Pretreatment method and detection method of tetrahydrocannabinol in cannabidiol raw material

Abstract

The invention discloses a pretreatment method of tetrahydrocannabinol in a cannabidiol raw material and a detection method thereof, and the method is characterized by comprising the following steps: placing a crushed CBD raw material sample in a centrifuge tube, adding an ammonium formate buffer solution, a mixed extracting solution formed by mixing acetonitrile and trichloromethane, and normal hexane, carrying out vortex mixing for 1-3 min, then carrying out horizontal oscillation for 20-40 min, and carrying out centrifugation to obtain a supernatant; the CBD raw material sample, the ammonium formate buffer solution, the mixed extracting solution and the n-hexane are mixed according to a mixing ratio of 0.2 g: 12.5 mL: 20mL: 10mL, the CBD raw material sample, the ammonium formate buffer solution, the mixed extracting solution and the n-hexane are frozen in a refrigerator at the temperature of-20 DEG C for more than 1 hour, middle-layer clear liquid is taken through centrifugation and filtered through a 0.2-micron nylon filter membrane to be subjected to UPLC-MS / MS analysis, and the CBD raw material sample, the ammonium formate buffer solution, the mixed extracting solution and the n-hexane are mixed according to a mixing ratio of 0.2 g: 12.5 mL: 20mL: 10mL.

Description

Technical Field

[0001] The present invention relates to a method for detecting tetrahydrocannabinol, and in particular to a pretreatment method and a detection method for tetrahydrocannabinol in cannabidiol raw materials. Background Art

[0002] Cannabis (Cannabis sativa) is an annual herbaceous plant of the genus Cannabis in the family Cannabaceae. Cannabinoids are the main active components of cannabis. Cannabinoids are mainly divided into Δ9-tetrahydrocannabinol (Δ9-THC), which acts on the central nervous system and has hallucinogenic effects (abbreviated as Δ9-THC), and cannabidiol (CBD), which does not produce hallucinations (abbreviated as CBD), etc. CBD does not have psychoactive properties, does not cause excitement or addiction, but has many medical values similar to THC. However, THC is the main psychoactive chemical component in cannabis, which can cause excitement and addiction and is recognized as a drug worldwide. Δ9-THC exists in the original plant in the form of phenolic carboxylic acid body Δ9-tetrahydrocannabinolic acid (Δ9-THCA-A), and undergoes decarboxylation under the action of light, heat, etc. to change into the active form Δ9-THC. The content of Δ9-THC in cannabis-derived products is expressed as the sum of Δ9-THC and Δ9-THCA-A.

[0003] Currently, in production in places such as Yunnan, China, the THC component is removed by purifying cannabinoids, becoming a rapidly emerging industry. Therefore, it has become an urgent need to establish a sensitive and reliable detection method for THC in CBD raw materials. Since THC and CBD are isomers and their physical and chemical properties are very close, it poses a great challenge to detect trace amounts of THC in CBD by high performance liquid chromatography (HPLC-UV) or liquid chromatography-tandem mass spectrometry (LC-MS / MS). On the one hand, for HPLC-UV detection, as CBD with a content close to 100%, when the dilution factor does not exceed 100 times, a flat-top trailing peak will form on the chromatographic column, and the trailing phenomenon may cover the chromatographic peak of THC. If further diluted, although the trailing phenomenon can be reduced, for trace amounts of THC, the chromatographic peak may not be visible. On the other hand, when detected by LC-MS / MS method, there is also the problem that the trailing of CBD covers the chromatographic peak of THC, and sufficient sensitivity cannot be obtained by extensive dilution. Because the ion pair information of isomers is exactly the same, high-concentration CBD will generate strong background interference signals, restricting the sensitivity of THC. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a pretreatment method and a detection method for tetrahydrocannabinol in cannabidiol raw materials, which have strong purification effect, good specificity, high sensitivity, high accuracy and high recovery rate.

[0005] The technical solution adopted by the present invention to solve the above technical problems is as follows: A pretreatment method for tetrahydrocannabinol in cannabidiol raw materials, comprising the following steps: placing the crushed CBD raw material sample in a centrifuge tube, adding ammonium formate buffer solution, a mixed extraction solution composed of acetonitrile and chloroform, and n-hexane, vortex mixing for 1-3 min, then horizontally shaking for 20-40 min, freezing in a -20°C refrigerator for more than 1 h, centrifuging to take the middle-layer supernatant, and filtering through a 0.2-μm nylon filter membrane.

[0006] Further, the mixing ratio of the CBD raw material sample, the ammonium formate buffer solution, the mixed extraction solution, and the n-hexane is 0.2 g: 12.5 mL: 20 mL: 10 mL.

[0007] Further, the preparation method of the ammonium formate buffer solution is as follows: Dissolve 252.2 g of ammonium formate and 50 g of sodium chloride in water, then adjust the pH to 3.5 with formic acid, and add water to 1000 mL and mix by ultrasonic.

[0008] Further, the volume ratio of acetonitrile to chloroform in the mixed extraction solution is 17:3.

[0009] The present invention also provides a detection method for tetrahydrocannabinol in cannabidiol raw materials using the above pretreatment method, comprising the following steps:

[0010] Step 1, LC-MS / MS analysis

[0011] Gas phase conditions: The chromatographic column is Waters C 18 and C 18 Connected in series in turn; Mobile phase A is water containing 5 vt% methanol and 2.5 mmol / L ammonium acetate, adjusted to pH 7.9 with ammonia water, mobile phase B is methanol, the flow rate is 0.3 μL / min; The chromatographic column temperature is 45°C; The injection volume is 5 μL, and the mobile phase gradient elution is shown in Table 1,

[0012] Table 1. Mobile phase gradient elution

[0013]

[0014]

[0015] Mass spectrometry conditions: Data acquisition was carried out by multiple reaction monitoring. The capillary voltage in the negative ion mode was 3.0 kV; the ion source temperature was 150 °C; the desolvation gas temperature was 500 °C; the desolvation flow rate was 950 L / h; the cone orifice flow rate was 150 L / h; the collision gas was argon 3.3×10 -3 mba, and the monitored ions are shown in Table 2,

[0016] Table 2 Retention time, characteristic ions, cone orifice voltage and ion pairs under positive and negative ion switching modes

[0017]

[0018] Step 2. Concentration calculation method: In the formula,

[0019] X - Content of the analyte in the sample, in micrograms per kilogram, with the unit of mg / kg;

[0020] C - Concentration of the analyte in the sample treatment solution, in ng / mL;

[0021] V - Volume of constant volume, in milliliters;

[0022] m - Mass of the sample, in grams;

[0023] F - Dilution factor;

[0024] The total amount calculation method of the analyte in the sample is calculated according to the following formula:

[0025] Δ 9 Total amount of Δ9-THC (mg / kg) = Δ 9 -THC + 0.877×Δ 9 -THCA-A, where: 0.877 - Coefficient for converting tetrahydrocannabinolic acid to tetrahydrocannabinol, and the unit of the total amount of Δ9-THC is mg / kg.

[0026] Furthermore, it also includes the preparation of a matrix-added standard calibration curve as follows: Weigh 0.2 g of blank CBD raw material sample into 6 test tubes, add 50 μL of mixed internal standard solution and 0, 0.05 μg, 0.2 μg, 1 μg, 2 μg, 10 μg of the second mixed standard working solution respectively. After sample extraction and purification according to the pretreatment method in Claim 1, use the concentration as the abscissa and the response value of the analyte at the corresponding added concentration as the ordinate to make a matrix-added standard curve, and use the internal standard method, with the added calibration curve as the basis for quantification.

[0027] Furthermore, the preparation method of the mixed internal standard solution is as follows: Take 2000 μL of 2 μg / mL tetrahydrocannabinol-D3 and 800 μL of 100 μg / mL diethylstilbestrol-D8, mix them and make the volume constant to 10.0 mL with methanol to obtain the mixed internal standard solution.

[0028] Further, the method for preparing the second mixed standard working solution is as follows: Pipette 100 μL of 1.0 mg / mL tetrahydrocannabinolic acid standard solution and 100 μL of 1.0 mg / mL tetrahydrocannabinol standard solution, mix them, and make up the volume to the 10 mL scale with ethanol to obtain the first mixed standard working solution with the concentration of each component being 10.0 μg / mL. Pipette 1000 μL of the first mixed standard working solution into a 10 mL volumetric flask and make up the volume to the scale with ethanol to obtain the second mixed standard working solution with the concentration of each component being 1000 ng / mL.

[0029] Compared with the prior art, the advantages of the present invention are

[0030] I. In terms of pretreatment: The three-phase extraction method is adopted to complete extraction and purification in one step.

[0031] 1. The pH of the soaking solution is 3.5 high-concentration ammonium formate, which can remove alkaline impurities and promote the target substance to enter the organic phase.

[0032] 2. The extraction solution is acetonitrile + chloroform with a volume of 17 + 3, which can ensure that the target substance will not be carried away by n-hexane during n-hexane degreasing, thus avoiding losses.

[0033] 3. Only through the three-phase extraction method that simultaneously includes extraction and purification steps, the pretreatment can be quickly and simply completed in one step, without the need for cumbersome means such as solid-phase extraction purification and solid-phase dispersion extraction purification.

[0034] II. In terms of instrumental analysis: Through the following four combined means, the analytical requirements for analyzing THC in CBD raw materials by LC-MS / MS are met.

[0035] 1. By extending the length of the liquid chromatography column, the separation time distance between CBD and THC is increased to reduce the interference of the background signal. The separation time distance on a common 100 mm chromatographic column is about 1 min, and even on a 250 mm long chromatographic column, it is generally only about 3 min. Even with a gentle mobile phase gradient, the background interference caused by peak tailing is still serious. In the present invention, two chromatographic columns of 150 mm and 250 mm are connected in series, greatly extending the difference in retention time. Then, through a more gentle mobile phase gradient, the distance between the two chromatographic peaks is further increased, and finally it reaches about 9 min.

[0036] 2. By switching the flow path of the instrument, the time period from 1 min before the elution peak of CBD to 1.5 min before the elution peak of THC is switched to the waste liquid to avoid entering the mass spectrometer, greatly weakening the interference signal of the background ions.

[0037] 3. For the traditional analysis of THC and CBD, both are analyzed in the positive ion mode with strong response signals. In contrast, the present invention conducts the analysis in the negative ion mode with weakened signals, combined with a milder mobile phase gradient elution, to obtain a lower and flatter baseline and a higher signal-to-noise ratio for the target peaks, with better separation from impurity peaks.

[0038] 4. By adjusting the pH of the aqueous phase - ammonium acetate to 7.8 - 7.9 with ammonia water, ensure that the peak time of THCA is more than 1 minute before that of CBD, ensuring that the switching of the flow path does not affect the simultaneous determination of THCA. Without adjusting the pH, the peak time of THCA is close to or even about 1 minute after that of CBD, and the flow path switching cannot be carried out. Meanwhile, the weakly alkaline environment can promote the ionization efficiency of negative ions. Description of the Drawings

[0039] Figure 1 are chromatograms of different LC-MS / MS analysis methods, where A is the positive ion mode, B is the negative ion mode, and C is the negative ion mode with a milder mobile phase;

[0040] Figure 2 is the MRM spectrum of the CBD raw material sample;

[0041] Figure 3 is the MRM spectrum of the CBD sample with 10 mg / kg of THC and THCA added. Tetrahydrocannabinolic acid is at 28.98 min, and the corresponding internal standard is cis - hexestrol at 28.2 min. Tetrahydrocannabinol is at 38.94 min, and the corresponding internal standard is D3 - tetrahydrocannabinol at 38.85 min. Detailed Embodiments

[0042] The following further describes the present invention in detail with reference to the embodiments in the drawings.

[0043] Ammonium formate buffer solution: Add 252.2 g of ammonium formate and 50 g of sodium chloride to 600 mL of water, adjust the pH to 3.5 with about 200 mL of formic acid, add water to 1000 mL, and mix by ultrasonic.

[0044] Mixed extraction solution: Take 850 mL of acetonitrile and 150 mL of chloroform and mix them.

[0045] The first mixed standard working solution: Pipette 100 μL of 1.0 mg / mL tetrahydrocannabinolic acid standard solution and 100 μL of 1.0 mg / mL tetrahydrocannabinol standard solution, mix them, and make up the volume to 10 mL with ethanol to obtain the first mixed standard working solution with a concentration of 10.0 μg / mL for each component.

[0046] Second mixed standard working solution: Pipette 1000 μL of the first mixed standard working solution into a 10 mL volumetric flask, and make up the volume to the mark with ethanol to obtain the second mixed standard working solution with the concentration of each component being 1000 ng / mL.

[0047] Mixed internal standard solution: Take 2000 μL of 2 μg / mL tetrahydrocannabinol-D3 and 800 μL of 100 μg / mL diethylstilbestrol-D8, mix them, and make up the volume to 10.0 mL with methanol to obtain the mixed internal standard solution.

[0048] Specific Example 1: A method for detecting tetrahydrocannabinol in cannabidiol raw materials.

[0049] After the CBD raw material sample is crushed and sieved, add the internal standard, soak it ultrasonically with the buffer solution, add acetonitrile + chloroform and n-hexane, mix well, freeze, centrifuge, take 5 mL of the middle layer, purify it by matrix solid-phase dispersion extraction, and perform multiple reaction monitoring (MRM) determination by LC-MS / MS in the negative ion mode (both THC and THAA are in the negative ion mode), retaining two columns with a total length of 400 mm in series. The specific steps are as follows:

[0050] Step 1: Sample pretreatment

[0051] After crushing the CBD raw material sample with a kitchen blender, remove large particles with a 100-mesh sieve, and store the fine powder in a sealed and dry manner. Weigh 0.2 g of the crushed CBD raw material sample into a 50 mL graduated centrifuge tube, add 50 μL of the mixed internal standard solution, 12.5 mL of ammonium formate buffer solution, 20.0 mL of the mixed extraction solution, and 10 mL of n-hexane, vortex mix for 2 min, then shake horizontally for 30 min, freeze in a -20°C refrigerator for more than 1 h, centrifuge at 4500 r / min for 10 min, take 1 mL of the middle layer supernatant, and filter it through a 0.2 μm nylon filter membrane for LC-MS / MS determination.

[0052] Step 2: Matrix-added standard calibration curve

[0053] Weigh 0.2 g of blank CBD samples into 6 50 mL test tubes, add 50 μL of the mixed internal standard solution and 0, 0.05 μg, 0.2 μg, 1 μg, 2 μg, 10 μg of the second mixed standard working solution respectively. After extracting and purifying the samples according to the above pretreatment method, take the concentration as the abscissa and the response value of the analyte at the corresponding added concentration as the ordinate to make a matrix-added standard curve. Use the internal standard method and take the added calibration curve as the basis for quantification.

[0054] Step 3: LC-MS / MS analysis

[0055] Gas phase conditions: The chromatographic column is Waters C 18(2.1 mm × 150 mm, 3 μm) and C 18 (4.6 mm × 250 mm, 5 μm) are connected in series in turn; Mobile phase A: water containing 5% methanol and 2.5 mmol / L ammonium acetate, adjusted to pH 7.9 with ammonia water, Mobile phase B: methanol, flow rate is 0.3 μL / min; Column temperature: 45 °C; Injection volume: 5 μL; The mobile phase gradient elution of the target is shown in Table 1,

[0056] Table 1. Mobile phase gradient elution of the target

[0057]

[0058] Mass spectrometry conditions: Data acquisition is carried out by multiple reaction monitoring (MRM), the capillary voltage in the negative ion mode is 3.0 kV; Ion source temperature: 150 °C; Desolvation gas temperature: 500 °C; Desolvation flow rate: 950 L / h; Cone hole flow rate: 150 L / h; Collision gas: argon 3.3×10 -3 mba, monitoring ions: see Table 3,

[0059] Table 3. Retention time, characteristic ions, cone hole voltage, collision energy and detection limit (ordinary samples) under positive and negative ion switching mode

[0060]

[0061]

[0062] Note: * represents the quantitative ion pair. When performing sample determination, if the retention time of the detected chromatographic peak is consistent with that of the standard product, it is confirmed by the MS / MS mode. Select one parent ion and two daughter ions generated by this parent ion. When the selected ions all appear and the ion ratios are consistent, then the presence of this compound can be judged.

[0063] Step 4. Concentration calculation method

[0064]

[0065] In the formula:

[0066] X - content of the analyte in the sample, unit is micrograms per kilogram (mg / kg);

[0067] C - concentration of the analyte in the sample treatment solution, unit is micrograms per liter (ng / mL);

[0068] V - constant volume, unit is milliliters (mL);

[0069] m - mass of the sample, unit is grams (g);

[0070] F - dilution factor;

[0071] The total amount calculation method of the analyte in the sample is obtained according to the calculation formula (2).

[0072] Δ 9 Total amount of Δ-THC (mg / kg) = Δ 9 -THC + 0.877×Δ 9 -THCA-A………………………(2)

[0073] Where: 0.877—the coefficient for converting tetrahydrocannabinolic acid to tetrahydrocannabinol.

[0074] Specific Example 2. Verification of the detection method for tetrahydrocannabinol in cannabidiol raw materials established in Specific Example 1.

[0075] 1. Optimization of the LC-MS / MS analysis method

[0076] Method of Comparative Example 1: The same as Specific Example 2 above, except that in step 3, the capillary voltage in the negative ion mode is 3.0 kV, which is replaced with a capillary voltage of 3.0 kV in the positive ion mode.

[0077] Method of Comparative Example 2: The same as Specific Example 2 above, except that the mobile phase gradient elution method is replaced as follows:

[0078] Mobile phase A: 95% - 0% (0 - 20 min), held for 18 min, 0% - 95% (38 - 42 min).

[0079] The results are as Figure 1 shown. As can be seen from Figure 1 A in it, when collected in the positive ion mode, the signal-to-noise ratio is low. As can be seen from Figure 1 B and C in it, when collected in the negative ion mode, the signal-to-noise ratio is high. However, the mobile phase of the detection method established in Specific Example 1 uses a milder gradient elution, prolonging the retention time, which can further reduce the baseline noise and has a better separation degree from impurity peaks.

[0080] 2. Specificity

[0081] There are no interfering impurity peaks in the THC and THCA chromatographic peaks, and the specificity is good. See Figure 2 and Figure 3 .

[0082] 3. Standard curve and detection limit

[0083] The detection limits of THC and THCA-A are 2 mg / kg and 0.5 mg / kg respectively. Matrix-added standard curves are used for quantification, and the linear regression coefficient R > 0.99. The standard addition curve has good linearity (regression coefficient > 0.990).

[0084] 4. Average recovery rate and precision

[0085] For CBD raw materials, the quantitative limits of THC and THCA-A are 5 mg / kg and 1.0 mg / kg, respectively. The average recovery rates at three levels of 5, 10, and 25 mg / kg are in the range of 70% to 110%, and the relative standard deviation is less than 10%.

[0086] The above description is not a limitation of the present invention, nor is the present invention limited to the above examples. Changes, modifications, additions, or substitutions made by those of ordinary skill in the art within the scope of the essence of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A method for pre-treating tetrahydrocannabinol in cannabidiol raw material, characterized in that The method comprises the following steps: placing a crushed CBD raw material sample in a centrifuge tube, adding an ammonium formate buffer, a mixed extract prepared by mixing acetonitrile and chloroform, and n-hexane, vortex mixing for 1-3 minutes, then horizontally shaking for 20-40 minutes, freezing in a -20°C refrigerator for more than 1 hour, centrifuging to obtain a middle clear liquid, and filtering through a 0.2 μm nylon filter membrane.

2. The method for pre-treating tetrahydrocannabinol in a cannabidiol raw material according to claim 1, characterized in that: The mixing ratio of the CBD raw material sample, the ammonium formate buffer, the mixed extract and the n-hexane is 0.2 g: 12.5 mL: 20 mL: 10 mL.

3. The method for pre-treating tetrahydrocannabinol in a cannabidiol raw material according to claim 1, characterized in that The preparation method of the ammonium formate buffer is as follows: 252.2 g of ammonium formate and 50 g of sodium chloride are dissolved in water, the pH is adjusted to 3.5 with formic acid, water is added to 1000 mL, and ultrasonic mixing is performed.

4. The method for pre-treating tetrahydrocannabinol in a cannabidiol raw material according to claim 1, characterized in that: The volume ratio of acetonitrile to chloroform in the mixed extract is 17:

3.

5. A method for detecting tetrahydrocannabinol in a cannabidiol raw material using the pre-treatment method according to any one of claims 1 to 4, characterized in that The following steps are involved: Step 1: LC-MS / MS analysis Gas phase conditions: chromatographic column is Waters C 18 and C 18 The mobile phase A was water containing 5vt% methanol and 2.5mmol / L ammonium acetate, and the pH was adjusted to 7.9 with ammonia water. The mobile phase B was methanol, and the flow rate was 0.3μL / min. The column temperature was 45℃. The injection volume was 5μL, and the mobile phase gradient elution was as shown in Table 1. Table 1. Mobile phase gradient elution Mass spectrometry conditions: data acquisition was performed by multiple reaction monitoring, capillary voltage in negative ion mode was 3.0 kV; ion source temperature: 150 °C; desolvation temperature: 500 °C; desolvation flow rate: 950 L / h; cone flow rate: 150 L / h; collision gas: argon 3.3 × 10 -3 mba, monitoring ions: see Table 2, Table 2 Retention time, characteristic ions, cone voltage and ion pairs in positive and negative ion switching mode Step 2: Concentration calculation method: In the formula, X - the content of the substance to be tested in the sample, in micrograms per kilogram, mg / kg; C - concentration of the substance to be tested in the sample treatment solution, in ng / mL; V - constant volume, in milliliters; m - sample mass, in grams; F – dilution factor; The total amount of the analyte in the sample is calculated according to the following formula: Δ 9 -Total THC (mg / kg) = Δ 9 -THC+0.877×Δ 9 -THCA-A, where: 0.877 — coefficient for converting tetrahydrocannabinolic acid into tetrahydrocannabinol, Δ9 — total amount of THC in mg / kg.

6. The method for detecting tetrahydrocannabinol in a cannabidiol raw material according to claim 5, characterized in that It also includes the preparation of a matrix addition standard calibration curve as follows: 0.2g of blank CBD raw material sample is weighed into 6 test tubes, 50μL of mixed internal standard solution and 0, 0.05μg, 0.2μg, 1μg, 2μg, and 10μg of the second mixed standard working solution are added respectively, and after the sample is extracted and purified according to the pretreatment method of claim 1, the concentration is used as the horizontal axis and the response value of the analyte at the corresponding addition concentration is used as the vertical axis to make a matrix addition standard curve, using the internal standard method, and the addition calibration curve is used as the basis for quantification.

7. The method for detecting tetrahydrocannabinol in a cannabidiol raw material according to claim 6, characterized in that The mixed internal standard solution is prepared as follows: 2000 μL of 2 μg / mL tetrahydrocannabinol-D3 and 800 μL of 100 μg / mL diethylstilbestrol-D8 are mixed and then fixed to 10.0 mL with methanol to obtain a mixed internal standard solution.

8. The method for detecting tetrahydrocannabinol in a cannabidiol raw material according to claim 6, characterized in that The preparation method of the second mixed standard working solution is as follows: 100 μL of 1 mg / mL tetrahydrocannabinolic acid standard solution and 100 μL of 1 mg / mL tetrahydrocannabinol standard solution are mixed and diluted to 10 mL mark with ethanol to obtain a first mixed standard working solution in which the concentration of each component is 10 μg / mL; 1000 μL of the first mixed standard working solution is drawn into a 10 mL volumetric flask, and diluted to the mark with ethanol to obtain a second mixed standard working solution in which the concentration of each component is 1000 ng / mL.

Images