An Analytical Quality by Design (AQbD) Approach to Separating Cannabinoid Stereoisomers

The cannabis market has been rapidly growing with the list of identified cannabinoids constantly increasing. Analytical methods for detecting cannabinoids by means of HPLC (High Pressure Liquid Chromatography) analysis have been explored and currently there are methods to separate 21 cannabinoids in a single run. Expanding the list of cannabinoids is possible, however stereoisomers challenge the current methods and, therefore, further method optimization is needed. The process of method development was made easier and faster with the use of an analytical method development software. The software uses “Analytical Quality by Design (AQbD)” concepts to help determine the optimal method for cannabinoid separation. 

Six Daicel chiralpak columns (IA-U, IB-U, IC-U, ID-U, IG-U, IH-U: 3.0mmx100mmx1.6µm) with varying stationary phase chemistries were used with a method scouting system and AQbD software to separate the (6aR,9R)-Δ10-THC, (6aR,9S)-Δ10-THC, 9(R)- Δ6a,10a-THC, and 9(S)- Δ6a,10a-THC stereoisomers along with twenty other cannabinoids. Standards were diluted in methanol to prepare a 25µg/mL mix standard for analysis. The mobile phase was 0.085% phosphoric acid in water and 0.085% phosphoric acid in acetonitrile.

Utilizing the software, the six columns were used to run mobile phase concentrations ranging from 45% to 65% organic to determine the optimal column and mobile phase combination. Of the six columns IA-U (stationary phase: amylose tris(3,5-dimethyl-phenylcarbamate) and IG-U (stationary phase: amylose tris(3-chloro-5-methyl-phenylcarbamate) were the only two columns with separation of all compounds. Using the optimization step, it was determined that the best mobile phase condition was a stepwise gradient using a 60:40 ratio for a hold time of two minutes and then a 50:50 ratio for 13 minutes. The resolution between the 9(R)- Δ6a,10a-THC and 9(S)- Δ6a,10a-THC isomers, which were not separated on the other columns, was 1.285 and 1.298 for the IG-U and IA-U column, respectively. In the robustness stage, the desired flow rate and column oven temperature was determined to be 0.8 mL/min and 35℃. This method can also be applied to more complex cannabinoid mixtures. 

Learning Objectives:

1. Observe the difference in cannabinoid separation between chiral and reverse phase stationary phases.

2. Discuss the benefits of using AQbD principles for method development.

3. Discuss software techniques to separate overlapping peaks.