The most labor-intensive part of determining the potency of samples containing cannabinoids is extraction. Due to a plethora of matrices, from candy, baked goods, lotions, and so on, selecting the most suitable extraction method can also be a challenge. The following video and technical article will guide you through that initial process:
Let’s focus on the analytical part of the analysis.
Restek scientists showed that separation of regulated and some of the most frequent cannabinoids can be performed by a very simple isocratic analysis. Isocratic elution is typically effective in the separation of sample components that are very different in their affinity for the stationary phase. Raptor ARC-18 column chemistry compliments the cannabinoids’ structures, and the three analysis methods were developed on this column. The main difference is column dimensions and particle size, which will affect the analysis time:
1. Starting point isocratic method.
- Starting Isocratic analysis (150x4.6mm, 2.7µm, Catalog # 9314A65)
- 16 component isocratic.
- 9 minutes; flow rate 1.5mL/min
2. Faster analysis using a UHPLC system.
- Fast Analysis (100x3.0mm, 1.8µm, Catalog # 931421E)
- 16 component isocratic.
- 4 minutes; flow rate 1.0mL/min
3. Saving Solvent? The reduced ID column used in the application requires a lower flow rate.
- Solvent Savings Analysis (150x2.1mm, 2.7µm, Catalog # 9314A62)
- 19 component isocratic.
- 10 minutes; flow rate 0.4mL/min
A new Raptor ARC-18 column is stored in a reverse phase mobile phase 45/55 water/acetonitrile and should always be conditioned before use:
- Our suggestion is to run approximately 10-20% less organic content than the storage solvent for about 10-20 column volumes. For example, if the HPLC column is stored in 45/55 water/acetonitrile, a good starting solvent would be approximately 60/40 water/acetonitrile. This step is crucial, mainly because our method mobile phase contains salts (buffer). Preconditioning the column will prevent salts from precipitating out of the solution, possibly clogging the column and increasing backpressure.
- Then, introduce the method mobile phase containing buffer for 10-20 column volumes. You are now ready to start your analysis. Check out this video for more details: LC Column Conditioning
Tips and Tricks to help prevent variability:
- Buffer Strength/pH: Buffering the mobile phase is crucial in keeping retention time consistent in this analysis. In our application, we used a 5mM ammonium formate buffer. It was found adjusting between 5mM and 10mM ammonium formate can fine-tune the separation. Our scientists didn’t see any noticeable difference (resolution/selectivity) in chromatography when switching from 5 to 10mM buffer concentration, you can adjust compounds like CBNA and THCA-A without losing the overall selectivity. Also, if you don’t use an acid modifier in the mobile phase, the retention time decreased drastically for acidic cannabinoids. Little chromatography change was noticed adjusting pH with 0.1% acetic acid or 0.1% formic acid.
- Sample Solvent: Are you seeing poor peak shape, especially with the early eluting analytes? Injecting a 100% organic solvent sample will cause peak distortion, peak broadening, poor sensitivity, and shortening of retention times. This happens because some analytes will tend to travel too quickly through the column, instead of eluting in a symmetrical band. After all, they don’t mix evenly with the mobile phase. This is more pronounced in isocratic analysis than gradient analysis. The sample solvent should be as similar to the starting conditions as possible (aqueous/organic composition). Even if extracting in 100% organic solvent, consider doing a dilution into a more equivalent solvent to your starting conditions. In the applications mentioned above, we use 25:75 water/methanol as the diluent.
- Injection volume: Seeing peak distortion? The injection volume leads to many of the same problems that the sample solvent can cause. Higher-than-recommended injection volume on an isocratic analysis can cause peak shape distortion. Please see this FAQ for the correct injection volume based on the column ID: HPLC FAQ's
- Guard columns: It is strongly recommended to use a guard column or pre-column filter for this analysis. This will help to increase column lifetime due to removing particulate contaminants and strongly retained compounds that are inherent with this analysis. Sample filtration is a step that shouldn’t be forgotten in the analysis. Sample-Filtration.
- Column regeneration: How do I extend the lifetime of my HPLC column? At the end of the day or analytical run, best practice is to run an increasing organic gradient up to 95-100% organic solvent to dissolve the non-polar particulates trapped at the head of the column. If drastic increasing pressure and degrading peak shape is noticed the last resort before buying a new column, try a cleaning procedure (backflushing not recommended for Raptor ARC-18 columns); LC Cleaning Recommendations
- Wavelength: Our applications use 228nm, but this is something that can be optimized instrument-to-instrument to get the best column sensitivity and low baseline distortion. Please note that interferences can come from possible terpenes in the matrix. Most terpenes do not respond well at UV wavelength ≥ 220nm. Minor terpene interferences could potentially impact the quantitation of CBGA and THCVA if present in high enough concentrations.
To conclude I would like to bring to your attention an excellent article about organic modifiers as it pertains to this analysis. Selecting a different organic modifier is a good way to change the selectivity and resolution of your analysis. The article shows the difference between a protic solvent like methanol (better resolution of later eluting cannabinoids) and an aprotic solvent like acetonitrile (better resolution of the earlier eluting cannabinoids), and how this can also change your chromatography.