A pretreatment method and detection method of tetrahydrocannabinol in cannabidiol raw material

Abstract

The application discloses a pretreatment method and a detection method of tetrahydrocannabinol in cannabidiol raw materials, and has the characteristics that the method comprises the following steps: placing a crushed CBD raw material sample in a centrifugal tube, adding ammonium formate buffer solution, mixed extraction liquid mixed by acetonitrile and chloroform, and n-hexane, vortex mixing for 1-3 min, then horizontal oscillation for 20-40 min, freezing in a-20 DEG C refrigerator for more than 1 h, centrifuging to obtain a middle layer clear liquid, filtering through a 0.2 mu m nylon filter membrane into a sample bottle for UPLC-MS / MS analysis, wherein the mixing ratio of the CBD raw material sample, the ammonium formate buffer solution, the mixed extraction liquid and the n-hexane is 0.2 g:12.5 mL:20 mL:10 mL, and the method has the advantages of strong purification effect, good specificity, high sensitivity, high accuracy and high recovery rate.

Description

Technical Field

[0001] This invention relates to a method for detecting tetrahydrocannabinol (THC), and more particularly to a pretreatment method for THC in cannabidiol (CBD) raw materials and a method for detecting THC therein. Background Technology

[0002] Cannabis (Annabissativa) is an annual herbaceous plant belonging to the genus Cannabis in the family Cannabinaceae. Cannabinoids are the main active components of cannabis. Cannabinoids are mainly divided into two categories: Δ9-tetrahydrocannabinol (Δ9-THC), which acts on the central nervous system and has hallucinogenic effects, and cannabidiol (CBD), which does not produce hallucinations. CBD is not psychoactive and does not cause excitement or addiction, but it has many medicinal values ​​similar to THC. However, THC is the main psychoactive chemical component in cannabis, capable of causing excitement and addiction, and is recognized worldwide as a drug. Δ9-THC exists in the original plant as the phenolic carboxylate form Δ9-tetrahydrocannabinolic acid (Δ9-THCA-A), which undergoes decarboxylation under the influence of light and heat, transforming 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, the purification of cannabinoids to remove THC has become a rapidly emerging industry in Yunnan and other regions of China. Therefore, establishing a sensitive and reliable method for detecting THC in CBD raw materials has become an urgent need. Since THC and CBD are isomers with very similar physicochemical properties, detecting trace amounts of THC in CBD using high-performance liquid chromatography (HPLC-UV) or liquid chromatography-mass spectrometry (LC-MS / MS) poses a significant challenge. On the one hand, for HPLC-UV detection, even with CBD at a concentration close to 100%, a flat-topped, tailed peak will form on the column at dilutions not exceeding 100 times. This tailing phenomenon may mask the THC peak. Further dilution can reduce the tailing phenomenon, but for trace amounts of THC, the peak may not be visible. On the other hand, LC-MS / MS detection faces the same problem of CBD tailing masking the THC peak, and sufficient sensitivity cannot be obtained through large-scale dilution. Because ion pairs that are isomers have completely identical information, high concentrations of CBD will generate strong background interference signals, which limits 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 with 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-mentioned technical problems is as follows: a pretreatment method for tetrahydrocannabinol in cannabidiol raw materials, comprising the following steps: placing the pulverized CBD raw material sample into a centrifuge tube, adding ammonium formate buffer, a mixed extract composed of acetonitrile and chloroform, and n-hexane, vortexing for 1-3 min, then horizontally shaking for 20-40 min, freezing in a -20℃ freezer for more than 1 h, centrifuging to obtain the middle layer clear liquid, and filtering through a 0.2 μm nylon filter membrane.

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

[0007] Furthermore, the preparation method of the ammonium formate buffer is as follows: dissolve 252.2g of ammonium formate and 50g of sodium chloride in water, adjust the pH to 3.5 with formic acid, add water to 1000mL and sonicate to mix.

[0008] Furthermore, the volume ratio of acetonitrile to chloroform in the mixed extract is 17:3.

[0009] This invention also provides a method for detecting tetrahydrocannabinol (THC) in cannabidiol raw materials prepared using the above-described pretreatment method, comprising the following steps:

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

[0011] Gas chromatography conditions: Waters C10 column 18 and C 18 The chromatography columns were connected in series; mobile phase A consisted of water containing 5 wt% methanol and 2.5 mmol / L ammonium acetate, with the pH adjusted to 7.9 using ammonia; mobile phase B consisted of methanol; the flow rate was 0.3 μL / min; the column temperature was 45℃; the injection volume was 5 μL; and the mobile phase gradient elution was shown in Table 1.

[0012] Table 1. Mobile phase gradient elution

[0013]

[0014]

[0015] Mass spectrometry conditions: Data acquisition was performed via multiple reaction monitoring (MRM); capillary voltage in negative ion mode was 3.0 kV; ion source temperature was 150 °C; desolvent gas temperature was 500 °C; desolvent flow rate was 950 L / h; orifice flow rate was 150 L / h; collision gas was argon 3.3 × 10⁻⁶. -3 MBA, monitored ions: see Table 2.

[0016] Table 2 shows the retention time, characteristic ions, cone voltage, and ion pairs under positive and negative ion switching modes.

[0017]

[0018] Step 2, Concentration Calculation Method: In the formula,

[0019] X – The content of the analyte in the sample, expressed in micrograms per kilogram (mg / kg);

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

[0021] V – Isovolute volume, in milliliters;

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

[0023] F – Dilution factor;

[0024] The total amount of the analyte in the sample is calculated using the following formula:

[0025] Δ 9 -Total THC (mg / kg) = Δ 9 -THC+0.877×Δ 9 -THCA-A, where: 0.877—the coefficient for converting tetrahydrocannabinol (THC) to tetrahydrocannabinol (THC), Δ9—total THC in mg / kg.

[0026] Furthermore, the method 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, and 50μL of mixed internal standard solution and 0, 0.05μg, 0.2μg, 1μg, 2μg, and 10μg of second mixed standard working solution are added respectively. After sample extraction and purification according to the pretreatment method of claim 1, a matrix addition standard curve is plotted with concentration as the abscissa and the response value of the analyte at the corresponding addition concentration as the ordinate. The internal standard method is used, and the addition calibration curve is used as the basis for quantification.

[0027] Furthermore, the method for preparing 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 then dilute to 10.0 mL with methanol to obtain the mixed internal standard solution.

[0028] Further, the preparation method of the second mixed standard working solution is as follows: Pipette 100 μL of 1.0 mg / mL tetrahydrocannabinol standard solution and 100 μL of 1.0 mg / mL tetrahydrocannabinol standard solution, mix them, and then dilute to 10 mL with ethanol to obtain a first mixed standard working solution with a concentration of 10.0 μg / mL for each component. Pipette 1000 μL of the first mixed standard working solution into a 10 mL volumetric flask and dilute to the mark with ethanol to obtain a second mixed standard working solution with a concentration of 1000 ng / mL for each component.

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

[0030] I. Pretreatment: The three-phase extraction method is used to complete extraction and purification in one step.

[0031] 1. The soaking solution has a pH of 3.5 and a high concentration of ammonium formate, which can remove alkaline impurities and promote the entry of the target substance into the organic phase.

[0032] 2. The extract is acetonitrile + chloroform, with a volume of 17 + 3, which ensures that the target substance is not carried away by hexane during hexane defatting and thus is not lost.

[0033] 3. The pretreatment can be completed quickly and easily in one step using a three-phase extraction method that includes both extraction and purification steps, without the need for cumbersome methods such as solid phase extraction purification or solid phase dispersion extraction purification.

[0034] II. Instrumental Analysis: The following four combined methods were used to achieve the analytical requirements for LC-MS / MS analysis of THC in CBD raw materials.

[0035] 1. By extending the length of the liquid chromatography column, the separation time distance between CBD and THC is increased, thereby reducing background signal interference. On a typical 100mm column, the separation time distance is approximately 1 minute, and even on a 250mm column, it is generally only about 3 minutes. Even with a gentle mobile phase gradient, background interference from peak tailing remains significant. This invention uses two columns, 150mm and 250mm, connected in series, greatly extending the retention time difference. Then, through a gentler mobile phase gradient, the distance between the two chromatographic peaks is further increased, ultimately reaching approximately 9 minutes.

[0036] 2. By switching the flow path of the instrument, the time period from 1 minute before the CBD peak to 1.5 minutes before the THC peak is switched to waste liquid to avoid entering the mass spectrometer, which greatly reduces the background ion interference signal.

[0037] 3. Traditional analyses of THC and CBD are performed in a positive ion mode with a strong response signal. This invention, however, uses a negative ion mode with a weaker signal. Combined with a gentler mobile phase gradient elution, it achieves a flatter baseline and a higher signal-to-noise ratio for the target peak, resulting in better separation from impurity peaks.

[0038] 4. Adjust the pH of the aqueous phase-ammonium acetate to 7.8-7.9 using ammonia water to ensure that the THCA elution time is more than 1 minute before the CBD peak, thus ensuring that flow path switching will not affect the simultaneous determination of THCA. If the pH is not adjusted, the THCA elution time will be close to or even about 1 minute after the CBD peak, making flow path switching impossible. Simultaneously, a weakly alkaline environment can promote the ionization efficiency of negative ions. Attached Figure Description

[0039] Figure 1 Chromatograms for different LC-MS / MS analytical methods are shown, where A represents positive ion mode, B represents negative ion mode, and C represents negative ion mode with a milder mobile phase.

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

[0041] Figure 3 MRM spectra of THC and THCA added to CBD samples at 10 mg / kg were obtained. Tetrahydrocannabinol was 28.98 min, with the corresponding internal standard being cis-dextrin at 28.2 min. Tetrahydrocannabinol was 38.94 min, with the corresponding internal standard being D3-tetrahydrocannabinol at 38.85 min. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

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

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

[0045] First mixed standard working solution: Take 100 μL of 1.0 mg / mL tetrahydrocannabinol standard solution and 100 μL of 1.0 mg / mL tetrahydrocannabinol standard solution, mix them, and then dilute 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 dilute to the mark with ethanol to obtain the second mixed standard working solution with a concentration of 1000 ng / mL for each component.

[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 then dilute to 10.0 mL with methanol to obtain the mixed internal standard solution.

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

[0049] After pulverizing the CBD raw material sample, the sample was sieved, an internal standard was added, and the sample was ultrasonically soaked in buffer solution. Acetonitrile, chloroform, and n-hexane were added and mixed thoroughly. The sample was then frozen, centrifuged, and 5 mL of the middle layer was collected. The sample was purified by matrix dispersion solid-phase extraction and retained by LC-MS / MS in negative ion mode (THC and THAA, both in negative ion mode) using two columns with a total length of 400 mm in series. Multiple reaction monitoring (MRM) analysis was performed, including the following steps:

[0050] Step 1: Sample Pretreatment

[0051] The CBD raw material sample was pulverized using a food processor, and large particles were removed by sieving through a 100-mesh sieve. The fine powder was then sealed, dried, and stored. 0.2 g of the pulverized CBD raw material sample was weighed into a 50 mL graduated centrifuge tube, and 50 μL of mixed internal standard solution, 12.5 mL of ammonium formate buffer, 20.0 mL of mixed extract, and 10 mL of n-hexane were added. The mixture was vortexed for 2 min, followed by horizontal shaking for 30 min. The mixture was then frozen at -20℃ for at least 1 h, centrifuged at 4500 r / min for 10 min, and 1 mL of the middle layer supernatant was collected and filtered through a 0.2 μm nylon membrane for LC-MS / MS analysis.

[0052] Step 2: Matrix Addition Standard Calibration Curve

[0053] Weigh 0.2g of blank CBD sample into six 50mL test tubes, add 50μL of mixed internal standard solution and 0, 0.05μg, 0.2μg, 1μg, 2μg, and 10μg of second mixed standard working solution respectively. After sample extraction and purification according to the above pretreatment method, plot the matrix addition standard curve with concentration as the x-axis and the response value of the analyte at the corresponding addition concentration as the y-axis. Use the internal standard method and the addition calibration curve as the basis for quantification.

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

[0055] Gas chromatography conditions: Waters C10 column 18(2.1mm×150mm, 3μm) and C 18 (4.6 mm × 250 mm, 5 μm) columns were connected in series; mobile phase A: water containing 5% methanol and 2.5 mmol / L ammonium acetate, pH adjusted to 7.9 with ammonia; mobile phase B: methanol, flow rate 0.3 μL / min; column temperature: 45℃; injection volume: 5 μL; gradient elution of the target analyte is shown in Table 1.

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

[0057]

[0058] Mass spectrometry conditions: Data acquisition was performed using multiple reaction monitoring (MRM) with a capillary voltage of 3.0 kV in negative ion mode; ion source temperature: 150 °C; desolvent gas temperature: 500 °C; desolvent flow rate: 950 L / h; cone flow rate: 150 L / h; collision gas: argon 3.3 × 10⁻⁶ -3 MBA, monitored ions: see Table 3.

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

[0060]

[0061]

[0062] Note: * represents quantitative ion pairs. When performing sample analysis, if the retention time of the detected chromatographic peak is consistent with that of the standard, confirm the result using MS / MS mode. Select a precursor ion and its two daughter ions. If both selected ions appear and their proportions are consistent, the presence of this compound can be confirmed.

[0063] Step 4: Concentration Calculation Method

[0064]

[0065] In the formula:

[0066] X – The content of the analyte in the sample, expressed in micrograms per kilogram (mg / kg);

[0067] C – The concentration of the analyte in the sample treatment solution, expressed in micrograms per liter (ng / mL);

[0068] V – Fixed volume, in milliliters (mL);

[0069] m – Sample mass, in grams (g);

[0070] F – Dilution factor;

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

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

[0073] In the formula: 0.877—the coefficient for converting tetrahydrocannabinol (THC) to tetrahydrocannabinol (THC).

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

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

[0076] Comparative Example 1 Method: Same as the above specific embodiment 2, except that the capillary voltage of negative ion mode in step 3 is 3.0kV, and the capillary voltage of positive ion mode is 3.0kV.

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

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

[0079] The results are as follows Figure 1 As shown, by Figure 1 As can be seen from point A, the signal-to-noise ratio is low when collected in positive ion mode. Figure 1 As can be seen from B and C, the signal-to-noise ratio is high when collected in negative ion mode. However, in a specific embodiment, the mobile phase of the detection method uses a gentler gradient elution and extends the retention time, which can further reduce baseline noise and improve the separation from impurity peaks.

[0080] 2. Specificity

[0081] The THC and THCA chromatographic peaks showed no interfering impurity peaks, indicating good specificity. (See...) Figure 2 and Figure 3 .

[0082] 3. Standard curve and detection limit

[0083] The limits of detection for THC and THCA-A were 2 mg / kg and 0.5 mg / kg, respectively. Quantification was performed using a matrix spiking standard curve, with a linear regression coefficient R > 0.99. The standard spiking curve showed good linearity (regression coefficient > 0.990).

[0084] 4. Average recovery rate and precision

[0085] For CBD feedstock, the limits of quantitation for THC and THCA-A were 5 mg / kg and 1.0 mg / kg, respectively. The average recoveries at the three spiking levels of 5, 10, and 25 mg / kg ranged from 70% to 110%, with relative standard deviations of less than 10%.

[0086] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A pretreatment method for tetrahydrocannabinol (THC) in cannabidiol raw materials, characterized in that... The process includes the following steps: Place the pulverized CBD raw material sample in a centrifuge tube, add ammonium formate buffer, a mixed extract solution consisting of acetonitrile and chloroform, and n-hexane. Vortex for 1-3 minutes, then shake horizontally for 20-40 minutes. Freeze at -20°C for at least 1 hour. Centrifuge and collect the middle layer. Filter through a 0.2 μm nylon membrane. The mixing ratio of the CBD raw material sample, the ammonium formate buffer, the mixed extract solution, and the n-hexane is 0.2 g: 12.5 mL: 20 mL: 10 mL. The ammonium formate buffer is prepared as follows: Dissolve 252.2 g of ammonium formate and 50 g of sodium chloride in water, adjust the pH to 3.5 with formic acid, add water to 1000 mL, and sonicate. The volume ratio of acetonitrile to chloroform in the mixed extract solution is 17:

3.

2. A method for detecting tetrahydrocannabinol (THC) in cannabidiol raw materials prepared using the pretreatment method described in claim 1, characterized in that... Includes the following steps: Step 1: LC-MS / MS analysis Liquid chromatography conditions: Column: Waters C10 18 and C 18 The chromatography columns were connected in series; mobile phase A consisted of water containing 5 wt% methanol and 2.5 mmol / L ammonium acetate, with the pH adjusted to 7.9 using ammonia; mobile phase B consisted of methanol; the flow rate was 0.3 μL / min; the column temperature was 45℃; the injection volume was 5 μL; and the gradient elution of the mobile phases was shown in Table 1. Table 1. Mobile phase gradient elution Mass spectrometry conditions: Data acquisition was performed via multiple reaction monitoring (MRM); capillary voltage in negative ion mode was 3.0 kV; ion source temperature was 150 °C; desolvent gas temperature was 500 °C; desolvent flow rate was 950 L / h; orifice flow rate was 150 L / h; collision gas was argon 3.3 × 10⁻⁶. -3 mbar, monitored ions: see Table 2. Table 2 shows the retention time, characteristic ions, cone voltage, and ion pairs under positive and negative ion switching modes. ， Step 2, Concentration Calculation Method: In the formula, X－ The content of the analyte in the sample, in mg / kg; C－ The concentration of the analyte in the sample treatment solution, in ng / mL; V－ Volume at constant volume, in milliliters; m－ Sample mass, in grams; F－ Dilution factor; The total amount of the analyte in the sample is calculated using the following formula: Δ 9 -Total THC (mg / kg) = Δ 9 -THC+ 0.877 ×Δ 9 -THCA-A, where: 0.877—the coefficient for converting tetrahydrocannabinol (THCA) to tetrahydrocannabinol (THC), Δ 9 -The total THC is measured in mg / kg. The matrix addition standard calibration curve is prepared as follows: 0.2 g of blank CBD raw material sample is weighed into 6 test tubes, and 50 μL of mixed internal standard solution and 0, 0.05 μg, 0.2 μg, 1 μg, 2 μg, and 10 μg of second mixed standard working solution are added respectively. After sample extraction and purification according to the pretreatment method described in claim 1, a matrix addition standard curve is plotted with concentration as the abscissa and the response value of the analyte at the corresponding addition concentration as the ordinate. The internal standard method is used, and the addition calibration curve is used as the basis for quantification.

3. The method for detecting tetrahydrocannabinol in cannabidiol raw materials according to claim 2, characterized in that... The method for preparing 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 then dilute to 10.0 mL with methanol to obtain the mixed internal standard solution.

4. The method for detecting tetrahydrocannabinol in cannabidiol raw materials according to claim 2, characterized in that... The preparation method of the second mixed standard working solution is as follows: 100 μL of 1 mg / mL tetrahydrocannabinol standard solution and 100 μL of 1 mg / mL tetrahydrocannabinol standard solution are mixed and then diluted to 10 mL with ethanol to obtain the first mixed standard working solution with a concentration of 10 μg / mL for each component. 1000 μL of the first mixed standard working solution is then transferred to a 10 mL volumetric flask and diluted to the mark with ethanol to obtain the second mixed standard working solution with a concentration of 1000 ng / mL for each component.

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