A method for detecting the content of fluocinolone hydroquinone retinoic acid cream

Abstract

The present invention belongs to the field of drug analysis technology, and is specifically related to a content detection method of a fluocinolone hydroquinone retinoic acid cream and its use for measuring the drug release amount in the in vitro release test of fluocinolone hydroquinone retinoic acid cream. The content detection method described in the present invention is to adopt high performance liquid chromatography, with phosphate buffer solution as mobile phase A and methanol as mobile phase B, and gradient elution is performed on the chromatographic column of octadecylsilane bonded silica gel, and the content of three active ingredients of fluocinolone, hydroquinone and retinoic acid can be detected simultaneously, and the running time is short and operability is strong. In addition, the content detection method provided by the present invention can be applied to the detection of drug content in the fluocinolone hydroquinone retinoic acid cream test solution obtained by sampling with physiological saline 50% ethanol as the receiving medium, selecting a specific artificial membrane, and using an in vitro release device to measure the cumulative release of drugs in the in vitro release test of fluocinolone hydroquinone retinoic acid cream.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and specifically relates to a method for detecting the content of fluocinolone hydroquinone retinoic acid cream and an application thereof in determining the drug release amount in an in vitro release test of fluocinolone hydroquinone retinoic acid cream. Background Art

[0002] Fluocinolone Hydroquinone Retinoic Acid Cream is a commonly used compound preparation for external use on the skin, mainly containing three medicinal active ingredients: 0.01% fluocinolone, 4% hydroquinone and 0.05% retinoic acid. Among them, fluocinolone is a corticosteroid that reduces inflammation and irritation, hydroquinone is a skin whitening agent that inhibits melanin production, and retinoic acid is a retinoic acid that promotes skin cell renewal and exfoliation. Fluocinolone Hydroquinone Retinoic Acid Cream is a first-line and preferred treatment method, and is clinically used for the treatment of moderate to severe melasma under sun protection measures.

[0003] At present, the in vitro release method of fluocinolone hydroquinone retinoic acid cream (including in vitro release sample preparation method and in vitro release sample detection method) is not included in the pharmacopoeia of various countries, and it is impossible to detect the content of fluocinolone, hydroquinone and retinoic acid components released by fluocinolone hydroquinone retinoic acid cream through an in vitro release instrument. There is no content detection method in the prior art that can simultaneously detect the three active ingredients of fluocinolone, hydroquinone and retinoic acid. If a new method is not developed, the samples need to be detected using three liquid phase methods. Not only is the sample inspection complicated and cumbersome, but the content detection method of the single-prescription preparation or raw material drug takes too long to run, and the extraction solvents used are different, which is not suitable for the content detection of fluocinolone hydroquinone retinoic acid cream. In addition, there are many excipients in the fluocinolone hydroquinone retinoic acid cream itself, which will cause great interference to the detection of the active ingredient content.

[0004] Therefore, it is necessary to develop a method for detecting the content of fluocinolone hydroquinone retinoic acid cream. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention aims to provide a method for detecting the content of fluocinolone hydroquinone retinoic acid cream. The method is time-saving, highly sensitive, and has a good separation effect, and can effectively improve the shape of the chromatographic peak.

[0006] The technical solution described in the present invention is as follows:

[0007] In the first aspect, the present invention provides a method for detecting the content of fluocinolone acetonide, hydroquinone and retinoic acid cream, wherein the content detection method is a reverse high performance liquid chromatography method, wherein a fluocinolone acetonide, hydroquinone and retinoic acid cream test solution is subjected to gradient elution on a chromatographic column filled with alkylsilane bonded silica gel using mobile phase A and mobile phase B as eluents; the conditions of the chromatography method include:

[0008] Detection wavelength: 238~240nm and 348~352nm,

[0009] Column temperature: 25~35℃,

[0010] Mobile phase A: 0.005~0.015mol / L phosphate buffer solution, and adjust the pH value to 2.50~3.50,

[0011] Mobile phase B: methanol,

[0012] Flow rate: 0.8~1.2mL / min,

[0013] Injection volume: 5~15μL,

[0014] The procedure of the gradient elution is: .

[0015] Preferably, the chromatographic column is ZORBAX SB-Aq 4.6mm×150mm, 3.5μm.

[0016] Preferably, the phosphate in the mobile phase A includes at least one of potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and ammonium dihydrogen phosphate, more preferably potassium dihydrogen phosphate.

[0017] Preferably, the concentration of fluocinolone acetonide in the test solution of the fluocinolone hydroquinone tretinoin cream is 0.00125~0.005 mg / L, the concentration of hydroquinone is 0.5~2 mg / L, and the concentration of tretinoin is 0.00625~0.025 mg / L, more preferably the concentration of fluocinolone acetonide is 0.0025 mg / L, the concentration of hydroquinone is 1 mg / L, and the concentration of tretinoin is 0.0125 mg / L.

[0018] In the second aspect, the present invention provides a method for detecting the content of fluocinolone hydroquinone retinoic acid cream for use in determining the drug release amount in an in vitro release test of fluocinolone hydroquinone retinoic acid cream. The in vitro release test uses a diffusion cell device, with normal saline-50% ethanol as the receiving medium, and the fluocinolone hydroquinone retinoic acid cream is smeared on an artificial membrane with a quantitative loop placed on it, and samples are taken at a fixed time to obtain a fluocinolone hydroquinone retinoic acid cream test solution.

[0019] Preferably, the artificial membrane used in the in vitro release test is a microporous organic filtration membrane with a diameter of 50 mm and a pore size of 0.45 μm.

[0020] Preferably, in the in vitro release test, the temperature of the receiving medium in the diffusion cell device during sample loading is 32° C., the volume of the receiving cell is 12 mL, and the stirring speed is 400-600 rpm.

[0021] Preferably, the loading amount of fluocinolone hydroquinone retinoic acid cream in the in vitro release test is 0.2-0.4 g.

[0022] Preferably, the specific operation of fixed time sampling in the in vitro release test is to take out all the receiving medium and replenish an equal amount of constant temperature receiving medium at 0.5h, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h, 12h, and 24h.

[0023] Specifically, the content detection method used for determining the drug release amount in the in vitro release test is as described in the first aspect, using reverse HPLC, using mobile phase A and mobile phase B as eluents to perform gradient elution on a chromatographic column filled with alkylsilane bonded silica gel; the conditions of the chromatography include:

[0024] Detection wavelength: 238~240nm and 348~352nm,

[0025] Column temperature: 25~35℃,

[0026] Mobile phase A: 0.005~0.015mol / L phosphate buffer solution, and adjust the pH value to 2.5~3.5,

[0027] Mobile phase B: methanol,

[0028] Flow rate: 0.8~1.2mL / min,

[0029] Injection volume: 5~15μL,

[0030] The procedure of the gradient elution is: ; The preparation of the reference solution involved in the determination of drug release in the in vitro release test is as follows: weigh the tretinoin reference substance and the fluocinolone acetonide reference substance respectively, dissolve them in methanol, prepare tretinoin reference substance stock solution and fluocinolone acetonide reference substance stock solution, then weigh the hydroquinone reference substance, dissolve it in normal saline-50% ethanol, add appropriate amounts of tretinoin reference substance stock solution and fluocinolone acetonide reference substance stock solution, and dilute with normal saline-50% ethanol to prepare a solution containing 12.5±10%μg / mL of tretinoin, 2.5±10%μg / mL of fluocinolone acetonide, and 1±10%mg / mL of hydroquinone, which is the reference solution.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The content detection method provided by the present invention can simultaneously detect the contents of three active ingredients, fluocinolone, hydroquinone and tretinoin, in fluocinolone hydroquinone tretinoin cream, and can be applied to the content detection of fluocinolone hydroquinone tretinoin cream and the detection of in vitro release samples, and has the characteristics of good specificity and short operation time;

[0033] 2. The content detection method provided by the present invention can be used to determine the drug release amount in the in vitro release test of fluocinolone acetonide, hydroquinone and retinoic acid cream. The receiving medium in the in vitro release test method can meet the sink conditions of the three active ingredients of fluocinolone acetonide, hydroquinone and retinoic acid, has good sample stability and is conducive to a better chromatographic peak shape in drug detection. Under the conditions of selecting a suitable artificial membrane, the content detection method provided by the present invention can obtain a complete release curve (linear part R 2 ≥0.9). BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a chromatogram of hydroquinone and fluocinolone acetonide at a wavelength of 239 nm under standard detection conditions of the fluocinolone hydroquinone retinoic acid cream of Example 2;

[0035] Figure 2 is a chromatogram of retinoic acid in Example 2 fluocinolone hydroquinone retinoic acid cream under standard detection conditions at a wavelength of 350 nm;

[0036] Figure 3 is a square root graph of the cumulative drug release of fluocinolone acetonide-hydroquinone retinoic acid cream of Example 5 versus time;

[0037] Figure 4 is a square root graph of the cumulative drug release of hydroquinone from the fluocinolone hydroquinone retinoic acid cream of Example 5 versus time;

[0038] Figure 5 is a square root graph of the cumulative drug release of retinoic acid from the fluocinolone hydroquinone retinoic acid cream of Example 5 versus time;

[0039] Figure 6 is a graph of the cumulative drug permeability of fluocinolone acetonide-hydroquinone retinoic acid cream of Example 5 over time;

[0040] Figure 7 is a graph of the cumulative drug permeability of hydroquinone of the fluocinolone hydroquinone retinoic acid cream of Example 5 versus time;

[0041] Figure 8 is a graph of the cumulative drug permeability of retinoic acid versus time of the fluocinolone hydroquinone retinoic acid cream of Example 5;

[0042] The reference numerals in the above chromatogram are: 1: hydroquinone peak; 2: fluocinolone peak; 3: retinoic acid peak. DETAILED DESCRIPTION

[0043] The present invention is further described in detail in conjunction with the following specific examples and drawings, and the protection content of the present invention is not limited to the following examples. Without departing from the spirit and scope of the inventive concept, the changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims are the scope of protection. The process, conditions, and test methods for implementing the present invention, except for the contents specifically mentioned below, are common knowledge and common common sense in the art; the materials, reagents, etc. for implementing the present invention, unless otherwise specified, can be obtained from commercial channels, and the present invention has no special restrictions.

[0044] Example 1 Content Detection Method Development

[0045] Determine the detection method: When there are many samples to be tested, it is troublesome to use a UV spectrophotometer to detect the content, and there will be interference during the detection. In addition, fluocinolone acetonide and retinoic acid are detected by C18 columns and UV detectors in the pharmacopoeias of various countries. Therefore, a UV detector is selected, and a C18 column is used as the chromatographic column, and the sample is tested by high performance liquid chromatography.

[0046] Determine the collection wavelength: Using full wavelength scanning, the maximum absorption wavelength of fluocinolone is 239nm, and hydroquinone also has good absorption at this wavelength; the maximum absorption wavelength of retinoic acid is 350nm, so the detection wavelength is determined to be 238~240nm and 348~352nm.

[0047] Determine the elution system: Optimize the related substance detection method of fluocinolone EP Pharmacopoeia. The three active ingredients have good solubility in methanol, and methanol is used to dissolve the reference substance. The related substance detection methods of fluocinolone acetonide and hydroquinone all use phosphate buffer as the mobile phase, so phosphate buffer is used as mobile phase A and methanol is used as mobile phase B. When the three active ingredients of fluocinolone acetonide, hydroquinone and retinoic acid are determined at pH <3.4, ​​the LogD curve is a horizontal straight line. Generally, the pH value of the phosphate solution is adjusted to 2.90~3.10 (within the buffer range of dihydrogen phosphate-phosphate buffer solution), but the pH value is adjusted to 2.5~3.5. The conditions are still met; in order to reduce the running time, the buffer salt concentration is reduced to 0.005~0.015mol / L to reduce its buffering capacity. Do not use a 250mm column to reduce retention time, use a 3.5μm column instead of a 5μm column to enhance the separation ability of the column. In summary, a 4.6 mm×150mm 3.5μm column is used; according to the literature, when 75% methanol is used as the mobile phase, the running time of retinoic acid is too long, exceeding 80 minutes before the peak appears. When the methanol ratio is increased to 90%, retinoic acid can peak in about 15 minutes, so the mobile phase methanol ratio is increased to 85%~90% for elution.

[0048] The specific elution program is: gradient elution program: 0~3min, the volume proportion of mobile phase A changes from 85~95% to 5~95%, 3~6min, the volume proportion of mobile phase A changes from 5~95% to 5~15%, 6~10min, the volume proportion of mobile phase A is 5~15%, 10~10.1min, the volume proportion of mobile phase A changes from 5~15% to 85~95%, 10.1~15min, the volume proportion of mobile phase A is 85~95%.

[0049] Example 2 Detection of the content of fluocinolone hydroquinone retinoic acid cream

[0050] Based on the content detection method determined in Example 1, a fluocinolone hydroquinone retinoic acid cream (containing 0.1 mg of fluocinolone, 40 mg of hydroquinone and 0.5 mg of retinoic acid per gram of cream) was prepared. The concentration of fluocinolone in the test solution was 0.0025 mg / L, the concentration of hydroquinone was 1 mg / L, and the concentration of retinoic acid was 0.0125 mg / L, and a reference solution with the same concentration level was prepared. The content detection method was reverse high performance liquid chromatography. The fluocinolone hydroquinone retinoic acid cream test solution was subjected to gradient elution on a chromatographic column filled with alkylsilane bonded silica gel using mobile phase A and mobile phase B as eluents; the conditions of the chromatography method included:

[0051] Detection wavelength: 238~240nm and 348~352nm,

[0052] Column temperature: 25~35℃,

[0053] Mobile phase A: 0.005~0.015mol / L phosphate buffer solution, and adjust the pH value to 2.50~3.50,

[0054] Mobile phase B: methanol,

[0055] Flow rate: 0.8~1.2mL / min,

[0056] Injection volume: 5~15μL,

[0057] The procedure of the gradient elution is: ; Table 1 is used as the standard test condition. By changing a single test parameter, the relative deviation (RSD) of the results of the content of each active ingredient described in Table 2 for the same test solution is less than 1%, which proves that the repeatability and specificity of the content detection method are good; the chromatogram under standard conditions is shown in Figure 1~Figure 2 .

[0058] Table 1 Standard test conditions

[0059]

[0060] Table 2 Content test results of fluocinolone hydroquinone retinoic acid cream

[0061]

[0062] Example 3 Determination of receptor medium for in vitro release assay

[0063] Experimental method: The saturated solubility of the raw materials in different receiving media was investigated. About 10 mg of fluocinolone raw material, about 10 mg of retinoic acid raw material, and about 250 mg of hydroquinone raw material were accurately weighed and placed in sample bottles with 25 mL of receiving medium. Three parallel samples were placed in a constant temperature oscillator (32±1°C) with lids on. The solution concentrations were measured after 4h, 24h, and 26h.

[0064] Acceptor medium: The acceptor medium was selected from 0.1M hydrochloric acid, pH 4.5 acetate buffer, pH 6.5 phosphate buffer, pH 7.4 phosphate buffer, and normal saline. Because retinoic acid and fluocinolone have low solubility in water and can be dissolved to a certain extent in ethanol, a certain proportion of ethanol was added to the acceptor medium to improve the solubility.

[0065] Test results: The results show that in the receptor medium with 50% ethanol, except for 0.1M hydrochloric acid, other receptor media can reach the sink condition for fluocinolone, hydroquinone and retinoic acid. Considering that the main clinical application site of this product is the face, physiological saline-50% ethanol was selected as the receptor medium.

[0066] Example 4 Determination of artificial membrane for in vitro release test

[0067] In vitro release tests require the selection of a suitable artificial membrane, which should have no obstruction to the active ingredients, no obvious adsorption effect, and no compatibility issues with the active ingredients.

[0068] Retinoic acid stock solution: Weigh about 25 mg of tretinoin, place in a 100 mL volumetric flask, add appropriate amount of methanol to dissolve it, dilute to the scale with methanol, shake well, and obtain.

[0069] Fluocinolone stock solution: Weigh about 10 mg of fluocinolone, place in a 200 mL volumetric flask, add appropriate amount of methanol to dissolve it, dilute to the scale with methanol, shake well, and obtain.

[0070] Hydroquinone stock solution: Weigh about 1000 mg of hydroquinone, place it in a 50 mL volumetric flask, add an appropriate amount of receiving solution to dissolve it, dilute it to the scale with the receiving solution, shake well, and you have it.

[0071] Inactive ingredient membrane solution: Pipette 12 mL of the receptor medium solution into a volumetric bottle, place an artificial membrane in each bottle as the inactive ingredient membrane solution, and place in a water bath at 32°C for 24 hours. Repeat 3 times.

[0072] Membraneless solution: Pipette 10 mL of retinoic acid stock solution, fluocinolone stock solution, and hydroquinone stock solution into a 200 mL volumetric flask, add the receptor medium to the mark, and pipette 12 mL of each solution into the volumetric flask as the membraneless solution, and place in a water bath at 32°C for 24 hours. Repeat 3 times.

[0073] Membrane solution: Pipette 10 mL of retinoic acid stock solution, fluocinolone stock solution, and hydroquinone stock solution into a 200 mL volumetric flask, add the receptor medium to the mark, pipette 12 mL of the solution into a vial, place an artificial membrane in each of them as the membrane solution, and place in a water bath at 32°C for 24 hours. Repeat 3 times.

[0074] The results showed that the recovery rate of active ingredients in the membrane solution relative to the membrane-free solution was between 95.0% and 105.0% within 24 hours, and the inertness of the artificial membrane (microporous organic filtration membrane) met the requirements. In addition, the in vitro release test preferred an artificial membrane with a diameter of 50 mm and a pore size of 0.45 μm.

[0075] Example 5 In vitro release test of fluocinolone hydroquinone retinoic acid cream

[0076] Based on the content detection method determined in Examples 1-4 and the receptor medium and artificial membrane required for the in vitro release test method, the in vitro release test of fluocinolone hydroquinone retinoic acid cream includes the following steps:

[0077] Step 1: Prepare the test solution

[0078] An in vitro release apparatus was used, and the receiving medium was saline-50% ethanol for the test. The artificial membrane was a microporous filter membrane (organic, Jinteng, diameter: 50mm, pore size: 0.45μm). The temperature of the receiving medium in the diffusion cell device was 32°C, the receiving cell volume was 12mL, and the stirring speed was 400-600rpm. A quantitative ring was placed on the artificial membrane, and about 0.3g of fluocinolone hydroquinone retinoic acid cream was evenly applied on the artificial membrane, weighed, and placed in the diffusion cell device. All the receiving medium was taken out at 0.5h, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h, 12h, and 24h, and an equal amount of constant temperature receiving medium was added. The taken out receiving medium was the test solution.

[0079] Step 2: Prepare reference solution

[0080] Retinoic acid reference stock solution: accurately weigh 25 mg of tretinoin reference, place in a 100 mL volumetric flask, add appropriate amount of methanol to dissolve, dilute to scale with methanol, shake well, and prepare two portions in parallel.

[0081] Fluocinolone acetonide reference stock solution: accurately weigh 10 mg of fluocinolone acetonide reference solution, place in a 200 mL volumetric flask, add appropriate amount of methanol to dissolve, dilute to scale with methanol, shake well, and prepare two portions in parallel.

[0082] Reference solution: accurately weigh 20 mg of hydroquinone reference substance, place in a 20 mL volumetric flask, add an appropriate amount of receptor medium to dissolve, pipette 1 ml of retinoic acid reference substance stock solution and fluocinolone reference substance stock solution respectively, dilute to scale with receptor medium, shake well, and prepare two in parallel.

[0083] Step 3: Content detection by HPLC

[0084] Reverse phase high performance liquid chromatography was used, octadecylsilane bonded silica gel was used as the chromatographic column filler, the specific chromatographic column was ZORBAX SB-Aq 4.6mm×150mm, 3.5μm, mobile phase A and mobile phase B were used as eluents, and gradient elution was performed; the chromatographic conditions included:

[0085] Detection wavelength: 238~240nm and 348~352nm,

[0086] Column temperature: 25~35℃,

[0087] Mobile phase A: 0.005~0.015mol / L potassium dihydrogen phosphate solution, and adjust the pH value to 2.50~3.50 with phosphoric acid.

[0088] Mobile phase B: methanol,

[0089] Flow rate: 0.8~1.2mL / min,

[0090] Injection volume: 5~15μL,

[0091] The procedure of the gradient elution is: .

[0092] According to the obtained chromatographic peak areas of the test solution and the reference solution, the drug content, cumulative drug release and cumulative permeability of the in vitro release of fluocinolone hydroquinone retinoic acid cream at each time point were calculated. The results are as follows: Figure 3~Figure 8 As shown, the content detection method provided by the present invention can describe the release curve of the duration of fluocinolone hydroquinone retinoic acid cream, and the cumulative drug amount released per unit area of ​​fluocinolone hydroquinone retinoic acid cream in the in vitro release test is linearly related to the square root of time, wherein fluocinolone: ​​y=2.2003x+1.1941, R 2 =0.9936; Hydroquinone: y=1133.3839x+553.5195, R 2=0.9764; Retinoic acid: y=3.3524x-2.2380, R 2 =0.9863, with good discrimination and specificity (R 2 ≥0.9).

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for detecting the content of fluocinolone hydroquinone retinoic acid cream, characterized in that: The content detection method is a reverse phase high performance liquid chromatography method, wherein the fluocinolone hydroquinone retinoic acid cream test solution is subjected to gradient elution on a chromatographic column filled with alkylsilane bonded silica gel using mobile phase A and mobile phase B as eluents; the conditions of the chromatography method include: Detection wavelength: 238~240nm and 348~352nm, Column temperature: 25~35℃, Mobile phase A: 0.005~0.015mol / L phosphate buffer solution, and adjust the pH value to 2.50~3.50, Mobile phase B: methanol, Flow rate: 0.8~1.2mL / min, Injection volume: 5~15μL, The procedure of the gradient elution is: ； The concentration of fluocinolone acetonide, hydroquinone and tretinoin cream test solution is 0.00125-0.005 mg / L, the concentration of hydroquinone is 0.5-2 mg / L, and the concentration of tretinoin is 0.00625-0.025 mg / L; the chromatographic column is ZORBAX SB-Aq 4.6 mm×150 mm, 3.5 μm.

2. The content detection method according to claim 1, characterized in that: The phosphate in the mobile phase A includes at least one of potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and ammonium dihydrogen phosphate.

3. A method for detecting the content of fluocinolone acetonide hydroquinone retinoic acid cream as described in any one of claims 1 to 2, for use in determining the drug release amount in an in vitro release test of fluocinolone acetonide hydroquinone retinoic acid cream, characterized in that: The in vitro release test adopts a diffusion cell device, with normal saline-50% ethanol as the receiving medium, and the fluocinolone hydroquinone retinoic acid cream is applied to the artificial membrane with a quantitative loop placed on it, and samples are taken at a fixed time to obtain the fluocinolone hydroquinone retinoic acid cream test solution.

4. The use according to claim 3, characterized in that The artificial membrane used in the in vitro release test is a microporous organic filtration membrane with a diameter of 50 mm and a pore size of 0.45 μm.

5. The use according to claim 3, characterized in that: In the in vitro release test, the temperature of the receiving medium in the diffusion cell device during sample loading was 32° C., the volume of the receiving cell was 12 mL, and the stirring speed was 400-600 rpm.

6. The use according to claim 3, characterized in that: The loading amount of fluocinolone hydroquinone retinoic acid cream in the in vitro release test was 0.2-0.4 g.

7. The use according to claim 3, characterized in that: The specific operation of fixed time sampling in the in vitro release test is to take out all the receiving medium and replenish the same amount of constant temperature receiving medium at 0.5h, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h, 12h and 24h.

8. The use according to any one of claims 3 to 7, characterized in that: The preparation of the reference solution involved in the determination of drug release in the in vitro release test is as follows: weigh the tretinoin reference substance and the fluocinolone acetonide reference substance respectively, dissolve them in methanol, prepare tretinoin reference substance stock solution and fluocinolone acetonide reference substance stock solution, then weigh the hydroquinone reference substance, dissolve it in normal saline-50% ethanol, add appropriate amounts of tretinoin reference substance stock solution and fluocinolone acetonide reference substance stock solution, and dilute with normal saline-50% ethanol to prepare a solution containing 12.5±10%μg / mL of tretinoin, 2.5±10%μg / mL of fluocinolone acetonide, and 1±10%mg / mL of hydroquinone, which is the reference solution.

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