Analysis and detection method for simultaneously detecting tretinoin and tranexamic acid and use of analysis and detection method

Abstract

Provided in the present invention is an analysis and detection method for simultaneously detecting tretinoin and tranexamic acid, and the use of the analysis and detection method. The method comprises: analyzing a sample to be tested comprising tretinoin and tranexamic acid by using an ultra-high performance liquid chromatography-triple quadrupole mass spectrum, so as to achieve the qualitative and quantitative detection of tretinoin and tranexamic acid. The analysis and detection method provided in the present invention has the characteristics of a high sensitivity and high precision, good repeatability, high accuracy, high stability, etc. The detection method of the present invention can be relatively easily transferred and applied to the detection of the two compounds in biological samples such as plasma and tissue homogenates, and can satisfy the requirements for drug concentration monitoring.

Description

Analytical method for simultaneous detection of retinoic acid and tranexamic acid and its application

[0001] This application claims priority to a prior application, patent application number 2023107952866, filed with the State Intellectual Property Office of China on June 30, 2023, entitled “Analytical Detection Method for Simultaneous Detection of Retinoic Acid and Tranexamic Acid and Its Application.” The entire text of the prior application is incorporated herein by reference. Technical Field

[0002] The present 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

[0003] Tretinoin is a vitamin A derivative, also known as all-trans retinoic acid (RA). It is widely used in dermatology and beauty fields, such as treating acne, anti-aging, and reducing pigmentation. Its main function is to promote the proliferation and regeneration of epidermal cells by regulating gene expression and cell proliferation and differentiation, thereby increasing the rate of cell renewal, thereby improving skin texture and reducing spots and pigmentation.

[0004] Tranexamic acid (TA), also known as tranexamic acid, inhibits fibrinolysis caused by plasmin, thereby exerting a hemostatic effect. Therefore, TA is widely used in cardiovascular surgery, surgical procedures, obstetric surgery, dental surgery, and other fields to alleviate postoperative bleeding. Furthermore, due to its pharmacological effects of inhibiting melanin production, reducing pigmentation, and suppressing inflammatory responses, TA is also widely used in dermatology and cosmetic surgery.

[0005] In short, tranexamic acid reduces melanin formation, while retinoic acid accelerates skin metabolism and promotes melanin excretion. Therefore, combining these two drugs to create a compound preparation can exert a synergistic effect and be used to reduce skin spots and pigmentation, showing promising application prospects in dermatology and cosmetics.

[0006] The IVPT test, or in vitro permeation test, is primarily used to evaluate drug permeability and the permeability of topical skin preparations. This test simulates drug absorption by human skin and is crucial for quality control and efficacy evaluation of topical preparations. The skin acts as a natural barrier, and the permeation rate of topical preparations is often very low. Furthermore, the IVPT test uses skin samples, which have a complex composition and a matrix that may interfere with detection, posing a significant challenge to the sensitivity and accuracy of quantitative analysis methods.

[0007] 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 ion and specific fragment ions of the compound in the spectrum, the compound can be quantified and identified. Therefore, it is very suitable to apply HPLC / UPLC-MS technology to the analysis and detection of IVPT samples.

[0008] However, because tretinoin is a lipid-soluble and highly hydrophobic drug, chromatographic separation typically requires a high proportion of organic solvents for elution. Tranexamic acid, on the other hand, is readily soluble in water and highly polar, requiring the addition of ion-pairing reagents to enhance retention or the use of hydrophilic interaction chromatography for reversed-phase chromatography. Consequently, currently reported detection methods cannot meet the requirements for simultaneous qualitative and quantitative analysis of tretinoin and tranexamic acid.

[0009] Summary of the Invention

[0010] In order to improve the above technical problems, the present invention provides an analytical detection method for simultaneously detecting tretinoin and tranexamic acid, which uses ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to analyze a sample containing tretinoin and tranexamic acid to achieve qualitative and / or quantitative detection of tretinoin and tranexamic acid.

[0011] The method comprises:

[0012] Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is used to analyze the test solution containing retinoic acid and tranexamic acid using formic acid solution or acetic acid solution as the aqueous phase and methanol, methanol solution or acetonitrile as the organic phase, thereby achieving qualitative and quantitative detection of retinoic acid and tranexamic acid.

[0013] 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 0.1-0.5% by volume, i.e., the concentration of formic acid in the formic acid solution is 0.1-0.5% by volume. For example, the concentration of the formic acid solution is 0.1%, 0.2%, 0.3%, 0.4%, or 0.5% by volume.

[0014] Preferably, the acetic acid solution is an aqueous acetic acid solution, and the concentration of the acetic acid solution is 0.1-0.5v%. For example, the concentration of the acetic acid solution is 0.1v%, 0.2v%, 0.3v%, 0.4v% or 0.5v%.

[0015] According to an embodiment of the present invention, the methanol solution is a methanol solution containing formic acid, for example, a methanol solution containing 0.1 v% formic acid.

[0016] According to an embodiment of the present invention, the method further comprises preparing a standard stock solution of tretinoin and a standard stock solution of tranexamic acid;

[0017] The method for preparing the retinoic acid standard stock solution is as follows: mixing retinoic acid with methanol to prepare the retinoic acid standard stock solution;

[0018] The preparation method of the tranexamic acid standard stock solution is: mixing tranexamic acid with water to prepare the tranexamic acid standard stock solution.

[0019] According to an embodiment of the present invention, the concentration of the tretinoin 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, and 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, and 1.5 mg / mL.

[0020] 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.

[0021] Preferably, the retinoic acid internal standard (RAIS) stock solution uses acitretin as the internal standard.

[0022] Preferably, the tranexamic acid internal standard (TAIS) stock solution uses 4-aminocyclohexane-1-carboxylic acid as the internal standard.

[0023] The preparation method of the tretinoin internal standard stock solution is as follows: dissolving acitretin in acetone and then diluting with methanol to prepare the tretinoin internal standard stock solution; preferably, the mass volume ratio of acitretin, acetone and methanol is 1 mg: 1-5 mL: 2-6 mL;

[0024] 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.

[0025] 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 tretinoin internal standard and tranexamic acid internal standard.

[0026] The preparation method of the internal standard working solution is as follows: 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;

[0027] Preferably, the concentration of the acetonitrile aqueous solution is 60-90 v%, exemplarily 60, 70, 80, 90 v%;

[0028] Preferably, the volume ratio of the tretinoin internal standard stock solution to the acetonitrile aqueous solution is 1:2-6, exemplarily 1:2, 1:3, 1:4, 1:5, or 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, or 1:6.

[0029] According to an embodiment of the present invention, the method further comprises preparing a standard curve, and the preparation of the standard curve can be performed according to conventional methods of those skilled in the art.

[0030] In some embodiments, preparing a standard curve comprises the following steps:

[0031] Prepare blank matrix solution, retinoic acid standard curve working solution, tranexamic acid standard curve working solution, and internal standard working solution (or diluent containing internal standard) to obtain a standard curve solution containing retinoic acid and tranexamic acid at 5 to 7 concentration gradient points; analyte ) is the horizontal axis, and the ratio of the analyte to the internal standard peak area (A analyte / A IS ) is the vertical axis, and the standard curve is fitted.

[0032] 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. The blank matrix liquid is obtained by subjecting a blank formulation (i.e., a formulation using the same excipients and preparation process but without an active ingredient) to IVPT under the same conditions to obtain a permeate blank matrix liquid or a skin extract blank matrix liquid.

[0033] According to an embodiment of the present invention, the internal standard method is used to detect and calculate the contents of tretinoin and tranexamic acid in the sample to be tested.

[0034] 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 used. analyte / A IS), and 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.

[0035] According to an embodiment of the present invention, the standard curve working solution includes a tretinoin standard curve working solution and a tranexamic acid standard curve working solution;

[0036] The method for preparing the retinoic acid standard curve working solution is as follows: mixing the retinoic acid standard stock solution with an acetonitrile aqueous solution to obtain the retinoic acid standard curve working solution;

[0037] Preferably, the concentration of the retinoic acid standard curve working solution is 200-10000 ng / mL, illustratively 200 ng / mL, 500 ng / mL, 1000 ng / mL, 2000 ng / mL, 5000 ng / mL, and 10000 ng / mL;

[0038] The preparation method of the tranexamic acid standard curve working solution is as follows: mixing the tranexamic acid standard stock solution with an acetonitrile aqueous solution to obtain the tranexamic acid standard curve working solution;

[0039] Preferably, the concentration of the tranexamic acid standard curve working solution is 10,000-750,000 ng / mL, exemplarily 10,000 ng / mL, 30,000 ng / mL, 75,000 ng / mL, 150,000 ng / mL, 300,000 ng / mL, and 750,000 ng / mL.

[0040] Preferably, the concentration of the acetonitrile aqueous solution is 60-90 v%, illustratively 60, 70, 80, 90 v%.

[0041] According to an embodiment of the present invention, the method further comprises preparing a receiver fluid standard curve solution and a skin extract standard curve solution;

[0042] The preparation method of the receiving solution standard curve solution is as follows: 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 the supernatant and diluting to obtain the receiving solution standard curve solution;

[0043] The preparation method of the skin extract standard curve solution is as follows: 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;

[0044] 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;

[0045] 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, or 750-1500 ng / mL.

[0046] 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;

[0047] Preferably, in the method for preparing the 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.

[0048] Preferably, the concentration of the internal standard substances (TAIS and RAIS) in the receiver fluid standard curve solution or the skin extract standard curve solution is 10-200 ng / mL; preferably, the concentration of the internal standard substances is 50-100 ng / mL.

[0049] According to an embodiment of the present invention, the protein precipitant is acetonitrile, methanol, trichloroacetic acid solution, etc.; preferably, the protein precipitant is acetonitrile.

[0050] According to an embodiment of the present invention, the volume ratio of the tretinoin working solution or the tranexamic acid working solution to the protein precipitant is 1:2-10, preferably 1:3-6.

[0051] According to an embodiment of the present invention, the internal standard-containing extract diluent comprises tretinoin internal standard and tranexamic acid internal standard.

[0052] 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.

[0053] Preferably, the concentration of the acetonitrile aqueous solution mixed with the tretinoin internal standard stock solution or the tranexamic acid internal standard stock solution is 60-90v%, exemplarily 60, 70, 80, 90v%.

[0054] According to an embodiment of the present invention, the method further includes processing 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 fluid produced by an IVPT test; the skin extract or the receiving fluid at least includes tretinoin and tranexamic acid. The receiving fluid 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 (such as at least one of acitretin (RAIS) and 4-aminocyclohexane-1-carboxylic acid (TAIS)) to eliminate errors.

[0055] 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 the supernatant to obtain a test sample containing tretinoin and tranexamic acid, i.e., an intradermal retention sample solution.

[0056] According to an embodiment of the present invention, the method for processing the receiving liquid is further specifically: mixing the receiving liquid sample with the internal standard working solution, then adding a protein precipitant, centrifuging to obtain the supernatant, adding an acetonitrile aqueous solution to dilute it, and obtaining a sample to be tested containing tretinoin and tranexamic acid, that is, a skin permeation sample solution.

[0057] Preferably, the concentration of the acetonitrile aqueous solution is 60-90 v%, illustratively 60, 70, 80, 90 v%.

[0058] Preferably, the concentration of the internal standard substances (TAIS and RAIS) in the intradermal retention sample solution or the skin permeation sample solution is 10-200 ng / mL; preferably, the concentration of the internal standard substances is 50-100 ng / mL.

[0059] According to an embodiment of the present invention, the test conditions of the ultra-high performance liquid chromatography include:

[0060] Chromatographic column: Waters ACQUITY UPLC HSS T3 2.1*100mm, particle size 1.8μm;

[0061] or ThermoFisher ACCUCORE UREA-HILIC 2.1*100mm, with a filler particle size of 2.6μm; or Welch Ultimate XS-C18 2.1×100mm, with a filler particle size of 3.0μm; preferably Welch Ultimate XS-C18 2.1×100mm, with a filler particle size of 3.0μm;

[0062] Guard column: Welch Ultimate XS-C18 2.1 × 10 mm, particle size 3.0 μm;

[0063] 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;

[0064] Organic phase B - methanol, acetonitrile, methanol solution containing 0.1v% formic acid; gradient elution;

[0065] Preferably, the gradient elution program is as follows: 0-0.8 min, A is maintained at 98v%, and B is maintained at 2v%; 0.8-4 min, A decreases from 98v% to 5v%, and B increases from 2v% to 95v%; 4-6.5 min, A is maintained at 5v%, and B is maintained at 95v%; 6.5-7 min, A increases from 5v% to 98v%, and B decreases from 95v% to 2v%; 7-8.5 min, A is maintained at 98v%, and B is maintained at 2v%;

[0066] Flow rate: 0.3-0.5 mL / min, preferably 0.4 mL / min;

[0067] Injection volume: 2-20 μL, preferably 5 μL;

[0068] Column temperature: 30-60°C, preferably 40°C.

[0069] According to an embodiment of the present invention, the test conditions of the triple quadrupole mass spectrometer are as follows:

[0070] Ion source: electrospray ionization (ESI); Scan type: positive ion mode;

[0071] Spray voltage (IS): 5500V; temperature: 200-400°C, preferably 300°C;

[0072] Declustering voltage (DP): 20-50 V, preferably 20-40 V;

[0073] Impact voltage (CE): 12-40V, preferably 14-30V.

[0074] The present invention also provides application of the above method in biological samples such as plasma and tissue homogenate.

[0075] Beneficial effects of the present invention:

[0076] The analytical detection method provided by the present invention has the characteristics of high sensitivity and precision, good repeatability, high accuracy, and strong stability.

[0077] 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

[0078] Fig. 1 is tranexamic acid (TA) and its internal standard (TAIS) chromatogram;

[0079] FIG2 is a chromatogram of retinoic acid (RA) and its internal standard (RAIS);

[0080] Figure 3 is a chromatogram of the blank solution. DETAILED DESCRIPTION

[0081] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative 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 encompassed within the scope of protection that the present invention is intended to protect.

[0082] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0083] Example 1

[0084] Liquid phase method

[0085] Table 1 Liquid phase parameters

[0086] Table 2 Gradient elution table

[0087] Mass spectrometry

[0088] Scan type: Multiple reaction ion monitoring, positive ion mode (MRM positive)

[0089] Ion source: electrospray ionization (ESI)

[0090] Temperature parameter: 300℃

[0091] Spray voltage (IS): 5500V

[0092] Table 1 Analytes and IS mass spectrometry parameter settings

[0093] In Table 1, 2 and 3, or 4 and 5, or 6 and 7 are detected using different fragment ions.

[0094] 3. Specific steps for IVPT sample testing

[0095] 3.1. Preparation of Standard and Internal Standard Stock Solutions

[0096] 3.1.1. Retinoic acid standard stock solution

[0097] Accurately weigh 20 mg of retinoic acid standard into a 100 mL brown volumetric flask, dissolve it in methanol, and dilute to the mark.

[0098] 3.1.2. Tranexamic acid standard stock solution

[0099] Accurately weigh 15 mg of tranexamic acid standard into a 20 mL volumetric flask, dissolve in water, and dilute to the mark.

[0100] 3.1.3. Retinoic acid internal standard (RAIS) stock solution

[0101] Accurately weigh 10 mg of acitretin into a 50 mL brown volumetric flask, add approximately 10 mL of acetone to dissolve it, and then dilute to the mark with methanol.

[0102] 3.1.4. Tranexamic acid internal standard (TAIS) stock solution

[0103] Accurately weigh 10 mg of 4-aminocyclohexane-1-carboxylic acid into a 50 mL volumetric flask, dissolve it in water, and dilute to the mark.

[0104] All the above solutions were refrigerated at 2-8°C and returned to room temperature before use.

[0105] 3.2. Preparation of standard curve and internal standard working solution

[0106] 3.2.1. Internal standard working solution

[0107] Pipette 4 mL of each of two internal standard stock solutions (4 mL of retinoic acid internal standard stock solution and 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.

[0108] 3.2.2. Dilution of extract containing internal standard

[0109] 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 the extract dilution containing the internal standard.

[0110] 3.2.3. Standard curve working solution

[0111] 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.

[0112] The concentration series of the obtained TA standard curve working solution are: 10000, 30000, 75000, 150000, 300000, 750000 ng / ml.

[0113] The concentration series of the obtained RA standard curve working solutions were: 200, 500, 1000, 2000, 5000, and 10000 ng / ml.

[0114] 3.3. Preparation of standard curve solution

[0115] 3.3.1. Receiver (permeate) standard curve solution

[0116] Accurately pipette 160 μL of the permeate blank matrix, add 10 μL each of the RA standard curve working solution and the TA standard curve working solution of a series of concentrations, and 20 μL of the 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.

[0117] 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.

[0118] 3.3.2. Skin extract (intradermal retention) standard curve solution

[0119] Accurately pipette 160 μL of skin extract blank matrix, add 10 μL each of RA standard curve working solution and TA standard curve working solution of different concentrations, and 400 μL of the 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 this solution and dilute it with 900 μL of 80% acetonitrile to obtain the skin extract standard curve solution for analysis and detection.

[0120] In the obtained skin extract standard curve solutions (respectively designated 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.

[0121] 3.4. IVPT test sample processing

[0122] 3.4.1. The IVPT test samples used in the present invention are: a preparation containing a compound of two active ingredients, tretinoin and tranexamic acid, and samples produced by IVPT testing (including the receiving solution and skin extract obtained by the following treatment).

[0123] 3.4.2. Receiving fluid sample processing

[0124] 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.

[0125] 3.4.3. Skin extract (intradermal retention) sample processing

[0126] Skin samples were ground and extracted using a cryo-grinder. 100 μL of the skin extract was added to 400 μL of the extract diluent containing the internal standard and vortexed for 30 seconds to mix thoroughly. 100 μL of this solution was diluted with 900 μL of 80% acetonitrile and centrifuged. 5 μL of the supernatant was analyzed and the chromatogram recorded.

[0127] 3.5 IVPT test sample test results

[0128] 3.5.1 Compound Preparation 1:

[0129] Formula of compound preparation 1

[0130] Process: Weigh cetyl alcohol and medium-chain triglycerides into a beaker, heat in a water bath to 60°C and stir to melt the cetyl alcohol; 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 and maintain stirring in a water bath at 60°C; test the pH value and adjust the pH to 5.0-5.5 with an appropriate amount of sodium hydroxide aqueous solution; add water to the full amount; homogenize using a high shear homogenizer; naturally cool to obtain a light yellow ointment; fill into an aluminum tube and seal the end.

[0131] 3.5.2 Compound preparation 2: Batch number IDT-2212151

[0132] Formula of compound preparation 2

[0133] 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 isopropyl alcohol, 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; test the pH value and adjust the pH to 5.0-5.5 with an appropriate amount of triethanolamine; add water to the full amount; homogenize using a high shear homogenizer; naturally cool to obtain a light yellow ointment; fill into an aluminum tube and seal the end.

[0134] 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 fluid 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:

[0135] Table A TA and RA concentration test results in IVPT test samples of compound preparation 1 and compound preparation 2

[0136] 4. Preliminary Methodological Validation

[0137] System suitability

[0138] System suitability testing was performed to determine the RSD% of tretinoin and tranexamic acid standard preparations injected in duplicate for six times.

[0139] Table 8 System suitability results

[0140] in conclusion:

[0141] The RSD% of repeated injections of the standard preparation solution was greater than 2.0%, which met the requirements of the system suitability and system precision studies.

[0142] 4.2. Specificity

[0143] 4.2.1. Blank Interference

[0144] Specificity testing was performed to monitor the blank solution for interference with tretinoin and tranexamic acid. The blank sample chromatogram is shown in Figure 3.

[0145] in conclusion:

[0146] 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 times of tranexamic acid, tretinoin and their respective internal standards.

[0147] 4.3 Lower limit of quantification (LLOQ)

[0148] 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 10,000 ng / mL was further diluted to a concentration of 500 ng / mL using 80% acetonitrile to serve as the TA-LLOQ working solution.

[0149] Take the ex vivo pig skin and administer the blank sample percutaneously for 24 hours. Collect all the permeated liquid as the receiving liquid blank matrix solution. The skin after the test is processed according to 3.4.2 Skin Extract (Intradermal Retention) Sample to obtain the skin extract blank matrix.

[0150] 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 and add 900 μL of 80% acetonitrile to dilute to obtain the receiving liquid LLOQ solution. Repeat the injection 6 times and record the peak area of ​​the analyte and internal standard.

[0151] 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 this solution and dilute it with 900 μL of 80% acetonitrile to obtain the skin extract LLOQ solution. Repeat the injection 6 times and record the peak area of ​​the analyte and internal standard.

[0152] 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.

[0153] LLOQ results:

[0154] Table 9 LLOQ results

[0155] in conclusion:

[0156] 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, indicating that the analytical method has high sensitivity and can meet the quantitative requirements of IVPT analysis and detection.

[0157] 4.4. Accuracy and precision

[0158] Repeatability

[0159] Prepare six samples using the same preparation methods as for the receiving solution, standard curve sample solution No. 3 (JSBQ-3), and the skin extract standard curve sample solution No. 3 (TQBQ-3). Calculate the concentrations of tranexamic acid and tretinoin, compare them with the prepared concentrations, and calculate the recovery. The RSD% of the six injections should be between 90 and 110, and the RSD% of the six injections should be ≤ 5.0.

[0160] 4.4.2. Inter-batch precision

[0161] Reanalyze the JSBQ-3 and TQBQ-3 six times on different dates. Calculate the recovery and RSD%, and calculate the variability (CV%) of the results between dates. CV% should be ≤ 10.0.

[0162] Table 13.1 Repeatability and inter-assay precision results of JSBQ-3

[0163] Table 13.2 Repeatability and inter-batch precision results of TQBQ-3

[0164] in conclusion:

[0165] The accuracy and precision (repeatability and inter-batch precision) of this method meet the requirements of IVPT testing.

[0166] 4.5. Linearity

[0167] The determination 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); 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:

[0168] Table 14 Standard curve summary

[0169] in conclusion:

[0170] The analytes, tranexamic acid and tretinoin, showed good linearity in the receiving solution and skin extract, meeting the test requirements.

[0171] 5.1. Investigation of matrix effects

[0172] Matrix blank matrix (530 μL) was precisely aspirated into the permeate. 10 μL of low-concentration (200 ng / ml) or high-concentration (40,000 ng / ml) standard solutions (including low-concentration TA, high-concentration TA, low-concentration RA, and high-concentration RA) and 60 μL of internal standard working solution were added and vortex-mixed for 1 min to prepare matrix-test samples (n=6). 200 μL of matrix-test samples was added to 800 μL of acetonitrile, vortex-mixed for 3 min, and centrifuged at 12,000 rpm for 5 min. The supernatant was analyzed and the peak area was recorded as A. Standard solutions containing no matrix at the corresponding concentrations (low and high) were prepared using 80% acetonitrile as a diluent and injected (n=6). The peak area was recorded as B, and the B values ​​were averaged. The matrix factor (matrix factor = A / B) was calculated for each concentration of the analyte and internal standard. The internal standard-normalized matrix factor was calculated by dividing the matrix factor of the analyte by the matrix factor of the internal standard. The RSD was required to be ≤15%.

[0173] The skin extract blank matrix was used instead and the matrix effect of the skin extract was evaluated in the same manner.

[0174] The matrix effect results for two analytes (tranexamic acid and tretinoin) in two matrices (receiver fluid and skin extract) are as follows:

[0175] Table 2 Matrix effect determination results

[0176] in conclusion:

[0177] The normalized matrix factors of the two analytes (tranexamic acid and tretinoin) in both matrices (receiver fluid and skin extract) were approximately 100% (80%-120%), with RSDs < 15%; therefore, the matrix effect of this method meets the requirements of analytical detection.

[0178] 5.2. Screening of different chromatographic columns

[0179] Take 20 μL of each of 10 IVPT test samples (according to 3.4) and each of the receiver fluid and skin extract standard solution samples, vortex mix, and use them as QC samples. Analyze and test the QC samples using different chromatographic columns, and compare the retention time and peak shape of the analytes using different chromatographic columns.

[0180] Column A: Waters ACQUITY UPLC HSS T3 (2.1*100mm, 1.8um)

[0181] Column B: ThermoFisher ACCUCORE UREA-HILIC (2.1*100mm, 2.6μm)

[0182] Column C: Welch Ultimate XS-C18 (2.1 × 100 mm, 3.0 μm)

[0183] Table 19 Screening results of different chromatographic columns

[0184] in conclusion:

[0185] Taking into account the peak shape and the availability of the chromatographic column, the preferred column is Welch Ultimate XS-C18 (2.1×100 mm, 3.0 μm).

[0186] 5.3. Investigation of flow rate

[0187] Different flow rates (0.3 mL / min, 0.4 mL / min) were set to analyze and detect the QC samples. The retention times of tranexamic acid and tretinoin are as follows:

[0188] Table 20 Screening results at different flow rates

[0189] in conclusion:

[0190] IVPT samples are relatively large, so a flow rate of 0.4 mL / min is preferred to shorten analysis and testing time. Furthermore, setting the flow rate too high (greater than 0.5 mL / min) increases the amount of liquid entering the MS system, leading to incomplete sample atomization, affecting the ionization of the analyte, and causing quantitative deviations. Therefore, experiments with higher flow rates were not conducted.

[0191] The above is an exemplary description of the embodiments of the present invention. However, the scope of protection of the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present invention shall be included in the scope of protection 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 is used to detect and 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 is 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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