Method for analyzing and detecting sirolimus impurity G

Abstract

The invention belongs to the technical field of medicine analysis, and particularly relates to an analysis and detection method of sirolimus impurity G, which can accurately separate and detect the impurity G. The analysis and detection method has the advantages of high detection speed, high specificity, high sensitivity and good repeatability.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to an analysis and detection method for sirolimus impurity G. Background Art

[0002] Sirolimus, also known as rapamycin, is a secondary metabolite secreted by soil Streptomyces that was first discovered by scientists in the soil of Easter Island, Chile in 1975. Its chemical structure belongs to the triene macrolide class of compounds. Due to the low fermentation yield of sirolimus and the complex extraction process, the drug was not developed and launched by Pfizer until September 15, 1999, and was subsequently launched in more than a dozen countries in Europe and the United States. Sirolimus has shown a strong immunosuppressive effect in clinical trials and can replace cyclosporine, which has a clinical history of more than 30 years. Compared with cyclosporine, sirolimus has a smaller oral dose, stronger anti-rejection effect, and fewer side effects. Therefore, sirolimus has quickly become a commonly used oral immunosuppressant for organ transplant recipients around the world since it was launched.

[0003] Sirolimus has a complex chemical structure and may produce multiple homologues and isomers during the fermentation process, such as 12-desmethylrapamycin and 29-O-desmethylsirolimus. Given the complexity of the impurity spectrum of sirolimus, it is urgent to establish a comprehensive and systematic quality control and impurity analysis method to better ensure the safety and effectiveness of clinical drugs.

[0004] Except for the United States Pharmacopoeia, no other national pharmacopoeia contains the relevant quality standards for sirolimus. The content analysis method of sirolimus published by the United States Pharmacopoeia only controls the content of sirolimus triketone isomers and oxirane isomers to be less than 0.5% and 5.0% respectively, and the content of other non-specific single impurities and total impurities is less than 0.15% and 5.0% respectively.

[0005] 1.0%. This quality standard does not strictly limit other specific impurities.

[0006] The "Technical Guidelines for the Study of Impurities in Chemical Drugs" issued by the State Food and Drug Administration of my country clearly states that the study of impurities is an important part of drug development, including the selection of appropriate analytical methods to accurately distinguish and determine the impurity content. This principle emphasizes that impurity research must run through the entire process of drug development; it also points out that the choice of analytical methods is directly related to the specificity and accuracy of impurity determination results.

[0007] The present invention systematically studies the relevant impurities of sirolimus, focuses on studying the specific impurity G with a significantly increased content in the stability study of the raw material drug, and systematically and deeply studies the analysis and detection method thereof. Summary of the invention

[0008] The object of the present invention is to provide an analytical detection method for sirolimus impurity G, which can accurately separate and detect impurity G. The analytical detection method has the advantages of fast detection speed, strong specificity, high sensitivity and good repeatability.

[0009] The technical scheme of the present invention is: a method for analyzing and detecting sirolimus impurity G, which adopts reversed-phase high performance liquid chromatography to detect a test solution, uses a mixed solution of citric acid aqueous solution and tetrahydrofuran as mobile phase A, and uses a mixed solution of acetonitrile and methanol as mobile phase B, and performs gradient elution.

[0010] The structure of the impurity G described in the present invention is shown in the following formula, and its molecular formula is C 50 H 77 NO 13 , molecular weight is 900.16:

[0011]

[0012] Preferably, in the detection method, the chromatographic column is selected from one of ZORBAX SB-C18, YMC Triart C18, WatersXbridge C18 or Welch XB C18.

[0013] Preferably, in the detection method, the specification of the chromatographic column is 4.6×250 mm, 5 μm.

[0014] Preferably, in the detection method, the column temperature of the chromatographic column is 35-50°C.

[0015] Preferably, the flow rate of the mobile phase is 1.0 to 2.0 mL / min.

[0016] Preferably, in the detection method, the concentration of sirolimus in the test solution is 0.8-2.0 mg / mL.

[0017] Preferably, the detection wavelength of the HPLC is 270-280 nm.

[0018] Preferably, the injection volume of the HPLC is 10 to 30 μL.

[0019] More preferably, the injection volume of the HPLC is 20 μL.

[0020] Preferably, in the mobile phase A, the volume ratio of 2% citric acid aqueous solution to tetrahydrofuran is 35:65.

[0021] More preferably, the citric acid aqueous solution with a mass concentration of 2% in the mobile phase A is adjusted to pH 4.5 with triethylamine.

[0022] Preferably, in the mobile phase B, the volume ratio of acetonitrile to methanol is 24:76.

[0023] Preferably, in the detection method, the gradient elution step is:

[0024]

[0025]

[0026] Further preferably, in the detection method, the gradient elution step is:

[0027]

[0028] In a preferred embodiment, the analysis and detection method comprises the following steps:

[0029] a. Sirolimus reference solution: accurately weigh an appropriate amount of sirolimus reference substance, add an appropriate amount of diluent 10% ethanol aqueous solution (volume ratio) to dissolve it until each 1mL contains 1.0 μg of the solution, shake well, and use it as the sirolimus reference solution;

[0030] b. Impurity reference stock solution: Weigh 10 mg of sirolimus impurity G and place them in 10 mL volumetric flasks, add diluent to dissolve and dilute to scale, shake well, and obtain;

[0031] c. Selective test solution: accurately weigh 10 mg of sirolimus reference substance and place it in a 10 mL volumetric flask, add 500 μL each of impurity G and reference substance stock solution, dilute to scale with diluent, shake well, and obtain;

[0032] d. Test solution: accurately weigh 10 mg of sirolimus sample to be tested, dissolve it in an appropriate amount of 10% ethanol aqueous solution (volume ratio) of diluent, and quantitatively dilute it to 1.0 mg / mL as the test solution;

[0033] e. Accurately measure each solution, inject it into the high performance liquid chromatograph respectively, and record the chromatogram.

[0034] Compared with the prior art, the present invention has the following outstanding advantages:

[0035] 1. The present invention provides a method for analyzing and detecting sirolimus and its impurities with fast detection speed, strong specificity, high sensitivity and good repeatability, which realizes the separation of sirolimus from other impurities, has high separation degree between peaks, and is conducive to accurately and quickly controlling the quality of sirolimus raw materials;

[0036] 2. The present invention provides an analytical detection method for sirolimus impurity G, which can accurately and quickly locate and detect, thereby ensuring the product quality of sirolimus and ensuring the safety and effectiveness of the drug. DETAILED DESCRIPTION

[0037] The following specific implementation modes are listed to further illustrate the present invention, but do not limit the scope of the present invention in any way. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0038] Example 1

[0039] (1) Solution preparation

[0040] a. Sirolimus reference solution: accurately weigh an appropriate amount of sirolimus reference substance (USP reference substance purity, calculated as 99.5%), add an appropriate amount of diluent 10% ethanol aqueous solution (volume ratio) to dissolve, and each 1mL contains 1.0mg sirolimus. Shake well to use as the sirolimus reference solution;

[0041] b. Impurity reference stock solution: accurately weigh 10 mg of sirolimus impurity G, place in a 10 mL volumetric flask, add diluent to dissolve and dilute to scale, shake well, and obtain;

[0042] c. Selectivity test solution: accurately weigh 10 mg of sirolimus reference substance and place it in a 10 mL volumetric flask, add 500 μL of impurity G reference substance stock solution, dilute to the mark with diluent, shake well, and obtain;

[0043] d. Accurately measure each solution, inject it into the high performance liquid chromatograph respectively, and record the chromatogram.

[0044] (2) HPLC conditions

[0045] Instrument: High performance liquid chromatography-ultraviolet detector;

[0046] Chromatographic column: ZORBAX SB-C18, 4.6×250 mm, 5 μm, column temperature 35 °C;

[0047] Mobile phase A: 2% citric acid aqueous solution and tetrahydrofuran (volume ratio 35:65), wherein the citric acid aqueous solution was adjusted to pH 4.5 with triethylamine;

[0048] B mobile phase: acetonitrile and methanol (volume ratio 24:76);

[0049] Flow rate: 1.5 mL / min;

[0050] Detection wavelength: 280nm;

[0051] Injection volume: 20 μL

[0052] The gradient elution steps are:

[0053]

[0054] The selective test solution was accurately measured, and the HPLC results were recorded according to the above chromatographic conditions. The results showed that the separation degree between impurity G and the next impurity peak was 1.77, and the separation effect was good.

[0055] Example 2

[0056] (1) Preparation of the solution: steps a. and b. are the same as those in Example 1, and step c. is as follows:

[0057] Selective test solution: accurately weigh 8 mg of sirolimus reference substance and place it in a 10 mL volumetric flask, accurately add 500 μL of impurity G reference substance stock solution, dilute to the scale with diluent, shake well, and obtain.

[0058] (2) HPLC conditions

[0059] Instrument: High performance liquid chromatography-ultraviolet detector;

[0060] Chromatographic column: YMC Triart C18, 4.6×250mm, 5μm, column temperature 45℃;

[0061] Mobile phase A: 2% citric acid aqueous solution and tetrahydrofuran (volume ratio 35:65), wherein the citric acid aqueous solution was adjusted to pH 4.5 with triethylamine;

[0062] B mobile phase: acetonitrile and methanol (volume ratio 24:76);

[0063] Flow rate: 1.0 mL / min;

[0064] Detection wavelength: 275nm;

[0065] Injection volume: 10 μL

[0066] The gradient elution steps are:

[0067]

[0068] The selective test solution was accurately measured and measured according to the above chromatographic conditions, and the HPLC results were recorded. The results showed that the separation degree between impurity G and the next impurity peak was 1.54, and the separation effect was good.

[0069] Example 3

[0070] (1) Preparation of the solution: steps a. and b. are the same as those in Example 1, and step c. is as follows:

[0071] Selective test solution: accurately weigh 20 mg of sirolimus reference substance and place it in a 10 mL volumetric flask, accurately add 500 μL of impurity G reference substance stock solution, dilute to the scale with diluent, shake well, and obtain.

[0072] (2) HPLC conditions

[0073] Instrument: High performance liquid chromatography-ultraviolet detector;

[0074] Chromatographic column: Waters Xbridge C18, 4.6×250 mm, 5 μm, column temperature 50 °C;

[0075] Mobile phase A: 2% citric acid aqueous solution and tetrahydrofuran (volume ratio 35:65), wherein the citric acid aqueous solution was adjusted to pH 4.5 with triethylamine;

[0076] B mobile phase: acetonitrile and methanol (volume ratio 24:76);

[0077] Flow rate: 2.0 mL / min;

[0078] Detection wavelength: 275nm;

[0079] Injection volume: 30 μL

[0080] The gradient elution steps are:

[0081]

[0082] The selective test solution was accurately measured and measured according to the above chromatographic conditions, and the HPLC results were recorded. The results showed that the separation degree between impurity G and the next impurity peak was 1.54, and the separation effect was good.

[0083] Example 4

[0084] (1) The solution was prepared in the same manner as in Example 1.

[0085] (2) HPLC conditions

[0086] Instrument: High performance liquid chromatography-ultraviolet detector;

[0087] Chromatographic column: ZORBAX SB-C18, 4.6×250 mm, 5 μm, column temperature 35 °C;

[0088] Mobile phase A: 2% citric acid aqueous solution and tetrahydrofuran (volume ratio 35:65), wherein the citric acid aqueous solution was adjusted to pH 4.5 with triethylamine;

[0089] B mobile phase: acetonitrile and methanol (volume ratio 24:76);

[0090] Flow rate: 1.5 mL / min;

[0091] Detection wavelength: 280nm;

[0092] Injection volume: 20 μL

[0093] The gradient elution steps are:

[0094]

[0095] The selective test solution was accurately measured and measured according to the above chromatographic conditions, and the HPLC results were recorded. The results showed that the separation degree between impurity G and the next impurity peak was 1.03, and the separation effect was relatively poor.

[0096] Comparative Example 1

[0097] (1) Preparation of solution is the same as in Example 1;

[0098] (2) HPLC conditions

[0099] Mobile phase A: acetonitrile and water in a volume ratio of 20:80;

[0100] Mobile phase B: methanol: acetonitrile volume ratio of 40:60;

[0101] Chromatographic column: Medium Spectrum Red RD-C18, specification: 4.6×250mm, 3.0μm;

[0102] Flow rate of mobile phase: 1.0 mL / min;

[0103] The column temperature is: 50°C;

[0104] The injection volume was: 10 μL;

[0105] Detection wavelength: 278nm;

[0106] Elution method: gradient elution, the specific procedure is as follows:

[0107] 0-33min: the volume of mobile phase A changed from 40% to 30%, and the volume of mobile phase B changed from 60% to 70%;

[0108] 33-40min: Mobile phase A volume is 30%, mobile phase B volume is 70%;

[0109] 40-50min: the volume of mobile phase A changed from 30% to 20%, and the volume of mobile phase B changed from 70% to 80%;

[0110] 50.1-60min: The volume of mobile phase A is 40%, and the volume of mobile phase B is 60%.

[0111] The selective test solution was accurately measured and measured under the above chromatographic conditions. The results showed that the impurity G and the two subsequent impurity peaks had poor separation and appeared as one chromatographic peak.

[0112] Comparative Example 2:

[0113] (1) Preparation of solution is the same as in Example 1;

[0114] (2) HPLC conditions

[0115] The high performance liquid chromatograph was Shimadzu LC2010, the mobile phase was 0.6% (V / V) glacial acetic acid aqueous solution: 1,4-dioxane = 42:58 (volume ratio); the pH of the glacial acetic acid aqueous solution was adjusted to 3.6 with triethylamine; the ultraviolet detection wavelength was 277 nm, the flow rate was 1 mL / min; the chromatographic column was Agilengt C8, and the particle size was 5 μm.

[0116] The above selectivity test solution was accurately measured and measured under the above chromatographic conditions. Impurity G and a subsequent impurity peak in the selectivity test solution were not completely separated, and the separation degree was 0.78.

[0117] Comparative Example 3:

[0118] (1) Preparation of solution is the same as in Example 1;

[0119] (2) HPLC conditions

[0120] The HPLC column was a Kinetex column (5um, F5 100A, 150x 21.2mm), and a BUCHI pure C-830Prep system was used. Gradient elution was performed at a flow rate of 5mL / min, with elution solvent A (water) and solvent B (methanol), and the gradient elution steps were: T=0min, 60%B; T=1min, 60%B; T=21min, 85%B; T=21min, 100%B; T=24min, 100%В; T=24min, 60%b; T=27min, 60%B.

[0121] The above selectivity test solution was accurately measured and measured under the above chromatographic conditions. Impurity G and a subsequent impurity peak in the selectivity test solution were not completely separated, and the separation degree was 0.74.

[0122] Experimental Example 1: Detection of API

[0123] (1) Solution preparation

[0124] Preparation of the test solution: accurately weigh 10 mg (content 100.4%) of pure sirolimus bulk drug, dissolve it in a proper amount of 10% ethanol aqueous solution (volume ratio) of diluent, and quantitatively dilute it to 1.0 mg / mL as the test solution. The preparation of other solutions is the same as in Example 1.

[0125] (2) HPLC conditions

[0126] Instrument: High performance liquid chromatography-ultraviolet detector;

[0127] Chromatographic column: ZORBAX SB-C18, 4.6×250 mm, 5 μm, column temperature 35 °C;

[0128] Mobile phase A: 2% citric acid aqueous solution and tetrahydrofuran (volume ratio 35:65), wherein the citric acid aqueous solution was adjusted to pH 4.5 with triethylamine;

[0129] B mobile phase: acetonitrile and methanol (volume ratio 24:76);

[0130] Flow rate: 1.5 mL / min;

[0131] Detection wavelength: 280nm;

[0132] Injection volume: 20 μL

[0133] The gradient elution steps are:

[0134]

[0135] The sample solution to be tested was accurately measured, and the test was performed according to the above chromatographic conditions, and the HPLC chromatogram was recorded. The results showed that the separation degree between impurity G and the next impurity peak was 1.98, and the separation effect was good.

[0136] Experimental Example 2: Stability Study of Sirolimus

[0137] The pure sirolimus API, a white crystalline powder, was placed under accelerated conditions (temperature 40±2°C; relative humidity 75±5%) and long-term conditions (temperature 25±2°C; relative humidity 60±5%) for 6 months. Samples were taken at the end of the 0th, 3rd and 6th months, and sirolimus and its impurities, moisture, content, etc. were determined. The results are shown in Tables 1 and 2.

[0138] Table 1 Stability test results under accelerated conditions

[0139]

[0140] Table 2 Stability test results under long-term conditions

[0141]

[0142] It can be seen from Table 1 and Table 2 that the impurity G in the sirolimus API shows a significant increasing trend under both accelerated and long-term conditions. Therefore, the analysis and detection of impurity G and its content are of great practical significance for the quality monitoring of APIs.

[0143] Experimental Example 3: Toxicology Study

[0144] Sirolimus impurity G was administered orally or intravenously to ICR mice weighing 18-22 g and SD rats weighing 170-260 g, 10 mice in each group, and the gastrointestinal toxicity of the mice and rats in each group was observed.

[0145] Group 1: ICR mice were given a single oral gavage and observed for 14 consecutive days. No obvious toxic reactions were found at doses of 250 and 500 mg / kg, while a dose of 750 mg / kg caused the death of mice.

[0146] Group 2: ICR mice were given a single intravenous injection and observed for 14 consecutive days. No obvious toxic reactions were observed at 50, 100, and 150 mg / kg. However, 250 mg / kg caused obvious toxic reactions in mice and death in some animals.

[0147] Group 3: SD rats were given a single oral gavage and observed for 14 consecutive days. No death occurred in the animals in the 200, 400, and 800 mg / kg groups, and no obvious toxic reactions were observed in the animals in the 200 and 400 mg / kg groups. The animals in the 800 mg / kg group had mild toxic reactions, but they quickly returned to normal.

[0148] Group 4: SD rats were given a single intravenous injection and observed for 14 consecutive days. No obvious toxic reactions were observed in the animals in the 50, 100, and 150 mg / kg groups, while obvious toxic reactions were observed in the animals in the 250 mg / kg group and some animals died.

[0149] Group 5: 28-day repeated administration toxicity test in SD rats, the NOAEL (no toxic effect dose) was found to be 10 mg / kg.

[0150] Group 6: The results of Ames test, chromosome aberration test and mouse in vivo micronucleus test were all negative.

[0151] Experimental Example 4: System Suitability Test

[0152] (1) Accurately weigh 25.47 mg and 25.64 mg of sirolimus reference substance, respectively, and place them in 25 mL volumetric flasks, dissolve them with 10 mL of ethanol by ultrasonication, dilute to the mark, and prepare solutions containing about 1.0 mg of sirolimus per 1 mL, which are sirolimus reference substance solutions 1 and 2, respectively. Take sirolimus reference substance solution 1, inject 5 injections continuously, and calculate the RSD of the retention time and peak area of ​​the main peak sirolimus in the chromatogram. Take sirolimus reference substance solution 2, inject 2 injections continuously, and calculate the average value of the sirolimus peak area in the chromatogram.

[0153] (2) Accurately weigh 10 mg of sirolimus API and place it in a 10 mL volumetric flask. Accurately add 500 μL of the impurity G reference substance stock solution, dilute to the mark with diluent, and shake well to obtain the system suitability solution.

[0154] (3) Accurately measure the above solution, measure it according to the above chromatographic conditions, and record the HPLC chromatogram.

[0155] (4) System suitability results

[0156] In sirolimus reference solution 1 and 2, the RSDs of the retention time and peak area of ​​the main peak of sirolimus were 0.23% and 0.076%, respectively, and the ratio of the response factors of the two reference solutions was 0.9956.

[0157] The RSDs of the peak areas of sirolimus and impurity G were 0.064% and 1.78%, respectively. The RSDs of the retention times were 0.29% and 0.17%, respectively. The separation degrees of the main peak and adjacent impurities were greater than 1.5, and the separation degrees of each known impurity and adjacent impurities were greater than 1.5.

[0158] Experimental Example 5: Accuracy Test

[0159] (1) Test method

[0160] Accurately weigh 249.59 mg of sirolimus reference substance, place it in a 50 mL volumetric flask, dissolve it with ultrasonic diluent and dilute it to the scale to prepare a reference substance stock solution containing 4.9668 mg of sirolimus per 1 mL.

[0161] Take 0.7mL, 1mL and 1.3mL of sirolimus reference stock solution in turn and place them in 10mL volumetric flasks respectively, dilute to the scale with diluent to prepare 70%, 100% and 130% accuracy solutions, and prepare 3 parallel copies of each.

[0162] Take the sirolimus reference solution and three portions of accuracy solution at each concentration respectively, perform sampling and testing according to the law, and calculate the recovery rate at each concentration.

[0163] Impurity reference substance stock solution: Accurately measure 5 mL of impurity G reference substance stock solution in sequence and place it in the same 50 mL volumetric flask, dilute to the scale with diluent, shake well, and you will get the 2-fold limit reference substance stock solution.

[0164] Impurity reference solution: Accurately measure 2.5 mL of the impurity reference stock solution into a 5 mL volumetric flask and dilute to the mark with diluent.

[0165] 50% accuracy solution: Accurately measure 1.25mL of the reference substance stock solution into a 5mL volumetric flask, and dilute to the mark with diluent. Prepare 3 portions in the same way.

[0166] 100% accuracy solution: Accurately measure 2.5mL of reference stock solution into a 5mL volumetric flask and dilute to the mark with diluent. Prepare 3 portions in the same way.

[0167] 150% accuracy solution: Accurately measure 3.75mL of the reference stock solution into a 5mL volumetric flask, and dilute to the mark with diluent. Prepare 3 portions in the same way.

[0168] Accurately measure 10 μl of each of the blank solution, reference solution and three accuracy solutions under each concentration item, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0169] (2) Acceptable standards

[0170] The single and average recoveries of sirolimus in 9 test solutions at each concentration were between 98% and 101%, with RSD ≤ 2%.

[0171] The single value and average value of the recovery rate of 9 accuracy solutions of impurity G should be between 90% and 108%, and the RSD of the 9 results should not exceed 10%.

[0172] (3) Results

[0173] The recovery of sirolimus was 98.90% to 100.20%, and the average recovery and RSD values ​​were 99.53% and 0.44%, respectively;

[0174] The recovery rate of sirolimus impurity G was 97.08% to 98.46%, and the average recovery rate and RSD were 97.63% and 0.46%, respectively; Experimental Example 6: Repeatability test

[0175] (1) Repeatable test operation

[0176] On the first day, experimenter A precisely pipetted 2 mL of sirolimus stock solution and 0.24 mL of impurity G reference stock solution into a 10 mL volumetric flask, diluted to the mark with diluent, shook well, and prepared 6 portions in parallel. Take the blank solution, sirolimus reference solution, and 6 portions of the test solution, measure according to the law, and record the chromatogram. Calculate the sirolimus content by the external standard method, and calculate the purity of each impurity by the area normalization method.

[0177] (2) Intermediate precision test operation

[0178] On the second day, experimenter A precisely pipetted 2 mL of sirolimus stock solution and 0.24 mL of impurity G reference stock solution into a 10 mL volumetric flask, diluted to the mark with diluent, shook well, and prepared 6 portions in parallel. Take the blank solution, sirolimus reference solution, and 6 portions of the test solution, measure according to the law, and record the chromatogram. Calculate the sirolimus content by the external standard method, and calculate the purity of each impurity by the area normalization method.

[0179] (3) Acceptable standards

[0180] The RSD of sirolimus content in the repeatability test of sirolimus test solution shall not be greater than 1.0%, and the RSD of sirolimus content in the precision test shall not be greater than 1.5%;

[0181] In the repeatability test of sirolimus test solution, the RSD of the purity of impurity G shall not be greater than 3.0% for 6 test solutions; in the precision test of 12 test solutions, the RSD of the main peak purity shall not be greater than 1.5%, and the RSD of the purity of impurity G shall not be greater than 5.0%.

[0182] (4) Test results

[0183] ① Repeatability test:

[0184] Six test solutions were prepared in parallel, and the average content and RSD of each substance were measured as follows:

[0185] Sirolimus = 0.9460 mg / mL, RSD value is 0.43%;

[0186] Impurity G = 0.51%, RSD 2.60%;

[0187] Total impurities = 4.45%, RSD 0.39%;

[0188] ②Intermediate precision test:

[0189] The average content and RSD of each substance in 12 test solutions were as follows:

[0190] Sirolimus solution:

[0191] Sirolimus = 0.9460 mg / mL, RSD value is 0.43%;

[0192] Impurity G = 0.51%, RSD 2.51%;

[0193] Total impurities = 4.42%, RSD 1.03%;

[0194] Experimental Example 7: Durability Test

[0195] (1) Test method

[0196] Under normal conditions, changing the column temperature (±2℃), flow rate (±0.1mL / min) and changing different chromatographic columns, blank, reference solution and selective test solution were injected respectively to investigate the changes in the main peak content, purity of each impurity, separation between the main peak and adjacent impurities, separation between known impurities and adjacent impurities, and RRT of each known impurity.

[0197] (2) Acceptable standards

[0198] Under each durability condition, the absolute value of the relative deviation (RD) of the sirolimus content from the normal condition shall not exceed 2%, and the absolute value of the relative deviation of the purity of impurity G shall not exceed 10%; the separation degree between the main peak and the adjacent impurities shall be greater than 1.5, and the separation degree between each impurity and the adjacent peak shall not be less than 1.2, and the relative standard deviation (RSD) of the RRT of each impurity shall not exceed 5%.

[0199] (3) Durability test results

[0200] Under each durability condition, the relative deviation absolute value (RD) of the sirolimus content and the normal condition is less than 2.0%, the relative deviation absolute value of the purity of impurity G is less than 10%; the RRT of impurity G is 0.68, and the relative standard deviation (RSD) of each impurity RRT is less than 5.0%. It meets the requirements and the method has good durability.

Claims

1. A method for analyzing and detecting sirolimus impurity G, characterized in that: The detection method adopts reverse phase high performance liquid chromatography to detect the solution to be tested, using a mixed solution of citric acid aqueous solution and tetrahydrofuran as mobile phase A and a mixed solution of acetonitrile and methanol as mobile phase B for gradient elution.

2. The detection method according to claim 1, characterized in that In the mobile phase A, the volume ratio of 2% citric acid aqueous solution to tetrahydrofuran is 35:

65.

3. The detection method according to claim 1, characterized in that In the mobile phase B, the volume ratio of acetonitrile to methanol is 24:

76.

4. The detection method according to claim 2, characterized in that The pH of the citric acid aqueous solution with a mass concentration of 2% in the mobile phase A was adjusted to 4.5 with triethylamine.

5. The detection method according to claim 1, characterized in that In the detection method, the flow rate of the mobile phase is 1.0 to 2.0 mL / min.

6. The detection method according to claim 1, characterized in that In the detection method, the chromatographic column is selected from one of ZORBAXSB-C18, YMC Triart C18, Waters Xbridge C18 or Welch XB C18.

7. The detection method according to claim 1, characterized in that In the detection method, the column temperature of the chromatographic column is 35-50°C.

8. The detection method according to claim 1, characterized in that In the detection method, the detection wavelength is 270-280 nm.

9. The detection method according to claim 1, characterized in that: In the detection method, the gradient elution step is:

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