Detection method of carfilzomib starting material related substances

Abstract

The invention discloses a method for detecting related substances of a carfilzomib starting material, which adopts a specific mobile phase and combines specific elution conditions to effectively separate the carfilzomib starting material from impurities thereof and ensure that the impurity content of the carfilzomib starting material is kept below a certain level. And the quality of the carfilzomib initial material is better controlled, so that a safe and effective crude drug finished product with low impurity content is prepared.

Description

Technical Field

[0001] The present invention relates to, but is not limited to, pharmaceutical analysis technologies, and particularly relates to a method for detecting related substances of a carfilzomib starting material. Background Art

[0002] Carfilzomib, with the chemical name (αS)-α-[(4-morpholinylacetyl)amino]benzoyl-L-leucyl-N-[(1S)-3-methyl-1-[[(2R)-2-methyloxiranyl]carbonyl]butyl]-L-phenylalaninamide, is a tetrapeptide epoxy backbone proteasome inhibitor. On July 20, 2012, the U.S. Food and Drug Administration (FDA) approved the marketing of the carfilzomib injection (trade name: Kyprolis) developed by Onyx Pharmaceuticals through the priority review process. The structural formula of carfilzomib is shown as follows:

[0003]

[0004] The structure of carfilzomib contains 5 chiral centers, and the control of its chiral purity is a quality control difficulty for this variety. SMA, SMB, SMC, and SMD are important starting materials for the production of carfilzomib, and their structures are as follows:

[0005]

[0006] The purity of the carfilzomib starting materials SMA, SMB, SMC, and SMD directly affects the purity of the carfilzomib raw material and the content of chiral impurities, thus directly affecting the effectiveness and safety of the formulated drug. Therefore, quality control and detection of the carfilzomib starting materials, especially the related substances of the starting materials, are beneficial to ensuring the quality controllability of chemically synthesized drugs.

[0007] Currently, there is no dedicated method for detecting related substances of carfilzomib starting materials. Therefore, it is necessary to establish a highly specific and accurate method for detecting related substances applicable to carfilzomib starting materials to achieve qualitative and / or quantitative detection of related substances, ensure the detection ability of the method for impurities, and ensure that the impurity content of the commercially purchased materials remains at a certain level, effectively controlling the quality of the starting materials, providing guidance for subsequent production processes, ensuring the safety and effectiveness of the raw materials, and further ensuring the effectiveness and safety of the marketed formulated drugs. Summary of the Invention

[0008] The present invention provides a method for detecting related substances of a carfilzomib starting material, which can better control the quality of the carfilzomib starting material. By combining a phosphoric acid aqueous solution or a phosphate buffer solution with acetonitrile (or a mixed solution of methanol and acetonitrile) and specific elution conditions, the carfilzomib starting material can be effectively separated from its impurities.

[0009] In an embodiment of the present invention, the carfilzomib starting materials include SMA, SMB, SMC, and SMD, and their structures are as follows:

[0010]

[0011] In an embodiment of the present invention, the related substances of the carfilzomib starting materials of the present invention refer to substances that are chemically similar or have a genetic relationship with the carfilzomib starting materials SMA, SMB, SMC, and SMD, and can be referred to as "related substances", "impurities", or "organic impurities" in the present invention. The presence of these substances will directly affect the purity of the carfilzomib API and the content of chiral impurities.

[0012] Specifically, in some embodiments, the "related substances of the carfilzomib starting materials" include, but are not limited to, one or more of the following substances: SMAZ1, SMAZ2, SMAZ3, SMAZ4, SMAZ5 shown in Table 1, SMBZ1, SMBZ2, SMBZ3, SMBZ4 shown in Table 2, SMCZ1, SMCZ2, SMCZ3 shown in Table 3, and SMDZ1, SMDZ2, SMDZ3, SMDZ4, SMDZ5, SMDZ6, SMDZ7, SMDZ8, SMDZ9 shown in Table 4;

[0013] In some embodiments, the "related substances of the carfilzomib starting materials" include SMAZ1, SMAZ2, SMAZ3, SMAZ4, SMAZ5 shown in Table 1, SMBZ1, SMBZ2, SMBZ3, SMBZ4 shown in Table 2, SMCZ1, SMCZ2, SMCZ3 shown in Table 3, and SMDZ1, SMDZ2, SMDZ3, SMDZ4, SMDZ5, SMDZ6, SMDZ7, SMDZ8, SMDZ9 shown in Table 4;

[0014] In some embodiments, the "related substances of the carfilzomib starting materials" consist of SMAZ1, SMAZ2, SMAZ3, SMAZ4, SMAZ5 shown in Table 1, SMBZ1, SMBZ2, SMBZ3, SMBZ4 shown in Table 2, SMCZ1, SMCZ2, SMCZ3 shown in Table 3, and SMDZ1, SMDZ2, SMDZ3, SMDZ4, SMDZ5, SMDZ6, SMDZ7, SMDZ8, SMDZ9 shown in Table 4.

[0015] Table 1 Related substances of the starting material SMA

[0016]

[0017]

[0018] Table 2 Related Substances of Starting Material SMB

[0019]

[0020] Table 3 Related Substances of Starting Material SMC

[0021]

[0022] Table 4 Related Substances of Starting Material SMD

[0023]

[0024]

[0025] On the one hand, the present invention provides a method for detecting related substances of carfilzomib starting materials, wherein the carfilzomib starting materials include SMA, SMB, SMC or SMD, and qualitative and / or quantitative detection is carried out by high performance liquid chromatography. The detection method includes the following detection conditions:

[0026] Chromatographic column: octadecylsilane bonded silica gel;

[0027] Mobile phase: gradient elution is carried out using mobile phase A and mobile phase B. Among them, mobile phase A is an aqueous phosphoric acid solution or a phosphate buffer solution; mobile phase B is acetonitrile or a mixed solution of methanol and acetonitrile.

[0028] In some embodiments, the gradient elution program is as follows:

[0029]

[0030] In some embodiments, mobile phase A is a phosphate buffer solution, wherein the phosphate buffer solution is a potassium dihydrogen phosphate buffer solution. Preferably, the pH of the potassium dihydrogen phosphate buffer solution is 4.0; mobile phase B is acetonitrile; preferably, the gradient elution program is as follows:

[0031]

[0032]

[0033] In some embodiments, mobile phase A is an aqueous phosphoric acid solution. Preferably, the concentration of the aqueous phosphoric acid solution is 0.05 wt%; mobile phase B is acetonitrile; preferably, the gradient elution program is as follows:

[0034]

[0035] In some embodiments, the mobile phase A is a phosphate buffer solution, wherein the phosphate buffer solution is a potassium dihydrogen phosphate buffer solution. Preferably, the pH of the potassium dihydrogen phosphate buffer solution is 2.5; the mobile phase B is acetonitrile; preferably, the gradient elution program is as follows:

[0036]

[0037] In some embodiments, the mobile phase A is a phosphate buffer solution, wherein the phosphate buffer solution is a sodium dihydrogen phosphate buffer solution. Preferably, the pH of the sodium dihydrogen phosphate buffer solution is 2.5; the mobile phase B is a mixed solution of methanol and acetonitrile. Preferably, the volume ratio of methanol to acetonitrile is 2:1;

[0038] Preferably, the gradient elution program is as follows:

[0039]

[0040] In some embodiments, the preparation method of the potassium dihydrogen phosphate buffer solution is as follows: Weigh 3.0 g of potassium dihydrogen phosphate, dilute it with water to 1000 ml, and adjust the pH to 4.0 or 2.5 with phosphoric acid.

[0041] In some embodiments, the preparation method of the sodium dihydrogen phosphate buffer solution is as follows: Weigh 6.0 g of sodium dihydrogen phosphate dihydrate, add 1000 ml of water, and adjust the pH to 2.5 with phosphoric acid.

[0042] In some embodiments, the detection conditions further include one or more of the following:

[0043] Column temperature: 25 - 40 °C. Preferably, the column temperature is 30 °C or 40 °C;

[0044] Flow rate: 1.0 ml / min;

[0045] Detection wavelength: 205 - 220 nm. Preferably, the detection wavelength is 205 nm or 210 nm;

[0046] Injection volume: 5 - 60 μl; Preferably, the injection volume is 5 μl, 20 μl or 50 μl.

[0047] In some embodiments, the specifications of the chromatographic column are: YMC-Pack ODS-AQ, 4.6×150 mm, 3 μm or Waters Symmetry C18 4.6×150 mm, 5 μm.

[0048] In some embodiments, the detection conditions further include using a trapping column, and the specifications of the trapping column are: Ghost Buster Column, 4.6 mm×50 mm.

[0049] In some embodiments, the detection conditions further include using the starting materials SMA, SMB, SMC or SMD of carfilzomib as the test samples; using one or more selected from impurities SMAZ1, SMAZ2, SMAZ3, SMAZ4, SMAZ5, SMBZ1, SMBZ2, SMBZ3, SMBZ4, SMCZ1, SMCZ2, SMCZ3 or SMDZ1, SMDZ2, SMDZ3, SMDZ4, SMDZ5, SMDZ6, SMDZ7, SMDZ8, SMDZ9 as the controls, and preparing the test sample solution and the control solution;

[0050] Preparation of the test sample solution: Take an appropriate amount of the test sample, dissolve and dilute it with a solvent to prepare a solution containing about 1.0 mg or 4.0 mg per 1 ml;

[0051] Preparation of the control solution: Accurately measure an appropriate amount of the test sample solution, and dilute it with a solvent to prepare a solution containing about 1 μg, 2 μg or 20 μg per 1 ml.

[0052] In some embodiments, the solvent is a mixed solution of water and acetonitrile with a volume ratio of 50:50 or a mixed solution of 0.05 wt% phosphoric acid aqueous solution and acetonitrile with a volume ratio of 9:1.

[0053] In some embodiments, the present invention provides a method for detecting related substances of the starting materials of carfilzomib, wherein the starting material SMA of carfilzomib is used as the test sample; one or more selected from impurities SMAZ1, SMAZ2, SMAZ3, SMAZ4, SMAZ5 are used as the controls, and the test sample and the control solution are prepared; the detection conditions include:

[0054] Chromatographic column: YMC-Pack ODS-AQ, 4.6×150 mm, 3 μm;

[0055] Mobile phase A: Potassium dihydrogen phosphate buffer solution (weigh 3.0 g of potassium dihydrogen phosphate, dilute it with water to 1000 ml, and adjust the pH to 4.0 with phosphoric acid);

[0056] Mobile phase B: Acetonitrile;

[0057] The gradient elution program is as follows:

[0058]

[0059] Column temperature: 30 °C;

[0060] Flow rate: 1.0 ml / min;

[0061] Detection wavelength: 210 nm;

[0062] Sample injection volume: 50 μl;

[0063] Trapping column: Ghost Buster Column, 4.6mm×50mm.

[0064] In some embodiments, the present invention provides a method for detecting related substances of carfilzomib starting materials, wherein the starting material SMB of carfilzomib is used as the test sample; one or more selected from impurities SMBZ1, SMBZ2, SMBZ3, and SMBZ4 are used as the control to prepare the test sample solution and the control solution; the detection conditions include:

[0065] Chromatographic column: YMC-Pack ODS-AQ, 4.6×150mm, 3μm;

[0066] Mobile phase A: 0.05wt% phosphoric acid aqueous solution;

[0067] Mobile phase B: acetonitrile;

[0068] The gradient elution program is as follows:

[0069]

[0070] Column temperature: 30°C;

[0071] Flow rate: 1.0ml / min;

[0072] Detection wavelength: 210nm;

[0073] Injection volume: 20μl;

[0074] Trapping column: Ghost Buster Column, 4.6mm×50mm.

[0075] In some embodiments, the present invention provides a method for detecting related substances of carfilzomib starting materials, wherein the starting material SMC of carfilzomib is used as the test sample; one or more selected from impurities SMCZ1, SMCZ2, and SMCZ3 are used as the control to prepare the test sample solution and the control solution; the detection conditions include:

[0076] Chromatographic column: Waters Symmetry C18 4.6×150mm, 5μm;

[0077] Mobile phase A: potassium dihydrogen phosphate buffer solution (weigh 3.0g of potassium dihydrogen phosphate, dilute with water to 1000ml, and adjust the pH to 2.5 with phosphoric acid);

[0078] Mobile phase B: acetonitrile;

[0079] The gradient elution program is as follows:

[0080]

[0081]

[0082] Column temperature: 30 °C;

[0083] Flow rate: 1.0 ml / min;

[0084] Detection wavelength: 210 nm;

[0085] Sample injection volume: 5 μl;

[0086] Trapping column: Ghost Buster Column, 4.6 mm × 50 mm.

[0087] In some embodiments, the present invention provides a method for detecting related substances of carfilzomib starting materials, wherein the starting material SMD of carfilzomib is used as the test sample; one or more selected from impurities SMDZ1, SMDZ2, SMDZ3, SMDZ4, SMDZ5, SMDZ6, SMDZ7, SMDZ8, SMDZ9 are used as controls, and the test sample solution and the control solution are prepared; the detection conditions include:

[0088] Chromatographic column: YMC-Pack ODS-AQ, 4.6 × 150 mm, 3 μm;

[0089] Mobile phase A: Sodium dihydrogen phosphate buffer solution (weigh 6.0 g of sodium dihydrogen phosphate dihydrate, add 1000 ml of water, and adjust the pH to 2.5 with phosphoric acid);

[0090] Mobile phase B: A mixed solution of methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 2:1;

[0091] The gradient elution program is:

[0092]

[0093] Column temperature: 40 °C;

[0094] Flow rate: 1.0 ml / min;

[0095] Detection wavelength: 205 nm;

[0096] Sample injection volume: 50 μl;

[0097] Trapping column: Ghost Buster Column, 4.6 mm × 50 mm.

[0098] Through continuous attempts and explorations, the inventor finally obtained the detection method of the present invention. By using a chromatographic column filled with octadecylsilane-bonded silica gel and combining phosphoric acid aqueous solution or phosphate buffer solution with acetonitrile (or a mixed solution of methanol and acetonitrile), effective separation of the starting material and its related substances can be achieved, and the separation effect is good.

[0099] Compared with the closest prior art, the detection method provided by the present invention can achieve effective separation of the starting material and its related substances.

[0100] Other features and advantages of the present invention will be described in the subsequent specification. And, in part, it will become obvious from the specification or be understood by implementing the present invention. Other advantages of the present invention can be achieved and obtained through the solutions described in the specification and the accompanying drawings. Description of the Drawings

[0101] The drawings are used to provide an understanding of the technical solution of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the technical solution of the present invention and do not constitute a limitation to the technical solution of the present invention.

[0102] Figure 1 Chromatogram of the test solution for Example 1;

[0103] Figure 2 Chromatogram of the test solution for Control Example 1;

[0104] Figure 3 Chromatogram of the test solution for Control Example 2;

[0105] Figure 4 Chromatogram of the test solution for Control Example 3;

[0106] Figure 5 Chromatogram of the test solution for Control Example 4;

[0107] Figure 6 Chromatogram of the test solutions for Control Example 5 and Control Example 6;

[0108] Figure 7 Chromatogram of the test solution for Example 2;

[0109] Figure 8 Chromatogram of the test solution for Control Example 7;

[0110] Figure 9 Chromatogram of the test solution for Example 3;

[0111] Figure 10 Chromatogram of the test solution for Control Example 8;

[0112] Figure 11Chromatogram of the test solution for Comparative Example 9;

[0113] Figure 12 Chromatogram of the test solution for Example 4;

[0114] Figure 13 Overlay chromatogram of the test solution for Comparative Example 10 and the impurity SMDZ1 and SMDZ2 localization solutions;

[0115] Figure 14 Overlay chromatogram of the test solution for Comparative Example 10 and the impurity localization solutions (SMDZ3 - SMDZ9, except SMDZ7);

[0116] Figure 15 Chromatogram of the impurity mixed solution for Comparative Example 10 (SMDZ1 - SMDZ9, except SMDZ7);

[0117] Figure 16 Chromatogram of the impurity mixed solution for Comparative Example 11 (SMDZ1 - SMDZ9, except SMDZ7);

[0118] Figure 17 Chromatogram of the mixed solution for Comparative Example 12 (main component and SMDZ1 - SMDZ8);

[0119] Figure 18 Chromatogram of the mixed solution for Comparative Example 13 (main component and SMDZ1 - SMDZ8);

[0120] Figure 19 Chromatogram of the mixed solution for Comparative Example 14 (main component and SMDZ1 - SMDZ8);

[0121] Figure 20 Chromatogram of the test solution for Comparative Example 15;

[0122] Figure 21 Chromatogram of the mixed solution for Comparative Example 15 (main component and SMDZ1 - SMDZ8). Detailed Description of the Invention

[0123] To make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the embodiments of the present invention will be described in detail below. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be arbitrarily combined with each other.

[0124] The carfilzomib starting materials and each impurity used in the specific embodiments of the present invention are all commercially available samples.

[0125] Among them,

[0126] System suitability requirements: Inject the reference solution continuously for 6 times, the RSD of the main peak peak area should not exceed 10.0%, and the RSD of the retention time should not exceed 1.0%.

[0127] Standard limit:

[0128] In the chromatogram of the test solution, if there are impurity peaks, the peak area of SMAZ4 should not be greater than 0.4 times (0.2%) of the main peak peak area of the reference solution, the peak areas of SMAZ2 and SMAZ3 should not be greater than 0.2 times (0.1%) of the main peak peak area of the reference solution, the area of other individual impurity peaks should not be greater than 0.2 times (0.1%) of the main peak peak area of the reference solution, and the sum of the areas of each impurity peak should not be greater than the main peak peak area of the reference solution (0.5%).

[0129] The calculation formula is:

[0130]

[0131]

[0132] Among them,

[0133] A 单杂 —— The peak area of a single impurity in the test solution;

[0134] A 对照 —— The main peak peak area in the reference solution;

[0135] 200 - Dilution factor of the reference solution;

[0136] In the chromatogram of the test solution, if there are impurity peaks, calculated according to the corrected peak area, the peak area of the SMBZ4 impurity peak should not be greater than 25 times (2.5%) of the main peak peak area of the reference solution, the area of other individual impurity peaks should not be greater than the main peak peak area of the reference solution (0.1%), and the sum of the areas of other impurity peaks should not be greater than 5 times (0.5%) of the main peak peak area of the reference solution.

[0137] The calculation formula is:

[0138]

[0139]

[0140] Among them:

[0141] A 单杂 —— The peak area of a single impurity in the test solution;

[0142] f - Impurity correction factor, the correction factor of unknown impurities is defaulted to 1;

[0143] A 对照 —— The main peak peak area in the reference solution;

[0144] 1000 - Dilution factor of the control solution;

[0145] Total of other impurities — The sum of the contents of other individual impurities in the test solution except SMBZ4.

[0146] If there are impurity peaks in the chromatogram of the test solution, the peak area of SMCZ3 shall not be greater than 3 times (0.3%) of the main peak area of the control solution, the peak area of any other individual impurity shall not be greater than the main peak area of the control solution (0.1%), and the sum of the peak areas of all impurity peaks shall not be greater than 5 times (0.5%) of the main peak area of the control solution.

[0147] The calculation formula is:

[0148]

[0149]

[0150] Where:

[0151] A 单杂 — The peak area of an individual impurity in the test solution;

[0152] f — Impurity correction factor, the correction factor for unknown impurities is defaulted to 1;

[0153] A 对照 — The peak area of the main peak in the control solution;

[0154] 1000 - Dilution factor of the control solution;

[0155] If there are impurity peaks in the chromatogram of the test solution, calculated by the corrected peak area, the peak areas of SMDZ2 and SMDZ3 shall not be greater than 1.5 times (0.3%) of the main peak area of the control solution, the peak areas of SMDZ8 and any other individual impurity peaks shall not be greater than the main peak area of the control solution (0.2%), and the sum of the peak areas of all impurity peaks shall not be greater than 5 times (1.0%) of the main peak area of the control solution.

[0156] The calculation formula is:

[0157]

[0158]

[0159] Where:

[0160] A 单杂 — The peak area of an individual impurity in the test solution;

[0161] f — Impurity correction factor, the correction factor for unknown impurities is defaulted to 1;

[0162] A 对照—— Peak area of the main peak in the control solution;

[0163] 500 - Dilution factor of the control solution.

[0164] Example 1

[0165] A method for detecting related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0166] Chromatographic conditions:

[0167] Chromatographic column: Octadecylsilane-bonded silica gel as the filler (YMC-Pack ODS-AQ, 4.6×150mm, 3μm);

[0168] Trapping column: Ghost Buster Column, 4.6mm×50mm;

[0169] Mobile phase A: Potassium dihydrogen phosphate buffer solution (weigh 3.0g of potassium dihydrogen phosphate, add water to dilute to 1000ml, adjust the pH to 4.0 with phosphoric acid);

[0170] Mobile phase B: Acetonitrile;

[0171] Gradient elution:

[0172]

[0173] Injection volume: 50μl;

[0174] Flow rate: 1.0ml / min;

[0175] Column temperature: 30°C;

[0176] Detection wavelength: 210nm;

[0177] Preparation of solutions:

[0178] Solvent: Water∶Acetonitrile (50∶50);

[0179] Test solution: Take an appropriate amount of starting material SMA, dissolve and dilute it with the solvent to make a solution containing about 4.0mg per 1ml;

[0180] Control solution: Accurately measure an appropriate amount of the test solution, and dilute it with the solvent to make a solution containing about 20μg per 1ml.

[0181] Determination method:

[0182] Accurately measure 50μl of each of the test solution and the control solution, inject them into the liquid chromatograph respectively, record the chromatogram, as Figure 1 shown.

[0183] As can be seen from the figure, after adjusting the pH value of mobile phase A to 4.0, the peak shapes of the main component and impurities are both good, and the separation effect between the main peak and the adjacent impurity peak, and between impurities and impurities is good (the resolution between SMAZ2 and the starting material SMA is 2.35).

[0184] Control Example 1

[0185] A method for detecting related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0186] Chromatographic conditions:

[0187] Chromatographic column: YMC-Pack ODS-AQ, 4.6×150mm, 3μm;

[0188] Mobile phase A: 0.05wt% phosphoric acid aqueous solution;

[0189] Mobile phase B: acetonitrile;

[0190] Gradient elution:

[0191]

[0192] Sample injection volume: 50μl;

[0193] Flow rate: 1.0ml / min;

[0194] Column temperature: 30℃;

[0195] Sample tray temperature: 10℃;

[0196] Detection wavelength: 210nm;

[0197] Preparation of solutions:

[0198] Solvent: 0.05mol / L potassium dihydrogen phosphate solution∶acetonitrile = 50∶50;

[0199] Test solution: Take an appropriate amount of the starting material SMA, weigh it accurately, dissolve it with the solvent and dilute it to a solution containing about 4mg per 1ml.

[0200] Control solution: Accurately measure an appropriate amount of the test solution and dilute it with the solvent to a solution containing about 20μg per 1ml.

[0201] Determination method:

[0202] Accurately measure the test solution and the control solution, inject them into the liquid chromatograph respectively, record the chromatogram, as Figure 2 shown.

[0203] As can be seen from the figure, SMA and SMAZ1 are isomers with the same peak elution time. The peak of SMAZ5 appears at the void volume and is interfered by the blank solvent peak. SMA and SMAZ2 fail to achieve separation.

[0204] Control Example 2

[0205] A method for detecting related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0206] Chromatographic conditions:

[0207] Chromatographic column: YMC-Pack ODS-AQ, 4.6×150mm, 3μm;

[0208] Mobile phase A: 0.05wt% phosphoric acid aqueous solution;

[0209] Mobile phase B: acetonitrile;

[0210] Gradient elution:

[0211]

[0212] Injection volume: 50μl;

[0213] Flow rate: 1.0ml / min;

[0214] Column temperature: 30°C;

[0215] Sample tray temperature: 10°C;

[0216] Detection wavelength: 210nm;

[0217] Preparation of solutions:

[0218] Solvent: 0.05mol / L potassium dihydrogen phosphate solution∶acetonitrile = 50∶50;

[0219] Test solution: Weigh an appropriate amount of the starting material SMA accurately, dissolve it with the solvent and dilute to a solution containing about 4mg per 1ml.

[0220] Control solution: Accurately measure an appropriate amount of the test solution and dilute it with the solvent to a solution containing about 20μg per 1ml.

[0221] Determination method:

[0222] Accurately measure the test solution and the control solution, inject them into the liquid chromatograph respectively, and record the chromatogram as Figure 3 shown.

[0223] As can be seen from the figure, after adjusting the gradient elution program, SMAZ2 and SMA fail to achieve separation, and the main peak and the impurity peaks have fronting shapes.

[0224] Control Example 3

[0225] A detection method for related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0226] Chromatographic conditions:

[0227] Chromatographic column: YMC-Pack ODS-AQ, 4.6×150mm, 3μm;

[0228] Mobile phase A: 0.05wt% phosphoric acid aqueous solution;

[0229] Mobile phase B: acetonitrile;

[0230] Gradient elution:

[0231]

[0232]

[0233] Injection volume: 50μl;

[0234] Flow rate: 1.0ml / min;

[0235] Column temperature: 30°C;

[0236] Sample tray temperature: 10°C;

[0237] Detection wavelength: 210nm;

[0238] Preparation of solutions:

[0239] Solvent: 0.05mol / L phosphoric acid aqueous solution∶acetonitrile = 50∶50;

[0240] Test solution: Take an appropriate amount of starting material SMA, weigh accurately, dissolve and dilute with the solvent to prepare a solution containing about 4mg per 1ml.

[0241] Control solution: Accurately measure an appropriate amount of the test solution and dilute it with the solvent to prepare a solution containing about 20μg per 1ml.

[0242] Determination method:

[0243] Accurately measure 50μl each of the test solution and the control solution, inject them into the liquid chromatograph respectively, record the chromatogram, as Figure 4 shown.

[0244] It can be concluded from the figure that after adjusting the gradient and changing the solvent, the main component and impurity peak shapes are good, and the solvent is applicable; however, the retention times of SMAZ2 and SMA are the same and separation cannot be achieved.

[0245] Control Example 4

[0246] A detection method for related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0247] Chromatographic conditions:

[0248] Chromatographic column: YMC-Pack ODS-AQ, 4.6×150mm, 3μm;

[0249] Mobile phase A: Potassium dihydrogen phosphate buffer solution with pH 3.0 (weigh 3.0 g of potassium dihydrogen phosphate, add 1000 ml of water, and adjust the pH to 3.0 with phosphoric acid);

[0250] Mobile phase B: Acetonitrile;

[0251] Gradient elution:

[0252]

[0253]

[0254] Injection volume: 50 μl;

[0255] Flow rate: 1.0 ml / min;

[0256] Column temperature: 30°C;

[0257] Sample tray temperature: 10°C;

[0258] Detection wavelength: 210 nm;

[0259] Preparation of solutions:

[0260] Solvent: 0.05 mol / L phosphoric acid aqueous solution∶acetonitrile = 50∶50;

[0261] Test solution: Take an appropriate amount of starting material SMA, weigh accurately, dissolve and dilute with the solvent to prepare a solution containing about 4 mg per 1 ml.

[0262] Reference solution: Accurately measure an appropriate amount of the test solution and dilute with the solvent to prepare a solution containing about 20 μg per 1 ml.

[0263] Determination method:

[0264] Accurately measure 50 μl each of the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, as Figure 5 shown.

[0265] It can be concluded from the figure that after changing the mobile phase A, the retention times of SMAZ2 and SMA are the same and separation cannot be achieved; SMAZ4 is interfered by unknown impurities; the baseline fluctuates significantly and there is a risk of interfering with the detection for the solvent peak (about 22 min).

[0266] Control Example 5

[0267] A detection method for related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0268] Chromatographic conditions:

[0269] Chromatographic column: YMC-Pack ODS-AQ, 4.6×150mm, 3μm;

[0270] Mobile phase A: Potassium dihydrogen phosphate buffer solution with pH 5.0 (weigh 3.0 g of potassium dihydrogen phosphate, add 1000 ml of water, and adjust the pH to 5.0 with phosphoric acid);

[0271] Trapping column: Ghost-Buster Column, 4.6mm×50mm;

[0272] Mobile phase B: Acetonitrile;

[0273] Gradient elution:

[0274]

[0275]

[0276] Sample injection volume: 50 μl;

[0277] Flow rate: 1.0 ml / min;

[0278] Column temperature: 30°C;

[0279] Sample tray temperature: 10°C;

[0280] Detection wavelength: 210nm;

[0281] Preparation of solutions:

[0282] Solvent: Water∶Acetonitrile (50∶50);

[0283] Test solution: Take an appropriate amount of starting material SMA, weigh it accurately, dissolve it with the solvent and dilute it to a solution containing about 4 mg per 1 ml.

[0284] Control solution: Accurately measure an appropriate amount of the test solution and dilute it with the solvent to a solution containing about 20 μg per 1 ml.

[0285] Determination method:

[0286] Accurately measure 50 μl each of the test solution and the control solution, inject them into the liquid chromatograph respectively, record the chromatogram, as Figure 6 shown.

[0287] Control Example 6

[0288] A detection method for related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0289] Chromatographic conditions:

[0290] Chromatographic column: YMC-Pack ODS-AQ, 4.6×150mm, 3μm;

[0291] Mobile phase A: Potassium dihydrogen phosphate buffer solution with pH 5.0 (weigh 3.0 g of potassium dihydrogen phosphate, add 1000 ml of water, and adjust the pH to 5.0 with phosphoric acid);

[0292] Trapping column: Ghost-Buster Column, 4.6mm×50mm;

[0293] Mobile phase B: Acetonitrile;

[0294] Gradient elution:

[0295]

[0296] Injection volume: 50 μl;

[0297] Flow rate: 1.0 ml / min;

[0298] Column temperature: 30°C;

[0299] Sample tray temperature: 10°C;

[0300] Detection wavelength: 210 nm;

[0301] Preparation of solutions:

[0302] Solvent: Potassium dihydrogen phosphate buffer solution with pH 5.0∶Acetonitrile = 50∶50;

[0303] Test solution: Take an appropriate amount of this product, weigh accurately, dissolve and dilute with the solvent to prepare a solution containing about 4 mg per 1 ml.

[0304] Reference solution: Accurately measure an appropriate amount of the test solution and dilute it with the solvent to prepare a solution containing about 20 μg per 1 ml.

[0305] Determination method:

[0306] Accurately measure 50 μl each of the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, as Figure 6 shown.

[0307] It can be seen from the figure that the baseline is stable after adding the trapping column. After the pH value of mobile phase A is adjusted from 3.0 to 5.0, the front of the main peak is obvious.

[0308] There was no significant difference in the peak shapes under the two solvent conditions of Comparative Example 5 and Comparative Example 6, and good separation was achieved among various impurities and between impurities and the main peak. However, the preparation of the solvent in Comparative Example 6 was more complex.

[0309] Example 2

[0310] A method for detecting related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0311] Chromatographic conditions:

[0312] Chromatographic column: Octadecylsilyl silica gel as the filler (YMC-Pack ODS-AQ, 4.6×150mm, 3μm);

[0313] Trapping column: Ghost Buster Column, 4.6mm×50mm;

[0314] Mobile phase A: 0.05wt% phosphoric acid aqueous solution;

[0315] Mobile phase B: Acetonitrile;

[0316] Gradient elution:

[0317]

[0318] Injection volume: 20μl;

[0319] Flow rate: 1.0ml / min;

[0320] Column temperature: 30°C;

[0321] Detection wavelength: 210nm;

[0322] Preparation of solutions:

[0323] Solvent: 0.05wt% phosphoric acid aqueous solution - acetonitrile (9:1);

[0324] Test solution: Take an appropriate amount of starting material SMB, dissolve it with the solvent and dilute it to make a solution containing about 1.0mg per 1ml.

[0325] Control solution: Accurately measure an appropriate amount of the test solution and dilute it with the solvent to make a solution containing about 1μg per 1ml.

[0326] Determination method:

[0327] Accurately measure the test solution and the control solution, inject them into the liquid chromatograph respectively, record the chromatogram, as Figure 7 shown, and the inspection results of the mixed solution are shown in Table 5.

[0328] Table 5 Inspection results of the mixed solution

[0329] Attribution Retention time (min) Resolution Signal-to-noise ratio SMBZ4 5.932 - 8453.45 SMBZ2 7.987 8.12 1287.03 SMB 10.229 4.61 77019.22 SMBZ3 15.165 17.70 7646.75

[0330] By increasing the injection volume, the signal-to-noise ratio of SMBZ2 at 10 μg / ml is about 1300. Theoretically, the signal-to-noise ratio of SMBZ2 at the limit concentration should be greater than 100, and the limit of quantification is about 10% of the limit concentration. The sensitivity is good, and the detection method can effectively separate SMB from related substances and between related substances.

[0331] Control Example 7

[0332] A method for detecting related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0333] Chromatographic conditions:

[0334] Chromatographic column: YMC-Pack ODS-AQ, 4.6×150 mm, 3 μm;

[0335] Mobile phase A: 0.05 wt% phosphoric acid aqueous solution;

[0336] Mobile phase B: acetonitrile;

[0337] Gradient elution ：

[0338]

[0339]

[0340] Injection volume: 10 μl;

[0341] Flow rate: 1.0 ml / min;

[0342] Column temperature: 30 °C;

[0343] Detection wavelength: 210 nm;

[0344] Preparation of solutions:

[0345] Solvent: 0.05 wt% phosphoric acid aqueous solution - acetonitrile (9:1);

[0346] Test solution: Take an appropriate amount of the starting material SMB, dissolve it with the solvent and dilute it to a solution containing about 1.0 mg per 1 ml.

[0347] Reference solution: Accurately measure an appropriate amount of the test solution and dilute it with the solvent to a solution containing about 1 μg per 1 ml.

[0348] Determination method:

[0349] Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram as Figure 8 shown.

[0350] Prepare a mixed solution containing approximately 1 mg / ml of carfilzomib SMB and approximately 10 μg / ml of each impurity (except SMBZ1), and inject the sample to examine the separation of each component. The results are shown in Table 6.

[0351] Table 6 Initial Conditions - Results of the Examination of the Mixed Solution

[0352] Attribution Retention time (min) Resolution Signal-to-noise ratio SMBZ4 5.659 - 4054.61 SMBZ2 7.506 8.48 665.49 SMB 10.205 7.34 52369.37 SMBZ3 15.117 17.77 3426.54

[0353] The results showed that the main component and the impurities, as well as the impurities and impurities, did not interfere with each other and the separation was good, but the response of SMBZ2 was relatively low.

[0354] Example 3

[0355] A method for detecting related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0356] Chromatographic conditions:

[0357] Chromatographic column: Octadecylsilyl silica gel as the filler (Waters Symmetry C18 4.6×150 mm, 5 μm);

[0358] Trapping column: Ghost Buster Column, 4.6 mm×50 mm;

[0359] Mobile phase A: Potassium dihydrogen phosphate buffer solution (weigh 3.0 g of potassium dihydrogen phosphate, dilute with water to 1000 ml, and adjust the pH to 2.5 with phosphoric acid);

[0360] Mobile phase B: Acetonitrile;

[0361] Gradient elution:

[0362]

[0363]

[0364] Injection volume: 5 μl;

[0365] Flow rate: 1.0 ml / min;

[0366] Column temperature: 30 °C;

[0367] Sample tray temperature: 10 °C;

[0368] Detection wavelength: 210 nm;

[0369] Preparation of solutions:

[0370] Solvent: Water∶Acetonitrile (50∶50);

[0371] Test solution: Take an appropriate amount of the starting material SMC, dissolve it with a solvent and dilute to make a solution containing about 1.0 mg per 1 ml;

[0372] Reference solution: Accurately measure an appropriate amount of the test solution, and dilute it with a solvent to make a solution containing about 1 μg per 1 ml.

[0373] Determination method:

[0374] Accurately measure the test solution and the reference solution, and inject them into the liquid chromatograph respectively, record the chromatogram, as Figure 9 shown.

[0375] It can be concluded from the figure that there is no interference between the starting material SMC and adjacent impurities, and between impurities in the test solution, and effective separation can be achieved.

[0376] Control Example 8

[0377] A method for detecting related substances of the starting material of carfilzomib, wherein the detection method includes the following detection conditions:

[0378] Chromatographic conditions:

[0379] Chromatographic column: Waters Symmetry C18 4.6×150 mm, 5 μm;

[0380] Mobile phase A: Potassium dihydrogen phosphate buffer solution (take 3.0 g of potassium dihydrogen phosphate, add 1000 ml of water, and adjust the pH value to 2.5 with phosphoric acid);

[0381] Mobile phase B: Acetonitrile;

[0382] Gradient elution:

[0383]

[0384]

[0385] Injection volume: 5 μl;

[0386] Flow rate: 1.0 ml / min;

[0387] Column temperature: 30 °C;

[0388] Detection wavelength: 210 nm;

[0389] Preparation of solution:

[0390] Solvent: Water∶Acetonitrile (50∶50);

[0391] Test solution: Take an appropriate amount of the starting material SMC, accurately weigh it, dissolve it with a solvent and dilute to make a solution containing about 4 mg per 1 ml.

[0392] Reference solution: Accurately measure an appropriate amount of the test solution and dilute it with the solvent to prepare a solution containing about 1 μg per 1 ml.

[0393] Using water∶acetonitrile (50∶50) as the diluent, prepare SMC, SMCZ2, and SMCZ3 solutions with a concentration of about 1 mg / ml to confirm the preliminary method. The results are as Figure 10 shown.

[0394] It can be seen from the figure that SMCZ2 and SMCZ3 show two unresolved chromatographic peaks, analyzed as peak shape splitting or unresolved adjacent impurities.

[0395] Control Example 9

[0396] A method for detecting related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0397] Chromatographic conditions:

[0398] Chromatographic column: Waters Symmetry C18 4.6×150 mm, 5 μm;

[0399] Trapping column: Ghost Buster Column, 4.6 mm×50 mm;

[0400] Mobile phase A: 0.05 wt% phosphoric acid aqueous solution;

[0401] Mobile phase B: acetonitrile;

[0402] Gradient elution:

[0403]

[0404] Injection volume: 5 μl;

[0405] Flow rate: 1.0 ml / min;

[0406] Column temperature: 30 °C;

[0407] Sample tray temperature: 10 °C;

[0408] Detection wavelength: 210 nm;

[0409] Preparation of solutions:

[0410] Solvent: water∶acetonitrile (50∶50);

[0411] Prepare a mixed solution of SMC (about 1 mg / ml) and each impurity (about 20 μg / ml) for injection and inspection, and add a trapping column to improve the baseline. The results are as Figure 11 shown.

[0412] As can be seen from the figure, the retention time of SMCZ3 is shifted to about 9 min, and the peak shape is good; SMCZ2 shows two unresolved chromatographic peaks.

[0413] Example 4

[0414] A method for detecting related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0415] Chromatographic conditions:

[0416] Chromatographic column: Octadecylsilyl-bonded silica gel as filler (YMC-Pack ODS-AQ, 4.6×150 mm, 3 μm);

[0417] Trapping column: Ghost Buster Column, 4.6 mm×50 mm;

[0418] Mobile phase A: Sodium dihydrogen phosphate buffer solution (take 6.0 g of sodium dihydrogen phosphate dihydrate, add 1000 ml of water, and adjust the pH to 2.5 with phosphoric acid);

[0419] Mobile phase B: Methanol∶acetonitrile (2∶1);

[0420] Gradient elution:

[0421]

[0422] Injection volume: 50 μl;

[0423] Flow rate: 1.0 ml / min;

[0424] Column temperature: 40 °C;

[0425] Detection wavelength: 205 nm;

[0426] Preparation of solutions:

[0427] Solvent: Water∶acetonitrile (50∶50);

[0428] Test solution: Take an appropriate amount of starting material SMD, dissolve it with the solvent and dilute it to make a solution containing about 1.0 mg per 1 ml;

[0429] Reference solution: Accurately measure an appropriate amount of the test solution, and dilute it with the solvent to make a solution containing about 2 μg per 1 ml.

[0430] Determination method:

[0431] Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, as Figure 12 shown.

[0432] It can be seen from the figure that baseline separation has been achieved, enabling interference-free detection, that is, good separation among components.

[0433] Control Example 10

[0434] A method for detecting related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0435] Chromatographic conditions:

[0436] Chromatographic column: Waters Altantis T3 4.6×150mm, 5μm;

[0437] Mobile phase A: Sodium dihydrogen phosphate buffer solution (take 3.1 g of sodium dihydrogen phosphate, add 1000 ml of water, and adjust the pH to 6.8 with 1 mol / L sodium hydroxide);

[0438] Mobile phase B: Acetonitrile;

[0439] Gradient elution:

[0440]

[0441] Injection volume: 50 μl;

[0442] Flow rate: 1.0 ml / min;

[0443] Column temperature: 40°C;

[0444] Detection wavelength: 205 nm;

[0445] Preparation of solutions:

[0446] Solvent: Mobile phase A∶Mobile phase B = 4∶1;

[0447] Prepare the test solution, the impurity localization solution and the impurity mixed solution with mobile phase A∶mobile phase B = 4∶1 as the solvent, and inject each solution for investigation. Among them, the high-concentration impurity solution uses water∶acetonitrile (50∶50) as the solvent, and the results are as Figures 13 - 15 shown in Table 7-8.

[0448] Table 7 Localization solution

[0449]

[0450]

[0451] Table 8 Method validation - Impurity mixed solution

[0452] Attribution Retention time (min) Relative retention time (RRT) Resolution Impurity SMDZ6 22.859 0.85 - Impurities SMDZ1, SMDZ4 24.774 0.92 5.49 Impurities SMDZ3, SMDZ8 26.071 0.97 2.73 Impurities SMDZ2, SMDZ5 27.687 1.03 1.57 Impurity SMDZ9 26.786 1.00 1.20

[0453] It can be concluded from the figure that in this method, the impurity SMDZ6 is baseline separated from the adjacent impurity, while the impurities SMDZ1 and SMDZ4, SMDZ2 and SMDZ5, and SMDZ3 and SMDZ8 overlap and peak pairwise in the mixed solution, failing to achieve separation. Additionally, the impurity SMDZ9 and the main component are enantiomers with the same retention time, thus failing to achieve separation.

[0454] Control Example 11

[0455] A method for detecting related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0456] Chromatographic conditions:

[0457] Chromatographic column: Waters Altantis T3 4.6×150mm, 5μm;

[0458] Mobile phase A: Sodium dihydrogen phosphate buffer solution (weigh 3.1 g of sodium dihydrogen phosphate, add 1000 ml of water, and adjust the pH to 6.8 with 1 mol / L sodium hydroxide);

[0459] Mobile phase B: Acetonitrile;

[0460] Gradient elution:

[0461]

[0462] Injection volume: 50 μl;

[0463] Flow rate: 1.0 ml / min;

[0464] Column temperature: 40°C;

[0465] Detection wavelength: 205 nm;

[0466] Preparation of solutions:

[0467] Using water:acetonitrile (50:50) as the solvent, prepare a mixed impurity solution of impurity SMD and impurities SMDZ1-6, SMDZ8-9. The results are as Figure 16 shown.

[0468] It can be seen from the figure that there is a separation trend between impurity SMDZ2 and impurity SMDZ5, but there is no separation trend between impurity SMDZ1 and impurity SMDZ4, and between impurity SMDZ3 and impurity SMDZ8.

[0469] Control Example 12

[0470] A method for detecting related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0471] Chromatographic conditions:

[0472] Chromatographic column: YMC-Pack ODS-AQ 4.6×150 mm, 3 μm;

[0473] Mobile phase A: Sodium dihydrogen phosphate buffer solution (weigh 3.1 g of sodium dihydrogen phosphate, add 1000 ml of water, adjust the pH to 2.5 with phosphoric acid);

[0474] Mobile phase B: Acetonitrile;

[0475] Gradient elution:

[0476]

[0477] Sample injection volume: 50 μl;

[0478] Flow rate: 1.0 ml / min;

[0479] Column temperature: 40 °C;

[0480] Detection wavelength: 205 nm;

[0481] Preparation of solution:

[0482] Solvent: Water∶Acetonitrile (50∶50);

[0483] Prepare a mixed solution of SMD and impurities SMDZ1 - SMDZ8, and inject the mixed solution for investigation. The results are as Figure 17 shown.

[0484] It can be concluded from the figure that the main component and 7 known impurities are detected in the mixed solution. Among them, there are unresolved double peaks at about RT 25 min and RT 27 min, and the sodium dihydrogen phosphate buffer solution with a pH value of 2.5 has no obvious differentiating effect on the peak emergence of each component.

[0485] Control Example 13

[0486] A method for detecting related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0487] Chromatographic conditions:

[0488] Chromatographic column: YMC-Pack ODS-AQ 4.6×150 mm, 3 μm;

[0489] Mobile phase A: Sodium dihydrogen phosphate buffer solution (weigh 3.1 g of sodium dihydrogen phosphate, add 1000 ml of water, adjust the pH to 5 with 1 mol / L sodium hydroxide);

[0490] Mobile phase B: Acetonitrile;

[0491] Gradient elution:

[0492]

[0493] Sample volume: 50 μl;

[0494] Flow rate: 1.0 ml / min;

[0495] Column temperature: 40 °C;

[0496] Detection wavelength: 205 nm;

[0497] Preparation of solution:

[0498] Solvent: water∶acetonitrile (50∶50);

[0499] Prepare a mixed solution of SMD and impurities SMDZ1 to SMDZ8, and inject the mixed solution for investigation. The results are as Figure 18 shown.

[0500] It can be concluded from the figure that the sodium dihydrogen phosphate buffer solution with a pH value of 5 has no obvious separation effect on the peak emergence of each component.

[0501] Control Example 14

[0502] A method for detecting related substances of carfilzomib starting materials, wherein the detection method includes the following detection conditions:

[0503] Chromatographic conditions:

[0504] Chromatographic column: YMC-Pack ODS-AQ 4.6×150 mm, 3 μm;

[0505] Mobile phase A: sodium dihydrogen phosphate buffer solution (take 3.1 g of sodium dihydrogen phosphate, add 1000 ml of water, and adjust the pH to 2.5 with phosphoric acid);

[0506] Mobile phase B: methanol∶acetonitrile (1∶1);

[0507] Gradient elution:

[0508]

[0509]

[0510] Sample volume: 50 μl;

[0511] Flow rate: 1.0 ml / min;

[0512] Column temperature: 40 °C;

[0513] Detection wavelength: 205 nm;

[0514] Preparation of solution:

[0515] Solvent: water∶acetonitrile (50∶50);

[0516] Test solution: Weigh an appropriate amount of starting material SMD accurately, dissolve and dilute it with the solvent to prepare a solution containing about 4 mg per 1 ml.

[0517] Reference solution: Accurately measure an appropriate amount of the test solution and dilute it with the solvent to prepare a solution containing about 2 μg per 1 ml.

[0518] Determination method:

[0519] Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram as Figure 19 shown.

[0520] The results showed that impurity SMDZ8 and impurity SMDZ3 failed to achieve baseline separation.

[0521] Control Example 15

[0522] A method for detecting related substances of carfilzomib starting material, wherein the detection method includes the following detection conditions:

[0523] Chromatographic conditions:

[0524] Chromatographic column: YMC-Pack ODS-AQ 4.6×150 mm, 3 μm;

[0525] Mobile phase A: Sodium dihydrogen phosphate buffer solution (weigh 3.1 g of sodium dihydrogen phosphate, add 1000 ml of water, adjust the pH to 2.5 with phosphoric acid);

[0526] Mobile phase B: Methanol∶acetonitrile (2∶1);

[0527] Gradient elution:

[0528]

[0529] Injection volume: 50 μl;

[0530] Flow rate: 1.0 ml / min;

[0531] Column temperature: 40°C;

[0532] Detection wavelength: 205 nm;

[0533] Preparation of solutions:

[0534] Solvent: water∶acetonitrile (50∶50);

[0535] Prepare a mixed solution of starting material SMD for investigation, and the results are as Figures 20 - 21 shown.

[0536] It can be concluded from the figure that the impurity SMDZ8 and the impurity SMDZ3 cannot be completely separated.

[0537] Although the embodiments disclosed in the present invention are as above, the above content is only the embodiments adopted for the convenience of understanding the present invention, and is not used to limit the present invention. Any person skilled in the art within the scope of the present invention can make any modifications and changes in the form and details of the implementation without departing from the spirit and scope disclosed by the present invention. However, the scope of patent protection of the present invention shall still be subject to the scope defined by the claims.

Claims

1. A method for detecting related substances of carfilzomib starting materials, characterized in that, Qualitative and / or quantitative detection is carried out by high performance liquid chromatography, and the detection method includes the following detection conditions: Chromatographic column: octadecylsilane bonded silica gel; Mobile phase: gradient elution is carried out using mobile phase A and mobile phase B. Among them, mobile phase A is an aqueous phosphoric acid solution or a phosphate buffer solution; mobile phase B is acetonitrile or a mixed solution of methanol and acetonitrile.

2. The detection method according to claim 1, wherein Mobile phase A is a phosphate buffer solution. Among them, the phosphate buffer solution is a potassium dihydrogen phosphate buffer solution. Preferably, the pH of the potassium dihydrogen phosphate buffer solution is 4.0; mobile phase B is acetonitrile; preferably, the gradient elution program is:

3. The detection method according to claim 1, wherein Mobile phase A is an aqueous phosphoric acid solution; preferably, the concentration of the aqueous phosphoric acid solution is 0.05 wt%; mobile phase B is acetonitrile; preferably, the gradient elution program is:

4. The detection method according to claim 1, wherein Mobile phase A is a phosphate buffer solution. Among them, the phosphate buffer solution is a potassium dihydrogen phosphate buffer solution. Preferably, the pH of the potassium dihydrogen phosphate buffer solution is 2.5; mobile phase B is acetonitrile; preferably, the gradient elution program is:

5. The detection method according to claim 1, characterized in that Mobile phase A is a phosphate buffer solution. Among them, the phosphate buffer solution is a sodium dihydrogen phosphate buffer solution. Preferably, the pH of the sodium dihydrogen phosphate buffer solution is 2.5; mobile phase B is a mixed solution of methanol and acetonitrile. Preferably, the volume ratio of methanol to acetonitrile is 2:1; Preferably, the gradient elution program is:

6. The detection method according to claim 2 or 4, characterized in that, The preparation method of the potassium dihydrogen phosphate buffer solution is: weigh 3.0 g of potassium dihydrogen phosphate, dilute it with water to 1000 ml, and adjust the pH to 4.0 or 2.5 with phosphoric acid.

7. The detection method according to claim 5, wherein The preparation method of the sodium dihydrogen phosphate buffer solution is: weigh 6.0 g of sodium dihydrogen phosphate dihydrate, add 1000 ml of water, and adjust the pH to 2.5 with phosphoric acid.

8. The detection method according to any one of claims 2-7, characterized in that, The detection conditions also include one or more of the following: Column temperature: 25 - 40 °C, preferably, column temperature: 30 °C or 40 °C; Flow rate: 1.0 ml / min; Detection wavelength: 205 - 220 nm, preferably, detection wavelength: 205 nm or 210 nm; Injection volume: 5 - 60 μl; preferably, injection volume: 5 μl, 20 μl or 50 μl.

9. The detection method according to claim 8, wherein The specifications of the chromatographic column are: YMC-Pack ODS-AQ, 4.6×150 mm, 3 μm or Waters Symmetry C18 4.6×150 mm, 5 μm; Preferably, the detection conditions also include the use of a trapping column. Optionally, the specifications of the trapping column are: GhostBuster Column, 4.6 mm×50 mm.

10. The detection method according to claim 9, wherein, The detection conditions further include using the starting materials SMA, SMB, SMC or SMD of carfilzomib as the test samples; using one or more selected from SMAZ1, SMAZ2, SMAZ3, SMAZ4, SMAZ5, SMBZ1, SMBZ2, SMBZ3, SMBZ4, SMCZ1, SMCZ2, SMCZ3 or SMDZ1, SMDZ2, SMDZ3, SMDZ4, SMDZ5, SMDZ6, SMDZ7, SMDZ8, SMDZ9 as the controls, and preparing the test sample solution and the control solution; Preparation of the test sample solution: Take an appropriate amount of the test sample, dissolve and dilute it with a solvent to prepare a solution containing about 1.0 mg or 4.0 mg per 1 ml; Preparation of the control solution: Accurately measure an appropriate amount of the test sample solution, and dilute it with a solvent to prepare a solution containing about 1 μg, 2 μg or 20 μg per 1 ml; Preferably, the solvent is a mixed solution of water and acetonitrile with a volume ratio of 50:50 or a mixed solution of 0.05 wt% phosphoric acid aqueous solution and acetonitrile with a volume ratio of 9:1.

Images