CN115792008A - Method for measuring content of cannabidiol in medical-grade high-purity cannabidiol - Google Patents
Method for measuring content of cannabidiol in medical-grade high-purity cannabidiol Download PDFInfo
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Abstract
The invention provides a method for measuring the content of cannabidiol in medical-grade high-purity cannabidiol, which comprises the following steps: step 1, weighing cannabidiol crystal powder, adding an organic solvent, performing ultrasonic dissolution, and diluting to obtain a solution to be detected; step 2, detecting the liquid to be detected by using a high performance liquid chromatograph, wherein the mobile phase of the high performance liquid chromatograph is formic acid water and formic acid acetonitrile, and performing gradient elution; step 3, quantifying by using an external standard method: preparing series of reference solutions with cannabidiol and tetrahydrocannabinol standard reference substances, and measuring the content of cannabidiol and tetrahydrocannabinol in the liquid to be measured. According to the invention, the cannabidiol and the tetrahydrocannabinol in the medical grade high-purity cannabidiol crystal powder are dissolved by using methanol in an ultrasonic manner, the experimental operation is simple, the processing time is short, and the detection result is accurate. The invention uses deionized water and acetonitrile as mobile phase, and has gradient elution, good target object peak shape and good separation degree.
Description
Technical Field
The invention relates to the technical field of detection, and particularly relates to a quantitative detection method for cannabidiol in pharmaceutical grade high-purity cannabidiol crystal powder.
Background
Industrial cannabis refers to cannabis with the Tetrahydrocannabinol (THC) content lower than 0.3%, china refers to industrial cannabis as han-hemp, which is an annual herbaceous plant in cannabis of cannabinaceae, the main active ingredient in cannabis is a cannabinoids compound, at present, 70 natural cannabinoids are known, and the industrial cannabis is mainly used for multiple sclerosis, motor nerve diseases, chronic intractable pain and drug induced vomiting in certain nervous system diseases, and in addition, has a certain effect on glaucoma, asthma and cardiovascular diseases. The most common components are Tetrahydrocannabinol (THC) and Cannabidiol (CBD), with different cannabis flowers and leaves containing different amounts of tetrahydrocannabinol and cannabidiol.
The pharmaceutical high-purity cannabidiol crystal powder refers to high-purity cannabidiol crystal extracted from natural industrial cannabis sativa flower leaves, white or off-white to yellowish crystal powder. Cannabidiol has high medicinal value, tetrahydrocannabinol has a neurohallucinogenic effect, can generate hallucinations, is listed as one of three major drugs in parallel with heroin and cocaine by the toxicity banning convention of the united nations, and becomes a reason for banning cannabis in many countries. With the intensive research on cannabis, cannabidiol is found to block the influence of tetrahydrocannabinol on the nervous system of a human body, has pharmacological activities of resisting spasm, rheumatic arthritis, anxiety and the like, and can effectively improve the sleep quality, relieve nerves, relax mood, relieve pressure and prevent depression. Cannabidiol has wide action and is not limited to single disease species or organ health care. Under the premise of not causing obvious damage to any cognitive system, the cannabidiol has the functions of relieving stress, resisting anxiety, resisting pressure and the like, and simultaneously has obvious positive effects on preventing various diseases such as senile dementia, epilepsy, infantile autism, cancer, liver cirrhosis and the like.
According to authoritative data statistics, about 1% of the world's population may become potential cannabidiol product users at some point in life in the next decade. The existing detection technology focuses on detection of industrial hemp flower leaves, full spectrum oil of cannabidiol, broad spectrum oil and cannabidiol crystals, and no detection method for accurately determining pharmaceutical grade high-purity cannabidiol crystal powder exists, along with the progress of the technology, the pharmaceutical grade high-purity cannabidiol crystal powder has higher and higher purity, the content of cannabidiol can reach 99.9 percent, even 99.99 percent, and based on the above, the development of the detection method capable of accurately determining the content of cannabidiol and tetrahydrocannabinol in the pharmaceutical grade high-purity cannabidiol crystal powder is necessary.
Disclosure of Invention
Aiming at the problems pointed out in the background technology, the invention provides a method for measuring the content of cannabidiol in medical-grade high-purity cannabidiol, which is characterized in that the content of cannabidiol and tetrahydrocannabinol in medical-grade high-purity cannabidiol crystal powder can be accurately and quickly detected by detecting with a high performance liquid chromatograph and quantifying with an external standard method.
The technical scheme of the invention is realized as follows:
a method for measuring the content of cannabidiol in pharmaceutical grade high-purity cannabidiol comprises the following steps: step (1), precisely weighing a medical grade high-purity cannabidiol crystal powder sample into a volumetric flask; adding an organic solvent, dissolving by ultrasonic, fixing the volume to a scale by using the organic solvent, and diluting by a proper time to obtain a solution to be detected;
detecting a liquid to be detected by using a high performance liquid chromatograph, wherein mobile phases of the high performance liquid chromatograph are formic acid water and formic acid acetonitrile, and performing gradient elution;
step (3), quantifying by using an external standard method: preparing a series of reference solutions with cannabidiol and tetrahydrocannabinol standard reference substances, and measuring the content of cannabidiol and tetrahydrocannabinol in the medical-grade high-purity cannabidiol crystal powder to-be-measured solution by an external standard method.
Preferably, the sample weighing amount in the step 1 is 10-100mg, and the dilution multiple is 500-10000 times.
Preferably, the sample weight in step 1 is 50mg, and the dilution factor is 1000 times.
Preferably, the organic solvent in step 1 is a chromatographic pure solvent selected from methanol, ethanol, n-hexane, and acetic acid
One or two of ethyl ester.
Preferably, the organic reagent in step 1 is chromatographically pure methanol.
Preferably, the ultrasonic time in the step 1 is 1min-5min.
Preferably, the ultrasonic time in step 1 is 3min, the ultrasonic power is 250W, and the working frequency is 40kHz.
Preferably, the brand model of the high performance liquid chromatograph in the step 2 is Agilent 1260; the mobile phase acetonitrile is chromatographically pure, the water is deionized water, and the proportion of the acetonitrile to the water is (60-80%): (20% -40%), gradient elution, flow rate is 0.8mL/min-1.2mL/min; the sample injection amount of the high performance liquid chromatograph is 8uL-12uL; the column temperature of the high performance liquid chromatograph is 20-40 ℃; the detection wavelength of the high performance liquid chromatograph is 210nm-230nm.
Preferably, the brand model of the high performance liquid chromatograph in the step 2 is Agilent 1260; the mobile phase acetonitrile is chromatographically pure, the water is deionized water, the example of the mobile phase is shown in the table, and the flow rate is 1.0mL/min; the sample injection amount of the high performance liquid chromatograph is 10uL; the column temperature of the high performance liquid chromatograph is 30 ℃; the detection wavelength of the high performance liquid chromatograph is 220nm.
TABLE 1 mobile phase gradient elution conditions
Preferably, the cannabidiol and tetrahydrocannabinol standard control solutions in step (4) are purchased from Sigma company of usa, cannabidiol and tetrahydrocannabinol standard solutions with concentrations of 1mg/mL are respectively diluted with chromatographic pure methanol to 10ug/mL, 30ug/mL, 50ug/mL, 70ug/mL and 100ug/mL series of control solutions, and are detected by a high performance liquid chromatograph, and a standard curve is drawn by taking the substance concentration as a horizontal coordinate and the peak area as a vertical coordinate.
By adopting the technical scheme, the invention has the beneficial effects that:
(1) According to the invention, the cannabidiol and the tetrahydrocannabinol in the medical grade high-purity cannabidiol crystal powder are ultrasonically dissolved by using the methanol, the experimental operation is simple, the processing time is short, and the detection result is accurate.
(2) The method uses deionized water and acetonitrile as mobile phases and performs gradient elution, and the target object has good peak shape and good separation degree. The mobile phase of the invention uses pure water, does not need to be provided with buffer salt, can prolong the service life of instrument components such as chromatographic columns and the like, and has simple instrument maintenance.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
Figure 1 cannabidiol standard control chromatogram.
Figure 2 cannabidiol standard curve.
FIG. 3 chromatogram of tetrahydrocannabinol standard control.
Figure 4 tetrahydrocannabinol standard curve.
FIG. 5 sample chromatogram of example 1.
FIG. 6 sample chromatogram of example 2.
FIG. 7 sample chromatogram of example 3.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention is described below with reference to fig. 1-4:
example 1:
32.04mg of a medical grade high-purity cannabidiol crystal powder sample is precisely weighed and put into a 100mL volumetric flask. Adding 20mL of methanol into the volumetric flask, oscillating with super-sonic waves until the sample is completely dissolved, then fixing the volume to the scale with the methanol, and uniformly mixing; precisely measuring 1mL of mixed solution in a 10mL measuring flask, adding methanol to a constant volume to scale, filtering with a 0.22 μm organic filter membrane, and measuring.
The instrument conditions of a high performance liquid chromatograph (Agilent 1260) are set as follows: the proportion of mobile phase chromatogram pure acetonitrile and deionized water is shown in the following table, gradient elution is carried out, and the flow rate is 1.0mL/min; the sample size was 10uL. The chromatographic column is Agilent ZORBAX Eclipse Plus C18, and the column temperature is 30 ℃; the detection wavelength was 220nm.
Cannabidiol and tetrahydrocannabinol standard reference substance solutions are purchased from Sigma company in America, cannabidiol and tetrahydrocannabinol standard substance solutions with the concentration of 1mg/mL are respectively diluted into 10ug/mL, 30ug/mL, 50ug/mL, 70ug/mL and 100ug/mL series reference substance solutions by using chromatographic pure methanol, and are detected by a high performance liquid chromatograph, and a standard curve is drawn by taking the substance concentration as a horizontal coordinate and the peak area as a vertical coordinate.
Quantifying by an external standard method: under the chromatographic conditions, a sample to be detected is detected by liquid chromatography, the corresponding retention time of standard reference substances of cannabidiol and tetrahydrocannabinol is compared, the peak area values of cannabidiol and tetrahydrocannabinol are recorded, the peak areas are brought into an external standard curve, the concentrations of cannabidiol and tetrahydrocannabinol in the liquid to be detected can be obtained, and the content of cannabidiol and tetrahydrocannabinol in the pharmaceutical grade high-purity cannabidiol crystal powder sample is calculated.
The external standard method is to measure separately under the same chromatographic conditions as the sample to be measured, and compare the obtained chromatographic peak area with the chromatographic peak area of the component to be measured to obtain the content of the component to be measured. The external standard substance and the component to be measured are both a substance but require certain purity, and the concentration of the external standard substance is close to that of the component to be measured during analysis, so that the accuracy of quantitative analysis is facilitated.
The external standard method can be divided into a correction curve method and an algorithm using a correction factor in operation and calculation.
The calibration curve method is to use a series of standard samples with different known contents to perform equal-quantity sample injection analysis, and then to make a relation curve between response signals and contents, namely a calibration curve. When the sample is quantitatively analyzed, the same amount of the same sample is added under the same condition of the calibration curve, the peak height or the peak area is measured from the chromatogram, and the content of the sample is searched from the calibration curve.
Example 2:
accurately weighing 10.89mg of medical grade high-purity cannabidiol crystal powder sample into a 100mL volumetric flask. Adding 20mL of methanol into the volumetric flask, oscillating with super-sonic waves until the sample is completely dissolved, then fixing the volume to the scale with the methanol, and uniformly mixing; filtering with 0.22 μm organic filter membrane, and testing.
The instrument conditions of a high performance liquid chromatograph (Agilent 1260) are set as follows: the proportion of mobile phase chromatogram pure acetonitrile and deionized water is shown in the following table, gradient elution is carried out, and the flow rate is 1.0mL/min; the sample size was 10uL. The chromatographic column is Agilent ZORBAX Eclipse Plus C18, and the column temperature is 30 ℃; the detection wavelength was 220nm.
Cannabidiol and tetrahydrocannabinol standard reference substance solutions are purchased from Sigma company in America, cannabidiol and tetrahydrocannabinol standard substance solutions with the concentration of 1mg/mL are respectively diluted into 10ug/mL, 30ug/mL, 50ug/mL, 70ug/mL and 100ug/mL series reference substance solutions by using chromatographic pure methanol, and are detected by a high performance liquid chromatograph, and a standard curve is drawn by taking the substance concentration as a horizontal coordinate and the peak area as a vertical coordinate.
Quantifying by an external standard method: under the chromatographic conditions, a sample to be detected is detected by liquid chromatography, the corresponding retention time of standard reference substances of cannabidiol and tetrahydrocannabinol is compared, the peak area values of cannabidiol and tetrahydrocannabinol are recorded, the peak areas are brought into an external standard curve, the concentrations of cannabidiol and tetrahydrocannabinol in the liquid to be detected can be obtained, and the content of cannabidiol and tetrahydrocannabinol in the pharmaceutical grade high-purity cannabidiol crystal powder sample is calculated.
Example 3:
30.07mg of industrial and medical grade high-purity cannabidiol crystal powder sample is precisely weighed into a 100mL volumetric flask. Adding 20mL of n-heptane into the volumetric flask, oscillating with super-sonic waves until the sample is completely dissolved, then adding n-heptane to fix the volume to a scale, and uniformly mixing; precisely measuring 1mL of mixed solution in a 10mL measuring flask, adding n-heptane to a constant volume to a scale, and filtering with a 0.22 μm organic filter membrane to be measured.
The instrument conditions of a high performance liquid chromatograph (Agilent 1260) are set as follows: the proportion of mobile phase chromatogram pure acetonitrile and deionized water is shown in the following table, gradient elution is carried out, and the flow rate is 1.0mL/min; the sample size was 10uL. The chromatographic column is Agilent ZORBAX Eclipse Plus C18, and the column temperature is 30 ℃; the detection wavelength was 220nm.
Cannabidiol and tetrahydrocannabinol standard reference substance solutions are purchased from Sigma company in America, cannabidiol and tetrahydrocannabinol standard substance solutions with the concentration of 1mg/mL are respectively diluted into 10ug/mL, 30ug/mL, 50ug/mL, 70ug/mL and 100ug/mL series reference substance solutions by using chromatographic pure methanol, and are detected by a high performance liquid chromatograph, and a standard curve is drawn by taking the substance concentration as a horizontal coordinate and the peak area as a vertical coordinate.
Quantifying by an external standard method: under the chromatographic conditions, a sample to be detected is detected by liquid chromatography, the corresponding retention time of standard reference substances of cannabidiol and tetrahydrocannabinol is compared, the peak area values of cannabidiol and tetrahydrocannabinol are recorded, the peak areas are brought into an external standard curve, the concentrations of cannabidiol and tetrahydrocannabinol in the liquid to be detected can be obtained, and the content of cannabidiol and tetrahydrocannabinol in the pharmaceutical grade high-purity cannabidiol crystal powder sample is calculated.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (5)
1. A method for measuring the content of cannabidiol in medical grade high-purity cannabidiol is characterized by comprising the following steps:
step 1, weighing cannabidiol crystal powder, adding an organic solvent, performing ultrasonic dissolution, and diluting to obtain a solution to be detected;
step 2, detecting the liquid to be detected by using a high performance liquid chromatograph, wherein the mobile phase of the high performance liquid chromatograph is formic acid water and formic acid acetonitrile, and performing gradient elution;
step 3, quantifying by using an external standard method: preparing series of reference solutions with cannabidiol and tetrahydrocannabinol standard reference substances, and measuring the content of cannabidiol and tetrahydrocannabinol in the liquid to be measured.
2. The method of claim 1, wherein the method comprises the steps of: the organic solvent is one or a mixture of methanol, ethanol, n-hexane and ethyl acetate.
3. The method of claim 1, wherein the method comprises the steps of: weighing cannabidiol crystal powder sample with the weight of 10-1000mg and the dilution multiple of 500-10000 times.
4. The method of claim 1, wherein the method comprises the steps of: the ultrasonic time is 1min-5min, the ultrasonic power is 250W, and the working frequency is 40kHz.
5. The method of claim 1, wherein the method comprises the steps of: the brand model of the high performance liquid chromatograph is Agilent 1260; the mobile phase acetonitrile is chromatographically pure, the formic acid is chromatographically pure, the water is deionized water, and the ratio of the formic acid to the water is (0.1-0.2%): (99.9% -99.8%), the ratio of formic acid to acetonitrile is (0.1% -0.2%): (99.9% -99.8%), the proportion of acetonitrile and water is (60% -80%): (20% -40%), gradient elution, flow rate is 0.8mL/min-1.2mL/min; the sample injection amount of the high performance liquid chromatograph is 8uL-12uL; the column temperature of the high performance liquid chromatograph is 20-40 ℃; the detection wavelength of the high performance liquid chromatograph is 210nm-230nm.
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