A sers detection system and method for skin depth resolved drug diffusion

Abstract

This invention provides a Skin-Resolved Diffusion (SERS) Detection System for Deep-Resolved Drug Diffusion, comprising a SERS-active microneedle array and a confocal Raman microscope. The SERS-active microneedle array is used for in-situ drug detection at multiple depths in the skin. It consists of polymer microneedles of varying lengths, with noble metal core-shell nanostars uniformly immobilized on their surfaces. Each noble metal core-shell nanostar comprises a noble metal nanostar core modified with an internal standard reporter molecule and an outer noble metal shell. The confocal Raman microscope records the SERS signal generated after the microneedles penetrate the skin. A quantitative model is established by comparing the peak ratio of the target drug characteristic peak to the internal standard characteristic peak in the SERS signal to determine the drug concentration at different depths. This invention enables quantitative analysis of the diffusion behavior of topical drugs at different skin types and depths.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection, specifically to a SERS detection system and method for deep-seated drug diffusion in the skin. Background Technology

[0002] Transdermal drug delivery systems (TDDS) are widely used in topical medications and transdermal formulations because they avoid the first-pass effect and improve medication adherence. However, the dense structure of the stratum corneum, the outermost layer of the skin, is a key barrier limiting transdermal drug absorption. Therefore, accurately assessing the diffusion behavior of drugs at different depths of the skin is crucial for ensuring the safety, consistency, and bioequivalence of topical formulations. Currently, the assessment methods commonly used by international regulatory systems (FDA, EMA, PMDA) mainly include in vitro release testing (IVRT) and in vitro permeation testing (IVPT).

[0003] Surface-enhanced Raman scattering (SERS) technology boasts advantages such as high sensitivity, molecular specificity, and non-destructive detection, enabling the quantitative detection of trace molecules through the electromagnetic enhancement effect generated by noble metal nanostructures. In recent years, combining SERS with microneedle (MN) platforms for the detection of components in in vivo or subcutaneous interstitial fluid (ISF) has gained increasing attention. Existing studies have utilized materials such as Au@Ag nanostructures and silver nanoparticles to detect glucose, H2O2, or antitumor drugs at a specific depth within the dermis. However, current work cannot achieve in-situ quantitative detection of drug concentrations at multiple depths in the skin for topical preparations; furthermore, a depth-resolved drug diffusion analysis method that can replace IVPT is lacking. Specific limitations are as follows:

[0004] 1. Limitations of traditional diffusion assessment methods: IVRT / IVPT can only measure the total flux of the drug across the entire skin, and cannot obtain information on the drug concentration distribution at different depths. This makes it difficult to reveal local skin pharmacokinetics and to effectively guide the quality consistency evaluation of generic topical formulations. Traditional tissue sampling, on the other hand, damages tissue structure. There is a lack of depth-based detection technologies that can simultaneously achieve localization and quantification.

[0005] 2. Current research on SERS-microneedles is limited to a single depth: it can only detect subcutaneous interstitial fluid or a single dermal layer, and cannot obtain the concentration gradient of drugs at multiple depths.

[0006] 3. Poor quantitative stability: The large curvature of the microneedle tip makes the signal highly susceptible to laser focus deviation, and the lack of an internal standard calibration mechanism leads to poor repeatability.

[0007] 4. Lack of an evaluation framework compatible with regulatory science: Currently, it cannot be used for prescription consistency evaluation, generic drug Q3 evaluation, or local skin pharmacokinetic studies. Therefore, there is an urgent need to develop a novel, non-destructive, quantitative, depth-resolution skin diffusion detection platform that meets regulatory requirements. Summary of the Invention

[0008] To address the aforementioned issues, this invention provides a SERS detection system and method for skin depth-based drug diffusion, enabling quantitative analysis of the diffusion behavior of topical drugs at different skin types and depths.

[0009] The specific plan is as follows:

[0010] A skin depth-resolved drug diffusion SERS detection system includes a SERS-active microneedle array and a confocal Raman microscope. The SERS-active microneedle array is used for in-situ drug detection at multiple depths in the skin. It is composed of polymer microneedles of different lengths, and noble metal core-shell nanostars are uniformly immobilized on the surface of the polymer microneedles. The noble metal core-shell nanostars include a noble metal nanostar core modified with an internal standard reporter molecule and an outer noble metal shell. The confocal Raman microscope is used to record the SERS signal generated after the microneedle array penetrates the skin, and a quantitative model is established by the peak ratio of the target drug characteristic peak to the internal standard characteristic peak in the SERS signal to measure the drug concentration at different depths.

[0011] Furthermore, the noble metal nanostar core is prepared by selecting materials such as pure gold, pure silver, or alloys to form a multi-branched nanostar structure according to the required enhancement factors.

[0012] Furthermore, the internal standard reporting molecule is adjusted according to the position of the Raman characteristic peak of the drug molecule to be tested, and is a Raman active molecule whose Raman characteristic peak does not overlap with the characteristic peak of the drug molecule to be tested.

[0013] Furthermore, the internal standard reporting molecule is 4-mercaptobenzonitrile (4-MBN).

[0014] Furthermore, the noble metal core-shell is a silver layer. By coating the product with a noble metal shell after modifying the internal standard molecule, the final product exhibits high SERS activity, stability, and characteristic peaks that can be used for internal standard correction.

[0015] Furthermore, the microneedle array is prepared using biocompatible polymer materials (such as PMMA), and the microneedle array is arranged in a repeating pattern with four different lengths of microneedles as a small unit, which is used to solve the problem that the needle tip is easy to bend or difficult to insert when the microneedle is inserted into the skin.

[0016] Furthermore, the length of the microneedles in the microneedle array ranges from 200 to 800 μm, and is selected according to the type of skin to be tested (mouse, pig skin, or human skin, etc.) and the target detection depth. This allows for flexible coverage from the epidermis to the deep dermis.

[0017] Furthermore, polymers were used to enhance the interaction between nanoparticles and the microneedle surface, and the microneedles were made to adsorb nanocolloids to obtain a SERS active microneedle array.

[0018] A method for in-situ drug detection at multiple depths in the skin includes the following steps:

[0019] S1. Place the skin tissue of the organism to be tested in a diffusion device and perform an in vitro diffusion experiment using the target topical formulation;

[0020] S2. After diffusion, the SERS active microneedle array is inserted into the diffused skin tissue and kept in the puncture state for a specific time for detection;

[0021] S3. Record the SERS signal of the array using a confocal Raman microscope;

[0022] S4. Establish a quantitative linear relationship based on the peak ratio of the characteristic peak of the targeted drug to that of the internal standard molecule, and calculate the drug concentration at different depths. Among these methods, compared with traditional single-peak intensity quantification, internal standard peak ratio correction can significantly improve the linearity and quantitative accuracy.

[0023] Furthermore, in step S1, the skin tissue includes human skin, animal skin, or an artificially constructed skin model; the diffusion device is a Franz diffusion cell.

[0024] The beneficial effects of this invention are as follows:

[0025] 1. High sensitivity and good repeatability

[0026] By introducing an internal standard calibration mechanism, the signal fluctuations caused by the curvature of the microneedle tip and the offset of the laser focus are effectively offset, significantly improving the anti-interference ability, detection sensitivity and quantitative repeatability of quantitative analysis from the technical level.

[0027] 2. Non-destructive, no tissue sectioning required

[0028] It maintains the integrity of the skin structure and achieves true in-situ multi-depth three-dimensional detection, which is significantly better than the limitation of traditional IVPT, which can only provide cross-skin throughput.

[0029] 3. A novel framework for evaluating skin spatial concentration was constructed.

[0030] The quantitative system of this invention can accurately capture the subtle differences in the spatial gradient of drugs on the local skin, and from a technical perspective, it realizes the refined characterization of the transdermal diffusion behavior of complex prescription formulations. This provides a regulatory science analysis tool with potential for widespread application for topical drug local PK studies and Q3 equivalence evaluation of generic drugs. Attached Figure Description

[0031] Figure 1 This is an SEM image of a PMMA microneedle array.

[0032] Figure 2This is a top view of a PMMA microneedle array SEM.

[0033] Figure 3 This is a SEM image of the synthesized AuNS@4-BMN@Ag nanoparticles.

[0034] Figure 4 This is a particle size distribution diagram of the synthesized AuNS@4-BMN@Ag nanoparticles.

[0035] Figure 5 This is a graph showing the changes in the Zeta potential of AuNS, AuNS@4-MBN, and AuNS@4-BMN@Ag during the synthesis process.

[0036] Figure 6 This is a top-view SEM image of AuNS@4-BMN@Ag nanoparticles attached to the surface of the PMMA microneedle tip.

[0037] Figure 7 This is a linear relationship graph of metronidazole content in agarose gel calibrated using internal standards.

[0038] Figure 8 The figure shows the results of detecting metronidazole content in isolated SD rat skin after in vitro transdermal diffusion via the prepared SERS substrate. (A), (B), and (C) represent the results of in vitro transdermal diffusion at 12, 18, and 24 hours after the drug dose of 100 mg was applied; (D), (E), and (F) represent the results of in vitro transdermal diffusion at 12, 18, and 24 hours after the drug dose of 200 mg was applied.

[0039] Figure 9 The image shows the intradermal metronidazole concentration distribution obtained by simulating the in vitro transdermal diffusion results. (A), (B), and (C) represent the drug dosage of 100 mg, and (D), (E), and (F) represent the results of in vitro transdermal diffusion of 200 mg for 12, 18, and 24 hours, respectively.

[0040] Figure 10 The figure shows the results of detecting metronidazole content in isolated SD rat skin after in vitro transdermal diffusion via the Franz pool using the prepared SERS substrate. (A)(B)(C) and (D)(E)(F) represent the results of transdermal diffusion of 200 mg of generic and original drugs for 12, 18, and 24 hours, respectively.

[0041] Figure 11 The image shows the intradermal metronidazole concentration distribution obtained by simulating the in vitro transdermal diffusion results. (A), (B), and (C) represent generic drugs, while (D), (E), and (F) represent active pharmaceutical ingredients. The results were obtained after 12, 18, and 24 hours of in vitro transdermal diffusion with an dosage of 200 mg. Detailed Implementation

[0042] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0043] As shown in the figure, this invention provides a SERS detection system for skin depth-resolved drug diffusion, comprising a SERS active microneedle array and a confocal Raman microscope. The SERS active microneedle array is used for in-situ drug detection at multiple depths in the skin. It is composed of polymer microneedles of different lengths, and noble metal core-shell structured nanostars are uniformly immobilized on the surface of the polymer microneedles. The noble metal core-shell structured nanostars include a noble metal nanostar core modified with an internal standard reporter molecule and an outer noble metal shell. The confocal Raman microscope is used to record the SERS signal generated after the microneedle array penetrates the skin, and a quantitative model is established by the peak ratio of the target drug characteristic peak to the internal standard characteristic peak in the SERS signal to measure the drug concentration at different depths.

[0044] The in-situ drug detection method at multiple depths in the skin includes the following steps:

[0045] S1. Place the skin tissue of the organism to be tested in a diffusion device and perform an in vitro diffusion experiment using the target topical formulation;

[0046] S2. After diffusion, the SERS active microneedle array is inserted into the diffused skin tissue and kept in the puncture state for a specific time for detection;

[0047] S3. Record the SERS signal of the array using a confocal Raman microscope;

[0048] S4. Establish a quantitative linear relationship based on the peak ratio of the characteristic peak of the targeted drug to the characteristic peak of the internal standard molecule, and calculate the drug concentration at different depths.

[0049] Example 1: Preparation of AuNS@4-MBN@Ag nanostructures

[0050] This embodiment details the preparation steps of the AuNS@4-MBN@Ag nanostructures used to construct SERS-microneedle arrays, including the synthesis of seed gold nanoparticles (Au Seed), the growth of multi-branched gold nanostars (AuNS), the modification of internal standard molecules, and the silver coating.

[0051] 1. Preparation of Au Seed

[0052] Add 100 mL of 0.01% (w / w) HAuCl4·3H2O solution to a 250 mL three-necked flask. Place the flask on a heating mantle and heat until boiling. Quickly add 4.5 mL of 1% sodium citrate solution while stirring vigorously. Continue boiling for approximately 15 min, then stop heating and allow to cool naturally to room temperature with magnetic stirring. Store the resulting AuSeed solution at 4 °C for later use.

[0053] The obtained Au Seeds were characterized by SEM, and their particle size was approximately 18.8 nm. They were uniformly distributed and their morphology was consistent with that reported in the literature.

[0054] 2. Growth of Au Nanostar (AuNS)

[0055] Preparation of AuNS growth medium: Add 240 mL of 0.25 mmol / L HAuCl4 solution to a beaker and stir at high speed at room temperature. Then add the following components sequentially: 240 μL of 1.0 mol / L HCl solution, 1.2 mL of 3.0 mmol / L AgNO3 solution, and 1.2 mL of 0.1 mol / L ascorbic acid (AA) solution. After thorough mixing, immediately add 2.4 mL of Au Seed solution. Within approximately 30 seconds, the system color rapidly changes from light red to dark green, indicating rapid formation of multi-branched AuNS. Centrifuge the reaction solution at 4000 rpm for 30 min, discard the supernatant, resuspend in ultrapure water, and wash twice more by centrifugation. Finally, redisperse with 60 mL of ultrapure water to obtain the AuNS colloidal solution for later use.

[0056] The obtained AuNS exhibited a multi-branched star-shaped structure in SEM, with a significantly increased particle size and high anisotropy.

[0057] 3. 4-MBN internal standard molecule modification

[0058] Prepare a 1.0 mmol / L DMSO stock solution of 4-MBN. Add 3.0 mL of this solution to 60 mL of AuNS colloid and sonicate for 1 h to allow 4-MBN molecules to firmly adsorb onto the AuNS surface. Then centrifuge at 4000 rpm for 30 min, discard the supernatant to remove unbound 4-MBN. Redisperse the nanoparticles in 120 mL of ultrapure water.

[0059] 4. Ag coating to form AuNS@4-MBN@Ag

[0060] Under thorough stirring, 600 μL of 1.0 mmol / L AgNO3 solution and 600 μL of 1.0 mmol / L ascorbic acid solution were rapidly added. Silver ions were reduced by AA and deposited layer by layer on the surface of the nanostars, forming a uniform Ag shell. After approximately 20 min of reaction, the generated AuNS@4-MBN@Ag nanostructures were collected by centrifugation at 4000 rpm for 30 min. After washing twice with ultrapure water, the nanostructures were resuspended in 10 mL of ultrapure water for later use.

[0061] Zeta potential measurements showed that AuNS (–19.1 mV) decreased to –26.3 mV after 4-MBN modification and increased to –22.2 mV after Ag coating, indicating that the three-step construction process was successful.

[0062] Example 2: Fabrication of AuNS@4-MBN@Ag / PMMA microneedle array

[0063] This embodiment describes in detail the pretreatment, surface molecular layer construction, and nanostructure modification process of PMMA microneedle array.

[0064] 1. Cleaning and pretreatment of microneedle arrays

[0065] A 10×10 microneedle array made of PMMA was selected, with each group containing microneedles of four lengths (200, 400, 600, and 800 μm). The microneedle array was sequentially immersed in anhydrous ethanol and ultrapure water, and washed three times each for 2 minutes, and then placed in a dust-free environment to air dry.

[0066] 2. Constructing PS-b-P4VP molecular brush layers

[0067] To enhance the interaction between nanoparticles and the microneedle surface, the dried microneedles were immersed in 200 μL of a 0.20 mg / mL PS-b-P4VP THF solution for 5 min. The P4VP in this block copolymer exhibits good coordination with the gold / silver surface, which is beneficial for the subsequent robust deposition of nanomaterials. After removing the microneedles, they were rinsed three times with ultrapure water to remove unadsorbed polymer.

[0068] 3. Nanomaterial Modification

[0069] The pretreated microneedles were completely immersed in AuNS@4-MBN@Ag colloidal solution and allowed to stand for 12 hours to allow the nanoparticles to fully adhere to the outer surface of the microneedles. After removal, they were gently rinsed with ultrapure water to remove any loosely bonded nanoparticles. This immersion-rinsing process was repeated twice to improve surface coverage. Finally, the microneedles were washed with anhydrous ethanol and water and allowed to air dry, yielding a SERS-activated PMMA microneedle array with a gray-black surface and uniform nanoparticle coverage.

[0070] SEM revealed highly dense nano-hotspot regions on the outer surface of the microneedles, which are beneficial for enhancing the Raman signal.

[0071] Example 3: Construction of a standard curve for agarose gel containing metronidazole

[0072] This example is used to construct the drug concentration standard curve required for SERS quantitative analysis.

[0073] 1. Preparation of drug-containing agar gel

[0074] Weigh an appropriate amount of agar powder, add it to deionized water, and heat until dissolved to obtain a 1–2% (w / v) agar solution. Cool the solution to approximately 50 °C, and add pre-prepared metronidazole aqueous solution sequentially to achieve final concentrations of 0.075, 0.5, 1.5, 2.5, 5.0, and 7.5 mg / mL. Mix thoroughly and pour into a mold to cool and form a gel.

[0075] 2. Microneedle insertion and SERS signal collection

[0076] AuNS@4-MBN@Ag microneedles were gently pressed into the gel surface to embed the needle tip, and left to stand for 10 min to allow metronidazole to fully diffuse into the nanohotspot region. The microneedle array was then inverted and attached to a quartz glass slide, and SERS scanning was performed using a 638 nm laser with an integration time of 30 s. Signals were collected from all 100 microneedles in each group, and the average value was used as the characteristic peak intensity at that concentration point.

[0077] 3. Plotting the standard curve

[0078] Select metronidazole 1270 cm -1 2230 cm (internal standard) -1 The peak value. Compare the two fitting methods:

[0079] Only 1270 cm -1 Strength → R 2 = 0.9459 using 1270 / 2230 cm -1 Peak ratio → R 2 =0.9943 (significant improvement)

[0080] The second linear result serves as the standard for subsequent calculations of skin drug concentrations.

[0081] Example 4: In vitro skin diffusion experiment in a Franz diffusion cell

[0082] 1. Skin preparation

[0083] Six-–7-week-old SD rats were sacrificed, their dorsal hair was shaved, and intact skin was harvested. After scraping off subcutaneous fat, the skin was washed with physiological saline and blotted dry on filter paper, then stored at -4 °C. Before the experiment, the skin was brought to room temperature and cut into 2×2 cm² pieces.

[0084] 2. Franz diffusion pool installation

[0085] Secure the skin, outer side up, between the donor and receptor in the diffusion chamber, ensuring no air bubbles are trapped. Add physiological saline to the receptor chamber and maintain the temperature at 37 ± 0.5 ℃.

[0086] 3. Drug application and diffusion

[0087] 100 mg metronidazole gel and 200 mg metronidazole gel were applied to the skin surface and allowed to diffuse for 12 h, 18 h, and 24 h, respectively. Each experiment was performed in duplicate.

[0088] 4. Microneedle detection after diffusion

[0089] Immediately after diffusion, the SERS-microneedle array was inserted into the skin with uniform pressure for approximately 10 minutes. It was then removed, and individual SERS measurements were performed on each needle. The microneedles were grouped according to different needle lengths (corresponding to different depths), and the average value of every 25 microneedles was taken as the depth concentration representative, and the RSD was calculated.

[0090] Experimental results showed that the diffusion of the drug from the surface to the depth layer was time-dependent, and the depth distribution differed significantly among different dose groups.

[0091] Example 5: Calculation and 3D visualization of depth-resolved skin drug concentration

[0092] Substituting the 1270 / 2230 peak ratio from Example 4 into the linear equation obtained in Example 3, the metronidazole concentration at each depth was calculated and summarized into six groups (dose × time).

[0093] The results showed that at 12 h, the drug concentration was mainly between 200–400 μm; at 24 h, the high concentration reached 600 μm; the high-dose group had a higher overall concentration and deeper penetration. A three-dimensional concentration map was created using software to show the spatial distribution characteristics of the drug within the skin.

[0094] Example 6: Consistency evaluation between generic and original topical formulations

[0095] Two batches of original drugs (A1, A2) and two batches of generic drugs (G1, G2) were uniformly applied to the skin (200 mg each) and diffused in a Franz diffusion cell for 18 h. After diffusion, the same SERS-microneedle detection procedure was used to obtain drug concentration profiles at depths of 200–800 μm.

[0096] The results showed that the skin depth distribution of the original A1 / A2 batches was highly consistent with that of the generic, and G1 / G2 also showed good intra-batch consistency. More importantly, the skin depth distribution of the original and generic almost overlapped.

[0097] This indicates that the method of the present invention can be used to evaluate the local PK consistency of topical generic drugs and can serve as a regulatory science tool to assist in BE assessment.

[0098] In summary, the present invention relates to the following key technical points:

[0099] 1. Design of multi-branched core-shell structure noble metal nanostars and internal standard reporter molecules: simultaneously providing high SERS enhancement and internal standard benchmark signals, and the materials and internal standard molecules can be flexibly replaced according to the targeted drug.

[0100] 2. Internal standard calibration system: solves the problem of unstable signal at the microneedle tip, and achieves high repeatability and accurate quantification.

[0101] 3. Innovative microneedle array arrangement pattern and multi-size customized microneedle array characterization technology: The repeated arrangement pattern of four different length microneedles as a small unit effectively improves the mechanical stability of the puncture structure, prevents microneedle bending and improves the success rate of puncture; and the size can be customized according to the skin thickness to achieve precise positioning of drugs in the skin at multiple depths.

[0102] 4. Deep resolution skin diffusion detection technology system: No slicing required, non-destructive operation, can replace some traditional IVPT.

[0103] 5. Quantitative methodologies for consistency evaluation of topical formulations: can be used for local PK equivalence analysis between original and generic drugs.

[0104] The technical means disclosed in this invention are not limited to those disclosed in the above embodiments, but also include technical solutions composed of any combination of the above technical features. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications are also considered within the scope of protection of this invention.

Claims

1. A SERS detection system for skin depth-resolved drug diffusion, characterized in that, The system includes a SERS-activated microneedle array and a confocal Raman microscope. The SERS-activated microneedle array is used for in-situ drug detection at multiple depths in the skin. It consists of polymer microneedles of different lengths, on the surface of which noble metal core-shell nanostars are uniformly immobilized. The noble metal core-shell nanostars include a noble metal nanostar core modified with an internal standard reporter molecule and an outer noble metal shell. The confocal Raman microscope is used to record the SERS signal generated after the microneedle array penetrates the skin, and to establish a quantitative model by comparing the peak ratio of the target drug characteristic peak to the internal standard characteristic peak in the SERS signal, thereby measuring the drug concentration at different depths.

2. The SERS detection system for skin depth-resolved drug diffusion according to claim 1, characterized in that, The core of the noble metal nanostar is made of pure gold, pure silver, or an alloy to form a multi-branched nanostar structure.

3. The SERS detection system for skin depth-resolved drug diffusion according to claim 1, characterized in that, The internal standard reporting molecule is a Raman-active molecule, and its Raman characteristic peaks do not overlap with the characteristic peaks of the drug molecule to be tested.

4. The SERS detection system for skin depth-resolved drug diffusion according to claim 1, characterized in that, The internal standard reporting molecule is 4-mercaptobenzonitrile.

5. The SERS detection system for skin depth-resolved drug diffusion according to claim 1, characterized in that, The precious metal core and shell are silver layers.

6. The SERS detection system for skin depth-resolved drug diffusion according to claim 1, characterized in that, The microneedle array is prepared using a biocompatible polymer material, and the microneedle array is arranged in a repeating pattern with four different lengths of microneedles as a small unit.

7. The SERS detection system for skin depth-resolved drug diffusion according to claim 1, characterized in that, The length of the microneedles in the microneedle array ranges from 200 to 800 μm, and is selected according to the type of skin to be tested and the target detection depth.

8. The SERS detection system for skin depth-resolved drug diffusion according to claim 1, characterized in that, By using polymers to enhance the interaction between nanoparticles and microneedle surfaces, and by enabling microneedles to adsorb nanocolloids, a SERS-active microneedle array is obtained.

9. A method for in-situ detection of drugs at multiple depths in the skin using the detection system described in any one of claims 1 to 7, characterized in that, Includes the following steps: S1. Place the skin tissue of the organism to be tested in a diffusion device and perform an in vitro diffusion experiment using the target topical formulation; S2. After diffusion, the SERS active microneedle array is inserted into the diffused skin tissue and kept in the puncture state for a specific time for detection; S3. Record the SERS signal of the array using a confocal Raman microscope; S4. Establish a quantitative linear relationship based on the peak ratio of the characteristic peak of the targeted drug to the characteristic peak of the internal standard molecule, and calculate the drug concentration at different depths.

10. The method for in-situ drug detection at multiple depths of the skin according to claim 9, characterized in that: In step S1, the skin tissue includes human skin, animal skin, or an artificially constructed skin model; the diffusion device is a Franz diffusion cell.

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