Reliable and robust method for the analysis of cannabinoids and terpenes in cannabis
The present invention teaches methods useful for accurately and precisely analyzing cannabis plants, plant parts, and extract samples. In particular, the present invention teaches methods of extracting, and quantifying cannabinoid and terpene constituents.
1. A method for determining a concentration of at least one cannabinoid and at least one terpene in a test sample, the method comprising the steps of:
a) extracting the test sample in an extraction solution, thereby producing an extracted test sample comprising the at least one cannabinoid and at least one terpene dissolved in the extraction solution, wherein the extraction solution comprises:
i) a solvent;
ii) a first internal standard with a known response ratio relative to the at least one cannabinoid;
iii) a second internal standard with a known response ratio relative to the at least one terpene; and
iv) a third internal standard;
b) diluting the extracted test sample produced by step a) one or more times with a dilution solution; wherein said dilution solution comprises the solvent, the first internal standard, and the second internal standard, but not the third internal standard;
c) subjecting the extracted test sample produced by step a), and/or the diluted test sample(s) produced by step b) to at least one chromatographic separation and detecting the separated sample(s) with at least one detector; and
d) determining the concentration of the at least one cannabinoid and the at least one terpene in the test sample; wherein the concentration of the at least one cannabinoid in the test sample is normalized for dilution errors based on the concentration of the third internal standard in the diluted test sample(s) produced by step b).
2. The method of claim 1 , wherein the at least one chromatographic separation is high performance liquid chromatography (HPLC).
3. The method of claim 2 , wherein the at least one detector is a photodiode array detector.
4. The method of claim 1 , wherein the at least one chromatographic separation is gas chromatography (GC).
5. The method of claim 4 , wherein the at least one detector is a flame ionization detector.
6. The method of claim 1 , wherein the at least one cannabinoid is selected from the group consisting of: tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), tetrahydrocannabinol (THC), cannabidiol (CBD), cannabiserol (CBG), cannabichromene (CBC), and delta-8-tetrahydrocannabinol (delta-8 THC).
7. The method of claim 1 , wherein the at least one cannabinoid is tetrahydrocannabinol (THC).
8. The method of claim 1 , wherein the at least one terpene is selected from the group consisting of: terpinolene, alpha phellandrene, beta ocimene, carene, limonene, gamma terpinene, alpha pinene, alpha terpinene, beta pinene, fenchol, camphene, alpha terpineol, alpha humulene, beta caryophyllene, linalool, caryophyllene oxide, and myrcene.
9. The method of claim 1 , wherein the solvent is ethanol.
10. The method of claim 1 , wherein the first internal standard is ibuprofen.
11. The method of claim 1 , wherein the second internal standard is n-nonane.
12. The method of claim 1 , wherein the third internal standard is 4-biphenyl carboxylic acid (BPCA).
13. The method of claim 1 , wherein the first internal standard is ibuprofen, the second internal standard is n-nonane, and the third internal standard is 4-biphenyl carboxylic acid.
14. A method for quantifying cannabinoids and terpenes from a test sample, said method comprising the steps of:
a) extracting the test sample in an extraction solution, thereby producing an analytical extract comprising the cannabinoids and terpenes dissolved in the extraction solution, said extraction solution comprising:
i. a solvent;
ii. a first internal standard, and;
iii. a second internal standard; and
b) performing a high performance liquid chromatography (HPLC), and a gas chromatography-flame ionization detector (GC-FID) analysis of the analytical extract of step a) to produce a signal for at least one cannabinoid and at least one terpene, and to produce a signal for each of the standards;
wherein the signal for the at least one cannabinoid is normalized based on the signal from the first internal standard to quantify said at least one cannabinoid in the test sample, and wherein the signal for the at least one terpene is normalized based on the signal from the second internal standard to quantify said at least one terpene in the test sample; wherein the first internal standard is ibuprofen at a known concentration, and the second internal standard is n-nonane at a known concentration.
15. The method of claim 14 wherein the quantification of cannabinoids is performed via high performance liquid chromatography (HPLC).
16. The method of claim 14 , wherein the quantification of terpenes is performed via gas chromatography-flame ionization detector (GC-FID).
17. The method of claim 14 , wherein the analytical extract is diluted in a dilution solution prior to undergoing the high performance liquid chromatography (HPLC) and/or the gas chromatography-flame ionization detector (GC-FID) analysis of step b); wherein the extraction solution comprises a third internal standard, and wherein the dilution solution is identical to the extraction solution except for the presence of the third internal standard.
18. The method of claim 17 , wherein the signal for the at least one cannabinoid is normalized based on the signal from the first internal standard and the third internal standard to quantify said at least one cannabinoid in the test sample, and/or wherein the signal for the at least one terpene is normalized based on the signal from the second internal standard and the third internal standard to quantify said at least one terpene in the test sample.
19. The method of claim 17 , wherein the third internal standard is 4-biphenyl carboxylic acid (BPCA).
20. The method of claim 14 , wherein the solvent is ethanol.