting the flexibility to avoid potential interferences.
References
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3. J. Steimling, T. Kahler, LCGC N. Amer. 36(6) (2018) 36.
4. W.M. Reuter, Chromatography Today. May/June (2018) 51.
5. A. James, Chromatography Today. Feb/March (2018) 40.
6. D.V. McCalley, J Chromatogr A. 1038 2004) 77.
7. D. Johnson, B. Boyes, R. Orlando, J Biomol Tech. 24 (2013) 187.
8. A. Klimek-Turek, T.H. Dzido, B. Misiolek, T. Kossowski, LCGC Europe. 27 (2014) 182.
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