JPH09243636A - Analysis of substance present on skin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To directly analyze a substance present on and in the skin of a human body even with the elapse of time by analyzing a sample collected from the skin by a pyrolytic mass spectrum method or a thermal detaching mass spectrum method to determine a specific substance present on and in the skin. SOLUTION: A sample is collected from the skin by a self-adhesive tape, adsorbing paper or an adhesive. A method using the self-adhesive tape is pref. from an aspect of sampling efficiency, a resistance feeling or simplicity. As pretreatment, a substance is decomposed at high temp. in a pyrolytic method and quantitatively analyzed on the basis of a decomposition product while a substance is heated to low or medium temp. in a thermal detaching method to be evaporated without decomposing an objective component to be quantitatively analyzed. In the next detection, a mass analyser is connected to a pyrolytic apparatus or a thermal detaching apparatus through gas chromatography or liquid chromatography or directly to perform mass analysis. By this method, change of the specific substance of the skin of a human body with the elapse of time can be accurately and simply analyzed and evaluated.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for analyzing a substance existing on the surface of the skin or inside the skin, and a method for evaluating the effectiveness of a skin external preparation or the like.

[0002]

2. Description of the Related Art When evaluating the transdermal absorbability from the skin surface of ultraviolet absorbents, physiologically active ingredients, preservatives, etc., which have been conventionally compounded in external preparations for skin, etc., the transdermal absorbability using Franz cell is used. A method using a model, a method using a radioisotope, etc. have been used. However, all of these methods have been problematic in that the transdermal absorbability from the skin surface of the human body is accurately evaluated, because they are used as test subjects of animals or are indirect evaluation methods.

And, as an example of actually tested on a human body,
Ceramide inside the skin is extracted from the skin with a solvent,
Although there are examples of analysis, this method has a problem in that it imposes a great burden on the subject and that evaluation over time is difficult to carry out.

[0004]

Therefore, an object of the present invention is to be able to directly analyze and evaluate percutaneous absorbability and the like on human skin, and to analyze and evaluate its change over time. In order to evaluate the effectiveness of external preparations for skin, etc., that the analysis method is simple, that the harmful effect on the human body is not exerted, that many kinds of substances existing on the skin can be quantified The purpose is to provide an analytical method that can be used.

[0005]

Means for Solving the Problems As a result of intensive studies in view of these problems, the present inventors have found that a sample collected from the skin with an adhesive tape or the like is subjected to a thermal decomposition mass spectrum method using a double shot pyrolyzer, or a thermal decomposition method. By quantifying specific substances present on the skin by desorption mass spectrometry, the percutaneous absorbability of the compounding ingredients in the external preparation for skin, or the analysis of the amount of change in the specific ingredient in skin tissue due to the effects of the external preparation for skin, etc. I found that I can do it.

That is, the present invention is characterized in that a sample collected from the skin is subjected to a thermal decomposition mass spectrum method or a thermal desorption mass spectrum method to quantify a specific substance present on the skin surface or inside the skin. It is in the analysis method of substances present in the skin.

[0007] This analysis method is applied to skin external preparations having a function of inhibiting the percutaneous absorption of substances which should not be percutaneously absorbed into the skin, such as organic UV absorbers, preservatives and antioxidants. Development, daily observation of changes in skin condition due to the use of physiologically active ingredients such as cerebroside and vitamins,
It is extremely useful for the development of external preparations for the skin that have a difficult scientific basis because conventional analysis cannot be performed, such as evaluation evaluation of effects and development of makeup cosmetics based on the observation of sebum secretion using squalene as an index. I found it to be effective.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The constitution of the present invention will be described in detail below.
The skin referred to in the present invention is intended for the skin of the whole body such as human face, arms and scalp, and also rabbits, guinea pigs, rats, dogs,
The skin of the whole body of animals such as pigs and cows is targeted.

As a method of collecting a sample used in the present invention,
The method of collecting a sample from the skin using adhesive tape, suction paper, adhesive, etc. is the most suitable method from the viewpoint of collection efficiency, resistance to peeling, simplicity, and reproducibility. preferable. Specific examples of the collection method include the following methods.

(1) Method of using adhesive tape An adhesive tape is applied to the skin site, a certain pressure is applied (for example, rubbing with a microspatel several times), the adhesive tape is peeled off, and a sample is taken. To do. At this time, as the size of the measurement sample, a square having a side of 2 × 2 mm or a circle having a diameter of 3 mm is preferably used. As the adhesive tape, cellophane tape is preferably used in terms of cost, simplicity, and homogeneity of the adhesive.

(2) Method using adsorption paper Adhesion paper is applied to the skin site, and after applying a constant pressure (eg 1 kg / cm ² ) for a certain period of time (eg 15 minutes), the adsorption paper is peeled off and a sample is collected To do. As the suction paper, filter paper, suction paper, or the like can be used.

(3) Method Using Adhesive An adhesive is applied to the sampled skin site, dried and then peeled off to collect a sample. As the adhesive, a cyanoacrylate adhesive or the like is preferable.

(4) Method using a replica A commercially available replica agent (silicone-based, epoxy-based, polyurethane-based, etc.) is applied and, after curing, peeled off and a sample is taken.

As a preferable point of these sampling methods,
Since the number of collection sites is small, it is possible to increase the number of collections as compared with the case where the collection site is large. Increasing the number of collections has the advantage of facilitating the measurement of changes over time and site-specific measurements. Further, there is an advantage that information in the depth direction of the skin can be obtained by changing the number of times of sampling of the same site.

The thermal decomposition method, which is the pretreatment of the sample in the analysis method of the present invention, refers to a method of decomposing a substance at a high temperature and quantitatively analyzing the substance by the decomposition product.
The thermal desorption method refers to a method of quantitatively analyzing a substance by heating the substance at low and medium temperatures and vaporizing the target component without decomposing it. As an example of the thermal decomposition method or the thermal desorption method, for example, a heating furnace type thermal decomposition apparatus represented by a double shot pyrolyzer (manufactured by Frontier Lab) is used, and the thermal decomposition / thermal desorption section temperature is room temperature to 800 ° C. It is preferable to set the interface temperature at room temperature to 400 ° C., preferably 250 to 400 ° C. at 300 to 600 ° C., and analyze. Within this range, the target component can be quantified with good reproducibility by thermal decomposition or thermal desorption.

Although a method of extracting a sample with a solvent to analyze a specific substance can be considered, this method has a drawback that the pretreatment step becomes complicated.

As a detection means after thermal decomposition or thermal desorption of the sample used in the present invention, a mass spectrometer (mass spectrum) is used.
Is used. As the separation means used in the present invention, a method of directly connecting the thermal decomposition / thermal desorption device and the mass spectrometer, or separation of gas chromatography, liquid chromatography or the like between the thermal decomposition / thermal desorption device and the mass spectrometer. There is a method of connecting devices.

Examples of the specific substance existing on the skin in the present invention include one or more of a compounding ingredient in an external preparation for skin, an intrinsic ingredient in skin tissue and a percutaneous absorption enhancer. Examples include squalene, organic ultraviolet absorbers, ceramides, physiologically active ingredients, preservatives, antioxidants, fragrances, surfactants, moisturizers such as hyaluronic acid, keratin, oils, and transdermal absorption enhancers. Furthermore, examples of substances that affect the skin include environmental pollutants and sensitizing substances.

The skin of the measurement site referred to in the present invention is the surface of the epidermis, dermis and the stratum corneum of the skin.

By using the above-mentioned analysis method of the present invention, a highly effective external preparation for skin can be developed. That is, according to this analysis method, percutaneous absorption of various components into the skin, changes in the amount of sebum, changes over time of substances in the body were analyzed and evaluated, and the results were developed based on the results, for example, percutaneous absorption was inhibited. Skin external preparation, skin external preparation that promotes percutaneous absorption, skin external preparation that responds to changes in the amount of sebum, skin external preparation that suppresses the amount of sebum,
It is possible to provide a skin external preparation that effectively demonstrates the effects of physiologically active ingredients, and a skin external preparation that protects the skin from environmental pollutants.

Examples of the external preparation for skin include ointment, poultice, hair-growth agent, hair-growth agent, sunscreen agent, foundation, white powder, lipstick, eye shadow, cheek, makeup base,
Examples include emulsions, lotions, creams, essences, cleansing, packs, shampoos, rinses, bath agents, fragrances, soaps, deodorants and the like.

As the compounding ingredients in the external preparation for skin, in addition to the above-mentioned substances, for example, oil agents, powders (pigments, dyes, resins), fluorine compounds, resins, which are usually used in medicines and cosmetics,
Surfactants, adhesives, preservatives, fragrances, ultraviolet absorbers (including organic and inorganic ones, which may correspond to UV-A or B), moisturizers, physiologically active ingredients, salts, solvent,
Components such as an antioxidant, a chelating agent, a neutralizing agent, and a pH adjusting agent can be mentioned.

[0023]

The present invention will be described in detail below with reference to examples and comparative examples.

Example 1 Analysis of Squalene By the method of the present invention, changes over time in the amount of sebum secreted outside the skin and the amount of squalene inside the skin were analyzed. The experimental method is as follows.

(1) Sampling method Panelists: 10 females Sampling site: face cheek and forehead Sampling method: Using commercially available cellophane tape, press the tape to the sampling site and use a microspatel 3 times. After rubbing, remove the tape and fold it in half. A circular sample having a diameter of 3 mm was cut out from this tape and used as a measurement sample. This operation was repeated several times. In addition, the measurement site was slightly moved, and the measurement was performed after 2, 4, and 6 hours.

(2) Analytical conditions The measurement was carried out by a mass spectrometer from a thermal decomposition / thermal desorption device through a gas chromatograph.

Thermal decomposition / thermal desorption device (double shot pyrolyzer) Thermal decomposition / thermal desorption part temperature: 300 ° C. Interface part temperature: 300 ° C. Gas chromatograph device (manufactured by Hewlett Packard) Injection port temperature: 300 ° C. Separation tube temperature: 230 ℃ (1min) → (35 ℃ / mi
n) → 300 ° C. → (10 ° C./min)→350° C. Separation tube: dimethyl polysiloxane-based column (Ultra Alloy UA-1 (manufactured by Frontier Laboratories)) Mass spectrometer: JMS DX-manufactured by JEOL Datum
303 Applied voltage: 70eV

(3) Analytical Results FIG. 1 shows the time-dependent change of squalene as an example of the analytical results.

The surface of the peeled tape in Example 1 was freeze-fractured and observed by a scanning electron microscope, and the boundary between the skin surface and the inside of the skin was set and analyzed. As a result, after washing the face, with the increase in the amount of squalene outside the skin over time, the amount of squalene inside the skin also increases, the amount of sebum increases when applying the foundation, it is possible to obtain previously unknown data. confirmed.

Example 2 Analysis of Ceramide The change in the amount of ceramide inside the skin was measured. With the right outer arm blank, a commercially available cerebroside-containing preparation was applied to the left outer arm for 1 month, and the amount of ceramide in the skin on the 0th and 1st month of application was measured.

(1) Sampling method Sampling method: Using a commercially available cellophane tape, the tape was pressure-bonded to the sampling site, rubbed three times with a microspatel, and then the tape was peeled off and folded in half. A circular sample having a diameter of 3 mm was cut out from this tape and used as a measurement sample. This operation was repeated several times.

(2) Analytical conditions Thermal decomposition / thermal desorption device (double shot pyrolyzer) Thermal decomposition / thermal desorption part temperature: 550 ° C. Interface part temperature: 350 ° C. Gas chromatograph device (manufactured by Hewlett Packard) Inlet temperature: 300 ℃ separation tube temperature: 100 ℃ → 340 ℃ separation tube: dimethylpolysiloxane-based column mass spectrometer: JEOL Datum JMS DX-
303 Applied voltage: 70eV

(3) Analytical results At the start of the measurement, no change was observed in the amount of ceramide inside the left and right skin. After application for 1 month, the amount of ceramide inside the skin at the cerebroside-applied site was significantly increased compared to the amount of ceramide at the non-applied site.

From the results of Example 2, it was confirmed that the method of the present invention can more easily evaluate the quantitative change of the specific substance existing in the skin tissue using the human body.

Example 3 Evaluation of skin external preparation (foundation) As a result of measuring the amount of sebum on the female face to which only lotion was applied by the measuring method of Example 1, it was found that the amount of sebum increased with time. It was Furthermore, the foundation A obtained by the formulation shown in Table 1 below was applied, and the amount of sebum was evaluated by the same method as in Example 1. As a result, a larger amount of sebum was obtained as compared with the case of only the lotion. It was confirmed that the amount was secreted. That is, it was found that by applying the foundation A, sebum was secreted in excess of the oil absorption amount of the foundation A, and phenomena such as shine and collapse occurred. From this, it was confirmed that even if the oil absorption amount of Foundation A was increased, makeup breakdown could not be prevented.

Therefore, a foundation B having a formulation shown in Table 2 was prepared, in which the sebum durability of the foundation itself was increased by blending a fluoroalkyl / polyoxyethylene co-modified silicone (hereinafter referred to as FPD modified silicone). However, when a trial test was actually carried out in the summer (8 females), all of the 8 females did not have makeup makeup and no shininess, and this idea could be verified.

[0037]

[Table 1]

(Preparation Method of Foundation A) After uniformly mixing all components shown in Table 1 and pulverizing with an atomizer,
The product was obtained by stamping on a metal plate using the mold.

[0039]

[Table 2]

(Preparation Method of Foundation B) After uniformly mixing all components shown in Table 2 and pulverizing with an atomizer,
The product was obtained by stamping on a metal plate using the mold.

[0041]

As described above, according to the present invention, the amount of a specific substance is quantified by using a thermal decomposition mass spectrum method or a thermal desorption mass spectrum method on a sample collected from the skin, so that the skin of a human body is actually measured. Accurate, simple analysis and evaluation of changes over time of specific substances can be provided, and it is possible to provide a method for analyzing substances that are present in the skin that are highly practical, and by using the method of the present invention, the effectiveness is improved. It is possible to develop a clear external preparation for skin.

[Brief description of drawings]

FIG. 1 shows the results of measurement of changes over time in the amount of squalene in Example 1 using the analysis method of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 30/06 G01N 30/06 G 30/88 30/88 E (72) Inventor Masafumi Imaseki Kanagawa 5-28, Kotobuki-cho, Odawara City, Kanebo Co., Ltd.

Claims (4)

[Claims] 1. A skin-existing substance characterized by quantifying a specific substance present on the skin surface or inside the skin by using a sample collected from the skin by a thermal decomposition mass spectrometry method or a thermal desorption mass spectrometry method. Analysis method. 2. The analysis method according to claim 1, wherein an adhesive tape is used when collecting a sample from the skin. 3. The analysis method according to claim 1, wherein a heating furnace type thermal decomposition apparatus is used as the thermal decomposition method. 4. The specific substance is one or more selected from the group consisting of a compounding ingredient in an external preparation for skin, an intrinsic ingredient in skin tissue, and a transdermal absorbent.
The analysis method according to any one of 3 to 3.