Evaluation location: New York, United States
HPLC protocol modified from Brownstein et al for in-house cannabinoid analysis.
Note that this assay only includes CBD, CBDA, THCd9, THCA, CBG, CBGA. Developed by Kevin Hernandez, Zachary Stansell, Tyler Gordon, Sadie Kraft, and Korey Brownstein. Will be implemented for routine compliance testing of regenerations and selections.
🧪HPLC cannabinoid evaluation🧪
Sample run time: 20 min.
Materials:
• Ascentis® Express C18, 2.7 μm HPLC Column
• Ascentis® Express Guard Cartridge Holder
• Ascentis® Express C18, 2.7 Micron Guard Cartridge
• No. 35 sieve
• No. 10 sieve
• Homogenizer
o Mortar and pestle
o Geno grinder
o Rolling pin on dry ice
o Hammer mill
o Ninja Blender
• 20 mL glass scintillation vial
• Analytical balance
• 200 proof Ethanol
• Sonicator
• 0.45 µm membrane filter
• 1ml plunger syringe
• 1000 ul pipette
• 100 ul pipette
• 10 ul pipette
• 1 ml Autosampler vials
• 1000ml Beaker
• Computer with Control Panel software
• HPLC instrument (The Robin)
• 4.0 L amber glass bottle or 1L bottle
• 18 M HPLC grade water
• 0.1% formic acid
• 95% formic acid
• 5mM Ammonium formate
• Acetonitrile
• Syringes (L-1000, green-taped 5000 mL, red-taped 5000 mL)
• Pipette (blue box tips)
• Labels or Marker
Standards:
• Cannabidiolic Acid (CBDA; CAS #: 1244-58-2)
• Cannabigerolic Acid (CBGA; CAS #: 25555-57-1)
• Cannabigerol (CBG; CAS #: 25654-31-3)
• Cannabidiol (CBD; CAS #: 13956-29-1)
• Δ9-Tetrahydrocannabinol (THCd9; CAS #: 1972-08-3)
• Δ9-Tetrahydrocannabinolic acid A (THCA or THCAA; CAS #: 23978-85-0)
Standard preparation
• All standard solutions are to be prepared at a concentration of 400 µg/mL.
• All standards are prepared in methanol.
Reagent preparation
• 1 L reagent for Mobile Phase A: combine 999 ml of HPLC grade water with 210 ul of formic acid and then add 0.3153g of ammonium formate. Lastly add 1000 ul of formic acid to solution.
• 1 L reagent for Mobile Phase B: add 999 ml of acetonitrile to 1 L bottle then add 1 ml of formic acid to solution.
Protocol
1. Dry inflorescences at room temperature (20 °C) to < 10% moisture.
2. Grind samples.
3. Sieve through a number 35 sieve (500 μm).
4. Sieve through a number 10 sieve (2000 μm)
5. Place 0.1000-0.1050 grams of ground hemp material in a 20 mL glass scintillation vial.
6. Prepare each sample in triplicate.
7. Add 20 mL of HPLC grade 200-proof ethanol to the sample.
8. Sonicate for 30 min at room temperature.
9. Remove from sonication bath.
10. Allow the sample to sit overnight (18 h) in the dark at room temperature.
11. The next day filter the supernatant through a 0.45 μm Nylon membrane filter into a sample vial for analysis.
12. Inject 1 µL sample volume.
13. Set flow rate to 0.350 mL/min.
14. HPLC gradient program: 0-2 min, 75% B (concave curve 9); 2-5 min, 90% B (concave curve 9); 5-5.1 min, 90% B (linear curve 5); 5.1-11.1 min, 100% B; 11.1-11.2 min, 100% B (linear curve 5); and 11.2-20, 75% B.
15. The column temperature is set to 10°C
16. Ten-point standard curves are developed for the cannabinoids using a setting of 228 nm from the diode array detector.
Methods provided by Berhow and Gude (2021) and modified by Dr. Korey Brownstein in 2023.
Edited 11/18/2024 by Kevin Hernandez.