A quantitative analytical method for tetrahydrocannabinol and cannabidiol

Abstract

This invention discloses a quantitative analysis method for tetrahydrocannabinol (THC) and cannabidiol (CBD). The analytical method includes the following steps: injecting the sample into a gas chromatograph, subjecting it to programmed temperature rise, and detecting the total content of CBD and / or THC using an external standard method; wherein the injection port temperature of the gas chromatograph is 200–300°C; the programmed temperature rise is from 150°C to 280°C; and the programmed temperature rise rate is 10–30°C / min. This analytical method can easily and accurately detect the total amount of usable THC and CBD in cannabis flower and leaf raw materials or extracts. Firstly, no special pretreatment of the sample is required; only a certain organic solvent is needed to achieve complete and effective extraction and simple filtration of the cannabinoid components in the sample. Secondly, no THCA and CBDA reference standards are needed to detect the actual total amount of THC and CBD. Thirdly, the quantitative analysis results are accurate and reliable.

Description

Technical Field

[0001] This invention relates to a quantitative analysis method for tetrahydrocannabinol (THC) and cannabidiol (CBD). Background Technology

[0002] Cannabis sativa L. is an annual herbaceous plant belonging to the genus Cannabis in the family Moraceae. It is found wild or cultivated all over the world and has a long history of medicinal use. In traditional Chinese medicine, "huomaren" refers to the seed kernel of cannabis after the shell has been removed. Some traditional medicines, including those in ancient China, also used cannabis seeds and female inflorescences (the part of the cannabis plant with the highest content of cannabinoids).

[0003] Cannabis flowers and leaves contain abundant cannabinoid bioactive substances. To date, more than 100 cannabinoid compounds have been isolated and identified from different cannabis plants, among which tetrahydrocannabinol (THC) and cannabidiol (CBD) are the most well-known.

[0004] However, at present, there is no simple and effective analytical method at the national and local levels for the actual total amount of THC and CBD in industrial hemp flower and leaf raw materials or extracts (including finished products).

[0005] THCA and CBDA (cannabidiol) in cannabis plants are the biosynthetic precursors of THC and CBD, respectively. Under certain conditions, they are decarboxylated and converted into THC and CBD. Therefore, the harvested cannabis flowers and leaves generally contain different proportions of THCA and CBDA.

[0006] Currently, the methods for determining the content of cannabinoid compounds in cannabis flower and leaf raw materials are all based on liquid chromatography. In the patent "A method for determining the CBD and THC content in industrial hemp flowers and leaves (CN 110780003 A)," Liu Shenggui et al. pulverized dried cannabis flowers and leaves, extracted them with methanol using ultrasound, and then used high-performance liquid chromatography (HPLC) with an external standard method to quantitatively determine the content of cannabidiol and tetrahydrocannabinol in the test solution. This method only determined the content of cannabidiol and tetrahydrocannabinol, failing to simultaneously determine the content of cannabidiol acid and tetrahydrocannabinol acid. In the patent "Qualitative and quantitative detection method for one or more substances in CBD, CBDA, and THC (CN 109725080 A)," Zhao Lining et al. used ultrasonic extraction of pulverized cannabis flowers and leaves with methanol, and employed high-performance liquid chromatography with a mixture of acetic acid aqueous solution and acetonitrile as the mobile phase for isocratic elution. The external standard method was used to detect the content of one or more substances in cannabidiol, cannabidiol acid, and tetrahydrocannabinol, but the content of tetrahydrocannabinol acid could not be detected simultaneously.

[0007] None of the above methods can accurately quantify the total amount of THC and CBD actually contained in cannabis plants or extracts (including finished products). Meanwhile, the preparation of THCA and CBDA standards is difficult and difficult to obtain. Therefore, the practical application of methods that simultaneously determine THCA and THC, CBDA and CBD in plants using liquid chromatography to accurately describe the total amount of available THC and CBD in plants is significantly limited. Summary of the Invention

[0008] The technical problem to be solved by the present invention is that there is a lack of methods in the prior art for determining the total amount of tetrahydrocannabinol (THC) and cannabidiol (CBD) in cannabis flowers and leaves. The present invention provides a quantitative analysis method for tetrahydrocannabinol and cannabidiol.

[0009] This invention provides a quantitative analysis method for tetrahydrocannabinol (THC) and cannabidiol (CBD), comprising the following steps: injecting the sample to be tested into a gas chromatograph, subjecting it to programmed temperature rise, and detecting the total content of CBD and / or THC using an external standard method; wherein the programmed temperature rise is from 150°C to 280°C; and the programmed temperature rise rate is 10–30°C / min.

[0010] In this invention, preferably, the injection port temperature of the gas chromatograph is 200–300°C.

[0011] When the injection port temperature of the gas chromatograph is 200-300℃, the total content of cannabidiol is defined as the content of cannabidiol and cannabidiol acid in the sample to be tested; the total content of tetrahydrocannabinol is the content of tetrahydrocannabinol and tetrahydrocannabinol acid.

[0012] In this invention, preferably, the programmed temperature rise can be from 150°C to 280°C within 40 minutes.

[0013] In this invention, preferably, the programmed temperature rise is divided into a first stage of temperature rise and a second stage of temperature rise.

[0014] Preferably, the first stage of heating can be a temperature increase from 150°C to 195°C.

[0015] Preferably, the heating rate of the first stage of heating can be 10℃ / min.

[0016] Preferably, the second stage of heating can be a temperature increase from 195°C to 280°C.

[0017] Preferably, the heating rate in the second stage is 30°C / min.

[0018] Preferably, in the programmed temperature rise, the column temperature is maintained for 10 to 20 minutes, for example, 18 minutes, after the first stage of temperature rise is completed.

[0019] In this invention, the temperature of the detector in the gas chromatograph can be 200–300°C.

[0020] In this invention, the hydrogen flow rate in the gas chromatograph can be 35-45 mL / min.

[0021] In this invention, the air flow rate in the gas chromatograph can be 350-450 mL / min.

[0022] In this invention, preferably, the temperature of the injection port can be 250–300°C.

[0023] In this invention, the sample to be tested can be an extract of cannabis flower and leaf raw materials or a mixture containing cannabidiol, cannabidiol acid, tetrahydrocannabinol and tetrahydrocannabinol acid.

[0024] In this invention, the gas chromatographic column can be an Agilent HP-5 (0.320 mm × 30 m, 0.25 μm).

[0025] In this invention, the injection volume of the sample to be tested can be 1 μL.

[0026] In this invention, the preparation method of the cannabis flower and leaf raw material extract includes the following steps: ultrasonic extraction of the pulverized cannabis flowers and leaves using an organic solvent.

[0027] In this invention, the organic solvent is a conventional organic solvent in the art, and the organic solvent is selected from one or more of methanol, acetone, diethyl ether, cyclohexane, petroleum ether and ethyl acetate; preferably methanol.

[0028] In this invention, the volume-to-mass ratio of the organic solvent to the cannabis flower and leaf raw material can be 50 mL / g.

[0029] In this invention, the analytical method employs either the internal standard method or the external standard method.

[0030] In this invention, the preparation method of cannabis flower and leaf raw material extract includes the following steps: at room temperature, the pulverized cannabis flower and leaf are ultrasonically extracted with an organic solvent, filtered through a 0.22μm microporous membrane, and an internal standard solution of n-eicosane is added.

[0031] In this invention, preferably, the programmed temperature rise is from 150°C to 195°C at a rate of 10°C / min; held for 18 min; then from 195°C to 200°C at a rate of 30°C / min; held for 30 min.

[0032] The inventors, while exploring analytical methods for cannabis flowers and leaves, discovered that increasing the temperature of the gas chromatograph inlet increased the total amount of tetrahydrocannabinol (THC) and cannabidiol (CBD). Based on this, the inventors further researched and found that cannabidiol and tetrahydrocannabinol transform into cannabidiol and tetrahydrocannabinol under heating conditions. Building upon this research, the inventors further developed the gas chromatographic quantitative analysis method of this invention.

[0033] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0034] The room temperature in this invention is generally 20-35℃.

[0035] Except for industrial hemp flower and leaf raw materials, all reagents and raw materials used in this invention are commercially available.

[0036] The positive and progressive effects of this invention are as follows:

[0037] This invention discovers that cannabidiol (CBD) and tetrahydrocannabinol (THC) are decarboxylated and converted to THC and CBD, respectively, when passed through a gas chromatograph. This invention provides a simple and accurate method for detecting the total amount of usable THC and CBD in cannabis flower and leaf raw materials or extracts (including finished products). Firstly, no special pretreatment of the sample is required; only a suitable organic solvent is needed to achieve complete and effective extraction of cannabinoid components and simple filtration. Secondly, no THCA and CBDA standards are needed to detect the actual total amount of THC and CBD. Thirdly, the quantitative analysis results are accurate and reliable. Therefore, the method described in this invention is simple to operate, stable, reproducible, and highly efficient, making it highly practical. Attached Figure Description

[0038] Figure 1 GC chromatogram of CBD reference standard;

[0039] Figure 2 GC chromatogram of THC reference standard;

[0040] Figure 3 Here is a GC image of Yunnan industrial hemp flower and leaf raw materials from Example 1;

[0041] Figure 4 This is a GC image of industrial hemp flower and leaf raw materials from Heilongjiang Province, as shown in Example 2. Detailed Implementation

[0042] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0043] The raw materials for industrial hemp flowers and leaves were provided by Yunnan Hangu Biotechnology Co., Ltd. and the Science and Technology Bureau of Sunwu County, Heihe City, Heilongjiang Province.

[0044] Example 1

[0045] Accurately weigh 0.2 g of Yunnan industrial hemp flower and leaf powder into an Erlenmeyer flask, add 10 mL of methanol, and extract by ultrasonication at room temperature for 30 minutes. Let it stand to room temperature. Add methanol to make up the weight, shake well, and filter through a 0.22 μm microporous membrane to obtain a clear and transparent filtrate. The internal standard is n-eicosane, so that the concentration of the internal standard solution is 0.12 mg / mL.

[0046] Preparation of standard solutions: Cannabidiol and cannabidiol (CBD) standard solutions were purchased from Sigma-Aldrich (USA). A 1 mg / mL tetrahydrocannabinol (THC) standard solution was diluted with chromatographically pure methanol to prepare a series of reference solutions at concentrations of 1 μg / mL, 4 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, and 100 μg / mL. An internal standard was simultaneously added to the reference solutions to achieve a final internal standard concentration of 10 μg / mL.

[0047] Preparation method of homemade cannabidiol (CBDA) reference standard:

[0048] Take 1 kg of industrial hemp flower and leaf powder (containing approximately 0.8% cannabidiol) and pack it into a 5-liter supercritical extraction vessel. Set the extraction temperature of the vessel to 50°C and the pressure to 35 MPa, and extract for 90 min. Gradually adjust the temperature and pressure of the separation vessel and collect the primary extract at a temperature of 45°C and a pressure of 7 MPa. The primary extract is a grayish-green semi-solid weighing 37 g with an HPLC purity of 75.1%, containing 20% ​​cannabidiol, and the separation and extraction rate is 92.5%.

[0049] Polystyrene microspheres with a particle size of 100 μm were soaked thoroughly in acetone-water, and 150 ml was packed into a column. 5 g of the transparent oily primary extract from Example 3 was dissolved in an appropriate amount of acetone and loaded onto the column. The column was eluted with 50% acetone-water. The concentrated eluent of CBDA was collected, and the solvent was recovered under reduced pressure to obtain crude CBDA with a content greater than 90%, a yield of 79%, and an HPLC purity of 90%.

[0050] 0.33 g of the prepared crude CBDA was dissolved in an appropriate amount of methanol and injected into a high-performance liquid chromatograph. A Waters Symmetry Prep C18 semi-preparative column (7.8 × 300 mm, 7 μm) was used, with acetonitrile-0.1% formic acid aqueous solution (68:32) as the mobile phase and a UV detector wavelength of 210 nm. The CBDA eluent was collected, the solvent was recovered under reduced pressure and evaporated to dryness to obtain 99% pure CBDA. A total of 0.27 g of pure product was obtained, with a yield of 91% (85.3%) and an HPLC purity of 99.5%.

[0051] Preparation method of cannabidiol (CBD) self-made reference standard: The preparation method is the same as that for cannabidiol acid. After extraction, impurities are removed by 200-300 mesh silica gel column (mobile phase ethyl acetate: petroleum ether = 1:99) to obtain crude CBD. Then, it is purified by high performance liquid chromatography (Shiseido UG80C18 column, specification 20mm I.D × 250mm 5μm, mobile phase acetonitrile:water = 68:32) to obtain pure CBD. The HPLC purity of pure CBD is 99.6%.

[0052] Preparation of reference solutions: Cannabidiol and cannabidiol acid reference standards were prepared in-house with a purity of over 99%. 10 mg of each reference standard was accurately weighed into a 10 mL volumetric flask, dissolved in methanol, and diluted to volume to obtain reference solutions with concentrations of 1.0277 mg / mL and 1.11 mg / mL, respectively. These reference solutions were then diluted with chromatographic-grade methanol to prepare a series of reference solutions with concentrations of 4 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 100 μg / mL, 200 μg / mL, and 500 μg / mL. An internal standard was added to achieve a final internal standard concentration of 0.06 mg / mL. The solutions were then analyzed by gas chromatograph under the aforementioned chromatographic conditions. A standard curve was plotted with the substance concentration on the x-axis and the peak area ratio of the reference standard to the internal standard on the y-axis.

[0053] Gas chromatography analysis conditions: injection port temperature 300℃, split ratio 2:1, detector heating temperature 300℃, air flow rate 400mL / min, hydrogen fuel flow rate 40mL / min, make-up gas flow rate 10mL / min, gas chromatography column flow rate 2.5mL / min, temperature program as shown in Table 1, injection volume 1μL.

[0054] Table 1. Column Temperature Program Heating Method

[0055] Heating rate (°C / min) Column temperature (°C) Hold time (min) 150 0 10 195 18 30 280 5

[0056] External standard quantification method: Under the above chromatographic conditions, the sample to be tested was detected by gas chromatography. The corresponding retention times of cannabidiol (CBD) and tetrahydrocannabinol (THC) standards were compared, and the ratio of the peak area of ​​CBD and THC to the peak area of ​​the internal standard was recorded. This peak area ratio was then substituted into the external standard curve to obtain the concentrations of CBD and THC in the test solution. The content of CBD and THC in the cannabis flower and leaf samples was then calculated. The results showed that the CBD content was 0.4% and the THC content was 0.025%. The test results were consistent with those obtained by the liquid chromatography method.

[0057] Example 2

[0058] Accurately weigh 0.2 g of Heilongjiang industrial hemp flower and leaf powder into an Erlenmeyer flask, add 10 mL of analytical grade methanol, and perform ultrasonic extraction at room temperature for 30 minutes. Allow to stand until the extract reaches room temperature. Make up the weight of the extract with analytical grade methanol, shake well, and let stand.

[0059] Draw 1.5 mL of the supernatant using a syringe, filter it through a 0.22 μm microporous membrane to obtain a clear and transparent test solution. Take 4 mL of the extract and add internal standard solution to make the concentration of internal standard solution 0.06 mg / mL.

[0060] The gas chromatography conditions were the same as in Example 1.

[0061] The preparation of the reference solution and the internal standard quantification method were the same as in Example 1. Results: Cannabidiol content 0.77%, Tetrahydrocannabinol content 0.043%. The results were consistent with those obtained using the liquid chromatography method.

[0062] Example 3: Investigation of different vaporization temperatures (inlet temperature)

[0063] CBD and CBDA reference standards with the same concentration were measured at vaporization temperatures of 150℃, 200℃, 250℃, 300℃, and 350℃, respectively. The injections were repeated twice, and the peak areas of the CBD and CBDA reference standards were recorded and their ratios were calculated. The specific data are shown in Table 1 below.

[0064] Table 1

[0065]

[0066] In the table, the retention times of both CBD and CBDA detected by gas chromatography are the same, and the conversion ratio is calculated as CBDA peak area / CBD peak area. The results show that the retention times of CBD and CBDA standards are consistent. With increasing vaporization temperature, the conversion ratio of CBDA to CBD gradually increases, stabilizing at 300 degrees Celsius. GC-MS results show that the retention times of CBD and CBDA standards are consistent, and the mass spectrometry fragmentation data are essentially identical, with a molecular ion peak at 314. It can be concluded that CBDA is converted to CBD under these conditions.

[0067] The gas phase test conditions and operations in Example 4 are the same as those in Example 1, with the differences shown in Table 2.

[0068] Table 2

[0069]

[0070]

[0071] The data in the table shows that when the initial column temperature in the column temperature ramp-up program is high, such as 230℃ or 210℃, the final detected total CBD content is higher. The main reason for this is that the CBD peak cannot be separated from the impurity peaks. When the injection port temperature is 250℃, the measured results are lower.

[0072] In this invention, the accuracy of the gas chromatography method is based on its consistency with the content determination value obtained by the liquid chromatography method. Generally, the relative error between the results of different methods is used for judgment (the relative error between the gas chromatography and liquid chromatography results in this invention is less than ±3%, which is within an acceptable range). When the gas chromatography content is lower than that in the liquid chromatography, it is highly likely that CBDA in the test sample was not completely converted to CBD; when the gas chromatography content is higher than that in the liquid chromatography, the analysis shows that the gas chromatography system failed to achieve complete and effective separation of CBD from other components in the sample during actual measurement, resulting in a higher measurement result.

[0073] Example 5: Comparison of gas chromatography and liquid chromatography results

[0074] The liquid chromatography column was an Agilent Extend C18 (5 μm 4.6 × 250 mm); mobile phase A was 0.1% phosphoric acid solution, and mobile phase B was acetonitrile. Gradient elution was performed according to the table below. Flow rate: 1 mL / min; column temperature: 25℃; sample tray temperature: 10℃; detection wavelength: 220 nm; injection volume: 10 μL.

[0075] The test solution from Example 1 was analyzed using liquid chromatography, and the CBD content was found to be 0.1679% and the CBDA content was 0.2634%. The total CBD content was calculated using the formula: CBD content + CBDA content × Mr(CBD) / Mr(CBDA), resulting in a total CBD content of 0.40%.

[0076] The gradient elution procedure is shown in Table 3.

[0077] Table 3

[0078]

Claims

1. A method for quantitative analysis of cannabidiol, characterized in that, The procedure includes the following steps: injecting the sample to be tested into a gas chromatograph, performing a programmed temperature increase, and detecting the total content of cannabidiol using the external standard method; wherein, the injection port temperature of the gas chromatograph is 300℃; the programmed temperature increase is from 150℃ to 280℃; the programmed temperature increase is divided into a first stage and a second stage; the first stage temperature increase is from 150℃ to 195℃; the programmed temperature increase rate is 10~30℃ / min; the programmed temperature increase is from 150℃ to 280℃ within 40 minutes; the first stage temperature increase rate is 10℃ / min; the second stage temperature increase is from 195℃ to 280℃; the second stage temperature increase rate is 30℃ / min; during the programmed temperature increase, the column temperature is maintained for 10~20 minutes after the first stage temperature increase; the chromatographic column used in the gas chromatograph is an Agilent HP-5.

2. The quantitative analysis method for cannabidiol as described in claim 1, characterized in that, The detector temperature of the gas chromatograph is 200~300℃.

3. The quantitative analysis method for cannabidiol as described in claim 1, characterized in that, In the gas chromatograph, the hydrogen flow rate is 35~45 mL / min.

4. The quantitative analysis method for cannabidiol as described in claim 1, characterized in that, In the gas chromatograph, the air flow rate is 350~450 mL / min.

5. The quantitative analysis method for cannabidiol as described in claim 1, characterized in that, After the first stage of heating is completed, the column temperature is maintained for 18 minutes.

6. The quantitative analysis method for cannabidiol as described in claim 1, characterized in that, The sample to be tested is an extract of cannabis flower and leaf raw materials or a mixture containing cannabidiol, cannabidiol acid, tetrahydrocannabinol and tetrahydrocannabinol acid.

7. The quantitative analysis method for cannabidiol as described in claim 1, characterized in that, The injection volume of the sample to be tested is 1 μL.

8. The quantitative analysis method for cannabidiol according to any one of claims 1-7, characterized in that, The programmed temperature rise is as follows: from 150°C to 195°C at a rate of 10°C / min; hold for 18 min; then rise from 195°C to 280°C at a rate of 30°C / min; hold for 5 min.

9. The quantitative analysis method for cannabidiol as described in claim 6, characterized in that, The preparation method of the cannabis flower and leaf raw material extract includes the following steps: ultrasonic extraction of the pulverized cannabis flowers and leaves using an organic solvent.

10. The quantitative analysis method for cannabidiol as described in claim 9, characterized in that, The organic solvent is selected from one or more of methanol, acetone, diethyl ether, cyclohexane, petroleum ether, and ethyl acetate.

11. The quantitative analysis method for cannabidiol as described in claim 10, characterized in that, The volume-to-mass ratio of the organic solvent to the cannabis flower and leaf raw material is 50 mL / g.

12. The quantitative analysis method for cannabidiol as described in claim 10, characterized in that, The organic solvent is methanol.

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