Method for separation and detection of erythromycin, tretinoin and bht in tretinoin erythromycin gel

Abstract

The application provides a separation method and detection method for erythromycin, isotretinoin and BHT in isotretinoin erythromycin gel, and the separation method comprises: the detection method adopts high performance liquid chromatography to separate erythromycin, isotretinoin and BHT in isotretinoin erythromycin gel; and the high performance liquid chromatography conditions comprise that a Shimadzu shim-pack Scepter C18-120 column is used as a chromatographic column; a mixed mobile phase of mobile phase A and mobile phase B is used for gradient elution; the content of erythromycin, BHT and isotretinoin can be determined at the same time, which is convenient, fast and practical.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for separating and detecting erythromycin, isotretinoin and BHT in isotretinoin erythromycin gel. Background Technology

[0002] Isotretinoin and erythromycin gel is a topical compound preparation used to treat skin diseases such as acne. Its main active ingredients include isotretinoin and erythromycin. Isotretinoin, as a stereoisomer of retinoic acid, regulates epidermal cell differentiation and inhibits sebum secretion; erythromycin is a macrolide antibiotic with antibacterial and anti-inflammatory effects. The combined use of these two ingredients has a synergistic effect, effectively improving symptoms of both inflammatory and non-inflammatory acne. Furthermore, BHT (butylated hydroxytoluene), as an antioxidant, is often added to gel formulations to prevent oxidative deterioration of the drug components, thereby extending the product's shelf life.

[0003] However, current methods for analyzing the components of isotretinoin erythromycin gel have many shortcomings. Traditional detection methods usually require separate analysis of the active pharmaceutical ingredient and BHT, 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 a certain extent, they often face problems such as insufficient resolution, low sensitivity, and cumbersome operation when simultaneously determining multiple components.

[0004] High-performance liquid chromatography (HPLC), as a highly efficient, sensitive, and specific analytical technique, has been 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. However, the determination of erythromycin content has traditionally relied on microbial assays, turbidimetric methods, and spectrophotometry. These methods are either cumbersome, time-consuming, and have poor repeatability, or they require harsh reaction conditions and have low sensitivity. In recent years, HPLC-ELSD (evaporative light scattering detector) coupled technology has provided a new approach for the determination of erythromycin content.

[0005] Nevertheless, no literature has yet reported a method for simultaneously detecting the contents of erythromycin, isotretinoin, and BHT in isotretinoin erythromycin gel. Therefore, developing a simple, efficient, and accurate method for simultaneously detecting the contents of erythromycin, isotretinoin, and BHT is of great significance for the quality control of isotretinoin erythromycin gel. Summary of the Invention

[0006] In view of this, one of the objectives of this invention is to provide a method for separating the antioxidant BHT and the active pharmaceutical ingredient from a complex isotretinoin erythromycin gel. This method has broad applicability and lays a solid foundation for the subsequent simultaneous detection of the contents of the three components.

[0007] To achieve the above objectives, the present invention adopts the following technical solution, namely, providing a method for separating erythromycin, isotretinoin, and BHT from isotretinoin erythromycin gel, wherein the separation method includes: separating erythromycin, isotretinoin, and BHT from isotretinoin erythromycin gel using high performance liquid chromatography:

[0008] The high-performance liquid chromatography conditions include: a Shimadzu shim-pack Scepter C18-120 column;

[0009] Gradient elution was performed using a mixed mobile phase A and mobile phase B.

[0010] The mobile phase A is a formic acid aqueous solution with a volume ratio of 0.08%~0.12%; the mobile phase B is a formic acid acetonitrile solution with a volume ratio of 0.08%~0.12%.

[0011] During the gradient elution process:

[0012] The initial ratio of mobile phase A to mobile phase B is 88–92:12–8;

[0013] Within 0 to 7 minutes, the ratio of mobile phase A to mobile phase B gradually and uniformly changes from the initial ratio to 5:95.

[0014] The ratio of mobile phase A to mobile phase B remains constant at 5:95 for 7 to 9 minutes.

[0015] Within 9 to 9.01 minutes, the ratio of mobile phase A to mobile phase B gradually changed from 5:95 to the initial ratio;

[0016] Within 9.01 to 12 minutes, the ratio of mobile phase A to mobile phase B remains unchanged from the initial ratio.

[0017] According to one embodiment of this application, in the gradient elution process, the initial ratio of mobile phase A to mobile phase B is 90:10;

[0018] The specific gradient elution process is as follows: within 0 to 7 minutes, the ratio of mobile phase A to mobile phase B gradually changes from 90:10 to 5:95 at a constant rate.

[0019] The ratio of mobile phase A to mobile phase B remains constant at 5:95 for 7 to 9 minutes.

[0020] Within 9 to 9.01 minutes, the ratio of mobile phase A to mobile phase B gradually changed from 5:95 to 90:10;

[0021] Within 9.01 to 12 minutes, the ratio of mobile phase A to mobile phase B remained constant at 90:10.

[0022] According to one embodiment of this application, the preparation method of mobile phase A is as follows: transfer 1 mL of formic acid to 1000 mL of water, shake well, and degas by ultrasonication; the preparation method of mobile phase B is as follows: transfer 1 mL of formic acid to 1000 mL of acetonitrile, shake well, and degas by ultrasonication.

[0023] According to one embodiment of this application, the high-performance liquid chromatography conditions include at least one of the following:

[0024] The length of the chromatographic column is 30~35mm;

[0025] The inner diameter is 2.5~3.5 mm.

[0026] The filler particle size is 2.5~3.5 mm;

[0027] The flow rate is 0.9~1.1 mL / min;

[0028] The column temperature is 30~40℃;

[0029] ELSD and PDA detectors are used, with detection wavelengths ranging from 190 to 400 nm and injection volumes ranging from 5 to 15 μL.

[0030] According to one embodiment of this application, the high-performance liquid chromatography conditions include:

[0031] The flow rate is 1.0 mL / min; or

[0032] The column temperature is 35℃; or

[0033] ELSD detector detects erythromycin, and BHT detection wavelength is 280±2nm; or

[0034] The detection wavelength for isotretinoin is 358±2nm; or

[0035] The injection volume was 10 μL.

[0036] According to one embodiment of this application, the high-performance liquid chromatography conditions include a needle washing solvent consisting of a mixed solution of acetonitrile and water.

[0037] According to one embodiment of this application, the needle washing solvent is acetonitrile:water = 50:50 by volume.

[0038] According to another aspect of this application, this application provides a method for detecting erythromycin, isotretinoin, and BHT in isotretinoin erythromycin gel, the detection method comprising:

[0039] Erythromycin, isotretinoin, and BHT were separated from isotretinoin erythromycin gel using any of the separation methods described above.

[0040] Prepare standard samples to obtain standard sample chromatograms; for test samples, calculate the concentrations of erythromycin, isotretinoin, and BHT in the isotretinoin erythromycin gel by peak area using the external standard method.

[0041] According to one embodiment of this application, erythromycin has a retention time of 2.6 ± 0.5 min, BHT has a retention time of 6.04 ± 0.5 min, and isotretinoin has a retention time of 6.24 ± 0.5 min.

[0042] According to one embodiment of this application, the erythromycin is in the range of 9.899~296.9694µg / mL, with y = 64.12x. 1.6384 ; and / or the BHT in the range of 0.2477~9.906µg / mL, y = 4941.1x + 160.1; and / or the isotretinoin in the range of 0.0493~9.8535µg / mL, y = 70036x + 2949.1; where y is the y-axis, representing the peak area, and x is the x-axis, representing the concentration.

[0043] The advantages of using the technical solution of this invention are as follows:

[0044] This invention provides a method for separating erythromycin, BHT, and isotretinoin from isotretinoin erythromycin gel and simultaneously detecting the content of each component. It belongs to reversed-phase high-performance liquid chromatography and can simultaneously separate erythromycin, BHT, and isotretinoin from isotretinoin erythromycin gel and determine their contents. The method of this invention has good reproducibility and accurate and reliable detection results, which is of great significance for achieving quality control of isotretinoin erythromycin gel.

[0045] The method for separating erythromycin, BHT and isotretinoin from isotretinoin erythromycin gel provided by the present invention can simultaneously determine the content of erythromycin, BHT and isotretinoin, which is convenient, fast and practical. Attached Figure Description

[0046] Figure 1 This is a blank matrix solution chromatogram (ELSD). Figure 2 This is the chromatogram of the blank matrix solution (280 nm); Figure 3 Chromatogram of blank matrix solution (358 nm); Figure 4 This is a chromatogram of erythromycin localization solution; Figure 5This is a chromatogram of the BHT localization solution; Figure 6 This is a chromatogram of isotretinoin localization solution; Figure 7 This is the chromatogram of the reference solution (ELSD). Figure 8 This is the chromatogram of the reference solution (280nm); Figure 9 This is the chromatogram of the reference solution (358 nm); Figure 10 This is the chromatogram of the sample solution (ELSD); Figure 11 This is the chromatogram of the sample solution (280nm); Figure 12 This is the chromatogram of the sample solution (358 nm); Figure 13 This is the limit of detection solution chromatogram (ELSD); Figure 14 This is the chromatogram of the solution at the detection limit (280 nm); Figure 15 This is the chromatogram of the solution at the detection limit (358 nm); Figure 16 This is a limit-of-quantity solution chromatogram (ELSD). Figure 17 This is the chromatogram of the limit of quantitation solution (280 nm); Figure 18 This is the chromatogram of the solution at the limit of quantitation (358 nm). Detailed Implementation

[0047] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0048] In this embodiment of the invention, the chromatographic conditions are as follows: the chromatographic column is a Shimadzu shim-pack Scepter C18-120 column; gradient elution is performed using a mixed mobile phase A and mobile phase B.

[0049] Preferably, the length of the chromatographic column is 30-35 mm;

[0050] Preferably, the inner diameter of the chromatographic column is 2.5~3.5 mm;

[0051] Preferably, the particle size of the chromatographic column packing is 2.5~3.5 mm;

[0052] Preferably, the flow rate of the chromatographic column is 0.9~1.1 mL / min;

[0053] Preferably, the column temperature of the chromatographic column is 30~40℃.

[0054] The mobile phase A is a formic acid aqueous solution with a volume ratio of 0.08%~0.12%; the mobile phase B is a formic acid acetonitrile solution with a volume ratio of 0.08%~0.12%.

[0055] Also, as a preferred option, an ELSD detector and a PDA detector are used, with a detection wavelength of 190~400nm and an injection volume of 5~15μL.

[0056] In one embodiment, an ELSD detector and a PDA detector were used for detection at wavelengths of 280 nm and 358 nm, respectively. The flow rate was 1.0 mL / min, the column temperature was 35 °C, and 10 μL of each of the test solution and the reference solution were accurately injected into the liquid chromatograph and the chromatograms were recorded.

[0057]

[0058] In this embodiment of the invention, 0.5 g of isotretinoin erythromycin gel sample was accurately weighed and placed in a 50 mL volumetric flask. An appropriate amount of tetrahydrofuran was added, and the mixture was sonicated for 3 min. After cooling to room temperature, water was added to dilute to the mark, shaken well, filtered, and the filtrate was used as the test solution. It is worth mentioning that the volume ratio of tetrahydrofuran to water is approximately 1:2 to 2:1.

[0059] Experiments have shown that isotretinoin erythromycin gel is dissolved in tetrahydrofuran instead of other solvents because isotretinoin erythromycin gel has better solubility in tetrahydrofuran.

[0060] In this embodiment of the invention, the reference stock solution is prepared as follows: take appropriate amounts of erythromycin, BHT and isotretinoin reference standards, accurately weigh them, add methanol and sonicate to dissolve them, and quantitatively dilute them to prepare solutions containing approximately 2000 μg of erythromycin, 500 μg of BHT and 500 μg of isotretinoin per ml, which are used as reference stock solutions for each component.

[0061] In this embodiment of the invention, the reference solution is prepared as follows: 1 mL of BHT and isotretinoin reference stock solutions and 10 mL of erythromycin reference stock solution are respectively placed in a 100 mL volumetric flask containing an appropriate amount of methanol, diluted to the mark with diluent, and shaken well to obtain the reference solution.

[0062] In this embodiment of the invention, the method for determining the content is as follows: accurately measure the sample solution and the reference solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the content by peak area using the external standard method.

[0063] Example 1: Specificity

[0064] Take an appropriate amount of blank excipient (excluding erythromycin, BHT, and isotretinoin), prepare a blank matrix solution according to the method for preparing the test solution, inject it into the liquid chromatograph, and record the chromatogram; the chromatogram is as follows. Figure 1 , Figure 2 and Figure 3 As shown.

[0065] Accurately weigh 101.01 mg of erythromycin reference standard, place it in a 10 mL volumetric flask, dissolve it in methanol, and dilute to the mark to prepare the erythromycin reference standard stock solution. Measure 2 mL of the erythromycin reference standard stock solution, place it in a 50 mL volumetric flask, and dilute to the mark with methanol to prepare the erythromycin positioning solution. Inject the solution into the liquid chromatograph and record the chromatogram; the chromatogram is shown below. Figure 4 As shown.

[0066] Accurately weigh 24.77 mg of BHT reference standard and place it in a 100 mL volumetric flask. Dissolve and dilute to the mark with methanol to prepare the BHT reference standard stock solution. Measure 2 mL of the BHT reference standard stock solution into a 10 mL volumetric flask and 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 5 As shown.

[0067] Accurately weigh 24.89 mg of isotretinoin reference standard and place it in a 100 mL amber volumetric flask. Dissolve and dilute to the mark with methanol via sonication to prepare the isotretinoin reference standard stock solution. Measure 2 mL of the isotretinoin reference standard stock solution into a 10 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 6 As shown.

[0068] Take 1 mL of each of the aforementioned BHT and isotretinoin reference stock solutions and 10 mL of erythromycin reference stock solution, and place them in a 100 mL volumetric flask containing an appropriate amount of methanol. Dilute to the mark with diluent and mix well to obtain the reference solutions. Inject the solutions into the liquid chromatograph and record the chromatograms; the chromatograms are shown below. Figure 7 , Figure 8 and Figure 9 As shown.

[0069] Accurately weigh 107.1 mg of isotretinoin erythromycin gel, prepare a sample solution, inject it into the liquid chromatograph, and record the chromatogram; the chromatogram is as follows. Figure 10 , Figure 11 and Figure 12 As shown.

[0070] Table 2 Specificity Results

[0071]

[0072] The results showed that under these chromatographic conditions, the baseline was stable, and there was no interference at the peak positions of erythromycin, BHT, and isotretinoin in the blank matrix solution chromatogram. In both the reference solution and the sample solution, the minimum resolution between erythromycin and its adjacent peak was 1.684, and the minimum resolution between BHT and isotretinoin and their adjacent peaks was 1.746, which met the separation requirements.

[0073] Example 2: System Applicability

[0074] 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.

[0075] Table 3 System precision results

[0076]

[0077] The results showed that the RSD of retention time for each component was ≤2%, and the RSD of peak area was ≤5%, indicating good system precision.

[0078] Example 3: Sensitivity Test

[0079] 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 4, LOQ data are shown in Table 5, and the LOQ chromatogram is shown below. Figure 13 , Figure 14 and Figure 15 As shown, the chromatogram for the limit of quantitation is as follows: Figure 16 , Figure 17 and Figure 18 As shown.

[0080] Table 4 Detection Limit Test Data

[0081]

[0082] Table 5. Limit of Quantitation Test Data

[0083]

[0084] Example 4: Linear (Power Function) and Range

[0085] Accurately weigh 101.01 mg of erythromycin, place it in a 10 mL volumetric flask, add methanol to dissolve and dilute to the mark, and use it as the erythromycin reference stock solution;

[0086] Accurately weigh 24.77 mg of BHT and place it in a 100 mL volumetric flask. Add methanol and sonicate to dissolve and dilute to the mark to prepare BHT reference stock solution.

[0087] Accurately weigh 24.89 mg of isotretinoin reference standard, place it in a 100 mL amber volumetric flask, add methanol and sonicate to dissolve and dilute to the mark, and use it as the stock solution of isotretinoin reference standard.

[0088] Accurately measure appropriate amounts of each reference standard stock solution to prepare a series of solutions with gradient concentrations of 1%, 5%, 10%, 50%, 100%, 150%, and 200% of the limit, respectively. Perform linear regression with solution concentration as the x-axis and peak area as the y-axis, and derive the linear (power function) equation. The results are shown in Tables 6, 7, and 8.

[0089] Table 6 Results of the power function test for erythromycin

[0090]

[0091] Table 7 Results of BHT linearity test

[0092]

[0093] Table 8. Results of linearity test for isotretinoin

[0094]

[0095] The results showed that erythromycin exhibited a good power-law relationship between concentration and peak area within the test concentration range of 9.899 μg / mL to 296.9694 μg / mL; BHT and isotretinoin showed good linear relationships between concentration and peak area within the test concentration range of 0.2477 μg / mL to 9.906 μg / mL and retinoic acid within the test concentration range of 0.0493 μg / mL to 9.8535 μg / mL, respectively, meeting the requirements for content determination by high performance liquid chromatography.

[0096] Example 5: Accuracy Test

[0097] Accurately weigh 250.85 mg of erythromycin reference standard, place it in a 25 mL volumetric flask, add methanol to dissolve and dilute to the mark, shake well, and label it as erythromycin reference standard stock solution.

[0098] Accurately weigh 25.44 mg of BHT reference standard, place it in a 100 mL volumetric flask, add methanol to dissolve and dilute to the mark, shake well, and label it as BHT reference standard stock solution.

[0099] Accurately weigh 26.25 mg of isotretinoin reference standard, place it in a 100 mL amber volumetric flask, add methanol to dissolve and dilute to the mark, shake well, and label it as isotretinoin reference standard stock solution.

[0100] Take 1 mL each of the aforementioned erythromycin reference stock solution, BHT reference stock solution, and isotretinoin reference stock solution and place them in a 50 mL amber volumetric flask. Dilute to the mark with methanol, shake well, and label as reference solutions.

[0101] Accurately transfer 0.25 mL of isotretinoin reference stock solution, 1.25 mL of erythromycin reference stock solution, and 1.25 mL of BHT reference stock solution into 50 mL amber volumetric flasks containing an appropriate amount of methanol solution. Dilute to the mark with methanol and shake well to obtain the limit of quantitation stock solutions.

[0102] Preparation of 150% accuracy solutions: Accurately weigh 0.262 g, 0.263 g, and 0.258 g of blank excipients into 25 mL volumetric flasks. Add 0.75 mL each of erythromycin reference stock solution, BHT reference stock solution, and isotretinoin reference stock solution, respectively. Add an appropriate amount of diluent, sonicate for 3 min, and after cooling to room temperature, continue to dilute to the mark with diluent and shake well. Label as A-150%-1~3.

[0103] The diluent is a mixed solution of tetrahydrofuran and water in a volume ratio of 1:1.

[0104] Preparation of 100% accuracy solutions: Accurately weigh 0.251 g, 0.259 g, and 0.248 g of blank excipients into 25 mL volumetric flasks. Add 0.5 mL each of erythromycin reference stock solution, BHT reference stock solution, and isotretinoin reference stock solution, respectively. Add an appropriate amount of diluent, sonicate for 3 min, and after cooling to room temperature, continue to dilute to the mark with diluent and shake well. Label as A-100%-1~3.

[0105] Preparation of Quantitation Limit Accuracy Solutions: Accurately weigh 0.248 g, 0.252 g, and 0.255 g of blank excipients into 25 mL volumetric flasks, add 1 mL of Quantitation Limit Stock Solution, then add an appropriate amount of diluent, sonicate for 3 min, and after cooling to room temperature, continue to dilute to the mark with diluent and shake well. Label as A-LOQ-1~3.

[0106] The above reference solution and accuracy solution were injected into the liquid chromatograph, the chromatograms were recorded, and the recovery rate was calculated. The results are shown in Tables 9, 10 and 11.

[0107] Table 9 Recovery rate of erythromycin

[0108]

[0109] Table 10 BHT Detection Recovery Rate

[0110]

[0111] Table 11. Recovery rate of isotretinoin

[0112]

[0113] Example 6: Repeatability Test

[0114] Six sample solutions were prepared for testing, and the results are shown in Tables 12, 13 and 14 below.

[0115] Table 12 Results of Erythromycin Repeatability Tests

[0116]

[0117] Table 13 Results of BHT Repeatability Tests

[0118]

[0119] Table 14 Results of repeatability test of isotretinoin

[0120]

[0121] The results showed that the RSD% of erythromycin, BHT and isotretinoin in the six sample solutions were all less than 5%, which met the requirements for content determination by high performance liquid chromatography.

[0122] Example 7: Solution Stability Test

[0123] Stability was investigated using the reference solution and the test solution under the repeatability test. The tests were conducted at 0h, 12h and 18h, respectively. The test results are shown in Tables 15 and 16.

[0124] Table 15 Stability results of the reference solution

[0125]

[0126] The results showed that after the reference solution was prepared and stored at room temperature in the dark for 18 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 in the dark for 18 hours.

[0127] Table 16. Results of sample solution stability

[0128]

[0129] The results showed that after the sample solution was prepared and placed at room temperature in the dark for 18 hours, the ratio of the peak area of ​​each impurity to that at 0 hours was between 95% and 105%, indicating that the test solution was stable within 18 hours at room temperature in the dark.

[0130] Example 8: Durability Test

[0131] To examine the tolerance of this method to changes in conditions, a robustness test was conducted. The chromatographic parameters were adjusted: column temperature (35±2℃), flow rate (1.0±0.1mL / min), and initial proportion of mobile phase B (10%±2%). Reference solution and test solution were injected separately, and the contents of erythromycin, BHT, and isotretinoin in the test solution were compared under different parameter conditions.

[0132] 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 17.

[0133] Table 17 Durability Test Results

[0134]

[0135] The results showed that the flow rate of 0.9–1.1 mL / min, the column temperature of 33–37 °C, and the initial proportion of mobile phase B of 8%–12% all met the requirements for the detection of each component.

[0136] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications may still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions may be made to some of the technical features. Such 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 the present invention.

Claims

1. A method for separating erythromycin, isotretinoin, and BHT from isotretinoin erythromycin gel, characterized in that, The separation method includes: separating erythromycin, isotretinoin, and BHT from isotretinoin erythromycin gel using high performance liquid chromatography. The high-performance liquid chromatography conditions include: a Shimadzu shim-pack Scepter C18-120 column; Gradient elution was performed using a mixed mobile phase A and mobile phase B. The mobile phase A is a formic acid aqueous solution with a volume ratio of 0.08%~0.12%; the mobile phase B is a formic acid acetonitrile solution with a volume ratio of 0.08%~0.12%. During the gradient elution process: The initial ratio of mobile phase A to mobile phase B is 88–92:12–8; Within 0 to 7 minutes, the ratio of mobile phase A to mobile phase B gradually and uniformly changes from the initial ratio to 5:

95. The ratio of mobile phase A to mobile phase B remains constant at 5:95 for 7 to 9 minutes. Within 9 to 9.01 minutes, the ratio of mobile phase A to mobile phase B gradually changed from 5:95 to the initial ratio; Within 9.01 to 12 minutes, the ratio of mobile phase A to mobile phase B remains unchanged from the initial ratio.

2. The method for separating erythromycin, isotretinoin, and BHT from isotretinoin erythromycin gel according to claim 1, characterized in that, During the gradient elution process, the initial ratio of mobile phase A to mobile phase B is 90:10; The specific gradient elution process is as follows: within 0 to 7 minutes, the ratio of mobile phase A to mobile phase B gradually changes from 90:10 to 5:95 at a constant rate. The ratio of mobile phase A to mobile phase B remains constant at 5:95 for 7 to 9 minutes. Within 9 to 9.01 minutes, the ratio of mobile phase A to mobile phase B gradually changed from 5:95 to 90:10; Within 9.01 to 12 minutes, the ratio of mobile phase A to mobile phase B remained constant at 90:

10.

3. The method for separating erythromycin, isotretinoin, and BHT from isotretinoin erythromycin gel according to claim 1, characterized in that, The preparation method of mobile phase A is as follows: transfer 1 mL of formic acid to 1000 mL of water, shake well, and degas by sonication. The preparation method of mobile phase B is as follows: transfer 1 mL of formic acid to 1000 mL of acetonitrile, shake well, and degas by sonication.

4. The method for separating erythromycin, isotretinoin, and BHT from isotretinoin erythromycin gel according to claim 1, characterized in that, The high-performance liquid chromatography conditions include at least one of the following: The length of the chromatographic column is 30~35mm; The inner diameter is 2.5~3.5 mm. The filler particle size is 2.5~3.5 mm; The flow rate is 0.9~1.1 mL / min; The column temperature is 30~40℃; ELSD and PDA detectors are used, with detection wavelengths ranging from 190 to 400 nm and injection volumes ranging from 5 to 15 μL.

5. The method for separating erythromycin, isotretinoin, and BHT from isotretinoin erythromycin gel according to claim 4, characterized in that, The high-performance liquid chromatography conditions include: The flow rate is 1.0 mL / min; or The column temperature is 35℃; or ELSD detector detects erythromycin, and BHT detection wavelength is 280±2nm; or The detection wavelength for isotretinoin is 358±2nm; or The injection volume was 10 μL.

6. The method for separating erythromycin, isotretinoin, and BHT from isotretinoin erythromycin gel according to claim 1, characterized in that, The high-performance liquid chromatography conditions include using a mixture of acetonitrile and water as the needle washing solvent.

7. The method for separating erythromycin, isotretinoin, and BHT from isotretinoin erythromycin gel according to claim 6, characterized in that, The solvent for washing needles is acetonitrile:water = 50:50 by volume.

8. A method for detecting erythromycin, isotretinoin, and BHT in isotretinoin erythromycin gel, characterized in that, The detection method includes: Erythromycin, isotretinoin, and BHT were separated from isotretinoin erythromycin gel by any of the separation methods described in claims 1-7; Prepare standard samples to obtain standard sample chromatograms; for test samples, calculate the concentrations of erythromycin, isotretinoin, and BHT in the isotretinoin erythromycin gel by peak area using the external standard method.

9. The method for detecting erythromycin, isotretinoin, and BHT in isotretinoin erythromycin gel according to claim 8, characterized in that, Retention time of 2.6±0.5 min indicates erythromycin, retention time of 6.04±0.5 min indicates BHT, and retention time of 6.24±0.5 min indicates isotretinoin.

10. The method for detecting erythromycin, isotretinoin, and BHT in the isotretinoin erythromycin gel according to claim 8, characterized in that, The erythromycin was in the range of 9.899~296.9694µg / mL, y = 64.12x 1.6384 ; and / or the BHT in the range of 0.2477~9.906µg / mL, y = 4941.1x + 160.1; and / or the isotretinoin in the range of 0.0493~9.8535µg / mL, y = 70036x + 2949.1; where y is the y-axis, representing the peak area, and x is the x-axis, representing the concentration.

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