Cosmetic functional component transdermal absorption analysis method based on mass spectrum imaging and application

Abstract

The invention provides a transdermal absorption partition identification method based on mass spectrum imaging. The invention also provides a mass spectrum imaging-based cosmetic functional component transdermal absorption analysis method. The invention provides a mass spectrum imaging-based cosmetic functional component transdermal absorption analysis method and application. The invention relates to a novel method for high-precision and high-specificity positioning analysis of functional components, functional component metabolites and skin endogenous components in cosmetics in complex skin tissues and application of the novel method based on a transdermal experiment method for simulating cosmetic in-vitro transdermal on the basis of human in-vitro skin and a mass spectrum imaging technology. And a reliable technical platform is provided for research on a cosmetic transdermal absorption mechanism.

Description

Technical Field

[0001] The present invention belongs to the technical field of mass spectrometry analysis and detection, and relates to a method for analyzing the transdermal absorption of cosmetic active ingredients based on mass spectrometry imaging and its application. Background Art

[0002] Traditional methods (such as in vitro skin diffusion models, chromatography / spectroscopy techniques, Raman spectroscopy, and fluorescence labeling) have significant drawbacks in the transdermal research of cosmetics. Taking the diffusion cell method as an example, it mainly evaluates the transdermal absorption by measuring the mass flow of substances through the skin. However, this method can only provide overall absorption data and cannot accurately reflect the distribution and concentration changes of cosmetic ingredients in different skin layers, resulting in poor accuracy. In vitro skin requires mechanical separation of the epidermis and dermis layers, which destroys the tissue structure and loses metabolic activity, making it impossible to trace metabolite products or the interaction with endogenous substances; Raman spectroscopy is greatly interfered by the matrix and has low specificity; fluorescence labeling affects the authenticity of transdermal behavior due to labeling modification and signal interference. In addition, traditional methods generally lack spatial resolution, cannot obtain in-situ information on the dynamic distribution of components (such as follicle targeting), and are difficult to support multi-dimensional analysis of complex formulations. Summary of the Invention

[0003] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a method for analyzing the transdermal absorption of cosmetic active ingredients based on mass spectrometry imaging and its application, which can quickly identify metabolites specifically distributed in the skin, and based on these metabolites, quickly partition the epidermis and dermis layers of the skin, and at the same time achieve high-precision and high-specificity localization analysis and application of cosmetic active ingredients, metabolite products of active ingredients, and skin endogenous components in human in vitro skin in complex skin tissues.

[0004] To achieve the above object and other related objects, the first aspect of the present invention provides a method for identifying transdermal absorption and partitioning based on mass spectrometry imaging, including the following steps:

[0005] 1) After smearing and loading the surface of the in vitro skin sample with the test substance mother liquor for transdermal absorption, collecting the sample, embedding, freezing and thawing, fixing and sectioning, the obtained tissue section is adhered and fixed on a glass slide to obtain a sample of the section to be tested;

[0006] 2) Respectively take the sample of the section to be tested as the first sample of the section to be tested and the second sample of the section to be tested. The first sample of the section to be tested is subjected to mass spectrometry imaging analysis in positive and negative ion modes using an imaging platform tandem mass spectrometer to obtain mass spectrometry imaging data and its imaging map of the transdermal absorption components, and the second sample of the section to be tested is stained with HE and then photographed using a scanner to obtain a stained section map;

[0007] 3) Preprocess the original data of the mass spectrometry imaging data of the transdermal absorption components to obtain processed data;

[0008] 4) Register the stained section image with the imaging map of the transdermal absorption component, partition the skin tissue of the ex vivo skin sample into the epidermis layer and the dermis layer, and determine the corresponding processing data for the epidermis layer and the dermis layer;

[0009] 5) Compare the processing data of the epidermis layer region with the processing data of the dermis layer region, screen for ions specifically expressed in the epidermis layer and the dermis layer in the skin tissue, identify metabolites according to database matching, and obtain metabolites specifically expressed in the epidermis layer and metabolites specifically expressed in the dermis layer as partition identification components;

[0010] 6) Repeat step 2) for the other ex vivo skin samples to obtain the mass spectrometry imaging data of the transdermal absorption component for the test section samples obtained in step 1), and then compare the processing data obtained in step 3) with the partition identification components to determine the partition where the transdermal absorption component is located in the other ex vivo skin samples.

[0011] The second aspect of the present invention provides a method for analyzing the transdermal absorption of cosmetic efficacy components based on mass spectrometry imaging, including the following steps:

[0012] A) Adopt step 1) of the method for transdermal absorption partition identification based on mass spectrometry imaging provided in the first aspect of the present invention to obtain test section samples;

[0013] B) Directly perform transdermal absorption on the surface of the ex vivo skin sample according to step 1) of the method for transdermal absorption partition identification based on mass spectrometry imaging provided in the first aspect of the present invention, collect the sample, embed it, freeze and thaw it, section it after fixation, and adhere and fix the obtained tissue section on a glass slide to obtain blank section samples;

[0014] C) Adopt steps 2)-4) of the method for transdermal absorption partition identification based on mass spectrometry imaging provided in the first aspect of the present invention for the test section samples and the blank section samples to obtain the corresponding processing data for the test section samples and the blank section samples in the epidermis layer and the dermis layer;

[0015] D) Compare the processing data of the epidermis layer and the dermis layer, and perform database matching according to the change multiple of the processing data of the test section samples and the processing data of the blank section samples to determine the skin endogenous components, transdermal components, and / or transdermal component metabolites in the ex vivo skin sample.

[0016] As described above, the method for analyzing the transdermal absorption of cosmetic efficacy components based on mass spectrometry imaging and its application provided by the present invention have the following beneficial effects:

[0017] (1) The transdermal absorption analysis method and application of cosmetic active ingredients based on mass spectrometry imaging provided by the present invention realize a new method and application for high-precision and high-specificity localization analysis of active ingredients, metabolite of active ingredients and skin endogenous components in complex skin tissues through a transdermal experiment method simulating in vitro transdermal absorption of cosmetics on ex vivo human skin and mass spectrometry imaging technology, providing a solid and scientific basis for the screening of cosmetic active substances and the in-depth study of their action mechanisms, thereby guiding the development and optimization of new products and promoting the continuous progress of cosmetics science and technology.

[0018] (2) The transdermal absorption analysis method and application of cosmetic active ingredients based on mass spectrometry imaging provided by the present invention realize that the ex vivo skin tissue maintains physiological metabolic activity for more than 72 hours in an in vitro environment by precisely regulating the culture medium components and culture parameters, providing an ex vivo skin in vitro simulation system based on metabolic activity maintenance technology. This technology successfully overcomes the defect of metabolic inactivation of traditional ex vivo skin models and establishes an evaluation system that truly reflects the dynamic penetration law and metabolic transformation process of cosmetic active ingredients in living skin, providing a reliable technical platform for the study of cosmetic transdermal absorption mechanisms.

[0019] (3) The transdermal absorption analysis method and application of cosmetic active ingredients based on mass spectrometry imaging provided by the present invention provide a method for identifying ion groups specifically distributed in the skin and quickly and specifically separating the epidermis and dermis layers with the help of these ion groups. Compared with the traditional HE staining section registration method, this technology improves the layering efficiency by about 240 - 300 times (from an average of 5 - 8 minutes / sample to 1 - 2 seconds), and at the same time avoids tissue section loss, enabling the sample to be completely retained for subsequent analysis.

[0020] (4) The transdermal absorption analysis method and application of cosmetic active ingredients based on mass spectrometry imaging provided by the present invention provide a method for panoramic visualization of skin components. Compared with the traditional Franz diffusion cell combined with chromatography or chromatography hyphenation method, this method has the advantages of simple pretreatment operation without the need to separate the epidermis and dermis layers; efficient analysis and intuitive results, and the distribution of all transdermal components and their metabolites, as well as skin endogenous metabolites in the epidermis and dermis layers can be directly seen through only one scan, realizing the simultaneous identification of endogenous components, original cosmetic components and metabolites. Description of the Drawings

[0021] Figure 1 Shown is the HE staining diagram of the cosmetic administration group XC of the present invention.

[0022] Figure 2 Shown is the ion diagram specifically expressed in the epidermis layer of the skin of the cosmetic administration group XC of the present invention.

[0023] Figure 3 shows the HE staining of the transdermal components of the present invention and their cosmetics in the epidermal and dermal regions of skin tissue Figure 3a 、 3b 、mass spectrometry imaging Figure 3c 、 3d 、3e, 3f, 3g, 3h, 3i, 3j, 3k, 3l, 3m, 3n, wherein

[0024] Figure 3a is the HE staining image of the blank control group Blank;

[0025] Figure 3b is the HE staining image of the cosmetic administration group XC;

[0026] Figure 3c is the mass spectrometry imaging map of the transdermal component Luteolin in the blank control group Blank;

[0027] Figure 3d is the mass spectrometry imaging map of the transdermal component Luteolin in the cosmetic administration group XC;

[0028] Figure 3e is the mass spectrometry imaging map of the transdermal component Morin in the blank control group Blank;

[0029] Figure 3f is the mass spectrometry imaging map of the transdermal component Morin in the cosmetic administration group XC;

[0030] Figure 3g is the mass spectrometry imaging map of the transdermal component Isorhamnetin in the blank control group Blank;

[0031] Figure 3h is the mass spectrometry imaging map of the transdermal component Isorhamnetin in the cosmetic administration group XC;

[0032] Figure 3i is the mass spectrometry imaging map of the transdermal component Chlorogenic acid in the blank control group Blank;

[0033] Figure 3j is the mass spectrometry imaging map of the transdermal component Chlorogenic acid in the cosmetic administration group XC;

[0034] Figure 3k is the mass spectrometry imaging map of the transdermal component Luteolin 4'-sulfate in the blank control group Blank;

[0035] Figure 3lMass spectrometry imaging of the transdermal component Luteolin 4'-sulfate in the cosmetic administration group XC;

[0036] Figure 3m Mass spectrometry imaging of the transdermal component 8-Hydroxyluteolin 8-sulfate in the blank control group Blank;

[0037] Figure 3n Mass spectrometry imaging of the transdermal component 8-Hydroxyluteolin 8-sulfate in the cosmetic administration group XC.

[0038] Figure 4 shows the distribution of the transdermal components in the skin of the present invention Figure 4a 、 4b 、4c, 4d, 4e, 4f, where

[0039] Figure 4a Bar charts of the relative contents of the transdermal component Luteolin in the epidermis, dermis and whole of the skin in the Blank and XC groups;

[0040] Figure 4b Bar charts of the relative contents of the transdermal component Morin in the epidermis, dermis and whole of the skin in the Blank and XC groups;

[0041] Figure 4c Bar charts of the relative contents of the transdermal component Isorhamnetin in the epidermis, dermis and whole of the skin in the Blank and XC groups;

[0042] Figure 4d Bar charts of the relative contents of the transdermal component Chlorogenic acid in the epidermis, dermis and whole of the skin in the Blank and XC groups;

[0043] Figure 4e Bar charts of the relative contents of the transdermal component Luteolin 4'-sulfate in the epidermis, dermis and whole of the skin in the Blank and XC groups;

[0044] Figure 4f Bar charts of the relative contents of the transdermal component 8-Hydroxyluteolin 8-sulfate in the epidermis, dermis and whole of the skin in the Blank and XC groups. Detailed implementation methods

[0045] The inventors of the present application have developed a method and application for analyzing the transdermal absorption of cosmetic efficacy components based on mass spectrometry imaging, which are specifically described as follows.

[0046] In the first aspect of the present invention, a method for zonation identification of transdermal absorption based on mass spectrometry imaging is provided, including the following steps:

[0047] 1) After smearing and loading the surface of the ex vivo skin sample with the test substance mother liquor, perform transdermal absorption, collect the sample, embed it, freeze it, thaw it after freezing, fix it, and then section it. The obtained tissue section is adhered and fixed on a glass slide to obtain a sample slice to be measured;

[0048] 2) Take the sample slice to be measured as the first sample slice to be measured and the second sample slice to be measured respectively. Perform mass spectrometry imaging analysis on the first sample slice to be measured in positive and negative ion modes using an imaging platform tandem mass spectrometer to obtain the mass spectrometry imaging data and its imaging map of the transdermal absorption components. After HE staining the second sample slice to be measured, take a picture using a scanner to obtain a stained slice map;

[0049] 3) Perform preprocessing on the original data of the mass spectrometry imaging data of the transdermal absorption components to obtain the processed data;

[0050] 4) Register the stained slice map with the imaging map of the transdermal absorption components, divide the skin tissue of the ex vivo skin sample into the epidermis layer and the dermis layer, and determine the processed data corresponding to the epidermis layer and the dermis layer;

[0051] 5) Compare the processed data of the epidermis layer region with the processed data of the dermis layer region, screen the ions specifically expressed in the epidermis layer and the dermis layer of the skin tissue, and perform metabolite identification according to database matching to obtain the metabolites specifically expressed in the epidermis layer and the metabolites specifically expressed in the dermis layer as the zonation identification components;

[0052] 6) Repeat step 1) for other ex vivo skin samples to obtain the sample slices to be measured, perform the mass spectrometry imaging data of the transdermal absorption components obtained in step 2), and then compare the processed data obtained in step 3) with the zonation identification components to determine the partition where the transdermal absorption components are located in other ex vivo skin samples.

[0053] In the above step 1), the ex vivo skin sample is obtained after processing the ex vivo skin.

[0054] In one embodiment, the ex vivo skin is fresh human ex vivo skin, which can be obtained from a hospital.

[0055] In one embodiment, the processing includes: disinfecting and cleaning the ex vivo skin, then peeling off the subcutaneous tissue, obtaining the skin tissue for gas-liquid culture, and obtaining the ex vivo skin sample.

[0056] In a specific embodiment, the treatment is carried out in a laminar flow hood.

[0057] In a specific embodiment, the disinfectant used for the disinfection treatment is alcohol. The alcohol is medical alcohol. Specifically, the excised skin can be placed in a laminar flow hood and disinfected with medical alcohol.

[0058] In a specific embodiment, the cleaning agent used for the cleaning is phosphate buffered saline (PBS). The amount of the cleaning agent used is sufficient to clean the excised skin.

[0059] In a specific embodiment, the number of times of cleaning is at least 3 times, preferably 3 times.

[0060] In a specific embodiment, the subcutaneous tissue includes but is not limited to subcutaneous fat, etc.

[0061] In a specific embodiment, the skin tissue is selected from at least one of the epidermis layer or the dermis layer of the skin, preferably the epidermis layer and the dermis layer of the skin. The skin tissue should maintain the integrity of the epidermis layer and the dermis layer of the skin.

[0062] In a specific embodiment, the gas-liquid culture is carried out in an activity maintenance system.

[0063] In a preferred embodiment, the activity maintenance system is a CO2 incubator with a serum medium inside.

[0064] In a further preferred embodiment, the temperature of the CO2 incubator is 37 ± 0.5 °C.

[0065] In a further preferred embodiment, the serum concentration added in the serum medium is 2 - 10 wt%.

[0066] In the above step 1), the test substance mother liquor is an extract of cosmetic raw materials. The cosmetic raw material is specifically an extract of Lonicera japonica. The Lonicera japonica is the dried flower buds or the flowers to be opened of the plant Lonicera japonica Thunb. of the genus Lonicera in the family Caprifoliaceae.

[0067] In an embodiment, the extract of cosmetic raw materials is an aqueous solution of cosmetic raw materials.

[0068] In an embodiment, the concentration of the cosmetic raw material in the extract of cosmetic raw materials is 10 - 100 mg / mL (1 - 10%), preferably 10 - 50 mg / mL (1 - 5%), more preferably 50 mg / mL (5%).

[0069] In the above step 1), the dosage of the test substance mother liquor applied is 15 - 25 μL, preferably 20 μL.

[0070] In the above step 1), the transdermal absorption is carried out by gas-liquid culture in an active retention system.

[0071] In the above step 1), the administration time of the transdermal absorption is 22 - 26 hours, preferably 24 hours.

[0072] In the above step 1), when collecting the sample, the surface moisture of the sample is wiped dry with blotting paper.

[0073] In the above step 1), the embedding is to add an embedding agent in an embedding mold to fix the sample.

[0074] In one embodiment, the embedding agent is a commonly used embedding agent, specifically, for example, Cryo-Gel embedding gel (Leica Cryo-Gel Embedding Medium, Item No. 14020108926).

[0075] In one embodiment, the embedding is a commonly used embedding method, that is, adding an embedding agent in an embedding mold, then putting the sample in and filling the embedding agent to completely cover it, and adding dry ice to solidify the embedding agent for fixation.

[0076] In one embodiment, air bubbles should be avoided during the embedding.

[0077] In the above step 1), the cryopreservation is to freeze and store the embedded sample in a refrigerator.

[0078] In the above step 1), the temperature of the cryopreservation is -85 to -75 °C, preferably -80 °C.

[0079] In the above step 1), the temperature of the thawing is -15 to -25 °C, preferably -20 °C.

[0080] In the above step 1), the thawing time is overnight. The above thawing means taking out the embedded sample and the blank embedded sample from a -80 °C ultra-low temperature refrigerator and placing them in a -20 °C refrigerator for overnight thawing.

[0081] In the above step 1), the fixation is a conventional method for fixing samples. Specifically, it is to add an embedding agent on the bottom tray of the embedding mold to fix the embedded sample.

[0082] In the above step 1), the sectioning is a conventional sectioning process step. Specifically, the sectioning is to section the fixed embedded sample with a microtome to cut out frozen tissue sections of a set thickness.

[0083] In one embodiment, the microtome is a commonly used microtome, such as the CM1950 cryostat produced by Leica Microsystems, Germany.

[0084] In one embodiment, when embedding the sample slices, they are placed on the sample head of the microtome.

[0085] In one embodiment, the sectioning conditions of the microtome are as follows: the temperature of the chamber is -20 to -25 °C; the temperature of the sample head is -10 to -20 °C.

[0086] In one embodiment, the section thickness after sectioning is 8 - 12 μm, preferably 10 μm.

[0087] In one embodiment, the size of the section after sectioning is 10 - 14 mm 2 。

[0088] In the above step 1), the glass slide is a positively charged anti - detachment glass slide.

[0089] In one embodiment, the positively charged anti - detachment glass slide is the Thermo positively charged anti - detachment glass slide.

[0090] In the above step 2), the number of the section samples to be tested is not less than 10, preferably 10. The section samples other than the first and the second section samples to be tested are used as backup samples.

[0091] In the above step 2), the number of the first section samples to be tested is not less than 2, preferably 2. They are used for mass spectrometry imaging analysis in positive and negative ion modes on an imaging platform tandem mass spectrometer.

[0092] In the above step 2), the number of the second section samples to be tested is not less than 1, preferably 1. They are used for HE staining of tissues.

[0093] In the above step 2), the first and the second section samples to be tested are cryopreserved before use.

[0094] In one embodiment, the cryopreservation is to freeze and store the first and the second section samples to be tested in a refrigerator.

[0095] In one embodiment, the cryopreservation temperature is -85 to -75 °C, preferably -80 °C.

[0096] In the above step 2), the imaging platform is the DESI XS imaging platform (Waters).

[0097] In the above step 2), the data acquisition and scanning conditions of the imaging platform include: the spray solvent is an aqueous solution of methanol and / or acetonitrile, preferably an aqueous solution of acetonitrile, more preferably an aqueous solution of acetonitrile with a volume percentage of 75-95% (V / V), and most preferably an aqueous solution of acetonitrile with a volume percentage of 80% (V / V). The appropriate spray solvent can be selected according to the physicochemical properties of the target active ingredient; the spray flow rate is 1.0-3.0 μL / min, preferably 2.0 μL / min; the spray voltage is ±0.2-0.5 kV, preferably ±0.3 kV; the spatial resolution is 10*10~50*50 μm 2 , preferably 20*20 μm 2 .

[0098] In the above step 2), the mass spectrometer is a Synapt XS mass spectrometer (Waters).

[0099] In the above step 2), the measurement conditions of the mass spectrometer include: the acquisition ion mode is positive / negative ion mode; the mass scanning range is 70-1200; the mass resolution is in high-resolution mode, with a resolution of 18000-22000, preferably 20000.

[0100] In the above step 2), the HE staining is performed by the commonly used HE staining method. The HE staining method, namely hematoxylin-eosin staining, is an existing conventional staining method.

[0101] In the above step 2), the scanner is a commonly used scanner for photographing tissue sections, such as a 3D HISTECH scanner produced by Pannoramic MIDIFL company.

[0102] In the above step 2), the resolution of the scanner for photographing is 4-6 μm, preferably 5 μm.

[0103] In the above step 3), the preprocessing of the original data is to convert the format of the mass spectrometry imaging data by the first processing software and then perform data preprocessing by the second processing software.

[0104] In one embodiment, the first processing software is the imzMLConverter processing software.

[0105] In one embodiment, the format conversion is to convert the mass spectrometry imaging data from the raw format file to the imzML format file.

[0106] In one embodiment, the second processing software is the Cardinal processing software (version v3.8).

[0107] In one embodiment, the data preprocessing includes but is not limited to background subtraction, peak alignment, peak screening, etc.

[0108] In step 4) above, the registration includes the following steps:

[0109] 41) Select ions reflecting the sample contour from the imaging map of the transdermal absorption component and adjust the obtained imaging map of the ions.

[0110] 42) Perform contour registration on the stained section map and the imaging map of the ions.

[0111] In step 41) above, the ions reflecting the sample contour include positive ions and negative ions. The mass-to-charge ratio of the positive ions is m / z 104.1075 (choline, M+), and the mass-to-charge ratio of the negative ions is m / z 303.2330 (arachidonic acid, M-H).

[0112] In step 41) above, the imaging map of the ions is adjusted in the optical imaging software. Specifically, enter this ion in "MS Navigation" in the optical imaging software, and after pressing Enter, adjust the imaging map to be displayed as "jet".

[0113] In step 42) above, the image format of the stained section map is TIFF. Specifically, select a 600 dpi scanned file and save it in the TIFF image format.

[0114] In step 42) above, the contour registration is performed by clicking "Launch the image overlay tool" in the optical imaging software.

[0115] In step 42) above, the contour registration needs to adjust the transparency, size and orientation of the stained section map to match the contour of the imaging map of the ions.

[0116] In step 42) above, the time for the contour registration is 5 - 8 min.

[0117] In step 4) above, the epidermis and dermis partitioning is performed by clicking "Enable polygon tool for ROI selection" in the optical imaging software. According to the epidermis contour and dermis contour presented in the stained section map, the epidermis and dermis regions are selected.

[0118] In step 4) above, the processing data corresponding to the epidermis and dermis needs to be processed such as peak data extraction and missing value interpolation.

[0119] In step 4) above, the processed data corresponding to the epidermis layer and the dermis layer are saved by clicking "Export ROI Location Info" in the optical imaging software. After finding a suitable storage location, name the file as "sample - region - mode", such as "Experimentalgroup - EPI - NEG".

[0120] In step 5) above, the screening is selected from any of the following:

[0121] 51) When the average signal intensity of the processed data in the epidermis layer region / the average signal intensity of the processed data in the dermis layer region ≥ 10, an ion group specifically expressed in the epidermis layer is obtained;

[0122] 52) When the average signal intensity of the processed data in the epidermis layer region / the average signal intensity of the processed data in the dermis layer region ≤ 0.1, an ion group specifically expressed in the epidermis layer is obtained; the " / " above means ratio.

[0123] In step 5) above, when performing database matching, an annotation algorithm is used to match metabolites of ions specifically expressed in the epidermis layer and the dermis layer with the database, obtaining epidermis - layer - specifically - expressed metabolites and dermis - layer - specifically - expressed metabolites.

[0124] In one embodiment, the annotation framework used in the annotation algorithm is the PySM and mass2adduct annotation frameworks.

[0125] In one embodiment, the database is selected from at least one of an endogenous skin - specific database, a cosmetic efficacy ingredient database, or a cosmetic efficacy ingredient metabolite database.

[0126] In one specific embodiment, the endogenous skin - specific database is a metabolite database in the skin built by the company itself. For details, refer to Patent No. ZL202210439119.3, "A Method for Establishing a Spatial Metabolome Database with Multidimensional Annotation".

[0127] In one specific embodiment, the cosmetic efficacy ingredient database and the cosmetic efficacy ingredient metabolite database are databases built by the company itself. For details, refer to Application No. 202310129751.2, "A Method for Establishing a High - Throughput Traditional Chinese Medicine and Its Metabolite Database LutMet - TCM and Its Application", and Application No. 202411453328.9, "A Method and System for Structure Annotation of Parent Metabolites Based on Mass Spectrometry".

[0128] In step 5) above, the conditions for database matching are: the mass error tolerance is 4 - 6 ppm, preferably 5 ppm; adduct ions are allowed, and the positive ions are selected from [M + H] + 、[M + Na]+ 、 [M + K] + 、 [M + H - H2O] + At least one of, the negative ions are selected from [M - H] - 、 [M + Cl] - 、 [M + HCOO] - At least one of; retain the annotation results with a retention score ≥ 0.8.

[0129] The above database matching compares the mass-to-charge ratio (m / z) corresponding to the signals detected by the ions specifically expressed in the epidermis and dermis with the mass-to-charge ratio of the corresponding metabolite components in the database to determine the corresponding epidermis-specifically expressed metabolites and dermis-specifically expressed metabolites.

[0130] In the above step 5), the epidermis-specifically expressed metabolites include but are not limited to Cholesterolsulfate (cholesterol ester, CAS number 1256 - 86 - 6, HMDB0000653, C18043), L-Aspartic acid (aspartic acid, CAS number 56 - 84 - 8, HMDB0000191, C00049), Glutamate (glutamic acid, CAS number 56 - 86 - 0, HMDB0000148, C00025), Histidine (histidine, CAS number 71 - 00 - 1, HMDB0000177, C00135), Ascorbic acid (ascorbic acid, CAS number 50 - 81 - 7, HMDB0000044, C00072), fatty acid family metabolites such as Linoleic acid (linoleic acid, CAS number 60 - 33 - 3, HMDB0000673, C01595), 5-HETE (5-hydroxyicosatetraenoic acid, CAS number 330796 - 62 - 8, HMDB0011134, C04805), Arachidonic acid (arachidonic acid, CAS number 506 - 32 - 1, HMDB0001043, C00219), Elaidic acid (elaidic acid, CAS number 112 - 79 - 8, HMDB0000573, C01712), 9-OxoODE (9-oxo-10E,12Z-octadecadienoic acid, CAS number 54232 - 59 - 6, HMDB0004669, C14766), etc.

[0131] In one embodiment, the epidermis-specifically expressed metabolites are selected from one or more of cholesterol ester, aspartic acid, glutamic acid, histidine, ascorbic acid, linoleic acid, 5-hydroxyicosatetraenoic acid, arachidonic acid, elaidic acid, 9-oxo-10E,12Z-octadecadienoic acid.

[0132] In the above step 5), the dermis layer-specifically expressed metabolites include, but are not limited to, Threonine (threonine, CAS No. 72-19-5, HMDB0000167, C00188), Malic acid (malic acid, CAS No. 6915-15-7, HMDB0000156, C00149), Hypoxanthine (hypoxanthine, CAS No. 68-94-0, HMDB code HMDB0000157, C00262), Glutamine (glutamine, CAS No. 56-85-9, HMDB0000641, C00064), Pyridoxine (pyridoxine, CAS No. 65-23-6, HMDB0000239, C00314), Glucose (glucose, CAS No. 50-99-7, HMDB0304632, C00031), etc.

[0133] In one embodiment, the dermis layer-specifically expressed metabolites are selected from one or more of threonine, malic acid, hypoxanthine, glutamine, pyridoxine, and glucose.

[0134] In the above step 6), the time for determining the partition area of the transdermal absorption components in other ex vivo skin samples is 1 to 2 s. It can quickly identify the epidermis layer and dermis layer of the skin.

[0135] The above method can be used to quickly partition the epidermis layer and dermis layer by replacing HE staining during subsequent transdermal absorption experiments. The above method verifies that the ex vivo skin used has metabolic activity, can be quickly partitioned, and can realize the partitioning of the epidermis layer and dermis layer of components through mass spectrometry imaging data.

[0136] The second aspect of the present invention provides a method for analyzing the transdermal absorption of cosmetic efficacy components based on mass spectrometry imaging, including the following steps:

[0137] A) Using step 1) of the transdermal absorption partition identification method based on mass spectrometry imaging provided in the first aspect of the present invention to obtain a sample slice to be measured;

[0138] B) Directly performing transdermal absorption on the surface of the ex vivo skin sample according to step 1) of the transdermal absorption partition identification method based on mass spectrometry imaging provided in the first aspect of the present invention, collecting samples, embedding, freezing and then thawing, sectioning after fixation, and adhering and fixing the obtained tissue sections on glass slides to obtain blank slice samples;

[0139] C) Using steps 2)-4) of the transdermal absorption partition identification method based on mass spectrometry imaging provided in the first aspect of the present invention for the sample slice to be measured and the blank slice sample to obtain the corresponding processing data of the sample slice to be measured and the blank slice sample in the epidermis layer and dermis layer;

[0140] D) Compare the processed data of the epidermis and dermis, and perform database matching based on the fold change of the processed data of the test section sample compared to that of the blank section sample to determine the skin endogenous components, transdermal components, and / or metabolites of transdermal components in the ex vivo skin sample.

[0141] In the above step B), the corresponding conditions of the preparation steps of the blank section sample are the same as those in step 1) of the transdermal absorption partition identification method based on mass spectrometry imaging provided in the first aspect of the present invention.

[0142] In the above step C), the test section sample is the cosmetic administration group (XC), and the blank section sample is the blank control group (Blank). The blank section sample is obtained by performing a blank experiment on an ex vivo skin sample without applying the test substance mother liquor for sampling.

[0143] In the above step D), the fold change is calculated according to formula (1).

[0144] The formula (1) is: fold change = (average signal intensity of the processed data of the test section sample) / (average signal intensity of the processed data of the blank section sample).

[0145] In the above step D), the transdermal component and / or metabolite of the transdermal component is selected from any of the following:

[0146] D1) When matching the skin endogenous exclusive database, if the fold change is greater than or equal to 2 and there is a statistical difference (P-value < 0.05), it is determined as a transdermal component.

[0147] D2) When matching the cosmetic efficacy component database, if the fold change is greater than or equal to 2 and the average signal intensity of the processed data of the blank section sample is less than 1000, and there is a statistical difference (P-value < 0.05), it is determined as a transdermal component.

[0148] D3) When matching the cosmetic efficacy component metabolite database, if the fold change is greater than or equal to 2 and the average signal intensity of the processed data of the blank section sample is less than 1000, and there is a statistical difference (P-value < 0.05), it is determined as a metabolite of the transdermal component.

[0149] The above transdermal component or metabolite of the transdermal component can be absorbed by the skin.

[0150] In the above step D), the skin endogenous components include, but are not limited to, Glyoxylic acid (glyoxylic acid, CAS No. 298-12-4), Glycolic acid (glycolic acid, CAS No. 79-14-1), L-Lactic acid (lactic acid, CAS No. 79-33-4), Glyceric acid (glyceric acid, CAS No. 473-81-4), Taurine (taurine, CAS No. 473-81-4), L-Aspartic acid (aspartic acid, CAS No. 56-84-8), Malic acid (malic acid, CAS No. 97-67-6), L-Glutamine (glutamine, CAS No. 5959-95-5), L-Glutamic acid (glutamic acid, CAS No. 6893-26-1), L-Arginine (L-arginine, CAS No. 74-79-3), Ascorbic acid (ascorbic acid, CAS No. 134-03-2), Glucose (glucose, CAS No. 50-99-7), Citric acid (citric acid, CAS No. 77-92-9), FA(18:0) (stearic acid, CAS No. 57-11-4), FA(22:6) (docosahexaenoic acid, CAS No. 6217-54-5), Adenosine monophosphate (adenosine monophosphate, CAS No. 61-19-8), ADP (adenosine diphosphate, CAS No. 58-64-0), N-Acetylneuraminic acid (sialic acid, CAS No. 19342-33-7), NAD (nicotinamide adenine dinucleotide, CAS No. 53-84-9), etc.

[0151] In one embodiment, the skin endogenous component is selected from one or more of glyoxylic acid, glycolic acid, lactic acid, glyceric acid, taurine, aspartic acid, malic acid, glutamine, glutamic acid, L-arginine, ascorbic acid, glucose, citric acid, stearic acid, docosahexaenoic acid, adenosine monophosphate, adenosine diphosphate, sialic acid, and nicotinamide adenine dinucleotide.

[0152] In the above step D), the transdermal components include, but are not limited to, Luteolin (luteolin, CAS No. 491-70-3), Morin (morroniside, CAS No. 480-16-0), Isorhamnetin (isorhamnetin, CAS No. 480-19-3), Chlorogenic acid (chlorogenic acid, CAS No. 202650-88-2), etc.

[0153] In one embodiment, the transdermal component is selected from one or more of luteolin, morroniside, isorhamnetin, and chlorogenic acid.

[0154] In the above step D), the metabolites of the transdermal component include but are not limited to Luteolin 4'-sulfate (CAS No. 60889-07-8), 8-Hydroxyluteolin 8-sulfate, etc. The structural formula of 8-Hydroxyluteolin 8-sulfate is:

[0155] In one embodiment, the metabolites of the transdermal component are selected from one or two of Luteolin 4'-sulfate and 8-Hydroxyluteolin 8-sulfate.

[0156] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the protection scope of the present invention.

[0157] The following specific examples illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0158] In the following examples, the reagents or instruments not indicated by the manufacturer can be all conventional products that can be purchased from the market. For example, the DESI XS imaging platform (Waters, USA) and Synapt XS mass spectrometer (Waters, USA) used for sample analysis and detection.

[0159] Example 1

[0160] Fresh human ex vivo skin is obtained from the hospital. The skin tissue is disinfected with medical alcohol in a laminar flow hood, and then the skin tissue is washed 3 times with PBS buffer solution. Then, subcutaneous tissues such as subcutaneous fat are peeled off in a laminar flow hood to obtain skin tissue including the skin epidermis and the skin dermis, and attention should be paid to maintaining the integrity of the skin epidermis and the skin dermis. The skin tissue is placed in a CO2 incubator at 37°C containing a serum medium with 5 wt% serum for air-liquid culture to obtain an ex vivo skin sample.

[0161] The ex vivo skin sample is taken out from the CO2 incubator and divided into two.

[0162] An in vitro skin sample was directly and evenly applied with 20 μL of the test substance stock solution on the surface of the in vitro skin sample for drug administration. The test substance stock solution was an aqueous solution of a cosmetic raw material, the cosmetic raw material was honeysuckle extract, and the concentration of the cosmetic raw material was 50 mg / mL. Then, it was placed in a CO2 incubator at 37 °C containing a serum medium with 5 wt% serum for gas-liquid culture for 24 hours of transdermal absorption. After that, the sample was taken out, and the water on the surface of the sample was dried with absorbent paper. The embedding agent was added to the embedding mold to fix the sample, and care was taken to avoid the generation of air bubbles. The embedded sample was stored frozen in an -80 °C refrigerator and then taken out and placed in a -20 °C refrigerator for overnight thawing. The embedding agent was added to the bottom tray of the embedding mold to fix it, and it was placed on the sample head of the microtome. The temperature of the chamber was -23 °C; the temperature of the sample head was -15 °C, and frozen tissue sections with a set thickness of 10 μm and a size of 12 mm 2 were cut and adhered and fixed on positively charged adhesive-free glass slides to obtain the test section samples. The number of test section samples was 10, numbered XC as the experimental drug administration group. Among them, the first test section sample was 2, the second test section sample was 1, and the remaining 7 were backup samples.

[0163] Another in vitro skin sample was directly placed in a CO2 incubator at 37 °C containing a serum medium with 5 wt% serum for gas-liquid culture for 24 hours of transdermal absorption. After that, the sample was taken out, and the water on the surface of the sample was dried with absorbent paper. The embedding agent was added to the embedding mold to fix the sample, and care was taken to avoid the generation of air bubbles. The embedded sample was stored frozen in an -80 °C refrigerator and then taken out and placed in a -20 °C refrigerator for overnight thawing. The embedding agent was added to the bottom tray of the embedding mold to fix it, and it was placed on the sample head of the microtome. The temperature of the chamber was -23 °C; the temperature of the sample head was -15 °C, and frozen tissue sections with a set thickness of 10 μm and a size of 12 mm 2 were cut and adhered and fixed on positively charged adhesive-free glass slides to obtain the blank section samples. The number of blank section samples was 10, numbered Blank as the blank control group. Among them, the first blank section sample was 2, the second blank section sample was 1, and the remaining 7 were backup samples.

[0164] The experimental drug administration group and the blank control group were stored frozen in an -80 °C refrigerator for subsequent analysis.

[0165] Example 2

[0166] 1. Mass spectrometry imaging analysis

[0167] The first test section sample in the experimental drug administration group obtained in Example 1 was taken out, and mass spectrometry imaging analysis was carried out in positive and negative ion modes using an imaging platform tandem mass spectrometer to obtain the mass spectrometry imaging data and its imaging map of the transdermal absorption components.

[0168] The imaging platform is the DESIXS imaging platform. The data acquisition scanning conditions of the imaging platform include: the spraying solvent is an aqueous solution of acetonitrile with a volume percentage of 80% (V / V); the spraying flow rate is 2.0 μL / min; the spraying voltage is ±0.3 kV; the acquisition ion mode is positive / negative ion mode; the spatial resolution is 20*20 μm 2 .

[0169] The mass spectrometer is a Synapt XS mass spectrometer. The measurement conditions of the mass spectrometer include: the acquisition ion mode is positive / negative ion mode; the mass scanning range is 70 - 1200; the mass resolution is in high-resolution mode with a resolution of 20000.

[0170] 2. HE staining

[0171] Take out the second test section sample in the experimental drug administration group obtained in Example 1, stain it by the HE staining method, and then take a stained section image with a scanner.

[0172] The scanner is a 3D HISTECH type scanner, and the resolution of the scanner for taking pictures is 5 μm.

[0173] 3. Pretreatment of raw data

[0174] For the mass spectrometry imaging data of the transdermal absorption components obtained in Step 1, use the imzMLConverter processing software to convert the original.raw format file into an.imzML format file, and perform mass spectrometry data pretreatment on the above data through Cardinal v3.8, including operations such as background subtraction, peak alignment, and peak screening, to obtain processed data.

[0175] 4. Registration and partitioning

[0176] Register the stained section image obtained in Step 2 with the imaging map of the transdermal absorption components obtained in Step 1. For specific details, see Figure 1 、 2Specifically, open the MSiReader software and import the.imzML format file of the first processed data. Find an ion in the imaging map of the first processed data that can best reflect the sample contour. Select positive ions with m / z 104.1075 (choline, M+), and negative ions with m / z 303.2330 (arachidonic acid, M-H). Enter this ion in "MS Navigation" in the software, and after pressing Enter, adjust the imaging map to be displayed as "jet". Then use the optical imaging software to export the stained section image and save it in the TIFF image format (600 dpi scanned file). Perform contour registration on the stained section image and the ion imaging map. In the optical imaging software, click "Launch the image overlay tool". In the newly popped-up window, load the stained section image and adjust the transparency of the stained section image to facilitate observing the matching situation between the HE screenshot and the imaging map. Then adjust the size and orientation of the HE stained image to perform contour registration with the imaging map. This process takes about 6 minutes. Then select the "Enable polygon tool for ROI selection" function, and according to the epidermal layer contour and dermal layer contour presented in the HE staining, select the epidermal layer and dermal layer regions to achieve the partitioning of the epidermal layer and dermal layer of the skin tissue of the sample. At the same time, determine the processed data corresponding to the epidermal layer and dermal layer, and perform processing such as peak data extraction and missing value interpolation on the processed data. The processed data can be right-clicked, and there is an option "Export ROI Location Info". After clicking it, a new window will pop up. After finding a suitable storage location, name the file in the format of "sample-region-mode", such as "Experimentalgroup-EPI-NEG".

[0177] 5. Screening of Ions

[0178] Compare the processed data of the epidermal layer region with the processed data of the dermal layer region to screen for ions specifically expressed in the epidermal layer and dermal layer of the skin tissue. When the average signal intensity of the second processed data in the epidermal layer region / the average signal intensity of the second processed data in the dermal layer region ≥ 10, an ion group specifically expressed in the epidermal layer is obtained; when the average signal intensity of the second processed data in the epidermal layer region / the average signal intensity of the second processed data in the dermal layer region ≤ 0.1, an ion group specifically expressed in the epidermal layer is obtained.

[0179] Identify metabolites by matching the ion groups specifically expressed in the epidermis and the ion groups specifically expressed in the epidermis according to the database, that is, use annotation algorithms and databases to match metabolites for the ions specifically expressed in the epidermis and dermis. The annotation frameworks used in the annotation algorithms are the PySM and mass2adduct annotation frameworks, and the databases are the skin endogenous exclusive database, the cosmetic efficacy ingredient database, and the cosmetic efficacy ingredient metabolite database. The conditions for database matching are: the mass error tolerance is 5 ppm; adduct ions are allowed, and the positive ions are selected from [M+H] + 、[M+Na] + 、[M+K] + 、[M+H-H2O] + , and the negative ions are selected from [M-H] - 、[M+Cl] - 、[M+HCOO] - ; retain the annotation results with a retention score ≥ 0.8. Obtain the metabolites specifically expressed in the epidermis and the metabolites specifically expressed in the dermis as partition identification components. The specific metabolites specifically expressed in the epidermis and the metabolites specifically expressed in the dermis are shown in Tables 1 and 2 below.

[0180] Table 1 Information Table of Metabolites Specifically Expressed in the Epidermis

[0181]

[0182] Table 2 Information Table of Metabolites Specifically Expressed in the Dermis

[0183]

[0184] Example 3

[0185] Take other ex vivo skin samples, prepare the experimental drug administration group by the same method and conditions as in Example 1. Take the first test section sample in the experimental drug administration group and perform mass spectrometry imaging analysis according to Step 1 of Example 2. Compare the processed data obtained from the mass spectrometry imaging data of the transdermal absorption components according to Step 3 of Example 2 with the partition identification components obtained in Step 5 of Example 2. There is no need to perform HE staining again, and it only takes 1.5 s. The metabolites specifically expressed in the epidermis and the metabolites specifically expressed in the dermis can respectively identify the epidermis and dermis of the skin, and can quickly determine the partition where the transdermal absorption components are located in other ex vivo skin samples.

[0186] Example 4

[0187] Take out the first test section sample and the second test section sample in the experimental drug administration group obtained in Example 1, and the first blank section sample and the second blank section sample in the blank control group, and perform analysis according to Steps 1-4 of Example 2 to obtain the processed data corresponding to the test section samples and the blank section samples in the epidermis and dermis.

[0188] Compare the processing data of the epidermis and dermis, and perform database matching according to the change multiple of the processing data of the test section sample and the blank section sample to determine the skin endogenous components, transdermal components, and / or transdermal component metabolites in the ex vivo skin sample. The specific skin endogenous components are shown in Table 3 below, and the transdermal components and transdermal component metabolites are shown in Table 4 below.

[0189] Among them, the change multiple is calculated according to formula (1), and formula (1) is: change multiple = (average signal intensity of the processing data of the test section sample) / (average signal intensity of the processing data of the blank section sample).

[0190] The database matching conditions are the same as those in step 5 of Example 2. Specifically, when matching to the skin endogenous exclusive database, if the change multiple is greater than or equal to 2 and there is a statistical difference (P-value < 0.05), it is determined as a transdermal component; when matching to the cosmetic efficacy component database, if the change multiple is greater than or equal to 2 and the average signal intensity of the processing data of the blank section sample is lower than 1000 and there is a statistical difference (P-value < 0.05), it is determined as a transdermal component; when matching to the cosmetic efficacy component metabolite database, if the change multiple is greater than or equal to 2 and the average signal intensity of the processing data of the blank section sample is lower than 1000 and there is a statistical difference (P-value < 0.05), it is determined as a transdermal component metabolite.

[0191] Table 3. Information Table of Skin Endogenous Metabolites

[0192]

[0193] Table 4. Information Table of Mass Spectrometry Imaging Analysis of Transdermal Efficacy Components

[0194]

[0195] Example 5

[0196] Use characteristic ions to identify the epidermis and dermis regions, and draw mass spectrometry imaging maps of transdermal components and transdermal component metabolites in the epidermis and dermis regions of the skin tissue. The specific results are shown in Figure 3. From Figure 3 Figure 3c 、 3dAs can be seen from 3e, 3f, 3g, 3h, 3i, 3j, 3k, 3l, 3m, 3n, the transdermal components are only distributed in the XC group, and it can be intuitively determined that Luteolin, Morin, Isorhamnetin, Chlorogenic acid, Luteolin 4'-sulfate, and 8-Hydroxyluteolin 8-sulfate are transdermal components and transdermal component metabolites, and they can only exert their effects by penetrating the skin. The accuracy of the analysis method results in Example 4 can be verified.

[0197] Example 6

[0198] The relative contents (mean values) of the overall, epidermal layer, and dermal layer penetration of the active ingredients were compared and analyzed, and the specific results are shown in Figure 4. From Figure 4a , 4b , 4c, 4d, 4e, 4f in Figure 4, it can be seen that Luteolin, Morin, Isorhamnetin, Chlorogenic acid, Luteolin 4'-sulfate, and 8-Hydroxyluteolin 8-sulfate are mainly enriched in the epidermal layer region, can exert their effects in the epidermal layer region, and are transdermal components and transdermal component metabolites. The accuracy of the analysis method results in Example 4 can be verified.

[0199] In summary, the present invention provides a method and application for analyzing the transdermal absorption of cosmetic active ingredients based on mass spectrometry imaging. Based on the in vitro transdermal experiment method of simulating the transdermal penetration of cosmetics using human ex vivo skin and mass spectrometry imaging technology, a new method and application for high-precision and high-specificity localization analysis of cosmetic active ingredients, active ingredient metabolites, and skin endogenous components in complex skin tissues are realized, providing a reliable technical platform for the study of the transdermal absorption mechanism of cosmetics. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

[0200] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A method for identifying transdermal absorption zones based on mass spectrometry imaging, comprising the following steps: 1) Applying the test substance mother solution to the surface of the ex vivo skin sample for transdermal absorption, collecting the sample, embedding, freezing, thawing, fixing, and then slicing. The obtained tissue slices are adhered and fixed on a glass slide to obtain the slice sample to be tested; 2) taking a first slice sample to be tested and a second slice sample to be tested, respectively, and performing mass spectrometry imaging analysis on the first slice sample in positive and negative ion modes using an imaging platform tandem mass spectrometer to obtain mass spectrometry imaging data and an imaging image of the transdermal absorbed component; and performing HE staining on the second slice sample to be tested and then photographing it using a scanner to obtain a stained slice image; 3) preprocessing the raw data of the mass spectrometry imaging data of the transdermal absorption component to obtain processed data; 4) aligning the stained slice image with the image of the transdermal absorption component, partitioning the skin tissue of the ex vivo skin sample into the epidermis and dermis, and determining the corresponding processing data for the epidermis and dermis; 5) Compare the processed data of the epidermis region with the processed data of the dermis region, screen the ions specifically expressed in the epidermis and dermis in the skin tissue, perform metabolite identification based on database matching, and obtain epidermis-specifically expressed metabolites and dermis-specifically expressed metabolites as partition identification components; 6) Repeat step 1) for other ex vivo skin samples to obtain the slice samples to be tested, perform step 2) to obtain mass spectrometry imaging data of the transdermal absorption components, and then perform step 3) to compare the processed data obtained with the partition identification components to determine the partitions of the transdermal absorption components in other ex vivo skin samples.

2. The method for identifying transdermal absorption regions based on mass spectrometry imaging according to claim 1, wherein, Step 1) includes any one or more of the following conditions: 11) The ex vivo skin sample is obtained by processing the ex vivo skin; the processing includes: disinfecting the ex vivo skin and then cleaning it, then peeling off the subcutaneous tissue, obtaining the skin tissue for air-liquid culture, and obtaining the ex vivo skin sample; Preferably, the disinfectant used in the disinfection treatment is alcohol; and / or, the cleaning agent used in the cleaning is phosphate buffer solution; and / or, the gas-liquid culture is carried out in an activity maintenance system; and the activity maintenance system is a CO2 incubator with a built-in serum culture medium. 12) The test substance mother solution is a cosmetic raw material extract, and the cosmetic raw material is specifically a honeysuckle extract; preferably, the concentration of the cosmetic raw material in the cosmetic raw material extract is 10 to 100 mg / mL; 13) The amount of the test substance stock solution used for smearing is 15-25 μL; 14) The transdermal absorption is carried out in an air-liquid culture in an active maintenance system; 15) The transdermal absorption administration time is 22-26 hours; 16) When collecting the sample, use absorbent paper to wipe the surface moisture of the sample; 17) The freezing temperature is -85 to -75°C; 18) The thawing temperature is -15 to -25°C; the thawing time is overnight; 19) The glass slide is a positively charged anti-shedding slide.

3. The method for identifying transdermal absorption partitions based on mass spectrometry imaging according to claim 1, wherein Step 2) includes any one or more of the following conditions: 21) The first and second slice samples to be tested are frozen before use; 22) The imaging platform is the DESIXS imaging platform; 23) The data acquisition and scanning conditions of the imaging platform include: the spray solvent is an aqueous solution of methanol and / or acetonitrile; the spray flow rate is 1.0 - 3.0 μL / min; the spray voltage is ±0.2 - 0.5 kV; the spatial resolution is 10*10~50*50 μm 2 ; 24) The mass spectrometer is the Synapt XS mass spectrometer; 25) The measurement conditions of the mass spectrometer include: the ion collection mode is positive / negative ion mode; the mass scanning range is 70 - 1200; the mass resolution is in high resolution mode with a resolution of 18000 - 22000; 26) The resolution of the scanner for shooting is 4 - 6 μm.

4. The method for identifying transdermal absorption regions based on mass spectrometry imaging according to claim 1, wherein In step 3), the pre - processing of the original data is to convert the format of the mass spectrometry imaging data through the first processing software and then perform data pre - processing using the second processing software; preferably, the first processing software is the imzMLConverter processing software; the second processing software is the Cardinal processing software.

5. The method for identifying transdermal absorption regions based on mass spectrometry imaging according to claim 1, wherein In step 4), the registration includes the following steps: 41) Select ions reflecting the sample contour in the imaging map of the transdermal absorption component and adjust the obtained ion imaging map; 42) Perform contour registration on the stained section map and the ion imaging map; Preferably, in 41), the ions reflecting the sample contour include positive ions and negative ions, the mass - to - charge ratio of the positive ions is m / z 104.1075, and the mass - to - charge ratio of the negative ions is m / z 303.2330.

6. The method for identifying transdermal absorption regions based on mass spectrometry imaging according to claim 1, wherein In step 5), it includes any one or more of the following conditions: 51) The screening is selected from any of the following: 511) When the average signal intensity of the processed data in the epidermis region / the average signal intensity of the processed data in the dermis region ≥ 10, obtain the ion group specifically expressed in the epidermis; 512) When the average signal intensity of the processed data in the epidermis region / the average signal intensity of the processed data in the dermis region ≤ 0.1, obtain the ion group specifically expressed in the epidermis. 52) When performing database matching, use an annotation algorithm to match the metabolites of the ions specifically expressed in the epidermis and dermis with the database to obtain the metabolites specifically expressed in the epidermis and the metabolites specifically expressed in the dermis; The conditions for database matching are as follows: the mass error tolerance is 4 - 6 ppm; adduct ions are allowed, and the positive ions are selected from + [M + H] + [M + Na] + [M + K] + [M + H - H2O] - and at least one of them, and the negative ions are selected from [M - H] - [M + Cl] - and at least one of them; retain the annotation results with a retention score ≥ 0.8; 54) The metabolites specifically expressed in the epidermis are selected from one or more of cholesterol esters, aspartic acid, glutamic acid, histidine, ascorbic acid, linoleic acid, 5 - hydroxy - eicosatetraenoic acid, arachidonic acid, elaidic acid, 9 - oxo - 10E,12Z - octadecadienoic acid; 55) The metabolites specifically expressed in the dermis are selected from one or more of threonine, malic acid, hypoxanthine, glutamine, pyridoxol, glucose.

7. The method for identifying transdermal absorption regions based on mass spectrometry imaging according to claim 6, wherein It includes any one or more of the following conditions: 521) In item 52), the annotation framework used in the annotation algorithm is the PySM and mass2adduct annotation frameworks; 522) In item 52), the database is selected from at least one of the skin endogenous exclusive database, the cosmetic efficacy ingredient database, or the cosmetic efficacy ingredient metabolite database.

8. A method for analyzing the transdermal absorption of cosmetic efficacy ingredients based on mass spectrometry imaging, comprising the following steps: A) Adopt step 1) of the transdermal absorption partition identification method based on mass spectrometry imaging according to any one of claims 1 - 7 to obtain a sample of the slice to be measured; B) Directly perform transdermal absorption on the surface of the ex vivo skin sample according to step 1) of the method for identifying transdermal absorption partition based on mass spectrometry imaging as described in any one of claims 1-7, collect the sample, embed it, freeze and store it, thaw it, fix it and then section it. The obtained tissue section is adhered and fixed on a glass slide to obtain a blank section sample; C) Use steps 2)-4) of the method for identifying transdermal absorption partition based on mass spectrometry imaging as described in any one of claims 1-7 for the test section sample and the blank section sample to obtain the corresponding processing data of the test section sample and the blank section sample in the epidermis and dermis; D) Compare the processing data of the epidermis and dermis, and perform database matching according to the change multiple of the processing data of the test section sample and the processing data of the blank section sample to determine the skin endogenous components, transdermal components and / or transdermal component metabolites in the ex vivo skin sample.

9. The method for analyzing transdermal absorption of cosmetic active ingredients based on mass spectrometry imaging according to claim 8, wherein In step D), the change multiple is calculated according to formula (1), The formula (1) is: change multiple = (average signal intensity of the processing data of the test section sample) / (average signal intensity of the processing data of the blank section sample).

10. The method for analyzing transdermal absorption of cosmetic active ingredients based on mass spectrometry imaging according to claim 8, characterized in that, Including any one or more of the following conditions: D1) The transdermal component and / or transdermal component metabolite is selected from any one of the following: D11) When matching to the skin endogenous exclusive database, if the change multiple is greater than or equal to 2 and there is a statistical difference with P-value < 0.05, it is determined as a transdermal component; D12) When matching to the cosmetic efficacy ingredient database, if the change multiple is greater than or equal to 2 and the average signal intensity of the processing data of the blank section sample is lower than 1000, and there is a statistical difference with P-value < 0.05, it is determined as a transdermal component; D13) When matching to the cosmetic efficacy ingredient metabolite database, if the change multiple is greater than or equal to 2 and the average signal intensity of the processing data of the blank section sample is lower than 1000, and there is a statistical difference with P-value < 0.05, it is determined as a transdermal component metabolite; D2) The skin endogenous component is selected from one or more of glyoxylic acid, glycolic acid, lactic acid, glyceric acid, taurine, aspartic acid, malic acid, glutamine, glutamic acid, L-arginine, ascorbic acid, glucose, citric acid, stearic acid, docosahexaenoic acid, adenosine monophosphate, adenosine diphosphate, sialic acid, nicotinamide adenine dinucleotide; D3) The transdermal component is selected from one or more of luteolin, morroniside, isorhamnetin, chlorogenic acid; D4) The transdermal component metabolite is selected from one or two of luteolin-4'-sulfate or 8-hydroxyluteolin 8-sulfate.

Images