CN110133148A - A kind of hemp HPLC finger print measuring method - Google Patents
A kind of hemp HPLC finger print measuring method Download PDFInfo
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- CN110133148A CN110133148A CN201910463271.3A CN201910463271A CN110133148A CN 110133148 A CN110133148 A CN 110133148A CN 201910463271 A CN201910463271 A CN 201910463271A CN 110133148 A CN110133148 A CN 110133148A
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
This application provides a kind of hemp HPLC finger print measuring methods, it is characterized by comprising the following steps: the preparation of (1) test solution: taking hemp medicinal material, crush, sieving, baking, the hemp medicinal material precise powder after taking baking is weighed, and extractant is added, ultrasonic extraction, it puts to room temperature, crosses miillpore filter, take subsequent filtrate to obtain the final product；(2) preparation of reference substance solution: cannabidiolic acid, cannabidivarin, cannabigerol, cannabidiol, Δ 9- tetrahydro time cannabinol, cannabichromene, tetrahydrocannabinol, geranyl flavone A reference substance are taken respectively, methanol dissolution is added, reference substance solution is made；(3) measuring method: it is accurate respectively to draw reference substance solution and test solution injection high performance liquid chromatograph, measurement, and record chromatogram.
Description
Technical field
The invention belongs to drug detection and HPLC detection technique fields, and specifically, the present invention provides a kind of hemp HPLC
Finger print measuring method.
Background technique
Hemp Cannabis sativa L., also known as fiery fiber crops, Chinese fiber crops, hemp, flax, wild flax, are Moraceae annual herb
Plant.Contain cannabinol compounds and its derivative, flavones and its glycosides compound, alkaloid compound, perfume (or spice) in hemp
Beans chlorins compound, terpene and phytosterin compound, fatty acid compound, luxuriant and rich with fragrance class compound and other compounds.Cannabinol
Constituents are the main actives of hemp floral leaf, mainly include THC (tetrahydrocannabinol, tetrahydrocannabinol),
CBD (cannabidiol, cannabidiol), CBC (cannabichromene, cannabichromene), CBN (cannabinol, hemp
Phenol), CBG (cannabigerol, cannabigerol) and its propyl homologue THCV, CBDV, CBCV and CBGV etc..These cannabinols
The separating property of constituents and solid component, dissolubility, extraction conditions have difference, same sample under same sample processing mode
Also often there is difference in the content of each hemp phenols component, and difference is often more significant in different batches sample.
It is single to the quality evaluating method of hemp at present, hemp polyphenol in hemp is predominantly detected using HPLC-UV
Content.The measuring method of HPLC finger-print is widely used for the measurement of Chinese medicine, plant, but the original as described in upper section
Cause, HPLC finger-print always exists the problem that map degree of fitting is poor, measurement effect is unstable for hemp, therefore it is not enough to
System, the quality for completely evaluating hemp.It finds the method for being able to achieve universal, the stable extraction of each hemp phenols component and then establishes
Reliable hemp HPLC finger print measuring method, on the whole evaluates hemp quality, in agricultural product quality evaluation, food
There is demand in product drug inspection.
Summary of the invention
In order to overcome the deficiencies of the prior art, the present invention is intended to provide a kind of hemp HPLC finger print measuring method, energy
The quality for effectively reflecting hemp is conducive to carry out integral monitoring to hemp, avoids the unicity and one-sidedness of quality control.
This method is stable, reliable.
On the one hand, this application provides a kind of hemp HPLC finger print measuring methods, which is characterized in that including walking as follows
It is rapid:
(1) preparation of test solution: taking hemp medicinal material, crushes, and is sieved, baking, the hemp medicinal powder after taking baking
It is accurately weighed, extractant is added, ultrasonic extraction puts to room temperature, crosses miillpore filter, take subsequent filtrate to obtain the final product；
(2) preparation of reference substance solution: CBDA, CBDV, CBG, CBD, THCV, CBC, THC, geranyl flavone A are taken respectively
Reference substance is added methanol dissolution, reference substance solution is made；
(3) measuring method: it is accurate respectively to draw reference substance solution and test solution injection high performance liquid chromatograph, it surveys
It is fixed, and record chromatogram.
Further, hemp medicinal material described in step (1) is the mature industry hemp medicinal material picked the 7-9 month.
Further, hemp medicinal material described in step (1) is selected from: marihuana, cannabis, cannabis root, hemp stalk and big
One or more of numb seed is with the combination of arbitrary proportion；Preferably, the hemp medicinal material is cannabis and/or hemp
Leaf.
Further, hemp medicinal material described in step (1) baking temperature be 100-140 DEG C (such as 100,110,115,
120,125,130,140℃)；In one embodiment of the invention, the baking temperature is 120 DEG C.
Further, hemp medicinal material described in step (1) baking time be 60-150 minutes (such as 60,80,90,100,
120,150 minutes)；In one embodiment of the invention, the baking time is 90 minutes.
Further, extractant described in step (1) is organic solvent, wherein the organic solvent can be selected from: ethyl alcohol, first
One or more of alcohol, isopropanol, ethyl acetate and acetone are with the combination of arbitrary proportion.
In an embodiment of the invention, the organic solvent is ethyl alcohol.
Further, the concentration of extractant described in step (1) be 90-98% (v/v, such as 90%, 92%, 94%,
95%, 96%, 98%)；In one embodiment of the invention, the concentration of the extractant is 95%.
In one embodiment of the invention, the extractant is 95% ethyl alcohol.
Further, the dosage of extractant described in step (1) be 50-200ml/g hemp medicinal powder (such as 50,60,
70,80,100,150,200ml/g hemp medicinal powder)；In one embodiment of the invention, the dosage of the extractant is
70ml/g hemp medicinal powder.
Further, the power of ultrasonic extraction described in step (1) be 100-600W (e.g., 100,200,300,400,
500,600W), frequency is 10-50kHz (e.g., 10,20,30,40,50kHz)；In one embodiment of the invention, described super
The power that sound extracts is 400W, frequency 30kHz.
Further, the time of ultrasonic extraction described in step (1) be 5-60 minutes (such as 5,10,15,20,30,60 points
Clock)；In one embodiment of the invention, the time of the ultrasonic extraction is 10 minutes.
In one embodiment of the invention, the step (1) are as follows: hemp floral leaf is taken, is crushed, sieving, 120 DEG C of bakings 90
Minute, hemp floral leaf precise powder 95% ethyl alcohol of weighed addition after taking baking, the dosage of 95% ethyl alcohol is 70ml/g cannabis
Leaf powder ultrasonic extraction 10 minutes, is put to room temperature, crosses 0.22 μm of miillpore filter, take subsequent filtrate to obtain the final product.
Further, the high performance liquid chromatograph use mixed type silica matrix efficient liquid phase chromatographic stuffing, as C18,
C8, CN bonded silica gel filler.
Further, the measurement uses gradient elution program.
Further, the gradient elution program is as follows:
Elution time be 0-15 minutes when, the volumetric concentration of mobile phase: mobile phase A: 25-30% (such as 25%, 26%,
27%, 28%, 29%, 30%), Mobile phase B: surplus；
When elution time is 15-30 minutes, the volumetric concentration of mobile phase: mobile phase A: being 20- by 25-30% change of gradient
25% (such as 20%, 22%, 23%, 24%, 25%), Mobile phase B: surplus；
Elution time be 30-45 minutes when, the volumetric concentration of mobile phase: mobile phase A: 20-25% (such as 20%, 22%,
23%, 24%, 25%), Mobile phase B: surplus；
When elution time is 45-46 minutes, the volumetric concentration of mobile phase: mobile phase A: being 25- by 20-25% change of gradient
30%, Mobile phase B: surplus；
When elution time is 46-56 minutes, the volumetric concentration of mobile phase: mobile phase A: 25-30%, Mobile phase B: surplus.
In one embodiment of the invention, the gradient elution program is as follows:
When elution time is 0-15 minutes, the volumetric concentration of mobile phase: mobile phase A: 30%, Mobile phase B: surplus；
When elution time is 15-30 minutes, the volumetric concentration of mobile phase: mobile phase A: being 23% by 30% change of gradient,
Mobile phase B: surplus；
When elution time is 30-45 minutes, the volumetric concentration of mobile phase: mobile phase A: 23%, Mobile phase B: surplus；
When elution time is 45-46 minutes, the volumetric concentration of mobile phase: mobile phase A: being 30% by 23% change of gradient,
Mobile phase B: surplus；
When elution time is 46-56 minutes, the volumetric concentration of mobile phase: mobile phase A: 30%, Mobile phase B: surplus.
Further, the change of gradient of the mobile phase A concentration be linear gradient variation (mobile phase concentration variation with
Time is linear), exponential gradient variation (concentration variation and the time exponent function relation of mobile phase) or broken line change of gradient
(concentration of mobile phase changes and the time is at random relationship)；In one embodiment of the invention, the flowing phase concentration
Change of gradient be linear gradient variation.
Further, the mobile phase A be 0.01-0.5% (v/v, such as 0.01%, 0.05%, 0.1%, 0.15%,
0.2%, 0.5%) aqueous formic acid；In one embodiment of the invention, the mobile phase A is 0.1% aqueous formic acid.
Further, the Mobile phase B be 0.01-0.5% (v/v, such as 0.01%, 0.05%, 0.1%, 0.15%,
0.2%, 0.5%) formic acid acetonitrile solution；In one embodiment of the invention, the Mobile phase B is that 0.1% formic acid acetonitrile is molten
Liquid.
Further, the flow velocity of the high performance liquid chromatograph is 1.0-1.2ml/min (such as 1.0,1.1,1.2ml/
min)；In one embodiment of the invention, the flow velocity of the high performance liquid chromatograph is 1.0ml/min.
Further, the Detection wavelength of the high performance liquid chromatograph be 208-220nm (such as 208,210,215,
220nm)；In one embodiment of the invention, the Detection wavelength of the high performance liquid chromatograph is 210nm.
Further, the column temperature of the high performance liquid chromatograph is 25-40 DEG C (such as 25,28,30,32,35,40 DEG C)；?
In one embodiment of the present of invention, the column temperature of the high performance liquid chromatograph is 30 DEG C.
On the other hand, the present invention provides the above methods applies in hemp quality evaluation.
On the other hand, the application the present invention provides the above method in food, drug, port or judicial detection.
Hemp in the present invention can be the hemp from each subspecies in each place of production, include but is not limited to originate from India, the Central Asia,
Bhutan, Nepal, Sillim, the hemp of China, sativa and indica subspecies.
Various reagents in the present invention can according to need selection and meet detection, medical, medicinal standard domestic or import
Reagent.
Method of the invention can be used for agricultural product, the drug quality evaluation of hemp, be can also be used for after improving to containing
Food, drug, port or the judicial detection of hemp component (such as cannabinol compounds).
Compared with prior art, improvement of the invention is:
Fingerprint analysis method is analyzed on the whole for multicomponent feature in hemp medicinal material, convenient for big
The whole control of numb quality；
The detection that hemp finger-print is carried out using efficient liquid phase, is the supplement to the existing analysis method of hemp；
The sample processing conditions for devising suitable hemp, for more than this characteristic component of hemp and plant that extraction conditions are different
Article kind realizes reliable and blanket detection.
Compared with prior art, the invention has the following advantages that
The present invention uses HPLC method, shares 19, peak in the hemp finger-print of foundation, and calibrate 8 chromatographic peaks (such as
Shown in attached drawing 3), peak 7 is CBDV, and peak 9 is CBDA, and peak 10 is geranyl flavone A, and peak 11 is CBG, and peak 12 is CBD, and peak 13 is
THCV, peak 17 are THC, and peak 19 is CBC, can effectively characterize the quality of each ingredient in hemp medicinal material.
Finger-print focuses on each correlation for constituting fingerprint characteristic peak, focuses on whole facial feature, both avoided because
Measure individual chemical ingredient and caused by total quality one-sidedness, and a possibility that reduce for requisite quality and artificially handle.
The peak of hemp HPLC finger-print obtained by the method for the present invention is more, and peak shape is good, is easy to identify, and similitude is high, accurately and reliably.
This method has the advantages that be applicable in universal, precision, stability, reproducible and easy, quick, accurate, steady
It is fixed, reliable, it can be used for the control of hemp quality.
This method can quickly and accurately identify the type of hemp.
Detailed description of the invention
Fig. 1 show the HPLC spectrogram of the reference substance of the embodiment of the present invention 1.
Fig. 2 show the fingerprint image of more batches of hemp floral leaf test samples in the embodiment of the present invention 1 according to scheme (1) preparation
Spectrum.
Fig. 3 show the reference fingerprint of embodiment 1.
Fig. 4 show the HPLC spectrogram of test sample of the experiment (1) of embodiment 2 under the Detection wavelength of 210nm.
Fig. 5 show the HPLC spectrogram of test sample of the experiment (1) of embodiment 2 under the Detection wavelength of 220nm.
Fig. 6 show the HPLC spectrogram of test sample of the experiment (1) of embodiment 2 under the Detection wavelength of 230nm.
Fig. 7 show the HPLC spectrogram of test sample of the experiment (1) of embodiment 2 under the Detection wavelength of 254nm.
Fig. 8 show the HPLC spectrogram of test sample of the experiment (1) of embodiment 2 under the Detection wavelength of 280nm.
The experiment (2) that Fig. 9 show embodiment 2 uses 0.1% formic acid acetonitrile solution as the test sample of Mobile phase B
HPLC spectrogram.
The experiment (2) that Figure 10 show embodiment 2 uses 0.1% aqueous formic acid as the test sample of Mobile phase B
HPLC spectrogram.
Figure 11 show the HPLC spectrogram of test sample of the experiment (3) of embodiment 2 under the flow velocity of 0.8ml/min.
Figure 12 show the HPLC spectrogram of test sample of the experiment (3) of embodiment 2 under the flow velocity of 1.0ml/min.
Figure 13 show the HPLC spectrogram of test sample of the experiment (3) of embodiment 2 under the flow velocity of 1.2ml/min.
Figure 14 show the HPLC spectrogram of test sample of the experiment (4) of embodiment 2 under 25 DEG C of column temperature.
Figure 15 show the HPLC spectrogram of test sample of the experiment (4) of embodiment 2 under 30 DEG C of column temperature.
Figure 16 show the HPLC spectrogram of test sample of the experiment (4) of embodiment 2 under 35 DEG C of column temperature.
Figure 17 show the experiment (5) of embodiment 2 using mobile phase A: 35%, Mobile phase B: 65% mobile phase originates ratio
The HPLC spectrogram of the test sample of example.
Figure 18 show the experiment (5) of embodiment 2 using mobile phase A: 30%, Mobile phase B: 70% mobile phase originates ratio
The HPLC spectrogram of the test sample of example.
Figure 19 show the experiment (5) of embodiment 2 using mobile phase A: 25%, Mobile phase B: 75% mobile phase originates ratio
The HPLC spectrogram of the test sample of example.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical solution of the present invention is clearly and completely described, it is clear that institute
The embodiment of description is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, originally
Field those of ordinary skill every other embodiment obtained without creative efforts, belongs to the present invention
The range of protection.
Definition
Unless otherwise defined, all technical and scientific terms used herein have with the present invention relates to the technologies in field
The normally understood identical meaning of personnel, the following abbreviation such as occurred in the present invention and its corresponding substance are as follows:
CBDA cannabidiolic acid
CBDV cannabidivarin
CBD cannabidiol
CBG cannabigerol
CBC cannabichromene
THCV Δ 9- tetrahydro time cannabinol
THC tetrahydrocannabinol
Instrument and reagent
Hundred a ten thousandth balances (Sartorius MSA6.6S-0CE-DM)；
Shimadzu LC-16 high performance liquid chromatograph；
4.6 × 25mm of Welch XB-C18,5 μm of chromatographic columns；
CBDA reference substance, CBDV reference substance, CBG reference substance, CBD reference substance, THCV reference substance, CBC reference substance, THC pairs
According to product, geranyl flavone A, methanol, formic acid, acetonitrile, ethyl alcohol, HPLC grades, it is purchased from U.S. Sigma Aldrich；
Similarity evaluation uses the Chinese Pharmacopoeia committee " chromatographic fingerprints of Chinese materia medica similarity evaluation software " (2012
Version) it carries out.
1 test sample treatment conditions of embodiment are selected
Scheme (1)
The preparation of test solution: taking hemp floral leaf appropriate, crushes, and crosses No. 2 sieves, and 120 DEG C are toasted 90 minutes, after taking baking
Hemp floral leaf powder 0.5g, it is accurately weighed, be placed in conical flask, be added 95% ethyl alcohol 35ml, ultrasonic extraction (power: 400W,
Frequency: 30kHz) 10 minutes, it puts to room temperature, crosses 0.22 μm of miillpore filter, take subsequent filtrate to obtain the final product.
Scheme (2)
The preparation of test solution: taking hemp floral leaf appropriate, crushes, and crosses No. 2 sieves, and 120 DEG C are toasted 90 minutes, after taking baking
Hemp floral leaf powder 0.5g, it is accurately weighed, be placed in conical flask, be added 95% ethyl alcohol 50ml, ultrasonic extraction (power: 400W,
Frequency: 30kHz) 20 minutes, it puts to room temperature, crosses 0.22 μm of miillpore filter, take subsequent filtrate to obtain the final product.
Scheme (3)
The preparation of test solution: taking hemp floral leaf appropriate, crushes, and crosses No. 2 sieves, and 120 DEG C are toasted 120 minutes, takes baking
Hemp floral leaf powder 0.5g afterwards, it is accurately weighed, it is placed in conical flask, is added 95% ethyl alcohol 35ml, ultrasonic extraction (power:
400W, frequency: 30kHz) 30 minutes, it puts to room temperature, crosses 0.22 μm of miillpore filter, take subsequent filtrate to obtain the final product.
The preparation of reference substance solution: CBDA, CBDV, CBG, CBD, THCV, CBC, THC, the control of geranyl flavone A are taken respectively
Appropriate product are added methanol and reference substance solution are made；The concentration of each reference substance be respectively 10 μ g/ml of CBDA, 10 CBDV μ g/ml,
10 μ g/ml of CBG, 150 CBD μ g/ml, 10 THCV μ g/ml, 10 CBC μ g/ml, 10 THC μ g/ml, 10 μ of geranyl flavone A
g/ml。
Measuring method: it is accurate respectively to draw reference substance solution and each 10 μ l injection high performance liquid chromatograph of test solution,
Through high effective liquid chromatography for measuring, and record chromatogram.
High performance liquid chromatography: use octadecyl silane for filler chromatographic column；Mobile phase A is 0.1% formic acid
Aqueous solution, Mobile phase B are 0.1% formic acid acetonitrile solution；Gradient elution, flow velocity 1.0ml/min, Detection wavelength 210nm, column temperature
30℃。
Gradient elution program is as follows:
When elution time is 0-15 minutes, the volumetric concentration of mobile phase: mobile phase A: 30%, Mobile phase B: 70%；
When elution time is 15-30 minutes, the volumetric concentration of mobile phase: mobile phase A: being 23% by 30% change of gradient,
Mobile phase B: being 77% by 70% change of gradient；
When elution time is 30-45 minutes, the volumetric concentration of mobile phase: mobile phase A: 23%, Mobile phase B: 77%；
When elution time is 45-46 minutes, the volumetric concentration of mobile phase: mobile phase A: being 30% by 23% change of gradient,
Mobile phase B: being 70% by 77% change of gradient；
When elution time is 46-56 minutes, the volumetric concentration of mobile phase: mobile phase A: 30%, Mobile phase B: 70%.
The spectrogram of each reference substance as shown in Figure 1, multiple batches of (S1-S11 batches) test sample according to scheme (1) preparation HPLC
Finger-print is as shown in Figure 2.Using chromatographic fingerprints of Chinese materia medica similarity evaluation software to the multiple batches of hemp HPLC fingerprint of gained
Map is analyzed, and has been carried out Supplements and Auto-matching to each finger-print chromatographic peak using median method, has been calculated similar
Reference fingerprint is spent and generates, as shown in Figure 3.Wherein for the fingerprint similarity of 11 batches of test samples 0.9 or more, this is right
It is standard finger-print according to finger-print.Chromatographic peak 19 is shared in finger-print, and wherein 8 chromatographies are selected according to reference substance
Peak: peak 7 is CBDV, and peak 9 is CBDA, and peak 10 is geranyl flavone A, and peak 11 is CBG, and peak 12 is CBD, and peak 13 is THCV, peak 17
For THC, peak 19 is CBC, for characterizing the quality of each ingredient in hemp floral leaf.
The test sample for preparing 10 batches according to scheme (1)-(3) respectively, is measured, analyzes and similarity-rough set, knot
Fruit finds that baking degree, solvent usage and ultrasound condition influence measurement result very big in test sample processing, especially for
The peak CBG, THCV and CBC, in 10 batch samples of scheme (2) and (3), even whether there is or not great Cheng all occur for the size at these three peaks
Degree ground difference, causes the similarity difference of each sample map very big, therefore, compared with scheme (2) and (3), scheme (1) is more suitable for
For hemp HPLC finger print measuring method.The test sample processing mode of scheme (1) (will crush, the hemp floral leaf of sieving exists
120 DEG C are toasted 90 minutes, and 95% ethyl alcohol 35ml, ultrasonic extraction 10 minutes is added in hemp floral leaf powder 0.5g after baking) it can be with
Guarantee that each batch has suitable similarity (0.9 or more), is the suitable condition of hemp HPLC determining fingerprint pattern.
2 high-efficient liquid phase chromatogram condition of embodiment is selected
(1) Detection wavelength is investigated
Test solution is prepared according to the method for 1 scheme of embodiment (1).
The measuring method of reference implementation example 1 and the high-efficient liquid phase chromatogram condition in addition to Detection wavelength, respectively 210,220,
230, it 254, is measured under the Detection wavelength of 280nm, records chromatogram, successively as shown in Figure 4-8.
By Fig. 4-8 it is found that under the Detection wavelength of 210 and 220nm, 8 chromatographic peaks demarcated in embodiment 1 are (shown in Fig. 4
Peak 7,9,10,11,12,13,17,19) it is clear and legible, under other Detection wavelengths, certain wave crests be difficult to distinguish even much at
Divide non-appearance.And relative to 220nm, under the Detection wavelength of 210nm, peak shape more preferably, therefore selectes 210nm as suitable inspection
Survey wavelength.
(2) mobile phase is investigated
Test solution is prepared according to the method for 1 scheme of embodiment (1).
The measuring method of reference implementation example 1 and the high-efficient liquid phase chromatogram condition in addition to Mobile phase B, are respectively adopted 0.1% first
Sour acetonitrile solution, 0.1% aqueous formic acid are measured as Mobile phase B, record chromatogram, respectively as shown in figs. 9-10.
By Fig. 9-10 it is found that using 0.1% aqueous formic acid as Mobile phase B when, spectrogram medium wave peak is not easy to distinguish, and supply
The non-appearance of many ingredients in test product, is not used to the HPLC determining fingerprint pattern of hemp.Therefore, it is molten to select 0.1% formic acid acetonitrile
Liquid is as Mobile phase B.
(3) flow velocity is investigated
Test solution is prepared according to the method for 1 scheme of embodiment (1).
The measuring method of reference implementation example 1 and the high-efficient liquid phase chromatogram condition in addition to flow velocity, be respectively adopted 0.8,1.0,
The flow velocity of 1.2ml/min, is measured, and records chromatogram, respectively as figs 11-13.
By Figure 11-13 it is found that under the flow velocity of 1.0ml/min, the wave crest of each ingredient is clear and legible in test sample,
Under the flow velocity of 0.8ml/min, when detecting between the interior peak CBC (60min) (peak 19 shown in Fig. 4) non-appearance, in the stream of 1.2ml/min
Under speed, 8 equal appearances of chromatographic peak of calibration, but not as good as clear under the flow velocity of 1.0ml/min.Therefore, 1.0ml/min conduct is selected
Suitable flow velocity.
(4) column temperature is investigated
Test solution is prepared according to the method for 1 scheme of embodiment (1).
The measuring method of reference implementation example 1 and the high-efficient liquid phase chromatogram condition in addition to column temperature are respectively adopted 25,30,35 DEG C
Column temperature, be measured, record chromatogram, respectively as illustrated in figures 14-16.
By Figure 14-16 it is found that under 25,30,35 DEG C of column temperature, 8 equal appearances of chromatographic peak of calibration, in 30 DEG C of column temperature
Lower appearance is relatively apparent, and retention time is more appropriate, therefore, selectes 30 DEG C and is used as suitable column temperature.
(5) mobile phase original ratio is investigated
Test solution is prepared according to the method for 1 scheme of embodiment (1).
The measuring method of reference implementation example 1 and the high-efficient liquid phase chromatogram condition in addition to mobile phase original ratio, are respectively adopted
Following mobile phase original ratio: (a) mobile phase A: 35%, Mobile phase B: 65%；(b) mobile phase A: 30%, Mobile phase B: 70%；
(c) mobile phase A: 25%, Mobile phase B: 75%, it is measured, records chromatogram, respectively as in figs. 17-19.
By Figure 17-19 it is found that in (a) mobile phase A: 35%, Mobile phase B: interior when detecting under 65% original ratio
The peak CBC (peak 19 shown in Fig. 4) non-appearance, in (b) mobile phase A: 30%, Mobile phase B: 70% and (c) mobile phase A: 25%, flowing
Under the original ratio of phase B:75%, the wave crest of each ingredient is clear and legible in test sample.Therefore, mobile phase A can be used: 25-
30%, Mobile phase B: surplus is as suitable mobile phase original ratio.
3 Precision Experiment of embodiment
Test solution is prepared according to the method for 1 scheme of embodiment (1).
It takes with a test solution, continuously repeats 6 needle of sample introduction, the chromatographic condition selected using embodiment 2 is investigated accurate
Degree.The results are shown in Table 1 and 2, shared peak retention time RSD is respectively less than 0.18%, peak area RSD no more than 0.95%, repeat into
Similarity between 6 needle of sample is 1.000, and precision is good.
1 precision experiment result of table (retention time)
2 precision experiment result of table (peak area)
4 repeated experiment of embodiment
Test solution is prepared according to the method for 1 scheme of embodiment (1).
It weighs with a collection of 6 parts of test sample, according to sample solution preparation method, operation repetitive is selected using embodiment 2
Chromatographic condition investigates repeatability.It the results are shown in Table 3 and 4, shared peak retention time RSD is respectively less than 0.36%, and peak area RSD is not
Similarity greater than 1.86%, 6 part of sample room is 0.999, and repeatability is good.
3 repeated experiment result (retention time) of table
4 repeated experiment result (peak area) of table
5 stability experiment of embodiment
Test solution is prepared according to the method for 1 scheme of embodiment (1).
Same test solution is taken, respectively at 0h, 2h, 4h, 6h, 8h, 16h, liquid chromatograph is injected for 24 hours, using implementation
The selected chromatographic condition measurement of example 2.It the results are shown in Table 5 and 6, shared peak retention time RSD is respectively less than 0.82%, and peak area RSD is equal
Less than 2.0%, similarity is 0.999, and test solution is stablized interior for 24 hours.
5 stability experiment result (retention time) of table
6 stability experiment result (peak area) of table
Embodiment 3-5's the experimental results showed that method of the invention in addition to have between each batch good similitude it
Outside, repeatability and precision show well, and sample is also able to maintain testing result within a certain period of time and stablizes, and are suitable for practical inspection
It surveys and uses.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, made any modification, equivalent replacement etc. be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of hemp HPLC finger print measuring method, which comprises the steps of:
(1) preparation of test solution: taking hemp medicinal material, crushes, and is sieved, baking, the hemp medicinal material precise powder after taking baking
It is weighed, extractant is added, ultrasonic extraction puts to room temperature, crosses miillpore filter, take subsequent filtrate to obtain the final product；
(2) preparation of reference substance solution: cannabidiolic acid, cannabidivarin, cannabigerol, cannabidiol, Δ 9- tetra- are taken respectively
Time cannabinol, cannabichromene, tetrahydrocannabinol, geranyl flavone A reference substance are hydrogenated, methanol dissolution is added, reference substance is made
Solution；
(3) measuring method: accurate absorption reference substance solution and test solution injection high performance liquid chromatograph respectively, measurement, and
Record chromatogram.
2. according to the method described in claim 1, wherein hemp medicinal material described in step (1) is selected from: marihuana, cannabis, big
One or more of snippings, hemp stalk and cannabis seeds are with the combination of arbitrary proportion, preferably cannabis and/or big
Sesame slices；
Extractant described in step (1) is organic solvent, wherein the organic solvent is selected from: ethyl alcohol, methanol, isopropanol, acetic acid
One or more of ethyl ester and acetone are with the combination of arbitrary proportion, preferably ethyl alcohol.
3. according to the method described in claim 1, wherein hemp medicinal material described in step (1) baking temperature be 100-140 DEG C,
Baking time is 60-150 minutes；
The concentration of the extractant is 90-98%；
The dosage of the extractant is 50-200ml/g hemp medicinal powder；
The power of the ultrasonic extraction is 100-600W, frequency 10-50kHz；
The time of the ultrasonic extraction is 5-60 minutes.
4. being toasted according to the method described in claim 1, wherein the temperature of the baking of hemp medicinal material described in step (1) is 120 DEG C
Time is 90 minutes；The extractant is 95% ethyl alcohol；The dosage of the extractant is 70ml/g hemp medicinal powder；It is described super
The power that sound extracts is 400W, and frequency 30kHz, the time of the ultrasonic extraction is 10 minutes.
5. method according to claim 1-4, wherein the high performance liquid chromatograph uses mixed type silica gel base
Matter efficient liquid phase chromatographic stuffing, preferably C18, C8 or CN bonded silica gel filler；
The measurement uses gradient elution program, and the gradient elution program is as follows:
When elution time is 0-15 minutes, the volumetric concentration of mobile phase: mobile phase A: 25-30%, Mobile phase B: surplus；
When elution time is 15-30 minutes, the volumetric concentration of mobile phase: mobile phase A: being 20- by 25-30% change of gradient
25%, Mobile phase B: surplus；
When elution time is 30-45 minutes, the volumetric concentration of mobile phase: mobile phase A: 20-25%, Mobile phase B: surplus；
When elution time is 45-46 minutes, the volumetric concentration of mobile phase: mobile phase A: being 25- by 20-25% change of gradient
30%, Mobile phase B: surplus；
When elution time is 46-56 minutes, the volumetric concentration of mobile phase: mobile phase A: 25-30%, Mobile phase B: surplus；
Wherein, the mobile phase A is 0.01-0.5% aqueous formic acid, and the Mobile phase B is 0.01-0.5% formic acid second
Nitrile solution.
6. according to the method described in claim 5, wherein the gradient elution program is as follows:
When elution time is 0-15 minutes, the volumetric concentration of mobile phase: mobile phase A: 30%, Mobile phase B: 70%；
When elution time is 15-30 minutes, the volumetric concentration of mobile phase: mobile phase A: being 23% by 30% change of gradient, flowing
Phase B: surplus；
When elution time is 30-45 minutes, the volumetric concentration of mobile phase: mobile phase A: 23%, Mobile phase B: 77%；
When elution time is 45-46 minutes, the volumetric concentration of mobile phase: mobile phase A: being 30% by 23% change of gradient, flowing
Phase B: surplus；
When elution time is 46-56 minutes, the volumetric concentration of mobile phase: mobile phase A: 30%, Mobile phase B: 70%.
7. according to the method described in claim 5, it is characterized in that, the mobile phase A is 0.1% aqueous formic acid；And/or
The Mobile phase B is 0.1% formic acid acetonitrile solution；
The change of gradient of the mobile phase A concentration is linear gradient variation, exponential gradient variation or broken line change of gradient.
8. according to right want 5 described in method, wherein the flow velocity of the high performance liquid chromatograph be 1.0-1.2ml/min, preferably
For 1.0ml/min；
The Detection wavelength of the high performance liquid chromatograph is 208-220nm, preferably 210nm；
The column temperature of the high performance liquid chromatograph is 25-40 DEG C, preferably 30 DEG C.
9. method according to claim 1-8 is applied in hemp quality evaluation.
10. application of the method according to claim 1-8 in food, drug, port or judicial detection.
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| CN110632224A (en) * | 2019-09-16 | 2019-12-31 | 福建省中科生物股份有限公司 | Establishment method of hemp plant fingerprint spectrum |
| CN111592448A (en) * | 2020-04-20 | 2020-08-28 | 周宇平 | Process for separating and purifying hypocannabidiol from industrial hemp |
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| CN112730697A (en) * | 2020-09-15 | 2021-04-30 | 中国标准化研究院 | Method for simultaneously detecting 5 cannabinol compounds by using HPLC-MS/MS |
| CN113390993A (en) * | 2021-06-15 | 2021-09-14 | 中国标准化研究院 | A method for simultaneously detecting various cannabinoids in a sample containing folium Cannabis extract by HPLC |
| CN114062526A (en) * | 2020-08-05 | 2022-02-18 | 汉义生物科技（北京）有限公司 | Detection method for quantifying cannabinoids based on relative correction factor method |
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| CN111592448A (en) * | 2020-04-20 | 2020-08-28 | 周宇平 | Process for separating and purifying hypocannabidiol from industrial hemp |
| CN114062526A (en) * | 2020-08-05 | 2022-02-18 | 汉义生物科技（北京）有限公司 | Detection method for quantifying cannabinoids based on relative correction factor method |
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| CN112034059A (en) * | 2020-08-21 | 2020-12-04 | 滇麻生物科技(曲靖)有限公司 | Method for detecting cannabinoids in industrial hemp flowers and leaves and extract thereof by high performance liquid chromatography |
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| CN112730696A (en) * | 2020-09-15 | 2021-04-30 | 中国标准化研究院 | Method for detecting 5 cannabinol compounds in cannabis sativa oil by using HPLC (high performance liquid chromatography) method |
| CN112710768A (en) * | 2020-12-10 | 2021-04-27 | 安徽瑞达健康产业有限公司 | Method for measuring content of dehydroxycannabidiol (DH-CBD) |
| CN113390993A (en) * | 2021-06-15 | 2021-09-14 | 中国标准化研究院 | A method for simultaneously detecting various cannabinoids in a sample containing folium Cannabis extract by HPLC |
| CN114814009A (en) * | 2022-04-07 | 2022-07-29 | 国家烟草质量监督检验中心 | Analysis method simultaneously suitable for phenolic compounds in novel tobacco products and metabolites of phenolic compounds in urine |
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