CN112730679A - Method for determining CBD, THC and CBN content in hemp based on GC-MS technology-isotope internal standard method - Google Patents

Method for determining CBD, THC and CBN content in hemp based on GC-MS technology-isotope internal standard method Download PDF

Info

Publication number
CN112730679A
CN112730679A CN202011549569.5A CN202011549569A CN112730679A CN 112730679 A CN112730679 A CN 112730679A CN 202011549569 A CN202011549569 A CN 202011549569A CN 112730679 A CN112730679 A CN 112730679A
Authority
CN
China
Prior art keywords
thc
cbd
cbn
internal standard
hemp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202011549569.5A
Other languages
Chinese (zh)
Inventor
李智宁
魏悦
李飞飞
李晓
范毅
马艳妮
张丽先
宁二娟
李自红
宋梦娇
张桃桃
王学方
刘冠华
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Henan Napu Biotechnology Co ltd
Original Assignee
Henan Cogo Testing International Co ltd
Henan Academy of Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Henan Cogo Testing International Co ltd, Henan Academy of Sciences filed Critical Henan Cogo Testing International Co ltd
Priority to CN202011549569.5A priority Critical patent/CN112730679A/en
Publication of CN112730679A publication Critical patent/CN112730679A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • 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
    • 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
    • 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
    • G01N30/14—Preparation by elimination of some components
    • 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
    • G01N30/72—Mass spectrometers
    • 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
    • G01N30/8624—Detection of slopes or peaks; baseline correction
    • G01N30/8631—Peaks
    • G01N30/8634—Peak quality criteria
    • 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
    • G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • 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
    • G01N2030/042—Standards
    • G01N2030/045—Standards internal
    • 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
    • G01N30/14—Preparation by elimination of some components
    • G01N2030/146—Preparation by elimination of some components using membranes
    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Engineering & Computer Science (AREA)
  • Quality & Reliability (AREA)
  • Library & Information Science (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

The invention relates to a method for simultaneously determining the content of three effective components, namely CBD, THC and CBN in hemp based on GC/MS technology-isotope internal standard method, and discussing the chemical phenotype; extracting a target component in a sample by using methanol, and preparing a sample solution after ultrasonic extraction and centrifugation; a qualitative and quantitative analysis method for determining the contents of three components including Tetrahydrocannabinol (THC), Cannabinol (CBN) and Cannabidiol (CBD) in hemp is established by selecting reasonable chromatographic and mass spectrum conditions and applying an isotope internal standard method by using a gas chromatography-mass spectrometer (GC/MS). The results show that: the method is obtained through some methodology researches, and has the advantages of good specificity, high accuracy and high sensitivity. The linear relation in the selected concentration range is good, the linear range is wide, the stability is good, and the precision is high.

Description

Method for determining CBD, THC and CBN content in hemp based on GC-MS technology-isotope internal standard method
Technical Field
The invention belongs to the technical field of determination of CBD, THC and CBN content in China hemp, and particularly relates to a method for determining CBD, THC and CBN content in China hemp based on GC-MS technology-isotope internal standard method.
Background
Hemp, i.e., industrial hemp (Cannabis sativa L), is an annual herb plant of the genus Cannabis of the family Cannabaceae, also known as Cannabis sativa, Coxseed, and the like, and refers to non-narcotic types of hemp having a Tetrahydrocannabinol (THC) content of less than 0.3% in the plant. China has a history of planting and using hemp raw materials for thousands of years, and the application of the hemp raw materials relates to a plurality of fields of medicines, foods, health care products, cosmetics and the like. Modern pharmacological studies show that: the hemp has biological activities of analgesia, intraocular pressure reduction, tumor resistance, vomiting resistance, thrombus resistance, bacteria resistance, inflammation resistance and the like. The chemical components of hemp are extremely complex, and the active ingredients can be mainly divided into two main classes of Cannabinoids and non-Cannabinoids, wherein Cannabinoids (Cannabinoids) are a class of secondary metabolites which are specific to cannabis plants and contain alkyl and monoterpene group molecular structures. At present, more than 100 cannabinoids are isolated from dry hemp material and fresh hemp leaves, mainly including THC, Cannabidiol (CBD), Cannabinol (CBN), Cannabichromene (CBC), Cannabigerol (CBG) and propyl homologues thereof, among which the highest content is THC, CBD and CBN. Meanwhile, hemp also contains non-cannabinoid compounds such as flavone and its glycosides, alkaloids, coumarins, polyphenols, polysaccharides, sterols, fatty acids, phenanthrene compounds, etc.
At present, the conventional methods for detecting cannabinoids mainly comprise TLC (thin layer chromatography), GC (gas chromatography) and HPLC (high performance liquid chromatography), and some detection methods, particularly newly-emerging GC-MS and LC-MS methods are mainly applied to biological products such as blood, urine, tissues and the like of cannabis eaters and test animals. The thin-layer chromatography has complicated procedures, long analysis time and easy occurrence of false positive results; the sensitivity of GC and HPLC methods is low, the content of cannabinoids in different parts of the hemp plant is possibly low, and the requirement on the sensitivity is high; the GC-MS technology is a currently recognized forensic scientific analysis method at home and abroad, and a commonly used EI source has stronger selectivity, specificity, accuracy and higher sensitivity, and can effectively perform qualitative and quantitative detection on various components of hemp plants and hemp drugs. The hemp related line marking detection method (GAT 1008.3-2013 common drug inspection method for public safety line marking, the third part of hemp) adopts GC-FID technology, uses two quantitative methods of external standard method and internal standard method (selecting tribenzylamine as internal standard) to determine THC content, and simultaneously adopts GC-MS technology to qualitatively screen and confirm three components of THC, CBD and CBN; however, the method still has the defects of low detection limit, low sensitivity, poor specificity and the like.
Based on this, the application provides an isotope internal standard (THC-D) based on GC/MS technology3) The method for simultaneously, qualitatively and quantitatively detecting the contents of the three components including THC, CBD and CBN by an internal standard method is carried out, the deuterated isotope of the substance to be detected has extremely consistent molecular properties with the molecular properties of the substance to be detected, and has completely the same encounter with the substance to be detected in the pretreatment process, the chromatographic separation process and the ionization process, and the isotope can be separated by mass spectrometry, so that the interference of matrix effect can be greatly eliminated, errors generated in the analysis method process can be more effectively corrected, and the quantitative determination is more accurate and reliable.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for determining the content of CBD, THC and CBN in hemp based on a GC-MS technology-isotope internal standard method.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for measuring the content of CBD, THC and CBN in hemp based on GC-MS technology-isotope internal standard method comprises the following steps:
1) preparation of a series of mixed standard working solutions containing an internal standard:
accurately weighing CBD, CBN, THC and internal standard THC-D respectively3Preparing a reference substance, namely preparing a CBD, CBN and THC series mixed standard working solution containing an internal standard by using methanol as a solvent;
the concentrations of CBD, CBN and THC in each mixed standard working solution are the same, and the internal standard THC-D3The concentration of (A) is 2.0 mu g/ml; the concentrations of CBD in the series of mixed standard working solutions are 32.0, 16.0, 8.0, 4.0, 0.8, 0.4 and 0.2 mu g/ml respectively;
2) preparation of a test solution:
precisely weighing 20mg of hemp sample powder, accurately adding 20mL of methanol, and simultaneously adding 1.0mg/mL of THC-D3Measuring 40 μ L of internal standard solution, weighing and recording as m; standing at room temperature for 20-40min, ultrasonic treating for 5-20min, cooling to room temperature, weighing again, and adding methanol to balance weightCentrifuging, and filtering with organic filter membrane to obtain test solution;
3) GCMS measurement:
performing GCMS determination on the series of mixed standard working solutions containing the internal standards prepared in the step 1), measuring and recording CBD, THC, CBN and the internal standard THC-D3The chromatographic peak area of (A) is respectively CBD, THC, CBN and internal standard THC-D3The ratio of peak areas of (A) to (B) is ordinate, and CBD, THC, CBN and internal standard THC-D are taken as3The concentration ratio of the CBD to the THC to the CBN is an abscissa, standard curves of the CBD, the THC and the CBN are respectively drawn, and corresponding linear equations are obtained through calculation; performing GCMS measurement on the sample solution obtained in the step 2) and recording CBD, THC, CBN and internal standard THC-D3Substituting the chromatographic peak areas into corresponding linear equations to calculate the content of CBD, THC and CBN in the test sample.
Specifically, the GC conditions of the gas chromatograph are as follows: a chromatographic column: rtx-5MS capillary column, injection port temperature: 280 ℃; the carrier gas is He gas; the flow rate of the carrier gas is 1.0-1.5 ml/min. And (3) sample introduction mode: constant linear velocity shunting sample introduction; linear velocity: 38.7 cm/sec; the split ratio is as follows: 5:1 or 2: 1; sample introduction amount: 1 mul; temperature programming: maintaining at 200 deg.C for 2min, heating to 240 deg.C at 10 deg.C/min, maintaining for 18min, heating to 280 deg.C at 20 deg.C/min, and maintaining for 2 min.
Specifically, the mass spectrum MS conditions are as follows: an ion source: EI, electron energy 70eV, detector voltage: 0.93-1.20kv, ion source temperature 240 ℃; the interface temperature was 250 ℃ and the solvent delay was 9.0 min. Scanning mode: scan mode and Sim mode; detector voltage: 0.93 kV; resolution ratio: scan range: 40-600 amu; in Sim mode, the mass spectrometer selected ion monitoring parameters as follows:
Figure BDA0002857324840000031
the research aims to establish a GC-MS universal quantitative analysis method for simultaneously determining three main active ingredients, namely THC, CBD and CBN, in hemp plants through systematic, efficient and rigorous steps, and isotope internal standard (THC-D) is adopted3) The method can be used for quantification to obtain more accurate, reliable, sensitive, efficient and popularAnd a quantitative detection method of the same. Meanwhile, the research utilizes the method to accurately measure hemp samples of different producing areas respectively, and preliminarily discusses the content characteristics and the chemical phenotypes of three cannabinoids, namely THC, CBD and CBN. The establishment and determination results of the research method provide data reference for later-stage system deep research on the content and distribution characteristics of more cannabinoids, so that the method can be more widely applied; also lays an experimental foundation for reasonably utilizing the plant resources of the species and developing diversified products with high added values, and is important for guiding the production and control work of industrial hemp. Compared with the prior art, the invention has the following beneficial effects:
the invention establishes a method for simultaneously determining the contents of three effective components THC, CBD and CBN in hemp based on GC/MS technology-isotope internal standard method, and discusses the content characteristics and chemical phenotypes of hemp in different production areas. The method comprises the steps of firstly extracting target components in a sample by using methanol, preparing a test solution after standing extraction, ultrasonic extraction and centrifugal purification, and then utilizing a gas chromatography-mass spectrometer (GC/MS) to select reasonable chromatographic and mass spectrum conditions and establish qualitative and quantitative analysis for determining the content of three components including Tetrahydrocannabinol (THC), Cannabinol (CBN) and Cannabidiol (CBD) in the hemp by utilizing an isotope internal standard method. The results show that: the method has the advantages of good specificity, high accuracy and high sensitivity through some methodology investigation; the linear relation in the selected concentration range is good, the linear range is wide, the stability is good, and the precision is high. The content of the THC, the CBD and the CBN in the hemp can be simultaneously detected by the determination method, the operation is simple and easy, and the result is reliable. Through analyzing and discussing the content distribution characteristics and chemical phenotypes of the THC, CBD and CBN in different producing areas, the content characteristics are obviously different, and 18 batches of samples are preliminarily judged to be fiber type hemp.
Drawings
FIG. 1 shows CBD (Peak 1) and THC-D in Scan mode3Total ion flow graph (TIC graph, in the figure, a: mixed standard; b: sample) of THC (peak 2, 3) and CBN (peak 4);
FIG. 2 shows the mode of SimCBD (Peak 1), THC-D3(Peak 2), THC (Peak 3) and CBN (Peak 4) (MIC plot, in which a: superscript; b: sample);
FIG. 3 is a standard graph of CBD, THC, CBN;
FIG. 4 shows CBD and THC-D3Typical mass ion flow diagrams for/THC and CBN (MIC plot, mixed standard-8.0 μ g/ml), the lower plot is an exploded view of the upper plot;
FIG. 5 shows CBD and THC-D in hemp samples3Typical mass ion flow diagrams (MIC diagrams) for/THC and CBN;
fig. 6 shows a non-split-flow mode sample injection (a) for a non-split-flow liner tube and a non-split-flow mode sample injection (B) for a split-flow liner tube.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.
1 Material
1.1 drugs and reagents
CBD control (1.0mg/ml, lot: FE10071912), CBN control (1.0mg/ml, lot: FE01202013), THC control (1.0mg/ml, lot: FE 04222001); tetrahydrocannabinol-D3(THC-D31.0mg/ml, batch number: FE06111401), four control stock solutions were purchased from Cerilliant corporation. Methanol (LC/MS grade, Fisher Scientific).
Sample preparation: selecting 6 hemp samples from Yunnan (Kunming, Yuxi), inner Mongolia (Jining), Heilongjiang (Qinggang), Gansu (Longxi) and Qinghai (Xining) in different producing areas, and counting 18 batches. The samples were collected and provided by the biotechnology development center in Henan province and identified as annual herbaceous plants of the cannabis genus of the Cannabaceae family by Zhao Tianzeng researchers, academy of sciences in Henan province. The specimens are stored in a natural product focus laboratory of academy of sciences of Henan province, and the serial numbers of the specimens are 201908001 and 201908018 in sequence.
1.2 instrumentation
Gas chromatography mass spectrometer (GCMS-QP2010ULtra, Shimadzu, Japan); an electronic analytical balance model AUW220D (Shimadzu corporation, japan); ME 204/one ten thousandth balance (Mettler-Torlado, Switzerland); a liquid transferring gun: 100. mu.l, 200. mu.l, 1000. mu.l (Eppendorf); KQ-5WE ultrasonic pool, Kunshan ultrasonic instruments Inc.; model H1850r desk refrigerated centrifuge (chanshaxiang instruments limited, hunan).
2 methods and results
2.1 chromatographic conditions
The gas chromatography GC conditions were: a chromatographic column: rtx-5MS capillary column (30X 0.25mm,0.25 μm); sample inlet temperature: 280 ℃; carrier gas: he gas; the flow rate of the carrier gas is 1.0 ml/min; total flow rate: 9.0 ml/min; pressure: 1110.5 kPa. And (3) sample introduction mode: constant linear velocity shunting sample introduction; linear velocity: 38.7 cm/sec; the split ratio is as follows: 2: 1; sample introduction amount: 1 μ l. Temperature programming: maintaining at 200 deg.C for 2min, heating to 240 deg.C at 10 deg.C/min, maintaining for 18min, heating to 280 deg.C at 20 deg.C/min, and maintaining for 2 min.
2.2 Mass Spectrometry conditions
The MS conditions of the mass spectrum are as follows: an ion source: EI, electron energy 70eV, detector voltage: 0.93kv, ion source temperature: 240 ℃; interface temperature: 250 ℃; scanning mode: scan mode and Sim mode; detector voltage: 0.93 kV; solvent retardation: 9.0 min. Resolution ratio: scan range: 40-600 amu; in Sim mode: 9.5min-11.5 min: 231 → 246/314, 11.5min-13.5 min: 302 → 234/317, 299 → 231/314, 13.5min-16.0 min: 295 → 238/310; namely, the mass spectrometer selected ion monitoring parameters are detailed in the following table:
Figure BDA0002857324840000051
the application provides CBD and THC-D obtained by GC-MS analysis3Total ion chromatograms (TIC chart) of the four components THC and CBN are shown in figure 1. Selected quantitative qualitative ion pairs: CBD: 231 → 246/314; THC-D3: 302 → 234/317; THC: 299 → 231/314; and (3) CBN: 295 → 238/310; the qualitative and quantitative multi-ion flow monitoring chromatogram (MIC chart) of the four components is shown in figure 2, so as to prove the correctness of the mass spectrum condition selected by the application.
2.3 preparation of a series of Mixed Standard working solutions containing an internal Standard
Respectively and precisely measuring 1mL of CBD, CBN and THC reference product mother liquor with the concentration of 1.0mg/mL in three 10mL volumetric flasks, accurately fixing the volume to the scales by using chromatographic methanol, and using the standard stock solutions as CBD, CBN and THC so that the concentrations of three components in the standard stock solutions are respectively: CBD: 100. mu.g/ml, CBN: 100. mu.g/ml, THC: 100. mu.g/ml. Precisely measuring standard stock solutions with the same amounts of CBD, CBN and THC in the same volumetric flask (so as to ensure that the concentrations of CBD, CBN and THC in the volumetric flask are the same), and gradually diluting the standard stock solutions with chromatographic methanol to obtain mixed working solutions with the concentrations of CBD, THC and CBN of 40.0, 20.0, 10.0, 5.0, 1.0, 0.5 and 0.25 mu g/mL respectively (namely, the concentrations of CBD, THC and CBN in each mixed working solution are the same, for example, the concentrations of CBD, THC and CBN in the mixed working solution of 40.0 mu g/mL are the same and are 40.0 mu g/mL).
Accurately measuring THC-D with concentration of 1.0mg/ml3Adding 1mL of reference mother liquor into a 10mL volumetric flask, and accurately metering to the scale with chromatographic methanol to obtain THC-D3The internal standard stock solution of (1), so that THC-D in the internal standard stock solution3The concentration of (2) is 100. mu.g/ml. Then, an appropriate amount of internal standard stock solution is precisely measured and placed in a volumetric flask, and the internal standard stock solution is diluted into an internal standard solution with the concentration of 10.0 mu g/ml by chromatographic methanol.
Then, 4.0mL of series mixed working solutions of 40.0, 20.0, 10.0, 5.0, 1.0, 0.5 and 0.25 mu g/mL are respectively mixed with 1.0mL of internal standard solution of 10.0 mu g/mL, so as to obtain series mixed standard working solutions containing the internal standard; internal standard THC-D in series of mixed standard working solutions containing internal standard3The concentrations of (A) were all 2.0. mu.g/ml, and the concentrations of CBD, THC and CBN were all 32.0, 16.0, 8.0, 4.0, 0.8, 0.4, 0.2. mu.g/ml, respectively. The above solutions were stored in a refrigerator at-20 ℃ for further use.
Performing GCMS determination on the serial mixed standard working solutions containing the internal standards obtained by the preparation, measuring and recording CBD, THC, CBN and the internal standard THC-D3The chromatographic peak area of (A) is respectively CBD, THC, CBN and internal standard THC-D3The ratio of peak areas of (A) to (B) is ordinate, and CBD, THC, CBN and internal standard THC-D are taken as3The concentration ratio of (c) is plotted on the abscissa, standard curves of CBD, THC, and CBN (see fig. 3) are respectively plotted and calculated to obtain corresponding linear equations, which are detailed in table 1 below.
TABLE 1 Linear equation obtained by measuring CBD, THC and CBN by internal standard method
Figure BDA0002857324840000061
2.4 preparation and measurement of test solutions
Precisely weighing 20mg of hemp sample powder in a volumetric flask with a stopper, accurately adding 20mL of chromatographic methanol, and simultaneously adding 1.0mg/mL of THC-D3Measuring 40 μ L of internal standard solution, weighing and recording as m; standing at room temperature for 30min, performing ultrasonic treatment for 10min, cooling to room temperature, weighing again, adding chromatographic methanol to balance weight to m, transferring into 5ml EP tube, centrifuging at 10000r/min at high speed, and filtering with organic filter membrane with pore diameter of 0.22 μm to brown sample injection vial to obtain test solution.
Accurately weighing hemp leaf sample powder of 18 batches in total from 6 different production places (3 batches in each production place), preparing a test solution according to the method, determining according to the GC-MS condition of the chromatographic mass spectrum, and recording CBD, THC, CBN and internal standard THC-D3Substituting the chromatographic peak areas into the linear equation in the table 1 to calculate the content of CBD, THC and CBN in the test sample. The measurements were performed 3 times per batch and the sample measurements averaged and the results are shown in table 2 below.
The results in table 2 show: the content of the 18 batches of samples is quantitatively calculated by adopting an isotope internal standard method, the content differences of three index components of CBD, THC and CBN in different production places are obvious, the content of CBD in Kunming and Gansu is higher and is between 1.237 and 3.429 percent, the content of THC in inner Mongolia Gning is higher and reaches between 0.198 and 0.286 percent and is close to the critical value of drug type hemp (the content of THC is more than 0.3 percent), and the content of the three components in the hemp sample in Qinggang production place of Heilongjiang is the lowest. This may be due to differences in hemp varieties or regional environments. Therefore, the cannabinoids and other chemical components in hemp are influenced by genetic factors and may be influenced by other factors such as the environment (light exposure length, temperature, humidity, soil fertility, ultraviolet intensity, etc.).
Furthermore, the chemical phenotype of the hemp plant is mainly classified into drug type hemp (type i) and fiber type hemp (type ii), and reference is made to the literature (grand vitamin, zheng xiao yu, zhao powerful, etc.. analysis of 3 components and chemical phenotype in hemp plant based on ultra performance liquid chromatography-mass spectrometry [ J ] analytical chemistry, 2017 (7)):
judging according to the THC content. If THC content is greater than 0.3%, it is drug type cannabis sativa; if the content of the delta 9-THC is less than 0.3 percent, the hemp is fiber type hemp;
and judging according to a phenotype index calculation formula (THC + CBN)/CBD. If the phenotype index is more than 1, the drug type cannabis sativa is obtained; if the phenotype index is less than 1, the hemp is fiber type hemp;
thirdly, judging according to the two phenotype indexes THC/CBD and CBN/CBD. If one of the two phenotypic indexes is more than 1, the marijuana is drug type marijuana; and if both are less than 1, the fiber type hemp is obtained. Furthermore, it is reported in the literature that Δ 9-THC can degrade to produce CBN, and these 2 ratios are the phenotypic index — (split).
Therefore, the chemical phenotype of the hemp in different producing areas is divided according to the method of the phenotype index. The results in table 2 show that the 18 batches of the 6 production areas are all fiber type hemp, but China hemp in inner Mongolia and Heilongjiang production areas has THC/CBD ratio of more than 1 and CBN/CBD of less than 1, but according to the phenotype index (the THC + CBN)/CBD of less than 1 and THC content of less than 0.3 percent, the production area hemp is also judged to be fiber type hemp.
TABLE 2 determination of CBD, CBN and THC content and determination of chemical phenotype in hemp samples from different origins (n-3)
Figure BDA0002857324840000071
Figure BDA0002857324840000081
2.5 methodological validation
2.5.1 specialization examination
Under the selected chromatographic and mass spectrum conditions, CBD and THC-D3/Typical ion chromatogram of THC and CBN mixed standard sample and four components in the sample are shown in FIG. 4 and FIG. 5. As can be seen from FIGS. 4 and 5, CBD, THC-D3/The retention time of the four main peaks of THC and CBN is 10.390min, 12.240min/12.305min and 13.910min respectively, and the isotope internal standard THC-D3Although the method is combined with THC, the method adopts the selection of characteristic target ions for quantification, selects a reference ion pair and the abundance ratio thereof for qualitative determination, has no interference between target peaks and is more accurate in qualitative and quantitative determination. Therefore, the method has higher specificity.
2.5.2 detection and quantitation limits
The serial mixed standard working solutions (concentration 4.0 μ g/ml) containing the internal standard in "2.3" were precisely pipetted and diluted stepwise with chromatographic methanol, and then subjected to measurement and analysis under the conditions of chromatographic mass spectrometry under "2.1" and "2.2", and the lowest limit of detection (LOD) was calculated as 3-fold signal-to-noise ratio (S/N: 3:1) and the lowest limit of quantitation (LOQ) was calculated as 10-fold signal-to-noise ratio (S/N: 10:1), and the results are shown in table 3. The results in table 3 show that: the linear range of CBD, CBN and THC in the sample in the selected concentration range is wide, the linear relation is good, the detection limit is lower, the stability is good, and the accuracy is higher.
TABLE 3 internal Standard method for determination of detection limits and quantitation limits
Figure BDA0002857324840000082
2.5.3 sample recovery test
Accurately weighing 10mg (namely, the sampling amount is halved) of hemp sample powder into a volumetric flask with a plug, accurately adding 20mL of chromatographic methanol, simultaneously and accurately adding CBD, CBN and THC reference substances (marked as quality control samples, each addition value is 3 parts, and the result is averaged) with different low, medium and high addition values respectively, after processing according to the method under the preparation item of the sample solution, reversely calculating the actual measurement concentration by a standard curve and converting the actual measurement value, calculating the recovery rate and precision by comparing the actual measurement value with the addition value after subtracting the base value, and showing the recovery rate and precision results of different quality control samples in Table 4. The data in table 4 shows: the RSD of the method is 2.77-6.23%, and the recovery rate of the method is 93.20-112.67%. The results show that the addition recovery rate of CBD, CBN and THC in the sample under the test condition meets the requirement, and the method has good precision.
Table 4 recovery, precision (n ═ 3) of CBD, CBN and THC in the sample matrix
Figure BDA0002857324840000091
2.5.4 precision test
Precisely absorbing the sample solution of the same sample, determining according to the above GC-MS conditions, continuously injecting sample 6 needles, and recording CBD, THC, CBN and internal standard THC-D3Substituting the chromatographic peak area into the linear equation in the table 1 to calculate the content of CBD, THC and CBN in the test sample. RSD for CBD, THC and CBN content were 1.81%, 2.10% and 4.49%, respectively, all less than 5% (see table 5), indicating good precision.
Table 5 precision test results (n ═ 6)
Figure BDA0002857324840000092
2.5.5 repeatability test
Precisely weighing 6 parts of the same sample (Qinghai Xining) powder 20mg, preparing according to the method of preparing 2.4 test sample solution, measuring under the conditions of the chromatographic mass spectrum GC-MS, feeding two needles for each part of sample, and recording CBD, THC, CBN and internal standard THC-D3Substituting the chromatographic peak area into the linear equation in the table 1 to calculate the content of CBD, THC and CBN in the test sample. The RSD of the measurement results is less than 5 percent (see Table 6), which indicates good repeatability.
Table 6 results of the repeatability tests (n ═ 6)
Figure BDA0002857324840000093
2.5.6 stability test
In order to ensure the reliability and repeatability of the analysis result, the stability of the sample needs to be inspected, and the stability of the sample solution placed at room temperature is mainly inspected in the experiment. After the test solution is respectively placed for 0h, 4h, 8h, 12h, 24h and 48h at room temperature, the RSD of the values measured by CBD, CBN and THC in the sample is respectively between 3.09-4.78%, 2.49-5.86% and 2.68-5.32%, and the stability is respectively between 9.57-100.78%, 95.08-100.55% and 95.74-97.87% by measuring under the condition of the chromatographic mass spectrum GC-MS, which shows that the test solution has good stability when placed for 48h at room temperature, and the detailed results are shown in Table 7. Therefore, the hemp or the methanol extract thereof can be stably stored if the hemp is stored at room temperature and in a dark place, and can better guide the sampling, transportation, storage and identification of the hemp inspection material in a short period of time in judicial practice activities.
Table 7 stability test results (n ═ 3)
Figure BDA0002857324840000101
3 results and discussion
3.1 selection of chromatographic Mass Spectrometry conditions
In order to obtain the optimal chromatographic mass spectrum conditions under the items of 2.1 and 2.2 by changing the parameters of a constant linear velocity/constant pressure mode, column flow, a temperature rise program, a split flow ratio, a sample inlet temperature, an ion source temperature, an interface temperature and the like in research of obtaining the optimal chromatographic mass spectrum conditions under the items of 2.1 and 2.2, wherein the optimal chromatographic mass spectrum conditions have the advantages of better separation degree, proper peak emergence time, symmetrical and sharp peak shapes, good response signals and better system adaptability.
3.2 selection of the splitting ratio
Because the content of 3 components in samples at different parts can be very low, the sensitivity of the method has certain requirements, and the lower the limit of the method is, the better the limit is. In order to reduce the quantitative limit and improve the signal response, a non-shunting liner tube is adopted in research, and the target component response is improved by sampling in a non-shunting mode. Meanwhile, a shunting liner tube can be adopted, and when the shunting ratio is set to be-1, the sampling is equivalent to non-shunting mode sampling under the shunting liner tube. However, the results (see fig. 6) indicate that the split liner no-split mode response is reduced compared to the no-split liner no-split mode response, but both result in the target component peak splitting phenomenon. The reason may be that non-split feeding may cause secondary feeding of the sample, resulting in peak-shaped bifurcation. Therefore, in order to obtain a symmetrical and sharp peak shape and meet the sensitivity requirement, a shunting liner tube shunting mode is finally selected for sample injection, and the shunting ratio is 2: 1.
3.3 selection of Peak area ratio and Peak height ratio quantification
The research respectively adopts peak area ratio and peak height ratio to establish a standard curve of an isotope internal standard method to quantify the content of three effective components of CBD, CBN and THC. The results (table 8) show that the peak area ratio method is more stable and linear (the correlation coefficient R is closer to 1) relative to the peak height ratio method, which may be due to the fact that the peak height increases with the concentration due to the instrument itself and the chromatographic column, the instrument generates a certain supersaturation phenomenon, so that the direct proportional relationship between the peak height and the concentration changes to a certain extent, the peak area is more stable, and the reproducibility is good.
TABLE 8 Linear comparison of two quantification modes (peak area ratio to peak height ratio)
Figure BDA0002857324840000111
3.4 conclusion
The invention establishes a method for measuring the content of CBD, THC and CBN in hemp in different producing areas based on GC/MS technology-isotope internal standard method and carries out preliminary judgment on the chemical phenotype. The method is obtained through methodology investigation, and has the advantages of good specificity, high accuracy, low detection limit, simple and easy operation, sensitivity, high efficiency and reliable result. The isotope internal standard method is one of the internal standard methods, and a deuterated standard product of an object to be detected is used as an internal standard, because the molecular structure is the same, the isotope internal standard usually has chemical properties very close to the object to be detected, and is basically consistent with the influence of the object to be detected in the pretreatment process and the chromatographic separation process in the ionization process, so that the interference of matrix effect can be greatly eliminated, and errors generated in the analysis method process can be more effectively corrected. Therefore, the method can be more widely applied to different hemp inspection materials, drugs and other products added with three components, such as food, medicines, health care products and the like. Through simple chemical phenotype judgment and analysis of hemp samples of different batches, the samples of 18 batches of 6 producing areas are all fiber type hemp, however, researches show that the distribution rule of the hemp components in plants is as follows according to the increasing sequence: the root, stem, branch, lower leaf and upper leaf may cause the difference between the measured result and the actual result due to the limited number of sample batches, and the fact that some hemp leaves are more and some stems are more in the sample collection process and the different distribution of the hemp content is also influenced by different collection periods. In addition, there is no uniform division standard for the chemical phenotype judgment of hemp so far. Therefore, the chemical phenotype of the hemp plant cannot be judged only by means of the measurement result and the judgment mode of the batch of samples, comprehensive judgment is still needed for the chemical phenotype analysis of the hemp, and further research is needed for samples at different parts and different harvesting periods in the hemp plant.
In summary, the content characteristics of CBD, THC and CBN in hemp in different production areas are different, and the difference is influenced by other factors such as environment besides the genetic internal factors, and whether other cannabinoids have similar characteristics, and further statistics and verification are needed. The establishment and determination results of the method provide data reference and method reference for later-stage system deep research on the content characteristics of more cannabinoids and the influence rule of the environment on the components; also lays an experimental foundation for reasonably utilizing the plant resources of the species and developing diversified products with high added values, and has theoretical and guiding significance for industrial hemp production and control work.

Claims (5)

1. A method for measuring the content of CBD, THC and CBN in hemp based on GC-MS technology-isotope internal standard method is characterized by comprising the following steps:
1) preparation of a series of mixed standard working solutions containing an internal standard:
accurately weighing CBD, CBN, THC and internal standard THC-D respectively3A reference substance is prepared by the following steps of,preparing CBD, CBN and THC series mixed standard working solution containing internal standard by using methanol as a solvent;
the concentrations of CBD, CBN and THC in each mixed standard working solution are the same, and the internal standard THC-D3The concentration of (A) is 2.0 mu g/ml; the concentrations of CBD in the series of mixed standard working solutions are 32.0, 16.0, 8.0, 4.0, 0.8, 0.4 and 0.2 mu g/ml respectively;
2) preparation of a test solution:
precisely weighing 20mg of hemp sample powder, accurately adding 20mL of methanol, and simultaneously adding 1.0mg/mL of THC-D3Measuring 40 μ L of internal standard solution, weighing and recording as m; standing at room temperature for 20-40min, performing ultrasonic treatment for 5-20min, cooling to room temperature, weighing again, adding methanol to balance weight to m, centrifuging, and filtering with organic filter membrane to obtain test solution;
3) and (3) GC-MS determination:
performing GC-MS determination on the series of mixed standard working solutions containing the internal standard prepared in the step 1), measuring and recording CBD, THC, CBN and the internal standard THC-D3The chromatographic peak area of (A) is respectively CBD, THC, CBN and internal standard THC-D3The ratio of peak areas of (A) to (B) is ordinate, and CBD, THC, CBN and internal standard THC-D are taken as3The concentration ratio of the CBD to the THC to the CBN is an abscissa, standard curves of the CBD, the THC and the CBN are respectively drawn, and corresponding linear equations are obtained through calculation; performing GC-MS measurement on the sample solution obtained in the step 2) and recording CBD, THC, CBN and internal standard THC-D3Substituting the chromatographic peak areas into corresponding linear equations to calculate the content of CBD, THC and CBN in the test sample.
2. The method for determining the content of CBD, THC and CBN in hemp based on GC-MS technology-isotope internal standard method as claimed in claim 1, wherein the GC conditions of gas chromatography are as follows: a chromatographic column: rtx-5MS capillary column, injection port temperature: 280 ℃; the carrier gas is He gas; the flow rate of the carrier gas is 1.0-1.5 ml/min.
3. The method for determining the content of CBD, THC and CBN in hemp based on GC-MS technology-isotope internal standard method as claimed in claim 2, wherein the sample injection mode is as follows: constant linear velocity shunting sample introduction; linear velocity: 38.7 cm/sec; the split ratio is as follows: 5:1 or 2: 1; sample introduction amount: 1 mul; temperature programming: keeping the temperature at 200 ℃ for 2min, heating to 240 ℃ at 10 ℃/min, keeping the temperature for 18min, heating to 280 ℃ at 20 ℃/min, and keeping the temperature for 2 min.
4. The method for determining the content of CBD, THC and CBN in hemp based on GC-MS technology-isotope internal standard method as claimed in claim 1, wherein the MS conditions of mass spectrum are as follows: an ion source: EI, electron energy 70eV, detector voltage: 0.93-1.20kv, ion source temperature 240 ℃; the interface temperature was 250 ℃ and the solvent delay was 9.0 min.
5. The method for determining the content of CBD, THC and CBN in hemp based on GC-MS technique-isotope internal standard method as claimed in claim 4, wherein the scanning mode is: scan mode and Sim mode; detector voltage: 0.93 kV; resolution ratio: scan range: 40-600 amu; in Sim mode, the mass spectrometer selected ion monitoring parameters as follows:
Figure 442669DEST_PATH_IMAGE001
。
CN202011549569.5A 2020-12-24 2020-12-24 Method for determining CBD, THC and CBN content in hemp based on GC-MS technology-isotope internal standard method Pending CN112730679A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202011549569.5A CN112730679A (en) 2020-12-24 2020-12-24 Method for determining CBD, THC and CBN content in hemp based on GC-MS technology-isotope internal standard method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202011549569.5A CN112730679A (en) 2020-12-24 2020-12-24 Method for determining CBD, THC and CBN content in hemp based on GC-MS technology-isotope internal standard method

Publications (1)

Publication Number Publication Date
CN112730679A true CN112730679A (en) 2021-04-30

Family

ID=75605415

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202011549569.5A Pending CN112730679A (en) 2020-12-24 2020-12-24 Method for determining CBD, THC and CBN content in hemp based on GC-MS technology-isotope internal standard method

Country Status (1)

Country Link
CN (1) CN112730679A (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5036014A (en) * 1989-01-31 1991-07-30 Elsohly Mahmoud A Deuterated cannabinoids as standards for the analysis of tetrahydrocannabinol and its metabolites in biological fluids
CN108760947A (en) * 2018-09-05 2018-11-06 翟纹静 The method of a variety of anesthetic in triple level four bars gas chromatography mass spectrometry detection aquatic products
CN109187830A (en) * 2018-09-29 2019-01-11 云南中烟工业有限责任公司 The method of 9 kinds of alcohol compound contents in GC-MS method Rapid Simultaneous Determination edible essence fragrance
KR102132810B1 (en) * 2020-03-05 2020-07-13 대한민국 Method for analysis of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid using injection port derivatization gc-ms

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5036014A (en) * 1989-01-31 1991-07-30 Elsohly Mahmoud A Deuterated cannabinoids as standards for the analysis of tetrahydrocannabinol and its metabolites in biological fluids
CN108760947A (en) * 2018-09-05 2018-11-06 翟纹静 The method of a variety of anesthetic in triple level four bars gas chromatography mass spectrometry detection aquatic products
CN109187830A (en) * 2018-09-29 2019-01-11 云南中烟工业有限责任公司 The method of 9 kinds of alcohol compound contents in GC-MS method Rapid Simultaneous Determination edible essence fragrance
KR102132810B1 (en) * 2020-03-05 2020-07-13 대한민국 Method for analysis of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid using injection port derivatization gc-ms

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
EUNYOUNG JANG ET AL.: "Concentrations of THC, CBD, and CBN in commercial hemp seeds and hempseed oil sold in Korea", 《FORENSIC SCIENCE INTERNATIONAL》 *
向平等: "生物样品分析方法的有效性验证", 《法医学杂志》 *
杜鸿雁等: "氘代内标在法庭毒物分析的应用", 《中国法医学杂志》 *
王占良等: "气相色谱-质谱法同时分析运动营养品中的大麻酚、大麻二酚和Δ9-四氢大麻酚", 《中国运动医学杂志》 *
肖培云等: "工业大麻研究.Ⅲ.不同生长期工业大麻中四氢大麻酚和大麻二酚含量的测定方法比较", 《中国医药工业杂志》 *
艾斯凯尔，艾尔肯等: "气相色谱-质谱联用(GC-MS)法检测人体血液、尿液中大麻及其主要代谢物的含量", 《新疆医科大学学报》 *
郭宇姝等: "GC-MS法测定火麻中△~9-四氢大麻酚的含量", 《中国药房》 *

Similar Documents

Publication Publication Date Title
Sakunphueak et al. Simultaneous determination of three naphthoquinones in the leaves of Impatiens balsamina L. by reversed‐phase high‐performance liquid chromatography
CN102692462A (en) Danhong injection quality control method
CN108169375B (en) Method for identifying authenticity of propolis by combining fingerprint spectrum and antioxidant activity
CN116429943A (en) Quality analysis method of traditional Chinese medicine radix scutellariae based on dual-wavelength equal-baseline differential fusion map
CN110455961B (en) High-flux detection method for multiple components in health-care wine
CN108152425B (en) Method for detecting lignanoids in sesame oil by high performance liquid chromatography
CN112730679A (en) Method for determining CBD, THC and CBN content in hemp based on GC-MS technology-isotope internal standard method
CN103399111B (en) Method for selectively measuring ethylene glycol monoethyl ether acetate in dry food packaging paper based on headspace-gas chromatography/mass spectrometry
CN106442750B (en) A kind of method of free gossypol detoxification situation in evaluation cottonseed oil
CN114217002B (en) Method for detecting contents of chamomile azulene and sabinene in chamomile essential oil
CN115629144A (en) Extraction and determination method of liliflorin A and liliflorin H in lily medicinal material
CN111579684B (en) Method for measuring content of total capsaicin in capsule wall material of capsule
CN112255339B (en) Method for rapidly determining contents of three sesame lignans in sesame oil
CN108693289A (en) The content assaying method of magnoflorine in a kind of herringbone fruit medicinal material
CN104458931B (en) A kind of detection method of chromatogram of Radix Isatidis
CN111721875A (en) Method for identifying rhizoma coptidis deltoideae and rhizoma coptidis
Qin et al. Quantitative RP–LC analysis of mangiferin and homomangiferin in Mangifera indica L. leaves and in Mangifera persiciforma CY Wu et TL Ming Leaves
CN112240914A (en) Method for detecting flavone components in anoectochilus formosanus with different appearance phenotypes
CN110361489A (en) Amitriptyline monitor drug concentration kit and its detection method in a kind of blood
RU2782620C1 (en) Method for quantifying syringin in the bark of common lilac
CN114942296B (en) Detection method for removing heavy oil component of mugwort in monoterpene type
CN114778730B (en) Method for simultaneously detecting 6 organic acids in aster medicinal material or aster product
CN115219602B (en) Method for measuring volatile organic acid in tobacco and tobacco products
CN115372521B (en) Method for separating and identifying phytosphingosine and/or N-acetylphytosphingosine
CN113533607B (en) Method for evaluating quality of peony leaf medicinal material

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
TA01 Transfer of patent application right

Effective date of registration: 20230607

Address after: 450000 No.1, 3rd floor, building 1, 58 Hongzhuan Road, Jinshui District, Zhengzhou City, Henan Province

Applicant after: Henan Napu Biotechnology Co.,Ltd.

Address before: No.58, Hongzhuan Road, Jinshui District, Zhengzhou City, Henan Province, 450000

Applicant before: Henan Academy of Sciences

Applicant before: HENAN COGO TESTING INTERNATIONAL Co.,Ltd.

TA01 Transfer of patent application right