Method for rapidly detecting 10 cannabinoids in industrial hemp fresh floral leaves based on HPLC (High Performance Liquid Chromatography) method

Abstract

The invention discloses a method for rapidly detecting ten cannabinoids in industrial hemp fresh floral leaves based on an HPLC (High Performance Liquid Chromatography) method. The method comprises the following steps: S1, preparing a mixed standard solution; s2, pretreatment of a fresh industrial cannabis sativa floral leaf sample: liquid nitrogen grinding wall breaking, solvent ultrasonic extraction and low-temperature centrifugation are carried out, and a supernatant is taken for sample preparation; and S3, high performance liquid chromatography determination. The ten cannabinoids disclosed by the invention comprise cannabidiol (CBD), cannabidiol acid (CBDA), hypocannabidiol (CBDV), tetrahydrocannabinol acid (THCA), delta 9-tetrahydrocannabinol (delta 9-THC), delta 8-tetrahydrocannabinol (delta 8-THC), hypocannabinol (THCV), cannabinoid phenol (CBG), cannabinol (CBN) and cannabinocene (CBC). According to the method, the situation that carboxylic acid cannabinoid in fresh floral leaves is prone to decarboxylation conversion in the pretreatment process is fully considered, low-temperature extraction is conducted through liquid nitrogen grinding wall breaking, and it is ensured that carboxylic acid cannabinoid is stable. The detection method disclosed by the invention realizes high-throughput detection of 10 cannabinoids, can rapidly, objectively, accurately and synchronously detect various cannabinoids including carboxylic acid cannabinoids in fresh industrial hemp floral leaves, and has the advantages of short detection time (not exceeding 15 minutes), accurate quantification, reliable result, high efficiency and the like; and a reliable basis can be provided for industrial hemp characteristic variety breeding and scientific harvesting.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and specifically relates to a method for rapidly detecting 10 cannabinoids in fresh flowers and leaves of industrial hemp based on HPLC. Background Art

[0002] Industrial cannabis (industrial cannabis) is a type of cannabis plant with a tetrahydrocannabinol (THC) content of less than 0.3% (w / w). Its plants contain a variety of medicinally valuable compounds, including terpenes, flavonoids, alkaloids, and cannabinoids. Cannabinoids, among others, have both chemoprotective and pharmacological properties and are primarily accumulated in the glandular trichomes of female plants, with highest concentrations occurring in the top floral leaves during the initial fruiting phase. Currently, over 100 cannabinoids have been isolated from cannabis, including cannabidiol (CBD), cannabidiolic acid (CBDA), cannabidivarin (CBDV), Δ9-tetrahydrocannabinol (Δ9-THC), Δ8-tetrahydrocannabinol (Δ8-THC), tetrahydrocannabinolic acid (THCA), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), and cannabivarin (THCV).

[0003] British pharmaceutical company GW has developed Epidiolex, a plant-based anti-epileptic drug with fewer side effects, and Sativex, a prescription cannabis-based treatment for multiple sclerosis. Both are now on the market and have demonstrated excellent efficacy. With the development of CBD-related pharmaceuticals and the growth of the medical cannabis industry, the medicinal value of industrial hemp has steadily increased. In recent years, increasing research has focused on phytocannabinoids beyond CBD and THC. Studies have shown that CBN improves sleep, CBDA has anticonvulsant, anti-inflammatory, antiemetic, and migration-inhibiting effects on highly invasive MDA-MB-231 human breast cancer cells, CBG has potential efficacy in treating glaucoma, enteritis, and prostate cancer, and CBC has analgesic, brain cell growth-stimulating, and gastrointestinal hypermotility-correcting effects. Studies have shown that combining multiple cannabinoids can have synergistic effects, and that the synergistic effects of a "full spectrum" of cannabinoids are crucial to the effectiveness of cannabis products.

[0004] With the in-depth study of cannabinoids and the exploration of their pharmacological effects, high-content medicinal industrial hemp varieties are highly sought after. Currently, the main target traits for the selection and breeding of medicinal hemp varieties are high CBD and low THC content (less than 0.3%). As the medical value of other cannabinoids is discovered, the market demand for specialty varieties with other high cannabinoid content will also become increasingly strong.

[0005] Throughout the growth period of industrial hemp, the existence and distribution of cannabinoids vary greatly. Fresh flowers and leaves mainly exist in the form of carboxylic acids (such as CBDA, THCA, etc.). Improper treatment (such as light and heating) can easily lead to their decarboxylation. When testing flower and leaf raw materials, the conventional sample pretreatment method is to sun-dry or oven-dry the flowers and leaves and then extract them with solvents. However, whether it is low-temperature drying or shade drying, it will cause the decarboxylation of carboxylic acid cannabinoids in fresh flowers and leaves. It is impossible to objectively and truly assess the content of various cannabinoids in industrial hemp flowers and leaves, and it is impossible to guide the scientific harvesting of flower and leaf raw materials. This will lead to a lack of real data support for variety selection.

[0006] At present, the analytical methods for detecting the content of cannabinoid components generally adopt gas chromatography-tandem mass spectrometry (GC-MS / MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), and high performance liquid chromatography (HPLC), which are mainly concentrated in the industrial hemp extraction industry, cosmetics, forensic medicine or public security evidence identification fields. Most existing test methods can only simultaneously measure a few cannabinoids, or there is improper sample pretreatment, resulting in the conversion of carboxylic acid cannabinoids, and the test results deviate from the true value. For example, patent CN113189244 discloses a method for detecting cannabinoids in industrial hemp flowers and leaves based on UPLC-MS / MS, which uses ultra-high performance liquid chromatography-tandem mass spectrometry to detect the content of four cannabinoids, CBGA, CBG, CBC, and CBCA, in industrial hemp flowers and leaves. The pretreatment of the flowers and leaves is to dry them in the shade and then extract them. It does not fully consider that carboxylic acid substances such as CBCA and CBGA will be decarboxylated and converted during the shade drying process, which leads to the measurement results being lower than the true value, and the method can only detect 4 cannabinoids. Patent CN112034059A discloses a method for detecting cannabinoids in industrial hemp flowers and leaves and their extracts using high-performance liquid chromatography. Although this method can detect 10 cannabinoids in industrial hemp flowers and leaves: CBD, CBDA, THC, THCA, CBG, CBGA, CBDV, CBC, CBN, and CBL, the detection time is long, requiring approximately 25 minutes. Furthermore, the invention pre-treats the flowers and leaves by drying them before extraction, similarly failing to consider the instability of carboxylic acid cannabinoids, which can be decarboxylated during heating. Furthermore, when calculating the cannabinoid content in the flowers and leaves, the invention fails to measure the moisture content of the flowers and leaves and does not deduct the moisture content, making the results unreliable.

[0007] Therefore, for those who need to accurately cultivate high-CBDA, low-THCA, and other special industrial hemp varieties, such as high-CBG+high-CBD+high-CBC+low-THC hemp varieties with excellent comprehensive performance, or those who need to closely track the content of multiple cannabinoids in fresh flowers and leaves of industrial hemp at different growth stages in order to achieve the purpose of scientific harvesting, it is very necessary to establish a method that can quickly, accurately and reliably determine multiple cannabinoids in fresh flowers and leaves of industrial hemp. Summary of the Invention

[0008] To address the problems in the prior art, the present invention provides a method for the rapid detection of 10 cannabinoids in fresh flowers and leaves of industrial hemp based on HPLC. This method can rapidly and accurately determine 10 cannabinoids, including carboxylic acid cannabinoids, in fresh flowers and leaves of industrial hemp. It has the advantages of high throughput, short detection time (no more than 15 minutes), accurate quantitative analysis, reliable results, and high efficiency.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] A method for rapidly detecting 10 cannabinoids in fresh flowers and leaves of industrial hemp based on HPLC, comprising the following steps:

[0011] S1. Preparation of mixed standard solution;

[0012] S2. Pre-treatment of fresh industrial hemp flower and leaf samples:

[0013] (1) Liquid nitrogen grinding and wall breaking: Accurately weigh an appropriate amount of fresh industrial hemp flowers and leaves, chop them into pieces, place them in a ceramic mortar, add an appropriate amount of liquid nitrogen, and quickly grind them into powder;

[0014] (2) Ultrasonic extraction: Transfer the ground sample to a centrifuge tube, accurately measure an appropriate amount of extraction solvent, clean the mortar in batches, transfer the cleaning solution to a centrifuge tube, add the remaining solvent, weigh, perform ultrasonic extraction, and let it stand at room temperature to re-weight;

[0015] (3) Sample preparation: The extract was mixed and centrifuged at low temperature. The supernatant was filtered through a 0.22 μm organic filter membrane and the filtrate was used as the test solution.

[0016] S3. High performance liquid chromatography detection:

[0017] (1) Standard curve drawing: HPLC was used to detect the mixed standard solution of each concentration prepared in step S1, and the retention time was used for qualitative analysis. A standard curve was drawn based on the corresponding relationship between the peak area measured at each concentration and its concentration;

[0018] (2) Sample testing: The test solution prepared in step S2 was subjected to HPLC testing, and the retention time was used for qualitative analysis and the standard curve regression equation was used for quantitative analysis to calculate the content of each cannabinoid in the test sample.

[0019] Preferably, the 10 cannabinoid components include: cannabidiol (CBD), cannabidiolic acid (CBDA), cannabidivarin (CBDV), tetrahydrocannabinolic acid (THCA), Δ9-tetrahydrocannabinol (Δ9-THC), Δ8-tetrahydrocannabinol (Δ8-THC), cannabigerol (THCV), cannabigerol (CBG), cannabinol (CBN), and cannabichromene (CBC).

[0020] Preferably, in step S1, the mass concentration gradient of the mixed standard solution of the 10 cannabinoids is: 0.8, 1.6, 4.0, 8.0, 24.0, 40.0 μg / ml.

[0021] Preferably, in step S2(1), the amount of liquid nitrogen added is just enough to cover the sample.

[0022] Preferably, in step S2, the extraction solvent is any one of methanol, ethanol, and acetonitrile, and the solid-liquid ratio is 1:5-20; the ultrasonic power is 30-50KHz, and the extraction time is 10-30min; the centrifugation temperature is 4-8°C, the speed is 8000-10000rpm, and the centrifugation time is 5-10min.

[0023] Preferably, in step S3, the HPLC conditions are:

[0024] Chromatographic column: C18, length 150 mm, inner diameter 4.6 mm, particle size 3-5 μm;

[0025] Mobile phase: acetonitrile and formic acid aqueous solution, volume ratio 60:40-80:20;

[0026] Column temperature: 25℃~45℃; flow rate: 0.5mL~1.5mL / min; injection volume: 1~10μL; detection wavelength: 220nm.

[0027] Preferably, the volume percentage of the formic acid aqueous solution in the mobile phase is 0.05%-0.5%; and the elution method is isocratic elution.

[0028] Preferably, the method further comprises determining the moisture content of fresh flowers and leaves of industrial hemp by a drying method: accurately weighing an appropriate amount of fresh flowers and leaves and placing them in a weighing dish, recording the initial net weight (M1), then drying them at 105°C to a constant weight, taking them out and placing them in a desiccator, waiting for the temperature to drop to room temperature, weighing them, and recording the net weight of the flowers and leaves when they are dried to a constant weight (M2). The moisture content (W) is calculated according to formula (1):

[0029]

[0030] Where:

[0031] W: moisture content of the test sample, %;

[0032] M1: initial net weight of the test sample before drying, in grams (g);

[0033] M2: Net weight of the test sample when dried to constant weight, in grams (g).

[0034] Preferably, in step S3, the cannabinoid content (X) in the fresh flowers and leaves of industrial hemp is calculated according to formula (2):

[0035]

[0036] Where:

[0037] X: content of the component being tested in the test sample, %;

[0038] C: The concentration of the component being tested in the test solution obtained from the standard working curve. The unit is micrograms per milliliter (μg / ml);

[0039] V: volume of the test solution, in milliliters (ml);

[0040] M: mass of the test sample, in grams (g);

[0041] W: moisture content of the test sample, %.

[0042] Beneficial effects of the present invention:

[0043] ① The detection method of the present invention realizes high-throughput detection of 10 cannabinoid components in fresh flowers and leaves of industrial hemp, including cannabidiol (CBD), cannabidiolic acid (CBDA), cannabidivarin (CBDV), tetrahydrocannabinolic acid (THCA), Δ9-tetrahydrocannabinol (Δ9-THC), Δ8-tetrahydrocannabinol (Δ8-THC), cannabigerol (THCV), cannabigerol (CBG), cannabinol (CBN), and cannabichromene (CBC).

[0044] ② The present invention fully considers that carboxylic acid cannabinoids (CBDA, THCA, etc.) in fresh flowers and leaves of industrial hemp are easily decarboxylated and converted during the pretreatment process, and adopts liquid nitrogen grinding and wall breaking for low-temperature extraction to ensure the stability of carboxylic acid cannabinoids and reliable detection results.

[0045] ③ The present invention can rapidly, objectively and accurately detect 10 cannabinoids, including carboxylic acid cannabinoids, in fresh flowers and leaves of industrial hemp. It has the advantages of short detection time (no more than 15 minutes), accurate quantification, reliable results and high efficiency, providing a reliable basis for the selection and scientific harvesting of characteristic varieties of industrial hemp. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a high performance liquid chromatogram of a mixed standard solution of 10 cannabinoids in the example;

[0047] Figure 2 4 is a high performance liquid chromatogram of the fresh flower and leaf extract of Yunma No. 8 in the embodiment. DETAILED DESCRIPTION

[0048] The present invention is further described below with reference to the following embodiments, comparative examples and accompanying drawings, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention fall within the scope of protection of the present invention.

[0049] Example

[0050] 1. Detection method

[0051] 1.1 Materials and Instruments

[0052] Cannabinoid standards: CBD, CBDA, CBDV, THCA, Δ9-THC, Δ8-THC, THCV, CBG, CBN, CBC standards (1 mg / ml, 1 ml) (Sigma, USA); methanol and acetonitrile (chromatographic grade, Merck, Germany); formic acid (analytical grade, Xilong); methanol (analytical grade, Xilong)

[0053] Flower and leaf raw materials: Fresh flowers and leaves of Yunma No. 8 grown by Yunnan Lvxin Biopharmaceutical Co., Ltd. at the mature stage.

[0054] Instruments: Agilent 1260II high-performance liquid chromatograph (Agilent); chromatographic column: Agilent ZORBAX SB-C18, length 150 mm, inner diameter 4.6 mm, particle size 5 μm (Agilent); water purifier (ELGA); AUW220D analytical balance (Shimadzu); desktop high-speed refrigerated centrifuge (Xiangyi); electric blast drying oven (Yiheng); ultrasonic cleaning apparatus (Goneng).

[0055] 1.2 Solution preparation

[0056] Mobile phase: acetonitrile and 0.1% formic acid aqueous solution, volume ratio 75:25;

[0057] Mixed standard stock solution: Accurately measure 0.8 ml of CBD (1 mg / ml), CBDA (1 mg / ml), CBDV (1 mg / ml), THCA (1 mg / ml), Δ9-THC (1 mg / ml), Δ8-THC (1 mg / ml), THCV (1 mg / ml), CBG (1 mg / ml), CBN (1 mg / ml), and CBC (1 mg / ml) standards and transfer them to a 10 ml volumetric flask. Add chromatographic methanol to make a mixed standard stock solution of 10 cannabinoids at a concentration of 80 μg / ml. Store at -20°C in the dark.

[0058] Standard working solution: Accurately measure 0.1, 0.2, 0.5, 1.0, 3.0, and 5.0 mL of the 10 cannabinoid mixed standard stock solutions into a 10 mL volumetric flask. Dilute to the mark with chromatographic methanol to prepare a series of standard working solutions with concentrations of 0.8, 1.6, 4.0, 8.0, 24.0, and 40.0 μg / mL. Prepare them before use.

[0059] 1.3 Determination of moisture content of fresh flowers and leaves of industrial hemp

[0060] The drying method was used: 5 g (accurate to 0.001 g) of fresh industrial hemp flowers and leaves were accurately weighed and placed in a weighing dish, and the initial net weight (M1) was recorded. The flowers and leaves were then dried at 105 °C to a constant weight. The flowers and leaves were taken out and placed in a desiccator. After the temperature dropped to room temperature, the flowers and leaves were weighed and the net weight (M2) of the flowers and leaves when they were dried to a constant weight was recorded. The moisture content (W) was calculated according to formula (1).

[0061]

[0062] 1.4 Pretreatment of fresh industrial hemp flower and leaf samples

[0063] (1) Liquid nitrogen grinding: Accurately weigh 3 g (accurate to 0.001 g) of fresh industrial hemp flowers and leaves, cut them into pieces and place them in a ceramic mortar. Add liquid nitrogen until the sample is just covered and quickly grind into powder.

[0064] (2) Ultrasonic extraction: Transfer the ground sample to a 50 ml centrifuge tube, accurately measure 30 ml of methanol at a material-liquid ratio of 1:10, rinse the mortar three times, transfer the rinse solution to a centrifuge tube, add the remaining solvent, weigh, and ultrasonically extract for 30 min. Let it stand at room temperature to re-weigh.

[0065] (3) Sample preparation: The extract was mixed and centrifuged at 4°C and 10,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm organic filter membrane and then tested for CBDV, CBG, CBD, THCV, CBN, Δ9-THC, Δ8-THC, CBC, and THCA content. 1 mL of the supernatant was transferred to a 50 mL volumetric flask, diluted to the mark with 100% methanol, filtered through a 0.22 μm organic filter membrane, and then tested for CBDA content.

[0066] 1.5 High-performance liquid chromatography detection

[0067] Liquid chromatography conditions: The chromatographic column was an Agilent ZORBAX SB-C18 column with a length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; the mobile phase was acetonitrile and 0.1% (v / v) formic acid aqueous solution in a volume ratio of 75:25; the column temperature was 30°C; the flow rate was 1.2 ml / min; the injection volume was 10 μL; and the detection wavelength was 220 nm.

[0068] 1.5.1 Standard Curve

[0069] The prepared 0.8, 1.6, 4.0, 8.0, 24.0, and 40.0 μg / ml series of standard working solutions were injected and analyzed according to the liquid chromatography conditions of this embodiment. Standard curves were drawn with the mass concentration (x, μg / ml) of the 10 cannabinoids as the abscissa and the peak area (y) as the ordinate. The curves were fitted linearly to obtain the linear regression equations of the standard curves of the 10 cannabinoids.

[0070] 1.5.2 Precision experiment

[0071] Take a standard working solution with a concentration of 8.0 μg / ml and repeat the injection 6 times according to the above liquid chromatography conditions (1.4). Substitute the peak area into the standard curve regression equation to calculate the relative standard deviation (RSD) of the measured concentration.

[0072] 1.5.3 Repeatability Experiment

[0073] Accurately weigh 3 g (accurate to 0.001 g) of 6 portions of fresh industrial hemp flowers and leaves, prepare samples according to the above sample pretreatment method (1.4), inject the prepared test solution into the above chromatographic conditions (1.5) and analyze. Measure the peak areas of 10 cannabinoids, substitute them into the standard curve regression equation, and calculate the relative standard deviation (RSD) of the measured concentrations.

[0074] 1.5.4 Stability test

[0075] Accurately weigh 3 g (accurate to 0.001 g) of fresh industrial hemp flowers and leaves, prepare the sample according to the above sample pretreatment method (1.4), and inject the prepared test solution into the above chromatographic conditions (1.5) for analysis. On the same day, measure once every 2 hours, for a total of 6 times. Substitute the peak area into the standard curve regression equation to calculate the relative standard deviation (RSD) of the measured concentration.

[0076] 1.5.5 Limit of Quantitation and Limit of Detection

[0077] The detection limit (LOD) was determined by using the standard working solution with the lowest concentration (0.8 μg / mL) and injecting the solution three times in a row through stepwise dilution. The concentration of the solution when the signal-to-noise ratio of the target component in each injection was ≥3:1 was determined as the limit of detection (LOD). The concentration of the solution when the signal-to-noise ratio of the target component in each injection was ≥10:1 was determined as the limit of quantification (LOQ) by injecting the solution six times in a row.

[0078] 1.5.6 Spike recovery

[0079] Accurately weigh 3 g (accurate to 0.001 g) of 9 portions of fresh flowers and leaves of industrial hemp with known content. Add three mixed standard solutions at low (1.6 μg / ml), medium (4.0 μg / ml), and high (8.0 μg / ml) levels to the flower and leaf samples for spike recovery. Each concentration is repeated three times. After spike addition, prepare the sample according to the sample pretreatment method (1.4) above. The prepared test solution is injected and analyzed according to the above liquid chromatography conditions (1.5) and the recovery rate is calculated.

[0080] 1.5.7 Testing of fresh flower and leaf samples of Yunma No. 8

[0081] Moisture content (W): Determine the moisture content of three fresh flower and leaf samples of Yunma No. 8 in parallel according to the method in 1.3, and calculate the moisture content according to formula (1) in 1.6, and calculate the average value.

[0082] Cannabinoids (X): Accurately weigh 3 g (accurate to 0.001 g) of fresh flowers and leaves of Yunma No. 8 in three portions. Prepare samples according to the above sample pretreatment method (1.4). Inject the prepared test solution according to the above chromatographic conditions (1.5) and analyze it. Substitute the concentration of each cannabinoid into the standard curve regression equation. Calculate the cannabinoid content according to formula (2) in 1.6 and obtain the average value.

[0083] 1.6 Data processing and result calculation

[0084] The linear regression equation and correlation coefficient of the standard curve of the present invention were obtained by Agilent OpenLab Data Analysis, and other data were processed using Excel 2010 software.

[0085] The moisture content is calculated according to formula (1):

[0086]

[0087] Where:

[0088] W: moisture content of the test sample, %;

[0089] M1: initial net weight of the test sample before drying, in grams (g);

[0090] M2: Net weight of the test sample when dried to constant weight, in grams (g).

[0091] The cannabinoid content in fresh flowers and leaves of industrial hemp is calculated according to formula (2):

[0092]

[0093] Where:

[0094] X: content of the component being tested in the test sample, %;

[0095] C: The concentration of the component being tested in the test solution obtained from the standard working curve. The unit is micrograms per milliliter (μg / ml);

[0096] V: volume of the test solution, in milliliters (ml);

[0097] M: mass of the test sample, in grams (g);

[0098] W: moisture content of the test sample, %.

[0099] 2. Methodological Evaluation

[0100] 2.1 Linear relationship, detection limit, and quantification limit

[0101] The technical solution of the present invention can effectively avoid baseline fluctuation by mixing acetonitrile and an aqueous solution containing 0.1% formic acid and then using single-channel isocratic elution. Figure 1 As can be seen from the mixed standard liquid chromatogram, under the chromatographic conditions of this embodiment, 10 cannabinoids can be separated within 15 minutes, and the retention time of the last cannabinoid THCA is 13.42 minutes. The separation effect and peak shape of each cannabinoid are good (see Figure 1 Agilent OpenLab Data Analysis was used to plot the standard curve regression equations for 10 cannabinoids. The linear range, correlation coefficient, LOD, and LOQ are shown in Table 1. The linear relationship was good, and the correlation coefficients r were all above 0.999.

[0102] Table 1 Retention time of 10 cannabinoids in sequence

[0103]

[0104]

[0105] 2.2 Precision

[0106] According to the above precision experiment operation, the mixed standard solution with a concentration of 8.0 μg / ml was measured in parallel 6 times. The measured concentrations and RSDs of the 10 cannabinoids are shown in Table 2.

[0107] As shown in Table 2, the RSD of the precision test results of the 10 cannabinoids ranged from 0.51% to 1.27%, indicating that the method has high accuracy.

[0108] Table 2 Precision test results (μg / ml)

[0109]

[0110] 2.3 Repeatability

[0111] According to the above repeatability experimental operation, 6 samples were measured in parallel. The concentrations and RSDs of the 10 cannabinoids in the measured samples are shown in Table 3.

[0112] As shown in Table 3, the RSD of the repeatability test results is between 0.52% and 1.91%, indicating that the method has good repeatability.

[0113] Table 3 Repeatability test results (μg / ml)

[0114]

[0115]

[0116] 2.4 Stability

[0117] Samples were prepared according to the stability test procedure described above and measured every 2 hours for 6 consecutive times. The concentrations and RSDs of the cannabinoids are shown in Table 4. As shown in Table 4, the RSDs of the stability test results for the 10 cannabinoids ranged from 0.62% to 1.48%, indicating that this method has good stability.

[0118] Table 4 Stability test results (μg / ml)

[0119]

[0120] 2.5 Spike recovery

[0121] Samples were prepared according to the aforementioned spike recovery experimental procedures, injected and analyzed under the same conditions, and recoveries and RSDs were calculated. The results are shown in Table 5. The spiked recoveries of the 10 cannabinoids ranged from 98.2% to 104.6%, meeting the experimental requirements. The relative standard deviations (RSDs) ranged from 0.9% to 2.6%, demonstrating the accuracy and reliability of this method.

[0122] Table 5 Spike recovery experimental results

[0123]

[0124] 2.6 Content of cannabinoids in fresh flower and leaf samples of Yunma No. 8

[0125] The average moisture content of fresh flower and leaf samples of Yunma No. 8 is 65%. The test results and RSDs of 10 cannabinoids in 3 parallel samples are shown in Table 6. The liquid chromatogram of the samples is shown in Figure 2 The relative standard deviations (RSDs) of the 10 cannabinoids were all <2.0%, indicating that the detection results of this method were accurate and reliable.

[0126] Table 6 Contents of 10 cannabinoids in Yunma No. 8 flowers and leaves in the embodiment

[0127]

[0128]

[0129] Comparative Example

[0130] Since carboxylic acid cannabinoids (such as CBDA, THCA, etc.) are unstable and easily deacidified and converted into CBD and THC after being heated, low-temperature drying or natural shade drying is usually used in the conventional pre-treatment process of flower and leaf raw materials. This easily leads to the decarboxylation of carboxylic acid cannabinoids in fresh flowers and leaves, thereby reducing the content and failing to objectively reflect the true content of various cannabinoids in the sample. Therefore, in this comparative example, the fresh flowers and leaves of Yunma No. 8 in the example are subjected to low-temperature drying and then ultrasonic extraction for sample preparation. The only difference between the comparative example and the example is the different pre-treatment, and the flower and leaf raw materials and liquid chromatography conditions are consistent with the example. The specific steps are as follows: 1. Determination of moisture content of fresh flowers and leaves of Yunma No. 8

[0131] The drying method was adopted: 5 g (accurate to 0.001 g) of fresh Yunma No. 8 flowers and leaves were accurately weighed and placed in a weighing dish, and the initial net weight (M1) was recorded. The flowers and leaves were then dried at 105°C to a constant weight. The flowers and leaves were taken out and placed in a desiccator. After the temperature dropped to room temperature, the flowers and leaves were weighed and the net weight (M2) when dried to a constant weight was recorded. The moisture content (W) was calculated in the same way as in the example.

[0132] 2. Pretreatment of fresh flower and leaf samples

[0133] (1) Low-temperature drying: Accurately weigh 3 g (accurate to 0.001 g) of fresh Yunma No. 8 flower and leaves (3 portions), chop them into small pieces, place them in a conical flask, and bake them at 60°C for 12 h.

[0134] (2) Ultrasonic extraction: Accurately measure 30 ml of methanol at a material-liquid ratio of 1:10, add it to the dried sample, weigh it, and ultrasonically extract it for 30 min. Let it stand at room temperature to re-weigh it;

[0135] (3) Sample preparation: The extract was mixed and allowed to stand for 10 min. The supernatant was filtered through a 0.22 μm organic filter membrane and then injected into HPLC to detect the contents of CBDV, CBG, CBD, THCV, CBN, Δ9-THC, Δ8-THC, CBC, and THCA. Another 1 mL of the supernatant was transferred to a 50 mL volumetric flask, made up to volume with 100% methanol, and then shaken. The supernatant was filtered through a 0.22 μm organic filter membrane and then injected into HPLC to detect the content of CBDA.

[0136] 3. High performance liquid chromatography detection: Liquid chromatography conditions are the same as those in the embodiment.

[0137] 4. Calculation of results: The calculation formula for the cannabinoid content in the sample is the same as in the example.

[0138] 5. Results and Analysis

[0139] The average moisture content of fresh Yunma No. 8 flower and leaf samples was determined to be 64%. The test results and RSDs for 10 cannabinoids in three replicate samples are shown in Table 7. In the Examples (see Table 6), CBDA and THCA were well-maintained in the flowers and leaves extracted using liquid nitrogen cryogenic extraction, reaching high levels of 2.650% and 0.140%, respectively, while CBD and Δ9-THC levels were relatively low, at 0.082% and 0.015%, respectively. Compared to the Examples, in the Comparative Examples (Table 7), the average CBDA and THCA contents decreased to 0.497% and 0.038%, respectively, while the average CBD and Δ9-THC contents increased to 1.952% and 0.113%, respectively.

[0140] Table 7 Contents of 10 cannabinoids in the flowers and leaves of Yunma No. 8 in the comparative example

[0141]

[0142] This indicates that carboxylic acid cannabinoids such as CBDA and THCA in industrial hemp flowers and leaves undergo decarboxylation during the pretreatment process, converting them into CBD and THC. Low-temperature drying is not suitable for the detection of carboxylic acid cannabinoids in fresh flowers and leaves, and cannot objectively and truly reflect the accurate content of cannabinoids in the sample, nor can it provide a reliable basis for cultivating distinctive and high-quality industrial hemp varieties. Therefore, the present invention selects liquid nitrogen wall-breaking low-temperature extraction as the pretreatment method for cannabinoid detection in fresh industrial hemp flowers and leaves.

[0143] Under the chromatographic conditions of the present invention, 10 cannabinoids can be completely separated within 15 minutes, with the retention time of the last cannabinoid, THCA, being 13.42 minutes. The separation effect and peak shape of each cannabinoid are ideal, the detection time is short, and the efficiency is high. The method also exhibits good stability, repeatability, precision, and spike recovery, indicating that the detection method is accurate and reliable.

[0144] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, under the guidance of the present invention, several improvements can be made without departing from the principles of the present invention. These improvements should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for rapid detection of 10 cannabinoids in fresh flowers and leaves of industrial hemp based on HPLC, characterized in that: The following steps are involved: S1. Preparation of mixed standard solution; S2. Pre-treatment of fresh industrial hemp flower and leaf samples: (1) Liquid nitrogen grinding and wall breaking: Accurately weigh an appropriate amount of fresh industrial hemp flowers and leaves, chop them into pieces, place them in a ceramic mortar, add an appropriate amount of liquid nitrogen, and quickly grind them into powder; (2) Ultrasonic extraction: Transfer the ground sample to a centrifuge tube, accurately measure an appropriate amount of extraction solvent, clean the mortar in batches, transfer the cleaning solution to a centrifuge tube, add the remaining solvent, weigh, perform ultrasonic extraction, and let it stand at room temperature to re-weight; (3) Sample preparation: The extract was mixed and centrifuged at low temperature. The supernatant was filtered through a 0.22 μm organic filter membrane and the filtrate was used as the test solution. S3. High performance liquid chromatography detection: (1) Standard curve drawing: HPLC was used to detect the mixed standard solution of each concentration prepared in step S1, and the retention time was used for qualitative analysis. A standard curve was drawn based on the corresponding relationship between the peak area measured at each concentration and its concentration; (2) Sample testing: The test solution prepared in step S2 was subjected to HPLC testing, and the retention time was used for qualitative analysis and the standard curve regression equation was used for quantitative analysis to calculate the content of each cannabinoid in the test sample.

2. The method for rapid detection of 10 cannabinoids in fresh flowers and leaves of industrial hemp based on HPLC according to claim 1, characterized in that: The 10 cannabinoid components include: cannabidiol (CBD), cannabidiolic acid (CBDA), cannabidivarin (CBDV), tetrahydrocannabinolic acid (THCA), Δ9-tetrahydrocannabinol (Δ9-THC), Δ8-tetrahydrocannabinol (Δ8-THC), cannabigerol (THCV), cannabigerol (CBG), cannabinol (CBN), and cannabichromene (CBC).

3. According to the method for rapid detection of 10 cannabinoids in fresh flowers and leaves of industrial hemp based on HPLC method in claim 1, in step S1, the mass concentrations of the mixed standard solution of the 10 cannabinoids are: 0.8, 1.6, 4.0, 8.0, 24.0, and 40.0 μg / ml.

4. The method for rapid detection of 10 cannabinoids in fresh flowers and leaves of industrial hemp based on HPLC according to claim 1, wherein the amount of liquid nitrogen added in step S2 is preferably enough to just cover the sample.

5. The method for rapid detection of 10 cannabinoids in fresh flowers and leaves of industrial hemp based on HPLC according to claim 1, characterized in that: In step S2, the extraction solvent is any one of methanol, ethanol, and acetonitrile, and the solid-liquid ratio is 1:5-20; the ultrasonic power is 30-50KHz, and the extraction time is 10-30min; the centrifugation temperature is 4-8°C, the speed is 8000-10000rpm, and the centrifugation time is 5-10min.

6. The method for rapid detection of 10 cannabinoids in fresh flowers and leaves of industrial hemp based on HPLC according to claim 1, characterized in that: In step S3, the HPLC conditions are: Chromatographic column: C18, length 150 mm, inner diameter 4.6 mm, particle size 3-5 μm; Mobile phase: acetonitrile and formic acid aqueous solution, volume ratio 60:40-80:20; Column temperature: 25℃~45℃; flow rate: 0.5mL~1.5mL / min; injection volume: 1~10μL; detection wavelength: 220nm.

7. The method for rapid detection of 10 cannabinoids in fresh flowers and leaves of industrial hemp based on HPLC according to claim 6, characterized in that: The volume percentage of the formic acid aqueous solution in the mobile phase is 0.05%-0.5%; and the elution method is isocratic elution.

8. The method for rapid detection of 10 cannabinoids in fresh flowers and leaves of industrial hemp based on HPLC according to claim 1, characterized in that: The method also includes determining the moisture content of fresh flowers and leaves of industrial hemp by a drying method: accurately weighing an appropriate amount of fresh flowers and leaves and placing them in a weighing dish, recording the initial net weight (M1), then drying them at 105°C to a constant weight, taking them out and placing them in a desiccator, waiting for the temperature to drop to room temperature, weighing them, and recording the net weight of the flowers and leaves when they are dried to a constant weight (M2). The moisture content (W) is calculated according to formula (1): Where: W: moisture content of the test sample, %; M1: initial net weight of the test sample before drying, in grams (g); M2: Net weight of the test sample when dried to constant weight, in grams (g).

9. A method for rapid detection of 10 cannabinoids in fresh flowers and leaves of industrial hemp based on HPLC according to claims 1-8, characterized in that: In step S3, the content of cannabinoids (X) in the fresh flowers and leaves of industrial hemp is calculated according to formula (2): Where: X: content of the component being tested in the test sample, %; C: The concentration of the component being tested in the test solution obtained from the standard working curve. The unit is micrograms per milliliter (μg / ml); V: volume of the test solution, in milliliters (ml); M: mass of the test sample, in grams (g); W: moisture content of the test sample, %.

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