CN114705763A - Method for simultaneously detecting CBD, CBN, THC and THCA by adopting high performance liquid chromatography - Google Patents
Method for simultaneously detecting CBD, CBN, THC and THCA by adopting high performance liquid chromatography Download PDFInfo
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- G—PHYSICS
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention belongs to the field of plant component detection, and particularly relates to a method for simultaneously detecting Cannabidiol (CBD), Cannabinol (CBN), Tetrahydrocannabinol (THC) and tetrahydrocannabinolic acid (THCA) by adopting a high performance liquid chromatography, which comprises the steps of chromatographic separation and detection, and is characterized in that the conditions of the chromatographic separation comprise: the mobile phase comprises acetonitrile, methanol and water; the volume ratio of the acetonitrile to the methanol to the water is 44:39: 17; the pH value of the mobile phase is 3.38-3.42; the elution mode is isocratic elution; the column temperature is 30 ℃; the chromatographic column for chromatographic separation is C18A column; the sample injection amount is 10 mu L; the flow rate of the mobile phase was 1.0 mL/min. The detection conditions include: the detector is a multi-wavelength ultraviolet detector or twoAn electrode tube array detector; the detection wavelengths were 210nm and 220 nm. The method provided by the invention has the advantages of stable baseline and good repeatability.
Description
Technical Field
The invention belongs to the field of plant component detection, and relates to a method for simultaneously detecting CBD, CBN, THC and THCA by adopting a high performance liquid chromatography.
Background
Industrial cannabis is becoming legislated in some countries and regions, where Cannabidiol (CBD) content is the focus of most interest to researchers. The standard for distinguishing industrial cannabis from toxic cannabis is the hallucinogenic component Tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA) can generate THC under the condition of heating, and the potential influence of the THCA content on the THC content detection is large. In recent years, the literature and standards have begun to identify industrial and toxic cannabis with THCA and THC content. Therefore, it is necessary to monitor the THCA content at the same time as the THC content. Furthermore, Cannabinol (CBN), which is a decomposition product of THC, is not normally found in live or freshly harvested cannabis plants, which is formed after exposure of the cannabis plants to light or oxygen for a prolonged period of time, and thus simultaneous monitoring of CBN content in long-standing samples is of reference value.
At present, the prior art "Fast Detection of 10Cannabinoids by RP-HPLC-UV Method in Cannabis sativa L" discloses a Method for simultaneously detecting THC, THCA, CBD and CBN by adopting high performance liquid chromatography, and acetonitrile and water are taken as mobile phases for gradient elution. However, the above detection method has problems of serious baseline drift and poor repeatability of detection results.
Disclosure of Invention
In view of the above, the present invention provides a method for simultaneously detecting CBD, CBN, THC and THCA by high performance liquid chromatography. The method provided by the invention has the advantages of stable baseline and good repeatability.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a method for simultaneously detecting CBD, CBN, THC and THCA by adopting a high performance liquid chromatography, which comprises the following steps of chromatographic separation and detection, wherein the chromatographic separation conditions comprise that: the mobile phase comprises acetonitrile, methanol and water; the volume ratio of the acetonitrile to the methanol to the water is 44:39: 17; the pH value of the mobile phase is 3.38-3.42;
the elution mode is isocratic elution; the column temperature is 30 ℃;
the conditions detected include: the detector is a multi-wavelength ultraviolet detector or a diode array detector; the detection wavelengths were 210nm and 220 nm.
Preferably, the chromatographic column for chromatographic separation is C18A column; the amount of sample was 10. mu.L.
Preferably, the column has a specification of 150mm x 4.6mm x 5 μm.
Preferably, the flow rate of the mobile phase is 1.0 mL/min.
Preferably, the pH adjuster of the mobile phase is phosphoric acid.
The invention also provides a preparation method of the computer sample, which comprises the following steps:
extracting a sample to be detected by using an extracting agent to obtain an extracting solution;
and filtering the extracting solution to obtain the upper computer sample.
Preferably, the extractant is a mixed solution of methanol and chloroform or methanol.
Preferably, the volume ratio of methanol to chloroform in the mixed solution of methanol and chloroform is 9: 1.
preferably, the extraction mode is oscillation extraction and/or ultrasonic extraction.
Preferably, the pore size of the filtration is 0.22 μm.
The invention provides a method for simultaneously detecting CBD, CBN, THC and THCA by adopting a high performance liquid chromatography, which comprises the steps of chromatographic separation and detection, and is characterized in that the chromatographic separation conditions comprise that: the mobile phase comprises acetonitrile, methanol and water; the volume ratio of the acetonitrile to the methanol to the water is 44:39: 17; the pH value of the mobile phase is 3.38-3.42; the elution mode is isocratic elution; the column temperature is 30 ℃; the conditions detected include: the detector is a multi-wavelength ultraviolet detector or a diode array detector; the detection wavelengths were 210nm and 220 nm. According to the method provided by the invention, a certain proportion of methanol is added into the mobile phase to ensure that the baseline is stable, so that the repeatability of the detection result is improved. On the other hand, the invention adopts a mode of isocratic elution of acetonitrile, methanol and water to improve the selectivity and the separation degree of the mobile phase and realize the separation of CBD, CBN and THC. Meanwhile, as THCA is an ionizable component, is greatly influenced by pH value, cannot detect THCA in a proper pH range or has long retention time, the pH value of the mobile phase is limited to be 3.38-3.42, so that THCA can be detected together with CBD, CBN and THC. In addition, the invention limits the detection wavelengths to 210nm and 220nm, and adopts a dual-signal detection mode to improve the sensitivity of the detection result. The results of the examples show that: the detection method can complete the simultaneous detection of CBD, CBN, THC and THCA within 20 min. In addition, the column temperature is adjusted, so that the peak output time is adjusted, and the detection efficiency is improved.
Further, the invention defines the chromatographic conditions as C18And the sample injection amount is 10 mu L, and the flow rate of the mobile phase is 1.0mL/min, so that the stability of a base line is further ensured, and the repeatability of a detection result is improved. The detection method is simple, the application range is wide, the accuracy is high, and the data of the embodiment shows that: the correlation coefficients of the standard curves are all 1, the RSD value (n is 3) is not more than 0.9%, and the recovery rate is 1.0-1.2.
In addition, the detection result of the embodiment shows that the detection method provided by the invention has very stable baseline and low requirement on the solvent of the sample to be detected, can well separate the complex components in the hemp extract, and is suitable for trace substance detection.
Drawings
FIG. 1 is a standard curve of CBD;
FIG. 2 is a standard graph of CBN;
FIG. 3 is a standard graph of THC;
FIG. 4 is a standard graph of THCA;
FIG. 5 is a real-time screenshot of the 3.3 sample recovery determination of example 3 during the test according to the test method of the present invention;
FIG. 6A is a chromatogram for detection of the labeled sample in comparative example 1;
FIG. 6B is a chromatogram of a mixed standard working solution having a concentration of 20. mu.g/mL, measured under the detection conditions in comparative example 1;
FIG. 7 is a chromatogram of the assay of 100. mu.g/mL standard according to the assay of the present invention;
FIG. 8 is a chromatogram of a labeled sample detected by the detection method of the present invention;
FIG. 9 is a chromatogram for detecting an unlabeled sample according to the detection method of the present invention.
Detailed Description
The invention provides a method for simultaneously detecting CBD, CBN, THC and THCA by adopting a high performance liquid chromatography, which comprises the steps of chromatographic separation and detection, and is characterized in that the chromatographic separation conditions comprise that: the mobile phase comprises acetonitrile, methanol and water; the volume ratio of the acetonitrile to the methanol to the water is 44:39: 17; the pH value of the mobile phase is 3.38-3.42; the elution mode is isocratic elution; the column temperature is 30 ℃; the conditions detected include: the detector is a multi-wavelength ultraviolet detector or a diode array detector; the detection wavelengths were 210nm and 220 nm.
In the present invention, unless otherwise specified, the reagents used are commercially available products well known to those skilled in the art.
In the present invention, the pH adjuster of the mobile phase is preferably phosphoric acid.
In the present invention, the column for chromatography is preferably C18The column, particularly preferred in the examples of the invention, is TC-C from Agilent18A column; the chromatographic columnThe specification of (2) is 150 mm. times.4.6 mm. times.5 μm.
In the invention, the sample amount in the chromatographic condition is 10 mu L; the flow rate of the mobile phase was 1.0 mL/min.
In the present invention, the method for preparing the computer sample preferably includes the following steps:
extracting a sample to be detected by using an extracting agent to obtain an extracting solution;
and filtering the extracting solution to obtain the upper computer sample.
The invention adopts an extracting agent to extract a sample to be detected to obtain an extracting solution.
In the present invention, the extractant is preferably a mixed solution of methanol and chloroform or methanol. In the present invention, the volume ratio of methanol to chloroform in the mixed solution of methanol and chloroform is preferably 9: 1. in the invention, when the extractant is a mixed solution of methanol and chloroform, the dosage ratio of the sample to be detected to the extractant is preferably 25-100 mg: 10 mL. In the present invention, when the extractant is methanol, the ratio of the sample to be tested to the extractant is preferably 100 mg: 10 mL.
In the present invention, the extraction is preferably performed by sequentially performing oscillation extraction and/or ultrasonic extraction. In the invention, the rotation speed of the oscillation extraction is preferably 200-300 rpm, and more preferably 280-300 rpm; the time for the oscillation extraction is preferably 10-15 min, and more preferably 10 min. In the invention, the ultrasonic extraction time is preferably 10-15 min, and more preferably 10 min.
After the extracting solution is obtained, the extracting solution is filtered to obtain the upper computer sample.
In the present invention, the pore size of the filtration membrane for filtration is preferably 0.22. mu.m.
And obtaining an upper computer sample, and directly processing the upper computer sample on a computer to perform high performance liquid chromatography detection.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the embodiment of the present invention, the specifications of the specific instrument, reagent and sample to be tested are as follows:
in the invention, the high performance liquid chromatograph specifically comprises: agilent 1260Infinity II System;
according to the invention, CBD, CBN, THC and THCA standard substances are purchased from Sigma company, and the concentration of the standard substances is 1mg/mL, wherein the solvent of the CBD, CBN and THC standard substances is methanol, and the solvent of the THCA standard substances is acetonitrile.
Example 1
1.1 preparation of Standard solution
By adopting a step-by-step gradient dilution method, CBD, CBN, THC and THCA standard substances with the concentration of 1mg/mL are respectively prepared into mixed standard solutions of CBD, CBN, THC and THCA with the concentrations of 50 mu g/mL, 25 mu g/mL, 5 mu g/mL, 1 mu g/mL, 0.5 mu g/mL, 0.2 mu g/mL and 0.02 mu g/mL by taking acetonitrile as a solvent.
1.2, linear regression equation
Testing the mixed standard working solution with the concentration of 50 mu g/mL, 25 mu g/mL, 5 mu g/mL, 1 mu g/mL, 0.5 mu g/mL, 0.2 mu g/mL, 0.02 mu g/mL according to the chromatographic condition and the detection condition in the high performance liquid chromatography to determine CBD, CBN, THC and THCA according to the retention time; and drawing a standard curve by taking the chromatographic peak areas of the obtained CBD, CBN, THC and THCA as vertical coordinates and the concentration of the mixed standard working solution as horizontal coordinates to obtain a standard curve regression equation, which is shown in Table 1 and figures 1-4.
TABLE 1 regression equation of standard curve
| Composition (I) | Linear Range ((μ g/mL) | Linear equation of equations | Correlation coefficient |
| CBD | 0.02～50 | y＝108.56x-5.7701 | 1.0000 |
| CBN | 0.02～50 | y＝92.703x-5.0026 | 1.0000 |
| THC | 0.02～50 | y＝120.63x-7.063 | 1.0000 |
| THCA | 0.02～50 | y＝40.06x-2.9371 | 1.0000 |
1.3 detection of detection Limit
Detecting with 0.02 μ g/mL standard solution according to the chromatographic condition and detection condition in the high performance liquid chromatography, and determining detection limit with the ratio of detected component signal (S) to baseline noise (N) not less than 3 as judgment standard. The results are shown in Table 2, and it is understood from Table 2 that the signal to noise ratio of the least sensitive THCA is 6.81087, which is greater than 3, and can be detected when the concentration of the standard substance is 0.02. mu.g/mL. Therefore, the detection limit of the detection method of the present invention was set to 0.02. mu.g/mL.
TABLE 2 detection results of detection limits of CBD, CBN, THC, THCA
Example 2
Sample extraction, example 2 included 2.1, 2.2 and 2.3.
2.1
Accurately weighing 25mg of sample powder into a centrifuge tube, adding 10mL of mixed solution of methanol and chloroform (the volume ratio of the methanol to the chloroform is 9: 1), oscillating and extracting for 10min under the condition that the rotation speed is 300rpm, then carrying out ultrasonic extraction for 10min, centrifuging for 10min by 1125 Xg to obtain supernatant and precipitate, extracting the obtained precipitate again, taking the supernatant, combining the supernatants obtained by two times of extraction, diluting the supernatant to 25mL by using the mixed solution of the methanol and the chloroform, drawing 2mL of diluted supernatant by using an injector, filtering the supernatant by using a 0.45 mu m filter membrane into a glass sample bottle, vacuumizing until the methanol and the chloroform are completely volatilized, adding 500 mu L of acetonitrile, fully dissolving, and filtering the solution by using a 0.22 mu m millipore filter into an automatic sample bottle to obtain the sample liquid to be detected.
2.2
Accurately weighing 100mg of sample powder in a centrifuge tube, adding 10mL of mixed solution of methanol and chloroform (the volume ratio of the methanol to the chloroform is 9: 1), oscillating and extracting for 10min under the condition of the rotation speed of 300rpm, then carrying out ultrasonic extraction for 10min, centrifuging for 10min at 1125 Xg to obtain supernatant and precipitate, extracting the obtained precipitate again, taking the supernatant, combining the supernatants obtained by two extractions, diluting the supernatant to 25mL by using the mixed solution of the methanol and the chloroform, diluting to a constant volume of 25mL, taking 500 mu L of extract, and filtering the extract by using a 0.22 mu m microporous filter to an automatic sample feeding bottle to obtain the sample liquid to be detected.
2.3
Accurately weighing 100mg of sample powder into a centrifuge tube, adding 10mL of methanol, performing oscillation extraction for 10min at the rotation speed of 300rpm, performing ultrasonic extraction for 10min, centrifuging for 10min to obtain supernatant and precipitate, performing re-extraction on the obtained precipitate, taking the supernatant, combining the supernatants obtained by two times of extraction, performing constant volume to 25mL of methanol, uniformly mixing, taking 500 mu L of extract, and filtering the 500 mu L of extract by using a 0.22 mu m microporous filter to an automatic sample feeding bottle to obtain the sample liquid to be detected.
Example 3
3.1 measurement of repetition Rate
Weighing 100mg of hemp mature flower and leaf sample, extracting and filtering according to '2.3 in example 2' to obtain a sample liquid to be detected, detecting the obtained sample liquid to be detected according to the chromatographic conditions and the detection conditions in the high performance liquid chromatography, repeating the detection for 3 times, and calculating the Relative Standard Deviation (RSD), wherein the results are shown in Table 3.
TABLE 3 results of measurement of repetition rate
(because the hemp mature mosaic sample contained no CBN, CBN data was obtained from spiked samples of this sample).
3.2 influence of Small-Range pH variation on Peak area
The invention also tests the influence of the pH value of the mobile phase on the detection result.
The test method comprises the following steps: taking a hemp mature flower and leaf sample 100mg, extracting according to '2.3 in example 2' to obtain a sample liquid to be detected, and detecting the obtained sample liquid to be detected according to chromatographic conditions and detection conditions in the high performance liquid chromatography, wherein the difference is as follows: the mobile phase had a pH of 3.38, 3.40, 3.42, respectively, and the Relative Standard Deviation (RSD) was calculated, and the results are shown in table 4.
TABLE 4 Effect of flow at different pH values on the peak area of CBD, CBN, THC and THCA
| Species of | pH＝3.38 | pH＝3.40 | pH＝3.42 | RSD(％) |
| CBD | 5210.602 | 5145.476 | 5146.44 | 0.72 |
| CBN | 3332.594 | 3315.349 | 3307.209 | 0.39 |
| THC | 4667.11 | 4637.944 | 4608.461 | 0.63 |
| THCA | 6831.32 | 6826.583 | 6805.593 | 0.20 |
As can be seen from Table 5, when the pH was varied within a range of 3.38 to 3.42, the influence on the detection results was small.
3.3 measurement of sample recovery
Weighing 100mg of hemp mature flower and leaf sample, extracting and filtering according to '2.3 in example 2' to obtain a sample solution to be detected; and adding standard substances of CBD, CBN, THC and THCA with the content shown in the table 4 into the obtained sample liquid to be detected, detecting according to the chromatographic conditions and the detection conditions in the high performance liquid chromatography, and calculating the recovery rate, wherein the test result of the recovery rate is shown in the table 5.
TABLE 5 recovery test results
| Cannabinoid class | Adding quantity of scalar | Recovery rate | Adding quantity of scalar | |
| CBD | ||||
| 30％ | 0.973991832 | 80％ | 1.082843196 | |
| CBN* | 0.5μg/mL | 1.071758196 | 2.5μg/ | 1.072817924 |
| THC | ||||
| 30％ | 1.026372655 | 60％ | 1.069117688 | |
| |
30％ | 1.123979031 | 90％ | 1.028947079 |
In the present invention, since the hemp mature mosaic sample does not contain CBN, the additive amount of CBN is a specific concentration value, and the additive amount percentage of CBD, THC and THCA is a percentage of the average value of 3 concentrations of each component in "determination of 3.1, repetition rate".
FIG. 5 is a real-time screenshot of "determination of sample recovery rate 3.3 in example 3" according to the detection method of the present invention, and it can be seen from FIG. 5 that: the base line is quite stable before the CBD peak of the first detection target sample appears, and the CBD, the CBN, the THC and the THCA are stable all the time in the detection process, thereby providing a foundation for improving the sensitivity and the repetition rate of the detection method.
Comparative example 1
Weighing 25mg of hemp mature flower and leaf sample, extracting according to '2.1 in example 2' to obtain an extracting solution, filtering the extracting solution through a 0.2-micron filter membrane to obtain a sample solution to be detected, adding 100 mu L of 20 mu g/mL mixed standard working solution into 500 mu L of the sample solution to be detected, and then carrying out high performance liquid chromatography detection and analysis on the obtained sample solution to be detected, wherein the chromatographic conditions are as follows: the mobile phase A is a mixed solution of water and 0.085 percent of phosphoric acid; the mobile phase B is a mixed solution of acetonitrile and 0.085 percent of phosphoric acid; the column temperature is 35 ℃, the sample injection is 5.0 mu L, the flow rate is 1.6mL/min, and the detection conditions are as follows: the detector is an ultraviolet detector, and the detection wavelength is 220 nm.
The elution is gradient elution; the elution procedure was:
0.0-3.0 min: the volume percentage content of the mobile phase B is 70 percent;
3.0-7.0 min: the volume percentage content of the mobile phase B is 85%;
7.0-8.0 min: the volume percentage content of the mobile phase B is 95 percent;
8.0-10.0 min: the volume percentage content of the mobile phase B is 70%.
A in FIG. 6 is the chromatogram for detecting the labeled sample in comparative example 1, and it can be seen from A in FIG. 6 that: the detection method in comparative example 1 was unstable in baseline at the time of detection.
In FIG. 6, B is a chromatogram of a mixed standard working solution having a concentration of 20. mu.g/mL, which was assayed under the assay conditions in comparative example 1, and which contained methanol in the solvent. As can be seen from B in fig. 6: baseline drift in comparative example 1 was severe when methanol was present in the solvent and the sample content was low.
The invention also detects the standard substance, and the detection method comprises the following steps: the mixed standard working solution with the concentration of 100 mug/mL is detected according to the chromatographic condition and the detection condition in the high performance liquid chromatography, and the detection result is shown in FIG. 7.
The invention also detects the sample containing the standard substance, and the detection method comprises the following steps:
taking 100mg of hemp mature flower and leaf sample, extracting and filtering according to '2.3 in example 2' to obtain a sample liquid to be detected, adding an equal amount of 1 mug/mL mixed standard working solution into the obtained sample liquid to be detected, and then detecting according to the chromatographic conditions and the detection conditions in the high performance liquid chromatography, wherein the detection result is shown in figure 8.
As can be seen from fig. 7 and 8: when the detected wavelengths are 210nm and 220nm, the peak areas of the detected components CBD, CBN, THC and THCA are obviously different, so that the sensitivity of the detection method is improved by adopting dual-wavelength detection.
FIG. 9 shows the detection result of the sample without the standard substance according to the detection method of the present invention, which comprises: taking 100mg of a hemp mature flower and leaf sample, extracting and filtering according to '2.3 in example 2' to obtain a sample liquid to be detected, and detecting the obtained sample liquid to be detected according to the chromatographic conditions and the detection conditions in the high performance liquid chromatography. From fig. 9 it can be derived that: the method has stable baseline and can realize the detection of trace components.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for simultaneously detecting CBD, CBN, THC and THCA by adopting high performance liquid chromatography comprises the steps of chromatographic separation and detection, and is characterized in that the conditions of the chromatographic separation comprise: the mobile phase comprises acetonitrile, methanol and water; the volume ratio of the acetonitrile to the methanol to the water is 44:39: 17; the pH value of the mobile phase is 3.38-3.42;
the elution mode is isocratic elution; the column temperature is 30 ℃;
the conditions detected include: the detector is a multi-wavelength ultraviolet detector or a diode array detector; the detection wavelengths were 210nm and 220 nm.
2. The method of claim 1, wherein the chromatographic column for chromatographic separation is C18A column; the amount of sample was 10. mu.L.
3. The method of claim 2, wherein the chromatography column has a specification of 150mm x 4.6mm x 5 μm.
4. The method of claim 1, wherein the flow rate of the mobile phase is 1.0 mL/min.
5. The method according to claim 1, wherein the pH adjusting agent of the mobile phase is phosphoric acid.
6. The method according to any one of claims 1 to 5, wherein the preparation method of the sample injection sample comprises the following steps:
extracting a sample to be detected by using an extracting agent to obtain an extracting solution;
and filtering the extracting solution to obtain the upper computer sample.
7. The method according to claim 6, wherein the extractant is a mixed solution of methanol and chloroform or methanol.
8. The method according to claim 7, wherein the volume ratio of methanol to chloroform in the mixed solution of methanol and chloroform is 9: 1.
9. the method according to claim 6, characterized in that the way of extraction is oscillatory extraction and/or ultrasonic extraction.
10. The method of claim 6, wherein the pore size of the filtration is 0.22 μm.
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