CN110954618A - Method for detecting Cannabigerol (CBG) in industrial cannabis sativa by using HPLC - Google Patents

Abstract

The invention relates to the technical field of detection, in particular to a quantitative detection method for detecting Cannabigerol (CBG) in industrial cannabis sativa by utilizing HPLC. The method comprises ultrasonic extracting Cannabigerol (CBG) from industrial folium Cannabis with methanol, and detecting the liquid to be detected with High Performance Liquid Chromatography (HPLC). And (3) quantitatively obtaining the content of Cannabigerol (CBG) in the flower leaf sample by adopting an external standard method. The method is simple to operate, accurate in result and good in stability, and enables Cannabigerol (CBG) in the cannabis sativa leaves to be quantified quickly and accurately.

Description

Method for detecting Cannabigerol (CBG) in industrial cannabis sativa by using HPLC

Technical Field

The invention relates to the technical field of detection, in particular to a method for detecting Cannabigerol (CBG) in industrial cannabis sativa by utilizing HPLC.

Background

Industrial hemp is becoming a global focus in recent times and is widely used in the industries of textile, paper making, functional health products, cosmetics, medicines, biological energy, building materials, new materials and the like.

Industrial cannabis refers to cannabis with a Tetrahydrocannabinol (THC) content of less than 0.3%, and is an annual herb plant of cannabis genus of cannabinaceae family, the main active ingredient of cannabis is cannabinoids, and the main cannabinoids include Tetrahydrocannabinol (THC), Cannabinol (CBN), Cannabidiol (CBD), Cannabigerol (CBG), cannabichromene (CBC), etc. Wherein cannabigerol (hereinafter referred to as CBG) has high medical value, has antibacterial, antitumor, antiinflammatory, antidepressant, antiemetic, and analgesic effects, and can be used for treating psoriasis. CBG has been considered to be a direct precursor of CBD and CBC in the transformation of cannabis plant matter.

CBG is a non-psychoactive ingredient and exists in the early stage of the hemp growth cycle, so that it is difficult to find a large amount of CBG, which means that the medical value can be obtained only by cultivating hemp, and it is important to accurately determine the content of CBG in flowers and leaves. However, there is no authoritative CBG detection standard, so it is necessary to develop a detection method capable of accurately determining the CBG content in industrial cannabis sativa leaves.

Disclosure of Invention

According to the method, the Cannabigerol (CBG) in the industrial cannabis sativa leaves is extracted by using methanol ultrasonic, the detection is carried out by using a High Performance Liquid Chromatography (HPLC), the quantification is carried out by an external standard method, and the content of the Cannabigerol (CBG) in the industrial cannabis sativa leaves can be accurately and rapidly detected.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention provides a method for measuring content of Cannabigerol (CBG) in industrial cannabis sativa leaves, which comprises the following steps:

① sample preparation, drying fresh industrial hemp flower and leaf sample in oven at 60 deg.C, pulverizing with pulverizer, and mixing.

② preprocessing the sample, accurately weighing the flower and leaf sample in a centrifuge tube, adding organic solvent, ultrasonic extracting, centrifuging, collecting supernatant, repeatedly ultrasonic extracting residues once, centrifuging, mixing supernatants, concentrating to constant volume to obtain sample processing solution to be detected.

③ Standard working solution is prepared by diluting standard solution of Cannabigerol (CBG) with methanol to obtain mixed standard working solution with concentration range of 1-100 μ g/mL, wherein the concentration range covers the content of the component to be detected in the sample.

④ High Performance Liquid Chromatography (HPLC) is carried out by detecting standard working solution and sample treatment solution by HPLC, regression analyzing corresponding concentration with chromatographic peak area of standard working solution target object to obtain standard curve, detecting prepared sample to obtain chromatographic peak area of detected target object, substituting into standard curve to obtain concentration of component to be detected in sample, calculating to obtain content, diluting and detecting if sample treatment solution concentration exceeds standard working solution concentration range.

The High Performance Liquid Chromatography (HPLC) conditions were as follows: the mobile phase is acetonitrile (chromatographic purity) and water (deionized water), and the proportion of the acetonitrile to the water is 65%: 35 percent, isocratic elution with the flow rate of 1 mL/min; the sample injection amount is 10 uL; the column temperature is 30 ℃; the detector detection wavelength is 220 nm.

Further, the hemp flower leaves in step ① are a leaf and bud composition of mature industrial hemp harvested at 9 months.

Further, in step ②, the organic solvent is chromatographically pure methanol, the mass ratio of the volume of the methanol to the flower and leaf sample is 10:1, the ultrasonic time is 20min, the ultrasonic power is 250W, the working frequency is 40kHz, the rotation speed of the centrifugation is 10000 r/min, and the time is 5 min.

Further, the step ③ of preparing the standard working solution of CBG series with concentration range of 1 μ g/mL-100 μ g/mL comprises:

① CBG standard substance (1 mg/mL, 1 mL) from Sigma, USA, was transferred to a 10mL volumetric flask, the flask and dropper were washed several times with chromatographic grade methanol, and the whole was transferred to the volumetric flask (note that it could not spill during the transfer), and the volume was fixed to obtain a CBG solution concentration of 100. mu.g/mL.

② mu g/mL CBG standard solutions of 40. mu.g/mL, 20. mu.g/mL and 10. mu.g/mL were prepared by pipetting 2mL, 1mL and 1mL of CBG standard solutions of 100. mu.g/mL to 5mL, 5mL and 10mL of volumetric flasks, respectively, and metering the volume with methanol.

③ pipette 40. mu.g/mL of CBG standard solution into a 1mL to 10mL volumetric flask and hold the volume with methanol to obtain 4. mu.g/mL of CBG standard solution.

④ pipette 20. mu.g/mL of CBG standard solution into a 1mL to 10mL volumetric flask and hold the volume with methanol to obtain 2. mu.g/mL of CBG standard solution.

⑤ pipette 10. mu.g/mL of CBG standard solution into a 1mL to 10mL volumetric flask and make up the volume with methanol to obtain 1. mu.g/mL of CBG standard solution.

Further, the brand model of the high performance liquid chromatograph is Agilent 1260; acetonitrile of a mobile phase is chromatographic pure, and water is deionized water; the chromatographic column is Agilent ZORBAX Eclipse Plus C18; the detector is a diode array detector (DAD detector for short).

The invention has the advantages that:

① the invention establishes a method for detecting Cannabigerol (CBG) in industrial hemp by using HPLC, can carry out qualitative and quantitative analysis on CBG compounds in industrial hemp, and provides scientific basis for accurate determination and rapid detection of cannabigerol components and contents.

② the invention uses methanol to carry out ultrasonic extraction of CBG in marihuana leaves, the experimental operation is simple, the processing time is short, the HPLC can complete the detection within 15 minutes, and the detection result is accurate and stable.

Drawings

FIG. 1 chromatogram of standard control of Cannabigerol (CBG).

FIG. 2 Standard Curve of Cannabigerol (CBG).

FIG. 3 chromatogram of the hemp flower and leaf sample of example 1.

Detailed Description

The following examples are merely illustrative of the present invention and do not limit the scope of the present invention in any way. It will be apparent to those skilled in the art that equivalent embodiments or modifications without departing from the technical spirit of the present invention are within the scope of the present invention.

Example 1

(1) Preparation of samples: drying fresh industrial hemp flower and leaf samples at 60 ℃ for 12 hours (the water content is less than 5%), crushing the samples to 20 meshes by a crusher, and uniformly mixing the samples to be tested.

(2) Pretreatment of a sample: accurately weighing 1.0g of a flower and leaf sample in a centrifuge tube, adding 10mL of chromatographic pure methanol, carrying out ultrasonic extraction for 20 minutes, then centrifuging for 5 minutes at 10000 rpm by using a centrifuge, transferring supernatant into a concentration bottle, repeatedly carrying out ultrasonic extraction on residues once by using 10mL of methanol, centrifuging, combining the supernatants, concentrating by using a rotary evaporator (at the temperature of 40 ℃) to remove part of solvent, transferring the concentrated solution into a 10mL volumetric flask, carrying out constant volume by using methanol, and filtering by using a 0.22 mu m organic filter membrane to obtain a solution to be detected.

(3) Preparation of standard working solution: preparing CBG series control solutions of 1. mu.g/mL, 2. mu.g/mL, 4. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL and 40. mu.g/mL by the following steps:

① CBG standard substance (1 mg/mL, 1 mL) from Sigma, USA, was transferred to a 10mL volumetric flask, the flask and dropper were washed several times with chromatographic grade methanol, and the whole was transferred to the volumetric flask (note that it could not spill during the transfer), and the volume was fixed to obtain a CBG solution concentration of 100. mu.g/mL.

② mu g/mL CBG standard solutions of 40. mu.g/mL, 20. mu.g/mL and 10. mu.g/mL were prepared by pipetting 2mL, 1mL and 1mL of CBG standard solutions of 100. mu.g/mL to 5mL, 5mL and 10mL of volumetric flasks, respectively, and metering the volume with methanol.

③ pipette 40. mu.g/mL of CBG standard solution into a 1mL to 10mL volumetric flask and hold the volume with methanol to obtain 4. mu.g/mL of CBG standard solution.

④ pipette 20. mu.g/mL of CBG standard solution into a 1mL to 10mL volumetric flask and hold the volume with methanol to obtain 2. mu.g/mL of CBG standard solution.

⑤ pipette 10. mu.g/mL of CBG standard solution into a 1mL to 10mL volumetric flask and make up the volume with methanol to obtain 1. mu.g/mL of CBG standard solution.

(4) Determination by high performance liquid chromatography (HPLC method): detecting the standard working solution and the sample treatment solution by using HPLC (high performance liquid chromatography), wherein the determination method is to perform regression analysis on the corresponding concentration of a target object of the standard working solution by using the chromatographic peak area of the target object to obtain a standard curve; measuring the prepared sample to obtain the chromatographic peak area of the detected target object, substituting the chromatographic peak area into a standard curve to obtain the concentration of the component to be detected in the sample, and further calculating to obtain the content of the component to be detected; and if the concentration of the sample treatment solution exceeds the concentration range of the standard working solution, diluting and detecting.

The High Performance Liquid Chromatography (HPLC) brand number is Agilent 1260 and the conditions are as follows: the mobile phase is acetonitrile and water, the acetonitrile is chromatographic pure, the water is deionized water, and the proportion of the acetonitrile to the water is 65%: 35 percent, isocratic elution with the flow rate of 1 mL/min; the sample injection amount is 10 uL; the chromatographic column is Agilent ZORBAX Eclipse Plus C18, and the column temperature is 30 ℃; the detector is a wavelength diode array detector (DAD detector for short) with a detection wavelength of 220 nm.

CBG peak time: 13.2min, standard curve: y =44.9911X-4.9173, R =0.99951, R²=0.99902。

Experimental example 1 repeatability experiment

The treated sample was prepared according to the method of example 1. Taking the same sample to make 6 parallel samples, carrying out parallel operation, repeating the operation for three days, and inspecting repeatability. The results are shown in Table 1, and the area of the peak area of the target CBG, namely the daily RSD and the daytime RSD are both less than 3%, which shows that the method has good repeatability.

TABLE 1 repeatability test results (Peak area)

Experimental example 2 standard recovery rate experiment

The treated sample was prepared according to the method of example 1. Weighing 4 parts of the same sample, using two parts as substrate samples, adding a CBG standard substance into the other two parts as a standard sample (the addition amount of the CBG is 20 mu g/g, adding the standard substance after the sample is weighed, balancing for 12h, and then processing), carrying out parallel operation on 4 samples, wherein the detection results and the standard recovery rate results of the samples are shown in a table 2. The average standard adding recovery rate of the two standard adding samples is 89.8 percent, which shows that the method has high accuracy and can meet the detection requirement.

TABLE 2 results of recovery test with addition of standard

Claims (6)

1. A method for detecting Cannabigerol (CBG) in industrial cannabis sativa by HPLC, comprising the steps of:

(1) preparation of samples: drying a fresh industrial hemp flower and leaf sample in an oven at 60 ℃, crushing by a crusher, and uniformly mixing to be tested;

(2) pretreatment of a sample: accurately weighing a flower and leaf sample in a centrifuge tube, adding an organic solvent, carrying out ultrasonic extraction, centrifuging to collect supernatant, repeatedly carrying out ultrasonic extraction on residues once, centrifuging, combining supernatants, concentrating and fixing volume to obtain a sample treatment solution to be detected;

(3) preparation of standard working solution: diluting the purchased Cannabigerol (CBG) standard solution with methanol to a standard working solution with the concentration range of 1-100 mug/mL, wherein the concentration range covers the content of the component to be detected in the sample;

(4) determination by high performance liquid chromatography (HPLC method): detecting the standard working solution and the sample treatment solution by using HPLC (high performance liquid chromatography), wherein the determination method is to perform regression analysis on the corresponding concentration of a target object of the standard working solution by using the chromatographic peak area of the target object to obtain a standard curve; measuring the prepared sample to obtain the chromatographic peak area of the detected target object, substituting the chromatographic peak area into a standard curve to obtain the concentration of the component to be detected in the sample, and further calculating to obtain the content of the component to be detected; and if the concentration of the sample treatment solution exceeds the concentration range of the standard working solution, diluting and detecting.

2. The method for detecting Cannabigerol (CBG) in industrial cannabis by HPLC as claimed in claim 1, wherein the High Performance Liquid Chromatography (HPLC) conditions are as follows: the mobile phase is acetonitrile (chromatographic purity) and water (deionized water), and the proportion of the acetonitrile to the water is 65%: 35 percent, isocratic elution with the flow rate of 1 mL/min; the sample injection amount is 10 uL; the column temperature is 30 ℃; the detector detection wavelength is 220 nm.

3. The method for detecting Cannabigerol (CBG) in industrial hemp by HPLC as claimed in claim 1, wherein the hemp flower leaves are a composition of leaves and buds of mature industrial hemp picked in 9 months, dried at 60 ℃ until the moisture is less than 5%, and pulverized to 20 mesh by a pulverizer.

4. The method for detecting Cannabigerol (CBG) in industrial cannabis sativa by HPLC as claimed in claim 1, wherein the organic solvent is chromatographically pure methanol, the ratio of volume amount of methanol to mass amount of the floral leaf sample is 10: 1; the ultrasonic time is 20min, the ultrasonic power is 250W, and the working frequency is 40 kHz; the rotation speed of the centrifugation is 10000 r/min, and the time is 5 min.

5. The method for detecting Cannabigerol (CBG) in industrial cannabis by HPLC as claimed in claim 1, wherein the step of preparing the CBG standard working solution with concentration ranging from 1 μ g/mL to 100 μ g/mL comprises:

① CBG standard substance (1 mg/mL, 1 mL) purchased from Sigma company in USA is transferred into a 10mL volumetric flask, the original flask and a dropper are washed for several times by chromatographic grade methanol, and all the substances are transferred into the volumetric flask (note that the substances cannot be spilled out in the transfer process), and the volume is fixed, so that the concentration of the CBG solution is 100 mug/mL;

② pipetting 100 μ g/mL CBG standard solution respectively to 2mL, 1mL to 5mL, 10mL volumetric flasks, and metering to volume with methanol to obtain 40 μ g/mL, 20 μ g/mL, 10 μ g/mL CBG standard solution;

③ sucking 40 mug/mL CBG standard solution by a pipette, and fixing the volume of the CBG standard solution in a volumetric flask of 1mL to 10mL by methanol to obtain 4 mug/mL CBG standard solution;

④ sucking 20 mug/mL CBG standard solution with a pipette, and fixing the volume of the CBG standard solution with methanol in a volumetric flask of 1mL to 10mL to obtain 2 mug/mL CBG standard solution;

⑤ pipette 10. mu.g/mL of CBG standard solution into a 1mL to 10mL volumetric flask and make up the volume with methanol to obtain 1. mu.g/mL of CBG standard solution.

6. The method for detecting Cannabigerol (CBG) in industrial cannabis by HPLC as recited in claim 1, wherein the HPLC is brand No. agilent 1260; the chromatographic column is Agilent ZORBAX eclipse C18; the detector is a diode array detector (DAD detector for short).

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