Kit and method for examining cannabinoids

Abstract

To provide a safely-storable examination kit for examining cannabinoids and a highly practical method for examining cannabinoids.SOLUTION: An examination kit for examining cannabinoids includes a base material and a flow passage on the base material. The flow passage includes: a first region containing a first pH adjusting reagent; a second region containing an aromatic amine compound and a first acid reagent; a third region containing a first reagent capable of generating diazonium ions from the aromatic amine compound by acting on the aromatic amine compound; and a fourth region for dropping a development solvent. The second region may be located between the first region and the fourth region and the third region may be located between the second region and the fourth region. The first region may be a region for dropping an extraction liquid containing an examination target object.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] One embodiment of the present invention relates to a test kit and a test method for inspecting cannabinoids.

Background Art

[0002] Cannabinoids such as THC (tetrahydrocannabinol) and THCA (tetrahydrocannabinolic acid) contained in cannabis are components regulated by the Narcotics Control Law because they have psychoactive effects. In Japan, the possession of cannabis itself is prohibited by the Cannabis Control Law. In recent years, due to the influence of the legalization of cannabis overseas, the number of arrests for possessing cannabis in Japan has also increased rapidly. At the site of crackdowns, a test kit that can quickly and accurately determine cannabis is required.

[0003] For the detection of cannabinoids, a colorimetric reagent using a diazonium compound is used. A method for quantifying cannabinoids is known in which a solution of a diazonium salt is prepared, and the diazonium ions dissociated from the salt cause a color reaction with cannabinoids under appropriate pH conditions (Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Diazonium salts and diazonium ions are significantly deteriorated by light and heat and lack stability. Test kits using diazonium salts and diazonium ions have limited storage methods and storage locations, and long-term storage is not possible.

[0006] In view of the above problems, an object of one embodiment of the present invention is to provide a test kit for inspecting cannabinoids with high storage stability and a test method for inspecting cannabinoids with high practicality.

Means for Solving the Problems

[0007] A test kit for inspecting cannabinoids according to one embodiment of the present invention includes a base material and a flow path on the base material. The flow path includes a first region containing a first pH adjusting reagent, a second region containing an aromatic amine compound and a first acidic reagent, and a third region containing a first reagent capable of generating diazonium ions from the aromatic amine compound by acting on the aromatic amine compound, and a fourth region for dropping a developing solvent.

[0008] The second region may be located between the first region and the fourth region, and the third region may be located between the second region and the fourth region.

[0009] The first region may be a region for dropping an extract containing the object to be inspected.

[0010] The base material may be paper.

[0011] The developing solvent may be an aqueous solution containing a polar solvent.

[0012] The first reagent may be a nitrite.

[0013] It may further include an extract for extracting the object to be inspected.

[0014] A test kit for inspecting cannabinoids according to one embodiment of the present invention includes a first vial containing an extract, a second vial containing a solution containing an aromatic amine compound and a solution containing a first acidic reagent, and a third vial containing a first reagent capable of generating diazonium ions from the aromatic amine compound by acting on the aromatic amine compound.

[0015] The extract may contain a first pH adjusting reagent and an organic solvent or an aqueous solution containing an organic solvent.

[0016] The first pH adjusting reagent may be an alkali metal-containing hydroxide.

[0017] The first acidic reagent may be a hydroxy acid.

[0018] The first reagent may be a nitrite.

[0019] A test method for inspecting cannabinoids according to an embodiment of the present invention extracts an object to be inspected contained in a sample with an extract, mixes the extract in which the object to be inspected is extracted with an aromatic amine compound-containing solution to prepare a first mixture, and mixes the first mixture with an aqueous solution containing a first reagent that can generate diazonium ions from the aromatic amine compound by acting on the aromatic amine compound to prepare a second mixture, adds an aqueous solution containing a first acidic reagent to the second mixture to prepare an acidic third mixture, adds an aqueous solution containing a first pH adjusting reagent to the third mixture to prepare a fourth mixture, and confirms the change in color tone between the color tone of the third mixture and the color tone of the fourth mixture.

[0020] A test method for inspecting cannabinoids according to an embodiment of the present invention extracts an object to be inspected contained in a sample with an extract, mixes the extract in which the object to be inspected is extracted with an aromatic amine compound-containing organic solvent to prepare a first mixture, mixes the first mixture with an aqueous solution containing a first acidic reagent to prepare a second mixture, and mixes the second mixture with an aqueous solution containing a first reagent that can generate diazonium ions from the aromatic amine compound by acting on the aromatic amine compound to prepare a third mixture, and confirms the change in color tone between the color tone of the second mixture and the color tone of the third mixture.

[0021] The object to be inspected may be cannabinoids.

[0022] The extract may contain an alkali metal-containing hydroxide and an aqueous alcohol solution.

[0023] The first pH adjustment reagent may be an alkali metal-containing hydroxide.

[0024] The first acidic reagent may be a hydroxy acid.

[0025] The first reagent may be a nitrite.

Advantages of the Invention

[0026] According to the present invention, it is possible to provide a test kit for testing cannabinoids with high storage stability. Further, according to the present invention, it is possible to provide a highly practical test method for testing cannabinoids.

Brief Description of the Drawings

[0027]

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Mode for Carrying Out the Invention

[0028] Hereinafter, the test method according to the present invention will be described in detail. However, the test method of the present invention is not construed as being limited to the description contents of the following embodiments and examples.

[0029] [First Embodiment] Hereinafter, a test kit which is one of the embodiments of the present invention will be described. The test kit is used for detecting cannabinoids contained in marijuana. In the embodiment of the present invention, cannabinoids refer to CBD, cannabinol (CBN), THC, THCA, etc., which are cannabinoids contained in marijuana.

[0030] <Test Kit 1> Referring to FIG. 1, the test kit 10 will be described. FIG. 1 is a plan view showing a test kit for testing cannabinoids according to an embodiment of the present invention.

[0031] The test kit 10 includes a base material 100, a flow path 102, a first region 104, a second region 106, a third region 108, and a fourth region 110.

[0032] The base material 100 is provided with a flow path 102 including the first region 104, the second region 106, the third region 108, and the fourth region 110. The base material 100 is provided with a coating layer 112.

[0033] The base material 100 is a base material on which a diazo coupling reaction and a color reaction are carried out. The base material 100 is a structure having a porous structure capable of causing capillary action in a sheet form. For the base material 100, for example, filter paper, qualitative filter paper, thin layer chromatography (TLC) plate or sheet, nitrocellulose paper, cloth, etc. can be used.

[0034] The developing solvent dropped in the fourth region 110 described later moves through the flow path 102. Specifically, the flow path 102 can function as a stationary phase through which a mobile phase such as a developing solvent moves. The flow path 102 is formed on the base material 100. The flow path 102 is located between the first region 104 and the fourth region 110. The flow path 102 may be formed by covering the base material 100 with the coating layer 112. The flow path 102 is a region not covered by the coating layer 112 of the base material 100.

[0035] The coating layer 112 is a wall for efficiently moving the developing solvent dropped in the fourth region 110 to the first region 104 through the flow path 102. The coating layer 112 can be provided on the base material 100. The coating layer 112 can cover the base material 100. The coating layer 112 is arranged to surround the flow path 102, the first region 104, the second region 106, the third region 108, and the fourth region 110. A hydrophobic substance can be used for the coating layer 112. The hydrophobic substance is a substance having an atomic group such as a hydrocarbon group in the molecule, and includes oils, petroleum, fats, alkanes, etc., and for example, wax can be mentioned. Also, in one embodiment, as long as the formed coating layer 112 is not dissolved by the developing solvent or the sample-containing solution, a hydrophilic polymer can also be used.

[0036] The first region 104 is a region where a sample or an analyte contained in the sample is dropped. The first region 104 is a region where the dropped sample or the analyte contained in the sample reacts with the color-forming reagent. The first region 104 is a region where the color development or color change phenomenon of the dropped sample or the analyte contained in the sample can be observed. The first region 104 is a region where it is possible to determine whether or not cannabinoids are present in the sample.

[0037] The first region 104 is formed in the flow path 102. The first region 104 is arranged at the end of the flow path 102. The shape of the first region 104 is circular in FIG. 1, but is not particularly limited. The size of the first region 104 only needs to be able to sufficiently contain the dropped sample or the analyte contained in the sample, and is not particularly limited.

[0038] The first region 104 is the region where the color reaction of cannabinoids occurs. The first region 104 contains a first pH adjusting reagent. The first pH adjusting reagent can adjust the first region 104 to an optimal pH for the sample or the analyte contained in the sample to react with the color reagent. The first pH adjusting reagent may be a non-volatile compound, and examples include hydroxides containing non-volatile alkali metals or alkaline earth metals and polyamines. Examples of the hydroxides containing non-volatile alkali metals or alkaline earth metals include sodium hydroxide, potassium hydroxide, and calcium hydroxide. Examples of polyamines include diethylenetriamine, tetraethylenepentamine, polyethylene polyamine, and polyethyleneimine. The first pH adjusting reagent may not be used.

[0039] As a method for supporting the first pH adjusting reagent on the first region 104, a method of dropping an aqueous solution containing the first pH adjusting reagent onto the first region 104 and drying the first region 104 can be mentioned. As a method for supporting the first pH adjusting reagent, for example, 1 μL of a 500 mM aqueous solution of sodium hydroxide is dropped onto the first region 104, and the first region 104 is dried at a temperature of about 50 °C for 5 minutes or more, or air-dried for 5 minutes or more.

[0040] The second region 106 is the region where the diazo coupling reaction occurs. The second region 106 is the region that generates the color reagent used in the color reaction occurring in the first region 104.

[0041] The second region 106 is formed in the flow path 102. The second region 106 is located between both ends of the flow path 102. The second region 106 is located between the first region 104 and the fourth region 110. The second region 106 is adjacent to the first region 104. The color reagent generated in the second region 106 moves to the adjacent first region 104 via the flow path 102 by the developing solvent. The shape and size of the second region 106 are not particularly limited, similar to the first region 104.

[0042] The second region 106 contains a precursor of a color reagent and a first acidic reagent. Generally, a diazonium salt is used as the color reagent for the color reaction of cannabinoids. The precursor of the color reagent is a precursor of the diazonium salt. The precursor of the diazonium salt is a more stable compound than the diazonium salt, for example, a compound in which reactions such as decomposition, synthesis, isomerization, oxidation, and polymerization are less likely to occur by light. The precursor of the diazonium salt is an aromatic amine compound, specifically, a derivative of aniline. As described above, the aromatic amine compound generally includes a precursor of a diazonium salt used in the color reaction of cannabinoids. Examples of the aromatic amine compound include precursors of diazonium salts described in, for example, Hygienic Chemistry, 25(6), 321-326(1979), Chem.Pharm.Bull., 16, 822-826(1968), International Publication No. 2021 / 068053, and Japanese Patent Application Laid-Open No. 2010-513857. Specifically, N-(4-amino-2,5-diethoxyphenyl)benzamide, which is a precursor of Fast Blue BB salt, o-dianisidine, which is a precursor of Fast Blue B salt, p-chloro-o-toluidine, which is a precursor of Fast Red TR salt, 4-nitro-o-anisidine, which is a precursor of Fast Red B salt, o-aminoazotoluene, which is a precursor of Fast Garnet GBC salt, and 2-amino-4-chloroanisole, which is a precursor of Fast Red RC salt, etc. are included.

[0043] The first acidic reagent can place the aromatic amine compound under acidic conditions. The first acidic reagent may be a non-volatile compound, and a hydroxy acid can be used. Examples of the non-volatile compound include non-volatile hydroxy acids, non-volatile oxo acids, sulfonic acids, carboxylic acids, and polymers obtained by polymerization of these acids. Examples of the non-volatile hydroxy acid or oxo acid include citric acid, succinic acid, fumaric acid, boric acid, benzenesulfonic acid, benzoic acid, polyacrylic acid, and polystyrene sulfonic acid. The first acidic reagent is preferably citric acid.

[0044] The first acidic reagent preferably has a pH value of 2 to 5.

[0045] As a method for supporting an aromatic amine compound and a first acidic reagent in the second region 106, examples include a method of mixing an organic solvent or an aqueous solution containing an organic solvent containing an aromatic amine compound, which is a precursor of a diazonium salt, with a solution containing a first acidic reagent, dropping the mixed solution onto the second region 106, and drying the second region 106. The organic solvent or the aqueous solution containing an organic solvent containing an aromatic amine compound only needs to dissolve the aromatic amine compound. For example, organic solvents such as methanol, ethanol, and acetonitrile, and aqueous solutions containing organic solvents can be used. For example, a 20 mM acetonitrile solution containing an aromatic amine compound and a 400 mM aqueous citric acid solution are mixed at a ratio of 1:1, 1 μL of the mixed solution is dropped onto the second region 106, and the second region 106 is dried at a temperature of about 50 °C for 5 minutes or more, or air-dried for 5 minutes or more.

[0046] The third region 108 is a region that supports a first reagent used in the reaction for generating diazonium ions from an aromatic amine compound.

[0047] The third region 108 is formed in the flow path 102. The third region 108 is located between the second region 106 and the fourth region 110. The third region 108 is adjacent to the second region 106. The first reagent contained in the third region 108 moves to the adjacent second region 106 through the flow path 102 by the developing solvent. The shape and size of the third region 108 are not particularly limited, similar to the first region 104.

[0048] The third region 108 contains a first reagent. As described above, by the action of the first reagent on the aromatic amine compound, diazonium ions can be generated from the aromatic amine compound. The first reagent is a compound that generates nitrosonium ions under acidic conditions. The first reagent is a non-volatile compound, and examples include nitrites. Specifically, as the first reagent sodium nitrite, potassium nitrite, etc. can be mentioned. The first reagent is preferably sodium nitrite.

[0049] The method of loading the first reagent onto the third region 108 is to drop an aqueous solution containing the first reagent onto the third region 108 and then dry the third region 108. For example, 1 μL of a 300 mM aqueous solution of sodium nitrite is dropped onto the third region 108, and the third region 108 is dried at a temperature of about 50 °C for 5 minutes or more, or air-dried for 5 minutes or more.

[0050] The fourth region 110 is a region where a solvent capable of moving substances or compounds contained in the third region 108 and the second region 106 to adjacent regions is dropped. Specifically, it is a region where a developing solvent that moves the first reagent contained in the third region 108 to the second region 106 and moves the diazonium ions generated in the second region 106 to the first region 104 is dropped.

[0051] The fourth region 110 is formed in the flow path 102. The fourth region 110 is disposed at the other end of the flow path 102 where the first region 104 is disposed. The shape and size of the fourth region 110 are the same as those of the first region 104, but it is preferably larger than the size of the first region 104 to the third region 108 in consideration of the amount of the solvent to be dropped.

[0052] The developing solvent dropped into the fourth region 110 is developed from the fourth region 110 toward the first region 104. Specifically, the developing solvent moves from the fourth region 110 to the first region 104 in the direction of the arrow shown in FIG. 1.

[0053] As the developing solvent dropped into the fourth region 110, a reaction solvent used for the diazo coupling reaction and the color development reaction can be used. As the developing solvent dropped into the fourth region 110, a solvent that does not dissolve the coating layer 112 can be used. As the developing solvent, an aqueous solution containing a polar solvent can be used. Examples of the developing solvent dropped into the fourth region 110 include an aqueous acetonitrile solution, an aqueous methanol solution, and an aqueous ethanol solution.

[0054] Here, in addition to the base material 100 provided with the flow path 102 including the first region 104, the second region 106, the third region 108, and the fourth region 110, the test kit 10 can further include an extraction solution for extracting the test object. The extraction solution for extracting the test object can be stored in a container capable of storing a liquid such as a vial. The extraction solution only needs to be able to extract the test object, and ethanol, methanol, acetonitrile, ethyl acetate, propanol, isopropyl alcohol, acetone, tetrahydrofuran (THF), N,N-dimethylformamide (DMF), etc., and an aqueous solution containing at least one of these can be used. For example, 1 mL of a 50% ethanol aqueous solution is used as the extraction solution.

[0055] <Judgment Procedure 1> The judgment procedure for cannabinoids using the test kit 10 will be described.

[0056] Put the sample into the extraction solution, and extract the test object contained in the sample with the extraction solution.

[0057] Drop 1 μL of the extraction solution in which the test object has been extracted onto the first region 104. The dropping amount of the extraction solution only needs to ensure an amount that can be sufficiently supported on the first region 104 and is not particularly limited.

[0058] Drop 40 μL of the developing solvent onto the fourth region 110. The dropping amount of the developing solvent only needs to ensure that the developing solvent spreads to the first region 104, and the amount can be appropriately changed according to the length of the flow path 102. For example, a 50% acetonitrile aqueous solution is used as the developing solvent.

[0059] After the developing solvent dropped onto the fourth region 110 spreads to the first region 104, compare the color tone of the first region 104 before and after the spreading of the developing solvent, and confirm the presence or absence of a color tone change and the color tone after the change. Or, compare the color tone of the first region 104 after spreading the developing solvent without including the test object in the first region 104 (reference sample) with the color tone of the first region 104 after spreading the developing solvent with the test object included in the first region 104, and confirm that the color tone is different from the reference sample and its color tone.

[0060] As a method for checking the color tone of the first region 104, for example, the first region 104 is photographed, and the photographed image is analyzed using image processing analysis software. By the image analysis, it is possible to identify the types of cannabinoids and perform a quantitative evaluation of the cannabinoids. The color space coordinates obtained by the image analysis are compared with the color space coordinates obtained by the image analysis of the basic image, and it is possible to determine whether the inspection object is cannabinoids. The basic image is a photographed image of the first region 104 when the inspection object is cannabinoids. As the image processing analysis software, for example, ImageJ software can be used.

[0061] In the analysis of the photographed image using the image processing analysis software, when using ImageJ software, from the photographed image of the first region 104, after measuring the (L*, a*, b*) coordinate values in the CIE L*a*b* color system of the first region 104 using ImageJ software, using the θ value of the following formula 1 with the coordinate values, the types of cannabinoids can be identified, and further, using the ΔE value of the following formula 4, the quantification of the cannabinoids can be performed.

[0062]

Equation

[0063] Here, Δa * and Δb * are defined by the following formula 2 and formula 3. Here, a * sample and b * sample are the a * coordinates and b * coordinates of the first region 104 containing the inspection object. a * Ref and b * Ref are the a * coordinates and b * coordinates of the first region 104 not containing the inspection object. Δa * / Δb *The value is specific to each cannabinoid.

[0064] [Number]

[0065] [Number]

[0066] [Number]

[0067] Here, L in the above formula 4 * sample is the L coordinate of the first region 104 containing the object to be inspected, and L * is the L coordinate of the first region 104 not containing the object to be inspected. * Ref is the L coordinate of the first region 104 not containing the object to be inspected. * is the coordinate.

[0068] By performing the above determination procedure, it is possible to identify and quantify the types of cannabinoids contained in the sample.

[0069] As described above, the inspection kit 10 according to an embodiment of the present invention includes a base material 100 and a flow path 102 on the base material 100. The flow path 102 includes a first region 104 containing a first pH adjustment reagent, a second region 106 containing an aromatic amine compound and a first acidic reagent, a third region 108 containing a first reagent, and a fourth region 110 for dropping a developing solvent. Thus, a diazo coupling reaction can be performed on the base material 100. Since an aromatic amine compound with high stability against light and heat is supported on the base material 100, the influence of light during the production of the inspection kit 10 is small. Furthermore, by using an aromatic amine compound with high light and heat stability, the restrictions on the storage method and storage location of the inspection kit 10 are relaxed, enabling long-term storage and providing an inspection kit with high storage stability.

[0070] (Modification 1) In the above-described embodiment, as a method for supporting the first pH adjusting reagent on the first region 104, a method of dropping an aqueous solution containing the first pH adjusting reagent onto the first region 104 and drying the first region 104 was used. However, the method of supporting the first pH adjusting reagent on the first region 104 is not necessarily limited to the method of dropping the aqueous solution containing the first pH adjusting reagent.

[0071] FIG. 2 is a plan view showing a test kit for inspecting cannabinoids according to a modified example of an embodiment of the present invention. In the test kit 20 according to Modified Example 1, a substrate capable of adjusting the pH for the test object to react with the coloring reagent is attached on the first region 104. As shown in FIG. 2, the pH-adjustable substrate 204 is attached on the first region 104. The substrate 204 is prepared separately from the substrate 100, for example, and an alkaline substrate 204 is attached to the first region 104. As the alkaline substrate 204, for example, alkaline filter paper can be used. As a method of attaching the alkaline substrate 204 on the first region 104, an adhesive having little influence on the color reaction can be applied to the substrate 204 and attached on the first region 104. For the adhesive, for example, a synthetic rubber-based material can be used, and the adhesive can be applied to the substrate 204 as an aerosol. In such a modified example, there is the same effect as that of the above-described embodiment.

[0072] Regarding the second region 106 and the third region 108 as well, similarly to the first region 104, substrates separately carrying each reagent may be prepared and attached to each region.

[0073] (Modified Example 2) The test kit 10 of the first embodiment includes a first region 104 containing a first pH adjusting reagent, a second region 106 containing an aromatic amine compound and a first acidic reagent, a third region 108 containing a first reagent, and a fourth region 110 for dropping a developing solvent. However, the test kit according to the present embodiment is not necessarily limited to four regions.

[0074] FIG. 3 is a plan view showing a test kit for inspecting cannabinoids according to a modified example of an embodiment of the present invention. In the test kit 30 according to the modified example, in addition to the first region 104 to the fourth region 110, a fifth region 114 can be provided. The fifth region 114 is a region to which the coloring reagent remaining in the first region 104 further moves.

[0075] As shown in FIG. 3, the fifth region 114 is located at one end of the flow path 102, and the fourth region 110 is located at the other end of the flow path 102. The fifth region 114 is provided on the flow path 102 such that the second region 106 and the third region 108 are located between the fifth region 114 and the fourth region 110. The fifth region 114 is arranged at a position beyond the first region 104 in the direction in which the developing solvent develops. The fifth region 114 is arranged at a position farther from the fourth region 110 than the first region 104.

[0076] By providing the fifth region 114 in the test kit 30, the coloring reagent that has moved from the second region 106 by the developing solvent can move to the fifth region 114 without remaining in the first region 104. Thus, when the coloring reagent moves to the fifth region 114, the color of the color reaction occurring in the first region 104 and the color of the coloring reagent do not mix. Since the color of the color reaction and the color of the coloring reagent do not mix, the variation in the color (color of the color reaction) of the first region 104 becomes smaller, and the determination of the color of the color reaction described later becomes more accurate. In such a modified example, there is the same effect as that of the above-described embodiment.

[0077] <Test Kit 2> An explanation of a test kit using a solution on a substrate will be given. The difference from the test kit 10 is that paper is not used for the substrate, but a solution is used. Note that the description of the configuration that is the same as or similar to that of the test kit 10 may be omitted.

[0078] The test kit includes a first vial, a second vial, and a third vial. As the vial, a container or bottle made of glass or plastic can be used. However, the material of the container or bottle only needs to be insoluble in the organic solvent used in the test kit, and is not limited thereto.

[0079] In the first vial, an extract for extracting the object to be tested contained in the sample is stored. The first vial contains the extract. The extract can contain a first pH adjusting reagent and an organic solvent or an aqueous solution containing an organic solvent. Note that the first vial may contain the first pH adjusting reagent and the organic solvent or the aqueous solution containing an organic solvent, or they may be contained in different vials. As the first pH adjusting reagent, the same first pH adjusting reagent as that in the test kit 10 can be used. However, a volatile compound can also be used as the first pH adjusting reagent used in this test kit. As the organic solvent or the aqueous solution containing an organic solvent, for example, ethanol, methanol, acetonitrile, or an aqueous solution containing at least one of these can be used. The first vial can contain, for example, sodium hydroxide as the first pH adjusting reagent and 1 mL of a 50% ethanol aqueous solution as the aqueous solution containing an organic solvent. Here, the first vial is not limited to a vial, and a container or bottle with a dropper or a container or bottle with a pipette can be used.

[0080] In the second vial, a diazo coupling reaction is carried out to generate a color - forming reagent. The second vial contains an organic solvent containing an aromatic amine compound. The second vial contains an aqueous solution containing a first acidic reagent. By including the aqueous solution containing the first acidic reagent in the second vial, the aromatic amine compound is kept stable, enabling a more stable color - forming reaction for cannabinoids. As the aromatic amine compound, an aromatic amine compound similar to that in Test Kit 10 may be used. As the organic solvent containing the aromatic amine compound, for example, ethanol, methanol, or acetonitrile can be used. As the first acidic reagent, a first acidic reagent similar to that in Test Kit 10 may be used. However, for the first acidic reagent used in this test kit, volatile compounds can also be used. The second vial contains, for example, 1 mL of a 50% ethanol aqueous solution containing a precursor of 5 mM Fast Blue BB and a 100 mM citric acid aqueous solution. Here, the second vial is not limited to a vial, and a container or bottle with a dropper or a container or bottle with a spout can also be used.

[0081] The third vial stores an aqueous solution containing a first reagent that acts on the aromatic amine compound in the diazo coupling reaction of the aromatic amine compound. The third vial contains the aqueous solution containing the first reagent. When the second vial does not contain the aqueous solution containing the first acidic reagent, the third vial can also contain the aqueous solution containing the first acidic reagent. Both the second vial and the third vial may contain the aqueous solution containing the first acidic reagent.

[0082] As the first reagent, a first reagent similar to that in Test Kit 10 may be used. Note that for the first reagent used in this test kit, volatile compounds can also be used. The third vial contains, for example, a 10 mM sodium nitrite aqueous solution. Here, the third vial is not limited to a vial, and a container or bottle with a dropper or a container or bottle with a spout can also be used.

[0083] <Determination Procedure 2> The determination procedure for cannabinoids in cannabis herb using a solution test kit is described.

[0084] Put about three pieces of the sample into the first vial, shake for about 1 minute, and extract the analyte into the extraction solution.

[0085] Drop 1 to 2 drops of the first reagent-containing aqueous solution contained in the third vial into the second vial, and gently mix the first reagent-containing aqueous solution, the aromatic amine compound-containing organic solvent, and the first acidic reagent-containing aqueous solution. By mixing the first reagent-containing aqueous solution, the alcohol aqueous solution containing the aromatic amine compound, and the first acidic reagent-containing aqueous solution, a coloring reagent is prepared.

[0086] Put the mixed solution in the second vial into the first vial and mix.

[0087] Compare the color tone of the mixed solution contained in the first vial with the color tone of the extraction solution before putting the mixed solution in the second vial, and confirm the color tone change.

[0088] As a method for confirming the color tone change, for example, there is a method of measuring the absorption spectrum of the mixed solution contained in the first vial and confirming the presence or absence of an absorption peak at a predetermined wavelength. Further, quantification is performed using the intensity of the absorption peak at a predetermined wavelength, for example, the absorption peak in the range of 450 nm to 590 nm. The absorption spectrum of the mixed solution can be measured using a spectrophotometer or the like.

[0089] By performing the above determination procedure, cannabinoids contained in the sample are determined.

[0090] As described above, the test kit according to an embodiment of the present invention includes a first vial containing an extraction solution, a second vial containing an organic solvent containing an aromatic amine compound and an aqueous solution containing a first acidic reagent, and a third vial containing a first reagent. By including these, a diazo coupling reaction using an aromatic amine compound can be carried out at the site of seizure to generate a coloring reagent. The aromatic amine compound exhibits higher stability against light and heat than the diazonium salt and diazonium ion. Thus, by using an aromatic amine compound having high stability against light and heat in the test kit, the restrictions on the storage method and storage location of the test kit are relaxed, long-term storage becomes possible, and a test kit with high storage stability can be provided.

[0091] [Second Embodiment] Hereinafter, a test method which is one of the embodiments of the present invention will be described. The test method is a test method for detecting cannabinoids contained in a sample.

[0092] <Test Method 1> FIG. 4 is a flowchart showing Test Method 1 in the second embodiment.

[0093] First, several pieces of a sample that is considered to contain the object to be tested are put into an extractant, shaken for about 1 minute, and the object to be tested is extracted (step S11). An organic solvent containing an aromatic amine compound is dropped into the extractant in which the object to be tested has been extracted, and the extractant and the organic solvent containing the aromatic amine compound are mixed to prepare a first mixed solution (step S12). An aqueous solution containing a first reagent is put into the first mixed solution, and the first mixed solution and the aqueous solution containing the first reagent are mixed to prepare a second mixed solution (step S13). An aqueous solution containing a first acidic reagent is added so that the second mixed solution becomes acidic, and an acidic third mixed solution is prepared (step S14). An aqueous solution containing a first pH adjustment reagent is added to the third mixed solution to prepare a fourth mixed solution (step S15). The color tone of the third mixed solution is compared with the color tone of the fourth mixed solution to confirm the change in color tone (step S16). The absorption spectrum of the fourth mixed solution is measured and compared with the wavelengths of characteristic absorption peaks of each cannabinoid to determine whether the object to be tested is a cannabinoid which is a component of cannabis (step S17).

[0094] Inspection method 1 is as follows. For example, 500 μL of a 50% aqueous ethanol solution containing a precursor of Fast Blue BB is dropped into 100 μL of an ethanol solution from which the object to be inspected has been extracted, and they are mixed to prepare a first mixed solution. 500 μL of a 1 mM aqueous sodium nitrite solution is dropped into the first mixed solution, and they are mixed to prepare a second mixed solution. 500 μL of a 100 mL aqueous citric acid solution is dropped to make the second mixed solution acidic, and they are mixed to prepare a third mixed solution. 500 μL of a 0.2 mM aqueous sodium hydroxide solution is added to the third mixed solution to prepare a fourth mixed solution. The color development of the fourth mixed solution is confirmed. The absorption spectrum of the fourth mixed solution is measured, and it is confirmed that the absorption spectrum of the fourth mixed solution has an absorption peak at 463 nm. Since the absorption peak at 463 nm coincides with the characteristic absorption peak of CBD, it is determined that the object to be inspected is CBD. Here, the object to be inspected may be quantified using the intensity of the absorption peak.

[0095] Note that it is also possible to visually determine whether the object to be inspected is a cannabinoid without measuring the absorption spectrum of the fourth mixed solution.

[0096] When visually determining whether the object to be inspected is a cannabinoid, it can be determined by comparing the first color tone of the third mixed solution containing the object to be inspected with the color tone of the fourth mixed solution. For example, when CBD is extracted into the extract, the color tone of the third mixed solution, which was yellow, changes to an orange color tone in the fourth mixed solution. Similarly, when CBN is extracted into the extract, the color tone of the third mixed solution, which was yellow, changes to a magenta color tone in the fourth mixed solution. Also, when THC is extracted into the extract, the color tone of the third mixed solution, which was yellow, changes to a vermilion color tone in the fourth mixed solution.

[0097] <Inspection method 2> Figure 5 is a flowchart showing inspection method 2 in the second embodiment.

[0098] First, several pieces of the sample that is considered to contain the object to be inspected are placed in an extraction solution, shaken for about 1 minute, and the object to be inspected is extracted (step S21). An aqueous alcohol solution containing an aromatic amine compound is dropped into the extraction solution from which the object to be inspected has been extracted, and the extraction solution and the solution containing the aromatic amine compound are mixed to prepare a first mixed solution (step S22). An aqueous solution containing a first acidic reagent is added so that the first mixed solution becomes acidic, and the first mixed solution and the aqueous solution containing the first acidic reagent are mixed to prepare a second mixed solution (step S23). An aqueous solution containing a first reagent is put into the second mixed solution, and the second mixed solution and the aqueous solution containing the first reagent are mixed to prepare a third mixed solution (step S24). The color tone of the second mixed solution is compared with the color tone of the third mixed solution, and the change in the color tone is confirmed (step S25). The change in the color tone is to measure the absorption spectrum of the third mixed solution, collate it with the wavelengths of the characteristic absorption peaks of each cannabinoid, and determine whether the object to be inspected is a cannabinoid that is a component of cannabis (step S26).

[0099] Inspection method 2 is, for example, to drop 500 μL of a 50% aqueous ethanol solution containing a precursor of Fast Blue BB into 100 μL of an ethanol solution from which the object to be inspected has been extracted, mix them, and prepare a first mixed solution. 500 μL of a 100 mM aqueous citric acid solution is put into the first mixed solution, and they are mixed to prepare a second mixed solution. 500 μL of a 20 mM aqueous sodium nitrite solution is put into the second mixed solution, and they are mixed to prepare a third mixed solution. The color development of the third mixed solution is confirmed. The absorption spectrum of the third mixed solution is measured, and it is confirmed that the absorption spectrum of the third mixed solution has an absorption peak near 460 nm. The absorption peak near 460 nm coincides with the characteristic absorption peak of CBD, and it is determined that the object to be inspected is CBD. Here, the quantification of the object to be inspected may be performed using the intensity of the absorption peak.

[0100] As described above, in the inspection method according to an embodiment of the present invention, an aromatic amine compound is mixed with an extract containing an object to be inspected, and a diazonium coupling reaction of the aromatic amine compound is carried out in a mixed solution containing the object to be inspected to generate a coloring reagent. Further, a coloring reaction between the coloring reagent and the object to be inspected can be carried out. Thereby, an aromatic amine compound that is stable to light and heat can be used without using a diazonium salt that is unstable to light and heat, and an inspection method with high practicality can be provided.

Example

[0101] Specific examples of the inspection kit according to the present invention described above are shown and described in more detail.

[0102] In Examples 1 to 8 and Reference Example 1, the inspection kit 30 was used. For the inspection kit 30, filter paper was used as the base material 100, 1 μL of a 500 mM aqueous sodium hydroxide solution was dropped onto the first region 104, and a mixed solution obtained by mixing a 40 mM acetonitrile solution of Azoic Diazo Componet 20 (Fast Blue BB precursor) and a 400 mM aqueous citric acid solution at a ratio of 1:1 was dropped onto the second region 106 at 1 μL, 1 μL of a 300 mM aqueous sodium nitrite solution was dropped onto the third region 108, and the inspection kit 30 was prepared by drying the base material 100 for 5 minutes or more. After preparing the inspection kit 30, it was stored for at least 1 day before being used for inspection.

[0103] <Reference Example 1> As Reference Example 1, 1 mL of 75% acetonitrile not containing the object to be inspected was dropped onto the first region 104 of the inspection kit 30, and 40 μL of 50% acetonitrile as a developing solvent was dropped onto the fourth region 110. Seven to eight minutes after dropping the developing solvent, the color tone of the first region 104 was confirmed. The results of Reference Example 1 are shown in FIG. 6. The color tone of the first region 104 was the same pale yellow as the color tone of the second region 106 before dropping the developing solvent.

[0104] <Examples 1 to 5> Cannabis sativa L. was used as the sample. Using 1 mL of 75% acetonitrile, the analyte was extracted from 20 mg of Cannabis sativa L., and the resulting extract was dropped onto the first region 104. 40 μL of 50% acetonitrile was dropped onto the fourth region 110. Seven to eight minutes after dropping 40 μL of 50% acetonitrile, the color tone of the first region 104 was compared with the color tone of the first region 104 in Reference Example 1. The test results of Examples 1 to 5 obtained from the above operations are shown in FIG. 6. As shown in FIG. 6, the color tone of the first region 104 changed to a unique purple color of THCA, which was different from the color tone of the first region 104 before dropping the developing solvent and the color tone of the second region 106. From this change and color tone of the color tone, it was possible to determine that the analyte was THCA contained in Cannabis sativa L.

[0105] <Example 6> As the cannabinoids, CBD was used. 75% acetonitrile in which CBD was dissolved to a concentration of 1 mg / mL was dropped onto the first region 104. As shown in FIG. 6, the color tone of the first region 104 changed to a unique orange color of CBD, which was different from the color tone of the first region 104 before dropping the developing solvent and the color tone of the second region 106. Also, it was different from the color tone of the first region 104 in Reference Example 1. From this change and color tone of the color tone, it was found that CBD could be determined by the test kit of this embodiment.

[0106] <Example 7> As the analyte, CBN was used. 75% acetonitrile in which CBN was dissolved to a concentration of 1 mg / mL was dropped onto the first region 104. In Example 7, the same operations as in Examples 1 to 5 were performed except that CBN was used as described above. The test results of Example 6 are shown in FIG. 6. As shown in FIG. 6, the color tone of the first region 104 changed to a unique magenta color of CBN, which was different from the color tone of the first region 104 before dropping the developing solvent and the color tone of the second region 106. Also, it was different from the color tone of the first region 104 in Reference Example 1. From this change and color tone of the color tone, it was found that CBN contained in Cannabis sativa L. could be determined by the test kit of this embodiment.

[0107] <Example 8> THC was used as the object to be inspected. 75% acetonitrile in which THC was dissolved to a concentration of 1 mg / mL was dropped onto the first region 104. In Example 8, the same operations as in Examples 1 to 5 were performed except that THC was used as described above. The inspection results of Example 6 are shown in FIG. 6. As shown in FIG. 6, the color tone of the first region 104 changed to a vermilion color peculiar to THC, different from the color tone of the first region 104 before the developing solvent was dropped and the color tone of the second region 106. Also, it was different from the color tone of the first region 104 in Reference Example 1. From this change and color tone, it was found that THC contained in marijuana can be determined by the inspection kit of this embodiment.

[0108] <Example 9> THCA was used as the object to be inspected. 75% acetonitrile in which THCA was dissolved to a concentration of 1 mg / mL was dropped onto the first region 104. In Example 9, the same operations as in Examples 1 to 5 were performed except that THCA was used as described above. The inspection results of Example 6 are shown in FIG. 6. As shown in FIG. 6, the color tone of the first region 104 changed to a purple color peculiar to THCA, different from the color tone of the first region 104 before the developing solvent was dropped and the color tone of the second region 106. Also, it was different from the color tone of the first region 104 in Reference Example 1. From this change and color tone, it was found that THCA contained in marijuana can be determined by the inspection kit of this embodiment.

[0109] The color tones of each cannabinoid in Examples 6 to 9 are shown in Table 1.

[0110]

Table 1

[0111] In Comparative Examples 1 to 4 and Reference Example 2, a diazonium salt was used as the coloring reagent supported on the second region 106.

[0112] <Reference Example 2> As Reference Example 2, 1 μL of a 500 mM aqueous sodium hydroxide solution was dropped onto the first region 104 of the test kit 30, 1 μL of a 20 mM acetonitrile solution of FastBlue BB salt was dropped onto the second region 106, and no reagent, solvent, etc. was dropped onto the third region 108. 40 μL of 50% acetonitrile was dropped onto the fourth region 110. Seven to eight minutes after the dropping of the developing solvent, the color tone of the first region 104 was confirmed. The test results of Reference Example 2 are shown in FIG. 7. The color tone of the first region 104 did not change before and after the dropping of the developing solvent.

[0113] <Comparative Examples 1 to 4> In Comparative Examples 1 to 4, FastBlue BB salt was used instead of the aromatic amine compound in the color-forming reagent. Specifically, 1 μL of a 500 mM aqueous sodium hydroxide solution was dropped onto the first region 104 of the test kit 30, 1 μL of a 20 mM acetonitrile aqueous solution of FastBlue BB salt was dropped onto the second region 106, and no reagent, solvent, etc. was dropped onto the third region 108. As the acetonitrile aqueous solution, a 50% acetonitrile aqueous solution was used. Similar to the test kit 30 used in the Examples, after preparing the test kit 30, it was stored for one day or more and used for the test.

[0114] As the test objects, CBD was used in Comparative Example 1, CBN was used in Comparative Example 2, THC was used in Comparative Example 3, and THCA was used in Comparative Example 4. 75% acetonitrile in which CBD, CBN, THC, and THCA were each dissolved to a concentration of 1 mg / mL was dropped onto the first region 104 of each test kit 30. 40 μL of 50% acetonitrile was dropped onto the fourth region 110. Seven to eight minutes after the dropping of 40 μL of 50% acetonitrile, the color tone of the first region 104 was compared with the color tone of the first region 104 of Reference Example 1. The test results of Comparative Examples 1 to 4 are shown in FIG. 7. The color tone of the first region 104 was the same as the color tone of the first region 104 of Reference Example 2 and did not change before and after the dropping of the developing solvent.

[0115] From the test results of Examples 1 to 9 and Comparative Examples 1 to 4, by using an aromatic amine compound (a precursor of a diazonium salt) as the color-developing reagent of the test kit, color development derived from cannabinoids was confirmed, and the cannabinoids contained in the sample were identified.

[0116] In Examples 10 to 13 and Reference Example 3, a test kit using a solution on a substrate was used. A 50% ethanol aqueous solution containing CBD was placed in the first vial A, a 0.2 mM sodium hydroxide aqueous solution was placed in the first vial B, a 50% acetonitrile aqueous solution of 5 mM Azoic Diazo Componet 20 (Fast Blue BB precursor) was placed in the second vial A, a 100 mM citric acid aqueous solution was placed in the second vial B, and a 1 mM sodium nitrite aqueous solution was placed in the third vial, thereby preparing a test kit using a solution.

[0117] <Reference Example 3> As Reference Example 3, 0.2 mL of a 50% acetonitrile aqueous solution of 5 mM Azoic Diazo Componet 20 in the second vial A was added to 2.5 mL of a 50% ethanol aqueous solution not containing CBD in the first vial A and mixed. Next, 0.1 mL of a 100 mM citric acid aqueous solution in the second vial B was dropped into the first vial A and mixed. Further, 0.1 mL of a 1 mM sodium nitrite aqueous solution in the third vial was dropped into the first vial A and mixed. Finally, 0.1 mL of a 0.2 mM sodium hydroxide aqueous solution in the first vial B was dropped into the first vial A, and the color development of the mixed solution contained in the first vial A was confirmed. The results of Reference Example 3 are shown in FIG. 8. Using a spectrophotometer (UV-1280, manufactured by Shimadzu Corporation), the absorption spectrum of the mixed solution obtained in the first vial A of Reference Example 3 was measured. The absorption spectrum of Reference Example 3 is shown in FIG. 9. As shown in FIG. 8, color development in Reference Example 3 was not confirmed. As shown in FIG. 9, an absorption peak appearing near 460 nm characteristic of CBD was not confirmed in Reference Example 3.

[0118] <Examples 10 to 13> In Examples 10 to 13, 50% ethanol aqueous solutions with CBD concentrations of 5 μg / mL, 10 μg / mL, 15 μg / mL, and 20 μg / mL were respectively included in the first vial A. In Examples 10 to 13, the same operations as in Reference Example 3 were performed except that CBD was included in the first vial A. The test results of Examples 10 to 13 are shown in FIG. 8. Using a spectrophotometer (UV-1280, manufactured by Shimadzu Corporation), the absorption spectra of the mixed solutions obtained in the first vial A of Examples 10 to 13 were measured. The absorption spectra of Examples 10 to 13 are shown in FIG. 9.

[0119] As shown in FIG. 8, the higher the concentration of CBD in the mixed solution obtained in the first vial A, the greater the intensity of the color development. Also, as shown in FIG. 9, the higher the concentration of CBD in the mixed solution obtained in the first vial A, the higher the intensity of the absorption peak that appears around 460 nm, which is characteristic of CBD.

[0120] Quantification of CBD was performed based on the intensity of the absorption peak that appears around 460 nm, which is characteristic of CBD as shown in FIG. 9. The results of the quantification of CBD are shown in FIG. 10. As shown in FIG. 10, since the absorption peak that appears around 460 nm, which is characteristic of CBD, is proportional to the concentration of CBD in the mixed solution obtained in the first vial A, a calibration curve for CBD can be created. From this, by using the test kit using the solution in the present invention, it is possible to calculate the concentration of cannabinoids.

[0121] <Reference Example 4> As Reference Example 4, 1 mL of 75% acetonitrile containing no test object was dropped into the first region 104 of the test kit 30, and 40 μL of 50% acetonitrile as a developing solvent was dropped into the fourth region 110. Seven to eight minutes after the dropping of the developing solvent, the color tone of the first region 104 was confirmed. The results of Reference Example 4 are shown in FIG. 11. The color tone of the first region 104 was the same light yellow as the color tone of the second region 106 before the dropping of the developing solvent.

[0122] <Example 14> As the object to be inspected, CBD was used. 75% acetonitrile in which CBD was dissolved to a concentration of 1 μg / mL was dropped into the first region 104. In Example 14, the same operations as in Examples 1 to 5 were performed, except that 75% acetonitrile in which CBD was dissolved to a concentration of 1 mg / mL was used for dropping into the first region 104. The inspection results of Example 14 are shown in FIG. 11.

[0123] <Image analysis> Image analysis was performed using the inspection results of Reference Example 4 and Example 14.

[0124] First, the fourth region 110 of Reference Example 4 was loaded into ImageJ, and the L * a * b * value at that location was read. Next, the image of the colored region of the fourth region 110 of Example 14 was loaded into ImageJ, and the L * a * b * value at that location was read. Finally, the cannabinoids were identified based on the θ value calculated by Equation (1). From the image of the colored region of the fourth region 110 of Example 14, the θ value was 1.4, and it could be identified as CBD. Also, a quantitative evaluation of the cannabinoids was performed based on the ΔE value calculated by Equation (4). From the image of the colored region of the fourth region 110 of Example 14, the ΔE value was 30, and the concentration of CBD was 1 mg / mL.

[0125] <Reference Example 5> In Reference Example 5, CBD, CBN, THC, and THCA were used, and the Δa * / Δb * value specific to each cannabinoid obtained by Equation (1) and the ΔE value of each cannabinoid obtained by Equation (4) were determined.

[0126] For each cannabinoid, the same operations as in Reference Example 4 and Example 14 were performed, except that 75% acetonitrile dissolved to have a concentration of 0 μg / mL (without cannabinoids), 10 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, and 1000 μg / mL was used. Δa * / Δb * values for each cannabinoid are shown in Fig. 12. In Fig. 12, the horizontal axis represents Δa * , and the vertical axis represents Δb * . Next, the ΔE values for each cannabinoid are shown in Fig. 13. In Fig. 13, the horizontal axis represents the concentration of each cannabinoid, and the vertical axis represents the ΔE value for each cannabinoid. By drawing an approximate curve of the ΔE values for each cannabinoid, a calibration curve for each cannabinoid was obtained. The obtained calibration curves for each cannabinoid are shown in Fig. 13.

[0127] In Examples 15 to 19 and Reference Examples 6 to 10, the test kit 30 was used. For the test kit 30, filter paper was used as the substrate 100, 1 μL of a 500 mM sodium hydroxide aqueous solution was dropped onto the first region 104, 1 μL of a poly(4-styrenesulfonic acid) solution adjusted to be a 25 vol% aqueous solution and 1 μL of a 20 mM acetonitrile solution of a Fast Blue BB precursor were dropped onto the second region 106, 1 μL of a 300 mM sodium nitrite aqueous solution was dropped onto the third region 108, and the substrate 100 was dried for 5 minutes or more to prepare the test kit 30. After preparing the test kit 30, it was stored for at least 1 day before use in the test.

[0128] <Reference Example 6> As Reference Example 6, 1 mL of 75% acetonitrile containing no test object was dropped into the first region 104 of the test kit 30, and 40 μL of 50% acetonitrile as a developing solvent was dropped into the fourth region 110. Seven to eight minutes after the dropping of the developing solvent, the color tone of the first region 104 was confirmed. The results of Reference Example 6 are shown in the Blank of Example 15 shown in FIG. 14. FIG. 14 shows the first region 104 of the test kit 30. The color tone of the first region 104 was the same light purple as the color tone of the second region 106 before the dropping of the developing solvent, and showed no change before and after the dropping of the developing solvent.

[0129] <Example 15> As a test object, CBD was used. 1 μL of 75% acetonitrile adjusted to have concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL of CBD respectively was dropped into the first region 104. In Example 15, the same operations as in Reference Example 6 were performed except that 1 mL of 75% acetonitrile containing the test object was dropped into the first region 104 of the test kit 30 as described above. The test results of Example 15 are shown in FIG. 14. In FIG. 14, the color tone of the first region 104 in the examples with concentrations of 500 μg / mL and 1000 μg / mL was different from the color tone of the first region 104 in Reference Example 6 and changed to the orange color specific to CBD.

[0130] <Reference Example 7> As Reference Example 7, the same operations as in Reference Example 6 were performed. The test results of Reference Example 7 are shown in the Blank of Example 16 shown in FIG. 14. The color tone of the first region 104 showed no change before and after the dropping of the developing solvent as in Reference Example 6.

[0131] <Example 16> As the object to be inspected, CBDA was used. 1 μL of 75% acetonitrile adjusted to have concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL of CBDA respectively was dropped into the first region 104. In Example 16, the same operations as in Example 15 were performed except that CBDA was used as described above. The inspection results of Example 16 are shown in FIG. 14. In FIG. 14, the color tone of the first region 104 in the examples with concentrations of 100 μg / mL, 500 μg / mL, and 1000 μg / mL changed to the characteristic orange color of CBDA, different from the color tone of the first region 104 in Reference Example 7.

[0132] <Reference Example 8> As Reference Example 8, the same operations as in Reference Example 6 were performed. The inspection results of Reference Example 8 are shown in the Blank of Example 17 shown in FIG. 14. The color tone of the first region 104 did not change before and after dropping the developing solvent as in Reference Example 6.

[0133] <Example 17> As the object to be inspected, CBN was used. 1 μL of 75% acetonitrile adjusted to have concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL of CBN respectively was dropped into the first region 104. In Example 17, the same operations as in Example 16 were performed except that CBN was used as described above. The inspection results of Example 17 are shown in FIG. 14. In FIG. 14, the color tone of the first region 104 in the examples with concentrations of 100 μg / mL, 500 μg / mL, and 1000 μg / mL changed to the characteristic magenta color of CBN, different from the color tone of the first region 104 in Reference Example 8.

[0134] <Reference Example 9> As Reference Example 9, the same operations as in Reference Example 6 were performed. The inspection results of Reference Example 9 are shown in the Blank of Example 18 shown in FIG. 14. The color tone of the first region 104 did not change before and after dropping the developing solvent as in Reference Example 6.

[0135] <Example 18> THC was used as the object to be inspected. 1 μL of 75% acetonitrile adjusted to have concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL of THC respectively was dropped into the first region 104. In Example 18, the same operations as in Example 16 were performed except that THC was used as described above. The inspection results of Example 18 are shown in FIG. 14. In FIG. 14, the color tone of the first region 104 in the examples with concentrations of 500 μg / mL and 1000 μg / mL changed to the vermilion color characteristic of THC, different from the color tone of the first region 104 in Reference Example 9.

[0136] <Reference Example 10> As Reference Example 10, the same operations as in Reference Example 6 were performed. The inspection results of Reference Example 10 are shown in the Blank of Example 19 shown in FIG. 14. The color tone of the first region 104 did not change before and after dropping the developing solvent, similar to Reference Example 6.

[0137] <Example 19> THCA was used as the object to be inspected. 1 μL of 75% acetonitrile adjusted to have concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL of THCA respectively was dropped into the first region 104. In Example 19, the same operations as in Example 16 were performed except that THC was used as described above. The inspection results of Example 19 are shown in FIG. 14. In FIG. 14, the color tone of the first region 104 in the examples with concentrations of 500 μg / mL and 1000 μg / mL changed to the purple color characteristic of THCA, different from the color tone of the first region 104 in Reference Example 10.

[0138] In Examples 20 to 24 and Reference Examples 11 to 15, the same test kit 30 as in Examples 15 to 19 and Reference Examples 6 to 10 was used, except that 1 μL of a polyacrylic acid solution adjusted to be a 25 vol% aqueous solution of a 0.1 g / mL aqueous polyacrylic acid solution and 1 μL of a 20 mM acetonitrile solution of a Fast Blue BB precursor were dropped into the second region 106.

[0139] <Reference Example 11> As Reference Example 11, 1 mL of 75% acetonitrile not containing the test object was dropped into the first region 104 of the test kit 30, and 40 μL of 50% acetonitrile, which is the developing solvent, was dropped into the fourth region 110. Seven to eight minutes after dropping the developing solvent, the color tone of the first region 104 was confirmed. The test result of Reference Example 11 is shown in the Blank of Example 20 shown in FIG. 15. FIG. 15 shows the first region 104 of the test kit 30. The color tone of the first region 104 was the same light yellow as the color tone of the second region 106 before dropping the developing solvent, and showed no change before and after dropping the developing solvent.

[0140] <Example 20> As the test object, CBD was used. 1 μL of 75% acetonitrile adjusted to have concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL of CBD respectively was dropped into the first region 104. In Example 20, the same operations as in Reference Example 11 were performed except that 1 mL of 75% acetonitrile containing the test object was dropped into the first region 104 of the test kit 30 as described above. The test result of Example 20 is shown in FIG. 15. In FIG. 15, the color tones of the first region 104 in the examples with concentrations of 500 μg / mL and 1000 μg / mL were different from the color tone of the first region 104 in Reference Example 11 and changed to the orange color characteristic of CBD.

[0141] <Reference Example 12> As Reference Example 12, the same operations as in Reference Example 11 were performed. The test result of Reference Example 12 is shown in the Blank of Example 21 shown in FIG. 15. The color tone of the first region 104 was the same light yellow as the color tone of the second region 106 before dropping the developing solvent, and showed no change before and after dropping the developing solvent.

[0142] <Example 21> As the object to be inspected, CBDA was used. 1 μL of 75% acetonitrile adjusted to have concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL of CBDA respectively was dropped into the first region 104. In Example 21, the same operations as in Example 20 were performed except that CBDA was used as described above. The inspection results of Example 21 are shown in Fig. 15. In Fig. 15, the color tones of the first region 104 in the examples with concentrations of 100 μg / mL, 500 μg / mL, and 1000 μg / mL changed to the characteristic orange color of CBDA, different from the color tone of the first region 104 in Reference Example 12.

[0143] <Reference Example 13> As Reference Example 13, the same operations as in Reference Example 11 were performed. The inspection results of Reference Example 13 are shown in the Blank of Example 22 shown in Fig. 15. The color tone of the first region 104 was the same light yellow as the color tone of the second region 106 before dropping the developing solvent and showed no change before and after dropping the developing solvent.

[0144] <Example 22> As the object to be inspected, CBN was used. 1 μL of 75% acetonitrile adjusted to have concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL of CBN respectively was dropped into the first region 104. In Example 17, the same operations as in Example 20 were performed except that CBN was used as described above. The inspection results of Example 22 are shown in Fig. 15. In Fig. 15, the color tones of the first region 104 in the examples with concentrations of 100 μg / mL, 500 μg / mL, and 1000 μg / mL changed to the characteristic magenta color of CBN, different from the color tone of the first region 104 in Reference Example 12.

[0145] <Reference Example 14> As Reference Example 14, the same operations as in Reference Example 11 were performed. The inspection results of Reference Example 11 are shown in the Blank of Example 23 shown in Fig. 15. The color tone of the first region 104 was the same light yellow as the color tone of the second region 106 before dropping the developing solvent and showed no change before and after dropping the developing solvent.

[0146] <Example 23> As the object to be inspected, THC was used. 1 μL of 75% acetonitrile adjusted to have concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL of THC respectively was dropped into the first region 104. In Example 18, the same operations as in Example 20 were performed except that THC was used as described above. The inspection results of Example 23 are shown in FIG. 15. In FIG. 15, the color tone of the first region 104 in the examples with concentrations of 500 μg / mL and 1000 μg / mL changed to the vermilion color peculiar to THC, different from the color tone of the first region 104 in Reference Example 14.

[0147] <Reference Example 15> As Reference Example 15, the same operations as in Reference Example 11 were performed. The inspection results of Reference Example 15 are shown in the Blank of Example 24 shown in FIG. 15. The color tone of the first region 104 was the same light yellow as the color tone of the second region 106 before dropping the developing solvent and did not show a change before and after dropping the developing solvent.

[0148] <Example 24> As the object to be inspected, THCA was used. 1 μL of 75% acetonitrile adjusted to have concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL of THCA respectively was dropped into the first region 104. In Example 24, the same operations as in Example 20 were performed except that THCA was used as described above. The inspection results of Example 24 are shown in FIG. 15. In FIG. 15, the color tone of the first region 104 in the examples with concentrations of 500 μg / mL and 1000 μg / mL changed to the purple color peculiar to THCA, different from the color tone of the first region 104 in Reference Example 15.

[0149] In Examples 25 to 29 and Reference Examples 16 to 20, except that 1 μL of 0.1 g / mL polyethyleneimine (average molecular weight Mn 600, Mw 1800) was dropped into the first region 104, and 1 μL of a 20 mM acetonitrile solution of Fast Blue BB precursor and 1 μL of a 200 mM aqueous solution of citric acid were dropped into the second region 106, the same test kit 30 as in Examples 15 to 19 and Reference Examples 6 to 10 was used.

[0150] <Reference Example 16> As Reference Example 16, 1 mL of 75% acetonitrile not containing the test object was dropped into the first region 104 of the test kit 30, and 40 μL of 50% acetonitrile as the developing solvent was dropped into the fourth region 110. Seven to eight minutes after the dropping of the developing solvent, the color tone of the first region 104 was confirmed. The test result of Reference Example 16 is shown as the Blank of Example 25 shown in FIG. 16. FIG. 16 shows the first region 104 of the test kit 30. The color tone of the first region 104 did not change before and after the dropping of the developing solvent.

[0151] <Example 25> As the test object, CBD was used. 1 μL of 75% acetonitrile adjusted to have concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL of CBD respectively was dropped into the first region 104. In Example 25, the same operation as in Reference Example 16 was performed except that 1 mL of 75% acetonitrile containing the test object was dropped into the first region 104 of the test kit 30 as described above. The test result of Example 25 is shown in FIG. 16. In FIG. 16, the color tone of the first region 104 in the examples with concentrations of 500 μg / mL and 1000 μg / mL changed to the orange color specific to CBD, different from the color tone of the first region 104 in Reference Example 16.

[0152] <Reference Example 17> As Reference Example 17, the same operation as in Reference Example 16 was performed. The test result of Reference Example 16 is shown as the Blank of Example 26 shown in FIG. 16. The color tone of the first region 104 did not change before and after the dropping of the developing solvent.

[0153] <Example 26> As the object to be inspected, CBDA was used. 1 μL of 75% acetonitrile adjusted to have concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL of CBDA respectively was dropped into the first region 104. In Example 26, the same operations as in Example 25 were performed except that CBDA was used as described above. The inspection results of Example 26 are shown in Fig. 16. In Fig. 16, the color tones of the first region 104 in the examples with concentrations of 100 μg / mL, 500 μg / mL, and 1000 μg / mL were different from the color tone of the first region 104 in Reference Example 12 and changed to the orange color peculiar to CBDA.

[0154] <Reference Example 18> As Reference Example 18, the same operations as in Reference Example 16 were performed. The inspection results of Reference Example 18 are shown in the Blank of Example 27 shown in Fig. 16. The color tone of the first region 104 did not change before and after dropping the developing solvent.

[0155] <Example 27> As the object to be inspected, CBN was used. 1 μL of 75% acetonitrile adjusted to have concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL of CBN respectively was dropped into the first region 104. In Example 17, the same operations as in Example 25 were performed except that CBN was used as described above. The inspection results of Example 27 are shown in Fig. 16. In Fig. 16, the color tones of the first region 104 in the examples with concentrations of 100 μg / mL, 500 μg / mL, and 1000 μg / mL were different from the color tone of the first region 104 in Reference Example 18 and changed to the magenta color peculiar to CBN.

[0156] <Reference Example 19> As Reference Example 19, the same operations as in Reference Example 16 were performed. The inspection results of Reference Example 19 are shown in the Blank of Example 28 shown in Fig. 16. The color tone of the first region 104 did not change before and after dropping the developing solvent.

[0157] <Example 28> As the object to be inspected, THC was used. 1 μL of 75% acetonitrile adjusted to have concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL of THC respectively was dropped into the first region 104. In Example 18, the same operations as in Example 25 were performed except that THC was used as described above. The inspection results of Example 28 are shown in FIG. 16. In FIG. 16, the color tone of the first region 104 in the examples with concentrations of 100 μg / mL, 500 μg / mL, and 1000 μg / mL changed to a vermilion color peculiar to THC, different from the color tone of the first region 104 in Reference Example 19.

[0158] <Reference Example 20> As Reference Example 20, the same operations as in Reference Example 16 were performed. The inspection results of Reference Example 20 are shown in the Blank of Example 29 shown in FIG. 16. The color tone of the first region 104 did not change before and after dropping the developing solvent.

[0159] <Example 29> As the object to be inspected, THCA was used. 1 μL of 75% acetonitrile adjusted to have concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL of THCA respectively was dropped into the first region 104. In Example 24, the same operations as in Example 20 were performed except that THC was used as described above. The inspection results of Example 29 are shown in FIG. 16. In FIG. 16, the color tone of the first region 104 did not change before and after dropping the developing solvent.

[0160] Examples 30 to 33 used the same test kit 30 as that used in Example 1.

[0161] <Examples 30 to 33> Marijuana was used as the sample. Samples of approximately 20 mg each were taken from five locations of dried marijuana, and the test objects were extracted using 1 mL of 75% acetonitrile for each, and then dropped into each of the first regions 104 of Examples 30 to 33. In Examples 30 to 33, the same operations as in Example 1 were performed except that samples of 20 mg each were taken from five locations of dried marijuana. The color tone of the first region 104 in Examples 30 to 33 changed to the characteristic purple of THCA.

[0162] <Image analysis> Using the test results of Examples 30 to 33, quantitative evaluations were performed by image analysis and HPLC (high performance liquid chromatography). For the image analysis, the same operations as the image analysis used in Reference Example 4 and Example 14 were performed. The ΔE value and calibration curve of THCA were those shown in the ΔE value and calibration curve of THCA in FIG. 13. The quantitative results by image analysis of Examples 30 to 33 are shown in Table 2 and FIG. 17. For the quantitative evaluation by HPLC, the extraction solutions used in Examples 30 to 33 were used. The quantitative results by HPLC of Examples 30 to 33 are shown in Table 2 and FIG. 17.

[0163]

Table 2

[0164] From Table 2 and FIG. 17, in Examples 30 to 33, the results of the quantitative evaluation by image analysis and the results of the quantitative evaluation by HPLC were correlated, although there were some errors.

Explanation of symbols

[0165] 10: Test kit, 100: Substrate, 102: Flow path, 104: First region, 106: Second region, 108: Third region, 110: Fourth region, 112: Coating layer, 114: Fifth region, 204: Substrate

Claims

1. A base material, and a flow path on the base material, and the flow path includes a first region containing a first pH adjusting reagent, a second region containing an aromatic amine compound and a first acidic reagent, a third region containing a first reagent capable of generating diazonium ions from the aromatic amine compound by acting on the aromatic amine compound, and a fourth region for dropping a developing solvent, a test kit for inspecting cannabinoids.

2. The second region is located between the first region and the fourth region, and the third region is located between the second region and the fourth region. The test kit according to claim 1.

3. The first region is a region for dropping an extract containing a test object. The test kit according to claim 1.

4. The base material is paper. The test kit according to claim 1.

5. The developing solvent is an aqueous solution containing a polar solvent. The test kit according to claim 1.

6. The test kit according to claim 1, further comprising an extract for extracting a test object.

7. The first reagent is a nitrite. The test kit according to claim 1.

8. A first vial containing an extract, a second vial containing an organic solvent containing an aromatic amine compound and an aqueous solution containing a first acidic reagent, and a third vial containing a first reagent capable of generating diazonium ions from the aromatic amine compound by acting on the aromatic amine compound, a test kit for inspecting cannabinoids.

9. The extract contains a first pH adjusting reagent and an organic solvent or an aqueous solution containing an organic solvent, The test kit according to claim 8.

10. The first pH adjusting reagent is a hydroxide containing an alkali metal. The test kit according to claim 1 or claim 9.

11. The first acidic reagent is a hydroxy acid. The test kit according to claim 1 or claim 7.

12. The first reagent is a nitrite. The test kit according to claim 8.

13. Extract the test object contained in the sample with an extract, mix the extract in which the test object is extracted with an organic solvent containing an aromatic amine compound to prepare a first mixture, mix the first mixture with an aqueous solution containing a first reagent capable of generating diazonium ions from the aromatic amine compound by acting on the aromatic amine compound to prepare a second mixture, manufacture. An aqueous solution containing a first acidic reagent is added to the second mixed solution to prepare an acidic third mixed solution, An aqueous solution containing a first pH adjusting reagent is added to the third mixed solution to prepare a fourth mixed solution The change in color tone between the color tone of the third mixed solution and the color tone of the fourth mixed solution is confirmed, A test method for testing cannabinoids.

14. The analyte contained in the sample is extracted with an extractant, The extractant in which the analyte is extracted and an organic solvent containing an aromatic amine compound are mixed to prepare a first mixed solution, The first mixed solution and an aqueous solution containing a first acidic reagent are mixed to prepare a second mixed solution, The second mixed solution and an aqueous solution containing a first reagent that can generate diazonium ions from the aromatic amine compound by acting on the aromatic amine compound are mixed to prepare a third mixed solution, The change in color tone between the color tone of the second mixed solution and the color tone of the third mixed solution is confirmed, A test method for testing cannabinoids.

15. The test method according to claim 13 or claim 14, wherein the analyte is cannabinoids.

16. The test method according to claim 13 or claim 14, wherein the extractant contains an alkali metal-containing hydroxide and an aqueous alcohol solution.

17. The test method according to claim 13, wherein the first pH adjusting reagent is an alkali metal-containing hydroxide.

18. The test method according to claim 13 or claim 14, wherein the first acidic reagent is a hydroxy acid.

19. The test method according to claim 13 or claim 14, wherein the first reagent is a nitrite.

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