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24 August / September 2016


Supercritical fluid chromatography (SFC), is the underlying principle behind UHPSFC technology, and presents an alternative and orthogonal solution for the separation of synthetic cannabinoids. UHPSFC takes advantage of smaller particle columns and allows for very efficient and rapid separations, up to four times faster than UHPLC. This is attributable to a mobile phase that is more diffusive and has lower viscosity, favouring the ability to run at higher linear mobile phase velocities than with UHPLC. In particular, since SFC can be operated under normal phase conditions, it generally offers greater selectivity for structural analogues and stereoisomers [2]. Here, simple method development will be demonstrated for the chiral analysis of selected synthetic cannabinoids, including separating HU-210 from its enantiomer HU- 211, and the stereoisomers of cis and trans (epi) CP 47,497, and CP 55,940. Structures for these compounds are shown in Figure 1, including stereochemical configurations.


Experimental


Federally exempt synthetic cannabinoid standards were obtained from Cayman Chemical (Ann Arbor, MI) including: HU-210, HU-211, (±)-CP 47,497, (±)-epi CP 47,497, (±)-CP 55,940 and (±)5-epi CP 55,940. The standards were received in solution at 5 mg in 500 µL methanol and were subsequently diluted to 1 mg/mL with ethanol. (-)-CP 47,497 and (-)-CP 55,940 standards were used to determine order of elution for those separations. 200 proof HPLC grade ethanol (Sigma-Aldrich) was used as the co-solvent and all injections were 1 µL.


All separations were performed on a Waters ACQUITY UPC2


System equipped with a


PDA (UV) detector and a QDa (MS) detector. Achiral analysis was performed using a 3x100 mm ACQUITY UPC2


Torus 1-AA column with


1.7 µm particles. This column was chosen based on a previously described achiral column screening of synthetic cannabinoids [2]. ACQUITY UPC2


Trefoil chiral columns,


including the amylose (AMY1) and cellulose (CEL1 & CEL2) based chiral stationary phases (Waters Corporation) were used for chiral screening and method development. All three Trefoil columns were 3x150 mm and were packed with 2.5 µm particles.


For screening, a generic gradient was used from 2% to 20% ethanol over 5 minutes, followed by a 1 minute hold at 20%, before returning to initial 2% conditions. The total flow was 2 mL/min, the back pressure was 2000 psi, and the column temperature was


Figure 2: Achiral separation of synthetic cannabinoids on the ACQUITY UPC2


Torus 1-AA column. Conditions


as follows: 1.5 mL/min total flow, 2-20% ethanol gradient over 5 minutes, temperature at 40°C, and pressure at 2000 psi


Figure 3: Chromatograms of the five chiral synthetic cannabinoids screened on the three Trefoil chiral columns. Conditions as follows: 2 mL/min total flow, 2-20% ethanol gradient over 5 minutes, temperature at 40°C, and pressure at 2000 psi


40°C. The PDA data was obtained using an absorbance compensated channel at 228 nm with a compensation reference from 500 to 600 nm. The same conditions were used for the achiral analysis, with the exception of the flow rate, which was 1.5 mL/min. After a review of initial column screening, optimised methods were developed. Those conditions are described in the respective figures.


Results and Discussion


Achiral separation of the five synthetic cannabinoids is displayed in Figure 2. The peaks are well separated under generic gradient conditions (2-20% ethanol) on the ACQUITY UPC2


Torus 1-AA column,


including the separation of two sets of diastereomers; cis and trans (epi) CP 47,497, and cis and trans (5-epi) CP 55,940.


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