CN117310021B - Method for separating and detecting related substances of ubenimex by high performance liquid chromatography - Google Patents
Method for separating and detecting related substances of ubenimex by high performance liquid chromatography Download PDFInfo
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- CN117310021B CN117310021B CN202311230832.8A CN202311230832A CN117310021B CN 117310021 B CN117310021 B CN 117310021B CN 202311230832 A CN202311230832 A CN 202311230832A CN 117310021 B CN117310021 B CN 117310021B
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- VGGGPCQERPFHOB-MCIONIFRSA-N Bestatin Chemical compound CC(C)C[C@H](C(O)=O)NC(=O)[C@@H](O)[C@H](N)CC1=CC=CC=C1 VGGGPCQERPFHOB-MCIONIFRSA-N 0.000 title claims abstract description 88
- 229950009811 ubenimex Drugs 0.000 title claims abstract description 88
- 238000000034 method Methods 0.000 title claims abstract description 65
- 239000000126 substance Substances 0.000 title claims abstract description 52
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 31
- 239000012535 impurity Substances 0.000 claims abstract description 365
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 110
- 239000003814 drug Substances 0.000 claims abstract description 39
- 229940079593 drug Drugs 0.000 claims abstract description 34
- 238000010828 elution Methods 0.000 claims abstract description 21
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 19
- 239000000243 solution Substances 0.000 claims description 99
- 239000002904 solvent Substances 0.000 claims description 81
- 238000001514 detection method Methods 0.000 claims description 57
- 238000007865 diluting Methods 0.000 claims description 48
- 239000000523 sample Substances 0.000 claims description 41
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 claims description 40
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 38
- 239000007788 liquid Substances 0.000 claims description 38
- 239000012488 sample solution Substances 0.000 claims description 29
- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 0.000 claims description 23
- OUCSEDFVYPBLLF-KAYWLYCHSA-N 5-(4-fluorophenyl)-1-[2-[(2r,4r)-4-hydroxy-6-oxooxan-2-yl]ethyl]-n,4-diphenyl-2-propan-2-ylpyrrole-3-carboxamide Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@H]2OC(=O)C[C@H](O)C2)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 OUCSEDFVYPBLLF-KAYWLYCHSA-N 0.000 claims description 22
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 19
- 239000012085 test solution Substances 0.000 claims description 16
- 239000007864 aqueous solution Substances 0.000 claims description 11
- 238000012937 correction Methods 0.000 claims description 10
- 238000002347 injection Methods 0.000 claims description 9
- 239000007924 injection Substances 0.000 claims description 9
- 230000007423 decrease Effects 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 230000003247 decreasing effect Effects 0.000 claims description 7
- 238000010812 external standard method Methods 0.000 claims description 7
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 5
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 5
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 5
- 230000014759 maintenance of location Effects 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 239000000463 material Substances 0.000 claims 3
- 238000004811 liquid chromatography Methods 0.000 claims 1
- 238000000926 separation method Methods 0.000 abstract description 68
- 230000000694 effects Effects 0.000 abstract description 5
- 239000012071 phase Substances 0.000 description 145
- 239000013558 reference substance Substances 0.000 description 93
- 239000011550 stock solution Substances 0.000 description 76
- 238000012360 testing method Methods 0.000 description 75
- 230000000052 comparative effect Effects 0.000 description 59
- 238000005303 weighing Methods 0.000 description 45
- 238000002360 preparation method Methods 0.000 description 42
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 20
- 239000003643 water by type Substances 0.000 description 17
- 239000008363 phosphate buffer Substances 0.000 description 16
- 238000003556 assay Methods 0.000 description 14
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 12
- 238000011084 recovery Methods 0.000 description 10
- 238000004090 dissolution Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 6
- 238000011160 research Methods 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000000337 buffer salt Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000012088 reference solution Substances 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- VMKOFRJSULQZRM-UHFFFAOYSA-N 1-bromooctane Chemical compound CCCCCCCCBr VMKOFRJSULQZRM-UHFFFAOYSA-N 0.000 description 1
- 125000006516 2-(benzyloxy)ethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])OC([H])([H])C([H])([H])* 0.000 description 1
- OCKGFTQIICXDQW-ZEQRLZLVSA-N 5-[(1r)-1-hydroxy-2-[4-[(2r)-2-hydroxy-2-(4-methyl-1-oxo-3h-2-benzofuran-5-yl)ethyl]piperazin-1-yl]ethyl]-4-methyl-3h-2-benzofuran-1-one Chemical compound C1=C2C(=O)OCC2=C(C)C([C@@H](O)CN2CCN(CC2)C[C@H](O)C2=CC=C3C(=O)OCC3=C2C)=C1 OCKGFTQIICXDQW-ZEQRLZLVSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 description 1
- 229940121948 Muscarinic receptor antagonist Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000000812 cholinergic antagonist Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000011258 core-shell material Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- -1 hydroxy diphenyl methyl Chemical group 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention provides a method for separating and detecting related substances of ubenimex by high performance liquid chromatography. The method adopts high performance liquid chromatography, and the chromatographic conditions include: the chromatographic column is Agilent InfinityLabPoroshell HPH-C18 chromatographic column; taking phosphate buffer solution as a mobile phase A, wherein the concentration of the phosphate buffer solution is 0.008 mol/L-0.012 mol/L, and the pH value is 2.5-3.1; acetonitrile is taken as a mobile phase B; gradient elution. The method can effectively detect 13 impurities at the same time, has good separation effect on each impurity, can accurately and sensitively quantitatively detect the impurity, and better monitors the quality of the ubenimex bulk drug.
Description
Technical Field
The invention relates to the technical field of medicine analysis, in particular to a method for separating and detecting related substances of ubenimex by using a high performance liquid chromatography.
Background
Ubenimex is a long-acting anticholinergic agent developed by the company glauca smith and used for the treatment of adult Chronic Obstructive Pulmonary Disease (COPD) patients. The chemical name is 1- [2- (benzyloxy) ethyl ] -4- (hydroxy diphenyl methyl) -1-azonia bicyclo [2.2.2] bromooctane, the molecular formula is C 29H34BrNO2, the molecular weight is 508.5, and the structural formula of the ubenimex is shown as formula I.
The impurity content in the medicine determines the quality of the medicine, and related substance research is one of the key problems of the quality research of the preparation, so that the preparation with high quality is required to be obtained, and the quality of the preparation is determined by starting from the raw material medicine, so that the detection level of the impurity in the raw material medicine is improved.
According to the synthesis route and possible degradation route analysis of the ubenimex, it is determined that 13 impurities possibly contained in the ubenimex bulk drug, namely, impurity A, impurity B, impurity C, impurity D, impurity E, impurity H, impurity J, impurity L, impurity M, impurity N, impurity iii-12, impurity I-5 and impurity I-11 have the following structural formulas:
at present, no detection method is found in the prior art, which can simultaneously and effectively detect the above 13 impurities, and the standard of the quality of the ubenimex is not received in European pharmacopoeia, british pharmacopoeia, united states pharmacopoeia, chinese pharmacopoeia and Japanese pharmacopoeia.
Therefore, research on a method for separating and detecting related substances of the ubenimex is very important for quality control of the ubenimex.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a method for separating and detecting related substances of ubenimex by using a high performance liquid chromatography. The method can effectively detect 13 impurities at the same time, has good separation effect on each impurity, can accurately and sensitively quantitatively detect the impurity, and better monitors the quality of the ubenimex bulk drug.
In order to achieve the above purpose, the present invention provides the following technical solutions:
A method for separating and detecting related substances of ubenimex by high performance liquid chromatography adopts high performance liquid chromatography, and chromatographic conditions comprise:
The chromatographic column is Agilent InfinityLabPoroshell HPH-C18 chromatographic column;
taking phosphate buffer solution as a mobile phase A, wherein the concentration of the phosphate buffer solution is 0.008 mol/L-0.012 mol/L, and the pH value is 2.5-3.1; acetonitrile is taken as a mobile phase B;
Gradient elution: at 0min, the volume ratio of the mobile phase A is 84% -86%, and the volume ratio of the mobile phase B is 14% -16%; from 0 to 40 minutes, the volume ratio of mobile phase A decreases linearly to 50% and the volume ratio of mobile phase B increases linearly to 50%; from 40 minutes to 60 minutes, the volume ratio of mobile phase A is linearly reduced to 20%, and the volume ratio of mobile phase B is linearly increased to 80%;60 minutes to 61 minutes, the volume ratio of the mobile phase A is linearly increased to 84% -86%, and the volume ratio of the mobile phase B is linearly decreased to 14% -16%;61 minutes to 70 minutes, the volume ratio of the mobile phase A is 84% -86%, and the volume ratio of the mobile phase B is 14% -16%.
According to the invention, the phosphate buffer is preferably prepared by the following steps: dissolving anhydrous sodium dihydrogen phosphate in water, and regulating pH with phosphoric acid.
According to preferred embodiments of the present invention, the chromatographic conditions include: the column temperature of the chromatographic column is 35-45 ℃, preferably 40 ℃; the flow rate of the mobile phase is 0.9ml/min to 1.1ml/min, preferably 1.0ml/min; the detector is an ultraviolet detector, and the detection wavelength is 210nm.
According to the invention, preferably, an aqueous acetonitrile solution is used as a solvent to prepare a sample solution; in the acetonitrile aqueous solution, the volume ratio of acetonitrile to water is 15:85, and the concentration of the ubenimex bulk drug in the test solution is 0.5mg/ml; diluting the test solution by 1000 times with a solvent to obtain a control solution; precisely measuring the solution of the sample and the control solution, respectively injecting into a high performance liquid chromatograph, and recording the chromatograms; the sample volume was 50. Mu.l.
According to the present invention, the content of each known impurity is preferably calculated according to the external standard method of the reference substance or the main component self-comparison method with the correction factor added, and the content of the unknown impurity is calculated according to the main component self-comparison method. The invention compares the impurity content calculated by the external standard method of each impurity with the impurity content calculated by the main component self-comparison method added with the correction factor, and no obvious difference exists between the two impurity content calculation results, which shows that the main component self-comparison method added with the correction factor can accurately quantify the content of each known impurity, and under the condition that the impurity reference substance is difficult to obtain, the main component self-comparison method added with the correction factor can quantify the content of each known impurity, thereby saving the cost.
According to the present invention, preferably, in the liquid chromatogram, the main component and each impurity in the ubenimum bromide are in the order of appearance of impurity J, impurity A, impurity D, impurity E, impurity H, impurity C, ubenimum bromide, impurity iii-12, impurity I-11, impurity N, impurity M, impurity B, impurity L and impurity I-5.
According to the present invention, it is preferable that the relative retention times of the impurity J, the impurity A, the impurity D, the impurity E, the impurity H, the impurity C, the impurity iii-12, the impurity I-11, the impurity N, the impurity M, the impurity B, the impurity L and the impurity I-5 in the liquid chromatogram be 0.50, 0.53, 0.67, 0.76, 0.89, 0.98, 1.08, 1.11, 1.38, 1.39, 1.43, 1.46 and 1.73, respectively. Each known impurity can be characterized by its relative retention time.
According to the present invention, the correction factors of impurity J, impurity A, impurity D, impurity E, impurity H, impurity C, impurity iii-12, impurity I-11, impurity N, impurity M, impurity B, impurity L and impurity I-5 are 1.2, 1.0, 1.3, 1.7, 0.9, 1.4, 0.5, 0.6, 0.9, 0.8 and 0.3, respectively.
The detection method provided by the invention is verified through a specificity test, and the detection method has good specificity, and the solvent, the main component, unknown impurities in the main component and the like do not interfere with the detection of known impurities.
The quantitative limit and the detection limit of the detection method and the linear range test are verified, and the method is high in applicability and high in sensitivity; repeatability tests prove that the method has high precision; the measurement of the recovery rate proves that the method has high accuracy. And can once only separate impurity J, impurity A, impurity D, impurity E, impurity H, impurity C, impurity iii-12, impurity I-11, impurity N, impurity M, impurity B, impurity L and impurity I-5, 13 kinds of known impurities altogether, inspection work efficiency is obviously improved.
Compared with the prior art, the invention has the beneficial effects that:
1. The detection method provided by the invention effectively solves the problems that the ubenimum bromide is completely overlapped with the impurity iii-12 and the impurity I-11 due to similar polarity, the impurity L, the impurity M and the impurity N are 3 positional isomers and are difficult to separate, and through screening of different chromatographic columns, including chromatographic columns with high carbon loading, chromatographic columns with different specifications, chromatographic columns with core-shell type and the like, the effects of low back pressure and Gao Zhuxiao on the common liquid phase can be achieved, and gradient program and pH optimization are carried out by using the chromatographic columns, the ubenimum bromide is effectively separated from the impurity iii-12 and the impurity I-11, the quantitative analysis of the 2 impurities can be accurately carried out, and the quality of the ubenimum bromide is better controlled. The separation problem of impurity L, impurity M and impurity N is solved, the separation of position isomers by using a C18 chromatographic column is realized, 3 position isomers can be separated without using a chiral chromatographic column, and the cost is saved.
2. According to the invention, by optimizing the proportion of acetonitrile and water in the solvent and the pH of the mobile phase A, the problems of improper pH of the mobile phase A and impurity pKa and poor peak shape of the impurity peak are solved, and after the peak shape is improved, the content of 13 impurities can be monitored more accurately and sensitively, and the efficiency is obviously improved.
3. According to the detection method provided by the invention, by improving the proportion of acetonitrile and the elution time in the gradient elution program, the problem that the impurity I-5 is difficult to elute due to the weak polarity is effectively solved, the risk of missing detection of the impurity is solved, and the content of the impurity I-5 is effectively monitored.
4. The method has the advantages of strong specificity and applicability, high detection sensitivity, good linear relation, high precision and accuracy, better durability, high detection efficiency, no influence on accurate detection and quantification of impurities when chromatographic conditions fluctuate to a certain extent, and capability of effectively controlling the quality of products.
5. The column temperature in the detection method is required to be proper, other column temperatures are compared, and the detection method is shown in comparative example 12, and the impurity C and the impurity iii-12 are found to have poorer separation degree from a main peak; the degree of separation of impurity B, impurity M and impurity N is poor. It can be seen that the separation degree between impurities becomes worse beyond the protection range of the column temperature of the invention, which indicates the importance of the preferred column temperature in the invention for the separation of the impurity of the ubenimex.
6. The pH of the phosphate buffer solution in the detection method is required to be proper, other pH values of the phosphate buffer solution are compared, and the comparison example 11 shows that the separation degree of the impurity C and the impurity iii-12 from the main peak is poor, and the detection method is not suitable for detecting substances related to the ubenimex.
7. The detection method of the invention has proper flow rate, and compared with other flow rates, see comparative example 13, the impurity C and the impurity iii-12 are found to have poorer separation degree from the main peak; the separation degree of the impurity B and the impurity L is poor; the degree of separation of impurity M and impurity N is poor. It can be seen that, beyond the flow rate protection range of the present invention, the degree of separation between impurities becomes worse, indicating the importance of the flow rate in the present invention for impurity separation of ubenimex.
8. The gradient elution procedure in the detection method has an important effect, and compared with other gradient elution procedures, see comparative example 6, comparative example 7 and comparative example 8, different elution procedures are found to influence the separation of impurity B, impurity L, impurity M and impurity N, and the gradient elution procedure is not suitable for the detection of substances related to ubenimex, and is critical.
9. In the detection method, phosphate buffer solution is used as a mobile phase A, and compared with phosphoric acid aqueous solution, see comparative example 9, the phenomenon that chromatographic peak splitting occurs due to poor peak shapes of the impurity J and the impurity A due to the buffer capacity of no buffer salt is found; the peak shapes of the impurity D and the impurity E are poor, trailing phenomenon exists, and the separation degree of the two impurities is poor; the ubenimex completely coincides with the impurity iii-12 and the impurity I-11, and no separation signs exist; the impurity M and the impurity N are completely overlapped, and the separation degree with the impurity L is poor. It can be seen that simple aqueous phosphoric acid solutions are not suitable for detection of the substances related to ubenimex, demonstrating the necessity of adding phosphate buffer in the mobile phase.
Drawings
FIG. 1 is a chromatogram of a hollow white solution of a specificity test;
FIG. 2 is a chromatogram of a control solution in a specificity test;
FIG. 3 is a chromatogram of a labeled test solution in a specificity test;
FIG. 4 is a chromatogram of a test solution in a specificity test;
FIG. 5 is a chromatogram of a system applicability solution at a flow rate of 0.9 ml/min;
FIG. 6 is a chromatogram of a system applicability solution at a flow rate of 1.1 ml/min;
FIG. 7 is a chromatogram of a system applicability solution at a column temperature of 35 ℃;
FIG. 8 is a chromatogram of a system applicability solution at a column temperature of 45 ℃;
FIG. 9 is a chromatogram of a system applicability solution at a phosphate buffer-acetonitrile volume ratio of 86:14;
FIG. 10 is a chromatogram of a system applicability solution at a phosphate buffer-acetonitrile volume ratio of 84:16;
FIG. 11 is a chromatogram of a system applicability solution at a phosphate buffer concentration of 8 mmol/L;
FIG. 12 is a chromatogram of a system applicability solution at a phosphate buffer concentration of 12 mmol/L;
FIG. 13 is a chromatogram of a system applicability solution at a phosphate buffer pH of 2.5;
FIG. 14 is a chromatogram of a system applicability solution at a phosphate buffer pH of 3.1;
fig. 15 is a chromatogram of comparative example 1.
Fig. 16 is a chromatogram of comparative example 2.
FIG. 17 is a chromatogram of comparative example 3.
FIG. 18 is a chromatogram of comparative example 4.
FIG. 19 is a chromatogram of comparative example 5.
FIG. 20 is a chromatogram of comparative example 6.
FIG. 21 is a chromatogram of comparative example 7.
FIG. 22 is a chromatogram of comparative example 8.
FIG. 23 is a chromatogram of comparative example 9.
FIG. 24 is a chromatogram of comparative example 10.
FIG. 25 is a chromatogram of phosphate buffer pH2.2 in comparative example 11.
FIG. 26 is a chromatogram of phosphate buffer pH3.5 in comparative example 11.
FIG. 27 is a chromatogram of comparative example 12 at a column temperature of 30 ℃.
FIG. 28 is a chromatogram of comparative example 12 at a column temperature of 50 ℃.
FIG. 29 is a chromatogram of comparative example 13 at a flow rate of 0.8 ml/min.
FIG. 30 is a chromatogram of comparative example 13 at a flow rate of 1.2 ml/min.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The raw materials used in the examples are all conventional products unless specified; the method is the prior art unless specified otherwise.
Example 1, a method for separating and detecting substances related to ubenimex by high performance liquid chromatography
Test instrument: waters high performance liquid chromatograph, model number is Acquity Arc. The detector used was a 2489 ultraviolet detector.
Chromatographic conditions:
Octadecylsilane chemically bonded silica is used as a filler (Agilent InfinityLabPoroshell HPH-C18 chromatographic column); taking 10mmol/L phosphate buffer solution (taking 1.2g of anhydrous sodium dihydrogen phosphate, adding 1000ml of water for dissolution, adjusting the pH value to 2.8 by phosphoric acid) as a mobile phase A, and acetonitrile as a mobile phase B, and performing gradient elution according to the following manner; the flow rate of the mobile phase is 1.0ml per minute; column temperature is 40 ℃; the detection wavelength is 210nm; the sample volume was 50. Mu.l.
At 0 minutes, the volume ratio of mobile phase A was 85% and the volume ratio of mobile phase B was 15%; from 0 to 40 minutes, the volume ratio of mobile phase A decreases linearly to 50% and the volume ratio of mobile phase B increases linearly to 50%; from 40 minutes to 60 minutes, the volume ratio of mobile phase A is linearly reduced to 20%, and the volume ratio of mobile phase B is linearly increased to 80%; from 60 minutes to 61 minutes, the volume ratio of mobile phase A increases linearly to 85% and the volume ratio of mobile phase B decreases linearly to 15%;61 minutes to 70 minutes, the volume ratio of mobile phase A was 85% and the volume ratio of mobile phase B was 15%.
Sample preparation:
solvent: water-acetonitrile (volume ratio 85:15).
Test solution: taking about 25mg of the crude drug of the ubenimex, putting the crude drug into a 50ml measuring flask, adding a solvent for dissolution, diluting to a scale, and shaking uniformly.
Control solution: precisely measuring 1ml of the sample solution, placing in a50 ml measuring flask, diluting to a scale with a solvent, shaking uniformly, precisely measuring 1ml, placing in a 20ml measuring flask, diluting to the scale with the solvent, and shaking uniformly.
Assay: precisely measuring the sample solution and the control solution, respectively injecting into a liquid chromatograph, and recording the chromatograms.
The results of the study showed that the degree of separation between the impurities was good when the impurity separation was performed according to this method, as shown in fig. 4. The method is suitable for detecting related substances of the ubenimex, and can effectively control the quality of the substances.
Example 2A verification test was performed on the detection method in example 1
2.1 Specificity test
Solvent: water-acetonitrile (volume ratio 85:15).
Impurity A control stock solution, impurity C control stock solution, impurity D control stock solution, impurity L control stock solution, impurity M control stock solution, impurity N control stock solution, impurity I-11 control stock solution, impurity iii-12 control stock solution): about 2.5mg of impurity A reference substance, about 2.5mg of impurity C reference substance, about 2.5mg of impurity D reference substance, about 2.5mg of impurity L reference substance, about 2.5mg of impurity M reference substance, about 2.5mg of impurity N reference substance, about 2.5mg of impurity I-11 reference substance and about 2.5mg of impurity iii-12 reference substance are taken, precisely weighed, respectively placed in 100ml measuring bottles, dissolved by adding solvent, diluted to scale and shaken uniformly.
Impurity B control stock solution: taking about 12.5mg of impurity B reference substance, precisely weighing, placing into a 50ml measuring flask, adding 10ml of acetonitrile for dissolution, diluting to scale with solvent, shaking, precisely weighing 1ml, placing into a 10ml measuring flask, diluting to scale with solvent, and shaking.
Impurity E control stock solution, impurity I-5 control stock solution: about 2.5mg of the impurity E reference substance and about 2.5mg of the impurity I-5 reference substance are taken, precisely weighed, placed in a 100ml measuring flask, dissolved and diluted to a scale by adding acetonitrile, and uniformly shaken.
Impurity H control stock solution, impurity J control stock solution: about 10mg of impurity H reference substance and about 7.5mg of impurity J reference substance are taken, precisely weighed, placed in a 100ml measuring flask, dissolved by adding solvent and diluted to scale, and shaken uniformly.
Each impurity localization solution: precisely measuring 1ml of each impurity reference substance stock solution, respectively placing into 50ml measuring flask, diluting to scale with solvent, and shaking.
Test solution: taking about 25mg of the crude drug of the ubenimex, putting the crude drug into a 50ml measuring flask, adding a solvent to dissolve and release the crude drug to a scale, and shaking the crude drug evenly.
Control solution: precisely measuring 1ml of each impurity reference substance stock solution, placing into the same 50ml measuring flask, diluting to scale with solvent, and shaking.
Adding a labeled test sample solution: taking about 25mg of the ubenimex bulk drug, precisely weighing, placing into a 50ml measuring flask, precisely weighing 1ml of each impurity reference substance stock solution, placing into the same 50ml measuring flask, adding solvent, dissolving, diluting to scale, and shaking uniformly.
Precisely measuring solvent (blank solution), sample solution, reference solution, standard sample solution and known impurity positioning solutions, respectively injecting into liquid chromatograph, and recording chromatogram under the same chromatographic conditions as in example 1. The results are shown in FIGS. 1 to 4.
The test result shows that the solvent, the main component and the unknown impurities in the main component do not interfere with the detection of all known impurities, the separation degree between all known impurities and adjacent impurity peaks meets the requirement, and the result shows that the method has good specificity.
2.2 Quantitative Limit and detection Limit test
Quantitative limiting solution: precisely measuring each proper amount of impurity monomer positioning solution, and adding solvent for quantitative dilution to prepare each solution with a signal to noise ratio of about 10.
Detection limit solution: precisely measuring each proper amount of impurity monomer positioning solution, and adding solvent for quantitative dilution to prepare each solution with a signal to noise ratio of about 3.
The quantitative limit solution or the detection limit solution is precisely measured, injected into a liquid chromatograph, and the chromatographic conditions are the same as in example 1, and a chromatogram is recorded.
TABLE 1 quantitative limit and detection limit test results
The test results showed that the lowest quantitative concentrations of impurity J, impurity A, impurity D, impurity E, impurity H, impurity C, ubenimum bromide, impurity iii-12, impurity I-11, impurity N, impurity M, impurity B, impurity L and impurity I-5 were approximately equivalent to 0.009%, 0.005%, 0.006%, 0.010%, 0.006%, 0.007%, 0.005%, 0.011%, 0.005% and 0.003% of the concentrations of the test samples, respectively. The minimum detection concentration of each impurity is about 0.004%, 0.002%, 0.003%, 0.004% of the concentration of the sample 0.002%, 0.003%, 0.002%, 0.003%, 0.002% and 0.001%.
2.3 Linear and Range test
Solvent: water-acetonitrile (85:15).
Linear solution: accurately measuring the respective proper amounts of the reference stock solutions of the impurity J and the impurity H, and quantitatively diluting with a solvent to prepare solutions with the concentration of about 0.09 mug/ml, 1.0 mug/ml, 1.6 mug/ml, 2.0 mug/ml, 3.0 mug/ml and 4.0 mug/ml.
Linear solution: precisely measuring impurity A, impurity D, impurity E, impurity C, impurity iii-12, impurity I-5, impurity I-11, impurity L, impurity N, impurity M, impurity B and the stock solution of the ubenimex reference substance, and quantitatively diluting with solvent to obtain solutions with the concentration of about 0.05 mug/ml, 0.2 mug/ml, 0.4 mug/ml, 0.5 mug/ml, 0.8 mug/ml and 1.0 mug/ml.
Each linear solution was precisely measured, and the solutions were injected into a high performance liquid chromatograph, and the chromatographic conditions were the same as in example 1, and the chromatograms were recorded. Linear regression was performed with concentration C (. Mu.g/ml) on the abscissa and peak area on the ordinate.
TABLE 2 impurity J Linear and Range test results
TABLE 3 results of impurity A linearity and Range test
TABLE 4 impurity D Linear and Range test results
TABLE 5 results of Linear and Range experiments with ubenimex
TABLE 6 impurity E Linear and Range test results
TABLE 7 impurity H Linear and Range test results
TABLE 8 results of impurity C linearity and Range test
TABLE 9 Linear and Range test results for impurities iii-12
TABLE 10 impurity I-5 Linear and Range test results
TABLE 11 impurity I-11 Linear and Range test results
TABLE 12 impurity N linearity and Range test results
TABLE 13 impurity M Linear and Range test results
TABLE 14 impurity B Linear and Range test results
TABLE 15 impurity L Linear and Range test results
The test results show that the linear relation among the impurities A, D, ubenimum bromide, E, C, iii-12, I-5, I-11, N, M, B, L, J and H is good.
2.4 Stability test
Solvent: water-acetonitrile (85:15).
Impurity B control stock solution: taking about 12.5mg of impurity B reference substance, precisely weighing, placing into a 50ml measuring flask, adding 10ml of acetonitrile for dissolution, diluting to scale with solvent, shaking, precisely weighing 1ml, placing into a 10ml measuring flask, diluting to scale with solvent, and shaking.
Mixing the control stock solution: taking about 2.5mg of each of an impurity A reference, an impurity C reference, an impurity D reference, an impurity E reference, an impurity L reference, an impurity M reference, an impurity N reference, an impurity I-5 reference, an impurity I-11 reference and an impurity iii-12 reference, about 10mg of an impurity H reference and about 7.5mg of an impurity J reference, precisely weighing, placing into the same 100ml measuring flask, dissolving in a solvent, diluting to a scale, and shaking uniformly.
Test solution: taking about 25mg of the crude drug of the ubenimex, precisely weighing, placing into a 50ml measuring flask, adding a solvent for dissolution, diluting to a scale, and shaking uniformly.
Control solution: precisely measuring 1ml of the sample solution, placing in a50 ml measuring flask, diluting to a scale with a solvent, shaking uniformly, precisely measuring 1ml, placing in a 20ml measuring flask, diluting to the scale with the solvent, and shaking uniformly.
Adding a labeled test sample solution: taking about 25mg of the ubenimex bulk drug, precisely weighing, placing into a 50ml measuring flask, precisely measuring 1ml of each of the impurity B reference substance stock solution and the mixed reference substance stock solution, placing into the same 50ml measuring flask, adding solvent, dissolving, diluting to scale, and shaking uniformly.
Taking a control solution, a test sample solution and a standard-added test sample solution, placing at room temperature, respectively carrying out sample injection analysis at the 0 th, 6 th, 12h, 18h and 24h ends after preparation, and recording a chromatogram under the same chromatographic conditions as in example 1.
TABLE 16 stability test results for control solutions
Time/h | 0 | 6 | 12 | 18 | 24 |
Percentage with 0h peak area (%) | N/A | 103.4 | 102.2 | 115.3 | 105.9 |
Table 17 test results of stability of the test solutions
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TABLE 18 test results of stability of test solutions
Remarks: N/A represents inapplicability
Verification result: in 12 hours at room temperature, in a chromatogram of the control solution, the peak area ratio of the ubenimex to the peak area ratio of 0 hour is in the range of 90-108%, which indicates that the stability of the control solution in 12 hours at room temperature is better; in the chromatogram of the sample solution under the room temperature condition, the difference between the content of each impurity at each time point and the content of each impurity at 0 hour is less than 0.02%, the difference between the total amount of the impurities and the total amount of the impurities at 0 hour is less than 0.05%, no new impurity which is more than the report limit is generated, and the stability of the sample solution is shown in at least 24 hours; and in the chromatogram of the standard sample solution, the peak area ratio of the impurity J, the impurity A, the impurity D, the impurity H, the impurity C, the impurity iii-12, the impurity I-11, the impurity N, the impurity M, the impurity B and the impurity L to the peak area ratio of 0H is within the range of 90-108%, which indicates that the standard sample solution is stable in 12H at the room temperature.
2.5 Repeatability test
Solvent: water-acetonitrile (85:15).
Impurity B control stock solution: about 12.5mg of impurity B reference substance is taken, precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to a scale by adding acetonitrile, and shaken well.
Mix control stock solution ①: and (3) accurately weighing about 2.5mg of each of the impurity A reference substance, the impurity C reference substance, the impurity D reference substance, the impurity L reference substance, the impurity M reference substance, the impurity N reference substance, the impurity I-11 reference substance and the impurity iii-12 reference substance, about 10mg of the impurity H reference substance and about 7.5mg of the impurity J reference substance, accurately measuring 2ml of the impurity B reference substance stock solution, placing into the same 100ml measuring flask, dissolving in a solvent, diluting to a scale, and shaking uniformly.
Mix control stock solution ②: taking about 2.5mg of each of the impurity E reference substance and the impurity I-5 reference substance, precisely weighing, placing into a same 100ml measuring flask, adding acetonitrile for dissolving and diluting to scale, and shaking uniformly.
Control solution: precisely measuring 1ml of each of the mixed reference stock solution ① and the mixed reference stock solution ②, placing into a 50ml measuring flask, diluting to scale with solvent, and shaking.
Adding a labeled test sample solution: taking about 25mg of the ubenimex bulk drug, precisely weighing, placing in a 50ml measuring flask, precisely measuring 1ml of each of the mixed reference substance stock solution ① and the mixed reference substance stock solution ②, placing in the same 50ml measuring flask, adding solvent, dissolving, diluting to scale, and shaking uniformly. 6 parts were prepared in parallel.
Test solution: taking about 25mg of the crude drug of the ubenimex, putting the crude drug into a 50ml measuring flask, adding a solvent for dissolution, diluting to a scale, and shaking uniformly.
Control solution: precisely measuring 1ml of the solution of the sample to be added with the standard, placing the solution into a 50ml measuring flask, diluting the solution to the scale with the solvent, shaking the solution uniformly, precisely measuring 1ml of the solution, placing the solution into a 20ml measuring flask, diluting the solution to the scale with the solvent, and shaking the solution uniformly.
Precisely measuring solvent, reference solution, test solution, and standard test solution, respectively injecting into liquid chromatograph, and recording chromatogram under the same chromatographic conditions as in example 1. The content of each known impurity is calculated according to the external standard method of the impurity reference substance and the main component self-comparison method added with the correction factor, the content of each unknown impurity is calculated according to the main component self-comparison method, and the test results of the two quantitative modes are compared.
TABLE 19 repeatability test results 1 (external standard method,%)
TABLE 20 repeatability test results 2 (principal component self-control method with correction factor added,%)
Verification result: preparing 6 parts of standard sample solution in parallel, calculating the content of each known impurity according to an external standard method and a main component self-comparison method added with correction factors, wherein the RSD values of impurities J, A, D, E, H, C, iii-12, I-11, N, M, B, L and I-5 are all less than 6%, and the RSD of the total amount of the impurities is 4.7% and less than 6%. The above results indicate that the method is reproducible.
2.6 Accuracy test
Solvent: water-acetonitrile (85:15).
The mixed control stock solution ①, the mixed control stock solution ②, the control solution, and the test solution prepared under the "repeatability test" item were taken.
Impurity H and impurity J control stock solutions: taking about 2.5mg of each of the impurity H reference substance and the impurity J reference substance, precisely weighing, putting into a 100ml measuring flask, adding a solvent for dissolution, diluting to a scale, and shaking uniformly.
Test solution: taking about 25mg of the crude drug of the ubenimex, precisely weighing, putting into a 50ml measuring flask, adding a solvent to dissolve and release to a scale, and shaking uniformly.
Recovery solution concentration 1: taking about 25mg of the ubenimex bulk drug, precisely weighing, placing into a 50ml measuring flask, precisely measuring 0.5ml of impurity H and impurity J reference substance stock solution, placing into the same 50ml measuring flask, adding solvent to dissolve and dilute to scale, and shaking uniformly. 3 parts were prepared in parallel.
Recovery solution concentration 2: taking about 25mg of the ubenimex bulk drug, precisely weighing, placing in a 50ml measuring flask, precisely measuring 0.5ml of each of the mixed reference substance stock solution ① and the mixed reference substance stock solution ②, placing in the same 50ml measuring flask, adding solvent, dissolving, diluting to scale, and shaking uniformly. 3 parts were prepared in parallel.
Recovery solution concentration 3: taking about 25mg of the ubenimex bulk drug, precisely weighing, placing in a 50ml measuring flask, precisely measuring 1ml of each of the mixed reference substance stock solution ① and the mixed reference substance stock solution ②, placing in the same 50ml measuring flask, adding solvent, dissolving, diluting to scale, and shaking uniformly. 3 parts were prepared in parallel.
Recovery solution concentration 4: taking about 25mg of the ubenimex bulk drug, precisely weighing, placing in a 50ml measuring flask, precisely measuring 1.5ml of each of the mixed reference substance stock solution ① and the mixed reference substance stock solution ②, placing in the same 50ml measuring flask, adding solvent, dissolving, diluting to scale, and shaking uniformly. 3 parts were prepared in parallel.
Precisely measuring the solvent, the reference substance solution, the sample solution and the recovery rate solutions with 4 concentrations, respectively injecting into a liquid chromatograph, recording the chromatogram under the same chromatographic conditions as in example 1, respectively calculating the detection amount of each known impurity according to the external standard method by using the peak area, and calculating the recovery rate.
Table 21 accuracy test results
Impurity name | Average recovery (%) | RSD(%) | Impurity name | Average recovery (%) | RSD(%) |
Impurity J | 103.7 | 1.5 | Impurity I-11 | 102.5 | 1.9 |
Impurity A | 104.3 | 0.9 | Impurity N | 106.2 | 1.6 |
Impurity D | 104.5 | 2.1 | Impurity M | 107.8 | 1.6 |
Impurity E | 108.4 | 3.9 | Impurity B | 101.7 | 2.3 |
Impurity H | 103.5 | 0.7 | Impurity L | 106.4 | 1.1 |
Impurity C | 103.7 | 3.4 | Impurity I-5 | 100.2 | 1.9 |
Impurity iii-12 | 106.8 | 1.7 | / | / | / |
Verification result: in the low, medium and high concentration ranges, the average recovery rate of the impurities J, A, D, E, H, C, iii-12, I-11, N, M, B, L and I-5 is 90-110%. The results show that the method is high in accuracy.
Comparative example 1 HPLC separation detection of the related substances of ubenimex.
Test instrument: waters high performance liquid chromatograph, model number is Acquity Arc. The detector used was a 2489 ultraviolet detector.
Chromatographic conditions:
chromatographic column: YMC-Pack pro C18 RS, 4.6mm.times.150mm, 3 μm;
Mobile phase: taking 0.1% phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B; gradient elution was performed as follows:
At 0 minutes, the mobile phase A volume ratio was 85% and the mobile phase B volume ratio was 15%; from 0 to 25 minutes, the mobile phase A volume ratio is linearly reduced to 50%, and the mobile phase B volume ratio is linearly increased to 50%;25 minutes to 25.1 minutes, the volume ratio of the mobile phase A is linearly increased to 85%, and the volume ratio of the mobile phase B is linearly decreased to 15%;25.1 minutes to 32 minutes, the volume ratio of mobile phase A is 85% and the volume ratio of mobile phase B is 15%.
Wavelength: 210nm; column temperature: 50 ℃; flow rate: 1.0ml/min; sample injection volume: 25 μl.
Sample preparation:
Solvent: acetonitrile-water (10:90).
Impurity B control stock solution: about 12.5mg of impurity B reference substance is taken, precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to a scale by adding acetonitrile, and shaken well.
Mix control stock solution ①: and (3) accurately weighing about 2.5mg of each of the impurity A reference substance, the impurity C reference substance, the impurity D reference substance, the impurity L reference substance, the impurity M reference substance, the impurity N reference substance, the impurity I-11 reference substance and the impurity iii-12 reference substance, about 10mg of the impurity H reference substance and about 7.5mg of the impurity J reference substance, accurately measuring 2ml of the impurity B reference substance stock solution, placing into the same 100ml measuring flask, dissolving in a solvent, diluting to a scale, and shaking uniformly.
Mix control stock solution ②: taking about 2.5mg of each of the impurity E reference substance and the impurity I-5 reference substance, precisely weighing, placing into a same 100ml measuring flask, adding acetonitrile for dissolving and diluting to scale, and shaking uniformly.
Control solution: precisely measuring 1ml of each of the mixed reference stock solution ① and the mixed reference stock solution ②, placing into a 50ml measuring flask, diluting to scale with solvent, and shaking.
Assay: precisely measuring the reference substance solution, injecting into a liquid chromatograph, and recording the chromatogram.
The results of the study showed that when impurity separation was performed according to this method, as shown in fig. 15, the degree of separation of impurity D and impurity E was poor; the ubenimex and the impurity C almost completely coincide, and no sign of separation exists; the impurity B and the impurity N are completely overlapped, and the separation degree of the impurity B and the impurity N from the impurity M is poor; impurity I-5 did not elute, and did not peak. It can be seen that this method is not suitable for detection of substances related to ubenimex, and the quality of the substance cannot be controlled.
Comparative example 2 HPLC separation detection of the related substances of ubenimex.
Test instrument: waters high performance liquid chromatograph, model number is Acquity Arc. The detector used was a 2489 ultraviolet detector.
Chromatographic conditions:
chromatographic column: YMC TRIART C18,4.6 mm. Times.150 mm,3 μm;
Mobile phase: taking 0.1% phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B; gradient elution was performed as follows:
At 0 minutes, the mobile phase A volume ratio was 85% and the mobile phase B volume ratio was 15%; from 0 to 40 minutes, the mobile phase A volume ratio is linearly reduced to 50%, and the mobile phase B volume ratio is linearly increased to 50%;40 to 50 minutes, the volume ratio of the mobile phase A is linearly reduced to 20%, and the volume ratio of the mobile phase B is linearly increased to 80%;50 to 60 minutes, 20% by volume of mobile phase A and 80% by volume of mobile phase B; 60 minutes to 61 minutes, the volume ratio of the mobile phase A is linearly increased to 85%, and the volume ratio of the mobile phase B is linearly decreased to 15%;61 minutes to 70 minutes, the volume ratio of mobile phase A is 85% and the volume ratio of mobile phase B is 15%.
Wavelength: 210nm; column temperature: 50 ℃; flow rate: 1.0ml/min; sample injection volume: 50 μl.
Sample preparation:
Solvent: water-acetonitrile (85:15).
Control solution: precisely measuring 1ml of each of the mixed reference stock solution ① (preparation method is the same as comparative example 1) and the mixed reference stock solution ② (preparation method is the same as comparative example 1), placing into a 50ml measuring flask, diluting to scale with solvent, and shaking.
Assay: precisely measuring the reference substance solution, injecting into a liquid chromatograph, and recording the chromatogram.
The results of the study showed that when impurity separation was performed according to this method, as shown in fig. 16, impurity J, impurity a, impurity D and impurity E were inferior in peak shape due to the solvent effect; the impurity B and the impurity N are completely overlapped, and the separation degree of the impurity B and the impurity N from the impurity M is poor. It can be seen that this method is not suitable for detection of substances related to ubenimex, and the quality of the substance cannot be controlled.
Comparative example 3 HPLC separation detection of the related substances of ubenimex.
Test instrument: waters high performance liquid chromatograph, model number is Acquity Arc. The detector used was a 2489 ultraviolet detector.
Chromatographic conditions:
Chromatographic column: waters XBiridge C18,4.6mm×250mm,5 μm;
mobile phase: taking phosphoric acid aqueous solution (taking 1000ml, adjusting pH value to 2.5 with phosphoric acid) as mobile phase A, and acetonitrile as mobile phase B; gradient elution was performed as follows:
At 0 minutes, the mobile phase A volume ratio was 85% and the mobile phase B volume ratio was 15%; from 0 to 40 minutes, the mobile phase A volume ratio is linearly reduced to 50%, and the mobile phase B volume ratio is linearly increased to 50%;40 to 50 minutes, the volume ratio of the mobile phase A is linearly reduced to 20%, and the volume ratio of the mobile phase B is linearly increased to 80%;50 to 60 minutes, 20% by volume of mobile phase A and 80% by volume of mobile phase B; 60 minutes to 61 minutes, the volume ratio of the mobile phase A is linearly increased to 85%, and the volume ratio of the mobile phase B is linearly decreased to 15%;61 minutes to 70 minutes, the volume ratio of mobile phase A is 85% and the volume ratio of mobile phase B is 15%.
Wavelength: 210nm; column temperature: 50 ℃; flow rate: 1.0ml/min; sample injection volume: 50 μl.
Sample preparation:
Solvent: water-acetonitrile (85:15).
Adding a labeled test sample solution: taking about 25mg of the crude drug of the ubenimex, precisely weighing, placing into a 50ml measuring flask, precisely weighing 1ml of each of the mixed reference substance stock solution ① (the preparation method is the same as that of comparative example 1) and the mixed reference substance stock solution ② (the preparation method is the same as that of comparative example 1), placing into the same 50ml measuring flask, dissolving with a solvent, diluting to a scale, and shaking uniformly.
Assay: precisely measuring the solution of the sample to be marked, injecting the solution into a liquid chromatograph, and recording the chromatogram.
As a result of the study, when the impurity separation is performed according to this method, as shown in fig. 17, the impurity M, the impurity N, the impurity L, and the impurity B overlap, and effective separation cannot be achieved. It can be seen that this method is not suitable for detection of substances related to ubenimex, and the quality of the substance cannot be controlled.
Comparative example 4 HPLC separation detection of the related substances of ubenimex.
Test instrument: waters high performance liquid chromatograph, model number is Acquity Arc. The detector used was a 2489 ultraviolet detector.
Chromatographic conditions:
Chromatographic column: waters XBiridge C18,4.6mm×250mm,5 μm;
mobile phase: taking phosphoric acid aqueous solution (taking 1000ml, adjusting pH value to 2.5 with phosphoric acid) as mobile phase A, and acetonitrile as mobile phase B; gradient elution was performed as follows:
At 0 minutes, the mobile phase A volume ratio was 85% and the mobile phase B volume ratio was 15%; from 0 to 40 minutes, the mobile phase A volume ratio is linearly reduced to 60%, and the mobile phase B volume ratio is linearly increased to 40%;40 to 60 minutes, the volume ratio of the mobile phase A is linearly reduced to 20%, and the volume ratio of the mobile phase B is linearly increased to 80%;60 minutes to 61 minutes, the volume ratio of the mobile phase A is linearly increased to 85%, and the volume ratio of the mobile phase B is linearly decreased to 15%;61 minutes to 70 minutes, the volume ratio of mobile phase A is 85% and the volume ratio of mobile phase B is 15%.
Wavelength: 210nm; column temperature: 50 ℃; flow rate: 1.0ml/min; sample injection volume: 50 μl.
Sample preparation:
Solvent: water-acetonitrile (85:15).
Adding a labeled test sample solution: taking about 25mg of the crude drug of the ubenimex, precisely weighing, placing into a 50ml measuring flask, precisely weighing 1ml of each of the mixed reference substance stock solution ① (the preparation method is the same as that of comparative example 1) and the mixed reference substance stock solution ② (the preparation method is the same as that of comparative example 1), placing into the same 50ml measuring flask, dissolving with a solvent, diluting to a scale, and shaking uniformly.
Assay: precisely measuring the solution of the sample to be marked, injecting the solution into a liquid chromatograph, and recording the chromatogram.
The results of the study show that when the impurity separation is performed according to the method, as shown in fig. 18, the separation degree of ubenimum bromide and the impurity C is poor, and the impurity M, the impurity N, the impurity L and the impurity B are overlapped, so that the effective separation cannot be achieved. It can be seen that this method is not suitable for detection of substances related to ubenimex, and the quality of the substance cannot be controlled.
Comparative example 5 HPLC separation detection of the related substances of ubenimex.
Test instrument: waters high performance liquid chromatograph, model number is Acquity Arc. The detector used was a 2489 ultraviolet detector.
Chromatographic conditions:
chromatographic column: YMC TRIART PFP,4.6 mm. Times.250 mm,5 μm;
mobile phase: taking phosphoric acid aqueous solution (taking 1000ml, adjusting pH value to 2.5 with phosphoric acid) as mobile phase A, and acetonitrile as mobile phase B; gradient elution was performed as follows:
At 0 minutes, the mobile phase A volume ratio was 85% and the mobile phase B volume ratio was 15%; from 0 to 40 minutes, the mobile phase A volume ratio is linearly reduced to 50%, and the mobile phase B volume ratio is linearly increased to 50%;40 to 50 minutes, the volume ratio of the mobile phase A is linearly reduced to 20%, and the volume ratio of the mobile phase B is linearly increased to 80%;50 to 60 minutes, the volume ratio of the mobile phase A is 20 percent, and the volume ratio of the mobile phase B is 80 percent; 60 minutes to 61 minutes, the volume ratio of the mobile phase A is linearly increased to 85%, and the volume ratio of the mobile phase B is linearly decreased to 15%;61 minutes to 70 minutes, the volume ratio of mobile phase A is 85% and the volume ratio of mobile phase B is 15%.
Wavelength: 210nm; column temperature: 35 ℃; flow rate: 1.0ml/min; sample injection volume: 50 μl.
Sample preparation:
Solvent: water-acetonitrile (85:15).
Adding a labeled test sample solution: taking about 25mg of the crude drug of the ubenimex, precisely weighing, placing into a 50ml measuring flask, precisely weighing 1ml of each of the mixed reference substance stock solution ① (the preparation method is the same as that of comparative example 1) and the mixed reference substance stock solution ② (the preparation method is the same as that of comparative example 1), placing into the same 50ml measuring flask, dissolving with a solvent, diluting to a scale, and shaking uniformly.
Assay: precisely measuring the solution of the sample to be marked, injecting the solution into a liquid chromatograph, and recording the chromatogram.
The results of the study showed that when the impurity separation was performed according to this method, as shown in fig. 19, effective separation was not achieved between 13 impurities, and only 9 impurity peaks were present, and the impurity peaks were not eluted, or overlapped with the main peaks, or overlapped with each other. It can be seen that this method is not suitable for detection of substances related to ubenimex, and the quality of the substance cannot be controlled.
Comparative example 6 chromatographic conditions (except for gradient elution procedure) of example 1 of the invention were used for HPLC separation detection of the related substances of ubenimum bromide.
Test instrument: waters high performance liquid chromatograph, model number is Acquity Arc. The detector used was a 2489 ultraviolet detector.
Chromatographic conditions were the same as in example 1, except that: gradient elution conditions were as follows:
At 0 minutes, the volume ratio of mobile phase A was 85% and the volume ratio of mobile phase B was 15%; from 0 to 30 minutes, the volume ratio of mobile phase A decreases linearly to 50% and the volume ratio of mobile phase B increases linearly to 50%;30 minutes to 60 minutes, the volume ratio of the mobile phase A is linearly reduced to 20%, and the volume ratio of the mobile phase B is linearly increased to 80%; from 60 minutes to 61 minutes, the volume ratio of mobile phase A increases linearly to 85% and the volume ratio of mobile phase B decreases linearly to 15%;61 minutes to 70 minutes, the volume ratio of mobile phase A was 85% and the volume ratio of mobile phase B was 15%.
Sample preparation:
Solvent: water-acetonitrile (85:15).
Adding a labeled test sample solution: taking about 25mg of the crude drug of the ubenimex, precisely weighing, placing into a 50ml measuring flask, precisely weighing 1ml of each of the mixed reference substance stock solution ① (the preparation method is the same as that of comparative example 1) and the mixed reference substance stock solution ② (the preparation method is the same as that of comparative example 1), placing into the same 50ml measuring flask, dissolving with a solvent, diluting to a scale, and shaking uniformly.
Assay: precisely measuring the solution of the sample to be marked, injecting the solution into a liquid chromatograph, and recording the chromatogram.
The results of the study showed that, when the impurity separation was performed according to this method, as shown in fig. 20, the impurity B and the impurity L were completely overlapped, and the impurity N and the impurity M were poor in separation degree. The method is not suitable for detecting related substances of the ubenimex, and the quality of the substances cannot be controlled.
Comparative example 7, chromatographic conditions (except for gradient elution procedure) of example 1 of the present invention were used for HPLC separation detection of the related substances of ubenimex.
Test instrument: waters high performance liquid chromatograph, model number is Acquity Arc. The detector used was a 2489 ultraviolet detector.
Chromatographic conditions were the same as in example 1, except that: gradient elution conditions were as follows:
At 0 minutes, the volume ratio of mobile phase A was 85% and the volume ratio of mobile phase B was 15%; from 0 minutes to 60 minutes, the volume ratio of mobile phase A is linearly reduced to 20%, and the volume ratio of mobile phase B is linearly increased to 80%; from 60 minutes to 61 minutes, the volume ratio of mobile phase A increases linearly to 85% and the volume ratio of mobile phase B decreases linearly to 15%;61 minutes to 70 minutes, the volume ratio of mobile phase A was 85% and the volume ratio of mobile phase B was 15%.
Wavelength: 210nm; column temperature: 40 ℃; flow rate: 1.0ml/min; sample injection volume: 50 μl.
Sample preparation:
Solvent: water-acetonitrile (85:15).
Adding a labeled test sample solution: taking about 25mg of the crude drug of the ubenimex, precisely weighing, placing into a 50ml measuring flask, precisely weighing 1ml of each of the mixed reference substance stock solution ① (the preparation method is the same as that of comparative example 1) and the mixed reference substance stock solution ② (the preparation method is the same as that of comparative example 1), placing into the same 50ml measuring flask, dissolving with a solvent, diluting to a scale, and shaking uniformly.
Assay: precisely measuring the solution of the sample to be marked, injecting the solution into a liquid chromatograph, and recording the chromatogram.
The results of the study showed that, when impurity separation was performed according to this method, as shown in fig. 21, the impurity N was less separated from the impurity M. The method is not suitable for detecting related substances of the ubenimex, and the quality of the substances cannot be controlled.
Comparative example 8 chromatographic conditions (except for gradient elution procedure) of example 1 of the invention were used for HPLC separation detection of the related substances of ubenimum bromide.
Test instrument: waters high performance liquid chromatograph, model number is Acquity Arc. The detector used was a 2489 ultraviolet detector.
Chromatographic conditions were the same as in example 1, except that: gradient elution conditions were as follows:
At 0 minutes, the volume ratio of mobile phase A was 85% and the volume ratio of mobile phase B was 15%; from 0 to 20 minutes, the volume ratio of mobile phase A is linearly reduced to 60%, and the volume ratio of mobile phase B is linearly increased to 40%; for 20 to 50 minutes, the volume ratio of the mobile phase A is linearly reduced to 30%, and the volume ratio of the mobile phase B is linearly increased to 70%; from 50 minutes to 60 minutes, the volume ratio of mobile phase A is linearly reduced to 20%, and the volume ratio of mobile phase B is linearly increased to 80%; from 60 minutes to 61 minutes, the volume ratio of mobile phase A increases linearly to 85% and the volume ratio of mobile phase B decreases linearly to 15%;61 minutes to 70 minutes, the volume ratio of mobile phase A was 85% and the volume ratio of mobile phase B was 15%.
Wavelength: 210nm; column temperature: 40 ℃; flow rate: 1.0ml/min; sample injection volume: 50 μl.
Sample preparation:
Solvent: water-acetonitrile (85:15).
Adding a labeled test sample solution: taking about 25mg of the crude drug of the ubenimex, precisely weighing, placing into a 50ml measuring flask, precisely weighing 1ml of each of the mixed reference substance stock solution ① (the preparation method is the same as that of comparative example 1) and the mixed reference substance stock solution ② (the preparation method is the same as that of comparative example 1), placing into the same 50ml measuring flask, dissolving with a solvent, diluting to a scale, and shaking uniformly.
Assay: precisely measuring the solution of the sample to be marked, injecting the solution into a liquid chromatograph, and recording the chromatogram.
The results of the study showed that, when the impurity separation was performed according to this method, as shown in fig. 22, impurity B overlapped with impurity L. The method is not suitable for detecting related substances of the ubenimex, and the quality of the substances cannot be controlled.
Comparative example 9 an experiment was performed using the chromatographic conditions (except for the mobile phase) of example 1 of the present invention, using 0.1% phosphoric acid aqueous solution as mobile phase a and acetonitrile as mobile phase B, for detecting the related substances of ubenimex.
Test instrument: waters high performance liquid chromatograph, model number is Acquity Arc. The detector used was a 2489 ultraviolet detector.
Chromatographic conditions were the same as in example 1, except that: 0.1% phosphoric acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B.
Sample preparation:
Solvent: water-acetonitrile (85:15).
Adding a labeled test sample solution: taking about 25mg of the crude drug of the ubenimex, precisely weighing, placing into a 50ml measuring flask, precisely weighing 1ml of each of the mixed reference substance stock solution ① (the preparation method is the same as that of comparative example 1) and the mixed reference substance stock solution ② (the preparation method is the same as that of comparative example 1), placing into the same 50ml measuring flask, dissolving with a solvent, diluting to a scale, and shaking uniformly.
Assay: precisely measuring the solution of the sample to be marked, injecting the solution into a liquid chromatograph, and recording the chromatogram.
The research result shows that when the chromatographic condition of the invention is used, and a 0.1% phosphoric acid solution is taken as a mobile phase A and acetonitrile is taken as a mobile phase B, as shown in fig. 23, the phenomenon of chromatographic peak splitting appears due to poor peak shapes of the impurity J and the impurity A without buffer salt; the peak shapes of the impurity D and the impurity E are poor, trailing phenomenon exists, and the separation degree of the two impurities is poor; the ubenimex completely coincides with the impurity iii-12 and the impurity I-11, and no separation signs exist; the impurity M and the impurity N are completely overlapped, and the separation degree with the impurity L is poor. It can be seen that a simple phosphoric acid solution is not suitable for detection of the substances related to ubenimex, demonstrating the necessity of adding phosphate buffer in the mobile phase.
Comparative example 10 the chromatographic conditions of example 1 of the present invention (except for the chromatographic column) were used for HPLC separation detection of the related substances of ubenimex.
Test instrument: waters high performance liquid chromatograph, model number is Acquity Arc. The detector used was a 2489 ultraviolet detector.
Chromatographic conditions were the same as in example 1, except that: chromatographic column: waters XBiridge C18, 4.6mm.times.250 mm,5 μm.
Sample preparation:
Solvent: acetonitrile-water (15:85).
Adding a labeled test sample solution: taking about 25mg of the crude drug of the ubenimex, precisely weighing, placing into a 50ml measuring flask, precisely weighing 1ml of each of the mixed reference substance stock solution ① (the preparation method is the same as that of comparative example 1) and the mixed reference substance stock solution ② (the preparation method is the same as that of comparative example 1), placing into the same 50ml measuring flask, dissolving with a solvent, diluting to a scale, and shaking uniformly.
Assay: precisely measuring the solution of the sample to be marked, injecting the solution into a liquid chromatograph, and recording the chromatogram.
The results of the study show that when the chromatographic conditions of the invention are used to replace chromatographic columns of different manufacturers, as shown in fig. 24, the impurity M and the impurity N are completely overlapped and the separation degree from the impurity L is poor. It can be seen that the preferred chromatographic column in the present invention is important for the separation of the impurity of ubenimex.
Comparative example 11, chromatographic conditions (except for pH of phosphate buffer) of example 1 of the present invention were used for HPLC separation detection of the related substances of ubenimum bromide.
Test instrument: waters high performance liquid chromatograph, model number is Acquity Arc. The detector used was a 2489 ultraviolet detector.
Chromatographic conditions were the same as in example 1, except that:
mobile phase 1: taking 10mmol/L phosphate buffer (anhydrous sodium dihydrogen phosphate 1.2g, adding 1000ml of water to dissolve, adjusting pH value to 2.2 with phosphoric acid) as mobile phase A, and acetonitrile as mobile phase B; gradient elution was performed as follows:
Mobile phase 2: 10mmol/L phosphate buffer (anhydrous sodium dihydrogen phosphate 1.2g, water 1000ml was added to dissolve, pH was adjusted to 3.5 with phosphoric acid) was used as mobile phase A, and acetonitrile was used as mobile phase B.
Sample preparation:
Solvent: acetonitrile-water (15:85).
Adding a labeled test sample solution: taking about 25mg of the crude drug of the ubenimex, precisely weighing, placing into a 50ml measuring flask, precisely weighing 1ml of each of the mixed reference substance stock solution ① (the preparation method is the same as that of comparative example 1) and the mixed reference substance stock solution ② (the preparation method is the same as that of comparative example 1), placing into the same 50ml measuring flask, dissolving with a solvent, diluting to a scale, and shaking uniformly.
Assay: precisely measuring the solution of the sample to be marked, injecting the solution into a liquid chromatograph, and recording the chromatogram.
The results of the study showed that, when the chromatographic conditions of the present invention were used in addition to the pH of the phosphate buffer, as shown in FIGS. 25 and 26, the impurity C and impurity iii-12 were found to have poor separation from the main peak. It can be seen that beyond the pH protection range of the present invention, the degree of separation between the main peak and the adjacent impurities becomes worse, indicating the importance of the preferred pH in the present invention for separation of the ubenimex impurities.
Comparative example 12, chromatographic conditions (except column temperature) of example 1 of the present invention were used for HPLC separation detection of the related substances of ubenimum bromide.
Test instrument: waters high performance liquid chromatograph, model number is Acquity Arc. The detector used was a 2489 ultraviolet detector.
Chromatographic conditions were the same as in example 1, except that: column temperature 1:30 ℃; column temperature 2:50 ℃.
Sample preparation:
Solvent: acetonitrile-water (15:85).
Adding a labeled test sample solution: taking about 25mg of the crude drug of the ubenimex, precisely weighing, placing into a 50ml measuring flask, precisely weighing 1ml of each of the mixed reference substance stock solution ① (the preparation method is the same as that of comparative example 1) and the mixed reference substance stock solution ② (the preparation method is the same as that of comparative example 1), placing into the same 50ml measuring flask, dissolving with a solvent, diluting to a scale, and shaking uniformly.
Assay: precisely measuring the solution of the sample to be marked, injecting the solution into a liquid chromatograph, and recording the chromatogram.
The results of the study showed that, when the chromatographic conditions of the present invention were used in addition to the column temperature, as shown in FIGS. 27 and 28, the impurity C and the impurity iii-12 were found to have poor separation from the main peak; the degree of separation of impurity B, impurity M and impurity N is poor. It can be seen that the separation degree between impurities becomes worse beyond the protection range of the column temperature of the invention, which indicates the importance of the preferred column temperature in the invention for the separation of the impurity of the ubenimex.
Comparative example 13, chromatographic conditions (except for flow rate) of example 1 of the present invention were used for HPLC separation detection of the related substances of ubenimex.
Test instrument: waters high performance liquid chromatograph, model number is Acquity Arc. The detector used was a 2489 ultraviolet detector.
Chromatographic conditions were the same as in example 1, except that: flow rate 1:0.8ml/min; flow rate 2:1.2ml/min.
Sample preparation:
Solvent: acetonitrile-water (15:85).
Adding a labeled test sample solution: taking about 25mg of the crude drug of the ubenimex, precisely weighing, placing into a 50ml measuring flask, precisely weighing 1ml of each of the mixed reference substance stock solution ① (the preparation method is the same as that of comparative example 1) and the mixed reference substance stock solution ② (the preparation method is the same as that of comparative example 1), placing into the same 50ml measuring flask, dissolving with a solvent, diluting to a scale, and shaking uniformly.
Assay: precisely measuring the solution of the sample to be marked, injecting the solution into a liquid chromatograph, and recording the chromatogram.
The results of the study showed that, when the chromatographic conditions of the present invention were used in addition to the flow rate, as shown in FIGS. 29 and 30, impurity C and impurity iii-12 were found to have poor separation from the main peak; the separation degree of the impurity B and the impurity L is poor; the degree of separation of impurity M and impurity N is poor. It can be seen that beyond the flow rate protection range of the present invention, the degree of separation between impurities becomes worse, indicating the importance of the preferred flow rates in the present invention for the separation of the ubenimex impurities.
Test example 1: the invention provides a detection test under different chromatographic conditions
Solvent: water-acetonitrile (volume ratio 85:15).
Impurity B control stock solution: about 12.5mg of impurity B reference substance is taken, precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to a scale by adding acetonitrile, and shaken well.
Mix control stock solution ①: taking about 2.5mg of each of an impurity A reference, an impurity C reference, an impurity D reference, an impurity L reference, an impurity M reference, an impurity N reference, an impurity I-11 reference and an impurity iii-12 reference, about 10mg of an impurity H reference and about 7.5mg of an impurity J reference, precisely weighing, precisely measuring 2ml of a stock solution of an impurity B reference, placing into the same 100ml measuring flask, dissolving in a solvent, diluting to a scale, and shaking uniformly.
Mix control stock solution ②: taking about 2.5mg of each of the impurity E reference substance and the impurity I-5 reference substance, precisely weighing, placing into a same 100ml measuring flask, adding acetonitrile for dissolving and diluting to scale, and shaking uniformly.
System applicability solution: taking about 25mg of the ubenimex bulk drug, precisely weighing, placing in a 50ml measuring flask, precisely measuring 1ml of each of the mixed reference substance stock solution ① and the mixed reference substance stock solution ②, placing in the same 50ml measuring flask, adding solvent, dissolving, diluting to scale, and shaking uniformly.
The test results under the conditions of different concentrations of the phosphate buffer in the mobile phase, different pH values of the phosphate buffer, different proportions of the phosphate buffer and acetonitrile, different flow rates and different column temperatures of the chromatographic column, namely, the conditions are changed, and other chromatographic conditions are compared with those of example 1 as follows:
In the test, the concentration of the phosphate buffer solution is 8mmol/L, 10mmol/L and 12mmol/L, the volume ratio of the phosphate buffer solution to acetonitrile is 86:14, 85:15 and 84:16, the flow rate of the mobile phase is 0.9ml, 1.0ml and 1.1ml per minute, the column temperature is 35 ℃,40 ℃, 45 ℃, the pH of the phosphate buffer solution is 2.5, 2.8 and 3.1, the influences of the conditions on the impurity content are respectively examined, and the durability of chromatographic conditions is examined. That is, the above conditions were changed under standard chromatographic conditions (chromatographic conditions in example 1). The flow rate of the system applicability solution is 0.9ml per minute, the flow rate of the system applicability solution is 1.1ml per minute, the column temperature of the system applicability solution is 35 ℃, the column temperature of the system applicability solution is 7, the column temperature of the system applicability solution is 45 ℃, the ratio of phosphate buffer solution to acetonitrile is 86:14, the ratio of phosphate buffer solution to acetonitrile is 84:16, the system applicability solution is 10, the concentration of phosphate buffer solution is 8mmol/L, the system applicability solution is 11, the concentration of phosphate buffer solution is 12mmol/L, the system applicability solution is 13, and the pH of phosphate buffer solution is 3.1. The impurity content test results are shown in the following table.
Table 22 durability test results of the influence of different variation conditions on the measurement results of the content of each impurity
The system applicability test shows that in the range of the chromatographic conditions provided by the invention, the separation between the impurities is good, the method has better durability, and the qualitative detection and quantitative detection results of the impurities are not affected by the fluctuation of the conditions in the range.
In conclusion, the invention establishes a detection method for separating the related substances of the ubenimex by using a high performance liquid chromatography and a detection method for quantifying impurities of the detection method. The invention adopts high performance liquid chromatography, and the sample treatment process is simple to operate, does not need a reference substance, and has low cost, accuracy and high efficiency; the sensitivity is high; the specificity is higher; the reagent used is acetonitrile, is easy to obtain, and has a wide application prospect.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A method for separating and detecting related substances of ubenimex by high performance liquid chromatography is characterized in that the high performance liquid chromatography is adopted, and chromatographic conditions comprise:
The chromatographic column is Agilent InfinityLabPoroshell HPH-C18 chromatographic column;
Taking phosphate buffer solution as a mobile phase A, wherein the concentration of the phosphate buffer solution is 0.008 mol/L-0.012 mol/L, and the pH is 2.5-3.1; acetonitrile is taken as a mobile phase B;
Gradient elution: at 0 min, the volume ratio of the mobile phase A is 84% -86%, and the volume ratio of the mobile phase B is 14% -16%; from 0 to 40 minutes, the volume ratio of mobile phase A decreases linearly to 50% and the volume ratio of mobile phase B increases linearly to 50%; from 40 minutes to 60 minutes, the volume ratio of mobile phase A is linearly reduced to 20%, and the volume ratio of mobile phase B is linearly increased to 80%;60 minutes to 61 minutes, the volume ratio of the mobile phase A is linearly increased to 84% -86%, and the volume ratio of the mobile phase B is linearly decreased to 14% -16%;61 minutes to 70 minutes, the volume ratio of the mobile phase A is 84% -86%, and the volume ratio of the mobile phase B is 14% -16%;
The chromatographic conditions include: the column temperature of the chromatographic column is 35-45 ℃; the flow rate of the mobile phase is 0.9ml/min to 1.1ml/min; the detector is an ultraviolet detector, and the detection wavelength is 210nm;
preparing a sample solution by using an acetonitrile aqueous solution as a solvent; in the acetonitrile aqueous solution, the volume ratio of acetonitrile to water is 15:85, and the concentration of the ubenimex bulk drug in the test solution is 0.5mg/ml; diluting the test solution by 1000 times with a solvent to obtain a control solution; precisely measuring the solution of the sample and the control solution, respectively injecting into a high performance liquid chromatograph, and recording the chromatograms; the sample injection volume is 50 mu l;
The related substances of the ubenimex have the following structures:
。
2. The method for separating and detecting the related substances of the ubenimex by using the high performance liquid chromatography as claimed in claim 1, wherein the phosphate buffer solution is prepared by the following steps: dissolving anhydrous sodium dihydrogen phosphate in water, and regulating pH with phosphoric acid.
3. The method for separating and detecting a material related to ubenimex by high performance liquid chromatography according to claim 1, wherein the column temperature of the chromatographic column is 40 ℃.
4. The method for separating and detecting a material related to ubenimex by high performance liquid chromatography according to claim 1, wherein the flow rate of the mobile phase is 1.0ml/min.
5. The method for separating and detecting a related substance of ubenimex by high performance liquid chromatography according to claim 1, wherein the content of each known impurity is calculated according to a reference external standard method or a main component self-comparison method added with a correction factor, and the content of an unknown impurity is calculated according to a main component self-comparison method.
6. The method for separating and detecting a material related to ubenimex by high performance liquid chromatography according to claim 1, wherein the main component and the respective impurities in ubenimex are in the order of appearance of impurity J, impurity a, impurity D, impurity E, impurity H, impurity C, ubenimex, impurity iii-12, impurity I-11, impurity N, impurity M, impurity B, impurity L and impurity I-5 in the liquid chromatography.
7. The method for separating and detecting a ubenimex-related substance according to claim 1, wherein the relative retention times of impurity J, impurity a, impurity D, impurity E, impurity H, impurity C, impurity iii-12, impurity I-11, impurity N, impurity M, impurity B, impurity L and impurity I-5 in the liquid chromatogram are 0.50, 0.53, 0.67, 0.76, 0.89, 0.98, 1.08, 1.11, 1.38, 1.39, 1.43, 1.46 and 1.73, respectively.
8. The method for separating and detecting a ubenimex-related substance by high performance liquid chromatography according to claim 1, wherein correction factors of impurity J, impurity a, impurity D, impurity E, impurity H, impurity C, impurity iii-12, impurity I-11, impurity N, impurity M, impurity B, impurity L and impurity I-5 are 1.2, 1.0, 1.3, 1.7, 0.9, 1.4, 0.5, 0.6, 0.9, 0.8 and 0.3, respectively.
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CN101173912A (en) * | 2007-02-09 | 2008-05-07 | 广东省保化检测中心有限公司 | High efficiency liquid chromatography detecting method of erythromycin in cosmetic |
CN101738444A (en) * | 2008-11-04 | 2010-06-16 | 上海科致生物科技有限公司 | Method for detecting melamine |
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JPH10206404A (en) * | 1997-01-27 | 1998-08-07 | Showa Denko Kk | Analysis method of anion and anion analysis set |
CN101173912A (en) * | 2007-02-09 | 2008-05-07 | 广东省保化检测中心有限公司 | High efficiency liquid chromatography detecting method of erythromycin in cosmetic |
CN101738444A (en) * | 2008-11-04 | 2010-06-16 | 上海科致生物科技有限公司 | Method for detecting melamine |
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