Method and system for detecting content of main drug and excipient in isotretinoin gel

Abstract

The application provides a content detection method and system of a main drug and excipients in isotretinoin gel, uses 0.1% formic acid aqueous solution as a mobile phase A and 0.1% formic acid-acetonitrile mixed solution as a mobile phase B, optimizes a gradient elution program for different components in the isotretinoin gel, so that the synchronous and accurate separation of multiple components in the isotretinoin gel is realized, and the content of each component is accurately determined through a chromatogram generated by a detector. The method is fast and efficient, and has wide applicability and high reliability. In addition, the application also provides a matched content detection system, which has the functions of synchronous separation and detection of the contents of four key components in the isotretinoin gel, and can flexibly cope with the detection requirements of various types and specifications of isotretinoin gel, and provides effective technical support for the quality inspection of isotretinoin gel.

Description

Technical Field

[0001] This patent application relates to the field of drug analysis technology, and further to a method and system for detecting the content of active pharmaceutical ingredient and excipients in isotretinoin gel. Background Technology

[0002] Isotretinoin gel is a topical medication used to treat skin conditions such as acne. Its preparation requires the addition of antibacterial agents such as benzyl alcohol, and antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) to maintain drug quality and stability. However, current methods for detecting the content of these components have limitations. Traditional detection methods typically require separate analysis of antibacterial agents, antioxidants, and the active pharmaceutical ingredient, which is not only time-consuming and labor-intensive but also prone to introducing errors. For example, while UV-Vis spectrophotometry and thin-layer chromatography can meet the quantitative analysis needs of single components to some extent, they often face problems such as insufficient resolution, low sensitivity, and cumbersome operation when simultaneously measuring multiple components.

[0003] High-performance liquid chromatography (HPLC), as a highly efficient, sensitive, and specific analytical technique, is now widely used in the field of pharmaceutical analysis. It separates and quantifies components in a mixture based on the different affinities of the sample mixture to the stationary and mobile phases, according to chromatographic principles.

[0004] Although HPLC technology has been proven effective, existing methods for the simultaneous detection of benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, and isotretinoin in isotretinoin gels have certain limitations, such as poor separation due to improper selection of mobile phase and insufficient sensitivity due to unreasonable selection of detection wavelength.

[0005] Therefore, there is an urgent need to develop a high-performance liquid chromatography (HPLC) method that can simultaneously detect the content of antibacterial agents, antioxidants, and active pharmaceutical ingredients in isotretinoin gels. Summary of the Invention

[0006] Current detection methods have limitations in simultaneously separating and detecting multiple components in isotretinoin gels, exhibiting low accuracy and efficiency, particularly in the separation and detection of benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, and isotretinoin. This application aims to provide a method and system for detecting the content of active pharmaceutical ingredient (API) and excipients in isotretinoin gels. The method employs high-performance liquid chromatography (HPLC) with a 0.1% formic acid aqueous solution as mobile phase A and a 0.1% formic acid-acetonitrile mixed solution as mobile phase B. The gradient elution program is optimized for different components in the isotretinoin gel to achieve precise simultaneous separation. Subsequently, a detector captures the signals as each component passes through, and the generated chromatogram is used to determine the content of each component. The content detection method provided in this application not only demonstrates rapid and efficient separation capabilities but also exhibits high accuracy and reliability. This application also provides a content detection system that matches the content detection method, which plays a crucial role in ensuring the quality control of isotretinoin gel.

[0007] To achieve the above objectives, this application adopts the following technical solution:

[0008] A method for detecting the content of active pharmaceutical ingredient (API) and excipients in isotretinoin gel, wherein the API is isotretinoin, and the excipients are an antibacterial agent and an antioxidant, wherein the antibacterial agent is benzyl alcohol, and the antioxidants are butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT); characterized in that the content detection method includes a separation step and a detection step: the separation step employs high performance liquid chromatography (HPLC), using a 0.1% (v / v) acid-water solution as mobile phase A and a 0.1% (v / v) acid-acetonitrile solution as mobile phase B, to perform gradient elution on the isotretinoin gel, thereby separating the antibacterial agent, the antioxidant, and the isotretinoin;

[0009] The detection steps include: the separated benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene and isotretinoin are respectively introduced into a detector, and the content of each component is determined according to the chromatogram output by the detector.

[0010] In some technical solutions, the initial volume ratio of mobile phase A to mobile phase B is (68-72):(32-28); the gradient elution process is as follows: within 0-8 minutes, the volume ratio of mobile phase A to mobile phase B is adjusted from the initial volume ratio to less than 70:30 and greater than or equal to 40:60; within 8-15 minutes, the volume ratio of mobile phase A to mobile phase B is adjusted to less than 40:60 and greater than or equal to 10:90; within 15-20 minutes, the volume ratio of mobile phase A to mobile phase B is maintained at less than 40:60 and greater than or equal to 10:90; within 20-20.1 minutes, the volume ratio of mobile phase A to mobile phase B is adjusted to the initial volume ratio; within 20.1-25 minutes, the ratio of mobile phase A to mobile phase B is maintained at the initial volume ratio.

[0011] In some technical solutions, the initial volume ratio is 70:30; the gradient elution process is as follows: within 0 to 8 minutes, the ratio of mobile phase A to mobile phase B is adjusted from 70:30 to 40:60; within 8 to 15 minutes, the volume ratio of mobile phase A to mobile phase B is adjusted from 40:60 to 10:90; within 15 to 20 minutes, the volume ratio of mobile phase A to mobile phase B is maintained at 10:90; within 20 to 20.1 minutes, the volume ratio of mobile phase A to mobile phase B is adjusted from 10:90 to 70:30; within 20.1 to 25 minutes, the ratio of mobile phase A to mobile phase B is maintained at 70:30.

[0012] In some technical solutions, the chromatographic column in the high-performance liquid chromatography method is a Shimadzu ShimNex HEC18-AQ column; the column length is 150 mm, the inner diameter is 4.6 mm, the packing particle size is 5 μm; the column temperature is 25~35℃; the injection flow rate is 0.9~1.1 mL / min; the injection volume is 5~15 μL; and the detection wavelength is 190~400 nm.

[0013] In some technical solutions, the acid is any one or a combination of formic acid, glacial acetic acid, trifluoroacetic acid, and phosphoric acid; and / or, the mobile phase A is prepared by transferring 1 mL of the acid to 1000 mL of water, shaking well, and ultrasonically degassing; and / or, the mobile phase B is prepared by transferring 1 mL of the acid to 1000 mL of acetonitrile, shaking well, and ultrasonically degassing.

[0014] In some technical solutions, the detection steps include: the detection wavelength of benzyl alcohol is 258±2nm; the detection wavelengths of butylated hydroxyanisole and butylated hydroxytoluene are 280±2nm; and the detection wavelength of isotretinoin is 358±2nm.

[0015] In some technical solutions, in the chromatogram obtained by testing the isotretinoin gel, the retention time is 3.421±0.5 min for benzyl alcohol, 10.069±0.5 min for butylated hydroxyanisole, 16.551±0.5 min for butylated hydroxytoluene, and 17.475±0.5 min for isotretinoin.

[0016] In some technical solutions, the determination of the content of each component based on the chromatogram output by the detector includes the following steps: preparing a standard to obtain a standard sample chromatogram; testing the chromatogram obtained from the isotretinoin gel, and calculating the content of benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, and isotretinoin by peak area using the external standard method.

[0017] In some technical solutions, the benzyl alcohol is in the range of 0.9926–198.5207 µg / mL, with y = 0.9276x - 1.684; and / or, the butylated hydroxyanisole is in the range of 0.2552–10.1667 µg / mL, with y = 7.0613x - 0.8458; and / or, the butylated hydroxytoluene is in the range of 0.2522–9.8640 µg / mL, with y = 4.8328x - 0.6542; and / or, the isotretinoin is in the range of 0.0486–9.7228 µg / mL, with y = 66.879x - 7.9836; where y is the y-axis, representing the peak area, and x is the x-axis, representing the concentration.

[0018] This application also discloses a content detection system for implementing the above-described content detection method, comprising: an injection system, a chromatographic column, and a detector; the injection system is used to provide the sample to be tested and the mobile phase to the chromatographic column; the detector is used to detect the components of the sample to be tested after separation by the chromatographic column, convert them into quantified signals, and record and analyze the signals to determine the concentration and content of each component.

[0019] Compared with the prior art, this application can bring the following beneficial effects:

[0020] 1. The content detection method provided in this application utilizes high-performance liquid chromatography (HPLC) technology and carefully optimizes the gradient elution program to achieve simultaneous and accurate separation of multiple components in isotretinoin gel. The detector captures the signals generated by each component during the separation process, thereby forming corresponding chromatograms. Based on this, the content of each component can be accurately quantified. This technology is not only convenient and efficient, completing the content determination in just 30 minutes, but also boasts high accuracy and reliability, ensuring the stability of the detection process and the credibility of the data. Furthermore, it has extremely high applicability, capable of meeting the detection needs of different types and specifications of isotretinoin gels.

[0021] 2. This application not only proposes a content detection method but also provides a corresponding content detection system. This system can simultaneously determine the content of four key components in isotretinoin gel. The operation is simple and rapid, significantly shortening the detection time while ensuring the accuracy of the results. It provides a powerful tool for accurately verifying the quality of isotretinoin gel and is of great significance for improving the overall quality control level of the gel. Attached Figure Description

[0022] The present application will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0023] Figure 1 The chromatogram of the blank matrix solution in Example 2 at a wavelength of 258 nm is shown.

[0024] Figure 2 The chromatogram of the blank matrix solution in Example 2 at a wavelength of 280 nm is shown.

[0025] Figure 3 The chromatogram of the blank matrix solution in Example 2 at a wavelength of 358 nm is shown.

[0026] Figure 4 The chromatogram of the benzyl alcohol positioning solution in Example 2;

[0027] Figure 5 The chromatogram of the butylated hydroxyanisole positioning solution in Example 2;

[0028] Figure 6 The chromatogram of the butylated hydroxytoluene positioning solution in Example 2 is shown below.

[0029] Figure 7 This is the chromatogram of the isotretinoin localization solution in Example 2;

[0030] Figure 8 The chromatogram of the reference solution in Example 2 at a wavelength of 258 nm is shown.

[0031] Figure 9The chromatogram of the reference solution in Example 2 at a wavelength of 280 nm is shown.

[0032] Figure 10 The chromatogram of the reference solution in Example 2 at a wavelength of 358 nm is shown.

[0033] Figure 11 The chromatogram of the sample solution in Example 2 at a wavelength of 258 nm;

[0034] Figure 12 The chromatogram of the sample solution in Example 2 at a wavelength of 280 nm;

[0035] Figure 13 The chromatogram of the sample solution in Example 2 at a wavelength of 358 nm;

[0036] Figure 14 The chromatogram of the detection limit solution in Example 4 at a wavelength of 258 nm is shown.

[0037] Figure 15 The chromatogram of the detection limit solution in Example 4 at a wavelength of 280 nm is shown.

[0038] Figure 16 The chromatogram of the detection limit solution in Example 4 at a wavelength of 358 nm is shown.

[0039] Figure 17 The chromatogram of the limit-of-quantity solution in Example 4 at a wavelength of 258 nm is shown.

[0040] Figure 18 The chromatogram of the limit-of-quantity solution in Example 4 at a wavelength of 280 nm is shown.

[0041] Figure 19 The chromatogram of the limit-of-quantity solution in Example 4 at a wavelength of 358 nm is shown. Detailed Implementation

[0042] The present application will be further described below with reference to specific embodiments, but these embodiments are merely exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that various improvements and modifications can be made without departing from the principles of the present application, and these improvements and modifications should also be considered within the scope of protection of the present application.

[0043] Example 1

[0044] This application proposes a method for detecting the content of active pharmaceutical ingredient (API) and excipients in isotretinoin gel. In addition to the API, isotretinoin gel typically contains various functional excipients, including but not limited to antibacterial agents and antioxidants. The specific formulation may vary depending on the brand and process. Some products may also contain other excipients such as humectants and thickeners. The content detection method provided in this application mainly targets the API (isotretinoin) and the content of antibacterial agents and antioxidants as key excipients. Specifically, antibacterial agents in retinoic acid gel include benzyl alcohol, and antioxidants include butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).

[0045] The content detection method includes a separation step and a detection step. The separation step uses high performance liquid chromatography (HPLC). A mobile phase A is a 0.1% (v / v) formic acid aqueous solution, and a mobile phase B is a 0.1% (v / v) formic acid-acetonitrile solution. The isotretinoin gel is subjected to gradient elution to separate the antibacterial agent, antioxidant, and isotretinoin. The antibacterial agent is benzyl alcohol, and the antioxidants are butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).

[0046] The detection process involves introducing the separated benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, and isotretinoin into the detector one by one, and accurately determining the content of each component by analyzing the chromatogram generated by the detector.

[0047] In some embodiments, the initial volume ratio of mobile phase A to mobile phase B is (68-72):(32-28), and the optional ratios include, but are not limited to, 68:32, 69:31, 70:30, 71:29 or 72:28.

[0048] In the separation step, the gradient elution process is as follows:

[0049] Within 0–8 minutes, the volume ratio of mobile phase A to mobile phase B is adjusted from the initial volume ratio to less than 70:30 and greater than or equal to 40:60. During this process, benzyl alcohol, the antibacterial agent, is the first component to be eluted due to its relatively high polarity. As the proportion of mobile phase B increases, other components with lower polarity will be eluted sequentially.

[0050] Within 8–15 minutes, the volume ratio of mobile phase A to mobile phase B is adjusted to less than 40:60 and greater than or equal to 10:90. This process further enhances the elution intensity, which helps to separate moderately polar components (antioxidant butylated hydroxyanisole and butylated hydroxytoluene). The increased proportion of mobile phase B accelerates the elution rate of these components, thereby achieving better separation results.

[0051] Within 15–20 minutes, the volume ratio of mobile phase A to mobile phase B is maintained at less than 40:60 and greater than or equal to 10:90. Maintaining this mobile phase ratio during this stage helps to achieve complete separation of the less polar component (isotretinoin). Simultaneously, this ensures that previously eluted components have sufficient time to pass through the detector for quantitative analysis.

[0052] Within 20–20.1 minutes, the volume ratio of mobile phase A to mobile phase B is adjusted to the initial volume ratio. This process rapidly adjusts the mobile phase ratio to restore the initial conditions of the column, preparing it for the next injection. Simultaneously, this step also helps clean the column, removing residual weakly polar components and reducing column contamination and memory effects.

[0053] Within 20.1–25 minutes, the ratio of mobile phase A to mobile phase B remains at its initial volume ratio. Maintaining a stable mobile phase ratio in the final stage helps the column equilibrate and regenerate, preparing it for the next separation process and improving the repeatability and accuracy of the analysis.

[0054] Preferably, the initial volume ratio of mobile phase A to mobile phase B is 70:30. Under this volume ratio, and referring to Table 1, the gradient elution process is as follows:

[0055] Within 0 to 8 minutes, the ratio of mobile phase A to mobile phase B is adjusted from 70:30 to 40:60.

[0056] Within 8 to 15 minutes, the volume ratio of mobile phase A to mobile phase B is adjusted from 40:60 to 10:90.

[0057] The volume ratio of mobile phase A to mobile phase B is maintained at 10:90 for 15 to 20 minutes.

[0058] Within 20 to 20.1 minutes, the volume ratio of mobile phase A to mobile phase B is adjusted from 10:90 to 70:30.

[0059] The ratio of mobile phase A to mobile phase B was maintained at 70:30 for 20.1 to 25 minutes.

[0060] Table 1 Gradient elution process

[0061]

[0062] Furthermore, the initial volume ratio of mobile phase A to mobile phase B can also be 80:20. Under this volume ratio setting, and referring to Table 2, the gradient elution process is as follows:

[0063] The gradient elution process is as follows:

[0064] Within 0 to 8 minutes, the ratio of mobile phase A to mobile phase B is adjusted from 80:20 to 50:50.

[0065] Within 8 to 15 minutes, the volume ratio of mobile phase A to mobile phase B is adjusted from 50:50 to 20:80.

[0066] The volume ratio of mobile phase A to mobile phase B is maintained at 20:80 for 15 to 20 minutes.

[0067] Within 20 to 20.1 minutes, the volume ratio of mobile phase A to mobile phase B is adjusted from 20:80 to 80:20.

[0068] The ratio of mobile phase A to mobile phase B was maintained at 80:20 for 20.1 to 25 minutes.

[0069] Table 2 Gradient elution process

[0070]

[0071] Furthermore, the initial volume ratio of mobile phase A to mobile phase B can also be 90:10. Under this volume ratio setting, and referring to Table 3, the gradient elution process is as follows:

[0072] Within 0 to 8 minutes, the ratio of mobile phase A to mobile phase B is adjusted from 90:10 to 60:40.

[0073] Within 8 to 15 minutes, the volume ratio of mobile phase A to mobile phase B is adjusted from 60:40 to 30:70.

[0074] The volume ratio of mobile phase A to mobile phase B is maintained at 30:70 for 15 to 20 minutes.

[0075] Within 20 to 20.1 minutes, the volume ratio of mobile phase A to mobile phase B was adjusted from 30:70 to 90:10.

[0076] The ratio of mobile phase A to mobile phase B was maintained at 90:10 for 20.1 to 25 minutes.

[0077] Table 3 Gradient elution process

[0078]

[0079] In this embodiment, the chromatographic column used in the above-mentioned high performance liquid chromatography method is a Shimadzu ShimNex HE C18-AQ column with a length of 150 mm, an inner diameter of 4.6 mm, a packing particle size of 5 μm, and a column temperature of 25~35℃, preferably 30℃.

[0080] The injection flow rate is 0.9–1.1 mL / min, preferably 1.0 mL / min; the injection volume is 5–15 μL, preferably 10 μL.

[0081] Furthermore, needle washing is required before and after each injection to ensure that the analytical process meets the prescribed quality control standards and to guarantee the accuracy and reliability of the analytical results. The needle washing condition uses a mixed solution of acetonitrile and water, preferably with a volume ratio of 50:50 (1:1), which effectively cleans the chromatographic needle.

[0082] In some embodiments, the acid in mobile phase A and mobile phase B is any one or a combination of formic acid, glacial acetic acid, trifluoroacetic acid, and phosphoric acid, preferably formic acid. Mobile phase A is prepared by transferring 1 mL of formic acid to 1000 mL of water, shaking well, and then degassing by sonication. Mobile phase B is prepared by transferring 1 mL of formic acid to 1000 mL of acetonitrile, shaking well, and then degassing by sonication.

[0083] In some embodiments, based on the separation steps described above, the detection steps provided in this application use a detection wavelength of 190~400nm for each component. Specifically: the detection wavelength for benzyl alcohol is 258±2nm (i.e., 256-260nm), the detection wavelengths for butylated hydroxyanisole and butylated hydroxytoluene are 280±2nm (i.e., 278-282nm), and the detection wavelength for isotretinoin is 358±2nm (i.e., 356-360nm).

[0084] Preferably, the detector is a diode array detector.

[0085] Furthermore, in the chromatograms obtained from testing isotretinoin gel, benzyl alcohol was retained at 3.421±0.5 min, butylated hydroxyanisole was retained at 10.069±0.5 min, dibutylated hydroxytoluene was retained at 16.551±0.5 min, and isotretinoin was retained at 17.475±0.5 min.

[0086] In some embodiments, the step of determining the content of each component based on the chromatogram output by the detector includes:

[0087] Prepare standard samples and obtain standard sample chromatograms;

[0088] Based on the obtained chromatogram, the contents of p-benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, and isotretinoin were calculated using the external standard method based on peak area.

[0089] Furthermore, for benzyl alcohol in the range of 0.9926–198.5207 µg / mL, y = 0.9276x -1.684.

[0090] For butylated hydroxyanisole, the value is y = 7.0613x -0.8458 in the range of 0.2552 to 10.1667 µg / mL.

[0091] In the range of 0.2522–9.8640 µg / mL, y = 4.8328x -0.6542.

[0092] For isotretinoin in the range of 0.0486–9.7228 µg / mL, y = 66.879x -7.9836.

[0093] In the above, y is the y-axis, representing the peak area, and x is the x-axis, representing the concentration.

[0094] Furthermore, the contents of benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, and isotretinoin in the isotretinoin gel are calculated using the external standard method based on the peak areas detected by the detector. Taking isotretinoin gel as an example, the specific steps include:

[0095] Step 1: Weigh 0.1-1g of isotretinoin gel sample accurately, place it in a volumetric flask (10-100mL), add an appropriate amount of diluent, sonicate for 10-30min, and after cooling to room temperature, continue to dilute to the mark with diluent, shake well, filter, and take the filtrate as the test solution.

[0096] The diluent mentioned above is a mixed solution of methanol and water, with a volume ratio of methanol:water = 50:50.

[0097] Step 2: Accurately weigh appropriate amounts of benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, and isotretinoin reference standards. Dissolve them in methanol by sonication and quantitatively dilute to prepare a solution containing approximately 10,000 μg benzyl alcohol, 500 μg butylated hydroxyanisole, 500 μg butylated hydroxytoluene, and 500 μg isotretinoin per mL. This solution is used as the reference standard stock solution. Accurately measure 1 mL of this solution into a 100 mL volumetric flask containing an appropriate amount of methanol, dilute to the mark with diluent, and shake well. This is used as the reference standard solution.

[0098] Step 3: Take 10 μL of the solutions from Step 1 and Step 2 and inject them into the liquid chromatograph. Record the chromatograms and calculate the contents of benzyl alcohol, butylated hydroxyanisole, dibutylated hydroxytoluene, and isotretinoin by peak area using the external standard method.

[0099] In order to gain a deeper understanding and effective application of the solution proposed in this application, and to fully verify its benefits, based on Example 1, this application also provides the following multiple embodiments to provide a detailed methodological demonstration of the content detection method provided in this application, so as to ensure the scientificity, accuracy and reproducibility of the method.

[0100] The chromatographic conditions were set as follows: the column was a Shimadzu ShimNex HE C18-AQ column, the flow rate was set at 1.0 mL / min, and the column temperature was 30 °C; the mobile phase A was 0.1% (v / v) formic acid aqueous solution, and the mobile phase B was A 0.1% (v / v) formic acid acetonitrile solution as a mixed mobile phase for gradient elution. The ratio of mobile phase A to mobile phase B was maintained at an initial volume ratio of 70:30. For the specific steps of gradient elution, please refer to the description in Example 1 and the data shown in Table 1.

[0101] Before proceeding with the following methodological demonstration, the test solution and the reference solution used to establish a standard curve for quantitative analysis are prepared, namely the reference stock solution and the reference solution.

[0102] (1) Preparation of test solution: Accurately weigh 0.5 g of isotretinoin gel sample and place it in a 50 mL volumetric flask. Add an appropriate amount of diluent, sonicate for 20 min, and after cooling to room temperature, continue to dilute to the mark with diluent. Shake well, filter, and take the filtrate as the test solution. The above diluent is a mixture of methanol and water in a volume ratio of methanol:water = 50:50.

[0103] (2) Preparation of reference stock solution: Take appropriate amounts of benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene and isotretinoin reference standards, accurately weigh them, add methanol and sonicate to dissolve them, and quantitatively dilute them to prepare a solution containing approximately 10,000 μg benzyl alcohol, 500 μg butylated hydroxyanisole, 500 μg butylated hydroxytoluene and 500 μg isotretinoin per 1 mL, as the reference stock solution.

[0104] (3) The reference solution is prepared as follows: accurately measure 1 mL of the reference stock solution and place it in a 100 mL volumetric flask containing an appropriate amount of methanol. Dilute with methanol to the mark, shake well, and prepare a solution containing approximately 100 μg of benzyl alcohol, 5 μg of butylated hydroxyanisole, 5 μg of dibutylated hydroxytoluene and 5 μg of isotretinoin per 1 mL. This solution is used as the reference solution.

[0105] Accurately measure 10 μL each of the above-mentioned test solution and reference solution, inject them into the liquid chromatograph, and detect them at wavelengths of 258 nm, 280 nm and 358 nm. Record the chromatograms and calculate the content by peak area according to the external standard method.

[0106] Example 2

[0107] This embodiment conducts specificity tests on the method.

[0108] Take an appropriate amount of blank excipient (excluding benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, and isotretinoin), prepare a blank matrix solution according to the above-described method for preparing the test solution, inject it into the liquid chromatograph, and record the chromatogram. The chromatogram is shown below. Figure 1 , Figure 2and Figure 3 As shown.

[0109] Accurately weigh 1028.27 mg of benzyl alcohol reference standard, place it in a 50 mL volumetric flask, dissolve it in methanol, and dilute to the mark to prepare the benzyl alcohol reference standard stock solution. Measure 5 mL of the benzyl alcohol reference standard stock solution, place it in a 50 mL volumetric flask, dilute to the mark with methanol, and prepare the benzyl alcohol positioning solution. Inject the solution into the liquid chromatograph and record the chromatogram. The chromatogram is shown below. Figure 4 As shown.

[0110] Accurately weigh 25.56 mg of butylated hydroxyanisole (BHA) reference standard and place it in a 50 mL volumetric flask. Dissolve and dilute to the mark with methanol to prepare the BHA stock solution. Measure 5 mL of the BHA stock solution and place it in a 50 mL volumetric flask. Dilute to the mark with methanol to prepare the BHA positioning solution. Inject the solution into the liquid chromatograph and record the chromatogram. The chromatogram is shown below. Figure 5 As shown.

[0111] Accurately weigh 25.23 mg of butylated hydroxytoluene (BHT) reference standard and place it in a 50 mL volumetric flask. Dissolve and dilute to the mark with methanol to prepare the BHT stock solution. Measure 5 mL of the BHT stock solution and place it in a 50 mL volumetric flask. Dilute to the mark with methanol to prepare the BHT positioning solution. Inject the solution into the liquid chromatograph and record the chromatogram. The chromatogram is shown below. Figure 6 As shown.

[0112] Accurately weigh 49.85 mg of isotretinoin reference standard and place it in a 100 mL amber volumetric flask. Dissolve the solution in methanol by sonication and dilute to the mark to prepare the isotretinoin reference standard stock solution. Measure 5 mL of the isotretinoin reference standard stock solution into a 50 mL volumetric flask and dilute to the mark with methanol to prepare the isotretinoin targeting solution. Inject the solution into the liquid chromatograph and record the chromatogram. The chromatogram is shown below. Figure 7 As shown.

[0113] Following the preparation methods for the reference stock solution and reference solution described above, accurately weigh 1002.63 mg of benzyl alcohol, 50.92 mg of butylated hydroxyanisole, 49.33 mg of butylated hydroxytoluene, and 49.12 mg of isotretinoin reference standard, respectively. Place them in a 100 mL amber volumetric flask, add methanol, sonicate for 20 min to dissolve, and dilute to the mark to prepare the reference stock solution. Measure 1 mL of the reference stock solution into a 100 mL volumetric flask, dilute to the mark with methanol to prepare the reference solution, inject into the liquid chromatograph, and record the chromatogram. The chromatogram is shown below. Figure 8 , Figure 9 and Figure 10 As shown.

[0114] Accurately weigh 113.1 mg of isotretinoin gel, prepare a sample solution, inject it into the liquid chromatograph, and record the chromatogram. The chromatogram is shown below. Figure 11 , Figure 12 and Figure 13 As shown.

[0115] Table 4 Specificity Test Results

[0116]

[0117] As shown in Table 4 above, the results indicate that under these chromatographic conditions, the baseline is stable, and there is no interference at the peak positions of benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, and isotretinoin in the blank matrix solution chromatogram. In both the reference solution and the sample solution, the minimum resolution between butylated hydroxyanisole and its adjacent peak is 2.32, and the minimum resolution between butylated hydroxytoluene and its adjacent peak is 7.57, which meets the separation requirements.

[0118] Example 3

[0119] This embodiment conducts a system suitability test on the method.

[0120] The reference solution was injected five times consecutively, and a final injection was made at the end of the sequence to assess the system precision.

[0121] Table 5 System precision results

[0122]

[0123] The results in Table 5 show that the RSD of retention time for each component is ≤2%, and the RSD of peak area is ≤5%, indicating good system precision.

[0124] Example 4

[0125] This embodiment examines the sensitivity of the method.

[0126] The reference solution was serially diluted to confirm the limits of detection (LOD) and quantitation (LOQ) for each component. Six injections of the LOQ solution and three injections of the LOQ solution were performed consecutively. The signal-to-noise ratio (SNR) for the LOQ should be no less than 10, and the SNR for the LOQ should be no less than 3. LOQ data are shown in Table 6, LOQ data are shown in Table 7, and the LOQ chromatogram is shown below. Figure 14 , Figure 15 and Figure 16 As shown, the chromatogram for the limit of quantitation is as follows: Figure 17 , Figure 18 and Figure 19 As shown.

[0127] Table 6 Detection Limit Test Data

[0128]

[0129] Table 7. Limit of Quantitation Test Data

[0130]

[0131] Example 5

[0132] This embodiment performs linearity and range evaluation on the method.

[0133] Accurately weigh 1002.63 mg of benzyl alcohol, 50.92 mg of butylated hydroxyanisole, 49.33 mg of butylated hydroxytoluene, and 49.12 mg of isotretinoin reference standard, respectively, and place them in a 100 mL amber volumetric flask. Dissolve them in methanol by sonication and dilute to the mark to prepare the reference standard stock solution. Accurately measure an appropriate amount of the reference standard stock solution to prepare a series of solutions with gradient concentrations of 1%, 10%, 50%, 100%, 150%, and 200% of the limit of quantitation (LOQ). Perform linear regression with solution concentration as the x-axis and peak area as the y-axis, and derive the linear equation. The results are shown in Table 8.

[0134] Table 8 Results of Linearity Tests

[0135]

[0136] The results in Table 8 show that, within the test concentration ranges of 0.9926 μg / mL to 198.5207 μg / mL, butylated hydroxyanisole (BHA) within the test concentration range of 0.2552 μg / mL to 10.1667 μg / mL, butylated hydroxytoluene (BHT) within the test concentration range of 0.2522 μg / mL to 9.8640 μg / mL, and isotretinoin (ART) within the test concentration range of 0.0486 μg / mL to 9.7228 μg / mL, the measured concentrations and peak areas exhibit a good linear relationship, meeting the requirements for content determination by high performance liquid chromatography.

[0137] Example 6

[0138] This embodiment conducts an accuracy test on the method.

[0139] Accurately weigh 1002.63 mg of benzyl alcohol, 50.92 mg of butylated hydroxyanisole, 49.33 mg of butylated hydroxytoluene, and 49.12 mg of isotretinoin reference standard, place them in a 100 mL amber volumetric flask, add methanol and sonicate to dissolve and dilute to the mark to prepare the reference standard stock solution.

[0140] Accurately transfer 1 ml of isotretinoin reference stock solution, 5 ml of butylated hydroxyanisole reference stock solution, 5 ml of butylated hydroxytoluene reference stock solution, and 0.5 ml of benzyl alcohol stock solution from the specificity test into 100 ml volumetric flasks containing an appropriate amount of methanol solution. Dilute with methanol to the mark, shake well, and use as the limit of quantitation stock solution.

[0141] Preparation of 200% accuracy solutions: Accurately weigh 0.2635 g, 0.2662 g, and 0.2598 g of blank excipients into 25 mL volumetric flasks, respectively. Add 0.5 mL of the reference standard stock solution to each flask, then add an appropriate amount of diluent. Sonicate for 20 min, and after cooling to room temperature, continue diluting with diluent to the mark. Shake well. Label as A-200%-1~3.

[0142] Preparation of 100% accuracy solutions: Accurately weigh 0.2575 g, 0.259 g, and 0.2584 g of blank excipients into 25 mL volumetric flasks, respectively. Add 25 mL of the reference standard stock solution to each flask, then add an appropriate amount of diluent. Sonicate for 20 min. After cooling to room temperature, continue diluting with diluent to the mark and mix well. Label as A-100%-1~3.

[0143] Preparation of Quantitation Limit Accuracy Solutions: Accurately weigh 0.2516 g, 0.255 g, and 0.2543 g of blank excipients into 25 mL volumetric flasks, respectively. Add 0.5 mL of the reference solution to each flask, then add an appropriate amount of diluent. Sonicate for 20 min, and after cooling to room temperature, continue diluting to the mark with diluent and mix well. Label as A-LOQ-1~3.

[0144] The above reference solution and accuracy solution were injected into the liquid chromatograph, the chromatograms were recorded, and the recovery rates were calculated. The results are shown in Tables 9, 10, 11 and 12.

[0145] Table 9 Detection recovery rate of benzyl alcohol

[0146]

[0147] Table 10 Recovery rate of butylated hydroxyanisole

[0148]

[0149] Table 11 Recovery rate of butylated hydroxytoluene

[0150]

[0151] Table 12 Recovery rate of isotretinoin

[0152]

[0153] Example 7

[0154] This embodiment demonstrates a repeatability test of the method.

[0155] Six sample solutions were prepared for testing, and the results are shown in Tables 13, 14, 15 and 16 below.

[0156] Table 13 Results of repeatability test of benzyl alcohol

[0157]

[0158] Table 14 Results of Repeatability Tests for Butylated Hydroxyanisole

[0159]

[0160] Table 15 Results of Repeatability Tests for Butylated Hydroxytoluene

[0161]

[0162] Table 16 Results of repeatability test of isotretinoin

[0163]

[0164] The results showed that the RSD% of benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, and isotretinoin in the six sample solutions were all less than 5%, which met the requirements for content determination by high performance liquid chromatography.

[0165] Example 8

[0166] This embodiment demonstrates a solution stability test for this method.

[0167] The stability of the reference solution and the test solution under the repeatability test was investigated, and the tests were conducted at 0h, 12h and 24h, respectively. The test results are shown in Tables 17 and 18.

[0168] Table 17 Stability results of the reference solution

[0169]

[0170] The results in Table 17 show that after the reference solution was prepared and left at room temperature for 24 hours, the ratio of the peak area of ​​each component to that at 0 h was between 95% and 105%, indicating that the reference solution was stable at room temperature for 24 hours.

[0171] Table 18. Results of sample solution stability

[0172]

[0173] The results in Table 18 show that after the sample solution was prepared and placed at room temperature for 24 hours, the ratio of the peak area of ​​each impurity to that at 0 h was between 95% and 105%, and no new impurities were generated. The test solution was stable at room temperature for 24 hours.

[0174] Example 9

[0175] This embodiment examines the robustness of the method under varying conditions through a robustness test. Chromatographic parameters were adjusted: column temperature (30±2℃), flow rate (1.0±0.1mL / min), and initial proportion of mobile phase B (30%±2%). Reference solution and test solution were injected separately, and the contents of benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, and isotretinoin in the test solution were compared under different parameter conditions.

[0176] The reference solution and sample solution used were those specified in the repeatability test. 10 µL of each solution was precisely transferred and injected into the liquid chromatograph under both standard and modified conditions, and the chromatograms were recorded. The results are shown in Table 19.

[0177] Table 19 Durability Test Results

[0178]

[0179] The results showed that the flow rate of 0.9–1.1 mL / min, the column temperature of 28–32 °C, and the initial proportion of mobile phase B of 28%–32%B all met the requirements for the detection of each component.

[0180] Example 10

[0181] Based on Example 1, this application also provides a system for detecting the content of active pharmaceutical ingredient and excipient in isotretinoin gel, including: an injection system, a chromatographic column and a detector, wherein the injection system includes an injection needle, an injection valve, a quantitative coil and a constant flow pump, etc., for introducing the sample to be tested and the mobile phase into the chromatographic column at a certain volume and speed, thereby starting the separation process.

[0182] The detector is used to detect the individual components of the sample after separation by the chromatographic column and convert them into quantitative signals (i.e., chemical signals are converted into electrical signals). These signals are then recorded and analyzed to determine the concentration and content of each component.

[0183] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit it. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they may still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features. However, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for detecting the content of the active pharmaceutical ingredient (API) and excipients in isotretinoin gel, wherein the API is isotretinoin, and the excipients are an antibacterial agent and an antioxidant, wherein the antibacterial agent is benzyl alcohol, and the antioxidants are butylated hydroxyanisole and butylated hydroxytoluene; characterized in that... The content detection method includes a separation step and a detection step: The separation step employs high-performance liquid chromatography (HPLC). In the HPLC, a Shimadzu ShimNex HE C18-AQ column is used, with a 0.1% (v / v) acid-water solution as mobile phase A and a 0.1% (v / v) acid-acetonitrile solution as mobile phase B. The isotretinoin gel is subjected to gradient elution to separate the antibacterial agent, the antioxidant, and the isotretinoin. The detection steps include: the separated benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene and isotretinoin are respectively introduced into a detector, and the content of each component is determined according to the chromatogram output by the detector; The initial volume ratio of the mobile phase A to the mobile phase B is (68-72):(32-28). The gradient elution process is as follows: Within 0 to 8 minutes, the volume ratio of mobile phase A to mobile phase B is adjusted from the initial volume ratio to less than 70:30 and greater than or equal to 40:60; Within 8 to 15 minutes, the volume ratio of mobile phase A to mobile phase B is adjusted to be less than 40:60 and greater than or equal to 10:

90. Within 15 to 20 minutes, the volume ratio of mobile phase A to mobile phase B is maintained at less than 40:60 and greater than or equal to 10:

90. Within 20 to 20.1 minutes, the volume ratio of mobile phase A and mobile phase B is adjusted to the initial volume ratio; Within 20.1 to 25 minutes, the ratio of mobile phase A to mobile phase B remains at the initial volume ratio.

2. The content detection method according to claim 1, characterized in that, The initial volume ratio is 70:30; The gradient elution process is as follows: Within 0 to 8 minutes, the ratio of mobile phase A to mobile phase B is adjusted from 70:30 to 40:60; Within 8 to 15 minutes, the volume ratio of mobile phase A to mobile phase B was adjusted from 40:60 to 10:90; The volume ratio of mobile phase A to mobile phase B is maintained at 10:90 for 15 to 20 minutes. Within 20 to 20.1 minutes, the volume ratio of mobile phase A to mobile phase B was adjusted from 10:90 to 70:30; The ratio of mobile phase A to mobile phase B was maintained at 70:30 for 20.1 to 25 minutes.

3. The content detection method according to claim 1, characterized in that, The chromatographic column is 150 mm long, has an inner diameter of 4.6 mm, and has a packing particle size of 5 μm. The column temperature is 25~35℃; The injection flow rate is 0.9–1.1 mL / min; The injection volume is 5~15μL; The detection wavelength is 190~400nm.

4. The content detection method according to claim 1, characterized in that, The acid is any one or a combination of formic acid, glacial acetic acid, trifluoroacetic acid, and phosphoric acid; and / or, The mobile phase A is prepared by transferring 1 mL of the acid to 1000 mL of water, shaking well, and then degassing by sonication; and / or, The mobile phase B is prepared by transferring 1 mL of the acid into 1000 mL of acetonitrile, shaking well, and then degassing by sonication.

5. The content detection method according to claim 1, characterized in that, In the detection steps: The detection wavelength of benzyl alcohol is 258±2nm; The detection wavelength for butylated hydroxyanisole and butylated hydroxytoluene is 280±2nm; The detection wavelength for isotretinoin is 358±2nm.

6. The content detection method according to claim 5, characterized in that, In the chromatogram obtained from the isotretinoin gel test, benzyl alcohol was retained at 3.421±0.5 min, butylated hydroxyanisole was retained at 10.069±0.5 min, dibutylated hydroxytoluene was retained at 16.551±0.5 min, and isotretinoin was retained at 17.475±0.5 min.

7. The content detection method according to claim 1, characterized in that, The steps for determining the content of each component based on the chromatogram output by the detector include: Prepare standard solutions and obtain standard sample chromatograms; The contents of benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, and isotretinoin were calculated by peak area using the external standard method based on the chromatogram obtained from the isotretinoin gel test.

8. The content detection method according to claim 7, characterized in that, The benzyl alcohol is in the range of 0.9926–198.5207 µg / mL, y = 0.9276x -1.684; and / or, The butyl hydroxyanisole is in the range of 0.2552–10.1667 µg / mL, y = 7.0613x -0.8458; and / or, The butylated hydroxytoluene is in the range of 0.2522–9.8640 µg / mL, with y = 4.8328x -0.6542; and / or, The isotretinoin has a value of y = 66.879x -7.9836 in the range of 0.0486 to 9.7228 µg / mL. Where y is the y-axis, representing the peak area, and x is the x-axis, representing the concentration.

9. A content detection system that implements the content detection method according to any one of claims 1-8. Its features are, include: Injection system, chromatographic column and detector, The injection system is used to provide the sample to be tested and the mobile phase to the chromatographic column; The detector is used to detect the components of the sample after separation by the chromatographic column, convert them into quantitative signals, and record and analyze the signals to determine the concentration and content of each component.

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