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This demonstrates the chromatographic efficiency of SFC using small particle size columns for separating closely related structural isomers, even while using achiral stationary phases under simple generic method conditions. However, each of the peaks in this chromatogram actually represents two different pharmaceutically active enantiomers. As such, the achiral separation alone is not adequate for comprehensive qualitative analysis of these synthetic cannabinoids.


In normal phase chromatography, and SFC in particular, the first step in method development involves column screening. In this case, the columns used in the screening were amylose (AMY1) and cellulose (CEL1 & CEL2) based chiral stationary phases, which are commonly used because of their wide range of applicability. In particular, the three Trefoil columns provide orthogonal selectivity, which is optimal for successful chiral method development. Much of the work involving cannabis quality control and post-extraction processing uses ethanol as the preferred solvent in order to avoid toxicity from solvent contamination in any final product or consumable. Ethanol has also been shown to be an effective co-solvent for the separation of natural cannabinoids (pending patent application). Consequently, ethanol was chosen as the sample diluent and co-solvent for method development. Methanol and isopropanol are also appropriate co-solvents for the analytical application; however for simplicity, ethanol was used throughout the study. As is typical in column screening, a generic


gradient from 2% to 20% ethanol was utilised. Figure 3 shows chromatograms of the column screen for the five synthetic cannabinoids. Many of the chiral separation screening results showed excellent peak shape and resolution.


A few observations that were made during the screen warrant some discussion. For the cyclohexylphenol (CP) compounds, specifically (±)-CP 47,497, (±)-epi CP 47,497, and (±)-CP 55,940, orthogonality was observed between the cellulose (CEL1 and CEL2) and amylose (AMY1) based stationary phases, where the elution order was reversed, but the enantiomers were still very well resolved. This has advantages for analysis of these compounds, especially in cases where there are matrix interferences. On the AMY1 column, the (±)-epi CP 47,497 cannabinoid enantiomers appear to be co- eluting. However, the peak at 5.07 is, in fact, only one of the enantiomers, the second peak failed to elute under the conditions of the gradient. Further investigation confirmed that the second peak eluted quite a bit later than the first, even under high co- solvent percentages. Another unexpected observation was that the (±)5-epi CP 55,940 sample appears to have a significant enantiomeric excess. Based on the UV and MS spectra, the small peak eluting at 4.72 on the CEL1 (4.79 on CEL2) appears to be the enantiomer of the larger peak in that particular sample.


Even though the generic screening conditions resulted in acceptable chromatography for many of these


compounds, in a fast paced analytical environment, it is always beneficial to decrease run times and simplify methodology. Also, since (±)-CP 47,497 and (±)-epi CP 47,497, and (±)-CP 55,940 and (±)5-epi CP 55,940 are actually diastereomer pairs, it would be more advantageous if all four stereoisomers (enantiomers and diastereomers) could be separated in a single run. To that end, the separations were optimised for speed and resolution of the four stereoisomers. Optimisation of the mobile phase conditions usually involves either focusing the gradient or running isocratically. Gradients usually result in better peak shape, however focusing gradients in SFC is more complicated than in HPLC because the effect of increasing co-solvent percentage (or %B in HPLC) on retention times is not linear, and the resulting chromatography is harder to predict. Isocratic methods are ideal because they are easy to develop based on the screening results and no equilibration is required between runs, improving productivity. Using retention times and gradient slope, and compensating for system and column volume delay, the co-solvent percentages at elution were determined for each compound. In SFC, usually the best starting point for optimisation is 5% below the calculated percentage.


Using the HU-210 and HU-211 separation on the AMY1 column (Figure 4A) as an example, with a gradient delay of 0.46 min, gradient slope of 3.6%/min, and 2% starting percentage, the co-solvent percentage at elution of the first peak at 4.12 minutes was calculated using the following equation:


%Co-solvent at Elution = (retention time – gradient delay) x gradient slope + starting %


%Co-solvent at Elution =


(4.12 min – 0.46 min ) x 3.6%/min + 2% %Co-solvent at Elution = 15%


Therefore, after subtracting the 5%, 10% isocratic co-solvent conditions were used as a starting point for optimisation (Figure 4B). The resulting chromatography showed good separation; but by increasing the co- solvent fraction of the mobile phase back to 15%, effective separation was achieved in approximately 2 minutes (Figure 4C).


Figure 4: Chromatograms showing optimization of the HU-210 and HU-211 separation on the Trefoil AMY1 column. Conditions are as follows: (A) 2%-20% ethanol gradient over 5 minutes (B) 10% ethanol isocratic method and (C) 15% ethanol isocratic method.


This same method optimisation strategy was used to develop fast methods to separate all four stereoisomers of the two cyclohexylphenol (CP) synthetic cannabinoids. For both sets of stereoisomers, the retention time of


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