Method for analyzing ingredients that have penetrated the skin

Abstract

The present invention aims to provide an analytical method that allows for a three-dimensional evaluation of the distribution of components that have penetrated the skin in a state closer to that of a living organism. [Solution] A method for analyzing components that have penetrated skin tissue, comprising the steps of: 1) bringing a drug into contact with skin tissue; 2) freezing the skin tissue from step 1; 3) scraping the frozen skin surface from step 2 using a gas cluster ion beam; and 4) performing TOF-SIMS measurement on the frozen skin surface scraped in step 3 to detect secondary ions of components derived from the drug and secondary ions of components derived from the skin. By performing TOF-SIMS measurement while scraping the frozen skin with a gas cluster ion beam, the ion distribution derived from the penetrated components can be analyzed, allowing for analysis of the skin permeability of components applied to the skin in a state closer to that of a living organism.

Description

Technical Field

[0001] The present invention relates to a method for analyzing components that have penetrated the skin.

Background Art

[0002] In the development of topical skin preparations, a technique for evaluating the skin permeability of components is necessary to confirm the pharmacological effects and safety of the topical preparation. The skin forms a brick-mortar-like structure of keratinocytes and intercellular lipids in the stratum corneum of the epidermis, preventing the entry of foreign substances from the external environment. Therefore, in evaluating the skin permeability of a substance, it is important to accurately measure the amount of the substance permeated and the permeation pathway in the stratum corneum having a barrier function.

[0003] Conventionally, when evaluating skin permeability, a test substance is applied to the skin of an animal, or a test substance is applied to excised skin or an artificial cultured skin model fixed in a Franz cell, and the amount of the test substance that has passed through or within the skin is measured. In addition, in order to observe the distribution of the test substance that has penetrated into the skin, a method of visualizing the penetrated component is used by using a test substance added with a radioisotope or a fluorescent label, or by using a method of recognizing the test substance by immunostaining or the like after penetration. However, these methods have problems that the test substances that can be visualized are limited and various substances cannot be evaluated, the molecular weight and structure of the test substance change due to the addition of a label and accurate permeability cannot be evaluated, and the distribution of the penetrated component changes due to the washing and drying processes in the pretreatment for visualization.

[0004] In addition, as a method of conducting a test on the human body, a method of quantifying the concentration of a compound in the stratum corneum peeled off by the tape stripping method using HPLC or a mass spectrometer is known (Patent Documents 1 and 2). However, there is a problem that it is necessary to solvent-extract or thermally decompose the compound from the stratum corneum peeled off by the tape stripping method, and quantification cannot be performed quickly. Furthermore, in these methods, although the amount of the component that has penetrated into the stratum corneum can be known, the distribution of the components in the continuous stratum corneum is unknown, so it is insufficient for evaluating the skin permeation pathway of the component.

[0005] Furthermore, methods have been devised to visualize components that have penetrated the skin using mass imaging techniques (Non-Patent Documents 1 and 2). This method has the advantage that, if specific secondary ions are released from the penetrating components, the distribution of the penetrating components can be directly confirmed without the need to label the penetrating components with isotopes or fluorescent labels. On the other hand, since mass imaging is performed under vacuum, it is not possible to measure highly volatile components, and it is necessary to use dried skin for measurement, which presents a challenge as the skin tissue used for measurement is in a different state from actual skin. In addition, mass imaging is a surface observation method, and there is a problem that it is not possible to confirm the three-dimensional distribution of components that have penetrated into the skin in an undried state similar to that of living tissue. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 9-243636 [Patent Document 2] Japanese Patent Publication No. 2013-15516 [Non-patent literature]

[0007] [Non-Patent Document 1] J.Controlled Release Vol.347(2022)78-88 [Non-Patent Document 2] Scientific Reports Vol8(2018)16683 [Overview of the project] [Problems that the invention aims to solve]

[0008] The present invention aims to provide an analytical method that allows for the three-dimensional evaluation of the distribution of components that have penetrated the skin in a state closer to that of a living organism. [Means for solving the problem]

[0009] To solve this problem, the inventors conducted extensive research and discovered that by measuring the amount and distribution of ions derived from the test substance using TOF-SIMS while the skin, which has been penetrated by the test substance, is being scraped with a gas cluster ion beam while the skin is frozen, the skin's permeability can be analyzed three-dimensionally in a state closer to that of a living organism. This led to the completion of the present invention.

[0010] In other words, the present invention encompasses the following inventions. (1) A method for analyzing components that have penetrated skin tissue, 1) The process of bringing the drug into contact with skin tissue, 2) Step 1: Freezing the skin tissue, 3) A step of scraping the frozen skin surface of step 2 using a gas cluster ion beam, 4) A step in which TOF-SIMS measurement is performed on the frozen skin surface scraped in step 3 to detect secondary ions of drug-derived components and secondary ions of skin-derived components. Analytical methods including those mentioned. (2) The method according to (1), wherein the TOF-SIMS measurement in step 4 is performed at multiple locations on the frozen skin surface scraped in step 3, and one-dimensional or two-dimensional information of the distribution of secondary ions of drug-derived components and secondary ions of skin-derived components on the frozen skin surface is obtained. (3) The method according to (2), which includes a step of repeating steps 3 and 4 two or more times to obtain two-dimensional or three-dimensional information of the distribution of drug components that have penetrated the skin and components derived from the skin. (4) The method according to (3), which includes a step of visualizing the internal distribution of components that have penetrated the skin tissue based on two-dimensional or three-dimensional information of the distribution of drug components that have penetrated the skin and components derived from the skin obtained. (5) The analytical method described in any of (1) to (4) above, wherein the skin tissue is excised skin or an artificial skin model. (6) Steps 3 and 4 are performed at a pressure of 10 ―7 ~10 ―6 Pascal, analytical method as described in (1) to (4) below, performed in an atmosphere at a temperature of -150 to -120 degrees Celsius. (7) The analytical method described in any of (1) to (4), wherein the skin-derived component in step 4 is selected from protein, water, fatty acids, and phospholipids. (8) The analytical method described in any of (1) to (4), wherein the mass number of the secondary anion of the skin-derived component in step 4 is one of the mass numbers selected from 26, 35, 42, 79, 140, 255, and 283. [Effects of the Invention]

[0011] According to the present invention, it is possible to analyze components that have penetrated skin tissue, and an analytical method can be provided that can evaluate the distribution of components that have penetrated the skin with greater accuracy. [Brief explanation of the drawing]

[0012] [Figure 1] This is a three-dimensional mass imaging image of ascorbic acid (m / z=175) that has penetrated into the skin, with the upper part of the Z-axis representing the skin surface and the lower part representing the deeper layers of the skin, as well as proteins (m / z=42), water (m / z=35.0), phosphoric acid (m / z=79), and palmitic acid (m / z=255) within the skin. [Figure 2] Figure 1 is a cross-sectional view of the skin in the horizontal plane (XY axis plane) after removing the imaging image from the top 7 to 10 times (corresponding to a depth of 11 to 16 μm from the skin surface). [Figure 3] This is a three-dimensional mass imaging image of pyridoxine hydrochloride (m / z=168) that has penetrated into the skin, with the upper part of the Z-axis representing the skin surface and the lower part representing the deeper layers of the skin, as well as proteins (m / z=42), water (m / z=35.0), phosphate (m / z=79), and palmitic acid (m / z=255) within the skin. [Figure 4] Figure 3 is a cross-sectional view of the skin in the horizontal plane (XY axis plane) after removing the imaging image from the top 1-2 times (corresponding to a depth of 3-6 μm from the skin surface). [Figure 5]This is a three-dimensional mass imaging image of ascorbic acid (m / z=175) that has penetrated into the skin, with the upper part of the Z-axis representing the skin surface and the lower part representing the deeper layers of the skin, as well as proteins (m / z=42), water (m / z=35.0), phosphoric acid (m / z=79), and palmitic acid (m / z=255) within the skin. [Figure 6] Figure 5 is a cross-sectional view of the skin in the horizontal plane (XY axis plane) after removing the imaging image from the top 7 to 10 times (corresponding to a depth of 11 to 16 μm from the skin surface). [Modes for carrying out the invention]

[0013] The present invention relates to an analytical method for analyzing components that have penetrated skin tissue, and includes the steps of: 1) bringing a drug into contact with skin tissue; 2) freezing the skin tissue from step 1; 3) scraping the frozen skin surface from step 2 using a gas cluster ion beam; and 4) performing TOF-SIMS measurement on the frozen skin surface scraped in step 3 to detect secondary ions of components derived from the drug and secondary ions of components derived from the skin.

[0014] In this invention, skin tissue can include excised skin or artificial skin models produced by artificial culture. The test subjects are typically humans. Furthermore, the skin permeability analysis method of this invention can be applied to humans or non-human animals that possess skin tissue. Specific examples of non-human animals include monkeys, dogs, cats, mice, rats, guinea pigs, and rabbits. For skin tissue, commercially available excised skin or artificial skin models for research purposes can be used.

[0015] The drug in this invention is not particularly limited, but examples include creams, lotions, gels, ointments, pastes, sprays, patches, and other substances applied to the skin. The drug contains components for evaluating its permeability. The method of contacting the skin is not particularly limited and may include direct application, spraying, patching, adhesion, or impregnation to the skin surface. The contact area, contact amount, contact time, and number of contacts can be freely selected. Skin tissue impregnated with the drug or skin tissue to which the drug has been intradermally injected may also be used.

[0016] The method for freezing skin tissue in this invention is not limited to a specific time, but in order to confirm permeability in a state closer to that of a living tissue, it is preferable to instantly freeze and fix the skin tissue at an extremely low temperature immediately after penetration. Specifically, this can be done with liquid nitrogen, dry ice, or a refrigeration device, with liquid nitrogen being preferred. Pretreatment of the sample after freezing is almost unnecessary, and the frozen skin surface is scraped using a gas cluster ion beam while the sample is still frozen. This prevents decomposition, scattering, and movement of components in the sample until the next TOF-SIMS analysis. If necessary, the skin tissue may be processed to change its shape, such as by slicing or shredding. Since frozen skin tissue can cause condensation at room temperature, it is preferably stored and processed at -79 degrees Celsius or lower, and more preferably at -196 degrees Celsius or lower. When introducing frozen skin tissue into the process of scraping the frozen skin surface using a gas cluster ion beam, the frozen skin tissue is maintained in a dry atmosphere to prevent condensation on the surface of the frozen skin tissue. Preferably, the humidity of the atmosphere is 20% or less, and more preferably 10% or less, and the tissue is introduced into the apparatus within 3 minutes.

[0017] The present invention relates to a process of ablating a frozen skin surface using a gas cluster ion beam, which involves ablating the skin surface to be measured using TOF-SIMS to a certain depth. Examples of gas cluster ion beams include Ar (argon), SF6, Cl, N, O2, CO2, etc., with Ar being preferred. The range of the skin surface ablated with the gas cluster ion beam is not particularly limited, but it is required to be larger than the range of the skin surface measured using TOF-SIMS. The depth to which the skin tissue is ablated can be appropriately selected according to the resolution to be accumulated as three-dimensional information. The ablative depth can be adjusted by changing the intensity and duration of irradiation with the gas cluster ion beam.

[0018] In the process of abrading a frozen skin surface using the gas cluster ion beam of the present invention and the process of mass spectrometry of secondary ions generated from skin tissue, the skin tissue to be measured is kept frozen under reduced pressure. As long as the skin tissue is kept frozen, the atmospheric pressure and temperature are not limited, but atmospheric pressure 10 ―8 ~10 -5 The process is preferably carried out in Pascals under an atmosphere of -150 to -50 degrees Celsius, and at a pressure of 10°C to minimize the effects of surface contamination, etc. ―7 ~10 ―6 Pascal, to suppress the sublimation of water, an atmosphere with a temperature of -150 to -120 degrees Celsius is even more preferable.

[0019] The components used to evaluate permeability can be either inorganic or organic. The concentration and number of components used to evaluate permeability are not particularly limited. It is preferable that the spectral results obtained by TOF-SIMS have a specific indicator secondary ion for the component being evaluated. By selecting a specific indicator secondary ion for the component being evaluated, the permeability of multiple components can be evaluated simultaneously.

[0020] In this invention, TOF-SIMS is a time-of-flight secondary ion mass spectrometry (TFSS). TOF-SIMS is a method that analyzes secondary ions emitted by irradiating a solid surface with primary ions using a TFS mass spectrometer, and performs structural analysis of the sample surface from the resulting spectrum. It allows observation of the distribution of molecules and atoms on the solid surface and visualization of the distribution. Because it is a TFS, it is possible to analyze a wide variety of ions simultaneously. Furthermore, both positive and negative ions can be detected, and can be appropriately selected depending on the component to be evaluated.

[0021] The measurement method of TOF-SIMS is not particularly limited and can be appropriately adjusted to enable optimal analysis. The primary ions to be used include bismuth, cesium, gold, gallium, manganese, argon, and oxygen, and preferably bismuth. The acceleration voltage, beam diameter, and pulse width of the primary ions can be appropriately adjusted according to the purpose of analysis, and preferably the acceleration voltage is 15 kV to 25 kV, the beam diameter is 100 nm to 1 μm, and the pulse width is 1 ns to 20 ns.

[0022] In the step of mass-analyzing the secondary ions generated from the frozen skin tissue of the present invention, it is possible to measure the intensity of the components of the drug penetrated into the skin and the secondary ions derived from the skin components contained in the skin tissue. From the intensities of the secondary ions of the components derived from the drug and the secondary ions of the components derived from the skin measured by TOF-SIMS at each point within a certain area on the skin surface, the distribution and intensity of the components of the drug penetrated into the skin and the skin components contained in the skin tissue can be obtained as one-dimensional or two-dimensional information.

[0023] The components derived from the skin for TOF-SIMS measurement are not particularly limited, and examples include proteins, water, lipids, inorganic salts, amino acids, saccharides, nucleic acids, etc., and preferably proteins, water, fatty acids, and phospholipids.

[0024] The mass number: m / z when detecting the secondary ions of the components derived from the skin as anions is not particularly limited, but 17(OH), 26(CN), 35(H3O2), 42(CNO), 79(PO3), 140(C2H7NPO4), 180(C5H 11 NPO4), 184(C5H 15 NPO4), 206(C5H 14 NPO4Na), 225(C 14 H 25 O2), 227(C 14 H 27 O2), 251(C 16 H 27 O2), 253(C 16 H 29 O2), 255(C 16 H 31 O2), 279(C18 H 31 O2), 281(C 18 H 33 O2), 283(C 18 H 35 O2), 311(C 20 H 39 O2), 339(C 22 H 43 O2), 353(C 23 H 45 O2), 367(C 24 H 47 O2), 383(C 27 H 43 O), 385(C 27 H 45 O), 395(C 26 H 51 O2) and 465(C 27 H 45 SO4 is one example, and preferably 26, 35, 42, 79, 140, 255 and 283.

[0025] When detecting secondary ions of skin-derived components as cations, the mass number (m / z) is not particularly limited, but is typically 19 (H3O), 30 (CH4N), 37 (H5O2), 43 (C3H7), 44 (C2H6N), 57 (C4H9), 70 (C4H8N), or 86 (C5H 12 N), 184(C5H 15 NPO4), 206(C5H) 14 NPO4Na), 224(C8H 19 NPO4), 313(C 19 H 37 O3), 337(C 21 H 37 O3), 339(C 21 H 39 O3), 341(C 21 H 41 O3), 367(C 27 H 43 ), 369(C 27 H 45 ), 384(C 27 H 44 O), 385(C 27 H 45 O), 414(C 26 H 56NO2), 549(C 35 H 65 O4), 551(C 35 H 67 O4), 577(C 37 H 69 O4) and 579(C 37 H 71 Examples include O4), preferably 37, 44, 70, 369, 414 and 577.

[0026] The m / z value used as the index can be set using the precise mass number of the secondary ion derived from the target component, and the index can also be a mass number that falls within any range centered on that precise mass number. The range of mass numbers used as the index can be adjusted to distinguish it from ions of other components that are close to the target index ion's mass number. The number of digits can also be set arbitrarily. For example, if an integer value is used for the index m / z, the secondary ions whose mass numbers result in the corresponding integer value when the measured value is rounded to the nearest integer can be summed and used. Also, if an m / z value with four decimal places is used as the index, the secondary ions whose mass numbers result in the corresponding index value when the fifth decimal place is rounded to the nearest integer can be summed and used.

[0027] By repeatedly performing the steps of scraping the frozen skin surface using the gas cluster ion beam of the present invention and detecting secondary ions of drug-derived components and skin-derived components using TOF-SIMS, it is possible to accumulate one-dimensional or two-dimensional distribution and intensity information of secondary ions of drug-derived components and skin-derived components at the depth of the skin in each step. Based on the acquired information, it becomes possible to visualize the three-dimensional distribution of components that have penetrated the skin tissue within the skin.

[0028] The analytical method of the present invention includes a step of visualizing the distribution of ions generated from drug components and skin-derived components in a planar or three-dimensional manner. The ions used for visualization can be selected from ions generated from drug components and skin-derived components. Preferably, specific indicator ions for drug components and skin-derived components are selected from the spectral results obtained by TOF-SIMS. The mass number, charge, and number of indicator ions are not particularly limited. The size of the field of view to be measured is not particularly limited, but 50 μm square to 1 mm square is preferred, and 100 μm square to 400 μm square is more preferred. The number of pixels in the image can be freely set, but 128 pixels square to 1024 pixels square is preferred. The measurement time can be in the range of several minutes to several hours and can be adjusted as appropriate while checking the detection status of each indicator ion.

[0029] Visualization is performed by printing markers at the locations where indicator ions of drug components and skin-derived components are detected. The size, type, color, and number of markers are not particularly limited and can be appropriately selected according to the detection sensitivity. Smaller markers result in higher resolution and are therefore preferable. [Examples]

[0030] The present invention will be described in detail below with reference to examples. The scope of the present invention is not limited to these examples.

[0031] Example 1: Evaluation of the permeability of ascorbic acid (Penetration and freezing process of chemicals) Human skin tissue (K.A.C. Co., Ltd.) was fixed to a Franz-type diffusion cell (PERMEGEAR), and 200 μL of an aqueous solution containing 5% ascorbic acid was applied to the epidermal side of the skin tissue. The solution was allowed to penetrate the skin for 24 hours while maintaining a temperature of 32°C. After that, the application site was washed, the skin tissue that had been penetrated was collected, and frozen in liquid nitrogen. The frozen skin tissue was then fixed in a fixture for TOF-SIMS measurement while still frozen.

[0032] (TOF-SIMS measurement) Frozen skin tissue impregnated with ascorbic acid was used as a specimen and analyzed by TOF-SIMS. The detection conditions are as follows. (1) Equipment: PHI TRIFT V nanoTOF (manufactured by ULVAC-FI) (2) Measurement conditions: High-resolution imaging mode Primary ion Bi3 ++ :30kV(current:0.5μA, pulse width 16ns) Detection: negative Measurement range: 100 × 100 μm 2 256 x 256 pixels, 2.06 minutes of cumulative processing (16 frames) Measured atmospheric pressure: 10 -7 Pascal Measurement temperature: -144℃

[0033] The indicator ion for ascorbic acid is m / z=175 (C6H7O6), the indicator ion for water is m / z=35.0 (H3O2), the indicator ion for protein (a component derived from skin tissue) is m / z=42 (CNO), the indicator ion for phosphate is m / z=79 (PO3), and the indicator ion for palmitic acid is m / z=255 (C 16 H 31 The locations detected by TOF-SIMS were mapped to O2.

[0034] (Cutting of the skin tissue surface using a gas cluster ion beam) Frozen skin tissue impregnated with ascorbic acid was used as a specimen, and the areas analyzed by TOF-SIMS were then cut using a gas cluster ion beam. In a single treatment, a depth of 1.6 μm from the surface of the skin tissue was removed. (1) Equipment: GCIB gun, model 06-2600 (manufactured by ULVAC-FI) (2) Measurement conditions Beam: 15kV Emission: 15mA Cutting area: 400 x 400 μm 2 , 8 minutes of irradiation Measured atmospheric pressure: 10 -7 Pascal Measurement temperature: -144℃ Gas cluster ion beam: Argon cluster

[0035] The process involved repeatedly performing mass spectrometry of the skin tissue surface using TOF-SIMS and cutting the skin tissue surface with gas cluster ions, for a total of 24 cycles. The obtained TOF-SIMS imaging images were accumulated and constructed as a 3D image.

[0036] The results are shown in Figures 1 and 2. Figure 1 is a three-dimensional mass imaging image of ascorbic acid (m / z=175) that has penetrated into the skin, with the upper part of the Z-axis being the skin surface and the lower part being the deeper part of the skin, along with proteins (m / z=42), water ions (m / z=35.0), phosphoric acid (m / z=79), and palmitic acid (m / z=255) inside the skin. All components can be visualized in three dimensions by freezing undried skin tissue, and the distribution and amount of components that have penetrated into the skin can be confirmed. Figure 2 is a three-dimensional mass imaging image of the layer obtained by scraping the imaging image from Figure 1 7 to 10 times from the top. The localization of ascorbic acid that has penetrated into the skin and the components inside the skin can be confirmed at various depths. It can be seen that ascorbic acid that has penetrated into the skin is localized in large quantities at the location where proteins (m / z=42) are present.

[0037] Example 2: Evaluation of the permeability of pyridoxine hydrochloride (Penetration and freezing process of chemicals) Human skin tissue (K.A.C. Co., Ltd.) was fixed to a Franz-type diffusion cell (PERMEGEAR), and 200 μL of an aqueous solution containing 20% ​​pyridoxine hydrochloride was applied to the epidermal side of the skin tissue. The solution was allowed to penetrate the skin for 3 hours while maintaining a temperature of 32 degrees Celsius. After that, the application site was washed, the skin tissue that had been penetrated was collected, and frozen in liquid nitrogen. The frozen skin tissue was fixed to a fixture for TOF-SIMS measurement, and TOF-SIMS measurement was performed.

[0038] (TOF-SIMS measurement) Frozen skin tissue impregnated with pyridoxine hydrochloride was used as a specimen and analyzed by TOF-SIMS. The detection conditions are as follows. (1) Equipment: PHI TRIFT V nanoTOF (manufactured by ULVAC-FI) (2) Measurement conditions: High-resolution imaging mode Primary ion Bi3 ++ :30kV(current:0.5μA, pulse width 16ns) Detection: negative Measurement range: 100 × 100 μm 2 256 x 256 pixels, 2.06 minutes of cumulative processing (16 frames) Measured atmospheric pressure: 10 -7 Pascal Measurement temperature: -144℃

[0039] The indicator ion for pyridoxine hydrochloride is m / z=168(C8H 10 NO3), the indicator ion for water is m / z=35.0 (H3O2), the indicator ion for protein as a component derived from skin tissue is m / z=42 (CNO), the indicator ion for phosphate is m / z=79 (PO3), and the indicator ion for palmitic acid is m / z=255 (C 16 H 31 The locations detected by TOF-SIMS were mapped to O2.

[0040] (Cutting of the skin tissue surface using a gas cluster ion beam) Frozen skin tissue impregnated with pyridoxine hydrochloride was used as a specimen, and the area analyzed by TOF-SIMS was then cut using a gas cluster ion beam. In a single treatment, a depth of 3.2 μm from the surface of the skin tissue was removed. (1) Equipment: GCIB gun, model 06-2600 (manufactured by ULVAC-FI) (2) Measurement conditions Beam: 15kV Emission: 15mA Cutting area: 400 x 400 μm 2 4 minutes of irradiation Measured atmospheric pressure: 10 -7 Pascal Measurement temperature: -144℃ Gas cluster ion beam: Argon cluster

[0041] The process involved repeatedly performing mass spectrometry of the skin tissue surface using TOF-SIMS and cutting the skin tissue surface with gas cluster ions, for a total of 36 cycles. The obtained TOF-SIMS imaging images were accumulated and constructed as a 3D image.

[0042] The results are shown in Figures 3-4. Figure 3 is a three-dimensional mass imaging image of pyridoxine hydrochloride (m / z=168) that has penetrated into the skin, with the upper part of the Z-axis being the skin surface and the lower part being the deeper part of the skin, along with proteins (m / z=42), water ions (m / z=35.0), phosphate (m / z=79), and palmitic acid (m / z=255) within the skin. All components can be visualized three-dimensionally by freezing undried skin tissue, allowing confirmation of the distribution and amount of components that have penetrated into the skin. Figure 4 is a three-dimensional mass imaging image of the layer obtained by scraping the top 1-2 times from the imaging image in Figure 3. The localization of pyridoxine hydrochloride that has penetrated into the skin and the components within the skin can be confirmed at various depths. It can be seen that pyridoxine hydrochloride that has penetrated into the skin is localized differently from the location of proteins (m / z=42).

[0043] Comparative Example 1: Evaluation of the permeability of ascorbic acid using dry skin (Penetration of the drug) Human skin tissue (K.A.C. Co., Ltd.) was fixed in a Franz-type diffusion cell (PERMEGEAR), and 200 μL of an aqueous solution containing 5% ascorbic acid was applied to the epidermal side of the skin tissue. The solution was allowed to penetrate the skin for 24 hours while maintaining a temperature of 32°C. After that, the application site of the drug was washed, the drug-penetrated skin tissue was collected, dried at 40°C for 48 hours, and then fixed to a fixture for TOF-SIMS measurement, and TOF-SIMS measurement was performed.

[0044] (TOF-SIMS measurement) Dry skin tissue infused with ascorbic acid was used as a sample and analyzed by TOF-SIMS. The detection conditions are as follows. (1) Equipment: PHI TRIFT V nanoTOF (manufactured by ULVAC-FI) (2) Measurement conditions: High-resolution imaging mode Primary ion Bi3 ++ : 30 kV (current: 0.5 μA, Pulse width 16 ns) Detection: negative Measurement range: 100×100 μm 2 , 256×256 pixel, 2.06 minutes integration (16 frames) Measurement pressure: 10 -7 Pascal Measurement temperature: 30 °C

[0045] The indicator ion of ascorbic acid is m / z = 175 (C6H7O6), the indicator ion of water is m / z = 35.0 (H3O2), the indicator ion of protein as a skin tissue-derived component is m / z = 42 (CNO), the indicator ion of phosphoric acid is m / z = 79 (PO3), and the indicator ion of palmitic acid is m / z = 255 (C 16 H 31 H2), and the locations detected by TOF-SIMS were mapped respectively.

[0046] (Cutting of the skin tissue surface by gas cluster ion beam) Using the dried skin tissue infiltrated with ascorbic acid as a specimen, the location where analysis was performed by TOF-SIMS was cut with a gas cluster ion beam. In one treatment, a depth of 1.6 μm was cut from the surface of the skin tissue. (1) Equipment: GCIB gun model 06-2600 (manufactured by ULVAC PHI, Inc.) (2) Measurement conditions Beem: 15 kV Emission: 15 mA Cutting range: 500×500 μm 2 , 2 minutes irradiation Measurement pressure: 10 -7 Pascal Measurement temperature: 30 °C Gas cluster ion beam: argon cluster

[0047] The process involved repeatedly performing mass spectrometry of the skin tissue surface using TOF-SIMS and cutting the skin tissue surface with gas cluster ions, for a total of 50 cycles. The obtained TOF-SIMS imaging images were accumulated and constructed as a 3D image.

[0048] The results are shown in Figures 5-6. Figure 1 is a three-dimensional mass imaging image of ascorbic acid (m / z=175) that penetrated into the skin, with the upper part of the Z-axis representing the skin surface and the lower part representing the deeper layers of the skin, as well as proteins (m / z=42), water ions (m / z=35.0), phosphate (m / z=79), and palmitic acid (m / z=255) within the skin. In the dry skin state, three-dimensional visualization of ascorbic acid, proteins, phosphate, and palmitic acid was possible, and the distribution and amount of components that penetrated into the skin could be confirmed, but the distribution of water could not be confirmed. Figure 6 is a three-dimensional mass imaging image of the layer obtained by scraping the imaging image from Figure 5 7-10 times from the top. Due to the deformation of the skin tissue caused by drying, the localization of ascorbic acid and skin components differed from the results for frozen skin tissue. [Industrial applicability]

[0049] According to the present invention, by measuring the amount and distribution of ions derived from the permeated components of a drug while the skin is frozen and then abraded with a gas cluster ion beam using TOF-SIMS, a method is provided that allows for three-dimensional analysis of the skin permeability of components applied to the skin in a state closer to that of a living organism.

Claims

1. A method for analyzing components that have penetrated skin tissue, 1) The process of bringing the drug into contact with skin tissue, 2) Step 1: Freezing the skin tissue, 3) A step of scraping the frozen skin surface of step 2 using a gas cluster ion beam, 4) A step in which TOF-SIMS measurement is performed on the frozen skin surface scraped in step 3 to detect secondary ions of drug-derived components and secondary ions of skin-derived components. Analytical methods including those mentioned.

2. The method according to claim 1, wherein TOF-SIMS measurement in step 4 is performed at multiple locations on the frozen skin surface scraped in step 3, and one-dimensional or two-dimensional information of the distribution of secondary ions of drug-derived components and secondary ions of skin-derived components on the frozen skin surface is obtained.

3. The method according to claim 2, further comprising the step of repeating steps 3 and 4 two or more times to obtain two-dimensional or three-dimensional information of the distribution of drug components that have penetrated the skin and components derived from the skin.

4. The method according to claim 3, further comprising the step of visualizing the internal distribution of components that have penetrated the skin tissue based on two-dimensional or three-dimensional information of the distribution of drug components that have penetrated the skin and components derived from the skin obtained.

5. The analytical method according to any one of claims 1 to 4, wherein the skin tissue is excised skin or an artificial skin model.

6. Steps 3 and 4 are performed at a pressure of 10 ―７ ~10 ―６ The analytical method according to any one of claims 1 to 4, performed in an atmosphere at a temperature of -150 to -120 degrees Celsius.

7. The analytical method according to any one of claims 1 to 4, wherein the skin-derived component in step 4 is selected from protein, water, fatty acids, and phospholipids.

8. The analytical method according to any one of claims 1 to 4, wherein the mass number of the secondary anion of the skin-derived component in step 4 is one of the mass numbers selected from 26, 35, 42, 79, 140, 255, and 283.

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