CN112730696A

CN112730696A - Method for detecting 5 cannabinol compounds in cannabis sativa oil by using HPLC (high performance liquid chromatography) method

Info

Publication number: CN112730696A
Application number: CN202110148255.2A
Authority: CN
Inventors: 兰韬; 席兴军; 云振宇; 初侨; 于聪聪; 孟玲玲; 张文焕
Original assignee: China National Institute of Standardization
Current assignee: China National Institute of Standardization
Priority date: 2020-09-15
Filing date: 2021-02-03
Publication date: 2021-04-30
Also published as: CN112730697A

Abstract

The invention belongs to the field of analytical chemistry, and relates to a method for detecting 5 cannabinoids in cannabis oil by using High Performance Liquid Chromatography (HPLC). The method adopts a C18 chromatographic column, uses ultrapure water as a mobile phase A and acetonitrile as a mobile phase B to carry out gradient elution, and a sample enters a detector for detection; the cannabinol compound is^Δ9-tetrahydrocannabinol, cannabidiol, cannabinol, tetrahydrocannabinolic acid and cannabidiolic acid. The technical scheme of the invention canThe method lays a methodological foundation for the establishment of relevant detection method standards, and has important significance for preventing cannabis oil with overproof cannabinol content from flowing into the market and promoting effective supervision of domestic and foreign industrial cannabis food.

Description

Method for detecting 5 cannabinol compounds in cannabis sativa oil by using HPLC (high performance liquid chromatography) method

Technical Field

The invention belongs to the field of analytical chemistry, and particularly relates to a method for detecting 5 cannabinoids in cannabis oil by using High Performance Liquid Chromatography (HPLC).

Background

The main effective components of Cannabis are cannabinol, including tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), Cannabinol (CBN), Cannabidiol (CBD),^△9The 5 Tetrahydrocannabinol (THC) contents are more, and the application is most extensive.

The five cannabinol substances, tetrahydrocannabinolic acid, cannabidiolic acid, cannabinol and cannabidiol, have certain pharmacological activity, and for example, cannabidiol has an obvious medical treatment effect on diseases such as epilepsy, depression, multiple sclerosis and the like. While^△9Tetrahydrocannabinol has a central nervous system affecting effect after ingestion or oral administration and is interconvertible with cannabinol and cannabidiol, and therefore needs to be strictly controlled.

The hemp seed oil is grease produced by cold pressing industrial hemp seeds. It contains up to 80% polyunsaturated fatty acids, with a ratio omega-6/omega-3 of 3:1, which is very close to the recommendations of the European food safety agency (3-5: 1). The hemp oil can effectively supplement alpha-linolenic acid which is an essential nutrient for human bodies after long-term eating, achieves the effects of moistening dryness, lubricating intestines, benefiting cardiovascular and cerebrovascular health, and has the effects of reducing blood fat, blood pressure, blood sugar and the like. Meanwhile, the health-care tea has an additional health-care effect because of containing cannabinoids such as CBD (CBD) and CBN (CBN). However, due to differences in preparation processes and maturity of raw materials, the cannabis oil may contain active substances affecting the central nervous system, such as THC. Policies and regulations are successively issued in many countries, such as the united states, germany, switzerland, belgium, European Industrial Hemp Association (EIHA), etc., which set an upper limit on the content of THC in hemp oil. However, China lacks corresponding management regulations for a large amount of cannabis oil products, and particularly lacks a detection method for THC, CBD and other main cannabinoids in the cannabis oil, so that manufacturers and supervision departments cannot determine the content of THC, CBD and other substances in the cannabis oil, and therefore the quality of the cannabis oil products cannot be controlled, and the cannabis oil products in the market are unsmooth. Therefore, it is necessary to establish stable and accurate detection method standards of THC, THCA, CBD, CBDA and CBN in the hemp seed oil.

However, due to the structural similarity of cannabinoids and the switching relationship between these substances, it is difficult to effectively separate various cannabinoids in the prior art, which puts high demands on the accuracy of the analysis method. Marinko Petrovic' and the like develop a detection method for the content of CBD, THC and CBN in the cannabis oil sample based on acetonitrile extraction and GC-MS detection means, and simultaneously explore the relationship between the content of cannabinol and the composition of fatty acid in the cannabis oil.

The patent number is CN107589203, the invention name is a method for simultaneously detecting three cannabinol compounds in hemp by SPE-HPLC, and provides a method for detecting three cannabinol compounds in hemp based on solid phase extraction-high performance liquid chromatography, and qualitative and quantitative analysis is carried out on the three cannabinol compounds. The three cannabinol compounds tested were CBD, CBN and THC, not containing THCA and CBDA.

In summary, no method for simultaneously analyzing and detecting THC, THCA, CBD, CBDA and CBN in the cannabis oil is reported at present, and the existing method lacks a systematic result verification process.

Disclosure of Invention

In view of the above, the present invention is centered on a method for simultaneously detecting 5 cannabinoids, and includes two sub-inventions (i.e., the invention a and the invention B), and respective developments based on the two inventions.

The invention aims to provide a method for simultaneously detecting 5 cannabinol compounds in cannabis sativa oil based on high performance liquid chromatography. The specific technical scheme is as follows.

A method for detecting 5 cannabinol compounds in cannabis sativa oil by using an HPLC method adopts a C18 chromatographic column, uses ultrapure water as a mobile phase A and acetonitrile as a mobile phase B to carry out gradient elution, and a sample enters a detector for detection; the cannabinol compound is^Δ9-tetrahydrocannabinol, cannabidiol, cannabinol, tetrahydrocannabinolic acid and cannabidiolic acid.

Further, the C18 chromatographic column is a Supersil-ODS2 chromatographic column (inner diameter of 4.6 mm. times.250 mm, and particle diameter of filler particles of 3-5 μm); the column temperature is 25-35 ℃; the detection wavelength is 200-230 nm.

Further, the flow rate of the mobile phase is 0.5-1.5 mL/min.

Further, the sample injection volume is 1-10 μ L.

Further, the conditions of the gradient elution are as follows:

0-20min, wherein the volume ratio of the mobile phase A to the mobile phase B is as follows: 30-10: 70-90;

20-22min, wherein the volume ratio of the mobile phase A to the mobile phase B is as follows: 10: 90;

22-24min, wherein the volume ratio of the mobile phase A to the mobile phase B is as follows: 10-30: 90-70;

24-30min, the volume ratio of the mobile phase A to the mobile phase B is as follows: 30:70.

Further, the detection method also comprises the steps of pretreating the sample before detection; the pretreatment comprises the operation steps of adding a sample into a mixed solvent of methanol and isopropanol, uniformly mixing in a vortex mode, then carrying out ultrasonic treatment and centrifugation, and taking supernate for filtration.

Further, the volume ratio of methanol to isopropanol in the mixed solvent is as follows: methanol: and (3) isopropanol is 50-80: 50-20.

Further, adding the sample into a mixed solution of methanol and isopropanol, vortexing for 1-5min, then ultrasonically extracting for 10-20min, centrifuging the extracting solution at 8000-12000rpm for 3-8min, and then taking the supernatant to pass through an organic filter membrane with the diameter of 0.1-0.5 μm.

Further, adding the sample into the mixed solution of methanol and isopropanol, vortexing for 2-5min, mixing, ultrasonically extracting for 10-15min, centrifuging the extract at 10000rpm for 1-5min, and filtering the supernatant with 0.22 μm organic filter membrane.

In one embodiment of the invention, 0.5g of hemp oil sample is accurately weighed into a 10ml centrifuge tube, 2.5ml of mixed solvent (methanol: isopropanol: 80:20, v/v) is added, vortex is carried out for 2min and mixing is carried out, ultrasonic extraction is carried out for 15min, the extracting solution is centrifuged for 5min at 10000rpm, and the supernatant is taken and passed through a 0.22 mu m organic filter membrane to be tested.

In one embodiment of the invention, the filtered supernatant is moved into an m-PFC rapid filtration type purification column for continuous filtration, an injection rod is slowly pushed, a liquid phase small bottle receives effluent, and the obtained filtrate is detected by an HPLC method.

The invention also provides a reagent composition for separating and detecting cannabinol compounds in the cannabis sativa oil, wherein the reagent composition adopts a C18 chromatographic column, ultrapure water is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the cannabinoids include^Δ9-one or more of tetrahydrocannabinol, cannabidiol, cannabinol, tetrahydrocannabinolic acid and cannabidiolic acid.

The invention also provides a standard method for simultaneously detecting 5 cannabinol compounds based on high performance liquid chromatography-tandem mass spectrometry. The specific technical scheme is as follows.

A method for simultaneously detecting 5 cannabinoids by using HPLC-MS/MS adopts a cannabinol special chromatographic column, uses formic acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B to carry out gradient elution, and enters a detector for detection; the cannabinol compound is tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), Cannabinol (CBN), Cannabidiol (CBD) and/or cannabinol compound^△9-Tetrahydrocannabinol (THC).

HPLC-MS/MS, namely high performance liquid chromatography-tandem mass spectrometry are combined, and the separation and analysis technology takes high performance liquid chromatography as a separation means and mass spectrometry as an identification tool. The HPLC method is limited by certain requirements when analyzing complex samples. And the HPLC-MS/MS combination can realize effective separation, qualitative and quantitative analysis on complex samples.

Further, the mass concentration of the formic acid aqueous solution is 0.1-0.5%.

Further, the formic acid aqueous solution has a mass concentration of 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%.

Further, the inner diameter of the chromatographic column special for the cannabinol is 4.6mm multiplied by 250mm, and the particle size of filler particles is 3-5 μm; the column temperature is 25-35 ℃.

Further, the flow rate of the mobile phase is 0.5-1.5 mL/min.

Further, the method employs a multiple reaction monitoring mode (MRM); the ionization modes of the 5 cannabinoids are all negative ion modes.

Further, gradient elution is carried out by adopting the method, and the gradient elution conditions are as follows: 0-15min, wherein the volume ratio of the mobile phase A to the mobile phase B is as follows: 30-5: 70-95; 15-15.1min, the volume ratio of the mobile phase A to the mobile phase B is as follows: 5-30: 95-70; 15.1-25min, the volume ratio of the mobile phase A to the mobile phase B is as follows: 30:70.

Further, the method also comprises the step of carrying out pretreatment operation on the sample before detection; the pretreatment operation comprises heating and melting a sample, dissolving the sample in an organic solvent, centrifuging the solution, and taking supernatant to pass through an organic filter membrane.

Further, the sample is chocolate and/or candy.

Further, adding the melted sample into methanol solution, whirling for 1-5min, centrifuging at 8000-12000rpm for 3-10min, and filtering the supernatant with 0.1-0.5 μm organic filter membrane. The purpose of this step is mainly to extract cannabinol compounds. Because chocolate contains grease, the extraction is carried out by using methanol, but the grease component is difficult to be extracted incompletely, and the detection of cannabinol is easily interfered by the presence of the grease. Therefore, centrifugation is adopted subsequently, and the supernatant is taken and filtered by an organic filter membrane for further purification. The final extract obtained is suitable for HPLC-MS/MS detection according to the invention.

The invention also provides a reagent composition for separating and detecting the cannabinol compounds in the chocolate, wherein the reagent composition adopts a cannabinol special chromatographic column, and takes a formic acid aqueous solution with the mass concentration of 0.1-0.5% as a mobile phase A and acetonitrile as a mobile phase B; the cannabinoids include tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), Cannabinol (CBN), Cannabidiol (CBD) or cannabinoids^△9-Tetrahydrocannabinol (THC).

Advantageous effects

The invention provides an analysis method for simultaneously detecting 5 cannabis phenolic substances in cannabis oil based on high performance liquid chromatography. The chromatographic conditions are optimized by adjusting the proportion of the mobile phase to realize effective separation of the chromatographic peak of the component to be detected and the impurity peak in the matrix, and a sample purification column is not needed, so that the analysis method is simple and convenient to operate, short in time consumption and economical. Meanwhile, the effectiveness of the method is verified through a precision experiment, a repeatability experiment, a standard addition recovery rate experiment and the like, and the result shows that the detection limit, the quantitative limit, the precision, the recovery rate and other results of the method are good, and the method can be used for the analysis and the verification of actual samples.

In addition, the hemp oil in five domestic places is detected, and the fact that the hemp oil contains THC is found, so that certain food safety risk exists. The technical scheme of the invention can lay a methodological foundation for establishing the standards of related detection methods, and has important significance for preventing cannabinol content from exceeding the standard from flowing into the market and promoting effective supervision of domestic and foreign industrial cannabis food.

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. It is obvious that the drawings in the following description are some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive exercise.

FIG. 1 shows the separation patterns of 5 cannabinol compounds in cannabis oil under two different chromatographic conditions (A is gradient procedure 1; B is gradient procedure 2);

FIG. 2 is a comparison of hemp oil samples before purification (A is a mixed standard with standard 1. mu.g/mL; B is a mixed standard with standard 5. mu.g/mL);

FIG. 3 is a high performance liquid chromatogram for testing chocolate samples.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, 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. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all 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.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

As used in this specification, the term "about" typically means +/-5% of the stated value, more typically +/-4% of the stated value, more typically +/-3% of the stated value, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, and even more typically +/-0.5% of the stated value.

In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description of "within a certain range" is merely for convenience and brevityAnd should not be construed as a rigid limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, the range

The description should be read as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, e.g., 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

The invention A comprises the following steps: method for detecting 5 cannabinol compounds in cannabis sativa oil by using HPLC (high performance liquid chromatography) method

Example one

Detection method

1) Brief introduction to materials and instruments

Cannabinol standards: CBDA, CBD, CBN, THC, THCA standards (1mg/mL, 1 mL); methanol, acetonitrile, isopropanol (chromatographic purity). The 5 productive places of cannabis sativa oil are respectively produced in Shanxi, Shaanxi, Guangxi, Heilongjiang and Gansu.

An iChrrom 5100 analytical liquid chromatograph which is provided with a DAD detector; Supersil-ODS2 column (4.6 mm. times.250 mm, 5 μm); a vortex oscillator; a centrifuge; an analytical balance; a water purifier; m-PFC rapid filtration column.

2) Solution preparation

Mobile phase: the mobile phase A is pure water, is self-made by a laboratory water purifier, has the conductivity of 18.2M omega, and is filtered by a water phase filter membrane and degassed by ultrasound before use. The mobile phase B is acetonitrile (chromatographic purity), and is subjected to suction filtration and ultrasonic degassing by an organic phase filter membrane before use.

Standard stock solutions: taking CBDA (1mg/mL), CBD (1mg/mL), CBN (1mg/mL), THC (1mg/mL) and THCA (1mg/mL) standard products, transferring the standard products into a 10mL volumetric flask, adding a mixed solvent (methanol: isopropanol: 80:20, v/v) for constant volume, preparing 5 cannabinol mixed standard stock solutions of 100 mu g/mL, refrigerating and storing in a refrigerator at 4 ℃, and keeping the shelf life of 1 month.

Standard working solution: accurately sucking 0.05 mL, 0.10 mL, 0.25 mL, 0.50 mL, 1.00 mL, 2.50 mL and 5.00mL of the mixed standard stock solution, respectively placing the mixed standard stock solution into a 10mL volumetric flask, fixing the volume by using a mixed solvent (methanol: isopropanol is 80:20, v/v), preparing a series of mixed standard working solutions with mass concentrations of 0.5, 1.0, 2.5, 5.0, 10.0, 25.0 and 50.0 mu g/mL, and preparing the mixed standard working solutions on site.

3) Sample pretreatment: accurately weighing 0.5g (accurate to 0.001g) of cannabis oil into a 10ml centrifuge tube, adding 2.5ml of mixed solvent (methanol: isopropanol: 80:20, v/v), whirling for 2min, mixing uniformly, performing ultrasonic extraction for 15min, centrifuging the extract for 5min at 10000rpm, taking supernatant, and filtering the supernatant with a 0.22 mu m organic filter membrane to be tested.

4) Sample pretreatment and purification: taking 2mL of the supernatant, filtering the supernatant with a 0.22-micron organic filter membrane, transferring the supernatant into the upper end of a m-PFC quick-filtration type small purification column, slowly pushing an injection rod, carrying the effluent liquid by a liquid phase small bottle, and waiting for detection on a computer.

5) Liquid chromatography conditions: a Supersil-ODS2 column (4.6 mm. times.250 mm, 5 μm) was used, the injection volume was 10. mu.L, the flow rate was 1mL/min, the column temperature: at 35 ℃, the detection wavelength is 220nm, the mobile phase A is ultrapure water, the mobile phase B is acetonitrile, and the gradient elution condition is 0-20min and 70% B-90% B; 20-22min, 90% B; 22-24min, 90% B-70% B; 24-30min, 70% B.

6) And (3) precision test: under the same instrument condition, taking a standard working solution with the concentration of 1 mu g/mL, continuously sampling for 6 times according to the liquid chromatogram condition, measuring the peak area, substituting into a standard curve linear regression equation to obtain a measured concentration value, and calculating the relative standard deviation RSD of the measured concentration.

7) And (3) repeatability test: accurately weighing 6 parts of 0.5g (accurate to 0.001g) cannabis oil under the same instrument condition, carrying out sample injection analysis on the 6 parts of processed liquid to be detected according to the liquid chromatography condition according to the method under the sample pretreatment, measuring the peak areas of 5 cannabinols, substituting the peak areas into a standard curve equation, and calculating the standard deviation RSD of the measured concentration.

8) Standard curve: and (3) carrying out sample injection analysis on the prepared standard working solutions of 0.5 mu g/mL, 1 mu g/mL, 2.5 mu g/mL, 5 mu g/mL, 10 mu g/mL, 25 mu g/mL and 50 mu g/mL according to the liquid chromatography conditions, respectively taking the concentrations of the 5 compounds as abscissa and the peak area response values as ordinate, drawing a series of standard curves, and carrying out linear regression to obtain a standard curve linear regression equation of the 5 cannabinols.

9) Quantification limit and detection limit: and (3) measuring the standard stock solution according to the liquid chromatography conditions, measuring peak areas of 5 cannabinols, and substituting the peak areas into a standard curve regression equation of corresponding substances. The LOD and LOQ of the method are calculated by taking the average signal-to-noise ratio of 3 times of the target component in the standard solution with the lowest level as the detection Limit (LOD) and the average signal-to-noise ratio of 10 times as the quantification Limit (LOQ).

10) And (3) standard addition recovery rate: accurately weighing 12 parts of cannabis sativa oil 0.5g (accurate to 0.01g), numbering 1-12, wherein No. 1-3 is used as a background, No. 4-6 is added to be labeled into a solution to be detected with a concentration of 0.5 mu g/mL, No. 7-9 is added to be labeled into a solution to be detected with a concentration of 1 mu g/mL, No. 10-12 is added to be labeled into a solution to be detected with a concentration of 5 mu g/mL, operating according to the method under the pretreatment of the sample, analyzing the treated background solution to be detected and low, medium and high 3 concentrations of labeled samples by HPLC, detecting a peak area, and calculating the recovery rate.

Results and analysis

1) Liquid chromatography condition optimization

In order to separate the five cannabinol species to be tested from the matrix components in the cannabis oil as much as possible, the mobile phase gradient procedure was first optimized. Taking methanol extract of Shanxi cannabis oil as a background solution, adding 5 microgram/mL mixed standard substance into the methanol extract, and performing chromatographic separation on the background solution and the added standard solution respectively by adopting two different mobile phase gradient programs, wherein the two mobile phase gradient programs are respectively as follows: (1)0-20min, 80% B-90% B; 20-22min, 90% B; 22-24min, 90% B-80% B; 24-30min, 80% B. (2)0-20min, 70% B-90% B; 20-22min, 90% B; 22-24min, 90% B-70% B; 24-30min, 70% B. The separation results are shown in FIG. 1, and the peak assignment was confirmed by the single-standard method. The results show that CBD is not separated from the cannabis oil matrix components and that THC and THCA are poorly separated under the gradient program (1) (fig. 1A). By reducing the composition of the initial organic phase, under the gradient program (2), the separation condition of each peak in the cannabis oil is better, and the impurity peak in the matrix and the chromatographic peaks of the five cannabinol substances to be detected are not mutually covered (fig. 1B), so the subsequent experiments are all optimized and verified by adopting the gradient program 2.

To summarize: as can be seen from the comparison between fig. 1A and fig. 1B, by adjusting the ratio of the mobile phase and changing the elution conditions, the effect of separating the impurity peak from the analyte chromatographic peak in the matrix can be achieved, and the possibility of interference of the impurity peak is reduced, so that the step of purifying the extract is omitted, and the operation is simple and convenient, and the time consumption is short.

2) Extraction solvent optimization

According to the physicochemical properties of 5 cannabinols, under the same instrument and experimental conditions, (1) 100% methanol was selected in the experiment; (2) methanol: 80 parts of isopropanol: 20; (3) methanol: isopropanol 50: 50, taking the same cannabis oil sample, adding 5 microgram/mL mixed standard substance, extracting with the 3 solvents respectively by adopting a standard stock solution extraction mode, and measuring the recovery rate of the added standard, wherein the results are shown in the following table 1. As can be seen from the table, the recovery of the sample after addition of isopropanol can be controlled between 70% and 100%, where the ratio of methanol: 80 parts of isopropanol: 20, the extraction is carried out, the result has better stability, and the RSD can be controlled to be less than 5 percent. Therefore methanol was used in the subsequent experiments: 80 parts of isopropanol: 20 of the extraction solvent.

TABLE 1 results of three different solvent extractions

3) Purification of extract

Because the hemp oil matrix contains a large amount of fatty acid, in order to reduce the influence of the matrix on the object to be measured and improve the sensitivity of the method, a sample matrix purification method is generally adopted to remove impurities in the sample matrix as much as possible on the premise of retaining the object to be measured.

The filtration purification method (m-PFC) is a method for purifying interfering substances in a matrix, which has been developed based on the QuEChERS method. The method comprises the steps of filling a filler containing multi-walled carbon nanotubes, N-propyl ethylenediamine (PSA), C18 and anhydrous magnesium sulfate into an injector, and enabling an extracting solution to pass through the filler in an extraction or pushing mode when the filler is used, so that the filler is enabled to act or adsorb with interference substances such as pigments, lipids, partial saccharides, sterols, tea polyphenols, organic acids, alkaloids and the like in a matrix, and the interference substances are not reacted with a target substance, and the purification of the matrix is completed. Compared with the traditional method, the method does not need to weigh the adsorbent, greatly shortens the purification time, can be used together with LC-MS, IMS, Raman and the like on line or off line, and can greatly improve the pretreatment speed and efficiency.

The invention inspects the influence of the m-PFC sample purification mode on the detection method, and the method respectively uses Shanxi hemp seed oil methanol extract added with mixed standard substances of 1 mug/mL and 5 mug/mL as a sample, selects an m-PFC rapid purification column as a purification column, purifies the sample by adopting the sample pretreatment purification method, and performs HPLC detection under the chromatographic conditions in the sample pretreatment, and the result is shown in figure 2. Wherein, FIG. 2A is the mixed standard substance with the added standard of 1 μ g/mL, and FIG. 2B is the mixed standard substance with the added standard of 5 μ g/mL.

To summarize: as can be seen from fig. 2, the m-PFC purification column selected in the experiment has a poor purification effect, not only is new impurities introduced, but also the component THCA to be detected is severely adsorbed, so that THCA is not detected, and therefore, no purification column is used for pretreatment in subsequent experiments. In the subsequent research, the physical and chemical properties of cannabinol can be referred to, the purification effects of various purification columns are compared, on the basis of not introducing impurities, various factors are combined, and the purification column capable of well absorbing other substances except the detected components is selected, so that the influence of the matrix effect on the detection result is reduced.

4) Standard curve, quantitative limit, detection limit

The series of 5 cannabinol mixed standard solutions were tested by drawing a standard curve regression equation from ichrom5100works, and the linear range, correlation coefficient, LOD and LOQ are shown in table 2 below.

TABLE 2 Linear Range, LOD and LOQ of five cannabinols

To summarize: as can be seen from the data in the table, the method has a good linear relation in the range of 0.5-50 mug/mL, and the correlation coefficients r2 are all greater than 0.998. The detection limit (LOD, S/N is 3) of 5 cannabinoids is 0.10-0.25 mu g/g, and the quantification limit (LOQ, S/N is 10) is 0.33-0.83 mu g/g. The method of the invention can reach standardization and can detect the concentration near the quantitative limit at the lowest.

5) Precision degree

The concentrations of five cannabinols CBDA, CBD, CBN, THC, THCA and RSD measured 6 times in parallel with 1. mu.g/mL mixed standard solution according to the precision experimental procedure described above are shown in Table 3 below.

TABLE 3 results of the precision test

To summarize: as can be seen from the data in the table, the precision experimental result RSD is between 0.7% and 3.9%, which shows that the method is stable and reliable and has high accuracy.

6) Repeatability of

The concentrations of five cannabinols, namely CBDA, CBD, CBN, THC and THCA, and RSD in the parallel 6 groups of cannabinol oils were determined by taking cannabis oil from Shaanxi as a sample according to the above-mentioned repetitive experimental procedures as shown in Table 4 below.

TABLE 4 results of repeated experiments

To summarize: as can be seen from the upper standard data, the RSD of 6 repeated experiments is between 1.9% and 3.2%, which indicates that the method has good repeatability.

7) Recovery rate of added standard

Accurately transferring a certain amount of mixed standard solution, adding the mixed standard solution into a sample, using a mixed solvent (methanol: isopropanol: 80:20, v/v) to perform constant volume preparation to prepare low, medium and high standard samples with concentration levels of 0.5 mu g/mL, 1 mu g/mL and 5 mu g/mL, using the experimental method to perform measurement, and calculating the recovery rate, wherein the results are shown in Table 5.

TABLE 5 results of recovery test with addition of standard

To summarize: as can be seen from the data in the table, the recovery rate of 5 cannabinols is 77.1-103.3%, and the relative standard deviation RSD is less than or equal to 4.7%, which shows that the method has good recovery rate and accurate and reliable results.

8) Determination of actual samples

The analytical test is carried out on the cannabis oil in 5 different producing areas of Shanxi, Guangxi, Shaanxi, Heilongjiang and Gansu according to the experimental formula, and the results are shown in Table 6.

TABLE 6 cannabinol content in cannabis sativa oil for different production areas

To summarize: the analysis result of a practical sample shows that 5 cannabinols in the cannabis oil of 4 production places such as Shaanxi, Guangxi, Heilongjiang, Gansu and the like are detected except that the Shanxi cannabis oil does not contain CBDA and THCA, and the content of addictive THC is more than 3.2 mug/g, wherein the THC content in the Shaanxi cannabis oil is even as high as 11.9 mug/g, which indicates that the commercially available cannabis oil has different degrees of cannabinol substance pollution and certain food safety risk.

The invention B comprises the following steps: method for simultaneously detecting 5 cannabinol compounds in chocolate by using HPLC-MS/MS

Example one

1) Sample pretreatment:

weighing 0.5g chocolate sample in a 10ml centrifuge tube, covering the cover, preheating in a 45 deg.C water bath for 5min to melt, adding 2.5ml methanol, vortex for 2min, centrifuging at 1000rmp for 5min, collecting supernatant, and filtering with 0.22 μm organic filter membrane.

2) And (3) detection:

the chromatographic conditions for the HPLC separation were: a column (4.6mm × 250i.d.,5.0 μm) dedicated to cannabinol, the column temperature being 30 ℃. The mobile phase A is 0.1% formic acid water solution, the mobile phase B is acetonitrile, the flow rate is 1.0mL/min, and the mobile phase is eluted in a gradient way. The gradient elution procedure was: 0-15min, 70% -95% B; 15-15.1min, 95% -70% B; 15.1-25min, 70% B.

3) Mass spectrum:

the mass spectrum part adopts a multiple reaction monitoring mode (MRM), and the mass spectrum parameters of five cannabinol substances are shown in the following table:

TABLE 7 Mass Spectrometry parameters for five cannabinoids

4) Results

The content of 5 cannabinol in the candy chocolate product is determined by an external standard method. The detection limit of the method is 10 mug/kg, the quantitative limit is 30 mug/kg, the recovery rate of 5 cannabinols is between 90 and 110 percent, and the RSD is less than 5 percent.

The total ion flux plot is shown in fig. 3. Wherein, according to the peak-appearing sequence, the corresponding peaks are CBDA, CBD, CBN, THC and THCA-A in turn.

While the present invention has been described with reference to the embodiments shown in the drawings, the present invention is not limited to the embodiments, which are illustrative and not restrictive, and it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for detecting 5 cannabinol compounds in cannabis sativa oil by using an HPLC method is characterized in that a C18 chromatographic column is adopted, ultrapure water is used as a mobile phase A, acetonitrile is used as a mobile phase B for gradient elution, and a sample enters a detector for detection; the above-mentionedCannabinol compounds are^Δ9-tetrahydrocannabinol, cannabidiol, cannabinol, tetrahydrocannabinolic acid and cannabidiolic acid.

2. The method of claim 1, wherein the C18 chromatography column is a Supersil-ODS2 chromatography column; the column temperature is 25-35 ℃; the detection wavelength is 200-230 nm.

3. The method of claim 1, wherein the flow rate of the mobile phase is 0.5 to 1.5 mL/min.

4. The method of claim 1, wherein the sample is injected in a volume of 1 to 10 μ L.

5. The method of any one of claims 1 to 4, wherein the gradient elution conditions are as follows:

6. The method of claim 1, further comprising pre-treating the sample prior to detecting; the pretreatment comprises the operation steps of adding a sample into a mixed solvent of methanol and isopropanol, uniformly mixing in a vortex mode, then carrying out ultrasonic treatment and centrifugation, and taking supernate for filtration.

7. The method according to claim 6, wherein the volume ratio of methanol to isopropanol in the mixed solvent is: methanol: and (3) isopropanol is 50-80: 50-20.

8. The method as claimed in claim 6, wherein the sample is added to the mixed solution of methanol and isopropanol, vortexed for 1-10min, then sonicated for 5-20min, the extract is centrifuged at 8000-12000rpm for 1-10min, and then the supernatant is passed through an organic filter of 0.1-0.5 μm.

9. The method of claim 8, wherein the sample is added into the mixed solution of methanol and isopropanol, vortexed for 2-5min, ultrasonically extracted for 10-15min, the extract is centrifuged at 10000rpm for 1-5min, and the supernatant is filtered through a 0.22 μm organic filter membrane to be tested.

10. A reagent composition for separating and detecting cannabinoids in cannabis sativa oil is characterized in that the reagent composition adopts a C18 chromatographic column, ultrapure water is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the cannabinoids include^Δ9-one or more of tetrahydrocannabinol, cannabidiol, cannabinol, tetrahydrocannabinolic acid and cannabidiolic acid.