JP2013015516A - Analysis method and analysis kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an analysis method which is capable of quickly analyzing components of a skin external preparation penetrating skin.SOLUTION: The analysis method is a method for analyzing components of a skin external preparation penetrating skin and includes: a step of applying the skin external preparation to skin; a step of peeling a plurality of horny layers from a region to which the skin external preparation has been applied, of the skin by using a plurality of adhesive tapes; a step of using a DART ion source 200 and successively introducing ions generated from the plurality of peeled horny layers into a mass spectrometer 300 to perform mass analysis.

Description

  The present invention relates to an analysis method and an analysis kit.

  Conventionally, as a method for analyzing a compound that has penetrated into the skin, a method is known in which the concentration of the compound in the stratum corneum peeled by a tape stripping method is quantified using HPLC (see Patent Document 1).

  However, since it is necessary to extract the compound from the stratum corneum peeled by the tape stripping method, there is a problem that rapid quantification cannot be performed.

  On the other hand, various methods are known as ionization methods in mass spectrometry. Recently, DART (Direct Analysis in Real Time) and DESI (Desorption Electrospray Ionization) have been attracting attention (see Patent Document 2).

As DART, a method is known in which atoms or molecules in an electronically excited state are collided with water in the atmosphere and subjected to Penning ionization, and protons generated are added to a sample for ionization. For example, when helium He (2 ³ S) in a metastable excited state is used, the sample M can be ionized as follows.

He (2 ³ S) + H ₂ O → H ₂ O ^{+ *} + He (1 ¹ S) + e ⁻
H ₂ O ^{+ *} + H ₂ O → H ₃ O ⁺ + OH ^*
H ₃ O ⁺ + nH ₂ O → [(H ₂ O) _n H] ⁺
[(H ₂ O) _n H] ⁺ + M → MH ⁺ + nH ₂ O
DESI is a method of desorbing ions by attaching an ionized solvent to a sample.

WO2007 / 136067 JP 2008-180659 A

  The present invention has been made in view of the above-described problems of the prior art, and an object of the present invention is to provide an analysis method capable of quickly analyzing components of a skin external preparation that has penetrated into the skin.

  The analysis method of the present invention is a method for analyzing a component of a skin external preparation that has penetrated into the skin, the step of applying the skin external preparation to the skin, and a plurality of layers from the area of the skin where the skin external preparation is applied. A step of peeling the stratum corneum, and a step of performing mass analysis by sequentially introducing ions generated from the peeled stratum corneum into a mass spectrometer using DART or DESI.

  The analysis kit of the present invention is an analysis kit used for analyzing components of a skin external preparation that has penetrated into the skin, and includes a plurality of adhesive tapes that are cut at predetermined intervals, and plate-like members or columnar members. Have.

  The analysis kit of the present invention is an analysis kit used for analysis of components of a skin external preparation that has penetrated into the skin, and includes a plurality of adhesive tapes, a first plate-like member, and a plurality of penetrations with a predetermined interval therebetween. A device having a second plate-like member in which a hole is formed is provided.

  ADVANTAGE OF THE INVENTION According to this invention, the analysis method which can analyze rapidly the component of the skin external preparation which permeate | transmitted skin can be provided.

It is a figure which shows an example of the adhesive tape in which the cut | interruption is made | formed by the predetermined space | interval. It is a top view which shows an example of the plate-shaped member to which the some adhesive tape cut | disconnected is being fixed. It is a schematic diagram which shows an example of the mass spectrometry method using the flat plate of FIG. It is a bottom view which shows the other example of the plate-shaped member to which the some adhesive tape cut | disconnected is fixed. It is a figure which shows an example of the columnar member to which the some adhesive tape cut | disconnected is being fixed. It is a schematic diagram which shows an example of the mass spectrometry method using the right isosceles triangular prism of FIG. It is sectional drawing which shows the modification of the right-angled isosceles triangular prism of FIG. It is sectional drawing which shows the modification of a columnar member. It is a figure which shows an example of the device by which the some adhesive tape is clamped. It is sectional drawing which shows the modification of the device with which the some adhesive tape cut | disconnected is clamped. It is sectional drawing which shows the modification of a 2nd plate-shaped member. It is a mass spectrum of the 1st stratum corneum peeled from the area | region where the cream agent of the upper arm inner side part of Example 1 was apply | coated. 2 is a mass chromatogram of Example 1 at m / z = 338.1. 2 is a mass chromatogram of Example 1 at m / z = 473.4. 4 is a mass chromatogram of Reference Example 1 at m / z = 473.4. 4 is a mass chromatogram of Reference Example 2 at m / z = 473.4.

  Next, the form for implementing this invention is demonstrated with drawing.

  The analysis method of the present invention is a method for analyzing components of a skin external preparation that has penetrated into the skin, the step of applying the skin external preparation to the skin, and a plurality of corners of the skin from the area where the skin external preparation is applied. A step of peeling the layers, and a step of sequentially introducing ions generated from the plurality of peeled stratum corneums into the mass spectrometer using DART or DESI for mass analysis.

  In the first embodiment of the analysis method of the present invention, the skin external preparation is applied using a step of applying the skin external preparation to the skin and a plurality of adhesive tapes that are cut at predetermined intervals. A step of peeling a plurality of stratum corneum from the region.

  Although it does not specifically limit as a skin external preparation, A solid preparation for external use, a liquid for external use, a spray agent, an ointment, a cream agent, a gel agent, a patch, etc. are mentioned.

  Although it will not specifically limit if it can apply to a tape stripping method as an adhesive tape, D-Squame (made by CuDerm), Cellotape (registered trademark) (made by Nichiban), PPS tape (made by Nichiban) Etc.

  FIG. 1 shows an example of an adhesive tape in which cuts are made at a predetermined interval. (A) and (b) are a top view and a cross-sectional view, respectively.

  The pressure-sensitive adhesive tape 10 has a circular shape when viewed from above, and has a pressure-sensitive adhesive layer 10b formed on the surface of the base material 10a, and three cuts 11 are formed at predetermined intervals. For this reason, the components of the external preparation for skin permeating into the skin can be analyzed a plurality of times while changing the conditions as necessary.

  The shape of the adhesive tape 10 is not limited to a circular shape when viewed from above, and includes a rectangular shape, an elliptical shape, and the like when viewed from above.

  Although it does not specifically limit as a method of making the cut | interruption 11 in the adhesive tape 10 with a predetermined space | interval, The method etc. which make a cut | interruption using a cutter are mentioned.

Spacing _{G 1} of the cut 11 is usually 0.5 to 5.0 mm, 1.0 to 3.0 mm is preferred. If the interval _{G 1} is less than 0.5 mm, sometimes the operability is lowered, and when it exceeds 5.0 mm, it may become unavailable adhesive tape 10 efficiently.

  Although the number of the cut | interruptions 11 is not specifically limited, Usually, it is 2-6 pieces.

  In addition, as for the adhesive tape 10 before use, the peeling material is hold | maintained on the surface of the adhesive layer 10b.

  The first embodiment of the analysis method of the present invention includes a step of cutting a plurality of adhesive tapes from which a plurality of stratum corneums have been peeled with a width corresponding to the interval between the cuts, and a plurality of cut adhesive tape stratums. The step of fixing the surface opposite to the fixed side to the plate-like member or the columnar member at a predetermined interval and the plate-like member or the columnar member to which the plurality of cut adhesive tapes are fixed are moved. Then, ions generated from a plurality of stratum corneum are sequentially introduced into a mass spectrometer and subjected to mass analysis.

  FIG. 2 shows a flat plate 20 as an example of a plate-like member to which a plurality of cut adhesive tapes are fixed.

  Two double-sided tapes 21 are fixed to the flat plate 20 substantially parallel to the length direction L. For this reason, the base material of the some adhesive tape 10 'cut | disconnected can be fixed.

  The material constituting the flat plate 20 is not particularly limited as long as it has heat resistance, but glass, quartz glass, ceramics, titanium, stainless steel, aluminum, iron, acrylic resin, polypropylene, fluororesin, vinyl chloride resin , Nylon, polystyrene, polycarbonate, silicone resin and the like.

  In addition, as for the flat plate 20 before use, the peeling material is hold | maintained on the surface of the adhesive layer of the double-sided tape 21. FIG.

Is fixed to the flat plate 20, the spacing _{G 2} of which are adjacent to cut the adhesive tape 10 'is typically a 1.0~50.0mm, 5.0~10.0mm are preferred. If the interval G ₂ is less than 1.0 mm, may be reduced quantitative of the components of the osmotic skin external preparation to the skin, when it exceeds 50.0 mm, it may become unavailable to flat plate 20 efficiently .

The flat plate 20 is determined according to the distance G ₁ and G _2, the position 1~N for fixing is displaying the cut adhesive tape 10 '. At this time, N is usually an integer of 1 to 90. Further, the flat plate 20 displays a position Blank for fixing the cut adhesive tape 10 ″ which is the same as the cut adhesive tape 10 ′ except that the stratum corneum is not peeled off.

  A standard sample layer 22 for mass calibration is formed on the flat plate 20 substantially parallel to the width direction W. For this reason, when mass analysis is performed using the flat plate 20 to which the plurality of cut adhesive tapes 10 ′ are fixed, the mass spectrometer can be calibrated and the sensitivity can be corrected.

  The calibration reagent is not particularly limited as long as ions can be generated using a DART ion source. However, in consideration of the accuracy of mass calibration, polyethylene glycol (mass spectrum peaks present at equal intervals) PEG60 to PEG2000), fatty acids having 4 to 36 carbon atoms, and the like, and two or more kinds may be used in combination.

  The method for forming the mass calibration standard sample layer 22 is not particularly limited, but there is a method for fixing the adhesive tape in which the solution of the mass calibration standard sample is applied to the substrate to the flat plate 20, and a mass calibration standard sample. Examples thereof include a method of drawing on the flat plate 20 using a pen filled with the added ink, and a method of applying ink to which the standard sample for mass calibration is added to the flat plate 20.

  The some adhesive tape 10 and the flat plate 20 can be used as an analysis kit used for the analysis of the component of the skin external preparation which permeated the skin.

  Note that the two double-sided tapes 21 do not have to be fixed to the flat plate 20, and a plurality of cut adhesive tapes 10 'may be fixed to the flat plate using an adhesive.

  Further, the standard sample layer 22 for mass calibration may not be formed on the flat plate 20.

  Furthermore, the flat plate 20 has a standard sample layer for quantification for quantifying the components of the external preparation for skin that has penetrated into the skin, instead of the standard sample layer 22 for mass calibration, or together with the standard sample layer 22 for mass calibration. It may be formed substantially parallel to the width direction W.

  The standard sample for quantification is a component of the external preparation for skin to be quantified, but may also serve as a standard sample for mass calibration.

  The method for forming the standard sample layer for quantification is not particularly limited, but after removing the stratum corneum from the area of the skin where the external preparation for skin is not applied, a predetermined amount of the standard sample solution for quantification is applied to the stratum corneum. A method of fixing the cut adhesive tape, which is the same as the cut adhesive tape 10 ', to the flat plate 20 except that it is applied to the flat plate 20, and a predetermined amount of a standard solution for quantification is applied to the adhesive layer. Except for the above, there is a method of fixing the cut adhesive tape identical to the cut adhesive tape 10 ″ to the flat plate 20.

  Further, instead of the adhesive tape 10, an adhesive tape without a cut may be used. In this case, the adhesive tape from which the stratum corneum has been peeled may be cut with a predetermined width or may not be cut.

  Furthermore, instead of peeling off the stratum corneum from the skin using the adhesive tape 10, an adhesive such as Aron Alpha (manufactured by Toagosei Co., Ltd.) may be applied to the skin to peel off the stratum corneum.

FIG. 3 shows an example of a mass spectrometry method using the flat plate 20. First, in a substantially horizontal plane, a flat plate 20 in which a plurality of cut adhesive tapes 10 ′ are fixed to a sample stage 100 that can be moved in the direction of the arrow in the figure is a length direction L of the flat plate 20. Place it so that it is approximately parallel to the arrow direction. Next, while moving the sample stage 100 in the direction of the arrow in the figure, using the DART ion source 200, helium He (2 ³ S) in a metastable excited state is collided with water in the atmosphere to be penning ionized. In the figure, protons generated by irradiating the flat plate 20 at a predetermined angle with respect to a substantially horizontal plane substantially perpendicular to the direction of the arrow are introduced into the mass spectrometer 300 and subjected to mass spectrometry. To do. At this time, the predetermined angle is usually 1 to 45 °. For this reason, two or more types of components of the external preparation for skin permeating into the skin can be analyzed simultaneously.

  At this time, since the resistance heating wire 311 is wound around the ion introduction tube 310 of the mass spectrometer 300, the ion introduction tube 310 is connected by applying a voltage to the resistance heating wire 311 using a power source (not shown). The generated ions can be subjected to mass spectrometry while being heated. Thereby, adhesion of the generated ions to the ion introduction tube 310 can be suppressed. As a result, it is possible to improve the quantitativeness of the component of the external preparation for skin that has penetrated into the skin. At this time, the inside of the ion introduction tube 310 is depressurized by a compressor (not shown).

  In addition, since the produced | generated ion tends to adhere to the ion introduction side of the ion introduction tube 310, the resistance heating wire 311 is usually wound around the ion introduction side of the ion introduction tube 310.

  The temperature of the inner wall of the ion introduction tube 310 when the ion introduction tube 310 is heated is usually 50 to 500 ° C, preferably 100 to 300 ° C. If the temperature of the inner wall of the ion introduction tube 310 is less than 50 ° C., ions generated on the ion introduction tube 310 may adhere, and if it exceeds 500 ° C., the mass spectrometer 300 may be adversely affected.

  Note that the method of heating the ion introduction tube 310 is not limited to the method of heating by heating the resistance heating wire 311, the method of heating using a ceramic fiber heater, the method of heating by irradiating microwaves, the hot air fan The method etc. which heat using are mentioned.

  Alternatively, the ion introduction tube 310 may be removed and the ion introduction port may be directly heated.

  Further, when ions generated on the ion introduction tube 310 are difficult to adhere, the ion introduction tube 310 may not be heated.

  The material constituting the ion introduction tube 310 is not particularly limited as long as it has heat resistance. Examples thereof include ceramics, glass, Teflon (registered trademark), stainless steel, niobium steel, and tantalum steel.

  The inner surface of the ion introduction tube 310 may be coated with fluorine resin, polyether ether ketone, silicone resin, or the like.

  The material forming the resistance heating wire 311 is not particularly limited, but a metal heating element such as an iron-chromium-aluminum alloy or a nickel-chromium alloy; a refractory metal heating element such as platinum, molybdenum, tantalum, or tungsten; Non-metallic heating elements such as silicon carbide, molybdenum-silicite, and carbon can be used.

  For example, when a nichrome wire having a diameter of 0.26 mm is used as the resistance heating wire 311, a current of 1 to 6 A is passed.

  Instead of the flat plate 20, a position corresponding to a position where the plurality of cut adhesive tapes 10 ′ on the surface opposite to the side where the plurality of cut adhesive tapes 10 ′ are fixed is fixed. A flat plate 20 ′ (see FIG. 4) identical to the flat plate 20 may be used except that the resistance heating wire 23 is installed. Thereby, in mass spectrometry, it can heat to predetermined temperature by applying a voltage to resistance heating wire 23 using a power supply (not shown). At this time, when a voltage is applied to the resistance heating wire 23 at a timing when the pressure-sensitive adhesive tape 10 ′ or 10 ″ obtained by cutting protons generated by Penning ionization is irradiated, the generated ions are efficiently desorbed. Can do.

  The material constituting the resistance heating wire 23 is not particularly limited, but a metal heating element such as an iron-chromium-aluminum alloy or nickel-chromium alloy; a refractory metal heating element such as platinum, molybdenum, tantalum, or tungsten; Non-metallic heating elements such as silicon carbide, molybdenum-silicite, and carbon can be used.

  A plurality of cut adhesive tapes 10 ′ are fixed to the flat plate 20 over a plurality of rows, and a sample stage that can be moved in the length direction and the width direction of the flat plate 20 is used instead of the sample stage 100. Mass spectrometry may be performed.

Further, instead of helium He (2 ³ S) in the metastable excited state, neon in the metastable excited state, argon in the metastable excited state, nitrogen in the metastable excited state, or the like may be used.

  Further, instead of the DART ion source 200, using a DESI ion source, an ionized solvent may be attached to the sample to desorb ions.

  Although it does not specifically limit as a solvent to ionize, Methanol, methanol aqueous solution, acetonitrile, acetonitrile aqueous solution etc. are mentioned.

  The solvent to be ionized may contain an acidic substance or a basic substance.

  FIG. 5 shows a right isosceles triangular prism 40 as an example of a columnar member to which a plurality of cut adhesive tapes are fixed. (A) and (b) are a perspective view and a sectional view, respectively.

One double-sided tape 41 is fixed to the surfaces P ₁ and P ₂ sandwiching the corner C of the right-angled isosceles triangular prism 40 so as to be substantially parallel to the height direction H. For this reason, the base material of several cut | disconnected adhesive tape 10 'can be fixed.

  The material constituting the right-angled isosceles triangular prism 40 is not particularly limited as long as it has heat resistance, but glass, quartz glass, ceramics, titanium, stainless steel, aluminum, iron, acrylic resin, polypropylene, fluororesin, Examples include vinyl chloride resin, nylon, polystyrene, polycarbonate, and silicone resin.

  The right isosceles triangular prism 40 before use has a release material held on the surface of the adhesive layer of the double-sided tape 41.

Is fixed to the right-angled isosceles triangular prism 40, gap _{G 2} of which are adjacent to cut the adhesive tape 10 'is typically a 1.0~50.0mm, 5.0~10.0mm are preferred. If the interval G ₂ is less than 1.0 mm, it may be reduced quantitative of the components of the osmotic skin external preparation to the skin, when it exceeds 50.0 mm, can not be utilized isosceles right triangular prism 40 efficiently Sometimes.

Positions 1 to N for fixing the cut adhesive tape 10 ′ determined according to the intervals G ₁ and G ₂ are displayed on the surfaces P ₁ and P ₂ sandwiching the corner C of the right isosceles triangular prism 40. ing. At this time, N is usually an integer of 1 to 90. Further, the cut adhesive tape 10 ′ that is the same as the cut adhesive tape 10 ′ is the same as the cut adhesive tape 10 ′ except that the stratum corneum is not peeled off on the surfaces P ₁ and P ₂ that sandwich the corner C of the right-angled isosceles triangular prism 40. A position Blank for fixing 'is displayed.

On the surfaces P ₁ and P ₂ sandwiching the corner C of the right-angled isosceles triangular prism 40, a mass calibration standard sample layer 42 is formed substantially parallel to the side directions S ₁ and S ₂ , respectively. For this reason, when mass analysis is performed using the right isosceles triangular prism 40 to which the plurality of cut adhesive tapes 10 ′ are fixed, the mass spectrometer can be mass calibrated and the sensitivity can be corrected.

  The calibration reagent is not particularly limited as long as ions can be generated using a DART ion source. However, in consideration of the accuracy of mass calibration, polyethylene glycol (mass spectrum peaks present at equal intervals) PEG60 to PEG2000), fatty acids having 4 to 36 carbon atoms, and the like, and two or more kinds may be used in combination.

  The method for forming the mass calibration standard sample layer 42 is not particularly limited, but a method for fixing an adhesive tape in which a solution of the mass calibration standard sample is applied to the base material to the right isosceles triangular prism 40, and for mass calibration. A method of drawing on a right isosceles triangular prism 40 using a pen filled with ink to which a standard sample is added, a method of applying ink to which a standard sample for mass calibration is added to a right isosceles triangular prism 40, and the like. Can be mentioned.

  The plurality of pressure-sensitive adhesive tapes 10 and right-angled isosceles triangular prisms 40 can be used as an analysis kit used for analyzing components of a skin external preparation that has penetrated into the skin.

Note that the surface P ₁ and P ₂ sandwiching the corners C of the right-angled isosceles triangular prism 40 may two-sided tape 41 is not be fixed, using an adhesive, a plurality of adhesive tape has been cut 10 'may be fixed.

Further, the mass calibration standard sample layer 42 may not be formed on the surfaces P ₁ and P ₂ sandwiching the corner portion C of the right isosceles triangular prism 40.

Further, on the surfaces P ₁ and P ₂ sandwiching the corner portion C of the right-angled isosceles triangular prism 40, instead of the mass calibration standard sample layer 42 or together with the mass calibration standard sample layer 42, the skin external use that penetrates the skin. The standard sample layer for quantification for quantifying the component of the agent may be formed substantially parallel to the side directions S ₁ and S ₂ , respectively.

  The standard sample for quantification is a component of the external preparation for skin to be quantified, but may also serve as a standard sample for mass calibration.

  The method for forming the standard sample layer for quantification is not particularly limited, but after removing the stratum corneum from the area of the skin where the external preparation for skin is not applied, a predetermined amount of the standard sample solution for quantification is applied to the stratum corneum. A method of fixing the cut adhesive tape identical to the cut adhesive tape 10 ′ to the right-angled isosceles triangular prism 40, except that it is applied to the adhesive tape, and a predetermined amount of the standard sample solution for quantification to the adhesive layer. A method of fixing the cut adhesive tape, which is the same as the cut adhesive tape 10 ″, to the right-angled isosceles triangular prism 40 except that it is applied may be used.

FIG. 6 shows an example of a mass spectrometry method using a right isosceles triangular prism 40. In FIG. 6, the same components as those in FIG. 3 are denoted by the same reference numerals, and description thereof is omitted. First, a right isosceles triangular prism 40 having a plurality of cut adhesive tapes 10 ′ fixed to a sample stage 100 that can be moved in the direction of the arrow in the drawing in a substantially horizontal plane is a right isosceles triangular prism 40. Is placed so that the height direction H is substantially parallel to the arrow direction. At this time, the right-angled isosceles triangular prism 40 is arranged so that the vertical plane faces the DART ion source 200 with respect to the sample stage 100 of the right-angled isosceles triangular prism 40. Next, while moving the sample stage 100 in the direction of the arrow in the figure, using the DART ion source 200, helium He (2 ³ S) in a metastable excited state is collided with water in the atmosphere to be penning ionized. The ions generated by irradiating the corner C of the right-angled isosceles triangular prism 40 with the proton generated in the horizontal plane approximately perpendicular to the direction of the arrow in the figure are introduced into the mass spectrometer 300. Perform mass analysis. For this reason, two or more types of components of the external preparation for skin permeating into the skin can be analyzed simultaneously. Moreover, the detection sensitivity of the component of the external preparation for skin which permeated the skin can be improved.

  Note that the right isosceles triangular prism 40 may be arranged so that the vertical plane faces the mass spectrometer 300 with respect to the sample stage 100 of the right isosceles triangular prism 40.

  Further, instead of the right-angled isosceles triangular prism 40, the right-angled isosceles triangular prism 40 is provided except that a resistance heating wire is installed at a position corresponding to the position where the plurality of adhesive tapes 10 'cut inside is fixed. The same right isosceles triangular prism may be used. Thereby, in mass spectrometry, it can heat to predetermined temperature by applying a voltage to a resistance heating wire using a power supply (not shown). At this time, when a voltage is applied to the resistance heating wire at the timing when the pressure-sensitive adhesive tape 10 ′ or 10 ″ obtained by cutting protons generated by Penning ionization is irradiated, the generated ions can be efficiently desorbed. it can.

  The material constituting the resistance heating wire is not particularly limited, but a metal heating element such as an iron-chromium-aluminum alloy or nickel-chromium alloy; a refractory metal heating element such as platinum, molybdenum, tantalum, or tungsten; Non-metallic heating elements such as silicon, molybdenum-silicite, and carbon can be used.

  Furthermore, instead of the right-angled isosceles triangular prism 40, a hollow right-angled isosceles triangular prism may be used, or a plate in which corners are formed by bending may be used.

Further, instead of the right-angled isosceles triangular prism 40, a columnar member (see FIG. 7A) in which the corner C of the right-angled isosceles triangular prism 40 is rounded, A trapezoidal column from which the equilateral triangular prism is removed (see FIG. 7B), and a rectangular column from which the triangular prism including the corner C is removed from the right-angled isosceles triangular prism 40 (see FIGS. 7C and 7D) may be used. . At this time, θ ₁ is usually 90 to 180 °, and θ ₂ is usually 136 to 180 °.

FIG. 8 shows a modification of the columnar member. At this time, θ ₃ is usually 90 to 135 °. Moreover, since the cross-section of the columnar member of FIGS. 8D to 8G is n-fold symmetric, it can be analyzed n times using one cut adhesive tape 10 ′.

  The second embodiment of the analysis method of the present invention is the same as the first embodiment of the analysis method of the present invention except that an adhesive tape that is not cut at predetermined intervals is used. A step of applying to the skin, and a step of peeling a plurality of stratum corneums from a region of the skin to which the external preparation for skin is applied, using a plurality of adhesive tapes.

  The second embodiment of the analysis method of the present invention uses a device having a first plate-like member and a second plate-like member in which a plurality of through holes are formed at a predetermined interval. A step of sandwiching the plurality of adhesive tapes such that the side on which the plurality of stratum corneum of the adhesive tape is fixed is opposed to the through hole.

  FIG. 9 shows a device 30 having a flat plate 31 and a flat plate 32 as an example of a device in which a plurality of adhesive tapes are sandwiched. 2A and 2B are top views of the flat plate 31 and the flat plate 32, respectively. FIG. 2C is a cross-sectional view of the device 30. FIG.

  The material constituting the flat plate 31 and the flat plate 32 is not particularly limited, but glass, quartz glass, ceramics, titanium, stainless steel, aluminum, iron, acrylic resin, polypropylene, fluororesin, vinyl chloride resin, nylon, polystyrene, polycarbonate And silicone resin.

  On the flat plate 31, positions 1 to 10 for sandwiching the adhesive tape 40 'from which the stratum corneum has been peeled and positions Blank for sandwiching the same adhesive tape 40 as the adhesive tape 40' are displayed except that the stratum corneum is not peeled off. ing. At this time, the adhesive tape 40 has a rectangular shape when viewed from above, and the positions 1 to 10 and Blank are determined according to the width of the adhesive tape 40.

  In addition, although the positions 1-10 are displayed on the flat plate 31, the positions 1-N should just be displayed similarly to the flat plate 20. FIG. At this time, N is usually an integer of 1 to 90.

  Further, a mass calibration standard sample layer 31 a is formed on the flat plate 31 substantially in parallel with the width direction W. For this reason, when mass spectrometry is performed using the device 30 in which a plurality of adhesive tapes are sandwiched, the mass spectrometer can be mass calibrated and sensitivity can be corrected.

  The calibration reagent is not particularly limited as long as ions can be generated using a DART ion source. However, in consideration of the accuracy of mass calibration, polyethylene glycol (mass spectrum peaks present at equal intervals) PEG60 to PEG2000), fatty acids having 4 to 36 carbon atoms, and the like, and two or more kinds may be used in combination.

  A method for forming the mass calibration standard sample layer 31a is not particularly limited, but a method of fixing an adhesive tape, in which a solution in which a mass calibration standard sample is dissolved in a solvent, is applied to a substrate, to the flat plate 31, mass Examples thereof include a method of drawing on the flat plate 31 using a pen filled with ink to which a calibration standard sample is added, a method of applying ink to which the standard sample for mass calibration is added to the flat plate 31, and the like.

  A rectangular parallelepiped through-hole 32a corresponding to the adhesive tapes 40, 40 'and the mass calibration standard sample layer 31a is formed in the flat plate 32 substantially parallel to the width direction W.

Through holes 32a spacing _{G 3} of the adjacent corresponding to the adhesive tape 40 and 40 'is usually 0.5~50.0mm, 5.0~10.0mm are preferred. When the spacing G ₃ is less than 0.5 mm, may be reduced quantitative of the components of the osmotic skin external preparation to the skin, sometimes it exceeds 50.0 mm, unavailable device 30 efficiently .

  A method of fixing the device 30 in which the plurality of adhesive tapes 40 and 40 ′ are sandwiched is not particularly limited, and examples thereof include a method of fixing using a fixing tool such as a clip.

  A device having a plurality of adhesive tapes 40, a flat plate 31 on which a mass calibration standard sample layer 31a is formed, and a flat plate 32 on which a through hole 32a is formed is used for analysis of components of a skin external preparation that has penetrated into the skin. The analysis kit can be used.

  The second embodiment of the analysis method of the present invention includes a step of sequentially introducing ions generated from a plurality of stratum corneums into a mass spectrometer and performing mass spectrometry while moving a device sandwiched between a plurality of adhesive tapes. Have. At this time, it is the same as the first embodiment of the analysis method of the present invention except that a device in which a plurality of adhesive tapes are sandwiched is used instead of the plate-like member to which the plurality of cut adhesive tapes are fixed. Thus, mass spectrometry can be performed.

  The flat plate 31 and the flat plate 32 may not have the mass calibration standard sample layer 31a and the through hole 32a.

  In addition, the flat plate 31 and the flat plate 32 contain components of a skin external preparation that penetrates the skin in place of the mass calibration standard sample layer 31a and the through hole 32a or together with the mass calibration standard sample layer 31a and the through hole 32a. A through-hole corresponding to the standard sample layer for quantification and the standard sample layer for quantification for quantification may be formed.

  The standard reagent for quantification is a component of the external preparation for skin to be quantified, but may also serve as a standard sample for mass calibration.

  The method for forming the standard sample layer for quantification is not particularly limited, but after removing the stratum corneum from the area of the skin where the external preparation for skin is not applied, a predetermined amount of the standard sample solution for quantification is applied to the stratum corneum. The method of using the same adhesive tape as the adhesive tape 40 ′ except that the adhesive tape 40 ′ is applied, and the adhesive tape 40 that has been cut, except that a predetermined amount of the standard sample solution for determination is applied to the adhesive layer. And the like using the same adhesive tape.

  Further, instead of the device 30, a flat plate 31 ′ in which a convex portion 31b is formed in a region where the adhesive tapes 40 and 40 ′ are sandwiched, a region where the mass calibration standard sample layer 31a is formed, and an adjacent convex portion 31b. A device 30 ′ having a flat plate 32 ′ in which a convex portion 32 b corresponding to a region between them is formed may be used (see FIG. 10). Thereby, it becomes easy to fix the device 30 ′ in which the plurality of cut adhesive tapes 10 ′ are sandwiched.

  Furthermore, a sample stage that can be moved in the length direction and the width direction of the device 30 instead of the sample stage 100 by installing a partition substantially parallel to the length direction L of the flat plate 32 in the through hole 32a. May be used to analyze the components of the external preparation for skin permeating into the skin a plurality of times while changing the conditions as necessary. Thereby, when analyzing several times, generation | occurrence | production of contamination can be suppressed.

  The number of partitions installed in the through hole 32a is usually 1 to 6.

  The width of the partition installed in the through hole 32a is usually 0.5 to 10.0 mm.

  Furthermore, instead of the through hole 32a, a flat plate 32 "having a trapezoidal columnar through hole 32a 'may be used (see FIG. 11).

  The taper angle α is usually 5 to 80 °, and preferably 10 to 50 °. When the taper angle α is less than 5 °, convection may occur when irradiating protons generated by Penning ionization. When the taper angle α exceeds 80 °, the flat plate 32 ″ may not be used efficiently.

  Further, the shape of the adhesive tape 40 is not limited to a rectangular shape when viewed from above, and includes a circular shape and an elliptical shape when viewed from above. When the adhesive tape 40 is circular when viewed from above, the shape of the through hole 32a may be cylindrical.

[Preparation of adhesive tape 10]
Using a cutter in which three blades are fixed at intervals of 2 mm, three cuts 11 were put in a circular adhesive tape D-Square (manufactured by CuDerm) having a diameter of 22 mm to obtain an adhesive tape 10.

[Preparation of coating solution for standard sample layer for mass calibration]
Artline Wetlite Refilling ink (red) (manufactured by Shachihata) 1 mL of 1 g / L methanol solution of polyethylene glycol 200 and 0.5 mL of 1 g / L methanol solution of polyethylene glycol 400 were added and stirred, mass A calibration standard layer coating solution was obtained.

[Fabrication of flat plate 20]
After fixing two pieces of double-sided tape 21 of 3 mm × 120 mm in parallel to the length direction of the glass plate of 50 mm × 200 mm so that the distance is 14 mm, a coating solution for standard sample layer for mass calibration is applied. A standard sample layer 22 for mass calibration was formed to obtain a flat plate 20 (N = 10).

[Production of right-angled isosceles triangular prism 40]
Parallel to the height direction H of a glass right angled isosceles triangular prism with a length of 10 mm and a height of 100 mm between two sides sandwiching a right angle, a double-sided tape 41 of 3 mm × 60 mm and an interval between corners C of 14 mm After fixing the two in such a manner, a mass calibration standard sample layer 42 was formed by applying a mass calibration standard sample layer coating solution to obtain a right isosceles triangular prism 40 (N = 5).

[Example 1]
(Cream application and exfoliation of stratum corneum)
The inner parts of the left and right upper arms were washed and then dried. Next, a cream containing 5% ufenamate and 0.5% tocopherol acetate as active ingredients was applied to the left and right upper arm inner parts.

  Five minutes after applying the cream, the inner part of the left upper arm was washed with soap, and using the adhesive tape 10, ten stratum corneum layers were peeled from the area where the cream was applied on the inner part of the left upper arm.

  Next, 30 minutes after applying the cream, the inside of the upper right arm is washed with soap, and the adhesive layer 10 is used to remove 10 stratum corneum from the area where the cream on the inside of the upper right arm is applied. It peeled.

(Fixing the adhesive tape with the stratum corneum peeled off)
The base part of the cut line 11 of the pressure-sensitive adhesive tape 10 from which the 10 stratum corneum layers were peeled was cut to obtain a 2 mm × 17 mm strip-shaped cut pressure-sensitive adhesive tape 10 ′. Next, the base-side surface of the cut adhesive tape 10 ′ from which the first to tenth layers were peeled was fixed to the positions 1 to 10 of the flat plate 20 at a pitch of 10 mm. Moreover, the base-side surface of the cut adhesive tape 10 ″ that was the same as the cut adhesive tape 10 ′ was fixed to the position Blank of the flat plate 20 except that the stratum corneum was not peeled off.

(Analysis of stratum corneum)
Next, using the mass spectrometry method of FIG. 3, the standard sample layer 22 for mass calibration of the flat plate 20 to which the plurality of cut adhesive tapes 10 ′ are fixed, the cut adhesive tape 10 ′, and the cut adhesive The ions generated from the tape 10 ″ were sequentially subjected to mass spectrometry. Specifically, first, the flat plate 20 was placed on the sample stage 100 such that the length direction L was substantially parallel to the arrow direction. Next, the DART ion source 200 is used to move helium He (2 ³ S) in a metastable excitation state while moving the sample stage 100 in a substantially horizontal plane at a rate of 0.2 mm / s in the direction of the arrow. Protons generated by penning ionization by colliding with water in the atmosphere are substantially perpendicular to the direction of the arrow in the figure, and the central part in the width direction W of the substrate 20 at an angle of 45 ° with respect to the substantially horizontal plane. Ions generated by irradiation were introduced into the mass spectrometer 300 for mass analysis. At this time, since the ion introduction tube 310 was heated by flowing a current of 2 A through the resistance heating wire 311, the temperature of the inner wall of the ion introduction tube 310 was 84 ° C.

  In addition, DART SVP (made by Ion Sense) was used as the DART ion source 200, and the set temperature of the gas heater was set to 400 ° C. Further, as the mass spectrometer 300, MicroTOFQII (manufactured by Bruker Dartonics) was used, and the measurement mode was set to positive ion mode. Further, as the ion introduction tube 310, a ceramic tube having an outer diameter of 6.2 mm, an inner diameter of 4.7 mm, and a length of 94 mm is used, and a resistance heating wire 311 is wound around a region 35 mm from the ion introduction side. It was. At this time, a nichrome wire having a diameter of 0.26 mm was used as the resistance heating wire 311.

  FIG. 12 shows the mass spectrum of the first stratum corneum peeled from the area where the cream agent on the inner right upper arm was applied. Note that m / z = 338.1 is the mass-to-charge ratio of ions generated from ufenamate, and m / z = 473.4 is the mass-to-charge ratio of ions generated from tocophenol acetate.

  FIG. 13 shows a mass chromatogram at m / z = 338.1. In addition, (a) and (b) are the mass chromatograms of the stratum corneum peeled from the area | region where the cream agent of the left upper arm inner part and the right upper arm inner part was apply | coated, respectively. It means the 10th stratum corneum.

  FIG. 14 shows a mass chromatogram at m / z = 473.4. In addition, (a) and (b) are the mass chromatograms of the stratum corneum peeled from the area | region where the cream agent of the left upper arm inner part and the right upper arm inner part was apply | coated, respectively. It means the 10th stratum corneum.

  From FIG. 13 and FIG. 14, in both ufenamate and tocophenol acetate, the concentration gradient between the 1st layer to the 10th layer of the stratum corneum between 5 minutes and 30 minutes after applying the cream. It can be seen that changes.

[Reference Example 1]
(Removal of stratum corneum)
The inner part of the upper arm was washed and then dried. Next, using the adhesive tape 10, the stratum corneum was peeled off from six locations on the inner side of the upper arm.

(Fixing the adhesive tape with the stratum corneum peeled off and applying tocophenol acetate)
The base part of the cut line 11 of the pressure-sensitive adhesive tape 10 from which the stratum corneum was peeled was cut to obtain a pressure-sensitive adhesive tape 10 ′ having a strip shape of 2 mm × 17 mm. Next, after fixing the base-side surface of the adhesive tape 10 ′ from which the cut stratum corneum has been peeled off to the positions 1 to 5 of the flat plate 20 at a 10 mm pitch, the pressure-sensitive adhesive tape 10 from which the cut stratum corneum has been peeled off To, 10 ng of tocopherol acetate was applied. Moreover, the base-side surface of the cut adhesive tape 10 ″ that was the same as the cut adhesive tape 10 ′ was fixed to the position Blank of the flat plate 20 except that the stratum corneum was not peeled off.

(Analysis of stratum corneum)
Next, using the mass spectrometry method of FIG. 3, the standard sample layer 22 for mass calibration of the flat plate 20 to which the plurality of cut adhesive tapes 10 ′ are fixed, the cut adhesive tape 10 ′, and the cut adhesive The ions generated from the tape 10 ″ were sequentially subjected to mass spectrometry. Specifically, first, the flat plate 20 was placed on the sample stage 100 such that the length direction L was substantially parallel to the arrow direction. Next, the DART ion source 200 is used to move helium He (2 ³ S) in a metastable excitation state while moving the sample stage 100 in a substantially horizontal plane at a rate of 0.2 mm / s in the direction of the arrow. Protons generated by penning ionization by colliding with water in the atmosphere are substantially perpendicular to the direction of the arrow in the figure, and the central part in the width direction W of the substrate 20 at an angle of 45 ° with respect to the substantially horizontal plane. Ions generated by irradiation were introduced into the mass spectrometer 300 for mass analysis. At this time, since the ion introduction tube 310 was heated by flowing a current of 2 A through the resistance heating wire 311, the temperature of the inner wall of the ion introduction tube 310 was 84 ° C.

  In addition, DART SVP (made by Ion Sense) was used as the DART ion source 200, and the set temperature of the gas heater was set to 500 ° C. Further, as the mass spectrometer 300, MicroTOFQII (manufactured by Bruker Dartonics) was used, and the measurement mode was set to positive ion mode. Further, as the ion introduction tube 310, a ceramic tube having an outer diameter of 6.2 mm, an inner diameter of 4.7 mm, and a length of 94 mm is used, and a resistance heating wire 311 is wound around a region 35 mm from the ion introduction side. It was. At this time, a nichrome wire having a diameter of 0.26 mm was used as the resistance heating wire 311.

  FIG. 15 shows a mass chromatogram at m / z = 473.4. In addition, 1-5 means the stratum corneum fixed to the positions 1-5 of the flat plate 20.

[Reference Example 2]
(Removal of stratum corneum)
The stratum corneum was peeled in the same manner as in Reference Example 1.

(Fixing the adhesive tape with the stratum corneum peeled off and applying tocophenol acetate)
The base part of the cut line 11 of the pressure-sensitive adhesive tape 10 from which the stratum corneum was peeled was cut to obtain a pressure-sensitive adhesive tape 10 ′ having a strip shape of 2 mm × 17 mm. Next, after fixing the surface of the adhesive tape 10 ′ from which the cut stratum corneum was peeled off to the positions 1 to 5 of the right isosceles triangular prism 40 at a pitch of 10 mm, each cut stratum corneum was peeled off. 10 ng of tocopherol acetate was applied to the adhesive tape 10 ′. Further, the base-side surface of the cut adhesive tape 10 ″, which is the same as the cut adhesive tape 10 ′, except that the stratum corneum is not peeled off, is fixed to the position Blank of the right-angled isosceles triangular prism 40. .

(Analysis of stratum corneum)
Next, using the mass spectrometry method of FIG. 6, the standard sample layer 42 for mass calibration of the right isosceles triangular prism 40 to which the plurality of cut adhesive tapes 10 ′ are fixed, the cut adhesive tape 10 ′ and the cut The ions generated from the pressure-sensitive adhesive tape 10 ″ were sequentially subjected to mass spectrometry. Specifically, first, the right isosceles triangular prism 40 was placed on the sample stage 100 so that the height direction H was substantially parallel to the arrow direction. At this time, the right-angled isosceles triangular prism 40 was arranged so that the vertical plane opposed to the DART ion source 200 with respect to the sample stage 100 of the right-angled isosceles triangular prism 40. Next, the DART ion source 200 is used to move helium He (2 ³ S) in a metastable excitation state while moving the sample stage 100 in a substantially horizontal plane at a rate of 0.2 mm / s in the direction of the arrow. Protons generated by penning ionization by colliding with water in the atmosphere are generated by irradiating the corner C of the right isosceles triangular prism 40 substantially perpendicular to the direction of the arrow in the figure in a substantially horizontal plane. Ions were introduced into the mass spectrometer 300 for mass analysis. At this time, since the ion introduction tube 310 was heated by flowing a current of 4 A through the resistance heating wire 311, the temperature of the inner wall of the ion introduction tube 310 was 110 ° C. However, the reason why the temperature of the inner wall of the ion introduction tube 310 is set to 110 ° C. is to reduce the occurrence of contamination, and the contribution to the improvement of detection sensitivity is small.

  In addition, DART SVP (made by Ion Sense) was used as the DART ion source 200, and the set temperature of the gas heater was set to 500 ° C. Further, as the mass spectrometer 300, MicroTOFQII (manufactured by Bruker Dartonics) was used, and the measurement mode was set to positive ion mode. Further, as the ion introduction tube 310, a ceramic tube having an outer diameter of 6.2 mm, an inner diameter of 4.7 mm, and a length of 94 mm is used, and a resistance heating wire 311 is wound around a region 35 mm from the ion introduction side. It was. At this time, a nichrome wire having a diameter of 0.26 mm was used as the resistance heating wire 311.

  FIG. 16 shows a mass chromatogram at m / z = 473.4. In addition, 1-5 means the stratum corneum fixed to the positions 1-5 of the right-angled isosceles triangular prism 40.

  15 and 16, it can be seen that the detection sensitivity of tocophenol acetate is improved when the mass spectrometry method of FIG. 6 is used instead of the mass spectrometry method of FIG. For this reason, in the analysis method of the present invention, the detection sensitivity can be improved by using the mass analysis method of FIG. 6 instead of the mass analysis method of FIG.

DESCRIPTION OF SYMBOLS 10 Adhesive tape 11 Break 10 'Cut | disconnected adhesive tape 20, 20' Flat plate 21 Double-sided tape 22 Standard sample layer for mass calibration 23 Resistance heating wire 30, 30 'Device 31, 31' Flat plate 31a Standard sample layer for mass calibration 32, 32 ′, 32 ″ flat plate 32a, 32a ′ through hole 40 triangular prism 41 double-sided tape 42 standard sample layer for mass calibration 100 sample stage 200 DART ion source 300 mass spectrometer 310 ion introduction tube 311 resistance heating wire

Claims (9)

A method for analyzing components of a skin external preparation that has penetrated into the skin,
Applying a topical skin preparation to the skin;
Peeling the plurality of stratum corneum from the area where the skin external preparation is applied;
An analysis method comprising a step of performing mass analysis by sequentially introducing ions generated from the separated stratum corneum into a mass spectrometer using DART or DESI. Using a plurality of adhesive tapes that are cut at a predetermined interval, the multi-layered stratum corneum is peeled from the area where the skin external preparation is applied,
Cutting the plurality of adhesive tapes from which the stratum corneum has been peeled with a width corresponding to the interval between the cuts;
A step of fixing the surface of the plurality of cut adhesive tapes on the side opposite to the side on which the stratum corneum is fixed to a plate-like member or a columnar member at a predetermined interval;
The mass generated by sequentially introducing ions generated from the plurality of stratum corneums into a mass spectrometer while moving the plate-like member or columnar member to which the plurality of cut adhesive tapes are fixed. The analysis method according to claim 1. The plate-like member or the columnar member is formed with a layer containing an adhesive, a layer containing a mass calibration standard sample and / or a layer containing a quantitative standard sample,
The analysis method according to claim 2, wherein ions generated from the layer including the mass calibration standard sample and / or the layer including the quantification standard sample are introduced into a mass spectrometer for mass analysis. Using a plurality of adhesive tapes, peeling a plurality of stratum corneum from the area where the skin external preparation is applied,
Using a device having a first plate-like member and a second plate-like member in which a plurality of through holes are formed at a predetermined interval, the side of the adhesive tape on which the horny layer is fixed Sandwiching a plurality of pressure-sensitive adhesive tapes from which the stratum corneum is peeled so as to face the through-hole,
2. The analysis according to claim 1, wherein ions generated from the plurality of stratum corneum are sequentially introduced into a mass spectrometer and mass spectrometry is performed while a device in which the plurality of adhesive tapes are sandwiched is moved. Method. The first plate-like member or the second plate-like member is formed with a layer containing a mass calibration standard sample and / or a layer containing a quantitative standard sample,
5. The analysis method according to claim 4, wherein ions generated from the layer including the mass calibration standard sample and / or the layer including the quantification standard sample are introduced into a mass spectrometer and subjected to mass analysis. The mass spectrometer has an ion introduction part for introducing the ions,
The analysis method according to claim 1, wherein the mass spectrometry is performed while heating the ion introduction part. The ion introduction part is a tube around which a resistance heating wire is wound,
The analysis method according to claim 6, wherein the ion introduction part is heated by applying a voltage to the resistance heating wire using a voltage application unit. An analysis kit used for analyzing components of a skin external preparation that has penetrated into the skin,
A plurality of adhesive tapes that are cut at predetermined intervals;
An analysis kit comprising a plate-like member or a columnar member. An analysis kit used for analyzing components of a skin external preparation that has penetrated into the skin,
Multiple adhesive tapes,
An analysis kit comprising a device having a first plate-like member and a second plate-like member in which a plurality of through holes are formed at a predetermined interval.

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