Method for analyzing active ingredients of cannabis and control program for liquid chromatograph

Patent metadata

Field	Value
Publication number	US-11099162-B2
Application number	US-201716338208-A
Country	US
Kind code	B2
Filing date	Aug 3, 2017
Priority date	Sep 30, 2016
Publication date	Aug 24, 2021
Grant date	Aug 24, 2021

Abstract

In an LC system using an ODS column (15) and UV detector (17), a cannabis-derived sample is analyzed by gradient elution using a phosphoric acid aqueous solution and phosphoric-acid-containing methanol. A control unit (3) regulates the openings of solenoid valves in a mixer (12) so that the increase rate of the mixture ratio of the phosphoric-acid-containing methanol in a second part of the analysis period is higher than in a first part. By this operation, ten active ingredients (including Total THC, Total CBD and CBN) contained in cannabis can be satisfactorily separated within an analysis time which is equal to or even shorter than approximately 30 minutes. Each ingredient separated by the column (15) is detected by the UV detector (17). An active ingredient identification processor (22) identifies the ten active ingredients based on the retention times of the peaks on a chromatogram created from the detection signals.

First claim

The invention claimed is:

1. A method for analyzing a plurality of active ingredients contained in cannabis using a liquid chromatograph, the method comprising:
a) a separation step, in which a plurality of components contained in a liquid sample are separated from each other by gradient elution using an ODS column as a column, with a phosphoric acid aqueous solution and phosphoric-acid-containing methanol as mobile phases;
b) a detection step, in which each component separated in the separation step is detected with a detector which is either an ultraviolet spectrometric detector or photodiode array detector; and
c) an identification step, in which a plurality of predetermined active ingredients are identified based on retention times of peaks observed on a chromatogram created based on a detection result obtained in the detection step, wherein:
the plurality of predetermined active ingredients to be identified include a following ten ingredients: tetrahydrocannabivarin (THCV), cannabidiol (CBD), cannabigerol (CBG), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabinol (CBN), Δ9-tetrahydrocannabinol (d9-THC), Δ8-tetrahydrocannabinol (Δ8-THC), a cannabichromene (CBC), and Δ9-tetrahydrocannabinolic acid A (THCA-A); and
a mixture ratio of the phosphoric-acid-containing methanol in the gradient elution in the separation step is changed so that the mixture ratio is increased at a first increase rate and subsequently at a second increase rate which is higher than the first increase rate.

2. The method for analyzing active ingredients of cannabis according to claim 1 , wherein:
at least two ingredients are separated from each other and sequentially eluted from the column within a period of time where the mixture ratio of the phosphoric-acid containing methanol is being increased at the first increase rate, whereas seven ingredients are separated from each other and sequentially eluted from the column within a period of time where the mixture ratio of the phosphoric-acid containing methanol is being increased at the second increase rate.

3. The method for analyzing active ingredients of cannabis according to claim 1 , wherein:
the second increase rate is 3-4 times as high as the first increase rate.

4. The method for analyzing active ingredients of cannabis according to claim 1 , wherein:
the column is an ODS column filled with a packing material having a particle diameter of 2.2 μm and a particle-pore size of 8 nm.

5. The method for analyzing active ingredients of cannabis according to claim 1 , wherein:
the detection wavelength of the detector is 220 nm.

6. A non-transitory computer-readable medium storing a control program for controlling an operation of a liquid chromatograph to perform the method of claim 1 , the liquid chromatograph including: a mobile phase mixer for regulating a mixture ratio of a first mobile phase and a second mobile phase; an injector for injecting a liquid sample into a mixed mobile phase prepared by the mobile phase mixer; a column for separating components contained in the injected liquid sample; and a detector for detecting a component in an eluate exiting from the column, so as to analyze a plurality of active ingredients contained in cannabis using this liquid chromatograph, the control program characterized by performing:
a) a sample injection step, in which the injector is operated so as to inject the liquid sample into the mixed mobile phase while the mobile phase mixer is controlled so that a mixture ratio of the first mobile phase which is the phosphoric acid aqueous solution and the second mobile phase which is phosphoric-acid-containing methanol is maintained in a predetermined state;
b) a gradient elution step, in which the mobile phase mixer is controlled so that, after the liquid sample is injected into the mixed mobile phase in the sample injection step, a mixture ratio of the second mobile phase is increased at a first increase rate for a predetermined period of time, and subsequently, the mixture ratio of the second mobile phase is increased at a second increase rate which is higher than the first increase rate.

7. The computer-readable medium according to claim 6 , wherein:
the second increase rate is 3-4 times as high as the first increase rate.

8. The method for analyzing active ingredients of cannabis according to claim 2 , wherein:
the second increase rate is 3-4 times as high as the first increase rate.

9. The method for analyzing active ingredients of cannabis according to claim 2 , wherein the at least two ingredients are THCV and CBD.

10. The method for analyzing active ingredients of cannabis according to claim 2 , wherein the seven ingredients are CBDA, CBGA, CBN, d9-THC, d8-THC, CBC and THCA-A.

Assignees

• Shimadzu Corp

Inventors

• Arao Yohei

Classifications

• C07C39/08

  Dihydroxy benzenes; Alkylated derivatives thereof · CPC title

• G01N2030/027

  Liquid chromatography · CPC title

• B01D15/325 Primary

  Reversed phase · CPC title

• G16C20/20

  Identification of molecular entities, parts thereof or of chemical compositions · CPC title

• G01N30/34

  of fluid composition, e.g. gradient (G01N30/36 takes precedence) · CPC title