Analysis and detection method for simultaneously detecting tretinoin and tranexamic acid and application thereof

Abstract

The invention provides an analysis and detection method for simultaneously detecting tretinoin and tranexamic acid and application thereof, and the method comprises the following steps: analyzing a to-be-detected sample containing tretinoin and tranexamic acid by using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to realize qualitative and quantitative detection of tretinoin and tranexamic acid. The analysis and detection method provided by the invention has the characteristics of high sensitivity and precision, good repeatability, high accuracy, strong stability and the like. The detection method disclosed by the invention can be easily transferred and applied to detection of two compounds in biological samples such as plasma and tissue homogenate, and can meet the requirement on drug concentration monitoring.

Description

Technical Field

[0001] The invention belongs to the field of analysis and detection, and particularly relates to an analysis and detection method for simultaneously detecting retinoic acid and tranexamic acid and an application thereof. Background Art

[0002] Retinoin is a vitamin A derivative, also known as all-transretinoic acid (RA), which is widely used in dermatology and cosmetology, such as treating acne, anti-aging, and reducing pigmentation. Its main function is to promote the proliferation and regeneration of epidermal cells and increase the rate of cell renewal by regulating gene expression and cell proliferation and differentiation, thereby improving skin texture and reducing spots and pigmentation.

[0003] Tranexamic acid (TA), also known as tranexamic acid, can inhibit the polymerization of fibrin and platelet aggregation, thereby reducing the formation of blood clots. Therefore, TA is widely used in cardiovascular surgery, surgical surgery, obstetric surgery, dental surgery and other fields to prevent and treat postoperative bleeding and thrombosis. In addition, since TA has pharmacological effects such as inhibiting the production of melanin, reducing pigmentation, and inhibiting inflammatory reactions, it is also widely used in dermatology and cosmetology.

[0004] In short, tranexamic acid can reduce the formation of melanin, while retinoic acid can accelerate skin metabolism and promote the excretion of melanin. Therefore, the combination of the two drugs to prepare a compound preparation can play a synergistic role in reducing skin spots and pigmentation, and has good application prospects in the fields of dermatology and cosmetology.

[0005] The IVPT test refers to the in vitro permeation test, which is mainly used to evaluate the permeability of drugs and the permeability of topical skin preparations. This test can simulate the absorption of drugs by human skin, and is of great significance to the quality control and efficacy evaluation of topical preparations. The skin has a natural barrier function, and the permeation rate of topical preparations is often very low; in addition, the IVPT test uses skin samples, which have complex components and the matrix may interfere with the detection, which is undoubtedly a challenge to the sensitivity and accuracy of the quantitative analysis method.

[0006] High performance / ultra-high performance liquid chromatography-triple quadrupole mass spectrometry detection technology has the characteristics of high sensitivity and high accuracy. After sample processing, it is separated by a high performance / ultra-high performance liquid chromatography (HPLC / UPLC) system to reduce the interference of complex components; the separated compounds enter the mass spectrometry (MS) system and are ionized into charged ions. The charged ions enter the first quadrupole (Q1) and produce a series of fragment ions through selective collision-induced dissociation (CID). The fragment ions are screened and separated by the secondary quadrupole (Q2) and the tertiary quadrupole (Q3) and reach the detector. After signal conversion and processing, the mass spectrum of the compound is obtained. By measuring the parent ions and specific fragment ions of the compound in the spectrum, the compound can be quantified and identified. Therefore, it is very appropriate to apply HPLC / UPLC-MS technology to the analysis and detection of IVPT samples.

[0007] However, since retinoic acid is a fat-soluble drug with strong hydrophobicity, it usually requires a high proportion of organic solvent elution to complete chromatographic separation; while tranexamic acid is easily soluble in water and has strong polarity, ion pair reagents are usually added in reverse phase chromatography to enhance retention or hydrophilic interaction chromatography separation and analysis is used. Therefore, the existing reported detection methods cannot meet the needs of simultaneous qualitative / quantitative analysis of retinoic acid and tranexamic acid. Summary of the invention

[0008] In order to improve the above technical problems, the present invention provides an analytical detection method for simultaneously detecting retinoic acid and tranexamic acid, which uses ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to analyze a sample containing retinoic acid and tranexamic acid to achieve qualitative and / or quantitative detection of retinoic acid and tranexamic acid.

[0009] The method comprises:

[0010] With formic acid solution or acetic acid solution as aqueous phase and methanol, methanol solution or acetonitrile as organic phase, ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is used to analyze the test solution containing retinoic acid and tranexamic acid, so as to achieve qualitative and quantitative detection of retinoic acid and tranexamic acid.

[0011] According to an embodiment of the present invention, the formic acid solution is an aqueous formic acid solution, or an aqueous formic acid-ammonium formate solution; the concentration of the formic acid solution is 0.1-0.5v%, that is, the concentration of formic acid in the formic acid solution is 0.1-0.5v%.

[0012] Preferably, the acetic acid solution is an aqueous acetic acid solution, and the concentration of the acetic acid solution is 0.1-0.5v%.

[0013] According to an embodiment of the present invention, the methanol solution is a methanol solution containing formic acid, such as a methanol solution containing 0.1 v% formic acid.

[0014] According to an embodiment of the present invention, the method further comprises the preparation of a standard stock solution of retinoic acid and a standard stock solution of tranexamic acid;

[0015] The method for preparing the retinoic acid standard stock solution is as follows: retinoic acid is mixed with methanol to prepare the retinoic acid standard stock solution;

[0016] The preparation method of the tranexamic acid standard stock solution is: mixing tranexamic acid with water to prepare the tranexamic acid standard stock solution.

[0017] According to an embodiment of the present invention, the concentration of the retinoic acid standard stock solution is 0.1-1 mg / mL, for example, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL. The concentration of the tranexamic acid standard stock solution is 0.5-1.5 mg / mL, for example, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL.

[0018] According to an embodiment of the present invention, the method further comprises the preparation of a retinoic acid internal standard (RAIS) stock solution and a tranexamic acid internal standard (TAIS) stock solution.

[0019] Preferably, the retinoic acid internal standard (RAIS) stock solution uses acitretin as the internal standard.

[0020] Preferably, the tranexamic acid internal standard (TAIS) stock solution uses 4-aminocyclohexane-1-carboxylic acid as the internal standard.

[0021] The method for preparing the retinoic acid internal standard stock solution is: dissolving acitretin in acetone, and then adding methanol to dilute, to prepare the retinoic acid internal standard stock solution; preferably, the mass volume ratio of acitretin, acetone and methanol is 1 mg: 1-5 mL: 2-6 mL;

[0022] The preparation method of the tranexamic acid internal standard stock solution is: dissolving 4-aminocyclohexane-1-carboxylic acid in water to prepare the tranexamic acid internal standard stock solution; preferably, the mass volume ratio of the 4-aminocyclohexane-1-carboxylic acid and water is 1 mg:2-10 mL.

[0023] According to an embodiment of the present invention, the method further comprises the preparation of an internal standard working solution; the internal standard working solution comprises a retinoic acid internal standard and a tranexamic acid internal standard.

[0024] The preparation method of the internal standard working solution is: mixing the retinoic acid internal standard stock solution, the tranexamic acid internal standard stock solution and an acetonitrile aqueous solution to obtain the internal standard working solution;

[0025] Preferably, the concentration of the acetonitrile aqueous solution is 60-90v%, exemplarily 60, 70, 80, 90v%;

[0026] Preferably, the volume ratio of the retinoic acid internal standard stock solution to the acetonitrile aqueous solution is 1:2-6, exemplarily 1:2, 1:3, 1:4, 1:5, 1:6. Preferably, the volume ratio of the tranexamic acid internal standard stock solution to the acetonitrile aqueous solution is 1:2-6, exemplarily 1:2, 1:3, 1:4, 1:5, 1:6.

[0027] According to an embodiment of the present invention, the method further comprises the preparation of a standard curve, and the preparation of the standard curve can be carried out according to conventional methods of those skilled in the art.

[0028] In some embodiments, the preparation of a standard curve comprises the following steps:

[0029] Prepare a blank matrix solution, a standard curve working solution of retinoic acid, a standard curve working solution of tranexamic acid, and an internal standard working solution (or a diluent containing an internal standard) to obtain a standard curve solution containing both retinoic acid and tranexamic acid at 5 to 7 concentration gradient points; analyte ) is the horizontal axis, the ratio of the peak area of ​​the analyte to the internal standard (A analyte / A IS ) is the ordinate, and the standard curve is fitted.

[0030] According to an embodiment of the present invention, the blank matrix liquid includes a permeate blank matrix liquid or a skin extract blank matrix liquid, wherein the blank matrix liquid is obtained by conducting an IVPT test under the same conditions using a blank preparation (i.e., a preparation using the same excipients and preparation process but without active ingredients) to obtain a permeate blank matrix liquid or a skin extract blank matrix liquid.

[0031] According to an embodiment of the present invention, the internal standard method is used to detect and calculate the contents of retinoic acid and tranexamic acid in the sample to be tested.

[0032] In some embodiments, the peak area ratio (A) of the analyte to the internal standard of the sample obtained by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry can be calculated. analyte / A IS ), and then the standard curves of the two analytes were inserted to obtain the analyte concentration (C analyt ), and further calculate the contents of retinoic acid and tranexamic acid in the sample to be tested.

[0033] According to an embodiment of the present invention, the standard curve working solution includes a retinoic acid standard curve working solution and a tranexamic acid standard curve working solution;

[0034] The preparation method of the retinoic acid standard curve working solution is: mixing the retinoic acid standard stock solution with an acetonitrile aqueous solution to obtain the retinoic acid standard curve working solution;

[0035] Preferably, the concentration of the retinoic acid standard curve working solution is 200-10000 ng / mL, exemplarily 200 ng / mL, 500 ng / mL, 1000 ng / mL, 2000 ng / mL, 5000 ng / mL, 10000 ng / mL;

[0036] The preparation method of the tranexamic acid standard curve working solution is: mixing the tranexamic acid standard product stock solution with an acetonitrile aqueous solution to obtain the tranexamic acid standard curve working solution;

[0037] Preferably, the concentration of the tranexamic acid standard curve working solution is 10000-750000 ng / mL, exemplarily 10000 ng / mL, 30000 ng / mL, 75000 ng / mL, 150000 ng / mL, 300000 ng / mL, 750000 ng / mL.

[0038] Preferably, the concentration of the acetonitrile aqueous solution is 60-90v%, exemplarily 60, 70, 80, 90v%.

[0039] According to an embodiment of the present invention, the method further comprises preparing a receiving fluid standard curve solution and a skin extract standard curve solution;

[0040] The preparation method of the receiving solution standard curve solution is: respectively mixing the retinoic acid standard curve working solution, tranexamic acid standard curve working solution, internal standard working solution, and permeate blank matrix solution, then mixing with a protein precipitant, centrifuging to obtain a supernatant and diluting to obtain the receiving solution standard curve solution;

[0041] The preparation method of the skin extract standard curve solution is: respectively mixing the retinoic acid standard curve working solution, the tranexamic acid standard curve working solution, the skin extract blank matrix solution and the extract diluent containing the internal standard, taking the supernatant and diluting it to obtain the skin extract standard curve solution;

[0042] Preferably, in the method for preparing the receiving solution standard curve solution, the concentration of the retinoic acid standard curve solution is 0.2-0.4 ng / mL, 0.5-1 ng / mL, 1-2 ng / mL, 2-4 ng / mL, 5-10 ng / mL, 10-20 ng / mL;

[0043] Preferably, in the method for preparing the receiving solution standard curve solution, the concentration of the tranexamic acid standard curve solution is 10-20 ng / mL, 30-40 ng / mL, 75-150 ng / mL, 150-300 ng / mL, 300-600 ng / mL, 750-1500 ng / mL.

[0044] Preferably, in the method for preparing the skin extract standard curve solution, the concentration of the retinoic acid standard solution is 0.2-0.4 ng / mL, 0.5-1 ng / mL, 1-2 ng / mL, 2-4 ng / mL, 5-10 ng / mL, 10-20 ng / mL;

[0045] Preferably, in the method for preparing a skin extract standard curve solution, the concentration of the tranexamic acid standard solution is 10-20 ng / mL, 30-40 ng / mL, 75-150 ng / mL, 150-300 ng / mL, 300-600 ng / mL, or 750-1500 ng / mL.

[0046] Preferably, the concentration of the internal standard (TAIS and RAIS) in the receiving solution standard curve solution or the skin extract standard curve solution is 10-200 ng / mL; preferably, the concentration of the internal standard is 50-100 ng / mL.

[0047] According to an embodiment of the present invention, the protein precipitant is acetonitrile, methanol, trichloroacetic acid solution, etc.; preferably, the protein precipitant is acetonitrile.

[0048] According to an embodiment of the present invention, the volume ratio of the retinoic acid standard curve working solution or the tranexamic acid standard curve working solution to the protein precipitant is 1:2-10, preferably 1:3-6.

[0049] According to an embodiment of the present invention, the internal standard-containing extract diluent comprises retinoic acid internal standard and tranexamic acid internal standard.

[0050] Preferably, the extract diluent containing the internal standard is obtained by mixing the retinoic acid internal standard stock solution, the tranexamic acid internal standard stock solution and an acetonitrile aqueous solution.

[0051] Preferably, the concentration of the acetonitrile aqueous solution mixed with the retinoic acid internal standard stock solution or the tranexamic acid internal standard stock solution is 60-90v%, exemplarily 60, 70, 80, 90v%.

[0052] According to an embodiment of the present invention, the method further includes processing of a sample to be tested, wherein the sample to be tested is a substance containing tretinoin and tranexamic acid. Exemplarily, the sample to be tested is, for example, a skin extract or a receiving solution produced by an IVPT test; the skin extract or the receiving solution at least includes tretinoin and tranexamic acid. The receiving solution contains a solubilizing agent added to increase the solubility of tretinoin, preferably a protein. Preferably, when tretinoin and tranexamic acid are detected by the internal standard method, the sample to be tested may also contain an internal standard (e.g., at least one of acitretin (RAIS) and 4-aminocyclohexane-1-carboxylic acid (TAIS)) to eliminate errors.

[0053] According to an embodiment of the present invention, the skin extract processing method is specifically as follows: the skin extract is mixed with an extract diluent containing an internal standard, the supernatant is obtained by centrifugation, and an acetonitrile aqueous solution is added to dilute it to obtain a test sample containing retinoic acid and tranexamic acid, i.e., an intradermal retention sample solution.

[0054] According to an embodiment of the present invention, the method for processing the receiving liquid is also specifically as follows: mixing the receiving liquid sample with the internal standard working solution, adding a protein precipitant, centrifuging to obtain the supernatant, adding an acetonitrile aqueous solution to dilute, and obtaining a sample to be tested containing retinoic acid and tranexamic acid, i.e., a skin permeation sample solution.

[0055] Preferably, the concentration of the acetonitrile aqueous solution is 60-90v%, exemplarily 60, 70, 80, 90v%.

[0056] Preferably, in the intradermal retention sample solution or the skin permeation sample solution, the concentration of the internal standard (TAIS and RAIS) is 10-200 ng / mL; preferably, the concentration of the internal standard is 50-100 ng / mL.

[0057] According to an embodiment of the present invention, the test conditions of the ultra-high performance liquid chromatography include:

[0058] Chromatographic column: Waters ACQUITY UPLC HSS T3 2.1*100mm, particle size of filler: 1.8μm;

[0059] Or ThermoFisher ACCUCORE UREA-HILIC 2.1*100mm, filler particle size is 2.6μm; or Welch Ultimate XS-C18 2.1×100mm, filler particle size is 3.0μm; preferably Welch Ultimate XS-C182.1×100mm, filler particle size is 3.0μm;

[0060] Guard column: Welch Ultimate XS-C18 2.1×10 mm, particle size 3.0 μm;

[0061] Mobile phase: aqueous phase A - 0.1v% to 0.5v% formic acid aqueous solution, or 0.15wt% formic acid-ammonium formate aqueous solution (pH 4.0±0.5), or 0.3v% acetic acid aqueous solution;

[0062] Organic phase B - methanol, acetonitrile, methanol solution containing 0.1v% formic acid; gradient elution;

[0063] Preferably, the gradient elution program is as follows: 0-0.8 min, A is maintained at 98 v%, and B is maintained at 2 v%; 0.8-4 min, A decreases from 98 v% to 5 v%, and B increases from 2 v% to 95 v%; 4-6.5 min, A is maintained at 5 v%, and B is maintained at 95 v%; 6.5-7 min, A increases from 5 v% to 98 v%, and B decreases from 95 v% to 2 v%; 7-8.5 min, A is maintained at 98 v%, and B is maintained at 2 v%;

[0064] Flow rate: 0.3-0.5 mL / min, preferably 0.4 mL / min;

[0065] Injection volume: 2-20 μL, preferably 5 μL;

[0066] Column temperature: 30-60°C, preferably 40°C.

[0067] According to an embodiment of the present invention, the test conditions of the triple quadrupole mass spectrometer are as follows:

[0068] Ion source: electrospray ionization source (ESI); Scan type: positive ion mode;

[0069] Spray voltage (IS): 5500V; temperature: 200-400°C, preferably 300°C;

[0070] Declustering voltage (DP): 20-50V, preferably 20-40V;

[0071] Impact voltage (CE): 12-40V, preferably 14-30V.

[0072] The present invention also provides application of the above method in biological samples such as plasma and tissue homogenate.

[0073] Beneficial effects of the present invention:

[0074] The analytical detection method provided by the present invention has the characteristics of high sensitivity and precision, good repeatability, high accuracy, strong stability, etc.

[0075] The detection method of the present invention can be easily transferred and applied to the detection of two compounds in biological samples such as plasma and tissue homogenate, and thus can be applied to the study of the pharmacokinetics and tissue distribution of the two compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 is the chromatogram of tranexamic acid (TA) and its internal standard (TAIS);

[0077] Figure 2 is the chromatogram of retinoic acid (RA) and its internal standard (RAIS);

[0078] Figure 3 is the blank solution chromatogram. DETAILED DESCRIPTION

[0079] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0080] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0081] Example 1

[0082] Liquid phase method

[0083] Table 1 Liquid phase parameters

[0084]

[0085]

[0086] Table 2 Gradient elution table

[0087] Time / min A / v% B / v% 0 98 2 0.8 98 2 4 5 95 6.5 5 95 7 98 2 8.5 98 2

[0088] Mass spectrometry

[0089] Scan type: Multiple reaction ion monitoring, positive ion mode (MRM positive)

[0090] Ion source: electrospray ionization (ESI)

[0091] Temperature parameters: 300℃

[0092] Spray voltage (IS): 5500V

[0093] Table 1 Analytes and IS mass spectrometry parameter settings

[0094]

[0095]

[0096] In Table 1, 2 and 3, or 4 and 5, or 6 and 7 are detected using different fragment ions.

[0097] 3. Specific steps for IVPT sample testing

[0098] 3.1. Preparation of standard and internal standard stock solutions

[0099] 3.1.1. Retinoic acid standard stock solution

[0100] Accurately weigh 20 mg of retinoic acid standard into a 100 mL brown volumetric flask, add methanol to dissolve and dilute to volume.

[0101] 3.1.2. Tranexamic acid standard stock solution

[0102] Accurately weigh 15 mg of tranexamic acid standard into a 20 mL volumetric flask, add water to dissolve and dilute to volume.

[0103] 3.1.3. Retinoic acid internal standard (RAIS) stock solution

[0104] Accurately weigh 10 mg of acitretin into a 50 mL brown volumetric flask, add about 10 mL of acetone to dissolve it, and then dilute it to the mark with methanol.

[0105] 3.1.4. Tranexamic acid internal standard (TAIS) stock solution

[0106] Accurately weigh 10 mg of 4-aminocyclohexane-1-carboxylic acid into a 50 mL volumetric flask, add water to dissolve and dilute to volume.

[0107] All the above solutions were refrigerated at 2-8°C and returned to room temperature before use.

[0108] 3.2. Preparation of standard curve and internal standard working solution

[0109] 3.2.1. Internal standard working solution

[0110] Pipette 4 mL of each of two internal standard stock solutions (4 mL of retinoic acid internal standard stock solution and 4 mL of tranexamic acid internal standard stock solution) into a 20 mL brown volumetric flask, and dilute to the mark with 80 v% acetonitrile to obtain the internal standard working solution.

[0111] 3.2.2. Dilution of extract containing internal standard

[0112] Pipette 250 μL of two internal standard stock solutions (250 μL each of retinoic acid internal standard stock solution and tranexamic acid internal standard stock solution), add to 50 mL volume, and dilute to the scale with 80v% acetonitrile to obtain an extract dilution containing the internal standard.

[0113] 3.2.3. Standard curve working solution

[0114] Accurately measure the RA or TA standard stock solution, use 80% acetonitrile as the diluent, and dilute it step by step to obtain a series of concentration standard curve working solutions.

[0115] The concentration series of the obtained TA standard curve working solutions were: 10000, 30000, 75000, 150000, 300000, 750000 ng / ml.

[0116] The concentration series of the obtained RA standard curve working solutions were 200, 500, 1000, 2000, 5000, and 10000 ng / ml, respectively.

[0117] 3.3. Preparation of standard curve solution

[0118] 3.3.1. Receiving solution (permeate) standard curve solution

[0119] Accurately pipette 160 μL of the permeate blank matrix, add 10 μL of RA standard curve working solution, TA standard curve working solution of a series of concentrations, and 20 μL of internal standard working solution, and vortex for 30 seconds to mix evenly; then add 800 μL of acetonitrile, vortex for 3 minutes, and centrifuge at 12000 rpm for 3 minutes to remove protein; take 100 μL of the supernatant, add 900 μL of 80% acetonitrile to dilute, and obtain the receiving solution standard curve solution for analysis and detection.

[0120] In the obtained receiving solution standard curve solutions (respectively recorded as JSBQ-1, JSBQ-2, JSBQ-3, JSBQ-4, JSBQ-5, and JSBQ-6), the TA concentrations were 10, 30, 75, 150, 300, and 750 ng / mL; the RA concentrations were 0.2, 0.5, 1, 2, 5, and 10 ng / mL; and the TAIS and RAIS concentrations were both 80 ng / mL.

[0121] 3.3.2. Skin extract (intradermal retention) standard curve solution

[0122] Accurately pipette 160 μL of blank matrix of skin extract, add 10 μL of RA standard curve working solution and TA standard curve working solution of different concentrations, and 400 μL of extract diluent containing internal standards (including retinoic acid internal standard and tranexamic acid internal standard) in 3.2.2, and vortex for 30 seconds to mix evenly; take 100 μL of the solution, add 900 μL of 80% acetonitrile to dilute, and obtain the skin extract standard curve solution for analysis and detection.

[0123] In the obtained skin extract standard curve solutions (respectively labeled as TQBQ-1, TQBQ-2, TQBQ-3, TQBQ-4, TQBQ-5, and TQBQ-6), the TA concentrations were 20, 60, 150, 300, 600, and 1500 ng / mL; the RA concentrations were 0.4, 1, 2, 4, 10, and 20 ng / mL; and the TAIS and RAIS concentrations were both 80 ng / mL.

[0124] 3.4. IVPT test sample processing

[0125] 3.4.1. The IVPT test samples used in the present invention are: a preparation of a compound of two active ingredients, retinoic acid and tranexamic acid, and samples produced by IVPT testing (including the receiving solution and skin extract obtained by the following treatment).

[0126] 3.4.2. Receiving fluid sample processing

[0127] Take 180 μL of the receiving liquid sample at each time point, add 20 μL of the corresponding internal standard working solution, and vortex for 30 seconds to mix; then add 800 μL of acetonitrile, vortex for 3 minutes, and centrifuge at 12000 rpm for 3 minutes; aspirate 100 μL of the supernatant, add 900 μL of 80% acetonitrile to dilute, centrifuge, take 5 μL of the supernatant for analysis, and record the chromatogram.

[0128] 3.4.3. Skin extract (intradermal retention) sample processing

[0129] Use a cryo-grinder to grind and extract the skin sample. Take 100 μL of the skin extract, add 400 μL of the extract diluent containing the internal standard, and vortex for 30 seconds to mix; take 100 μL of the solution, add 900 μL of 80% acetonitrile to dilute, centrifuge, take 5 μL of the supernatant for analysis, and record the chromatogram.

[0130] 3.5 IVPT test sample test results

[0131] 3.5.1 Compound preparation 1:

[0132] Formula of compound preparation 1

[0133] name Proportion 20g batch dosage Retinoic acid 0.1％ 0.02g Tranexamic acid 5％ 1.0g Twain 80 5％ 1.0％ Carbomer 980 2％ 0.4g Medium Chain Triglycerides 15％ 3.0g Cetyl Alcohol 10％ 2.0g Ethanol 5％ 1.0g Sodium hydroxide Adjust pH to 5.0-5.5 Adjust pH to 5.0-5.5 water Add to 100% Add to 20g

[0134] Process: Weigh hexadecanol and medium-chain triglycerides into a beaker, heat in a water bath to 60°C and stir to melt the hexadecanol; add carbomer 980 and stir to disperse to obtain an oil phase; weigh tranexamic acid and Tween 80, dissolve them in 50% of the prescribed amount of water, and heat to 60°C to obtain an aqueous phase; weigh retinoic acid, disperse it in ethanol, and add it to the oil phase and stir to disperse; then add the aqueous phase to the oil phase, maintain stirring in a 60°C water bath; detect the pH value, and use an appropriate amount of sodium hydroxide aqueous solution to adjust the pH to 5.0-5.5; add water to the full amount; use a high shear homogenizer to homogenize; obtain a light yellow ointment after natural cooling; fill it into an aluminum tube and seal it.

[0135] 3.5.2 Compound preparation 2: Batch number IDT-2212151

[0136] Formula of compound preparation 2

[0137] name Proportion 20g batch dosage Retinoic acid 0.1％ 0.02g Tranexamic acid 5％ 1.0g Caprylic / capric macrogol glycerides 3％ 0.6g Carbomer 980 1％ 0.2g Soybean Oil 15％ 3.0g Octadecanol 12％ 2.4g Isopropyl alcohol 5％ 1.0g Triethanolamine Adjust pH to 5.0-5.5 Adjust pH to 5.0-5.5 water Add to 100% Add to 20g

[0138] Process: Weigh octadecyl alcohol and soybean oil into a beaker, heat in a water bath to 60°C and stir to melt the octadecyl alcohol; add carbomer 980 and stir to disperse to obtain an oil phase; weigh tranexamic acid, dissolve it in 50% of the prescribed amount of water, heat to 60°C, add caprylic acid capric acid macrogol glyceride and disperse it evenly to obtain an aqueous phase; weigh retinoic acid, disperse it in isopropanol, and add it to the oil phase and stir to disperse it; then add the aqueous phase to the oil phase and maintain stirring in a water bath at 60°C; detect the pH value, and use an appropriate amount of triethanolamine to adjust the pH to 5.0-5.5; add water to the full amount; use a high shear homogenizer to homogenize; obtain a light yellow ointment after natural cooling; fill it into an aluminum tube and seal it.

[0139] An IVPT test was conducted to compare the transdermal performance of the two compound preparations. According to "3. Specific steps for IVPT sample testing", the receiving solution samples or skin extract (intradermal retention) samples obtained at each time point were tested to obtain the TA concentration and RA concentration in the IVPT test samples of compound preparation 1 and compound preparation 2. The test results are shown in Table A below:

[0140] Table A TA concentration and RA concentration test results of compound preparation 1 and compound preparation 2 IVPT test samples

[0141]

[0142] 4. Preliminary Methodological Validation

[0143] System suitability

[0144] System suitability testing was performed to determine the RSD% of 6 replicate injections of tretinoin and tranexamic acid standard preparations.

[0145] Table 8 System suitability results

[0146] System suitability Measured value RSD of the peak area response of tranexamic acid in the standard preparation solution 0.31％ RSD of the peak area response value of retinoic acid in the standard preparation solution 0.57％

[0147] in conclusion:

[0148] The RSD% of repeated injections of the standard preparation solution was greater than 2.0%, which met the requirements of system suitability and system precision studies.

[0149] 4.2. Exclusivity

[0150] 4.2.1. Blank Interference

[0151] Specificity testing was performed to monitor the blank solution for interference with retinoic acid and tranexamic acid. Figure 3 shown.

[0152] in conclusion:

[0153] The ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (LCMS) method of the present invention has excellent specificity, and the blank solution has no interference at the retention time of tranexamic acid and retinoic acid and their respective internal standards.

[0154] 4.3 Lower limit of quantitation (LLOQ)

[0155] The RA standard curve working solution with a concentration of 200 ng / mL was used as the RA-LLOQ working solution; the TA standard curve working solution with a concentration of 10000 ng / mL was further diluted to a concentration of 500 ng / mL using 80% acetonitrile to serve as the TA-LLOQ working solution.

[0156] Take the ex vivo pig skin and administer the blank sample percutaneously for 24 hours, then collect all the permeated liquid as the receiving liquid blank matrix solution; the skin after the test is processed according to the skin extract sample (intradermal retention) in 3.4.2 to obtain the skin extract blank matrix.

[0157] Take 160 μL of the receiving liquid blank matrix solution, add 10 μL each of the TA-LLOQ working solution and the RA-LLOQ working solution, and 20 μL of the internal standard working solution, and vortex for 30 seconds to mix; then add 800 μL of acetonitrile, vortex for 3 minutes, and centrifuge at 12000 rpm for 3 minutes to remove protein; take 100 μL of the supernatant, add 900 μL of 80% acetonitrile to dilute, and obtain the receiving liquid LLOQ solution. Repeat the injection 6 times, and record the peak areas of the analyte and the internal standard.

[0158] Accurately pipette 160 μL of skin extract blank matrix, add 10 μL each of TA-LLOQ working solution and RA-LLOQ working solution, and 400 μL of extract diluent containing internal standard, and vortex for 30 seconds to mix evenly; take 100 μL of the solution, add 900 μL of 80% acetonitrile to dilute, and obtain skin extract LLOQ solution. Repeat the injection 6 times, and record the peak areas of analyte and internal standard.

[0159] The peak area of ​​the blank sample at the retention time of the analyte should not be higher than 20% of the peak area of ​​the analyte in the LLOQ sample, and the peak area of ​​the internal standard at the retention time should not be higher than 5% of the peak area of ​​the internal standard.

[0160] LLOQ results:

[0161] Table 9 LLOQ results

[0162]

[0163] in conclusion:

[0164] The lower limits of quantification of TA and RA in the receiving fluid can be as low as 0.503 ng / mL and 0.198 ng / mL, respectively; the lower limits of quantification of TA and RA in the skin extract can be as low as 1.006 ng / mL and 0.396 ng / mL, respectively; this shows that the analytical method has high sensitivity and can meet the quantitative requirements of IVPT analysis and detection.

[0165] 4.4. Accuracy and precision

[0166] Repeatability

[0167] According to the preparation method of receiving solution No. 3 standard curve sample solution (JSBQ-3) and skin extract solution No. 3 standard curve sample solution (TQBQ-3), prepare 6 samples respectively and inject them for analysis. Calculate the concentration of tranexamic acid and retinoic acid, compare with the prepared concentration, get the recovery rate, and calculate the RSD% of 6 injections. The recovery rate% should be between 90-110; the RSD% of 6 injections should be ≤5.0.

[0168] 4.4.2.Batch-to-batch precision

[0169] Reanalyze JSBQ-3 and TQBQ-3 6 times on different dates. Calculate the recovery and RSD%, and calculate the variance (CV%) of the results on different dates. CV% should be ≤10.0.

[0170] Table 13.1 Repeatability and inter-batch precision results of JSBQ-3

[0171]

[0172] Table 13.2 Repeatability and batch precision results of TQBQ-3

[0173]

[0174]

[0175] in conclusion:

[0176] The accuracy and precision (repeatability and batch-to-batch precision) of this method meet the requirements of IVPT testing.

[0177] 4.5. Linear

[0178] The measurement results of the standard curve sample solution were statistically analyzed, and the ratio of the analyte peak area to the internal standard peak area (A analyte / A IS ) as the dependent variable (y); considering that the amount of internal standard added in the sample is consistent, in order to simplify the calculation, the sample solution concentration (C analyte ) as the independent variable (x); in order to improve the calculation accuracy of low-concentration samples, a linear regression was performed using a 1 / x weight (software: IBM SPSS Statistics 20); the linear equation was obtained:

[0179] Table 14 Standard curve summary

[0180] Serial number name Marking song R2 Linear range 1 Tranexamic acid receiving solution standard curve y=0.154x-0.020 0.997 10~750ng / ml 2 Tranexamic acid skin extract y=0.0145x-0.0225 0.998 20~1500ng / ml 3 Retinoic acid receiving solution standard curve y=0.771x-0.069 0.995 0.2~10ng / ml 4 Retinoic acid skin extract y=0.0838x-0.0142 0.987 0.4~20ng / ml

[0181] in conclusion:

[0182] The analytes, tranexamic acid and retinoic acid, showed good linear relationships in the receiving solution and the skin extract, meeting the test requirements.

[0183] 5.1. Investigation of matrix effects

[0184] Accurately draw 530 μL of the blank matrix of the permeate, add 10 μL of the standard solution of low concentration (200 ng / ml) or high concentration (40000 ng / ml) (including TA low concentration, TA high concentration, RA low concentration and RA high concentration) and 60 μL of the internal standard working solution, vortex mix for 1 min, and prepare the matrix inspection sample (n=6); take 200 μL of the matrix inspection sample, add 800 μL of acetonitrile, vortex mix for 3 min, and centrifuge at 12000 rpm for 5 min. Take the supernatant for detection, and record the peak area as A; use 80% acetonitrile as the diluent, prepare the corresponding concentration (low, high) matrix-free standard solution for injection (n=6), record the peak area as B, and take the average value of B; calculate the matrix factor of the analyte and the internal standard at each concentration (matrix factor = A / B). Further, divide the matrix factor of the analyte by the matrix factor of the internal standard to calculate the matrix factor normalized by the internal standard, and the RSD should be ≤15%.

[0185] The skin extract blank matrix was used instead and the matrix effect of the skin extract was evaluated in the same manner.

[0186] The matrix effect results for two analytes (tranexamic acid, tretinoin) in two matrices (receiving fluid, skin extract) are as follows:

[0187] Table 2 Matrix effect determination results

[0188]

[0189] in conclusion:

[0190] The normalized matrix factors of the two analytes (tranexamic acid and tretinoin) in the two matrices (receiving solution and skin extract) were approximately 100% (80% to 120%), with RSDs < 15%; therefore, the matrix effect of this method meets the requirements of analytical detection.

[0191] 5.2. Screening of different chromatographic columns

[0192] Take 20 μL of each of the 10 IVPT test samples of the present invention (according to 3.4) and the standard solution samples of each receiving solution and skin extract, vortex mix, and use them as QC samples. Use different chromatographic columns to analyze and detect the QC samples, and compare the retention time and peak shape of the analyte under different chromatographic columns. Chromatographic column A: Waters ACQUITY UPLC HSS T3 (2.1*100mm, 1.8um)

[0193] Column B: ThermoFisher ACCUCORE UREA-HILIC (2.1*100mm, 2.6μm) Column C: Welch Ultimate XS-C18 (2.1×100mm, 3.0μm)

[0194] Table 19 Screening results of different chromatographic columns

[0195]

[0196]

[0197] in conclusion:

[0198] Taking into account the peak shape and the availability of the chromatographic column, the preferred chromatographic column C is Welch Ultimate XS-C18 (2.1×100 mm, 3.0 μm).

[0199] 5.3. Investigation of flow rate

[0200] Different flow rates (0.3mL / min, 0.4mL / min) were set to analyze and detect the QC samples. The retention times of tranexamic acid and retinoic acid were as follows:

[0201] Table 20 Screening results at different flow rates

[0202] 0.3mL / min 0.4mL / min Tranexamic acid 1.12min 0.79min Retinoic acid 6.98min 5.67min

[0203] in conclusion:

[0204] IVPT samples are relatively large, so in order to shorten the analysis and testing time, a flow rate of 0.4 mL / min is preferred. In addition, if the flow rate is set too fast (greater than 0.5 mL / min), the liquid flow entering the MS system will increase, resulting in incomplete sample atomization, affecting the ionization of the analyte, and causing quantitative deviations. Therefore, no tests with higher flow rates were conducted.

[0205] The above is an exemplary description of the embodiments of the present invention. However, the protection scope of the present invention is not limited to the above embodiments. Any modification, equivalent substitution, improvement, etc. made by those skilled in the art within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An analytical detection method for simultaneously detecting retinoic acid and tranexamic acid, characterized in that, The method comprises: With formic acid solution or acetic acid solution as aqueous phase and methanol, methanol solution or acetonitrile as organic phase, ultra performance liquid chromatography-triple quadrupole mass spectrometry is used to analyze the sample solution containing retinoic acid and tranexamic acid, so as to achieve qualitative and quantitative detection of retinoic acid and tranexamic acid.

2. The method according to claim 1, characterized in that The formic acid solution is a formic acid aqueous solution or a formic acid-ammonium formate aqueous solution; the concentration of the formic acid solution is 0.1-0.5v%. Preferably, the acetic acid solution is an aqueous acetic acid solution, and the concentration of the acetic acid solution is 0.1-0.5v%. Preferably, the methanol solution is a methanol solution containing formic acid.

3. The method according to claim 1 or 2, characterized in that: The method also includes the preparation of a standard stock solution of retinoic acid and a standard stock solution of tranexamic acid; The method for preparing the retinoic acid standard stock solution is as follows: retinoic acid is mixed with methanol to prepare the retinoic acid standard stock solution; The preparation method of the tranexamic acid standard stock solution is: mixing tranexamic acid with water to prepare the tranexamic acid standard stock solution. Preferably, the method further comprises the preparation of a retinoic acid internal standard stock solution and a tranexamic acid internal standard stock solution; The method for preparing the retinoic acid internal standard stock solution is: dissolving acitretin in acetone, and then adding methanol to dilute, to prepare the retinoic acid internal standard stock solution; preferably, the mass volume ratio of acitretin, acetone and methanol is 1 mg: 1-5 mL: 2-6 mL; The preparation method of the tranexamic acid internal standard stock solution is: dissolving 4-aminocyclohexane-1-carboxylic acid in water to prepare the tranexamic acid internal standard stock solution; preferably, the mass volume ratio of the 4-aminocyclohexane-1-carboxylic acid and water is 1 mg:2-10 mL.

4. The method according to any one of claims 1 to 3, characterized in that: The method further comprises the preparation of an internal standard working solution; the internal standard working solution comprises a retinoic acid internal standard and a tranexamic acid internal standard; The preparation method of the internal standard working solution is: mixing the retinoic acid internal standard stock solution, the tranexamic acid internal standard stock solution and an acetonitrile aqueous solution to obtain the internal standard working solution; Preferably, the volume ratio of the retinoic acid internal standard stock solution to the acetonitrile aqueous solution is 1:2-6. Preferably, the volume ratio of the tranexamic acid internal standard stock solution to the acetonitrile aqueous solution is 1:2-6.

5. The method according to any one of claims 1 to 4, characterized in that: The internal standard method was used to calculate the content of retinoic acid and tranexamic acid in the sample to be tested, that is, the ratio of the peak area of ​​the analyte to the internal standard of the sample obtained by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry was A. analyte / A IS , insert the standard curves of the two analytes respectively, and obtain the analyte concentration C analyte , obtain the content of retinoic acid and tranexamic acid in the sample to be tested.

6. The method according to any one of claims 1 to 5, characterized in that: The standard curve working solution includes retinoic acid standard curve working solution and tranexamic acid standard curve working solution; The preparation method of the retinoic acid standard curve working solution is: mixing the retinoic acid standard stock solution with an acetonitrile aqueous solution to obtain the retinoic acid standard curve working solution; preferably, the concentration of the retinoic acid standard curve working solution is 200-10000 ng / ml; The preparation method of the tranexamic acid standard curve working solution is: mixing the tranexamic acid standard stock solution with an acetonitrile aqueous solution to obtain the tranexamic acid standard curve working solution; preferably, the concentration of the tranexamic acid standard curve working solution is 10000-750000 ng / mL.

7. The method according to any one of claims 1 to 6, characterized in that: The method further comprises preparing a receiving fluid standard curve solution and a skin extract standard curve solution; The preparation method of the receiving solution standard curve solution is: respectively mixing the retinoic acid standard curve working solution, tranexamic acid standard curve working solution, internal standard working solution, and permeate blank matrix solution, then mixing with a protein precipitant, centrifuging to obtain a supernatant and diluting to obtain the receiving solution standard curve solution; The preparation method of the skin extract standard curve solution is: respectively mixing the retinoic acid standard curve working solution, the tranexamic acid standard curve working solution, the skin extract blank matrix solution and the extract diluent containing the internal standard, taking the supernatant and diluting it to obtain the skin extract standard curve solution; In the method for preparing the receiving solution standard curve solution, the concentration of the retinoic acid standard curve solution is 0.2-0.4 ng / mL, 0.5-1 ng / mL, 1-2 ng / mL, 2-4 ng / mL, 5-10 ng / mL, 10-20 ng / mL; Preferably, in the method for preparing the receiving solution standard curve solution, the concentration of the tranexamic acid standard curve solution is 10-20 ng / mL, 30-40 ng / mL, 75-150 ng / mL, 150-300 ng / mL, 300-600 ng / mL, 750-1500 ng / mL. Preferably, in the method for preparing the skin extract standard curve solution, the concentration of the retinoic acid standard solution is 0.2-0.4 ng / mL, 0.5-1 ng / mL, 1-2 ng / mL, 2-4 ng / mL, 5-10 ng / mL, 10-20 ng / mL; Preferably, in the method for preparing a skin extract standard curve solution, the concentration of the tranexamic acid standard solution is 10-20 ng / mL, 30-40 ng / mL, 75-150 ng / mL, 150-300 ng / mL, 300-600 ng / mL, or 750-1500 ng / mL. Preferably, the concentration of the internal standard in the receiving solution standard curve solution or the skin extract standard curve solution is 10-200 ng / mL. Preferably, the protein precipitant is at least one of acetonitrile, methanol and trichloroacetic acid solution. Preferably, the internal standard-containing extract diluent comprises retinoic acid internal standard and tranexamic acid internal standard. Preferably, the extract diluent containing the internal standard is obtained by mixing the retinoic acid internal standard stock solution, the tranexamic acid internal standard stock solution and an acetonitrile aqueous solution.

8. The method according to any one of claims 1 to 7, characterized in that: The method further comprises processing a sample to be tested, wherein the sample to be tested is a substance containing retinoic acid and tranexamic acid. Exemplarily, the sample to be tested is a skin extract or a receiving fluid; the skin extract or the receiving fluid comprises at least retinoic acid and tranexamic acid. Preferably, the skin extract processing method is specifically as follows: mixing the skin extract with an extract diluent containing an internal standard, centrifuging to obtain the supernatant, adding an acetonitrile aqueous solution to dilute it, and obtaining a test sample containing retinoic acid and tranexamic acid, i.e., an intradermal retention sample solution. Preferably, the method for processing the receiving solution is further specifically as follows: mixing the receiving solution sample with the internal standard working solution, adding a protein precipitant, centrifuging to obtain the supernatant, adding an acetonitrile aqueous solution to dilute, and obtaining a sample to be tested containing retinoic acid and tranexamic acid, i.e., a skin permeation sample solution. Preferably, in the intradermal retention sample solution or the skin permeation sample solution, the concentration of the internal standard is 10-200 ng / mL.

9. The method according to any one of claims 1 to 8, characterized in that: The test conditions of the ultra-high performance liquid chromatography include: Chromatographic column: Waters ACQUITY UPLC HSS T3 2.1*100mm, particle size of filler: 1.8μm; Or ThermoFisher ACCUCORE UREA-HILIC 2.1*100mm, filler particle size is 2.6μm; or Welch Ultimate XS-C18 2.1×100mm, filler particle size is 3.0μm; preferably Welch Ultimate XS-C18 2.1×100mm, filler particle size is 3.0μm; Guard column: Welch Ultimate XS-C18 2.1×10 mm, particle size 3.0 μm; Mobile phase: aqueous phase A - 0.1v% to 0.5v% formic acid aqueous solution, or 0.15wt% formic acid-ammonium formate aqueous solution (pH = 4.0 ± 0.5), or 0.3v% acetic acid aqueous solution; Organic phase B - methanol, acetonitrile, methanol solution containing 0.1v% formic acid; gradient elution; Preferably, the gradient elution program is as follows: 0-0.8 min, A is maintained at 98 v%, and B is maintained at 2 v%; 0.8-4 min, A decreases from 98 v% to 5 v%, and B increases from 2 v% to 95 v%; 4-6.5 min, A is maintained at 5 v%, and B is maintained at 95 v%; 6.5-7 min, A increases from 5 v% to 98 v%, and B decreases from 95 v% to 2 v%; 7-8.5 min, A is maintained at 98 v%, and B is maintained at 2 v%; Flow rate: 0.3~0.5mL / min Injection volume: 2-20 μL; Column temperature: 30~60℃. Preferably, the test conditions of the triple quadrupole mass spectrometer are as follows: Ion source: electrospray ionization source (ESI); Scan type: positive ion mode; Spray voltage (IS): 5500V; Temperature: 200~400℃; Declustering voltage (DP): 20~50V, Impact voltage (CE): 12~40V.

10. Use of the method according to any one of claims 1 to 9 in plasma or tissue homogenate biological samples.

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