Analysis and detection method of carfilzomib starting material isomer

Abstract

The invention relates to a method for analyzing and detecting isomers of a carfilzomib starting material, which adopts a normal-phase chiral chromatographic column, takes a mixed solution containing normal hexane and C2-C3 alcohol as a mobile phase, effectively separates the isomers of the carfilzomib starting material from the carfilzomib starting material and impurities of the carfilzomib starting material, and has a good separation effect.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical analysis and quality control, and particularly to an analytical detection method for isomers of the starting material of carfilzomib. 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 developed by Onyx Pharmaceuticals through the priority review process, with the trade name: Kyprolis. Carfilzomib is the second proteasome inhibitor approved globally after bortezomib and is also the first therapeutic drug for multiple myeloma approved by the FDA in recent years. Carfilzomib is a new generation of protease inhibitor administered intravenously, selectively targeting the proteasome in hematological tumor cells and avoiding the toxic side effects caused by inhibiting the constitutive proteasome in non-malignant cells. Its structural formula is shown below:

[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.

[0005] SMA, SMC, and SMD are important starting materials for the production of carfilzomib, and their structures are as follows:

[0006]

[0007] Chiral centers exist in the molecules of the starting materials SMA, SMC, and SMD. The chiral purity of the starting materials and their isomers directly affects the purity of carfilzomib and the control of impurity content, thus directly affecting the efficacy of carfilzomib. Currently, there is no detection method for the isomers of the starting material of carfilzomib. Summary of the Invention

[0008] In view of the problems existing in the prior art, the present invention provides an analytical detection method for isomers of the starting material of carfilzomib, which can effectively separate the isomers of the starting material of carfilzomib from the starting material of carfilzomib and its impurities, thereby achieving better quality control of carfilzomib.

[0009] Specifically, the isomers referred to in the present invention are enantiomers.

[0010] Specifically, the present invention provides a method for analyzing and detecting the isomers of carfilzomib starting materials. The carfilzomib starting materials include SMA, SMC or SMD, and high performance liquid chromatography is used for qualitative and / or quantitative detection. The detection conditions include:

[0011] The chromatographic column is a normal phase chiral chromatographic column;

[0012] The mobile phase contains n-hexane and C2-C3 alcohol.

[0013] The present invention uses a normal phase chiral chromatographic column and a mixed solution containing n-hexane and C2-C3 alcohol as the mobile phase, which can effectively separate the isomers of carfilzomib starting materials from the carfilzomib starting materials and their impurities.

[0014] In some embodiments of the present invention, the isomer of the starting material SMA is SMAZ1. The impurities related to the starting material SMA are selected from one or more of SMAZ2, SMAZ3, SMAZ4 and SMAZ5.

[0015] In some embodiments of the present invention, the isomer of the starting material SMC is SMCZ1. The impurities related to the starting material SMC are selected from one or two of SMCZ2 and SMCZ3.

[0016] In some embodiments of the present invention, the isomer of the starting material SMD is SMDZ9. The impurities related to the starting material SMD are selected from one or more of SMDZ1, SMDZ2, SMDZ3, SMDZ4, SMDZ5, SMDZ6, SMDZ7, SMDZ8 and SMDZ10.

[0017] The structures of the carfilzomib starting materials, the isomers of the starting materials and the impurities related to the starting materials involved in the present invention are as follows:

[0018]

[0019]

[0020]

[0021] In some embodiments of the present invention, the mobile phase includes mobile phase A, and mobile phase A is a mixed solution of n-hexane and C2-C3 alcohol, and isocratic elution is carried out using mobile phase A.

[0022] In some embodiments of the present invention, the mobile phase includes mobile phase A and mobile phase B. Mobile phase A is a mixed solution of n-hexane and C2-C3 alcohol, and mobile phase B is a mixed solution of n-hexane and isopropanol. Gradient elution is carried out using mobile phase A and mobile phase B.

[0023] Preferably, the following elution procedure can be adopted for the gradient elution:

[0024]

[0025] In some embodiments of the present invention, in the case of isocratic elution using the mobile phase A, in the mobile phase A, the volume ratio of n-hexane to C2-C3 alcohol is (80-85):(15-20), such as 80:20, 81:19, 82:18, 83:17, 84:16 or 85:15.

[0026] Preferably, in the case of isocratic elution using the mobile phase A, the mobile phase A further comprises trifluoroacetic acid. Adding trifluoroacetic acid can adjust the pH of the eluent, and at the same time has a large ionic strength and a certain ion pair effect, which can enhance the separation effect and significantly improve the sample peak shape. In the mobile phase A, the volume ratio of n-hexane, C2-C3 alcohol and trifluoroacetic acid is preferably (80-85):(15-20):(0.1-0.2), such as 80:20:0.1, 81:19:0.1, 82:18:0.1, 83:17:0.1, 84:16:0.1, 85:15:0.1, 80:20:0.15, 81:19:0.15, 82:18:0.15, 83:17:0.15, 84:16:0.15, 85:15:0.15, 80:20:0.2, 81:19:0.2, 82:18:0.2, 83:17:0.2, 84:16:0.2 or 85:15:0.2.

[0027] Preferably, in the case of isocratic elution using the mobile phase A, the mobile phase A is a mixed solution of n-hexane, isopropanol and trifluoroacetic acid with a volume ratio of 85:15:0.1.

[0028] Preferably, in the case of isocratic elution using the mobile phase A, the mobile phase A is a mixed solution of n-hexane, isopropanol, ethanol and trifluoroacetic acid with a volume ratio of 80:15:5:0.1.

[0029] In some embodiments of the present invention, in the case of gradient elution using the mobile phase A and the mobile phase B, in the mobile phase A, the volume ratio of n-hexane to C2-C3 alcohol is (90-99):(1-10), and in the mobile phase B, the volume ratio of n-hexane to isopropanol is (90-99):(1-10).

[0030] In some specific embodiments, when gradient elution is performed using the mobile phase A and the mobile phase B, in the mobile phase A, the volume ratio of n-hexane to C2-C3 alcohol is 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2 or 99:1.

[0031] In some specific embodiments, when gradient elution is performed using the mobile phase A and the mobile phase B, in the mobile phase B, the volume ratio of n-hexane to isopropanol is 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2 or 99:1.

[0032] Preferably, when gradient elution is performed using the mobile phase A and the mobile phase B, the mobile phase A is a mixed solution of n-hexane and isopropanol with a volume ratio of (95-99):(1-5); and the mobile phase B is a mixed solution of n-hexane and isopropanol with a volume ratio of (90-94):(6-10).

[0033] More preferably, when gradient elution is performed using the mobile phase A and the mobile phase B, the mobile phase A is a mixed solution of n-hexane and isopropanol with a volume ratio of 99:1; and the mobile phase B is a mixed solution of n-hexane and isopropanol with a volume ratio of 90:10.

[0034] In some embodiments of the present invention, the starting material is SMA, the starting material isomer is SMAZ1, and the detection conditions include:

[0035] Chromatographic column: normal-phase chiral chromatographic column, preferably Chrial NQ(2) or its equivalent chromatographic column;

[0036] Mobile phase A: a mixed solution of n-hexane, isopropanol and trifluoroacetic acid;

[0037] Elution procedure: isocratic elution is performed using the mobile phase A.

[0038] Preferably, in the mobile phase A, the volume ratio of n-hexane, isopropanol, and trifluoroacetic acid is (80 - 85):(15 - 20):(0.1 - 0.2), such as 80:20:0.1, 81:19:0.1, 82:18:0.1, 83:17:0.1, 84:16:0.1, 85:15:0.1, 80:20:0.15, 81:19:0.15, 82:18:0.15, 83:17:0.15, 84:16:0.15, 85:15:0.15, 80:20:0.2, 81:19:0.2, 82:18:0.2, 83:17:0.2, 84:16:0.2, or 85:15:0.2, and preferably 85:15:0.1.

[0039] Preferably, the specification of Chrial NQ(2) or its equivalent chromatographic column is 4.6 mm × 250 mm, 3 μm - 5 μm, and preferably 5 μm.

[0040] Preferably, the column temperature is 30 - 40 °C, such as 30 °C, 32 °C, 34 °C, 36 °C, 38 °C, or 40 °C, and preferably 30 °C.

[0041] In some embodiments of the present invention, the starting material is SMC, and the starting material isomer is SMCZ1. The detection conditions include:

[0042] Chromatographic column: normal-phase chiral chromatographic column, preferably CHIRALPAK AD-H or its equivalent chromatographic column;

[0043] Mobile phase A: a mixed solution of n-hexane, isopropanol, ethanol, and trifluoroacetic acid;

[0044] Elution program: isocratic elution is carried out using the mobile phase A.

[0045] Preferably, in the mobile phase A, the volume ratio of n-hexane, isopropanol, ethanol, and trifluoroacetic acid is (80 - 85):(10 - 15):(5 - 10):(0.1 - 0.2), such as 80:15:5:0.1, 80:10:10:0.1, 80:5:15:0.1, 85:10:5:0.1, 85:5:10:0.1, 80:15:5:0.15, 80:10:10:0.15, 80:5:15:0.15, 85:10:5:0.15, 85:5:10:0.15, 80:15:5:0.2, 80:10:10:0.2, 80:5:15:0.2, 85:10:5:0.2, or 85:5:10:0.2, and preferably 80:15:5:0.1.

[0046] Preferably, the specifications of CHIRALPAK AD-H or its equivalent chromatographic column are 4.6 mm × 250 mm, 3 μm - 5 μm, preferably 5 μm.

[0047] Preferably, the flow rate is 0.5 - 1.2 mL / min, for example 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min or 1.2 mL / min, preferably 0.6 mL / min.

[0048] In some embodiments of the present invention, the starting material is SMD, the starting material isomer is SMDZ9, and the related impurities of the starting material are SMDZ3, SMDZ8 and SMDZ10. The detection conditions include:

[0049] Chromatographic column: normal-phase chiral chromatographic column, preferably Chrial MX(2) or its equivalent chromatographic column;

[0050] Mobile phase A: a mixed solution of n-hexane and isopropanol; the volume ratio of n-hexane to isopropanol is preferably (95 - 99):(1 - 5);

[0051] Mobile phase B: a mixed solution of n-hexane and isopropanol; the volume ratio of n-hexane to isopropanol is preferably (90 - 94):(6 - 10);

[0052] Elution program: gradient elution is carried out using the mobile phase A and the mobile phase B.

[0053] Preferably, the following elution program is used for the gradient elution:

[0054]

[0055] In some specific embodiments, in the mobile phase A, the volume ratio of n-hexane to isopropanol is 99:1, 98:2, 97:3, 96:4 or 95:5. Preferably, in the mobile phase A, the volume ratio of n-hexane to isopropanol is 99:1.

[0056] In some specific embodiments, in the mobile phase B, the volume ratio of n-hexane to isopropanol is 90:10, 91:9, 92:8, 93:7 or 94:6. Preferably, in the mobile phase B, the volume ratio of n-hexane to isopropanol is 90:10.

[0057] Preferably, the specifications of Chrial MX(2) or its equivalent chromatographic column are 4.6 mm × 250 mm, 3 μm - 5 μm, preferably 5 μm.

[0058] In some embodiments of the present invention, the C2-C3 alcohol includes one or more of ethanol, propanol, and isopropanol. Preferably, the C2-C3 alcohol includes one or two of ethanol and isopropanol.

[0059] In some embodiments of the present invention, the detection conditions further include one or more of the following:

[0060] The normal-phase chiral chromatographic column is Chrial NQ(2), CHIRALPAK AD-H, Chrial MX(2), or an equivalent chromatographic column; preferably, the specification of the normal-phase chiral chromatographic column is 4.6 mm × 250 mm, 3 μm - 5 μm, preferably 5 μm;

[0061] The column temperature is 20 - 40 °C;

[0062] The flow rate is 0.5 - 1.2 mL / min;

[0063] The detection wavelength is 200 - 250 nm;

[0064] The injection volume is 5 - 50 μL, preferably 8 - 20 μL.

[0065] In some specific embodiments, the column temperature is 20 °C, 22 °C, 24 °C, 26 °C, 28 °C, 30 °C, 32 °C, 34 °C, 36 °C, 38 °C, or 40 °C.

[0066] In some specific embodiments, the flow rate is 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, or 1.2 mL / min.

[0067] In some specific embodiments, the detection wavelength is 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, or 250 nm.

[0068] In some specific embodiments, the injection volume is 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, or 50 μL.

[0069] In some embodiments of the present invention, the detection conditions of the analytical detection method further include preparing one or more of the following solutions:

[0070] Diluent: n-hexane - ethanol, the volume ratio is preferably (80 - 100):(0 - 20);

[0071] Test solution: Weigh accurately the starting materials SMA, SMC or SMD of carfilzomib, dissolve and make up the volume with the diluent to obtain the test solution, the concentration of which is 1 - 10 mg / mL;

[0072] Control solution: Measure accurately the test solution, dilute and make up the volume with the diluent to obtain the control solution, the concentration of which is 0.001 - 0.2 mg / mL.

[0073] In some specific embodiments, in the diluent, the volume ratio of n - hexane to ethanol can be 80:20, 85:15, 90:10, 95:5 or 100:0.

[0074] In some specific embodiments, the concentration of the test solution is 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL or 10 mg / mL.

[0075] In some specific embodiments, the preparation of the test solution includes: Weigh accurately 25 - 100 mg of the starting materials SMA, SMC or SMD of carfilzomib, place in a 10 - 25 ml volumetric flask, add an appropriate amount of the diluent, shake to dissolve and dilute to the scale, and mix well.

[0076] In some specific embodiments, the concentration of the control solution is 0.001 mg / mL, 0.002 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, 0.1 mg / mL, 0.12 mg / mL, 0.14 mg / mL, 0.16 mg / mL, 0.18 mg / mL or 0.2 mg / mL.

[0077] In some specific embodiments, the preparation of the control solution includes: Measure accurately 1 mL of the test solution, place in a 50 - 100 ml volumetric flask, dilute to the scale with the diluent, mix well to obtain a diluted solution, then measure accurately 5 ml of the diluted solution, place in a 50 ml volumetric flask, dilute to the scale with the diluent, and mix well.

[0078] Compared with the prior art, the beneficial effects of the present invention:

[0079] The present invention provides an analytical detection method for the isomers of the starting materials of carfilzomib. Using a normal - phase chiral chromatographic column and a mixed solution containing n - hexane and C2 - C3 alcohol as the mobile phase, the isomers of the starting materials of carfilzomib are effectively separated from the starting materials of carfilzomib and their impurities, and the separation effect is good. Moreover, the retention time of the main peak is appropriate, and the impurities in the test solution are completely eluted. Description of the Drawings

[0080] Figure 1 Chromatogram of the detection method for Example 1;

[0081] Figure 2 Chromatogram of the detection method for Comparative Example 1;

[0082] Figure 3 Chromatogram of the detection method for Comparative Example 2;

[0083] Figure 4 Chromatogram of the detection method for Comparative Example 3;

[0084] Figure 5 Chromatogram of the detection method for Comparative Example 4;

[0085] Figure 6 Chromatogram of the detection method for Comparative Example 5;

[0086] Figure 7 Chromatogram of the detection method for Comparative Example 6;

[0087] Figure 8 Chromatogram of the detection method for Example 2;

[0088] Figure 9 Chromatogram of the detection method for Comparative Example 7;

[0089] Figure 10 Chromatogram of the detection method for Comparative Example 8;

[0090] Figure 11 Chromatogram of the detection method for Comparative Example 9;

[0091] Figure 12 Chromatogram of the detection method for Comparative Example 10;

[0092] Figure 13 Chromatogram of the detection method for Comparative Example 11;

[0093] Figure 14 Chromatogram of the detection method for Example 3;

[0094] Figure 15 Chromatogram of the detection method for Comparative Example 12;

[0095] Figure 16 Chromatogram of the detection method for Comparative Example 13;

[0096] Figure 17 Chromatogram of the detection method for Comparative Example 14;

[0097] Figure 18 Chromatogram of the detection method of Control Example 15;

[0098] Figure 19 Chromatogram of the detection method of Control Example 16. Detailed implementation manners

[0099] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0100] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. The experimental reagents used in the following embodiments are all conventional biochemical reagents unless otherwise specified; the raw materials, instruments and equipment used in the following embodiments can all be obtained through market purchase or can be obtained by existing methods; the dosage of the experimental reagents is the dosage of the reagents in conventional experimental operations unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified.

[0101] The starting materials of carfilzomib and each impurity used in the embodiments and control examples of the present invention are all commercially available samples.

[0102] Among them, the standard limit is:

[0103] In the chromatogram of the test solution, if there are isomer peaks, the peak area shall not be greater than the main peak area of the control solution (0.1% or 0.2%).

[0104] The calculation formula is:

[0105]

[0106] Among them,

[0107] A 异构体 represents the peak area of the isomer in the test solution;

[0108] A 对照 represents the main peak area in the control solution;

[0109] 500 or 1000 represents the dilution factor of the control solution.

[0110] The analysis and detection method of the present invention will be further described below through specific examples.

[0111] Example 1

[0112] A detection method for isomers of carfilzomib starting materials, the detection conditions adopted by the method include:

[0113] Chromatographic conditions:

[0114] Chromatographic column: normal-phase chiral chromatographic column (Chrial NQ(2) 4.6 mm × 250 mm, 5 μm);

[0115] Mobile phase: n-hexane - isopropanol - trifluoroacetic acid (volume ratio 85:15:0.1);

[0116] Sample injection volume: 20 μl;

[0117] Flow rate: 0.5 ml / min;

[0118] Column temperature: 35 °C;

[0119] Detection wavelength: 210 nm;

[0120] Running time: 40 min;

[0121] Preparation of solutions:

[0122] Diluent (blank solution): n-hexane - ethanol (volume ratio 90:10).

[0123] Test solution: Weigh accurately 100 mg of the starting material SMA of carfilzomib, place it in a 10-ml volumetric flask, add an appropriate amount of diluent, shake to dissolve SMA, and dilute to the mark with diluent, then mix well.

[0124] Reference solution: Pipette accurately 1 ml of the test solution, place it in a 100-ml volumetric flask, dilute to the mark with diluent, and mix well to obtain a diluted solution. Then pipette accurately 5 ml of the diluted solution, place it in a 50-ml volumetric flask, dilute to the mark with diluent, and mix well.

[0125] Determination method: Pipette accurately 20 μl each of the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, as Figure 1 shown.

[0126] As can be seen from Figure 1 it, by using a Chrial NQ(2) chromatographic column and a mobile phase selected from n-hexane, isopropanol and trifluoroacetic acid with a volume ratio of 85:15:0.1, effective separation of the starting material SMA from its isomer SMAZ1 can be achieved, with a resolution greater than 1.5 and the retention time of the main peak advanced.

[0127] Verification of the analytical method:

[0128] Perform a validation study on the analytical methodology for the starting material isomer SMAZ1 that needs to be controlled.

[0129] The methodological verification indicators mainly include: system suitability, specificity, detection limit and quantification limit, linearity, repeatability, accuracy, range, solution stability, durability, and sample detection. The verification results are shown in Table 1 below.

[0130] Table 1 Analytical Methodological Verification Results of Isomer SMAZ1 of Starting Material SMA

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137] Comparative Example 1 A detection method for isomers of carfilzomib starting material, and the detection conditions adopted by the method include:

[0138] Chromatographic conditions:

[0139] Chromatographic column: normal-phase chiral chromatographic column (AS-3, 4.6 mm × 150 mm, 3 μm);

[0140] Mobile phase A: n-hexane;

[0141] Mobile phase B: Take 1.0 ml of trifluoroacetic acid, add 1000 ml of ethanol, and mix well;

[0142] Elution program:

[0143]

[0144] Injection volume: 3 μl;

[0145] Flow rate: 0.5 ml / min;

[0146] Column temperature: 25 °C;

[0147] Detection wavelength: 210 nm;

[0148] Preparation of solution: The same as in Example 1.

[0149] The detection results are as Figure 2 shown.

[0150] As shown in the figure, under these chromatographic conditions, the starting material SMA has an early peak elution time and a too short retention time, which is relatively close to the blank peak. When the concentration of the main component increases, the peak may broaden and overlap with the blank negative peak, affecting the peak shape.

[0151] Control Example 2

[0152] A method for detecting the isomers of carfilzomib starting materials, the detection conditions adopted by the method include:

[0153] Chromatographic conditions:

[0154] Chromatographic column: normal-phase chiral chromatographic column (CHIRALPAK AD-H, 4.6 mm × 250 mm, 5 μm);

[0155] Mobile phase A: n-hexane;

[0156] Mobile phase B: Take 1.0 ml of trifluoroacetic acid, add 1000 ml of ethanol, and mix well;

[0157] Elution program:

[0158]

[0159] Injection volume: 3 μl;

[0160] Flow rate: 0.5 ml / min;

[0161] Column temperature: 25 °C;

[0162] Detection wavelength: 210 nm;

[0163] Preparation of solution: same as Example 1;

[0164] The detection results are as Figure 3 shown.

[0165] Control Example 3

[0166] A method for detecting the isomers of carfilzomib starting materials, the detection conditions adopted by the method include:

[0167] Chromatographic conditions:

[0168] Chromatographic column: normal-phase chiral chromatographic column (CHIRALPAK AD-H, 4.6 mm × 250 mm, 5 μm);

[0169] Mobile phase A: n-hexane;

[0170] Mobile phase B: Take 1.0 ml of trifluoroacetic acid, add 1000 ml of isopropanol, and mix well;

[0171] Elution program:

[0172]

[0173] Injection volume: 3 μl;

[0174] Flow rate: 0.5 ml / min;

[0175] Column temperature: 25 °C;

[0176] Detection wavelength: 210 nm;

[0177] Preparation of solution: The same as in Example 1.

[0178] The detection results are as Figure 4 shown.

[0179] From Figure 3 and Figure 4 it can be concluded that the separation of the starting material SMA from its enantiomer SMAZ1 can be achieved using an AD-H chromatographic column; however, the baseline of the chromatogram is unstable.

[0180] Comparative Example 4

[0181] A method for detecting the isomers of the starting material of carfilzomib, the detection conditions used in the method include:

[0182] Chromatographic conditions:

[0183] Chromatographic column: normal-phase chiral chromatographic column (CHIRALPAK AD-H, 4.6 mm × 250 mm, 5 μm);

[0184] Mobile phase: n-hexane - isopropanol - trifluoroacetic acid (volume ratio 80:20:0.1);

[0185] Sample injection volume: 3 μl;

[0186] Flow rate: 0.5 ml / min;

[0187] Column temperature: 25 °C;

[0188] Detection wavelength: 210 nm;

[0189] Preparation of solution: The same as in Example 1.

[0190] The detection results are as Figure 5 shown.

[0191] From Figure 5 it can be seen that the relevant impurities in the starting material SMA interfere with the detection of the enantiomer SMAZ1.

[0192] Comparative Example 5

[0193] A method for detecting the isomers of the starting material of carfilzomib, the detection conditions used in the method include:

[0194] Chromatographic conditions:

[0195] Chromatographic column: normal-phase chiral chromatographic column (Chrial NQ(2), 4.6 mm × 250 mm, 5 μm);

[0196] Mobile phase: n-hexane - ethanol - trifluoroacetic acid (volume ratio 95:5:0.1);

[0197] Sample injection volume: 3 μl;

[0198] Flow rate: 0.5 ml / min;

[0199] Column temperature: 25 °C;

[0200] Detection wavelength: 210 nm;

[0201] Preparation of solution: the same as in Example 1.

[0202] The detection results are as Figure 6 shown.

[0203] From Figure 6 it can be concluded that when using Chrial NQ(2) as the chromatographic column and n-hexane, ethanol and trifluoroacetic acid as the mobile phase, the separation of the starting material SMA and its enantiomer SMAZ1 cannot be achieved.

[0204] Control Example 6

[0205] A method for detecting the isomers of the starting material of carfilzomib, the detection conditions adopted by the method include:

[0206] Chromatographic conditions:

[0207] Chromatographic column: normal-phase chiral chromatographic column (Chrial NQ(2), 4.6 mm × 250 mm, 5 μm);

[0208] Mobile phase: n-hexane - ethanol - trifluoroacetic acid (volume ratio 85:15:0.1);

[0209] Sample injection volume: 3 μl;

[0210] Flow rate: 0.5 ml / min;

[0211] Column temperature: 25 °C;

[0212] Detection wavelength: 210 nm;

[0213] Preparation of solution: the same as in Example 1.

[0214] The detection results are as Figure 7 shown.

[0215] From Figure 7It can be obtained that when using Chrial NQ(2) as the chromatographic column, after adjusting the volume ratio of the mobile phase of n-hexane, ethanol and trifluoroacetic acid to 85:15:0.1, the separation of the starting material SMA and its enantiomer SMAZ1 can be achieved, but the retention time of the main peak is relatively late.

[0216] Example 2

[0217] A method for detecting the isomers of carfilzomib starting materials, the detection conditions adopted by the method include:

[0218] Chromatographic conditions:

[0219] Chromatographic column: polysaccharide derivative-coated chiral chromatographic column (CHIRALPAK AD-H 4.6mm×250mm, 5μm);

[0220] Mobile phase: n-hexane - isopropanol - ethanol - trifluoroacetic acid (volume ratio 80:15:5:0.1);

[0221] Injection volume: 8μl;

[0222] Flow rate: 0.6ml / min;

[0223] Column temperature: 35°C;

[0224] Detection wavelength: 210nm;

[0225] Running time: 40min;

[0226] Preparation of solutions:

[0227] Diluent: n-hexane - ethanol (volume ratio 80:20).

[0228] Test solution: Weigh accurately 25mg of the carfilzomib starting material SMC, place it in a 25ml volumetric flask, add an appropriate amount of diluent to dissolve SMC, and dilute to the scale with diluent, then shake well.

[0229] Control solution: Accurately measure 1ml of the test solution, place it in a 100ml volumetric flask, dilute to the scale with diluent, then shake well to obtain a diluted solution. Then accurately measure 5ml of the diluted solution, place it in a 50ml volumetric flask, dilute to the scale with diluent, and shake well.

[0230] Determination method:

[0231] Accurately measure the test solution and the control solution, inject them into the liquid chromatograph respectively, record the chromatogram, as Figure 8 shown.

[0232] From Figure 8 it can be obtained that the starting material SMC and its isomers and impurities can be effectively separated, and the retention time is advanced.

[0233] Verification of the analysis method:

[0234] A methodological verification study of the analysis method was conducted on the starting material isomer SMCZ1 that needs to be controlled.

[0235] The methodological verification indicators mainly include: system suitability, specificity, detection limit and quantification limit, linearity, repeatability, accuracy, range, solution stability, durability, and sample detection. The verification results are shown in Table 2 below.

[0236] Table 2 Methodological verification results of the isomer SMCZ1 of the starting material SMC

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243] Control Example 7

[0244] A detection method for the isomer of the carfilzomib starting material, and the detection conditions adopted by the method include: Chromatographic conditions:

[0245] Chromatographic column: normal-phase chiral chromatographic column AS-3, 4.6 mm × 150 mm, 3 μm;

[0246] Mobile phase A: n-hexane;

[0247] Mobile phase B: Take 1.0 ml of trifluoroacetic acid, add 1000 ml of ethanol, and mix well;

[0248] Elution program:

[0249]

[0250]

[0251] Injection volume: 3 μl;

[0252] Flow rate: 0.5 ml / min;

[0253] Column temperature: 25 °C;

[0254] Detection wavelength: 210 nm;

[0255] Preparation of solution: The same as in Example 2.

[0256] The test results are as Figure 9 shown.

[0257] It can be seen from Figure 9 that the separation of the starting material SMC of carfilzomib from its enantiomer SMCZ1 cannot be achieved using AS-3.

[0258] Comparative Example 8

[0259] A method for detecting the isomers of the starting material of carfilzomib, the detection conditions adopted by the method include:

[0260] Chromatographic conditions:

[0261] Chromatographic column: normal-phase chiral chromatographic column CHIRALPAK AD-H, 4.6 mm × 250 mm, 5 μm;

[0262] Mobile phase A: n-hexane;

[0263] Mobile phase B: Take 1.0 ml of trifluoroacetic acid, add 1000 ml of ethanol, and mix well;

[0264] Elution program:

[0265]

[0266] Sample injection volume: 3 μl;

[0267] Flow rate: 0.5 ml / min;

[0268] Column temperature: 25 °C;

[0269] Detection wavelength: 210 nm;

[0270] Preparation of solution: The same as in Example 2.

[0271] The test results are as Figure 10 shown.

[0272] It can be concluded from Figure 10 that the starting material SMC of carfilzomib can be separated from its enantiomer SMCZ1 using the CHIRALPAK AD-H chromatographic column, but the baseline is unstable.

[0273] Comparative Example 9

[0274] A method for detecting the isomers of the starting material of carfilzomib, the detection conditions adopted by the method include:

[0275] Chromatographic conditions:

[0276] Chromatographic column: normal-phase chiral chromatographic column CHIRALPAK AD-H, 4.6 mm × 250 mm, 5 μm;

[0277] Mobile phase: n-hexane - isopropanol - trifluoroacetic acid (volume ratio 82:18:0.1);

[0278] Sample injection volume: 20 μl;

[0279] Flow rate: 0.5 ml / min;

[0280] Column temperature: 35 °C;

[0281] Detection wavelength: 210 nm;

[0282] Running time: 30 min;

[0283] Preparation of solution: same as in Example 2.

[0284] The detection results are as Figure 11 shown.

[0285] From Figure 11 it can be concluded that when using the CHIRALPAK AD-H chromatographic column and n-hexane, isopropanol, and trifluoroacetic acid as the mobile phase, the starting material SMC of carfilzomib can be separated from its enantiomer SMCZ1, but the impurities in the starting material SMC interfere with the detection of the enantiomer SMCZ1.

[0286] Comparative Example 10

[0287] A method for detecting the isomers of the starting material of carfilzomib, the detection conditions adopted by the method include:

[0288] Chromatographic conditions:

[0289] Chromatographic column: normal-phase chiral chromatographic column CHIRALPAK AD-H, 4.6 mm × 250 mm, 5 μm;

[0290] Mobile phase: n-hexane - ethanol - trifluoroacetic acid (volume ratio 82:18:0.1);

[0291] Sample injection volume: 20 μl;

[0292] Flow rate: 0.5 ml / min;

[0293] Column temperature: 35 °C;

[0294] Detection wavelength: 210 nm;

[0295] Running time: 30 min;

[0296] Preparation of solution: same as in Example 1.

[0297] The detection results are as Figure 12 shown.

[0298] It can be concluded from Figure 12 that when using a CHIRALPAK AD-H chromatographic column and n-hexane, ethanol, and trifluoroacetic acid as the mobile phase, the starting material SMC of carfilzomib can be separated from its enantiomer SMCZ1, but the impurities in the starting material SMC interfere with the detection of the enantiomer SMCZ1.

[0299] Control Example 11

[0300] A method for detecting the isomers of the starting material of carfilzomib, the detection conditions adopted by the method include:

[0301] Chromatographic conditions:

[0302] Chromatographic column: normal-phase chiral chromatographic column CHIRALPAK AD-H, 4.6 mm × 250 mm, 5 μm;

[0303] Mobile phase: n-hexane - isopropanol - ethanol - trifluoroacetic acid (volume ratio 80:15:5:0.1);

[0304] Injection volume: 20 μl;

[0305] Flow rate: 0.5 ml / min;

[0306] Column temperature: 35 °C;

[0307] Detection wavelength: 210 nm;

[0308] Running time: 40 min;

[0309] Preparation of solution: same as Example 1.

[0310] The detection results are as Figure 13 shown.

[0311] It can be concluded from the figure that this method can effectively separate the starting material SMC from its enantiomer and related impurities, but the retention time of the main peak of the starting material SMC is relatively late.

[0312] Example 3

[0313] A method for detecting the isomers of the starting material of carfilzomib, the detection conditions adopted by the method include:

[0314] Chromatographic conditions:

[0315] Chromatographic column: Chrial MX(2) 4.6 × 250 mm, 5 μm;

[0316] Mobile phase A: n-hexane - isopropanol (volume ratio 99:1);

[0317] Mobile phase B: n-hexane - isopropanol (volume ratio 90:10);

[0318] The gradient elution program is as follows:

[0319]

[0320] Sample injection volume: 15 μl;

[0321] Flow rate: 0.8 ml / min;

[0322] Column temperature: 25 °C;

[0323] Detection wavelength: 210 nm;

[0324] Preparation of solutions:

[0325] Diluent: n-hexane.

[0326] Test solution: Weigh accurately 100 mg of the starting material SMD of carfilzomib, place it in a 10-ml volumetric flask, add an appropriate amount of n-hexane to dissolve SMD, and dilute to the mark with the diluent, then shake well.

[0327] Reference solution: Pipette accurately 1 ml of the test solution into a 50-ml volumetric flask, dilute to the mark with the diluent, and shake well to obtain a diluted solution. Then pipette accurately 5 ml of the diluted solution into a 50-ml volumetric flask, dilute to the mark with the diluent, and shake well.

[0328] Determination method:

[0329] Pipette accurately 15 μl each of the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram as Figure 14 shown.

[0330] Verification of the analytical method:

[0331] Carry out a validation study on the analytical methodology for the starting material isomer SMDZ9 that needs to be controlled.

[0332] The validation indicators of the methodology mainly include: system suitability, specificity, detection limit and quantitation limit, linearity, repeatability, accuracy, range, solution stability, robustness, and sample detection. The validation results are shown in Table 3 below.

[0333] Table 3 Validation study on the analytical methodology for the starting material isomer SMDZ9

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340] Control Example 12 A method for detecting the isomers of carfilzomib starting materials, the detection conditions adopted by the method include:

[0341] Chromatographic conditions:

[0342] Chromatographic column: normal-phase chiral chromatographic column AS-3, 4.6 mm × 150 mm, 3 μm;

[0343] Mobile phase A: n-hexane;

[0344] Mobile phase B: Take 1.0 ml of trifluoroacetic acid, add 1000 ml of ethanol, and mix well;

[0345] Gradient elution:

[0346]

[0347] Injection volume: 3 μl;

[0348] Flow rate: 0.5 ml / min;

[0349] Column temperature: 25 °C;

[0350] Detection wavelength: 210 nm;

[0351] Running time: 40 min;

[0352] Post-run time: 20 min;

[0353] Preparation of solution: The same as in Example 3.

[0354] The detection results are as Figure 15 shown.

[0355] From Figure 15 it can be concluded that the use of the AS-3 chromatographic column cannot achieve good separation of the enantiomers SMDZ9 and related impurities, and the starting material SMD has little retention on this chromatographic column.

[0356] Control Example 13

[0357] A method for detecting the isomers of carfilzomib starting materials, the detection conditions adopted by the method include:

[0358] Chromatographic conditions:

[0359] Chromatographic column: normal-phase chiral chromatographic column CHIRALPAK AD-H, 4.6 mm × 250 mm, 5 μm;

[0360] Mobile phase A: n-hexane;

[0361] Mobile phase B: Take 1.0 ml of trifluoroacetic acid, add 1000 ml of ethanol, and mix well;

[0362] Gradient elution:

[0363]

[0364] Injection volume: 3 μl;

[0365] Flow rate: 0.5 ml / min;

[0366] Column temperature: 25 °C;

[0367] Detection wavelength: 210 nm;

[0368] Running time: 40 min;

[0369] Post-run time: 20 min;

[0370] Preparation of solution: the same as in Example 3;

[0371] The test results are as Figure 16 shown.

[0372] From Figure 16 it can be concluded that the starting material SMD of carfilzomib and its enantiomer SMDZ9 cannot be well separated using the CHIRALPAK AD-H chromatographic column.

[0373] Comparative Example 14

[0374] A method for detecting an isomer of the starting material of carfilzomib, the detection conditions adopted by the method include:

[0375] Chromatographic conditions:

[0376] Chromatographic column: Chrial MX(2), 4.6 mm × 250 mm, 5 μm;

[0377] Mobile phase: n-hexane - isopropanol - trifluoroacetic acid (volume ratio 95:5:0.1);

[0378] Injection volume: 20 μl;

[0379] Flow rate: 0.5 ml / min;

[0380] Column temperature: 25 °C;

[0381] Detection wavelength: 210 nm;

[0382] Running time: 30 min;

[0383] Preparation of the solution: The same as in Example 13.

[0384] The test results are as Figure 17 shown.

[0385] From Figure 17 it can be concluded that the enantiomer SMDZ9 to be tested elutes too fast and peaks too early.

[0386] Comparing with Example 15

[0387] A method for detecting the isomers of carfilzomib starting materials, the detection conditions adopted by the method include the following steps:

[0388] Chromatographic conditions:

[0389] Chromatographic column: Chrial MX(2), 4.6 mm × 250 mm, 5 μm;

[0390] Mobile phase: n-hexane - isopropanol (volume ratio 98.5:1.5);

[0391] Injection volume: 20 μl;

[0392] Flow rate: 0.5 ml / min;

[0393] Column temperature: 25 °C;

[0394] Detection wavelength: 210 nm;

[0395] Running time: 30 min;

[0396] Preparation of the solution: The same as in Example 3.

[0397] The test results are as Figure 18 shown.

[0398] From Figure 18 it can be concluded that the impurities in the carfilzomib starting material SMD interfere with the detection of the enantiomer SMZD9.

[0399] Comparing with Example 16

[0400] A method for detecting the isomers of carfilzomib starting materials, the detection conditions adopted by the method include the following steps:

[0401] Chromatographic conditions:

[0402] Chromatographic column: Chrial MX(2), 4.6 mm × 250 mm, 5 μm;

[0403] Mobile phase: n-hexane - isopropanol (volume ratio 99:1);

[0404] Sample volume: 20 μl;

[0405] Flow rate: 0.5 ml / min;

[0406] Column temperature: 25 °C;

[0407] Detection wavelength: 210 nm;

[0408] Running time: 40 min;

[0409] Preparation of solution: The same as in Example 3.

[0410] The detection results are as Figure 19 shown.

[0411] From Figure 19 it can be concluded that when using the Chrial MX(2) chiral column with isopropanol - n - hexane (volume ratio 99:1), the retention time of the starting material is relatively late; and there are relatively many impurities in the starting material SMD of carfilzomib. It takes a very long time to elute all of them with isocratic elution of n - hexane - isopropanol (volume ratio 99:1), and the baseline is unstable.

[0412] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claimed rights.

Claims

1. An analytical detection method for the isomers of carfilzomib starting materials, characterized in that, The starting materials of carfilzomib include SMA, SMC or SMD, which are qualitatively and / or quantitatively detected by high performance liquid chromatography. The detection conditions include: The chromatographic column is a normal phase chiral chromatographic column; The mobile phase contains n-hexane and C2-C3 alcohol.

2. The analytical detection method according to claim 1, characterized in that, The mobile phase includes mobile phase A, which is a mixed solution of n-hexane and C2-C3 alcohol, and isocratic elution is carried out using mobile phase A; Alternatively, the mobile phase includes mobile phase A and mobile phase B. Mobile phase B is a mixed solution of n-hexane and isopropanol, and the compositions of mobile phase B and mobile phase A are different. Gradient elution is carried out using mobile phase A and mobile phase B.

3. The analytical detection method according to claim 2, characterized in that, In the case of isocratic elution using mobile phase A, in mobile phase A, the volume ratio of n-hexane to C2-C3 alcohol is (80-85):(15-20); Preferably, in the case of isocratic elution using mobile phase A, mobile phase A further includes trifluoroacetic acid; in mobile phase A, the volume ratio of n-hexane, C2-C3 alcohol and trifluoroacetic acid is preferably (80-85):(15-20):(0.1-0.2).

4. The analytical detection method according to claim 2, characterized in that In the case of gradient elution using mobile phase A and mobile phase B, in mobile phase A, the volume ratio of n-hexane to C2-C3 alcohol is (90-99):(1-10), and in mobile phase B, the volume ratio of n-hexane to isopropanol is (90-99):(1-10).

5. The analysis and detection method according to claim 3, characterized in that, The starting material is SMA, and the starting material isomer is SMAZ1. The detection conditions include: Chromatographic column: normal phase chiral chromatographic column, preferably Chrial NQ(2) or its equivalent chromatographic column; Mobile phase A: a mixed solution of n-hexane, isopropanol and trifluoroacetic acid; Elution program: isocratic elution is carried out using mobile phase A.

6. The analysis and detection method according to claim 3, characterized in that, The starting material is SMC, and the starting material isomer is SMCZ1. The detection conditions include: Chromatographic column: normal phase chiral chromatographic column, preferably CHIRALPAK AD-H or its equivalent chromatographic column; Mobile phase A: a mixed solution of n-hexane, isopropanol, ethanol and trifluoroacetic acid; Elution program: isocratic elution is carried out using mobile phase A.

7. The analysis and detection method according to claim 4, characterized in that The starting material is SMD, the starting material isomer is SMDZ9, and the related impurities of the starting material are SMDZ3, SMDZ8 and SMDZ10. The detection conditions include: Chromatographic column: normal phase chiral chromatographic column, preferably Chrial MX(2) or its equivalent chromatographic column; Mobile phase A: a mixed solution of n-hexane and isopropanol; the volume ratio of n-hexane to isopropanol is preferably (95-99):(1-5); Mobile phase B: a mixed solution of n-hexane and isopropanol; the volume ratio of n-hexane to isopropanol is preferably (90-94):(6-10); Elution program: gradient elution is carried out using mobile phase A and mobile phase B; Preferably, the following elution program is used for the gradient elution:

8. The analysis and detection method according to any one of claims 1-7, characterized in that The C2-C3 alcohol includes one or more of ethanol, propanol, and isopropanol; preferably, the C2-C3 alcohol includes one or two of ethanol and isopropanol.

9. The analysis and detection method according to any one of claims 1-7, characterized in that The detection conditions further include one or more of the following: The normal-phase chiral chromatographic column is Chrial NQ(2), CHIRALPAK AD-H, Chrial MX(2), or an equivalent chromatographic column; preferably, the specifications of the normal-phase chiral chromatographic column are 4.6 mm × 250 mm, 3 μm - 5 μm, preferably 5 μm; The column temperature is 20 - 40 °C; The flow rate is 0.5 - 1.2 mL / min; The detection wavelength is 200 - 250 nm; The injection volume is 5 - 50 μL, preferably 8 - 20 μL.

10. The analysis and detection method according to any one of claims 1-7, characterized in that, The detection conditions further include preparing one or more of the following solutions: Diluent: n-hexane - ethanol, with a volume ratio preferably (80 - 100):(0 - 20); Test solution: Weigh accurately the starting materials SMA, SMC, or SMD of carfilzomib, dissolve with the diluent and make up the volume to obtain the test solution, with a concentration of 1 - 10 mg / mL; Control solution: Measure accurately the test solution, dilute with the diluent and make up the volume to obtain the control solution, with a concentration of 0.001 - 0.2 mg / mL.

Images