A method for detecting impurities of azacitidine

Abstract

The present invention relates to a method for detecting azacitidine impurities, and belongs to the technical field of quality control of azacitidine. The present invention provides a method for detecting azacitidine impurities, using liquid chromatography, wherein the stationary phase is uncapped octadecylsilane bonded silica gel, the mobile phase is water-acetonitrile=(85-92):(8-15), v / v, and the ion pair reagent sodium octane sulfonate is added to the water phase. Compared with the prior art, the present detection method has the advantages of being able to simultaneously detect three azacitidine impurities, thereby saving analysis costs, high sensitivity, simple operation, separation and accuracy meeting requirements, good impurity peak symmetry, and greatly reducing the concentration of the test sample, thereby extending the tolerance and service life of the chromatographic column.

Description

Technical Field

[0001] The present invention relates to a method for detecting azacitidine impurities, and belongs to the technical field of quality control of azacitidine. Background Art

[0002] Azacitidine, also known as 5-azacytidine nucleoside and 5-azacytidine, is an organic compound with a chemical formula of C8H 12 N4O5, molecular weight is 244.205, chemical name is 1-(β-D-ribofuranosyl)-4-amino-1,3,5-triazine-2(1H)-one. Azacitidine is a cytosine analog, mainly used as an anti-tumor drug, clinically used to treat breast cancer, colon cancer, melanoma, acute myeloid leukemia, etc. Its structural formula is:

[0003]

[0004] Impurity detection is a very important link in controlling drug purity and improving drug quality. Impure injectable drugs can cause a variety of adverse drug reactions, such as allergies, drug fever, etc. Impure oral drugs may increase the incidence of adverse reactions, such as nausea, vomiting, abdominal pain, diarrhea, etc. Not only that, it may also affect the absorption of drugs, thereby affecting the efficacy of drugs. Therefore, it is particularly important to study the detection methods of drug impurities.

[0005] During the synthesis and storage of azacitidine, some impurities are inevitably generated, among which the common ones include impurity C, impurity G and impurity K with the following structures:

[0006]

[0007] Impurity C (furanosyl-3-guanyl urea) is a hydrolysis impurity produced by the degradation of azacitidine and is included in the draft quality standard for azacitidine in the United States Pharmacopeia USP43(5) In-Process Revision.

[0008] Impurity G (6-amino-5-azacytosine) is a common impurity in 5-azacytosine, the starting material for the synthesis of azacitidine. It is produced by the following route:

[0009]

[0010] Since 6-amino-5-azacytosine is the main impurity in the starting material 5-azacytosine, its solubility is similar to that of 5-azacytosine and it is not easy to remove during the refining process. The inventors have detected it in multiple batches of 5-azacytosine, indicating a residual risk. Moreover, 6-amino-5-azacytosine has extremely poor solubility and may not react completely during the protection process on HMDS. It may remain in the finished product in the form of 6-amino-5-azacytosine prototype (impurity G) or participate in the reaction to produce the derivative impurity 4,6-diamino-1-β-D-1,3,5-triazine-2(1H)-one (impurity K). The reaction process is as follows:

[0011]

[0012] Chinese patent application CN104672289A discloses the structures of impurities G and K, and mentions that when conducting quality research on azacitidine API, it is necessary to test the content of corresponding impurities in the API to ensure that the API that meets pharmaceutical standards and can be used to prepare safe and effective pharmaceutical preparations is prepared. However, the patent application does not disclose any detection method.

[0013] At present, there are few reports on the detection methods of azacitidine impurities, especially for impurity K. After searching, no research reports on its detection method have been found so far. Providing a method that can effectively detect azacitidine impurity K is of great significance for the quality control of azacitidine. The United States Pharmacopoeia USP43 (5) In-Process Revision azacitidine quality standard draft contains the detection method of azacitidine impurity C, which is as follows: Atlantis HILIC Silica 4.6*250mm, 5μm is used as the chromatographic column; acetonitrile-methanol-0.77g / L ammonium acetate buffer solution (75:20:5, v / v) is used as the mobile phase, and the mobile phase is adjusted to pH 4.0 with glacial acetic acid; the flow rate is 1.0ml per minute; the detection wavelength is 223nm; the column temperature is 30℃; the injection volume is 20μl; the test sample concentration is 50mg / ml. The above method is not suitable for the analysis and detection of impurities K and impurity G. Moreover, the inventors have found through experiments that even when the pharmacopoeia method is used to detect azacitidine impurity C, there are problems such as poor peak shape, low column efficiency, uneven baseline, and low sensitivity, which lead to inaccurate results when measuring the impurity chromatographic peak area, thereby affecting the accuracy of the impurity content detection in the test sample; in addition, the concentration of the test sample in the pharmacopoeia method is high, resulting in poor chromatographic column tolerance and large chromatographic column loss. Summary of the invention

[0014] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the first object of the present invention is to provide a method for effectively detecting azacitidine impurity K. The second object of the present invention is to provide a method for simultaneously separating and detecting azacitidine impurity C, impurity G and impurity K.

[0015] The present invention provides a method for detecting impurities of azacitidine, using liquid chromatography, wherein the stationary phase is uncapped octadecylsilane bonded silica gel, the mobile phase is water phase-acetonitrile=(85-92):(8-15), v / v, and the ion pair reagent sodium octane sulfonate is added to the water phase. Under the above conditions, the impurity K of azacitidine can be accurately detected.

[0016] The stationary phase in the detection method of the present invention is uncapped octadecylsilane bonded silica gel. "Capping" means that the bonded phase filler is capped with a reagent to obtain a deactivated silica gel surface. The stationary phase filler of the present invention is not capped, and there are exposed silanol groups on its surface.

[0017] Furthermore, the mobile phase is water phase-acetonitrile=(87-90):(10-13), v / v.

[0018] Preferably, the mobile phase is water phase-acetonitrile=(87-89):(11-13), v / v. Under the above conditions, azacitidine impurity C, impurity G and impurity K can be separated and detected simultaneously.

[0019] More preferably, the mobile phase is water-acetonitrile=88:12, v / v. Under the above conditions, the retention time is appropriate and the separation is good.

[0020] Furthermore, the mobile phase is used for isocratic elution.

[0021] Furthermore, the concentration of the sodium octane sulfonate solution in the aqueous phase is 8 to 12 mmol / L.

[0022] Preferably, the concentration of the sodium octane sulfonate solution in the aqueous phase is 9 to 11 mmol / L.

[0023] More preferably, the concentration of the sodium octane sulfonate solution in the aqueous phase is 10 mmol / L.

[0024] Furthermore, the pH of the aqueous phase is adjusted to 2.5-2.7.

[0025] Preferably, the aqueous phase is adjusted to pH 2.6.

[0026] Further preferably, phosphoric acid is used to adjust the pH of the aqueous phase.

[0027] Furthermore, the stationary phase is Agilent InfinityLab Poroshell 120SB-C18 or an equivalent chromatographic column.

[0028] Preferably, the filler particle size of Agilent InfinityLab Poroshell 120SB-C18 is 1.9-4 μm.

[0029] More preferably, the filler particle size of Agilent InfinityLab Poroshell 120SB-C18 is 4 μm.

[0030] Preferably, the inner diameter of the Agilent InfinityLab Poroshell 120SB-C18 is 2.1-4.6 mm.

[0031] Further preferably, the inner diameter of the Agilent InfinityLab Poroshell 120SB-C18 is 4.6 mm.

[0032] Preferably, the length of the Agilent InfinityLab Poroshell 120SB-C18 is 50-250 mm.

[0033] Further preferably, the length of the Agilent InfinityLab Poroshell 120SB-C18 is 250 mm.

[0034] Furthermore, the detection wavelength is 205-240 nm. Within the above wavelength range, the azacitidine impurity K can be accurately detected.

[0035] Preferably, the detection wavelength is 205-215 nm.

[0036] Further preferably, the detection wavelength is 210nm. According to the inventor's experimental investigation, impurity G has maximum absorption at 230nm and near ultraviolet end, impurity K has maximum absorption at 236nm and near ultraviolet end, but impurity C has weak absorption at about 230nm and has maximum absorption at near ultraviolet end. The detection wavelength adopted by the USP method is 223nm, where impurity C, impurity G and impurity K absorb weakly. The present invention preferably uses 210nm as the detection wavelength, thereby ensuring that impurity C, impurity G and impurity K all have higher detection sensitivity, which is conducive to the simultaneous detection of the above three impurities.

[0037] Furthermore, the flow rate of the mobile phase is 0.9-1.1 ml / min.

[0038] Preferably, the mobile phase flow rate is 1.0 ml / min.

[0039] Furthermore, the column temperature is 15-30°C.

[0040] Preferably, the column temperature is 18-22° C. After investigating the column temperature, it was found that compared with the condition of 30° C. adopted by the United States Pharmacopoeia, the column temperature was reduced to 18-22° C. At this time, the chromatogram baseline was smoother, the sensitivity was improved, and the detection result was more accurate.

[0041] More preferably, the column temperature is 20°C.

[0042] Furthermore, the impurity is an impurity K having the structure shown below:

[0043]

[0044] Preferably, the impurities further include at least one of impurity G and impurity C having the following structure:

[0045]

[0046] Furthermore, the concentration of the prepared test solution is 5 to 20 mg / ml. Compared with the United States Pharmacopoeia method, the present invention reduces the test solution concentration, improves the tolerance of the chromatographic column, and prolongs the service life of the chromatographic column by optimizing the conditions.

[0047] Preferably, the concentration of the test solution is prepared to be 5 to 15 mg / ml.

[0048] More preferably, the concentration of the test solution is prepared to be 10 mg / ml.

[0049] Furthermore, the concentration of impurity C in the prepared reference solution is 0.015-0.045 mg / ml.

[0050] Preferably, the concentration of impurity C in the prepared reference solution is 0.03 mg / ml.

[0051] Furthermore, the concentration of impurity G in the prepared reference solution is 0.0075-0.0225 mg / ml.

[0052] Preferably, the concentration of impurity G in the prepared reference solution is 0.015 mg / ml.

[0053] Furthermore, the concentration of impurity K in the prepared reference solution is 0.0075-0.0225 mg / ml.

[0054] Preferably, the concentration of impurity K in the prepared reference solution is 0.015 mg / ml.

[0055] Furthermore, the injection volume is 5 to 20 μL.

[0056] Preferably, the injection volume is 5 to 15 μL.

[0057] More preferably, the injection volume is 10 μL.

[0058] Furthermore, the diluent is dimethyl sulfoxide.

[0059] The present invention preferably adopts the external standard method to calculate the impurity peak content by peak area.

[0060] The present invention provides a method for detecting azacitidine impurities, which has at least the following beneficial technical effects:

[0061] 1. The detection method of the present invention can accurately detect the impurity K of azacitidine, filling the gap in the detection method of impurity K in the prior art, and is conducive to more comprehensive quality control of azacitidine.

[0062] 2. The present invention can simultaneously separate and detect azacitidine impurity C, impurity G and impurity K, thereby improving the efficiency of quality control and saving analysis costs.

[0063] 3. The method of the present invention is used to separate and detect azacitidine impurities C, G and K, and the separation and accuracy meet the requirements. Compared with the detection method of the United States Pharmacopoeia, the theoretical plate number of each impurity is greatly improved, and the column efficiency is higher. In addition, the peak shape of the impurity is improved and the sensitivity is increased. The peak shape of the spectrum obtained by the detection method of the present invention is sharp and symmetrical, and the baseline is smoother, so that the results of quantitative analysis of impurities are more accurate.

[0064] 4. Compared with the prior art, the detection method of the present invention greatly reduces the concentration of the test sample, improves the tolerance of the chromatographic column, and extends the service life of the chromatographic column.

[0065] 5. The liquid chromatography method of the present invention adopts an isocratic elution method to separate and detect azacitidine-related substances, which is simpler to operate than the prior art which adopts a gradient elution method. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 This is a chromatogram showing the location of picric acid in Example 1;

[0067] Figure 2 The chromatogram is a chromatogram showing the location of impurity C in Example 1;

[0068] Figure 3 The chromatogram is a chromatogram showing the location of impurity G in Example 1;

[0069] Figure 4 is a chromatogram for locating impurity K in Example 1;

[0070] Figure 5 The chromatogram of the test sample spiked solution in Example 1;

[0071] Figure 6 is a linear relationship diagram of impurity C in Example 4;

[0072] Figure 7 is a linear relationship diagram of impurity G in Example 4;

[0073] Figure 8 is a linear relationship diagram of impurity K in Example 4;

[0074] Fig. 9 The chromatogram of the test sample spiked solution in Example 9;

[0075] Fig.10 is the chromatogram of the test sample spiked solution in Example 10;

[0076] Fig.11 The chromatogram is a chromatogram showing the location of impurity C in Comparative Example 1;

[0077] Fig.12 The chromatogram is a chromatogram showing the location of impurity G in Comparative Example 1;

[0078] Fig.13 The chromatogram is a chromatogram showing the location of impurity K in Comparative Example 1;

[0079] Fig.14 The chromatogram of the test sample spiked solution in Comparative Example 1;

[0080] Fig.15 The chromatogram of the spiked solution of the test sample in Comparative Example 2;

[0081] Fig.16 The chromatogram of the test sample spiked solution in Comparative Example 3;

[0082] Fig.17 The chromatogram of the spiked solution of the test sample in Comparative Example 4;

[0083] Fig.18 The chromatogram of the spiked solution of the test sample in Comparative Example 5;

[0084] Fig.19 The chromatogram of the spiked solution of the test sample in Comparative Example 6;

[0085] Fig. 20 The chromatogram of the spiked solution of the test sample in Comparative Example 7;

[0086] Fig.21 The chromatogram of the spiked solution of the test sample in Comparative Example 8;

[0087] Fig. 22 The chromatogram is a chromatogram showing the location of impurity C in Comparative Example 9;

[0088] Fig.23 The chromatogram is a chromatogram showing the location of impurity G in Comparative Example 9;

[0089] Fig.24 The chromatogram is a chromatogram showing the location of impurity K in Comparative Example 9;

[0090] Fig.25 This is the chromatogram of the test sample spiked solution in Comparative Example 9. DETAILED DESCRIPTION

[0091] The scheme of the present invention will be further described below by specific implementation methods and experiments. It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0092] Since impurity C (furanose-3-guanyl urea) is unstable, it is usually prepared as a picric acid salt solution when used as a reference substance. The structure is shown below:

[0093]

[0094] The preparation method of the impurity C picrate is as follows: weigh about 15g of crude azacitidine and add it to a reaction bottle, then add 100ml of anhydrous ethanol, 100ml of water and 2g of sodium hydroxide, stir and react completely, adjust the pH to 6-7 with dilute hydrochloric acid, and concentrate to dryness. Add 150ml of methanol, stir at room temperature to dissolve it, filter, weigh about 16g of picric acid, add 200ml of anhydrous ethanol and 50ml of methanol, stir and dissolve to clarify, add to the above methanol solution, and a yellow solid begins to precipitate. Stir at room temperature for 30 minutes and then filter, wash the filter cake with anhydrous ethanol, and air dry at 40°C to obtain the impurity C picrate.

[0095] Impurity G: purchased from Shenzhen Feisi Biotechnology Co., Ltd., 12-DEC-19-15.

[0096] Impurity K: purchased from Shenzhen Zhenqiang Biotechnology Co., Ltd., A0390291.

[0097] Example 1: Using the method of the present invention to simultaneously separate and detect the three impurities C, G, and K of azacitidine

[0098] The chromatographic detection conditions are as follows:

[0099] Chromatographic column: an uncapped octadecylsilane bonded silica gel chromatographic column is used. In this embodiment, Agilent InfinityLab Poroshell 120SB-C18 is specifically selected, and the specific specifications are 4.6×250 mm, 4 μm;

[0100] Mobile phase: 10 mmol / L sodium octane sulfonate aqueous solution (pH adjusted to 2.6 with phosphoric acid)-acetonitrile (88:12, v / v);

[0101] Flow rate: 1ml / min;

[0102] Column temperature: 20°C;

[0103] Detection wavelength: 210nm;

[0104] Injection volume: 10 μl;

[0105] Test sample concentration: 10 mg / ml.

[0106] Blank solvent / diluent: DMSO.

[0107] Preparation of reference solution: Take appropriate amount of impurity C picrate, impurity G and impurity K reference substances respectively, weigh accurately, add diluent to dissolve and quantitatively dilute to make a solution containing approximately 0.03 mg of impurity C, 0.015 mg of impurity G and 0.015 mg of impurity K per 1 ml.

[0108] Preparation of test sample spiked solution: Take appropriate amount of impurity C picrate, impurity G and impurity K reference substances and azacitidine, accurately weigh them, dissolve them in diluent and quantitatively dilute them to make a solution containing approximately 10 mg of azacitidine, 0.03 mg of impurity C, 0.015 mg each of impurity G and impurity K per 1 ml.

[0109] Preparation of picric acid solution: Take an appropriate amount of picric acid, add dimethyl sulfoxide to dissolve it and quantitatively dilute it to make a solution containing about 0.1 mg of picric acid per 1 ml.

[0110] The chromatograms of picric acid solution, reference solution and test solution spiked with the sample are shown in Figure 1 to Figure 5 .

[0111] Depend on Figure 1 to Figure 5 It can be seen that the present invention can achieve the separation and detection of azacitidine impurities C, G and K. Compared with the detection method using the United States Pharmacopoeia (see Comparative Example 1), the chromatogram baseline obtained in this embodiment is smoother and the detection sensitivity is higher; the theoretical plate numbers of impurities C, G and K are greatly improved, and the column efficiency is higher; the separation of each impurity is greater than 1.5, especially the separation of impurities G and K is significantly improved compared with the United States Pharmacopoeia method, and the requirements for accurate detection can be met; the tailing factors of impurities C, G and K are between 1.0 and 1.2, and the tailing factor of impurity C is significantly lower than that of the United States Pharmacopoeia method, and the peak shape is more symmetrical.

[0112] The above results all indicate that the method of the present invention has a better separation effect on azacitidine impurity C, impurity G and impurity K, and the obtained detection results are more accurate. Moreover, compared with the United States Pharmacopoeia method, the detection method of the present invention has a higher sensitivity. For example, impurity C can be accurately detected at a low concentration level of 0.03 mg / ml, while the United States Pharmacopoeia method requires detection at a higher concentration of 0.15 mg / ml; based on this, the present invention can significantly reduce the concentration of the test solution, avoid irreversible damage to the chromatographic column, and help reduce the loss of the chromatographic column and extend its service life.

[0113] Example 2 Specificity and system applicability of the detection method of the present invention

[0114] The chromatographic detection conditions and blank solvent / diluent were the same as those in Example 1.

[0115] Preparation of test solution: Take an appropriate amount of azacitidine, weigh accurately, add diluent to dissolve and quantitatively dilute to make a solution containing approximately 10 mg per 1 ml.

[0116] Preparation of system suitability solution: Take appropriate amount of azacitidine and impurity C picrate, impurity G and impurity K reference substances, accurately weigh, add diluent to dissolve and quantitatively dilute to make a solution containing 10 mg of azacitidine, 0.03 mg of impurity C, 0.015 mg each of impurity G and impurity K per 1 ml.

[0117] Prepare a single localization solution: Take appropriate amount of impurities G and K respectively, add diluent to dilute to make a solution containing about 0.015 mg of each impurity per 1 ml, as the localization solution of impurities G and K. Take appropriate amount of impurity C picrate, add diluent to dilute to make a solution containing about 0.03 mg of impurity C per 1 ml, as the localization solution of impurity C.

[0118] The test results are shown in Table 1.

[0119] Table 1 Specificity and system suitability test results

[0120] name API (azacitidine) Impurity C Impurity G Impurity K Minimum separation Blank solvent interference no no no no N / A <![CDATA[RT 单个定位 ]]> 6.804 14.908 16.033 21.171 N / A <![CDATA[RT 系统溶液 ]]> 6.738 14.837 15.976 21.103 1.76 <![CDATA[RT 供试品溶液 ]]> 6.804 14.841 N / A N / A 1.76

[0121] From the above test results, it can be seen that each impurity is clearly located, and the blank solvent does not interfere with the detection of impurities C, impurity G and impurity K; the retention time of the impurities in the single impurity location and system suitability solution is basically consistent; the minimum separation of each impurity meets the requirements (separation ≥1.5), indicating that the method system has good applicability.

[0122] Example 3 Quantitative Limit and Detection Limit of the Detection Method of the Present Invention

[0123] The chromatographic detection conditions and blank solvent / diluent are the same as those in Example 1.

[0124] Take appropriate amounts of impurity C picrate, impurity G and impurity K reference substances, dissolve and dilute them in DMSO to make solutions of certain concentrations, and dilute them step by step. The signal-to-noise ratio is about 10 to 30 as the quantitative limit, and the signal-to-noise ratio is about 3 to 10 as the detection limit. The results of the quantitative limit and the detection limit are shown in Table 2 and Table 3, respectively.

[0125] Table 2 Quantitation limit results

[0126]

[0127] Table 3 Detection limit results

[0128]

[0129] From the results of the limit of quantification and the limit of detection, it can be seen that the signal-to-noise ratios of the limit of quantification solutions of 6 parallel samples of impurity C, impurity G, and impurity K are all greater than 10; the signal-to-noise ratios of the limit of detection solutions of 6 parallel samples of impurity C, impurity G, and impurity K are all greater than 3. The limit of quantification of impurity C is 0.003% of the concentration of the test sample, and the limit of detection is 0.002% of the concentration of the test sample; the limit of quantification of impurity G is 0.006% of the concentration of the test sample, and the limit of detection is 0.003% of the concentration of the test sample; the limit of quantification of impurity K is 0.006% of the concentration of the test sample, and the limit of detection is 0.003% of the concentration of the test sample; the detection method of the present invention adopts a test sample concentration of 10 mg / ml and an injection volume of 10 μl under conventional conditions, has high sensitivity, and can meet the requirements of accurate detection.

[0130] Example 4 Linearity and range of the detection method of the present invention

[0131] The chromatographic detection conditions and blank solvent / diluent are the same as those in Example 1.

[0132] Take appropriate amount of impurity C picrate, impurity G, and impurity K reference substances, dissolve them in DMSO and dilute them to prepare a certain concentration of stock solution, dilute the stock solution to prepare a series of linear solutions with different concentrations, record the chromatogram, perform linear regression on the concentration with the peak area of ​​each component, and calculate the correlation coefficient r and y-axis intercept to evaluate the linearity of each component in this method. The detection results of impurity C, impurity G, and impurity K are shown in Table 4, Table 5, and Table 6, respectively, and the linear relationship diagrams are shown in Table 6. Figure 6 , Figure 7 , Figure 8 .

[0133] Table 4 Impurity C linearity results

[0134]

[0135] Table 5 Impurity G linear results

[0136]

[0137]

[0138] Table 6 Impurity K linear results

[0139]

[0140] From the results in Table 4, Table 5 and Table 6, it can be seen that the y-axis intercepts of the linear regression equations of impurity C, impurity G and impurity K account for less than 25% of the limit concentration peak area, and the linear relationship of this method is good.

[0141] Example 5 Repeatability of the detection method of the present invention

[0142] The chromatographic detection conditions and blank solvent / diluent were the same as those in Example 1.

[0143] Prepare the test sample spike solution: Take appropriate amounts of impurity C picrate, impurity G and impurity K reference substances to make stock solutions, add them to the azacitidine test sample to make a solution containing approximately 10 mg of azacitidine, 0.03 mg of impurity C, 0.015 mg each of impurity G and impurity K per 1 ml of sample. Prepare 6 test sample spike solutions in the same way.

[0144] The test results are shown in Table 7.

[0145] Table 7 Repeatability test results

[0146]

[0147]

[0148] From the above test results, it can be seen that the RSDs of the impurity C, impurity G, and impurity K contents in the 6 reproducible solutions are 0.84%, 0.82%, and 1.04%, respectively, and the RSDs of the impurity contents are all <5%, so the detection method of the present invention has good repeatability.

[0149] Example 6 Accuracy of the detection method of the present invention

[0150] The chromatographic detection conditions and blank solvent / diluent are the same as those in Example 1.

[0151] Take appropriate amounts of impurity C picrate, impurity G, and impurity K reference substances to prepare stock solutions, and add them to the azacitidine sample at limit concentrations of 50%, 100%, and 150%, respectively, to prepare solutions containing 10 mg of azacitidine, 0.015 mg, 0.03 mg, and 0.045 mg of impurity C, and 0.0075 mg, 0.015 mg, and 0.0225 mg of impurity G and impurity K, respectively, per 1 ml of sample.

[0152] The accuracy test results of azacitidine impurity C, impurity G, and impurity K are shown in Table 8, Table 9, and Table 10, respectively.

[0153] Table 8 Impurity C Accuracy Results

[0154]

[0155] Table 9 Impurity G Accuracy Results

[0156]

[0157]

[0158] Table 10 Impurity K Accuracy Results

[0159]

[0160] From the above accuracy test results, it can be seen that the average recovery rates of 9 solutions of each concentration of impurity C, impurity G, and impurity K are all within the range of 90% to 108%, and the recovery RSDs are 0.58%, 1.99%, and 0.52%, respectively, all less than 10%, which meets the requirements. This shows that the accuracy of this method is good.

[0161] Example 7 Durability of the detection method of the present invention

[0162] In this example, the flow rate and column temperature were fine-tuned to examine the durability of the method. The durability examination conditions are shown in Table 11.

[0163] The preparation process of blank solvent / diluent and reference solution is as in Example 1.

[0164] The system suitability solution was prepared as described in Example 2.

[0165] Blank solvent, system suitability solution, and reference solution were sampled and tested under the conditions described in Table 11 to examine the separation and detection of impurities under each condition. The results are shown in Table 12.

[0166] Table 11 Durability inspection conditions

[0167]

[0168]

[0169] Table 12 Durability test results

[0170]

[0171] From the results in Table 12, it can be seen that under each durability test condition, each impurity is well separated, and the minimum separation degree is >1.5; the blank solvent does not interfere with the detection of impurities C, G and K in azacitidine; under each test condition, compared with the original conditions, the absolute difference in impurity content is within ±0.03%. Therefore, this method has good durability.

[0172] Example 8 Solution stability of the detection method of the present invention

[0173] The preparation of the reference solution is as in Example 1.

[0174] The preparation of the test solution is as in Example 2.

[0175] The test solution and the reference solution were placed at room temperature, respectively, and the samples were injected and analyzed at different time points. The chromatograms were recorded to examine the stability of the solutions. The stability results of the reference solution and the test solution are shown in Table 13 and Table 14, respectively.

[0176] Table 13 Reference solution stability results

[0177]

[0178]

[0179] Table 14 Stability results of test solution

[0180]

[0181] From the above stability results, it can be seen that within 16 hours, the peak area ratio change rate of each measured peak in the reference solution is compared with the peak area at 0 hour, which is within ±10%, and the solution is stable; only impurity C is detected in the test solution, and the absolute difference of impurity C content compared with 0 hour is within ±0.03%, and the solution is stable; the reference solution and the test solution are stable within 16 hours.

[0182] Example 9 Detection of azacitidine impurity K using the method of the present invention

[0183] The chromatographic detection conditions are as follows:

[0184] Column: Agilent InfinityLab Poroshell 120SB-C18, 4.6×250 mm, 4 μm;

[0185] Mobile phase: 10 mmol / L sodium octane sulfonate aqueous solution (pH adjusted to 2.6 with phosphoric acid)-acetonitrile (90:10, v / v);

[0186] Flow rate: 1ml / min;

[0187] Column temperature: 30°C;

[0188] Detection wavelength: 210nm;

[0189] Injection volume: 10 μl;

[0190] Test sample concentration: 10 mg / ml.

[0191] The blank solvent / diluent, picric acid solution and reference solution were prepared as described in Example 1.

[0192] Preparation of test sample spiked solution: Take appropriate amount of impurity C picrate, impurity G and impurity K reference substances and azacitidine, accurately weigh them, dissolve them in diluent and quantitatively dilute them to make a solution containing approximately 10 mg of azacitidine, 0.15 mg of impurity C, 0.02 mg each of impurity G and impurity K per 1 ml.

[0193] The chromatogram obtained from the spiked solution of the test sample is shown in Fig. 9 .

[0194] It can be seen that by adjusting the mobile phase ratio and column temperature based on the detection conditions of Example 1, the tailing factor of azacitidine impurity K is further reduced to 1.03, the peak shape is more symmetrical, the theoretical plate number is increased to 28459, and the column efficiency is higher, indicating that accurate detection of azacitidine impurity K can be achieved under the detection conditions of this example.

[0195] Example 10 Detection of azacitidine impurity K using the method of the present invention

[0196] The chromatographic detection conditions are as follows:

[0197] Column: Agilent InfinityLab Poroshell 120SB-C18, 4.6×250 mm, 4 μm;

[0198] Mobile phase: 10 mmol / L sodium octane sulfonate aqueous solution (pH adjusted to 2.6 with phosphoric acid)-acetonitrile (90:10, v / v);

[0199] Flow rate: 1ml / min;

[0200] Column temperature: 20°C;

[0201] Detection wavelength: 210nm;

[0202] Injection volume: 10 μl;

[0203] Test sample concentration: 10 mg / ml.

[0204] The blank solvent / diluent, picric acid solution and reference solution were prepared as described in Example 1.

[0205] Preparation of test sample spiked solution: Take appropriate amount of impurity C picrate, impurity G and impurity K reference substances and azacitidine, accurately weigh them, dissolve them in diluent and quantitatively dilute them to make a solution containing approximately 10 mg of azacitidine, 0.15 mg of impurity C, 0.02 mg each of impurity G and impurity K per 1 ml.

[0206] The chromatogram obtained from the spiked solution of the test sample is shown in Fig.10.

[0207] It can be seen that by adjusting the column temperature based on the detection conditions of Example 9, the baseline smoothness is improved, which is beneficial to improving the sensitivity and accuracy of the impurity K detection results, indicating that accurate detection of azacitidine impurity K can be achieved under the detection conditions of this example.

[0208] Comparative Example 1

[0209] Referring to the method described in the United States Pharmacopeia USP43(5) In-Process Revision, the chromatographic detection conditions are as follows:

[0210] Chromatographic column: Atlantis HILIC Silica 4.6*250mm, 5μm;

[0211] Mobile phase: acetonitrile-methanol-0.77 g / L ammonium acetate buffered saline solution (pH adjusted to 4.0 with glacial acetic acid) (75:20:5, v / v);

[0212] Flow rate: 1ml / min;

[0213] Column temperature: 30°C;

[0214] Detection wavelength: 223nm;

[0215] Injection volume: 20 μl;

[0216] Test sample concentration: 50 mg / ml.

[0217] The blank solvent / diluent, picric acid solution and reference solution were prepared as described in Example 1.

[0218] Preparation of test sample spiked solution: Take appropriate amount of impurity C picrate, impurity G and impurity K reference substances and azacitidine, accurately weigh, dissolve in diluent and quantitatively dilute to make a solution containing approximately 50 mg of azacitidine, 0.15 mg of impurity C, 0.1 mg each of impurity G and impurity K per 1 ml.

[0219] The chromatograms obtained from the reference solution and the test solution spiked with the test sample are shown in Figure 11 to Figure 14 .

[0220] Depend on Figure 11 to Figure 14It can be seen that the impurity C, impurity G and impurity K of azacitidine are detected by the method of the United States Pharmacopoeia USP43. The blank solvent (DMSO) and impurity K, impurity G and main component azacitidine peak at the same position and cannot be effectively separated; although the peaks of impurity C and API are separated, the impurity C peak is bifurcated, and the peak shape is asymmetric, its tailing factor is 3.03, the theoretical plate number is only 2807, and the column efficiency is low. Under this detection condition, due to the high concentration of the test sample, the baseline is not smooth, which will affect the accuracy of the results when the impurities are quantitatively analyzed. The above results all show that the impurity C, impurity G and impurity K of azacitidine cannot be accurately detected by this method. Moreover, it was found in the experiment that due to the high concentration of the test sample used, the chromatographic column had poor tolerance and large chromatographic column loss. When the inventor used the chromatographic column to detect other samples (the chromatographic column had been used to detect the content of impurity C in azacitidine using the method of the United States Pharmacopoeia), the peaks of other samples were bifurcated, indicating that the chromatographic column has caused irreversible damage, which will affect the detection of other samples.

[0221] Comparative Example 2

[0222] Based on the detection conditions of Comparative Example 1, the pH value of the ammonium acetate buffered saline solution was adjusted (from pH 4.0 to pH 3.0), and the chromatographic detection conditions were as follows:

[0223] Chromatographic column: Atlantis HILIC Silica 4.6*250mm, 5μm;

[0224] Mobile phase: acetonitrile-methanol-0.77 g / L ammonium acetate buffered saline solution (pH adjusted to 3.0 with glacial acetic acid) (75:20:5, v / v);

[0225] Flow rate: 1ml / min;

[0226] Column temperature: 30°C;

[0227] Detection wavelength: 223nm;

[0228] Injection volume: 20 μl;

[0229] Test sample concentration: 50 mg / ml.

[0230] The blank solvent / diluent, picric acid solution and reference solution were prepared as described in Example 1.

[0231] Preparation of test sample spiked solution: Take appropriate amount of impurity C picrate, impurity G and impurity K reference substances and azacitidine, accurately weigh, dissolve in diluent and quantitatively dilute to make a solution containing approximately 50 mg of azacitidine, 0.15 mg of impurity C, 0.1 mg each of impurity G and impurity K per 1 ml.

[0232] The chromatogram obtained from the spiked solution of the test sample is shown in Fig.15 .

[0233] Depend on Fig.15 It can be seen that, although the peak shape of impurity C was improved by optimizing pH based on the detection conditions in Comparative Example 1, the peaks of impurities K, G and API still completely overlapped and could not be effectively separated. At the same time, similar to the situation in Comparative Example 1, due to the high concentration of the test sample used, the chromatographic column tolerance was poor and the chromatographic column loss was large.

[0234] Comparative Example 3

[0235] Based on the detection conditions of Comparative Example 1, the pH value of the ammonium acetate buffered saline solution was adjusted (from pH 4.0 to pH 6.5), and the chromatographic detection conditions were as follows:

[0236] Chromatographic column: Atlantis HILIC Silica 4.6*250mm, 5μm;

[0237] Mobile phase: acetonitrile-methanol-0.77 g / L ammonium acetate buffered saline solution (pH adjusted to 6.5 with glacial acetic acid) (75:20:5, v / v);

[0238] Flow rate: 1ml / min;

[0239] Column temperature: 30°C;

[0240] Detection wavelength: 223nm;

[0241] Injection volume: 20 μl;

[0242] Test sample concentration: 50 mg / ml.

[0243] The blank solvent / diluent, picric acid solution and reference solution were prepared as described in Example 1.

[0244] Preparation of test sample spiked solution: Take appropriate amount of impurity C picrate, impurity G and impurity K reference substances and azacitidine, accurately weigh, dissolve in diluent and quantitatively dilute to make a solution containing approximately 50 mg of azacitidine, 0.15 mg of impurity C, 0.1 mg each of impurity G and impurity K per 1 ml.

[0245] The chromatogram obtained from the spiked solution of the test sample is shown in Fig.16 .

[0246] Depend on Fig.16It can be seen that based on the detection conditions of Comparative Example 1, the pH was adjusted to 6.5, and the peak of impurity C was still bifurcated, resulting in an asymmetric peak shape, thereby affecting the quantitative detection of impurities; impurity K, impurity G and the main component azacitidine still peaked at the same position and could not be effectively separated, and there was also the problem of poor chromatographic column tolerance and large chromatographic column loss due to the high concentration of the test sample used.

[0247] Comparative Example 4

[0248] Based on the detection conditions of Comparative Example 1, the pH value of the ammonium acetate buffer saline solution (from pH 4.0 to pH 3.0) and the mobile phase ratio (from 75:20:5 to 80:15:5) were adjusted, and the chromatographic detection conditions were as follows:

[0249] Chromatographic column: Atlantis HILIC Silica 4.6*250mm, 5μm;

[0250] Mobile phase: acetonitrile-methanol-0.77 g / L ammonium acetate buffered saline solution (pH adjusted to 3.0 with glacial acetic acid) (80:15:5, v / v);

[0251] Flow rate: 1ml / min;

[0252] Column temperature: 30°C;

[0253] Detection wavelength: 223nm;

[0254] Injection volume: 20 μl;

[0255] Test sample concentration: 50 mg / ml.

[0256] The blank solvent / diluent, picric acid solution and reference solution were prepared as described in Example 1.

[0257] Preparation of test sample spiked solution: Take appropriate amount of impurity C picrate, impurity G and impurity K reference substances and azacitidine, accurately weigh, dissolve in diluent and quantitatively dilute to make a solution containing approximately 50 mg of azacitidine, 0.15 mg of impurity C, 0.1 mg each of impurity G and impurity K per 1 ml.

[0258] The chromatogram obtained from the spiked solution of the test sample is shown in Fig.17 .

[0259] Depend on Fig.17It can be seen that on the basis of the detection conditions of Comparative Example 1, the pH value and the mobile phase ratio of the ammonium acetate buffer saline aqueous solution were adjusted, and the peaks of impurities C and impurity G and API were effectively separated, but the peaks of impurity K and API completely overlapped and could not be effectively separated; and the peak of impurity G was forked and the peak shape was asymmetric; due to the high concentration of the test sample used, the chromatographic column tolerance was poor and the chromatographic column loss was large.

[0260] Comparative Example 5

[0261] On the basis of the detection conditions of Comparative Example 4, the chromatographic column type was changed for the experiment (adjusted from Atlantis HILICSilica 4.6*250mm, 5μm to Agilent ZORBAX BONUS-RP, 4.6*250mm, 5μm), and the mobile phase ratio was adjusted (from 80:15:5→5:25:70) in combination with the use requirements of the chromatographic column. The chromatographic detection conditions are as follows:

[0262] Chromatographic column: Agilent ZORBAX BONUS-RP 4.6*250mm, 5μm;

[0263] Mobile phase: acetonitrile-methanol-0.77 g / L ammonium acetate buffered saline solution (pH adjusted to 3.0 with glacial acetic acid) (5:25:70, v / v);

[0264] Flow rate: 1ml / min;

[0265] Column temperature: 30°C;

[0266] Detection wavelength: 223nm;

[0267] Injection volume: 20 μl;

[0268] Test sample concentration: 50 mg / ml.

[0269] The blank solvent / diluent, picric acid solution and reference solution were prepared as described in Example 1.

[0270] Preparation of test sample spiked solution: Take appropriate amount of impurity C picrate, impurity G and impurity K reference substances and azacitidine, accurately weigh, dissolve in diluent and quantitatively dilute to make a solution containing approximately 50 mg of azacitidine, 0.15 mg of impurity C, 0.1 mg each of impurity G and impurity K per 1 ml.

[0271] The chromatogram obtained from the spiked solution of the test sample is shown in Fig.18 .

[0272] Depend on Fig.18It can be seen that azacitidine and its impurities C, G and K are not retained under this chromatographic condition, and all peak before 4 minutes, making them impossible to separate and detect.

[0273] Comparative Example 6

[0274] Based on the detection conditions of Comparative Example 5, the mobile phase ratio was adjusted (from 5:25:70→5:5:90), and the chromatographic detection conditions were as follows:

[0275] Chromatographic column: Agilent ZORBAX BONUS-RP 4.6*250mm, 5μm;

[0276] Mobile phase: acetonitrile-methanol-0.77 g / L ammonium acetate buffered saline solution (pH adjusted to 3.0 with glacial acetic acid) (5:5:90, v / v);

[0277] Flow rate: 1ml / min;

[0278] Column temperature: 30°C;

[0279] Detection wavelength: 223nm;

[0280] Injection volume: 20 μl;

[0281] Test sample concentration: 50 mg / ml.

[0282] The blank solvent / diluent, picric acid solution and reference solution were prepared as described in Example 1.

[0283] Preparation of test sample spiked solution: Take appropriate amount of impurity C picrate, impurity G and impurity K reference substances and azacitidine, accurately weigh, dissolve in diluent and quantitatively dilute to make a solution containing approximately 50 mg of azacitidine, 0.15 mg of impurity C, 0.1 mg each of impurity G and impurity K per 1 ml.

[0284] The chromatogram obtained from the spiked solution of the test sample is shown in Fig.19 .

[0285] Depend on Fig.19 It can be seen that, based on the detection conditions of Comparative Example 5, the mobile phase ratio was adjusted, but the retention of azacitidine and its impurities was not improved, and the peaks still appeared before 4 minutes, making it impossible to separate and detect them.

[0286] Comparative Example 7

[0287] In order to improve the sensitivity of the target impurities to be separated and reduce the effect of sample concentration on the chromatographic column, this experiment reduced the test sample concentration and injection volume, adjusted the detection wavelength from 223nm to 210nm, and used the ion pair reagent sodium octane sulfonate to enhance the retention behavior of the target peak to be measured. The chromatographic detection conditions are as follows:

[0288] Chromatographic column: Agilent ZORBAX BONUS-RP 4.6*250mm, 5μm;

[0289] Mobile phase: 10 mmol / L sodium octane sulfonate aqueous solution-acetonitrile (50:50, v / v);

[0290] Flow rate: 1ml / min;

[0291] Column temperature: 30°C;

[0292] Detection wavelength: 210nm;

[0293] Injection volume: 10 μl;

[0294] Test sample concentration: 10 mg / ml.

[0295] The blank solvent / diluent, picric acid solution and reference solution were prepared as described in Example 1.

[0296] Preparation of test sample spiked solution: Take appropriate amount of impurity C picrate, impurity G and impurity K reference substances and azacitidine, accurately weigh them, dissolve them in diluent and quantitatively dilute them to make a solution containing approximately 10 mg of azacitidine, 0.15 mg of impurity C, 0.02 mg each of impurity G and impurity K per 1 ml.

[0297] The chromatogram obtained from the spiked solution of the test sample is shown in Fig. 20 .

[0298] Depend on Fig. 20 It can be seen that the retention of each impurity is enhanced by adjusting the wavelength, sample concentration, injection volume and adding ion pair reagents to the mobile phase system. Although the peaks of impurity K and API are effectively separated, the theoretical plate number of impurity K is only 4159, the column efficiency is low, the tailing factor is 1.53, and the peak shape is asymmetric, which affects the accuracy of the results of quantitative analysis of azacitidine impurity K. In addition, the peaks of impurities C and G still overlap with the API, and effective separation is not achieved.

[0299] Comparative Example 8

[0300] Based on the detection conditions of Comparative Example 7, the mobile phase ratio was adjusted (from 50:50 to 90:10), and the chromatographic detection conditions were as follows:

[0301] Chromatographic column: Agilent ZORBAX BONUS-RP 4.6*250mm, 5μm;

[0302] Mobile phase: 10 mmol / L sodium octane sulfonate aqueous solution-acetonitrile (90:10, v / v);

[0303] Flow rate: 1ml / min;

[0304] Column temperature: 30°C;

[0305] Detection wavelength: 210nm;

[0306] Injection volume: 10 μl;

[0307] Test sample concentration: 10 mg / ml.

[0308] The blank solvent / diluent, picric acid solution and reference solution were prepared as described in Example 1.

[0309] Preparation of test sample spiked solution: Take appropriate amount of impurity C picrate, impurity G and impurity K reference substances and azacitidine, accurately weigh them, dissolve them in diluent and quantitatively dilute them to make a solution containing approximately 10 mg of azacitidine, 0.15 mg of impurity C, 0.02 mg each of impurity G and impurity K per 1 ml.

[0310] The chromatogram obtained from the spiked solution of the test sample is shown in Fig.21 .

[0311] Depend on Fig.21 It can be seen that after adjusting the ratio of the mobile phase to the organic phase based on the detection conditions of Comparative Example 7, the peaks of impurities C and G still overlap with the API, and effective separation is not achieved. In addition, the theoretical plate number of impurity K is only 3233, the column efficiency is low, and the separation degree is only 0.7, indicating that accurate detection of azacitidine impurity K cannot be achieved under the detection conditions of this embodiment.

[0312] Comparative Example 9

[0313] Based on the detection conditions of Comparative Example 8, the pH of the mobile phase was adjusted (pH 2.6), and the chromatographic detection conditions were as follows:

[0314] Chromatographic column: Agilent ZORBAX BONUS-RP 4.6*250mm, 5μm;

[0315] Mobile phase: 10 mmol / L sodium octane sulfonate aqueous solution (pH adjusted to 2.6 with phosphoric acid)-acetonitrile (90:10, v / v);

[0316] Flow rate: 1ml / min;

[0317] Column temperature: 30°C;

[0318] Detection wavelength: 210nm;

[0319] Injection volume: 10 μl;

[0320] Test sample concentration: 10 mg / ml.

[0321] The blank solvent / diluent, picric acid solution and reference solution were prepared as described in Example 1.

[0322] Preparation of test sample spiked solution: Take appropriate amount of impurity C picrate, impurity G and impurity K reference substances and azacitidine, accurately weigh them, dissolve them in diluent and quantitatively dilute them to make a solution containing approximately 10 mg of azacitidine, 0.15 mg of impurity C, 0.1 mg each of impurity G and impurity K per 1 ml.

[0323] The chromatograms obtained from the reference solution and the test solution spiked with the test sample are shown in Figure 22 to Figure 25 .

[0324] Depend on Fig.25 It can be seen that after the pH of the mobile phase was adjusted, the peaks of impurities C and G were still not effectively separated, and the separation degree was only 1.0; and the peak shapes of impurities C, impurity G, and impurity K were all asymmetric, among which the tailing factor of impurity K was 1.69, which affected the accuracy of the results during quantitative analysis; the theoretical plate numbers of impurities C, impurity G, and impurity K were all low, and the column efficiency was low.

[0325] It can be seen from Comparative Examples 1-9 that even if the pH value and the mobile phase ratio are continuously optimized, and even the retention behavior of the substance to be tested is enhanced by adding the ion pair reagent sodium octane sulfonate, the impurities C, G and K of azacitidine cannot be effectively detected by using a HILIC mode chromatographic column (Atlantis HILIC Silica) and a chromatographic column in which a polar amide group is embedded and capped in a long alkane chain (Agilent ZORBAX BONUS-RP). The target impurities to be separated cannot be effectively separated in the HILIC mode, and the chromatographic retention of the target impurities to be separated is weak under Agilent ZORBAX BONUS-RP.

Claims

1. A method for detecting impurities in azacitidine, characterized by: The liquid chromatography method is adopted, the stationary phase is uncapped octadecylsilane bonded silica gel, the mobile phase is water phase-acetonitrile=(85-92):(8-15), v / v, and the ion pair reagent sodium octane sulfonate is added to the water phase; Using the mobile phase to perform isocratic elution; The concentration of sodium octane sulfonate in the aqueous phase is 8 to 12 mmol / L; The aqueous phase is adjusted to a pH of 2.5 to 2.7; The impurities are impurity K, impurity G and impurity C with the following structures:

2. The detection method according to claim 1, characterized in that: The mobile phase was water-acetonitrile = (87-90): (10-13), v / v.

3. The detection method according to claim 1, characterized in that: The mobile phase was water-acetonitrile = (87-89): (11-13), v / v.

4. The detection method according to claim 1, characterized in that: The mobile phase was water-acetonitrile = 88:12, v / v.

5. The detection method according to claim 1, characterized in that: The concentration of sodium octane sulfonate in the aqueous phase is 9-11 mmol / L.

6. The detection method according to claim 1, characterized in that: The concentration of sodium octane sulfonate in the aqueous phase is 10 mmol / L.

7. The detection method according to claim 1, characterized in that: The pH of the aqueous phase was adjusted to 2.

6.

8. The detection method according to claim 1, characterized in that: The pH of the aqueous phase was adjusted with phosphoric acid.

9. The detection method according to claim 1, characterized in that: The stationary phase is Agilent InfinityLabPoroshell 120SB-C18 or an equivalent chromatographic column.

10. The detection method according to claim 9, characterized in that: The particle size of the filler of Agilent InfinityLab Poroshell120SB-C18 is 1.9 to 4 μm.

11. The detection method according to claim 9, characterized in that: The particle size of the packing material of Agilent InfinityLab Poroshell120SB-C18 is 4 μm.

12. The detection method according to claim 9, characterized in that: The inner diameter of Agilent InfinityLab Poroshell120SB-C18 is 2.1 to 4.6 mm.

13. The detection method according to claim 9, characterized in that: The inner diameter of the Agilent InfinityLab Poroshell120SB-C18 is 4.6 mm.

14. The detection method according to claim 9, characterized in that: The length of Agilent InfinityLab Poroshell120SB-C18 is 50 to 250 mm.

15. The detection method according to claim 9, characterized in that: The length of the Agilent InfinityLab Poroshell120SB-C18 is 250 mm.

16. The detection method according to claim 1, characterized in that: The detection wavelength is 205~240nm.

17. The detection method according to claim 1, characterized in that: The detection wavelength is 205~215nm.

18. The detection method according to claim 1, characterized in that: The detection wavelength is 210 nm.

19. The detection method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.9~1.1ml / min.

20. The detection method according to claim 1, characterized in that: The mobile phase flow rate was 1.0 ml / min.

21. The detection method according to claim 1, characterized in that: The column temperature is 15-30℃.

22. The detection method according to claim 1, characterized in that: The column temperature is 18-22°C.

23. The detection method according to claim 1, characterized in that: The column temperature was 20°C.

24. The detection method according to claim 1, characterized in that: The concentration of the test solution is 5-20 mg / ml, the concentration of impurity C in the reference solution is 0.015-0.045 mg / ml, the concentration of impurity G in the reference solution is 0.0075-0.0225 mg / ml, the concentration of impurity K in the reference solution is 0.0075-0.0225 mg / ml, the injection volume is 5-20 μL, and the diluent is dimethyl sulfoxide.

25. The detection method according to claim 1, characterized in that: The concentration of the test solution is 5-15 mg / ml, the concentration of impurity C in the reference solution is 0.03 mg / ml, the concentration of impurity G in the reference solution is 0.015 mg / ml, the concentration of impurity K in the reference solution is 0.015 mg / ml, and the injection volume is 5-15 μL.

26. The detection method according to claim 1, characterized in that: The concentration of the test solution was prepared to be 10 mg / ml and the injection volume was 10 μl.

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