JP6006548B2 - Component distribution visualization device, component distribution visualization method, and component distribution visualization program - Google Patents

Description

  The present application relates to a component distribution visualization device, a component distribution visualization method, and a component distribution visualization program.

  Conventionally, in order to evaluate how much an active ingredient contained in, for example, a transdermal preparation penetrates into the stratum corneum, it is required to visually indicate the degree of penetration of the active ingredient into the stratum corneum. Yes. On the other hand, the stratum corneum is extremely thin, about 0.001 mm per layer, and about 0.020 mm in all layers. Therefore, the method for clearly and easily visualizing the number of components existing in each layer constituting the stratum corneum is currently available. However, it does not exist.

  For example, as a conventional method for showing a rough distribution in the epidermis including the basement membrane from the stratum corneum / granular layer, a fluorescent labeled active ingredient is infiltrated into the skin, and the cut-out skin cross section is analyzed with a fluorescence microscope or a confocal laser microscope. A method of observing with (Conformal Laser Scanning Microscope: CLSM) and a surface measurement method such as Time of Flight Secondary Ion Mass Spectrometer (TOF-SIMS) are known.

  However, in the conventional method described above, the possibility that the transdermal absorbability changes due to the fluorescent label cannot be denied, and the cross-section of the cut-out skin is observed, so that it cannot be easily applied to human skin. In addition, the ionization efficiency is not good in TOF-SIMS, etc., and there is a clear image of the SN (Signal to noise) ratio because the absolute number of molecules present in the cross section is small and it is affected by endogenous molecules. It is rarely obtained. Furthermore, as described above, since the stratum corneum has a thin thickness of about 0.001 mm, there is no method for achieving both resolution and sensitivity capable of clearly distinguishing the molecular distribution difference between the stratum corneum.

  The disclosed technology has been made in view of the above problems, and aims to provide a component distribution visualization device, a component distribution visualization method, and a component distribution visualization program that easily visualize the component distribution in the stratum corneum. To do.

According to one aspect of the present invention, in order to achieve the above object, a component distribution visualization device for visualizing a component distribution in a stratum corneum, wherein each layer of the stratum corneum collected by a predetermined adhesive member is predetermined. Mass analysis means for obtaining mass analysis data including component amount information in the surface and depth directions of each layer, and the surface and depth of each layer obtained by the mass analysis means Image acquisition means for acquiring a component distribution image of each layer using mass spectrometry data including direction component amount information; compressing the component distribution image of each layer in a predetermined direction; and And a data processing unit that stacks the compressed images in correspondence with the depth direction of the stratum corneum to form a component distribution image in the stratum corneum .

  According to the disclosed technology, the component distribution in the stratum corneum can be easily visualized.

It is a figure which shows an example of the component distribution visualization apparatus which concerns on this embodiment. It is a figure which shows an example of the hardware constitutions which can implement | achieve the component distribution visualization process which concerns on this embodiment. It is a flowchart which shows an example of the component distribution visualization process which concerns on this embodiment. It is a figure for demonstrating the preparation method of the sample which evaluates transdermal absorbability. It is a figure which shows an example of the structure of the adhesive sheet for extract | collecting a stratum corneum. It is a figure which shows an example of the adhesive sheet affixed on the glass substrate. It is a figure for demonstrating DESI mass spectrometry. It is a figure for demonstrating acquisition of the component distribution image of each layer. It is a figure which shows an example of the component distribution image of each layer of a stratum corneum. It is a figure which shows the relationship between a heat map display and a mass chromatogram. It is a figure for demonstrating the method of visualizing the state of transdermal absorption. It is a figure for demonstrating the visualization method of the transdermal absorption state which concerns on this embodiment. It is a figure which shows an example of the component distribution in the stratum corneum obtained by the visualization method of this embodiment. It is a figure which shows the component distribution image in the stratum corneum corresponding to each example of FIG. It is a figure which shows an example of the variation of the component distribution image in a stratum corneum. It is FIG. (1) for demonstrating the quantification method using the component distribution image of a standard product. It is FIG. (2) for demonstrating the quantitative method using the component distribution image of a standard product.

  Hereinafter, embodiments of the present invention will be described in detail.

<About component distribution visualization device>
First, in this embodiment, a functional configuration example of a component distribution visualization apparatus that visualizes a component distribution in the stratum corneum will be described. FIG. 1 is a diagram illustrating an example of a component distribution visualization apparatus according to the present embodiment. As shown in FIG. 1, the image processing apparatus 10 according to the present embodiment includes an input unit 11, an output unit 12, a storage unit 13, a mass analysis unit 14, an image acquisition unit 15, and a data processing unit 16. , Quantification means 17, transmission / reception means 18, and control means 19.

  The input unit 11 accepts inputs such as start / end and setting of various instructions related to component distribution visualization processing from, for example, a user using the component distribution visualization device 10. The input unit 11 includes a pointing device such as a keyboard and a mouse if it is a general-purpose computer such as a PC (Personal Computer). The input unit 11 may be a voice input device such as a microphone that can be input by voice or the like.

  The output unit 12 outputs the content input by the input unit 11 and the content executed based on the input content. The output unit 12 includes, for example, a display and a speaker. The output unit 12 may include a printing device such as a printer. For example, when the component distribution visualization device 10 is a smartphone or a tablet terminal, the input unit 11 and the output unit 12 described above may have an input / output integrated configuration such as a touch panel.

  The storage unit 13 stores various information necessary in the present embodiment. Specifically, various programs for executing the component distribution visualization process in the present embodiment, various setting information, and the like are stored. In addition, the storage unit 13 can write or read the above-described various information at a predetermined timing as necessary.

  The storage means 13 is a collection of various types of information, and has a database function that is structured systematically so that information can be searched and extracted using, for example, keywords. Also good. Further, the information stored in the storage unit 13 may be obtained by accessing an external device connected to the communication network via the transmission / reception unit 18.

The mass analyzing means 14 performs a predetermined mass analysis (for example, DESI (Desorption Electrospray Ionization :) on each layer of the stratum corneum collected by a predetermined pressure-sensitive adhesive sheet (for example, Skin Checker (registered trademark)) according to a user instruction or the like. Mass spectrometry data including the component amount information in the plane and depth direction of each layer is obtained by analysis by desorption electric field spray ionization) or the like. An example of mass spectrometry data acquisition by the mass analyzer 14 will be described later. Further, the acquired mass spectrometry data and the like are stored in the storage means 13. The image acquisition means 15 includes mass analysis data including the component amount information in the surface and depth directions of each layer obtained by the mass analysis means 14. Is used to obtain a distribution image (component distribution image) of components contained in each layer of the stratum corneum (for example, active ingredients contained in a transdermally absorbed preparation or the like). The component distribution image acquired by the image acquisition unit 15 is displayed on the screen by the output unit 12 or stored in the storage unit 13.

  The data processing unit 16 bins the pixel data of the component distribution image of each layer obtained by the image acquisition unit 15 in a predetermined direction. Further, the data processing means 16 compresses the component distribution image of each layer on which the pixel data is binned at a predetermined compression rate in a predetermined direction, and generates a component distribution image in the stratum corneum using the compressed image.

  Here, the predetermined direction indicates, for example, a depth direction in which a component contained in the stratum corneum penetrates. The predetermined compression rate is, for example, a predetermined compression rate such as 1/5 or 1/10. For example, the compression rate can be set according to the number of layers acquired by the image acquisition unit 15. However, the present invention is not limited to this.

  Further, the data processing means 16 may generate the data using at least one of the expression variations of the component distribution in the plurality of stratum corneums set in advance. The data processing means 16 outputs the generated component distribution image in the stratum corneum to a screen such as the output means 12 and presents it to the user or the like, stores it in the storage means 13, or stores it in the external device via the transmission / reception means 18. Can be transmitted. An example of the component distribution image in the stratum corneum generated by the data processing means 16 will be described later.

  As described above, the data visualized in the stratum corneum is not limited to active ingredients contained in, for example, preparations.

  The quantification means 17 performs quantification using the image obtained by dropping a predetermined component on each layer of the stratum corneum collected by the image acquisition means 15. A specific quantification example by the quantification means 17 will be described later.

  The transmission / reception means 18 is an interface capable of transmitting / receiving an execution program for realizing the component distribution visualization process, information necessary for each process, and the like from an external device that can be connected using, for example, a communication network. Therefore, the transmission / reception means 18 can acquire the latest parameter information or the like from an external device or the like, or can transmit various information to the external device or the like.

  The control unit 19 controls the entire configuration of the image processing apparatus 10. For example, the control unit 19 controls at least one of mass spectrometry data acquisition, image acquisition and generation processing, and the like.

<Component distribution visualization device 10: hardware configuration example>
In the present embodiment, an execution program (component distribution visualization program) that causes a computer to execute each function of the component distribution visualization device 10 described above is generated and installed in, for example, a general-purpose PC, server, or the like, thereby realizing each process. . FIG. 2 is a diagram illustrating an example of a hardware configuration capable of realizing the component distribution visualization process according to the present embodiment.

  As shown in FIG. 2, the computer main body includes an input device 21, an output device 22, a drive device 23, an auxiliary storage device 24, a memory device 25, a CPU (Central Processing Unit) 26 that performs various controls, And a network connection device 27, which are connected to each other via a system bus B.

  The input device 21 includes, for example, a keyboard and a pointing device such as a mouse operated by a user, a voice input device such as a microphone, and the like, and inputs various operation signals such as execution of a program from the user.

  The output device 22 has a display for displaying various windows, data, and the like necessary for operating the computer main body that performs processing in the present embodiment, and displays the execution progress and results of the control program executed by the CPU 26.

  Here, the execution program installed in the computer main body in the present invention is provided by, for example, a portable recording medium 28 such as a USB (Universal Serial Bus) memory or a CD-ROM. The recording medium 28 can be set in the drive device 23, and an execution program included in the recording medium 28 is installed from the recording medium 28 to the auxiliary storage device 24 via the drive device 23.

  The auxiliary storage device 24 is a storage unit such as a hard disk, and stores an execution program according to the present embodiment, a control program provided in a computer, and the like, and performs input / output as necessary.

  The memory device 25 is a ROM (Read Only Memory), a RAM (Random Access Memory), or the like, and stores an execution program or the like read from the auxiliary storage device 24 by the CPU 26. Note that the auxiliary storage device 24 and the memory device 25 described above may be configured integrally as a single storage device.

  The CPU 26 controls processing of the entire computer, such as various operations and input / output of data with each hardware component, based on a control program such as an OS (Operating System) and an execution program stored in the memory device 25. Thus, the component distribution visualization process according to the present embodiment is realized. Various information necessary during the execution of the program may be acquired from the auxiliary storage device 24 and the execution result or the like may be stored.

  The network connection device 27 acquires an execution program from an external device connected to the communication network or executes the program by connecting to a communication network such as the Internet or a LAN (Local Area Network). The execution result obtained in this way or the execution program itself in this embodiment is provided to an external device or the like.

  With the hardware configuration described above, the component distribution visualization process according to the present embodiment can be executed. Further, an execution program may be installed and realized by a general-purpose personal computer or the like.

<Example of component distribution visualization>
Next, an example of the component distribution visualization process according to the present embodiment will be described. FIG. 3 is a flowchart illustrating an example of a component distribution visualization process according to the present embodiment.

  As shown in FIG. 3, the component distribution visualization apparatus 10 previously collects a stratum corneum from the upper arm portion with an adhesive sheet, analyzes each layer of the collected stratum corneum by DESI mass spectrometry, and analyzes the target region ( The mass spectrometry data of Region of Interest (ROI) is acquired (S01).

  Next, the component distribution visualization device 10 uses the mass analysis data of the target region obtained by the process of S01, for example, to determine the relationship between the position and intensity of ions of molecules of a predetermined component included in the target region of each layer. A component distribution image visualized with the color image is generated and acquired (S02).

  Next, the component distribution visualization apparatus 10 bins the pixel data of the component distribution image of each layer obtained by the process of S02 in a predetermined direction (S03).

  Next, the component distribution visualization device 10 compresses the component distribution image of each layer in which the pixel data has been binned by the processing of S03 at a predetermined compression rate in a predetermined direction, and uses the compressed image to determine the components in the stratum corneum. A distribution image is generated (S04).

  Next, the component distribution visualization apparatus 10 displays the component distribution image in the stratum corneum obtained by the process of S04 on the screen (S05).

  In addition, the component distribution visualization apparatus 10 quantifies using the image obtained by the process of S02, dripping a predetermined component on each layer of the stratum corneum collected by the process of S01 as needed (S06).

  Here, the component distribution visualization device 10 determines whether or not to end the process (S07), and ends the process when the process ends (for example, YES in S07) due to an end instruction from the user, for example. If the process is not completed (NO in S07), the process returns to S01.

  In the above-described process, the binned component distribution image of each layer is compressed at a predetermined compression rate in a predetermined direction in the process of S04. However, the present embodiment is not limited to this. For example, the compression may not be performed.

<Extracting stratum corneum by tape stripping>
FIG. 4 is a diagram for explaining a method for preparing a sample for evaluating transdermal absorbability. In the example of FIG. 4, for example, “IPSA (registered trademark) Metabolizer Moist White W1 (Cosmetic Liquid / Prototype)” (4MSK (4-methoxy salicic acid potassium salt), about 1% blended) is used.

First, wait about 30 minutes after washing the upper arm 31 with soap. Next, as shown in FIG. 4A, for example, about 0.7 g of the above-described sample is applied to the 40 × 40 mm ² region 32 of the upper arm portion 31, and left at room temperature for 1 hour. Next, the area | region 32 which apply | coated the sample is wash | cleaned with soap.

  Next, as shown in FIG. 4B, for example, the stratum corneum in the region 32 is collected with the adhesive sheet 40 such as Skin Checker (Promo Tool Co., Ltd.), and the stratum corneum attached to the adhesive portion of the adhesive sheet 40 is collected. For example, DESI mass spectrometry is performed. The adhesive sheet 40 is an example of an adhesive member. For collecting the stratum corneum using the adhesive member, for example, a general cellophane tape or the like may be used, or another member having adhesiveness may be used.

  FIG. 5 is a diagram showing an example of the structure of an adhesive sheet for collecting the stratum corneum. As shown in FIG. 5, the pressure-sensitive adhesive sheet 40 includes a support 41 made of, for example, a transparent resin and a stratum corneum peeling pressure-sensitive adhesive portion 42. Furthermore, the stratum corneum peeling adhesive part 42 has an adhesive part protecting release sheet 43. The stratum corneum peeling adhesive portion 42 is used by peeling off the adhesive portion protecting release sheet 43 during use.

  In addition, about the shape and size of the adhesive sheet shown in FIG. 5, it is not limited to this. Moreover, about the method of the percutaneous absorption test using an adhesive sheet, since a standard method can be used, description here is abbreviate | omitted.

  In this embodiment, for example, the subject is a healthy human (40's, one male), and the adhesive surface of the adhesive sheet 40 to which the stratum corneum of the subject is attached is cut out in a strip shape (for example, about 5 mm × 35 mm) from the center portion. For example, it is affixed on a glass substrate using a double-sided tape.

  FIG. 6 is a diagram illustrating an example of an adhesive sheet attached on a glass substrate. First, as shown in FIG. 6A, the human stratum corneum is sequentially collected from the outermost layer of the skin (first to fifth layers) by tape stripping operation using the adhesive sheet 40.

  Also, as shown in FIG. 6B, the adhesive surfaces of the adhesive sheets 40 are arranged on the glass substrate in the order of collection, and the target area (ROI) of each layer is set as, for example, 34 mm width × 5 mm height, (ROI) is analyzed by DESI mass spectrometry.

  FIG. 6C is an example image showing an adhesive sheet affixed on a glass substrate. In an actual example, for example, a double-sided tape is used on a 75 × 25 mm glass substrate (slide glass). Note that white dot portions shown in FIG. 6C indicate position markers.

  Here, the number of stratum corneum layers collected by one tape stripping operation as described above and the degree of uniformity depend on the collection site, age, degree of UV exposure, etc. However, it is assumed that, for example, one stratum corneum is collected per tape stripping operation.

<DESI mass spectrometry>
Next, DESI mass spectrometry performed on the target region described above will be described. FIG. 7 is a diagram for explaining DESI mass spectrometry.

As shown in FIG. 7A, a predetermined water / organic solvent of about 2 to 3 μL / min from the tip of the nozzle (Spray Tip) 51 with respect to the sample surface 50 included in the target region (ROI) described above. A mixed liquid (for example, formic acid 0.1%, acetonitol 50%) is sprayed with N ₂ gas. At this time, by applying a voltage of about 3 kV to the tip of the nozzle 51, a charged droplet is generated by an electrospray phenomenon, and this is sprayed onto the sample surface 50 in a jet form by N ₂ gas.

  Molecules knocked out of the sample surface 50 in the region of interest (ROI) by the sputtering phenomenon are ionized by charge exchange in the gas phase (and condensed phase), droplet pickup (Droplet Pickup), etc. An electric field (electric field gradient) under atmospheric pressure leads to an entrance (MS (Mass Spectrometry) Inlet (for example, an extended heating capillary for local sampling)) 52 of a mass spectrometer.

The molecular ions that have reached the entrance 52 are introduced into a high-vacuum (10 ⁻⁵ to 10 ⁻¹⁰ Torr) mass spectrometer, and the mass (strictly speaking, the ion mass divided by the charge m / z) and its mass Recorded as strength information. Since this m / z is unique to each molecule and its intensity is proportional to the amount of ions, it is possible to obtain qualitative / quantitative information of a plurality of molecules, for example, by one DESI mass analysis.

  In mass spectrometers that perform MS / MS (tandem mass spectrometry), target ions are isolated in the apparatus, and fragment ions that have been cleaved by collision with a gas are detected, so that qualities with high specificity can be obtained. Analysis and high-sensitivity quantification with a low detection limit are possible.

  As described above, by DESI mass spectrometry, it is possible to analyze (identify / quantify) multiple components contained in each layer of the stratum corneum attached to the sample surface 50 at a time. In addition, by controlling the composition and flow rate of the solvent to be sprayed, it is possible to extract molecules having a depth of about 0.001 to 0.1 mm, so in each layer of the stratum corneum tape stripped to the target region It is also possible to detect all the contained molecules. For example, if tape stripping is used, a sample can be collected almost non-invasively, and thus analysis on human skin is easy.

  In the present embodiment, the data obtained by the above-described DESI mass spectrometry is acquired by the mass analysis means 14 as mass analysis data.

Here, α shown in FIG. 7A indicates an incident angle, and β shown in FIG. 7 indicates a converging angle. D ₁ indicates the distance from the tip of the nozzle 51 to the sample surface 50, and d ₂ indicates the distance from the sample surface 50 to the center of the inlet portion 52. d ₃ indicates the distance from the tip of the nozzle 51 to the center of the inlet 52.

In order to improve the signal intensity (sensitivity) of the target ion using the above-described DESI mass spectrometry, the positional relationship among the sample surface 50, the nozzle 51, and the input unit 52 is important. For example, as a result of confirming the relationship between the signal intensity and stability of the target ion by experiment and examining the optimum conditions, the optimum values are d ₁ = 1.75 mm, d ₂ = 3.5 mm, d ₃ = 0.5 mm, Although α = 60 °, when d ₃ or α is changed, other parameters also change accompanyingly, so it is necessary to optimize each sample individually.

The optimum value varies depending on the droplets ejected from the nozzle 51, the N ₂ gas pressure (flow velocity), the state of the sample attached to the sample surface 50, the molecular weight / polarity of the sample molecule to be analyzed, etc. Correct as appropriate. Note that α, β, d ₁ , and d ₂ described above are ranges as shown in FIG. 7B (α = 0 to 90 °, β = 5 to 10 °, d ₁ = 1 to 10 mm, d ₂ = It is standard to set to 0-2 mm.

  In addition, since the sample surface 50 shown in FIG. 7 can be moved precisely and accurately at an arbitrary speed in the XY directions, the relative position between the nozzle 51 and the input unit 52 is kept constant while maintaining a predetermined time interval. By changing the sputtering position, it is possible to continuously acquire mass spectrometry data (such as the qualitative / quantitative information described above) at each point.

  Therefore, in this embodiment, the mass analysis unit 14 acquires the measurement point (XY) on the sample surface, the detected mass of the ion, and the total four-dimensional mass analysis data for each layer described above. The image acquisition means 15 visualizes the relationship between the position and intensity of ions of an arbitrary molecule on the sample surface and for each layer from the mass analysis data using a dedicated software described later (image of a predetermined color). Visualized component distribution images can be obtained. With this component distribution image, it is possible to simply and intuitively indicate the spatial position and amount of each of a plurality of molecules in each layer.

<Acquisition of component distribution image of each layer>
Next, the acquisition method of the component distribution image of each layer visualized using the mass spectrometry data in each layer mentioned above is demonstrated. FIG. 8 is a diagram for explaining the acquisition of the component distribution image of each layer. In addition, (1)-(6) shown to FIG. 8 (A), FIG. 8 (B), and FIG. 8 (D) respond | corresponds to each measurement position.

  As described above, it is possible to acquire mass spectrometry data for each measurement position by changing the sputtering position. Therefore, as shown in FIG. 8A, for example, XY coordinates are set for the target region (ROI), the moving speed and moving distance in the X direction, the number of steps in the Y direction, and the moving distance (step size). Set. In the example of FIG. 8A, for example, for a target area (ROI) of 5 mm × 5 mm, the moving speed in the X direction is 0.2 mm / second, the moving distance is 5 mm, the number of steps in the Y direction is 20, the step size is 0. It is set to 25 mm (number of steps 20 × 0.25 mm = movement distance 5 mm).

  In the example of FIG. 8A, for example, a mass spectrum (mass analysis data) acquired corresponding to the measurement mode in a line shape from, for example, the upper right starting point (for example, (1)), for example, for 25 seconds, Become. By repeating this step 19 times by 0.25 mm downward in the Y direction, a total of 20 sets of data can be obtained.

  FIG. 8B is an example of mass spectrometry data. For example, an MS spectrum including a mass chromatogram of m / z 167 obtained at the measurement position shown in FIG. 8A and a molecular ion corresponding to m / z 167. Is shown. The mass chromatogram is obtained by intensity tracing of predetermined ions.

  FIG. 8C is set by the user when the data of the mass chromatogram shown in FIG. 8B is converted by the data processing means 16 using software such as FireFly (registered trademark). An example of a screen is shown. Here, binning (division width) or the like in data conversion is set. For example, the number of data points in the Y direction shown in FIG. 8A is determined by the number of rows and the step size, whereas in the X direction shown in FIG. 8A, the measurement mass range, scan rate, automatic capture gain setting values, etc. The number of data points changes.

  Therefore, the data processing means 16 considers that the spatial resolution is the same in the XY direction, and when making the XY ratio of the target region (ROI) and the XY ratio of the measurement data constant, for example, the number of data points in the X direction is set to Y Binning processing is executed based on binning set to match the resolution of the direction (for example, setting the number of binning and segmentation time).

  Note that when the absolute intensity of the signal is weak (such as when the amount of ions entering the mass spectrometer is absolutely small), such as MS / MS measurement, the ion trap type mass spectrometer automatically increases the scan time and the X direction. Therefore, when binning is performed according to the resolution in the Y direction, data in the X direction may be lost. In such a case, for example, values such as Maximum Injection Time and Microscans may be adjusted to define the lower limit value of the measurement frequency.

  FIG. 8D shows an image obtained by visualizing the data converted by the image acquisition means 15 using, for example, the binning setting shown in FIG. 8C using software such as BioMap (registered trademark). The image shown in FIG. 8D is an image (component distribution image) in which the relationship between the position and intensity of ions of a predetermined m / z molecule is expressed by an image of a predetermined color, for example.

  In FIG. 8D, for example, the height of the signal intensity is indicated by a red-blue color (for example, a red-blue scale heat map in which high intensity is red, middle is green, and low intensity is blue) ). In this way, it is possible to indicate where and how much a component having a molecular specific mass exists in the two-dimensional space as imaging data of distribution and intensity.

  Note that since the heating capillary temperature of the input unit 52 in FIG. 7 described above affects the spatial resolution when generating the image shown in FIG. 8D, an optimum value is also set for the heating capillary temperature. And good.

<Specific example of component distribution image of each layer of stratum corneum>
Next, a specific example of the component distribution image of each layer acquired by the image acquisition unit 15 will be described using the mass spectrometry data described above. FIG. 9 is a diagram illustrating an example of a component distribution image of each layer of the stratum corneum.

  As shown in FIG. 9A, for example, starting from the upper right of the target region (ROI) having a width of 34 mm and a height of 5 mm, the control portion (product non-application portion), the fifth layer to the first layer are sequentially specified. A mass spectrum is acquired in a line shape at a moving speed (for example, 0.125 mm / second × 272 seconds), and a data set (mass spectrometry) is sequentially moved downward at a predetermined step size (for example, 0.2 mm). Data) is acquired for 25 lines.

  Here, in the measurement mode of mass spectrometry, in addition to MS scan measurement of m / z 50-250, MS / MS product ion measurement from m / z 167 is also performed for confirmation of specificity.

  FIG. 9B to FIG. 9D show component distribution images of each layer acquired by the image acquisition unit 15 using the mass spectrometry data acquired as described above. For example, FIG. 9 (B) and FIG. 9 (C) show an example of a component distribution image obtained by MS scan measurement of m / z 50-250, and FIG. 9 (D) shows the confirmation of specificity. An example of the component distribution image obtained by MS / MS product ion measurement from m / z 167 is shown.

  More specifically, FIG. 9B shows a component distribution image of m / z 167 corresponding to the above-described 4MSK, and FIG. 9C shows a component distribution image of m / z 187, for example. FIG. 9D shows a component distribution image of m / z 123 generated by MS / MS product ion measurement of m / z 167 corresponding to 4MSK.

  In the component distribution image shown in FIG. 9B, the abundance of 4MSK (m / z 167) transferred (percutaneously absorbed) from the above-described cosmetic liquid into the stratum corneum is clearly red in a red-blue scale on a two-dimensional plane. Is displayed. In addition, the difference between the stratum corneum layers and the difference in the two-dimensional distribution of the stratum corneum surface are clearly displayed.

M / z 123 displayed in a red-blue scale in the component distribution image of FIG. 9D is a characteristic fragment ion in which CO ₂ is desorbed from 4MSK. Therefore, the signal obtained by the MS / MS product ion measurement of m / z 167 → 123 can be almost uniquely assigned as being derived from 4MSK.

  Compared with the image of FIG. 9B and the image of FIG. 9D, the observed region of interest (ROI) is different, so the aspect of the two-dimensional distribution is different, but the amount trend between the stratum corneum is the same, It is shown that 4MSK is localized in the second and third layers under the present conditions.

  In addition, the sample described as “control” on the right side of the fifth layer is a stratum corneum collected from a site where the same washing operation was performed without applying the above-described cosmetic liquid. In the control region, no signal was observed for both m / z 167 and m / z 167 → 123. Therefore, both m / z 167 and m / z 167 → 123 shown in FIGS. 9 (B) and 9 (D) were in the stratum corneum. It can be said that 4MSK is specifically detected.

  FIG. 9C shows a component distribution image showing m / z 187 different from 4MSK. The structure of this molecule is not yet identified, but clearly shows a stratum corneum distribution different from 4MSK. Since the structure of this molecule was also detected from the stratum corneum at the control site described above, it was presumed to be an endogenous molecule derived from the skin, and the strength of the deeper stratum corneum was higher.

  As described above, a plurality of molecules can be analyzed by DESI mass spectrometry, and the percutaneous absorption kinetics of not only active ingredients but also endogenous molecules and base components can be analyzed simultaneously. The component distribution image visualized by using such DESI mass spectrometry data is visually appealing and easy to understand, and thus is useful for the development of a prescription with controlled transdermal absorbability.

<Relationship between component distribution image and mass chromatogram>
Next, the relationship between the heat map display of the component distribution image of each layer obtained by the image acquisition means 15 and the mass chromatogram obtained by DESI mass spectrometry will be described. FIG. 10 is a diagram showing the relationship between the heat map display and the mass chromatogram.

  FIG. 10A shows a distribution image of a component indicating m / z 167 in each layer, and FIG. 10B shows a mass chromatogram used to obtain the component distribution image shown in FIG. An example is shown. Lines 1 to 6 shown in FIG. 10 (A) correspond to the mass chromatograms of Lines 1 to 6 shown in FIG. 10 (B).

  For example, the strong part of the m / z 167 molecule in Line 1 shown in FIG. 10B is displayed in red on the component distribution image in FIG. 10A, and the m / z 167 shown in FIG. Corresponding to the intensity, a red-blue heat map is displayed on the component distribution image of FIG.

  Thus, since FIG. 10A is displayed in color based on the intensity information shown in FIG. 10B, the component distribution image shown in FIG. It is possible to display where and how much exists in the two-dimensional space.

<Method for visualizing transdermal absorption>
Next, a method for visualizing the percutaneous absorption state (component distribution) in the cross-sectional direction of the stratum corneum will be described. FIG. 11 is a diagram for explaining a method of visualizing the state of transdermal absorption. In the example of FIG. 11, in order to clarify the difference from the conventional method, FIG. 11A shows a conventional visualization method, and FIG. 11B shows the visualization method in the present embodiment.

  As shown in FIG. 11A, for example, in the conventional visualization method, the epidermis of a predetermined region is cut out, and a section of a slice including a stratum corneum of about 1 μm per layer is used as an observation plane (data acquisition plane). Visualize. In this method, only a low-sensitivity result is obtained because only the vicinity of the surface is observed, making it difficult to apply to human skin. In addition, the difference in percutaneous absorption between the stratum corneum cannot be grasped due to the limitation of observable resolution.

  On the other hand, in the visualization method of this embodiment shown in FIG. 11 (B), the plane (the surface seen from the arrow direction of FIG. 11 (B)) of the stratum corneum (about 1 μm of each layer) collected by the tape stripping operation described above. ) Is an observation plane (data acquisition plane), and information obtained from the observation plane is aggregated and stacked to obtain cross-sectional direction information, thereby visualizing the component distribution with high sensitivity.

  In other words, the information obtained from the observation surface of FIG. 11B detects the components contained in the layer of about 1 μm by the DESI mass spectrometry in the depth direction of the layer (for example, the direction of the arrow 60 that is the Z direction) as described above. Then, it is acquired as component distribution information of the entire surface (XYZ three-dimensional). Therefore, a result with higher sensitivity (good SN) can be obtained compared to the conventional visualization method in which only the vicinity of the surface in the cross-sectional direction of the conventional slice is observed. Hereinafter, the visualization method of the present embodiment will be specifically described.

  FIG. 12 is a diagram for explaining a method for visualizing a transdermal absorption state according to this embodiment. FIG. 12A shows a target region of the stratum corneum collected by tape stripping, and FIG. 12B shows a component distribution image of each layer described above. FIGS. 12C and 12D show component distribution images in the stratum corneum.

  First, for a predetermined target region (for example, 34 × 5 mm) shown in FIG. 12A, for example, the moving speed in the height direction (that is, the depth direction of the stratum corneum (cross-sectional direction)) is set to 0.125 mm / second. For example, scan measurement is performed with the step size in the width direction being 0.2 mm, and data is acquired with the number of data in the width direction being 25. At this time, the height / width ratio is 34/5.

  The data processing unit 16 acquires the pixels of the component distribution image of each layer generated by the image acquisition unit 15 from the data described above, and performs binning in the height direction of the data acquired from the target region shown in FIG. Thus, the component distribution image of each layer shown in FIGS. 12B and 12C is generated.

  In the example of FIG. 12B, the component distribution on the two-dimensional plane is represented by an XY ratio similar to that of the target region (ROI) shown in FIG. 12A, and an image similar to the observation plane is displayed. The number of bins (data points) in the height direction is set to 170.

  In the example of FIG. 12C, the binning number in the height direction (that is, the vertical direction) is set to 25, for example, because it is expressed as a component distribution on the cross section of the stratum corneum. Note that the number of data points in the width direction (that is, the horizontal direction) is 25, which is the number of data acquired in 0.2 mm steps in the horizontal direction in both FIG. 12 (B) and FIG. 12 (C). In the example of FIG. 12C, the number of pixels included in the width direction and the height direction of the image is aligned by the data processing unit 16, but the present invention is not limited to this.

  Here, for example, when performing binning in the height direction as shown in FIGS. 12B and 12C, the data processing unit 16 sets a predetermined number of adjacent pixels in the image in the height direction to one. Binning as a pixel is performed, for example, so that an average value of a predetermined number of adjacent pixel data becomes the data of the one pixel.

  That is, in the binning process, for example, one pixel is generated using a plurality of pixels, and is moved in each direction to generate an image in which the pixels are adjusted as a whole. Thereby, in the component distribution image shown in FIG. 12B or the component distribution image shown in FIG. 12C, it is possible to obtain a component distribution image obtained by smoothing the image in the height direction. In particular, since smoothing is performed using more pixels in FIG. 12C than in FIG. 12B, an image in which the component distribution is easy to recognize can be generated.

  Further, the component distribution image of each layer shown in FIG. 12B represents the three-dimensional component distribution information of each layer of the stratum corneum having a thickness of about 1 μm, as described in FIG. As shown in FIG. 12C, reducing the number of bins in the height direction (that is, the vertical direction) to 25 in the image shown in FIG. 12B approximates the image seen from the cross section of each stratum corneum layer. To do.

  Furthermore, in this embodiment, the data processing means 16 changes the XY ratio (X: height direction, Y: width direction) of the image and compresses it in the X direction with respect to the image shown in FIG. Thus, a component distribution image in the stratum corneum shown in FIG. In the example of FIG. 12D, the compression ratio is 1: 0.2 (height / width ratio: 5/25), but is not limited to this. By performing compression processing as shown in FIG. 12 (D), it can be grasped as a component distribution as the stratum corneum, to which layer in the stratum corneum the predetermined component penetrates, It is possible to visually and intuitively grasp whether a predetermined component is present in a layer at a high concentration.

  FIG. 13 is a diagram illustrating an example of a component distribution image in the stratum corneum obtained by the visualization method of the present embodiment. FIG. 13A shows an image obtained by simply compressing the component distribution image in which the number of data points in the height direction is binned so as to have the same XY ratio as that of the target region (ROI) as described above. 13B shows a component distribution image in the stratum corneum shown in FIGS. 12C and 12D described above, that is, a component distribution image subjected to binning to approximate the stratum corneum viewed from the cross-sectional direction. Show.

  In the example of FIG. 13B, as described above, since the compression is performed after adjusting the number of bins in the height direction to be smaller than usual, compared with the simple compressed image shown in FIG. Which layer in the stratum corneum has a high concentration of a given component is visually and intuitively expressed more naturally and is close to the image of each stratum corneum viewed from the cross-sectional direction. .

  Furthermore, FIG. 14 is a figure which shows the component distribution image in the stratum corneum corresponding to each example of FIG. 14A shows a component distribution image in the stratum corneum of m / z 167 corresponding to 4MSK in FIG. 9B, and FIG. 14B shows m / z corresponding to Unknown in FIG. 9C. The component distribution in the stratum corneum at z187 is shown. 14A and 14B are obtained by performing binning processing and compression processing similar to those in FIGS. 12C and 12D described above.

  In the component distribution image in the stratum corneum shown in FIG. 14A, the m / z 167 component is present in a higher concentration in the second and third layers of the stratum corneum corresponding to FIG. 9B. It is shown that. Similarly, in the component distribution image in the stratum corneum shown in FIG. 14B, the m / z 187 component is further increased in the fourth and fifth layers of the stratum corneum corresponding to FIG. 9C. It is shown to be present at high concentrations.

  As described above, the two-dimensional plane data (information on the position and amount of the component of each layer stripped by tape stripping) used to acquire the component distribution image of each layer of the stratum corneum is the depth when the stratum corneum is viewed as a cross section. Used as stacking information stacked in the vertical direction. At this time, it is possible to approximate the information when the stratum corneum is viewed from the cross-sectional direction by smoothing the data by the binning operation, instead of simply changing the XY ratio of the plane information and stacking.

  By visualizing the component distribution image in the stratum corneum in this way, it becomes possible to clearly distinguish and grasp the difference in percutaneous absorption between the stratum corneum without using a high-resolution device or method.

<Variation example of expression method>
Here, the component distribution image in the stratum corneum has been described using, for example, an image example by heat map display, but the image example generated in the present embodiment is not limited to this. For example, you may display as a variation imitating the detection method using a fluorescent probe.

  FIG. 15 is a diagram illustrating an example of a variation of the component distribution image in the stratum corneum. FIG. 15A is a diagram showing an example of a component distribution image in the stratum corneum in the heat map display described above, and FIG. 15B is a stratum corneum displayed in a blue-white scale. It is a figure which shows an example of an inside component distribution image.

  FIG. 15C is a diagram showing an example of a component distribution image in the stratum corneum displayed on a black-white scale, and FIG. 15D shows a red temperature scale (Red Temperature scale). It is a figure which shows an example of the component distribution image in the stratum corneum displayed by (scale).

Further, FIG. 15E shows a green-white scale (Green-White).
It is a figure which shows an example of the component distribution image in the stratum corneum displayed by (scale).

  15A to 15E show component distributions in the same stratum corneum, and the display method is changed. Moreover, the kind of variation is not limited to the example shown to FIG. 15 (A)-(E). In this embodiment, among the above-described variations, at least one expression method according to the content of the component is set in advance, and a component distribution image in the stratum corneum is generated and displayed on the screen with the set variation. Good.

<About quantification>
Here, an internal standard labeled with a stable isotope is indispensable for quantification by the above-described DESI mass spectrometry in order to correct a change in the intensity of the target signal depending on the state of the measurement site. On the other hand, when quantifying components in human skin, it is impossible to add an internal standard substance for safety.

  Therefore, in this embodiment, as an alternative method of quantification by the internal standard method, quantification is performed by the external standard method (absolute calibration curve method).

  FIGS. 16 and 17 are diagrams (No. 1 and No. 2) for explaining the quantitative method using the component distribution image of the standard product. In the example of FIG. 16A, quantitative results (first and second times) obtained from a component distribution image of 4MSK dropped on the stratum corneum stripped are shown.

  As shown in FIG. 16A, the dropped sample solution shows a good circular shape due to the moderate hydrophobicity of the stratum corneum. The component distribution image obtained from the spot after drying shows that each spot is moderately homogeneous, and the dropped sample is almost uniformly distributed.

In addition, as shown in FIG. 16B, the calibration curve shows good linearity with r ² = 0.996 to 0.998 between 2.5 and 250 ng. The reproducibility of 2 sets of repetition was also good.

Further, when the amount of 4MSK in the stratum corneum (FIG. 9 or FIG. 14) of tape stripping is estimated by an external standard method based on a calibration curve as shown in FIG. 16 (B), the first layer is 2.2 ng / mm. ² and the second to fifth layers were 4.2, 4.0, 2.7, and 1.9 ng / mm ² , respectively (FIG. 17).

Assuming that all 4MSKs are extracted and detected from the stratum corneum having a tape stripping of about 0.001 mm, a total of 14.9 ng / mm ² is percutaneously absorbed and transferred into the stratum corneum. As described in the sample preparation method of FIG. 4 described above, since the amount of 4MSK applied in this case is 4.38 μg / mm ² (1%, 4MSK × 0.7 g / 40 × 40 mm ² ), The transdermal absorption rate can be estimated as 0.34%.

  For example, the percutaneous absorption rate of 4MSK in a conventional method (for example, when the amount of 4MSK in a stratum corneum (multiple layers) peeled off by an instantaneous adhesive is measured by LC / MS) is about 1% or less. Thus, it can be said that the above-described method can also obtain a substantially reasonable quantitative value.

  As described above, the component distribution image can be used to realize highly accurate quantification of the component distribution. The above-described quantification can be realized by the quantification means 17, and the content to be quantified is not limited to this.

  As described above, according to the present embodiment, the component distribution in the stratum corneum can be easily visualized. Moreover, since it is possible to observe and evaluate not only the preparation but also a plurality of components such as a base and an active agent at a time, it is useful for formulation development. Furthermore, the two-dimensional plane data of each layer of the stratum corneum obtained by tape stripping is versatile because it can be used for a wide range of analysis data such as IR (infrared absorption spectrum) and Raman spectrum.

  The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific embodiments according to the present invention, and various modifications may be made within the scope of the gist of the present invention described in the claims. Can be changed.

DESCRIPTION OF SYMBOLS 10 Component distribution visualization apparatus 11 Input means 12 Output means 13 Recording means 14 Mass analysis means 15 Image acquisition means 16 Data processing means 17 Quantification means 18 Transmission / reception means 19 Control means 21 Input device 22 Output device 23 Drive device 24 Auxiliary storage device 25 Memory device 26 CPU
27 Network connection device 28 Recording medium 31 Upper arm portion 32 Region 40 Adhesive sheet 41 Support body 42 Adhesive portion for exfoliating stratum corneum 43 Adhesive portion protecting release sheet 50 Sample surface 51 Nozzle 52 Inlet portion

Claims (11)

A component distribution visualization device for visualizing the component distribution in the stratum corneum,
Mass analysis means for analyzing each layer of the stratum corneum collected by a predetermined adhesive member by predetermined mass analysis and obtaining mass spectrometry data including component amount information in the surface and depth directions of each layer;
Image acquisition means for acquiring a component distribution image of each layer using mass analysis data including component amount information in the surface and depth directions of each layer obtained by the mass analysis means ;
Data processing for compressing the component distribution image of each layer in a predetermined direction, laminating the compressed image with the predetermined direction corresponding to the depth direction of the stratum corneum, and forming the component distribution image in the stratum corneum component distribution visualizing device, characterized in that it comprises a means. Wherein the data processing means, component distribution visualizing device according to claim 1, characterized that you binning pixel data of the obtained layers of component distribution image by the image acquiring means in a predetermined direction. The data processing means includes
Component distribution visualizing device according to claim 2, characterized in that said pixel data is a spectrum distribution image of each layer are binned, compressed in Jo Tokoro compression ratio. The data processing means includes
The pixel data of the component distribution image of each layer, component distribution visualizing device according to claim 2 or 3, characterized in that binning the depth direction. The mass spectrometry means includes
5. The component distribution visualization apparatus according to claim 1, wherein each layer of the stratum corneum is analyzed by DESI mass spectrometry to obtain the mass spectrometry data. A component distribution visualization method for visualizing component distribution in the stratum corneum,
A mass spectrometry procedure for analyzing each layer of the stratum corneum collected by a predetermined adhesive member by predetermined mass spectrometry and obtaining mass spectrometry data including component amount information in the surface and depth directions of each layer,
An image acquisition procedure for acquiring a component distribution image of each layer using mass analysis data including component amount information in the surface and depth directions of each layer obtained by the mass analysis procedure ;
Data processing for compressing the component distribution image of each layer in a predetermined direction, laminating the compressed image with the predetermined direction corresponding to the depth direction of the stratum corneum, and forming the component distribution image in the stratum corneum component distribution visualization method characterized by having a step. Wherein the data processing procedure, component distribution visualizing method according to claim 6, characterized that you binning pixel data of the spectrum distribution image obtained layers by the image acquisition procedure in a predetermined direction. The data processing procedure is as follows:
Component distribution visualization method according to claim 7, characterized in that said pixel data is a spectrum distribution image of each layer are binned, compressed in Jo Tokoro compression ratio. The data processing procedure is as follows:
Component distribution visualization method according to claim 7 or 8, wherein the pixel data of the component distribution image of each layer, binning the depth direction. The mass spectrometric procedure is:
The component distribution visualization method according to any one of claims 6 to 9, wherein each layer of the stratum corneum is analyzed by DESI mass spectrometry to acquire the mass spectrometry data. Computer
The component distribution visualization program for functioning as each means which the component distribution visualization apparatus as described in any one of Claims 1 thru | or 5 has.