High performance liquid chromatography detection method and quality control method for index components in six-ingredient skin-moistening oil massage prescription

Abstract

The invention relates to a high performance liquid chromatography detection method for index components in a six-ingredient skin-moistening oil massage prescription, the six-ingredient skin-moistening oil massage prescription is used for treating atopic dermatitis, the index components are senkyunolide A, ligustilide and linoleic acid, and the high performance liquid chromatography method is used for simultaneously determining the contents of the three components, namely the senkyunolide A, the ligustilide and the linoleic acid. The invention further provides a quality control method for treating atopic dermatitis. The invention establishes a thin-layer identification method for six traditional Chinese medicines of the six-ingredient skin-moistening oil massage formula for treating atopic dermatitis to qualitatively identify the traditional Chinese medicines in a compound. The method for simultaneously determining the contents of the three components by high performance liquid chromatography is established by taking senkyunolide A, ligustilide, linoleic acid and other components in the formula as indexes through extraction and separation, and methodological investigation proves that the method is simple, convenient, rapid and accurate, has good precision, stability and repeatability, and can be widely applied to the field of clinical application. And a scientific basis is provided for multi-index quality control of the six-ingredient skin moistening oil formula.

Description

Technical Field

[0001] The present invention relates to the technical field of quality control of traditional Chinese medicine compound prescriptions, and in particular to a high performance liquid chromatography detection method and a quality control method for index components in a six-flavor moisturizing oil prescription. Background Art

[0002] The Six-ingredient Moisturizing Oil Massage Formula is a prescription for external use of atopic dermatitis based on the theory of “blood deficiency and wind dryness” in traditional Chinese medicine and Xia’s unique experience in using medicine. This formula is composed of six Chinese herbs, including hemp seed, which has the effect of “nourishing blood, moistening dryness, dispelling wind and relieving itching”[1]. It has been used clinically for many years and has proven to be effective. Related studies have also found that the main herbs, hemp seed and coix seed[2], can also be used alone to treat atopic dermatitis.

[0003] Currently, research on the Liuwei Moisturizing Oil formula has largely focused on the component analysis, content determination, and pharmacological activity of individual herbs. However, overall quality control of the formula remains limited, with few established methods for component analysis and content determination. On the one hand, different extraction processes or preparation methods used during the processing of the formula can lead to differences in the types and contents of active ingredients. On the other hand, traditional Chinese medicine preparations are complex in composition and difficult to control in terms of quality, and controlling their quality using single-component content determination also makes it difficult to characterize their overall quality level. Therefore, existing research is insufficient to serve as a basis for measuring the quality of the Liuwei Moisturizing Oil formula. Multi-index quality control of the active ingredients in traditional Chinese medicine preparations, or even analysis of the entire composition, is particularly important.

[0004] Chinese patent document CN112684053A discloses a method for simultaneously determining the contents of seven ingredients in a compound for treating chronic heart failure. The seven ingredients are gallic acid, hydroxysafflor yellow A, loganin, agarwood tetraol, psoralen, isopsoralen, and terglycidyl glycoside. The method is a high-performance liquid chromatography method using the following chromatographic conditions: a C18 column, a mobile phase of acetonitrile:water, gradient elution; a flow rate of 0.2-1.2 mL min-1, a column temperature of 20-40°C, and a detection wavelength of 220-360 nm. This method can simultaneously determine the contents of seven ingredients in a compound for treating chronic heart failure, exhibits good linearity, high precision, strong stability, good repeatability, high sample recovery, and is simple and easy to use, providing a basis for the formulation of quality standards for compound for treating chronic heart failure.

[0005] Chinese patent document CN 113567577A discloses a method for thin-layer chromatography identification and high-performance liquid chromatography detection of the indicative components in Shenshuai Yifang granules. The present invention further provides a method for high-performance liquid chromatography detection of the indicative components in Shenshuai Yifang granules. The method provided by the present invention for thin-layer chromatography identification and high-performance liquid chromatography detection of the indicative components in Shenshuai Yifang granules is accurate, reliable, simple, easy to perform, and highly specific, thereby improving the quality standard of the granules and being useful for quality control of Shenshuai Yifang granules.

[0006] However, there is currently no quality control research on the traditional Chinese medicine compound for treating atopic dermatitis as described in this application, nor is there a method for simultaneously measuring the contents of the three ingredients, ligustilide A, ligustilide, and linoleic acid, in the atopic dermatitis compound. Summary of the Invention

[0007] The purpose of the present invention is to provide a high performance liquid chromatography detection method and a quality control method for the index components in the six-flavor moisturizing oil formula in response to the deficiencies in the prior art.

[0008] In the first aspect, the present invention provides a high performance liquid chromatography detection method for the index components in the six-flavor moisturizing oil formula, the six-flavor moisturizing oil formula is used to treat atopic dermatitis, the index components are ligusticum lactone A, ligusticum lactone, and linoleic acid, the high performance liquid chromatography method simultaneously determines the contents of three components: ligusticum lactone A, ligusticum lactone, and linoleic acid, and the chromatographic conditions of the high performance liquid chromatography method are: using a C18 chromatographic column, mobile phase: 0.1% phosphoric acid aqueous solution-methanol, elution gradient of 0-20min, 50-30% 0.1% phosphoric acid aqueous solution, 50-70% methanol; 20-30min, 30-15% 0.1% phosphoric acid aqueous solution, 70-85% methanol; 30-35min, 15-8% 0.1% phosphoric acid aqueous solution , 85-92% methanol; 35-40min, 8-13% 0.1% phosphoric acid aqueous solution, 92%-87% phosphorus methanol; 40-45min, 13-30% 0.1% phosphoric acid aqueous solution, 87%-70% methanol; 45-50min, 30-50% 0.1% phosphoric acid aqueous solution, 70%-50% 0 methanol; 50-55min, 50% 0.1% phosphoric acid aqueous solution, 50% methanol; flow rate 0.8-1.2ml / min, column temperature 28-32℃, detection wavelength 200-230nm; the compound Chinese medicine for treating atopic dermatitis is composed of: 26-34 parts of hemp seed, 26-34 parts of angelica, 6-14 parts of Panax notoginseng, 9-21 parts of Chuanxiong, 26-34 parts of coix seed, and 26-34 parts of liquorice.

[0009] As a preferred example, the preparation method of the test solution of the high performance liquid chromatography method is: weighing hemp seed, angelica, Panax notoginseng, Chuanxiong, coix seed, and licorice, soaking them with soybean oil, stirring, filtering, and taking the filtrate to obtain.

[0010] As another preferred example, the preparation method of the reference solution of the HPLC method is as follows: take appropriate amounts of ligusticum lactone A, ligusticum lactone, and linoleic acid reference substances, add methanol to dissolve them, and prepare mixed reference solutions with concentrations of 0.2 mg / ml, 0.5 μl / ml, and 2.5 μl / ml, respectively.

[0011] As another preferred example, the detection wavelength is 210 nm.

[0012] As another preferred example, the injection volume is 10 μl.

[0013] As another preferred example, the column temperature is 30°C.

[0014] As another preferred example, the flow rate is 1.0 ml / min.

[0015] In a second aspect, the present invention provides a quality control method for a compound for treating atopic dermatitis, comprising the steps of the above-mentioned high performance liquid chromatography method.

[0016] The present invention has the advantage of establishing a thin-layer chromatography (TLC) identification method for the six Chinese medicinal herbs in the Liuwei Moisturizing Oil formula for treating atopic dermatitis, enabling qualitative identification of the compound Chinese medicinal herbs. Using multiple components in the formula, including ligustilide A, ligustilide, and linoleic acid, as indicators, a high-performance liquid chromatography method for simultaneously determining the contents of these three components was established through extraction and separation. Methodological investigations have demonstrated the simplicity, rapidity, and accuracy of this method, along with excellent precision, stability, and repeatability. This method provides a scientific basis for multi-index quality control of the Liuwei Moisturizing Oil formula. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 :Thin layer chromatography of hemp seed

[0018] 1-3. Test sample solution (batch numbers 20230801, 20230802, 20230803); 4. Linoleic acid reference solution 5. Negative control solution.

[0019] Figure 2 :Thin layer chromatogram of Chuanxiong and Angelica

[0020] 1-3. Test sample solutions (batch numbers are 20230801, 20230802, and 20230803); 4. Ligustilide reference solution; 5. Negative control solution.

[0021] Figure 3:Thin layer chromatography of coix seed

[0022] 1-3. Test sample solution (batch numbers 20230801, 20230802, 20230803); 4. Coix seed control medicinal material solution; 5. Negative control solution.

[0023] Figure 4 :Thin layer chromatography of Panax notoginseng

[0024] 1-3. Test sample solution (batch numbers 20230801, 20230802, 20230803); 4. Panax notoginseng control solution; 5. Negative control solution.

[0025] Figure 5 :Thin layer chromatogram of licorice

[0026] 1-3. Test sample solution (batch numbers 20230801, 20230802, 20230803); 4. Licorice control medicinal material solution 5. Negative control solution.

[0027] Figure 6 : HPLC chromatogram of the Six-flavor Moisturizing Oil Formula

[0028] 1: Ligusticum chuanxiong lactone A; 2: Ligusticum chuanxiong lactone; 3: Linoleic acid; A. Test solution B. Reference solution C. Negative control solution. DETAILED DESCRIPTION

[0029] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content of the present invention record, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the application's appended claims.

[0030] Example 1

[0031] 1. Instruments and reagents

[0032] 1.1 Instrument

[0033] Agilent 1260 high performance liquid chromatograph (Agilent Corporation, USA); BSA822-CW electronic balance (Beijing Sartorius Instrument Systems Co., Ltd.); CQ-250A-DST ultrasonic cleaner (Shanghai Yuejin Medical Instrument Co., Ltd.); SHZ-D(Ⅲ) circulating water vacuum pump (Zhengzhou Boco Equipment Co., Ltd.); RV-10 rotary evaporator (IKA Company, Germany); BD-Ⅱ multifunctional UV analyzer (Beijing Qihang Boda Technology Co., Ltd.); HWS28 electric constant temperature water bath (Shanghai Yiheng Scientific Experiment Co., Ltd.).

[0034] 1.2 Drug testing

[0035] Six-flavor moisturizing oil formula (Yueyang Hospital Preparation Room, batch numbers 20230801, 20230802, 20230803); ligustilide A reference substance (item number ST10580220, content ≥95.0%), purchased from Shanghai Shidande Standard Technology Service Co., Ltd.; ligustilide reference substance (batch number 111737-202311), linoleic acid reference substance (batch number 111622- 202105), coix seed (batch number 121254-202105), notoginseng (batch number 120941-201810), and licorice (batch number 120904-202021) were all purchased from the China Food and Drug Inspection Institute; silica gel G thin layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd., Shandong Province); methanol was chromatographic grade, and all other reagents were analytical grade; water was ultrapure water.

[0036] 2 Methods and Results

[0037] 2.1 Thin layer identification

[0038] Identification of hemp seed: Take 40ml of the six-flavor moisturizing oil rubbed formula, add 40ml of methanol, shake for 5 minutes, let it stand for 30 minutes, take the methanol layer solution, reflux and concentrate, and then make the volume to 2ml as the test solution; according to the prescription and process of the six-flavor moisturizing oil rubbed formula, prepare a negative sample without hemp seed, and prepare a negative control solution according to the test solution preparation method; take another linoleic acid reference standard, add methanol to make a solution containing 20μl per 1ml, as the reference solution. According to the thin layer chromatography method (General Rule 0502), take 3μl of each of the above solutions and spot them on the same silica gel G thin layer plate, use petroleum ether-ethyl acetate-formic acid (3:0.3:0.1) as the developing agent, develop, take out, dry, spray with 1% vanillin sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly colored. In the chromatogram of the test solution, spots of the same color appear at the corresponding positions of the chromatogram of the reference solution, and there is no interference from the negative control. See for details. Figure 1 .

[0039] Identification of Chuanxiong and Angelica: Take 40ml of the Six-flavor Moisturizing Oil rubbed formula, add 40ml of methanol, shake for 5 minutes, let it stand for 30 minutes, take the methanol layer solution, reflux and concentrate, and then make the volume to 2ml as the test solution; according to the prescription and process of the Six-flavor Moisturizing Oil rubbed formula, prepare a negative sample without Chuanxiong and Angelica, and prepare a negative control solution according to the test solution preparation method; take another ligustilide reference standard, add methanol to make a solution containing 50μl per 1ml, as the reference solution. According to the thin layer chromatography method (General Rule 0502), take 5μl of each of the above solutions and spot them on the same silica gel G thin layer plate, use petroleum ether-ethyl acetate (37:3) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm). In the chromatogram of the test solution, fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the reference solution. The spots are clear and there is no interference from the negative control. See for details. Figure 2 .

[0040] Identification of coix seed: Take 40ml of the six-flavor moisturizing oil rubbed formula, add 40ml of methanol, shake for 5 minutes, let it stand for 30 minutes, take the methanol layer solution, reflux and concentrate, and then make the volume to 2ml as the test solution; according to the prescription and process of the six-flavor moisturizing oil rubbed formula, prepare a negative sample without coix seed, and prepare a negative control solution according to the test solution preparation method; take another 10g of coix seed control medicinal material and prepare a control medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 5μl of each of the above solutions and spot them on the same silica gel G thin layer plate, use petroleum ether-ether-glacial acetic acid (83:17:1) as the developing agent, develop, take out, dry, spray with 1% vanillin sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly colored. In the chromatogram of the test solution, spots of the same color appear at the corresponding positions of the chromatogram of the reference solution. The spots are clear and there is no interference from the negative control. See for details. Figure 3 .

[0041] Identification of Panax notoginseng: Take 40ml of the Six-flavor Moisturizing Oil Formula, add 40ml of methanol, shake for 5 minutes, let it stand for 30 minutes, take the methanol layer solution, reflux and concentrate, and then make the volume to 2ml as the test solution; according to the prescription and process of the Six-flavor Moisturizing Oil Formula, prepare a negative sample that does not contain Panax notoginseng, and prepare a negative control solution according to the test solution preparation method; take another 5g of Panax notoginseng control medicinal material and prepare a control medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 5μl of each of the above solutions and spot them on the same silica gel G thin layer plate, use toluene-ethyl acetate-formic acid (15:1:0.3) as the developing agent, develop, take out, dry, spray with 1% vanillin sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly colored. In the chromatogram of the test solution, spots of the same color appear at the corresponding positions of the chromatogram of the reference solution. The spots are clear and there is no interference from the negative control. See for details. Figure 4 .

[0042] Identification of licorice: Take 40ml of the Six-flavor Moisturizing Oil Recipe, add 40ml of methanol, shake for 5 minutes, let it stand for 30 minutes, take the methanol layer solution, reflux and concentrate, and then make the volume to 2ml as the test solution; prepare a negative sample without licorice according to the prescription and process of the Six-flavor Moisturizing Oil Recipe, and prepare a negative control solution according to the test solution preparation method; take another 10g of licorice control medicinal material and prepare a control medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 5μl of each of the above solutions and spot them on the same silica gel G thin layer plate, use petroleum ether-ethyl acetate (37:3) as the developing solvent, develop, take out, dry, spray with 1% vanillin sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly colored. In the chromatogram of the test solution, spots of the same color appear at the corresponding positions of the chromatogram of the reference solution. The spots are clear and there is no interference from the negative control. See for details. Figure 5 .

[0043] 2.2 Content determination

[0044] 2.2.1 Chromatographic conditions

[0045] Chromatographic column: Agilent Zorbax Eclipse XDB-C 18 Column (250 mm × 4.6 mm, 5 μm); mobile phase: 0.1% phosphoric acid aqueous solution (A)-methanol (B), elution program see Table 1; flow rate: 1.0 ml / min; column temperature: 30°C; detection wavelength: 210 nm; injection volume: 10 μl.

[0046] Table 1 Gradient elution program

[0047]

[0048] 2.2.2 Solution preparation

[0049] (1) Reference solution: Take appropriate amount of ligustilide A, ligustilide, and linoleic acid reference substances, dissolve them in methanol, and prepare mixed reference solutions with concentrations of 0.2 mg / ml, 0.5 μl / ml, and 2.5 μl / ml, respectively.

[0050] (2) Test solution: Take 145g of sample (batch number 20230801: 30g of hemp seed, 30g of angelica, 10g of notoginseng, 15g of ligusticum chuanxiong, 30g of coix seed, 30g of liquorice, a total of 120 doses. 1. Extraction: Grind the above 17.4kg of medicinal materials into fine powder, soak with 2 times the volume of soybean oil (34.8L) for 7 days, stir from time to time, filter, and bottle. 2. Packaging: Pack according to 100mL per bottle, one person's dosage, 2 bottles.) 5ml, add 5ml of methanol, shake for 5 minutes, let it stand for 30 minutes, take the methanol layer, and the test solution is obtained.

[0051] (3) Negative sample solution: According to the formula and process of Liuwei Moisturizing Oil, a negative sample without Cannabis Sativa Seed, Chuanxiong Rhizome and Angelica Sinensis was prepared, and the negative control solution was prepared according to the preparation method of the test solution.

[0052] 2.3 Specificity test

[0053] Take 10 μl of each of the test solution, reference solution, and negative control solution and inject them according to the chromatographic conditions in 2.2.1. The results show that the theoretical plate number meets the requirements and the baseline separation is good; the negative control solution has no interfering peak at the same chromatographic retention time as the reference solution, indicating good specificity. Figure 6 .

[0054] 2.4 Methodological Investigation

[0055] 2.4.1 Linear relationship investigation

[0056] Take 1.25, 2.50, 5.00, 10.00, and 15.00 μl of the reference solution in 2.2.2, inject the solution according to the chromatographic conditions in 2.2.1, and record the peak areas. Plot a standard curve with injection volume as the abscissa (X) and peak area as the ordinate (Y). Determine the linear equation, correlation coefficient, and linear range for each component. The results are shown in Table 2. The experimental results demonstrate good linearity for each component within its respective concentration range.

[0057] Table 2 Results of linear relationship investigation of three principal components

[0058]

[0059] 2.4.2 Intra-day precision test

[0060] Accurately pipette 10 μl of the mixed reference solution under 2.2.2 and repeat the injection 6 times according to the chromatographic conditions under 2.2.1. Record the peak area and calculate the RSD value. The results are shown in Table 3, indicating that the precision of the instrument is good.

[0061] Table 3 Precision test results

[0062]

[0063] 2.4.3 Repeatability test

[0064] Prepare 6 test sample solutions according to the method under 2.2.3, inject 10 μl of each solution, record the peak areas of ligustilide A, ligustilide, and linoleic acid, convert their contents, and calculate the RSD values. The results are shown in Table 4, indicating that the method has good repeatability.

[0065] Table 4 Repeatability test results (mg / ml)

[0066]

[0067] 2.4.4 Stability test

[0068] Prepare the test solution as described in 2.2.2 and incubate at room temperature for 0 h, 4 h, 8 h, 12 h, 16 h, and 24 h. Inject 10 μl of the sample according to the chromatographic conditions in 2.2.1, determine the peak area, and calculate the RSD. The results are shown in Table 5, indicating that the test solution is stable within 24 h.

[0069] Table 5 Stability test results

[0070]

[0071] 2.4.5 Sample recovery test

[0072] Six samples from batch 20230801 with known index component contents were prepared by adding a certain amount of ligustilide A, ligustilide, and linoleic acid reference solution within the linear range. The contents were determined according to the chromatographic conditions in 2.2.1. The results are shown in Table 6, indicating that this method has good accuracy.

[0073] Table 6 Sample recovery test results

[0074]

[0075]

[0076] 2.5 Validation of content determination method

[0077] Three batches of Liuwei Moisturizing Oil (lot numbers 20230801, 20230802, and 20230803) were prepared for testing using the method in "2.2.3." The contents of ligustilide A, ligustilide, and linoleic acid were determined using the chromatographic conditions in "2.2.1." The results are shown in Table 7. The results demonstrate that this process is accurate, reliable, and feasible, suitable for industrial production.

[0078] Table 7 Determination results of the contents of ligustilide A, ligustilide and linoleic acid in the Liuwei Moisturizing Oil Formula

[0079]

[0080] This study established a thin-layer chromatography method for the identification of six Chinese medicinal herbs and the determination of three active ingredients in the Liuwei Moisturizing Oil Formula. This method is simple, feasible, highly sensitive, and reproducible, and can be used for quality control of the Liuwei Moisturizing Oil Formula. This provides a quality standard for the standardized clinical use of the Liuwei Moisturizing Oil Formula.

[0081] References:

[0082] [1] Cui Han, Chi Huiyan, She Yuanyao, et al. Meta-analysis of traditional Chinese medicine nourishing blood and moistening method for the treatment of atopic dermatitis[J]. World Journal of Integrated Traditional Chinese and Western Medicine,

[0083] 2023,18(02):249-255.

[0084] [2] Wang Junxia. Observation on the therapeutic effect of coix seed extract on BALB / c atopic dermatitis model mice[D], 2018.

[0085] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A high performance liquid chromatography detection method for the index components in the six-flavor moisturizing oil formula, characterized in that: The six-flavor moisturizing oil formula is used to treat atopic dermatitis. The index components are ligustilide A, ligustilide, and linoleic acid. The high-performance liquid chromatography method simultaneously determines the contents of ligustilide A, ligustilide, and linoleic acid. The chromatographic conditions of the high-performance liquid chromatography method are: using a C18 chromatographic column, mobile phase: 0.1% phosphoric acid aqueous solution-methanol, elution gradient 0-20min, 50-30% 0.1% phosphoric acid aqueous solution, 50-70% methanol; 20-30min, 30-15% 0.1% phosphoric acid aqueous solution, 70-85% methanol; 30-35min, 15-8% 0.1% phosphoric acid aqueous solution, 85-92% methanol; 35-40min ,8-13% 0.1% phosphoric acid aqueous solution, 92%-87% phosphorus methanol; 40-45min, 13-30% 0.1% phosphoric acid aqueous solution, 87%-70% methanol; 45-50min, 30-50% 0.1% phosphoric acid aqueous solution, 70%-50% 0 methanol; 50-55min, 50% 0.1% phosphoric acid aqueous solution, 50% methanol; flow rate 0.8-1.2ml / min, column temperature 28-32℃, detection wavelength 200-230nm; the compound Chinese medicine for treating atopic dermatitis is composed of 26-34 parts of hemp seed, 26-34 parts of angelica, 6-14 parts of Panax notoginseng, 9-21 parts of Chuanxiong, 26-34 parts of Coix seed, and 26-34 parts of licorice.

2. The high performance liquid chromatography method according to claim 1, characterized in that The preparation method of the test solution of the high performance liquid chromatography method is: weigh hemp seed, angelica, Panax notoginseng, Chuanxiong, coix seed, and liquorice medicinal materials, soak them with soybean oil, stir, filter, and obtain the filtrate.

3. The high performance liquid chromatography method according to claim 1, characterized in that The preparation method of the reference solution of the HPLC method is as follows: take appropriate amounts of ligustilide A, ligustilide, and linoleic acid reference substances, add methanol to dissolve them, and prepare mixed reference solutions with concentrations of 0.2 mg / ml, 0.5 μl / ml, and 2.5 μl / ml, respectively.

4. The high performance liquid chromatography method according to claim 1, characterized in that Detection wavelength: 210nm.

5. The high performance liquid chromatography method according to claim 1, characterized in that: The injection volume was 10 μl.

6. The high performance liquid chromatography method according to claim 1, characterized in that: Column temperature: 30℃.

7. The high performance liquid chromatography method according to claim 1, characterized in that: Flow rate: 1.0ml / min.

8. A quality control method for a compound for treating atopic dermatitis, characterized in that: The method comprises the steps of the high performance liquid chromatography method according to any one of claims 1 to 7.

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