Preparation and quantitative analysis method of 9-tetrahydrocannabinol cis-trans isomer

Abstract

The invention discloses a preparation and quantitative analysis method of a 9-tetrahydrocannabinol cis-trans isomer. The invention provides a preparation method of (+ / -)-9-trans-THC or (+ / -)-9-cis-THC. The preparation method comprises the following steps: separating a mixture of (+ / -)-9-trans-THC and (+ / -)-9-cis-THC by adopting chromatography; the separation conditions of the chromatography are as follows: a chromatographic column is a C18 chromatographic column; the mobile phase is acetonitrile and water; the volume ratio of the acetonitrile to the water is (7: 3)-(3: 1). The (+ / -)-9-trans-THC and (+ / -)-9-cis-THC prepared by the method disclosed by the invention are high in purity, and the quantitative analysis method provided by the invention realizes accurate quantification under the condition of no standard reference substance, and also has the advantages of no damage to samples, simple preparation, high analysis speed, good accuracy, good repeatability and the like.

Description

Technical Field

[0001] The present invention relates to a 9 -Preparation and quantitative analysis of tetrahydrocannabinol cis-trans isomers. Background Art

[0002] Cannabis sativa L. is an annual herbaceous plant of the genus Cannabis in the Moraceae family. It is distributed throughout the world and has a long history of medicinal use. Tetrahydrocannabinol (THC) is a cannabinoid isolated from the glandular hairs of the leaves and flowers of female cannabis plants. 9 -THC is the main psychoactive ingredient in cannabis. 9 -THC content in dry weight can be divided into drug hemp, intermediate hemp and industrial hemp. 9 -THC content is less than 0.3% by dry weight, and the risk of abuse is extremely low.

[0003] THC is C 21 Terpene phenolic skeleton compounds, there are many isomers in the cannabis plant that are completely different from THC in chemical structure and pharmacological activity, such as △ 8 -THC, cannabidiol (CBD), etc. 9 -THC has a unique tricyclic structure and contains two chiral carbon atoms, namely carbon atoms at positions 6a and 10a, so there are theoretically four stereoisomers, such as Figure 1 Although the World Health Organization clearly stated in its 2018 report that only (-)-trans-△ 9 -THC((-)-trans-△ 9 -THC), but studies published in 1977 and 2021 showed that cis- 9 -THC(cis-△ 9 -THC) is clearly present in fiber-type industrial hemp with low THC content.

[0004] Modern pharmacological research has confirmed that: (-)-trans-△ 9 -THC not only activates CB1 receptors and has certain psychoactive properties, but also acts on CB2 receptors. Moreover, at different dosage concentrations, it can act on multiple receptors and ion channels outside the endocannabinoid system, and has multiple biological effects such as anti-inflammatory, analgesic, and immune. In 1985, the US FDA approved the synthetic (-)-trans-△ 9Dronabinol (THC) is used to treat nausea and vomiting in cancer chemotherapy patients unrelieved by conventional antiemetics. It was later approved for anorexia nervosa and wasting syndrome in AIDS patients. However, extensive clinical experience indicates that the drug activity of stereoisomers containing chiral centers can differ significantly. When a mixture of stereoisomers, potentially caused by a chiral center, is used as a drug, it is crucial to understand the biological properties of the different isomers and the absolute content of the different stereoisomers in the mixture.

[0005] In my country, Yunnan Province passed local legislation in 2005 and Heilongjiang Province in 2017, allowing the cultivation, planting, and processing of industrial hemp under public security supervision. In order to maximize the utilization of hemp plant resources, the laboratory has previously studied the commercial industrial hemp flowers and leaves grown in the fields of Yunnan Province to clarify the cis-△ 9 -The presence of THC. At the same time, solving the problem of natural sources△ 9 - trans / cis-△ in THC 9 - Accurate quantification of THC is crucial for the subsequent development and rational utilization of industrial hemp plant sources. 9 -THC is crucial.

[0006] Summary of the Invention

[0007] The technical problem to be solved by this invention is to solve the problem caused by existing legal restrictions. 9 -To solve the problem of limited sources of standard or reference substances for the study of different stereoisomers of THC, a new △ 9 -THC cis-trans isomers 1 H qNMR quantitative analysis method.

[0008] The present invention solves the above technical problems through the following technical solutions.

[0009] The present invention provides a (±)-△ 9 -trans-THC or (±)-△ 9 -cis-THC preparation method, comprising the following steps: using chromatography to separate (±)-△ 9 -trans-THC and (±)-△ 9 -cis-THC mixture; the separation conditions of the chromatography are:

[0010] The chromatographic column is a C18 column;

[0011] The mobile phase was acetonitrile and water;

[0012] The volume ratio of the acetonitrile to the water is 13:7-3:1.

[0013] The chromatographic column can be a semi-preparative chromatographic column or a preparative chromatographic column, for example, a Waters Symmetry PrepC18 semi-preparative chromatographic column.

[0014] In the preparation method, the volume ratio of the acetonitrile to the water may be 7:3.

[0015] In the preparation method, the mobile phase is isocratic elution.

[0016] In the preparation method, the flow rate of the mobile phase of the semi-preparative chromatographic column may be 3 to 5 mL / min; and / or the flow rate of the mobile phase of the preparative chromatographic column may be 8 to 12 mL / min.

[0017] In the preparation method, the detector of the chromatography is a UV detector; the detection wavelength of the detector is preferably 210 nm.

[0018] In the preparation method, the (±)-△ 9 -trans-THC and (±)-△ 9 The mixture of -cis-THC is prepared by the following purification method, which comprises the following steps:

[0019] The raw materials were separated and purified by normal phase silica gel chromatography column, reverse phase silica gel chromatography column and Sephadex LH-20 chromatography column to obtain △ 9 -THC products;

[0020] The raw material is selected from an extract of industrial hemp flowers and leaves, such as a solvent extraction extract or a supercritical extraction extract;

[0021] The mobile phase of the normal phase silica gel chromatography column is petroleum ether and dichloromethane;

[0022] The mobile phase of the reversed-phase silica gel chromatography column is acetonitrile and water;

[0023] The mobile phase of the Sephadex LH-20 chromatography column is methanol and water.

[0024] In the purification method, the normal phase silica gel chromatography column removes most of the non-cannabinoid components (such as flavonoids and / or fatty acids) and cannabidiol (CBD) in the raw material.

[0025] In the purification method, the reverse phase silica gel chromatography column removes other cannabinoid components in the fraction, such as cannabidiol (CBDV).

[0026] In the purification method, in the normal phase silica gel chromatography column, the mass ratio of the raw material to the silica gel can be 1:20 to 1:30, preferably 1:20.

[0027] In the purification method, in the normal phase silica gel chromatography column, the mobile phase is gradient elution; the gradient elution conditions may be:

[0028]

[0029] In the purification method, in the normal phase silica gel chromatography column, the gradient elution may include the following steps: the mobile phase flow rate is 10 mL / min, in the first stage, the petroleum ether: dichloromethane (volume ratio) is 10:3, and the elution volume is 14 L; in the second stage, the petroleum ether: dichloromethane (volume ratio) is 10:4, and the elution volume is 9 L; in the third stage, the petroleum ether: dichloromethane (volume ratio) is 10:5, and the elution volume is 3 L.

[0030] In the purification method, in the reverse phase silica gel chromatography column, the mass ratio of the raw material purified by the normal phase silica gel chromatography column to the silica gel can be 1:20 to 1:30, preferably 1:20.

[0031] In the purification method, in the reverse-phase silica gel chromatography column, the volume fraction of acetonitrile in water can be 60-100%, preferably 70%.

[0032] In the purification method, in the reverse-phase silica gel chromatography column, the flow rate of the mobile phase can be 0.8 to 1.2 mL / min, preferably 1 mL / min.

[0033] In the purification method, in the Sephadex LH-20 chromatography column, the volume fraction of methanol in water can be 70-100%, preferably 80%.

[0034] In the purification method, in the Sephadex LH-20 chromatography column, the flow rate of the mobile phase can be 10 to 20 mL / h, preferably 10 mL / h.

[0035] In the purification method, the silica gel in the normal phase silica gel chromatography column and the reverse phase silica gel chromatography column can be 200-300 mesh silica gel.

[0036] In the purification method, the chromatography column separation can be the following steps: 9 -THC raw materials were mixed and then gradient eluted through normal phase silica gel chromatography column to remove the △ 9 The eluted fraction of -THC was mixed again and separated by reverse phase silica gel chromatography. 9 -THC fraction was separated by Sephadex LH-20 column chromatography, and the solvent in the fraction was removed by vacuum rotary evaporation to obtain △ 9 -THC products; the sample is a silica gel sample.

[0037] In the purification method, the temperature for separation and purification may be room temperature.

[0038] The present invention also provides a 9 -THC purification method, which comprises the following steps:

[0039] The raw materials were separated and purified by normal phase silica gel column, reverse phase silica gel column and Sephadex LH-20 column to obtain △ 9 -THC products;

[0040] The raw material is selected from the extract of industrial hemp flowers and leaves;

[0041] The mobile phase of the normal phase silica gel chromatography column is petroleum ether and dichloromethane;

[0042] The mobile phase of the reversed-phase silica gel chromatography column is acetonitrile and water;

[0043] The mobile phase of the Sephadex LH-20 chromatography column is methanol and water.

[0044] The extraction conditions and steps in the purification method are the same as those described in the previous purification method.

[0045] The present invention also provides a 9 -THC cis-trans isomer quantitative analysis method, comprising the following steps: using a nuclear magnetic resonance instrument to measure the nuclear magnetic spectrum of the test solution; calculating by an internal standard method to obtain (±)-trans-△ 9 -THC and (±)-cis-△ 9 -THC content;

[0046] The nuclear magnetic resonance apparatus 1 The H nuclear resonance frequency is 600 MHz;

[0047] The nuclear magnetic resonance instrument adopts zg30 pulse sequence;

[0048] The test solution is a mixed solution of the test substance, the internal standard substance and the deuterated solvent;

[0049] The analyte contains any proportion of (±)-trans-△ 9 -THC and (±)-cis-△ 9 -THC;

[0050] The internal standard substance is tetrachloronitrobenzene.

[0051] In the quantitative analysis method, the analyte is (±)-△ 9 -trans-THC or (±)-△ 9 -cis-THC.

[0052] In the quantitative analysis method, the concentration of the analyte in the test solution may be 0.1 to 20 mg / mL.

[0053] In the quantitative analysis method, the internal standard substance can be configured as a methanol solution of the internal standard substance; the concentration of the methanol solution can be 1 mg / mL.

[0054] In the quantitative analysis method, the deuterated solvent can be a conventional deuterated solvent in the art, which can completely dissolve the sample to be tested and the internal standard; preferably, it is deuterated methanol.

[0055] In the quantitative analysis method, the spectrum width (SWH) of the nuclear magnetic resonance instrument is preferably 12000 Hz.

[0056] In the quantitative analysis method, the sampling point number (TD) of the nuclear magnetic resonance instrument is preferably 6.4×10 4 .

[0057] In the quantitative analysis method, the sampling time (AQ) of the nuclear magnetic resonance instrument is preferably 2.75 s.

[0058] In the quantitative analysis method, the relaxation delay time (D1) of the nuclear magnetic resonance instrument is sufficient to restore the equilibrium of protons in different chemical environments in all compounds in the sample to be tested after transition, preferably 60s, which is sufficient to cover the longitudinal relaxation time of protons in different chemical environments.

[0059] In the quantitative analysis method, the sampling number (NS) of the nuclear magnetic resonance instrument is preferably at least 16.

[0060] In the quantitative analysis method, the number of empty scans (DS) of the nuclear magnetic resonance instrument is preferably 2.

[0061] In the quantitative analysis method, the test parameters of the nuclear magnetic resonance instrument can be obtained by using the zg30 pulse sequence at 298K. 1 H qNMR spectrum. The parameters were set as follows: spectrum width (SWH) 12 000 Hz, sampling point number (TD) 6.4×10 4 , sampling time (AQ) 2.75s, relaxation delay time (D1) 60s, number of sampling times (NS) 16, number of dry scans (DS) 2.

[0062] In the present invention, preferably, the detection temperature of the nuclear magnetic resonance apparatus is room temperature or below.

[0063] Preferably, the temperature of the sample chamber used for the measurement is lower than the boiling points of the analyte, the internal standard and the deuterated solvent in the solution to be measured.

[0064] In the present invention, the room temperature may be 20-32°C.

[0065] Except for △ 9 Except for THC-related materials, all reagents used in the present invention are commercially available.

[0066] The positive progress effect of the present invention is:

[0067] (1) The present invention provides 9 -THC product purification method, prepared △ 9 -High THC purity;

[0068] (2) (±)-△ obtained by separation of the present invention 9 -trans-THC and (±)-△ 9 -cis-THC has high purity;

[0069] (3) The quantitative analysis method provided by the present invention achieves accurate quantification in the absence of standard reference substances. In addition, it has the advantages of not destroying the sample, being simple to prepare, fast in analysis, and having good accuracy and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 △ 9 -HPLC spectra of THC cis and trans isomers;

[0071] Figure 2 This is the standard curve diagram of Example 2. DETAILED DESCRIPTION

[0072] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0073] The industrial hemp flower and leaf raw materials are provided by Yunnan Hangu Biotechnology Co., Ltd.

[0074] Example 1: Natural Source 9 - Preparation of THC products

[0075] The industrial hemp flower and leaf extract provided by Yunnan Hangu Company was used as raw material for column chromatography to further separate and purify the industrial hemp plant-derived △ 9-THC component. The following separation and purification processes are all completed at room temperature. First, the industrial hemp flower and leaf extract extract is mixed and passed through a normal phase silica gel (200:300 mesh) chromatography column. The mass ratio of the raw material to the silica gel is about 1:20. Petroleum ether: dichloromethane gradient elution is used to remove most of the non-cannabinoid components such as flavonoids, fatty acids and cannabidiol (CBD) in the raw material. Among them, the chromatographic fluid flow rate is 10mL / min, and the elution is carried out at a ratio of 10:3, 10:4, and 10:5, respectively. The elution volumes of the chromatographic fluid are 14L, 9L, and 3L, respectively, which are about 5.6, 3.6, and 1.2 column volumes. The target components in the eluted fractions are detected by high performance thin layer chromatography (HPTLC), where HPTLC uses HSGF 254 Silica gel plate, with n-hexane: dichloromethane (1:1) as the developing solvent, detected by iodine fumigation color development. After the effective components collected according to the HPTLC monitoring results were mixed with samples, 70% acetonitrile-water was used as the eluent for reverse phase silica gel chromatography, with a flow rate of about 1 mL / min and a mass ratio of effective components to silica gel of about 1:20, in order to remove other cannabinoid components in the fraction, such as cannabidiol (CBDV). The fractions were combined according to the HPTLC results and further purified by Sephadex LH-20 (80% methanol-water, flow rate 10 mL / h) column chromatography to obtain a fraction mainly containing natural △ 9 -THC fraction, after vacuum rotary evaporation to remove the solvent, a brownish-yellow translucent fluid extract was obtained, which is the natural source△ 9 -THC products. Natural origin detected by HPLC 9 -The purity of the target component in THC products, the results show that the main component △ 9 The purity of -trans-THC is 92.99%.

[0076] Example 2: △ 9 -THC cis-trans isomers 1 H Linearity investigation of qNMR quantitative method

[0077] △ 9 Preparation method of self-made reference substance of THC cis-trans isomers:

[0078] Take the natural source △ in Example 1 9 -THC products were dissolved in an appropriate amount of methanol and injected into a high performance liquid chromatograph using a Waters Symmetry PrepC18 semi-preparative column (7.8×300 mm, 7 μm) with an acetonitrile-water solution (70:30) as the mobile phase and a UV detector at a wavelength of 210 nm. (±)-△ 9 -trans-THC and (±)-△ 9 -cis-THC outflow part, the solvent is recovered under reduced pressure and evaporated to dryness to obtain △ 9 -THC cis-trans isomer working reference ( Figure 1 ).

[0079] Internal standard solution: Accurately weigh an appropriate amount of tetrachloronitrobenzene, dissolve it in methanol and prepare an internal standard solution containing approximately 1 mg of tetrachloronitrobenzene per 1 mL.

[0080] Working control solution: accurately weigh △ 9 -trans-THC and △ 9 -cis-THC working reference substance was dissolved in methanol so that each 1 mL contained △ 9 -trans-THC and △ 9 -cis-THC (approximately 20 mg and 2 mg). Dilute the above solution with methanol to create a series of working solutions with varying concentration gradients. Accurately measure 1 mL of each working solution and 1 mL of the internal standard solution into the same 10 mL round-bottom flask. After rotary evaporation, reconstitute with 1 mL of deuterated methanol and transfer an appropriate amount of the solution to a 5 mm NMR tube for analysis.

[0081] 1 H qNMR analysis conditions: 1H qNMR spectra were acquired at 298 K using the zg30 pulse sequence. The parameters were set as follows: spectrum width (SWH) 12 000 Hz, sampling number (TD) 6.4 × 10 4 , sampling time (AQ) 2.75s, relaxation delay time (D1) 60s, number of sampling times (NS) 16, number of dry scans (DS) 2.

[0082] The raw spectra were collected and Fourier transformed, zero-filled, windowed, phase-corrected, and baseline-corrected using TopSpin 3.6.0 software (Bruker Biospin, Rheinsteten, Germany). The solvent CD3OD was used as the chemical shift reference (3.310 ppm). The chemical shift of the internal standard tetrachloronitrobenzene was 8.15 ppm. δ6.43 and δ6.31 were used for Δ 9 -trans-THC and △ 9 Absolute quantification of -cis-THC.

[0083] △ 9 -cis-THC, 1H-NMR (CD3OD, 600MHz) δ6.31(m,1H),6.16(d,J=1.6,1H),6.05(d,J=1.6,1H),3.50(m,1H),2.39(t,J=7.7,2H),1.96(m,2H),1.88(m ,1H),1.70(m,1H),1.65(s,3H),1.55(m,2H),1.41(m,1H),1.34(s,3H),1.32(m,2H),1.28(m,2H),1.22(s,3H),0.90(t,J=6.9,3H).

[0084] △ 9 -trans-THC, 1 H-NMR (CD3OD, 600MHz) δ6.43(m,1H),6.16(d,J=1.6,1H),6.07(d,J=1.6,1H),3.15(m,1H),2.40(t,J=7.6,2H),2.14(m,2H),1.93(m ,1H),1.65(s,3H),1.60(m,1H),1.56(m,2H),1.41(m,1H),1.37(s,3H),1.33(m,2H),1.33(m,2H),1.04(s,3H),0.90(t,J=6.9,3H).

[0085] Linearity results: Under the above NMR data acquisition and processing conditions, the 9 -trans / cis-THC linearity test, record △ 9 -trans / cis-THC and internal standard peak area ratio, and plot it against different concentration gradients. 9 -trans / cis-THC standard curve ( Figure 2 ), the results show △ 9 -trans-THC and △ 9 -cis-THC were 5.133×10 -2 A good linear relationship was found in the range of 5.033×10-20.53 mg / mL and 5.033×10-2.013 mg / mL, and the correlation coefficient r was greater than 0.999.

[0086] Example 3: △ 9 -THC cis-trans isomers 1 H Repeatability investigation of qNMR quantitative method

[0087] Accurately weigh the natural source △ in Example 1 9 -THC product (extract) in appropriate amount, add methanol to dissolve so that each 1mL contains △ 9 -trans-THC and △ 9-cis-THC is approximately 1 mg and 0.1 mg. Accurately measure 1 mL of the above solution and 1 mL of internal standard solution into a 10 mL round-bottom flask. Rotary evaporate and reconstitute with 1 mL of deuterated methanol. Transfer to a 5 mm NMR tube for analysis.

[0088] Preparation method of internal standard solution and 1 The H qNMR analysis conditions were the same as in Example 2.

[0089] The repeatability results are shown in Table 1 below. The RSD of the content calculated from 6 tests is less than 2%, indicating good repeatability.

[0090] Table 1 Repeatability test results

[0091]

[0092] Example 4: △ 9 -THC cis-trans isomers 1 H Accuracy investigation of qNMR quantitative method

[0093] Natural source prepared according to Example 3 9 -THC product test solution was used as blank solution, and △ 9 -trans-THC and △ 9 -cis-THC working standard, each level of parallel preparation 3, determination, calculate the recovery rate of the sample. 1 The H qNMR analysis conditions were the same as in Example 2.

[0094] The accuracy results are shown in Table 2 below. The calculated recovery rates of 9 additions are between 90% and 110%, indicating good accuracy.

[0095] Table 2 Sample recovery determination results

[0096]

[0097] Example 5: 1 H Comparison of qNMR results with liquid chromatography results

[0098] Solution preparation:

[0099] 1 H qNMR solution: Accurately weigh the natural source △ in Example 1 9 -THC product, add methanol solvent to completely dissolve it to obtain the stock solution. Accurately pipette an appropriate amount of the above stock solution and 1 mL of internal standard solution into a 10 mL round-bottom flask, dry under reduced pressure until the solvent evaporates, then add 1 mL of deuterated methanol and dissolve the contents fully. Mix evenly to obtain a final product containing (±)-△ 9 -trans-THC and (±)-△ 9-cis-THC is approximately 1 mg / mL and 0.1 mg / mL. Transfer the above test solution into a 5mm NMR tube for testing.

[0100] HPLC solution: take 1 H qNMR solution was diluted with methanol to make the content of (±)-△ 9 -trans-THC and (±)-△ 9 -cis-THC test solution containing approximately 0.02 mg and 0.005 mg.

[0101] Analysis conditions:

[0102] 1 The H qNMR analysis conditions were the same as in Example 2.

[0103] HPLC analysis conditions: An Agilent Poroshell 120 EC-C18 (4.6 × 250 mm, 4 μm) analytical column was used at 25°C. The mobile phase consisted of 0.1% phosphoric acid solution in acetonitrile (30:70 v / v), with an isocratic elution time of 50 min. The flow rate was 1.0 mL / min, and the injection volume was 10 μL. The detection wavelength was 210 nm.

[0104] 1 The results of the H qNMR and HPLC assays are shown in Table 3. 1 The results of H qNMR and HPLC were basically consistent. 9 -trans-THC and (±)-△ 9 The relative deviations of the -cis-THC measurements were 0.32% and 3.74%, respectively. 1 Such deviation is acceptable due to unavoidable factors such as the relatively low sensitivity of the H qNMR method, the inherent deviation of the instrument, and the error caused by sample preparation.

[0105] Table 3 1 H qNMR and HPLC assay results

[0106]

Claims

1. A(±)-△ 9 -trans-THC or (±)-△ 9 -cis-THC preparation method, characterized in that, The method comprises the following steps: using chromatography to separate (±)-△ 9 -trans-THC and (±)-△ 9 -cis-THC mixture; the separation conditions of the chromatography are: The chromatographic column is a C18 column; The mobile phase was acetonitrile and water; The volume ratio of the acetonitrile to the water is 13:7-3:

1.

2. The preparation method according to claim 1, wherein It meets one or more of the following conditions: (1) The chromatographic column is a semi-preparative chromatographic column or a preparative chromatographic column, for example, a Waters Symmetry PrepC18 semi-preparative chromatographic column; (2) the volume ratio of the acetonitrile to the water is 7:3; (3) the mobile phase is isocratic elution; (4) The mobile phase flow rate of the semi-preparative chromatographic column is 3 to 5 mL / min; (5) The mobile phase flow rate of the preparative chromatographic column is 8 to 12 mL / min; (6) The detector of the chromatography method is a UV detector; the detection wavelength of the detector is preferably 210 nm.

3. The preparation method according to claim 1, wherein (±)-△ 9 -trans-THC and (±)-△ 9 The mixture of -cis-THC is prepared by the following purification method, which comprises the following steps: The raw materials were separated and purified by normal phase silica gel chromatography column, reverse phase silica gel chromatography column and Sephadex LH-20 chromatography column to obtain △ 9 -THC products; The raw material is selected from an extract of industrial hemp flowers and leaves, such as a solvent extraction extract or a supercritical extraction extract; The mobile phase of the normal phase silica gel chromatography column is petroleum ether and dichloromethane; The mobile phase of the reversed-phase silica gel chromatography column is acetonitrile and water; The mobile phase of the Sephadex LH-20 chromatography column is methanol and water.

4. The preparation method according to claim 3, wherein The normal phase silica gel chromatography column removes most of the non-cannabinoid components (such as flavonoids and / or fatty acids) and cannabidiol (CBD) in the raw material; and / or, the reverse phase silica gel column removes other cannabinoid components in the fraction, such as cannabidivarin (CBDV).

5. The preparation method according to claim 3, wherein It meets one or more of the following conditions: (1) The silica gel in the silica gel chromatography column is 200-300 mesh silica gel; (2) In the normal phase silica gel chromatography column, the mass ratio of the raw material to the silica gel is 1:20 to 1:30, preferably 1:20; (3) In the normal phase silica gel chromatography column, the mobile phase is gradient elution; preferably, the gradient elution conditions are: Preferably, the gradient elution comprises the following steps: the mobile phase flow rate is 10 mL / min, the first stage, petroleum ether: dichloromethane (volume ratio) is 10:3, and the elution volume is 14 L; the second stage, petroleum ether: dichloromethane (volume ratio) is 10:4, and the elution volume is 9 L; the third stage, petroleum ether: dichloromethane (volume ratio) is 10:5, and the elution volume is 3 L; (4) In the reversed-phase silica gel chromatography column, the volume fraction of acetonitrile in water is 60 to 100%, preferably 70%; (5) In the reverse phase silica gel chromatography column, the mass ratio of the raw material after purification by the normal phase silica gel chromatography column to the silica gel is 1:20 to 1:30, preferably 1:20; (6) In the reversed-phase silica gel chromatography column, the flow rate of the mobile phase is 0.8 to 1.2 mL / min, preferably 1 mL / min; (7) In the Sephadex LH-20 chromatography column, the volume fraction of methanol in water is 70-100%, preferably 80%; (8) In the Sephadex LH-20 chromatography column, the flow rate of the mobile phase is 10 to 20 mL / h, preferably 10 mL / h.

6. The preparation method according to any one of claims 3 to 5, characterized in that The chromatography column separation comprises the following steps: 9 -THC raw materials were mixed and then gradient eluted through normal phase silica gel chromatography column to remove the △ 9 The eluted fraction of -THC was mixed again and separated by reverse phase silica gel chromatography. 9 -THC fraction was separated by Sephadex LH-20 column chromatography, and the solvent in the fraction was removed by vacuum rotary evaporation to obtain △ 9 -THC products; the sample is a silica gel sample.

7. A kind of △ 9 -THC purification method, characterized in that, It includes the following steps: The raw materials were separated and purified by normal phase silica gel column, reverse phase silica gel column and Sephadex LH-20 column to obtain △ 9 -THC products; The raw material is selected from the extract of industrial hemp flowers and leaves; The mobile phase of the normal phase silica gel chromatography column is petroleum ether and dichloromethane; The mobile phase of the reversed-phase silica gel chromatography column is acetonitrile and water; The mobile phase of the Sephadex LH-20 chromatography column is methanol and water.

8. The purification method according to claim 7, wherein: The extraction conditions and steps in the purification method are as described in any one of claims 3 to 6.

9. A kind of △ 9 - A method for quantitative analysis of THC cis and trans isomers, characterized in that The method comprises the following steps: using a nuclear magnetic resonance instrument to measure the nuclear magnetic spectrum of the solution to be tested; calculating (±)-trans-△ 9 -THC and (±)-cis-△ 9 -THC content; The nuclear magnetic resonance apparatus 1 The H nuclear resonance frequency is 600 MHz; The nuclear magnetic resonance instrument adopts zg30 pulse sequence; The test solution is a mixed solution of the test substance, the internal standard substance and the deuterated solvent; The analyte is (±)-trans-△ 9 -THC and (±)-cis-△ 9 -THC; The internal standard substance is tetrachloronitrobenzene.

10. The quantitative analysis method according to claim 9, wherein It meets one or more of the following conditions: (1) The analyte is (±)-△ obtained by the preparation method according to any one of claims 1 to 6 9 -trans-THC or (±)-△ 9 -cis-THC; (2) The concentration of the analyte in the test solution is 0.1 to 20 mg / mL; (3) The internal standard substance is a methanol solution of the internal standard substance; preferably, the concentration of the methanol solution is 1 mg / mL; (4) The deuterated solvent is deuterated methanol; (5) The spectrum width (SWH) of the NMR instrument is 12000 Hz; (6) The sampling point number (TD) of the NMR instrument is 6.4×10 4 ; (7) The sampling time (AQ) of the NMR instrument is 2.75 s; (8) The relaxation delay time (D1) of the nuclear magnetic resonance instrument is 60s; (9) The sampling number (NS) of the nuclear magnetic resonance instrument is 16; (10) The number of empty scans (DS) of the NMR instrument is 2; (11) The test parameters of the NMR instrument are: using zg30 pulse sequence to obtain 1 H qNMR spectrum; the parameters were set as follows: spectrum width (SWH) 12 000 Hz, sampling point number (TD) 6.4×10 4 , sampling time (AQ) 2.75s, relaxation delay time (D1) 60s, number of sampling times (NS) 16, number of dry scans (DS) 2.

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