CN116559354A - Method for detecting carfilzomib enantiomer by adopting reversed phase chromatography - Google Patents
Method for detecting carfilzomib enantiomer by adopting reversed phase chromatography Download PDFInfo
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- CN116559354A CN116559354A CN202310814643.9A CN202310814643A CN116559354A CN 116559354 A CN116559354 A CN 116559354A CN 202310814643 A CN202310814643 A CN 202310814643A CN 116559354 A CN116559354 A CN 116559354A
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- carfilzomib
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- enantiomer
- acetonitrile
- phosphate buffer
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- BLMPQMFVWMYDKT-NZTKNTHTSA-N carfilzomib Chemical class C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)[C@]1(C)OC1)NC(=O)CN1CCOCC1)CC1=CC=CC=C1 BLMPQMFVWMYDKT-NZTKNTHTSA-N 0.000 title claims abstract description 100
- 238000000034 method Methods 0.000 title claims abstract description 35
- 238000004366 reverse phase liquid chromatography Methods 0.000 title claims abstract description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 89
- 229960002438 carfilzomib Drugs 0.000 claims abstract description 82
- 108010021331 carfilzomib Proteins 0.000 claims abstract description 82
- 238000001514 detection method Methods 0.000 claims abstract description 31
- 238000002360 preparation method Methods 0.000 claims abstract description 16
- 239000003814 drug Substances 0.000 claims abstract description 15
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 8
- 239000010452 phosphate Substances 0.000 claims abstract description 8
- 239000000945 filler Substances 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 53
- 239000012488 sample solution Substances 0.000 claims description 27
- 239000000523 sample Substances 0.000 claims description 24
- 238000002347 injection Methods 0.000 claims description 17
- 239000007924 injection Substances 0.000 claims description 17
- 239000013558 reference substance Substances 0.000 claims description 15
- 239000007864 aqueous solution Substances 0.000 claims description 13
- 238000012360 testing method Methods 0.000 claims description 12
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 11
- 230000004807 localization Effects 0.000 claims description 11
- 239000008363 phosphate buffer Substances 0.000 claims description 11
- 238000007865 diluting Methods 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- -1 3-chloro-5-methyl phenyl Chemical group 0.000 claims description 8
- 238000004090 dissolution Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000008055 phosphate buffer solution Substances 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 239000012085 test solution Substances 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- 230000005855 radiation Effects 0.000 claims 1
- 229940079593 drug Drugs 0.000 abstract description 10
- DPUFSSYRLDUXPL-UHFFFAOYSA-N (3-chloro-5-methylphenyl) carbamate Chemical compound CC1=CC(Cl)=CC(OC(N)=O)=C1 DPUFSSYRLDUXPL-UHFFFAOYSA-N 0.000 abstract description 5
- 239000007983 Tris buffer Substances 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 239000012535 impurity Substances 0.000 description 12
- 239000011550 stock solution Substances 0.000 description 12
- 238000011835 investigation Methods 0.000 description 11
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 9
- 229920000858 Cyclodextrin Polymers 0.000 description 8
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 8
- 229910052708 sodium Inorganic materials 0.000 description 8
- 239000011734 sodium Substances 0.000 description 8
- 238000000926 separation method Methods 0.000 description 7
- 239000001116 FEMA 4028 Substances 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 6
- 235000011175 beta-cyclodextrine Nutrition 0.000 description 6
- 229960004853 betadex Drugs 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 238000009472 formulation Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 239000008186 active pharmaceutical agent Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 229940088679 drug related substance Drugs 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 229960004543 anhydrous citric acid Drugs 0.000 description 1
- 150000007514 bases Chemical class 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000012069 chiral reagent Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- FOYNBVOYWCQRTP-UHFFFAOYSA-N ethanol;hexane;propan-2-ol Chemical group CCO.CC(C)O.CCCCCC FOYNBVOYWCQRTP-UHFFFAOYSA-N 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000010829 isocratic elution Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000012487 rinsing solution Substances 0.000 description 1
- 238000002604 ultrasonography 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
- G01N30/89—Inverse chromatography
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- 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)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention relates to a method for detecting carfilzomib enantiomers by using a reversed phase chromatography, which adopts the following chromatographic conditions: chromatographic column with amylose-tris (3-chloro-5-methylphenyl carbamate) as filler and mobile phase of 50mM phosphate buffer-acetonitrile with flow rate of 0.8-1.2ml/min and detection wavelength of 210nm, column temperature of 25-40 ℃ and ultraviolet detector, wherein the volume ratio of phosphate buffer-acetonitrile in mobile phase is 60:40-40:60. the method has the advantages of strong specificity, good accuracy, high sensitivity and good precision, and can be used for measuring the enantiomer of the carfilzomib in the bulk drug of the carfilzomib and the preparation, and also is suitable for detecting diastereoisomers.
Description
Technical Field
The invention relates to the field of medicine analysis, in particular to a method for detecting enantiomers in carfilzomib by using a reversed phase chromatography.
Background
Carfilzomib is a weakly basic compound with strong lipid solubility, is almost insoluble in water, and has 5 chiral centers in its structure (as shown in formula 1), so that a plurality of chiral isomers may be introduced during the synthesis of the bulk drug, wherein the specific structure of enantiomer is as shown in formula 2. The sulfobutyl beta cyclodextrin sodium is an anionic beta cyclodextrin derivative, has special affinity and inclusion effect on nitrogen-containing medicines, and has high water solubility, so that the carfilzomib and the sulfobutyl beta cyclodextrin sodium are almost opposite in solubility. Carfilzomib for injection is included with a high proportion of sodium sulfobutylβ cyclodextrin, thus exhibiting high water solubility characteristics similar to sodium sulfobutylβ cyclodextrin.
Current methods for detection of carfilzomib enantiomer impurities are chiral additive-reverse phase chromatography, as well as conventional normal phase chromatography. The carfilzomib auxiliary material for injection is mainly sulfobutyl beta cyclodextrin sodium, has strong ultraviolet terminal absorption and polarity, and is very weak to be retained under a reversed phase chromatographic system. In addition, in patent CN107402259a, normal phase chromatography is adopted, the chromatographic column is CHIRALPAK OX-H column, the mobile phase is n-hexane-isopropanol-ethanol system, and the content of 3 chiral isomers in carfilzomib is measured. When using normal phase chromatography to determine enantiomeric impurities in a formulation, the following disadvantages are mainly present: (1) sample solubility and extraction problems: the conventional normal phase solvent (such as n-hexane-alcohol solvent (isopropanol/methanol/ethanol) is not compatible with sulfobutyl beta cyclodextrin sodium in a sample, and the included carfilzomib and enantiomer thereof have risks in extraction efficiency, (2) the problems of column pressure, chromatographic peak shape and solvent effect are that the sulfobutyl beta cyclodextrin sodium concentration in a sample solution prepared by pure methanol is higher, the solution is not compatible with normal phase mobile phase, precipitation is carried out, so that the column pressure is continuously increased, and the main peak shape is poor, so that the solvent effect exists.
Since chiral isomer impurities may affect the quality of medicines, the detection of chiral isomers is needed in the quality research of bulk drugs and preparations of carfilzomib, but the existing normal phase chromatography is not suitable for the determination of enantiomers in carfilzomib for injection, and the chiral additive-reverse phase chromatography requires special chiral reagents, the development of a detection method suitable for enantiomers in carfilzomib for injection has important significance.
Disclosure of Invention
Aiming at the defects and shortcomings of the existing method for controlling the enantiomer impurities in the carfilzomib in normal phase chromatography, the invention provides a method for detecting the enantiomer impurities in the carfilzomib, which adopts reverse phase chromatography for detection and has the advantages of good specificity, high accuracy, good repeatability, high sensitivity and simple and rapid operation.
In order to achieve the above object, the present invention provides the following.
The invention provides a method for detecting a carfilzomib enantiomer by adopting a reversed phase chromatography, which adopts the following chromatographic conditions:
chromatographic column: a chromatographic column with amylose-tris (3-chloro-5-methylphenyl carbamate) covalently bonded on the surface of silica gel as a filler;
mobile phase: 50mM phosphate buffer-acetonitrile;
flow rate: 0.8-1.2ml/min;
detection wavelength: 210nm;
column temperature: 25-40 ℃;
sample injection amount: 50 μl;
the detector adopts an ultraviolet detector;
the elution mode is isocratic elution;
the volume ratio of phosphate buffer to acetonitrile in the mobile phase is 60:40-40:60;
the phosphate buffer solution in the mobile phase is potassium dihydrogen phosphate buffer solution, and the pH value is 3.0-4.5.
Further, the column packed with amylose-tris (3-chloro-5-methylphenyl carbamate) covalently bonded to the surface of silica gel was CHIRALPAK IG column, more preferably CHIRALPAK IG (Daicel, 4.6 mm. Times.250 mm,5 μm) column.
Further, in the mobile phase, the volume ratio of phosphate buffer to acetonitrile is 50:50.
further, in the mobile phase, the pH of the phosphate buffer was 3.5.
Further, the flow rate was 1.0ml/min.
Further, the column temperature was 30 ℃.
Further, the method comprises the steps of:
(1) Preparation of carfilzomib control solution: taking a proper amount of carfilzomib reference substance, adding a proper amount of acetonitrile, performing ultrasonic dissolution, and preparing a reference substance solution by using a 40% acetonitrile water solution;
(2) Preparation of carfilzomib enantiomer localization solution: taking a proper amount of a carfilzomib enantiomer reference substance, adding a proper amount of acetonitrile for ultrasonic dissolution, and preparing a carfilzomib enantiomer positioning solution by using a 40% acetonitrile aqueous solution;
(3) Preparation of test solution: taking a proper amount of carfilzomib for injection, re-dissolving the carfilzomib with water, and diluting the carfilzomib with 40% acetonitrile water solution to be used as a sample solution;
or, taking a proper amount of carfilzomib raw material medicine, adding a proper amount of acetonitrile for ultrasonic dissolution, diluting with 40% acetonitrile aqueous solution, and preparing a test sample solution;
or, taking a proper amount of other carfilzomib injections except for carfilzomib for injection, and preparing a sample solution;
(4) And (3) taking a carfilzomib reference substance solution, a carfilzomib enantiomer positioning solution and a test sample solution, injecting into a high performance liquid chromatograph, and performing high performance liquid chromatography analysis according to the chromatographic conditions.
The chromatographic column selected by the invention is CHIRALPAK IG column, the filler is amylose-tris (3-chloro-5-methylphenyl carbamate) covalently bonded on the surface of silica gel, and belongs to polysaccharide bonding chiral column, which is not only suitable for normal phase chromatographic systems, but also can be used under reversed phase chromatographic systems, the chiral compound has distinguishing force, the stability of the chromatographic column is high, the tolerance range of column temperature is 0-40 ℃, the tolerance range of pH is 2.0-9.0, and the service life is long.
According to the invention, by utilizing the chiral selectivity difference of chiral chromatographic columns on carfilzomib and enantiomers thereof, a reversed-phase liquid chromatographic analysis method which takes a chromatographic column with amylose-tris (3-chloro-5-methylphenyl carbamate) covalently bonded on the surface of silica gel as a filler as an analysis column is designed, and meanwhile, the separation of the carfilzomib and the enantiomers is realized by utilizing the hydrophobic effect of a system, so that the enantiomer content research is carried out, and the method is suitable for analyzing enantiomers in a carfilzomib bulk drug/preparation, wherein the detection limit reaches 0.05 mug/ml, and the quantitative limit reaches 0.1 mug/ml. Compared with the prior art, the method has obvious advantages, such as no interference of other impurities on enantiomer determination and good specificity; the sample solution is simple to prepare, the sample solubility and the compatibility with a mobile phase system are considered, and the operation is simple; the method prolongs the service life of the chromatographic column and equipment; the accuracy is high, and the repeatability is good; the peak shape of the enantiomer is improved, and the sensitivity is high; the reagent is simple and the detection cost is low. At the same time, the method is also suitable for detecting partial diastereoisomers.
Drawings
Fig. 1 shows that the volume ratio of phosphate buffer-acetonitrile in the mobile phase in example 1 is 50: carfilzomib-labeled test sample solution chromatogram for injection at 50;
FIG. 2 is a chromatogram of a hollow white adjuvant solution of example 5;
FIG. 3 is a chromatogram of the acid disruption solution of carfilzomib drug substance in example 5;
FIG. 4 is a chromatogram of a localization solution of carfilzomib diastereoisomer 1 in example 8;
FIG. 5 is a chromatogram of a localization solution of carfilzomib diastereoisomer 2 in example 8;
FIG. 6 is a chromatogram of a localization solution of carfilzomib diastereoisomer 3 in example 8;
FIG. 7 is a chromatogram of a localization solution of carfilzomib diastereoisomer 4 in example 8;
FIG. 8 is a chromatogram of a localization solution of carfilzomib diastereoisomer 5 in example 8.
Detailed Description
The following examples are provided to further illustrate and understand the spirit of the invention, but do not limit the scope of the invention in any way.
Example 1 flow phase investigation
Detector and chromatographic conditions
HPLC instrument: shimadzu, LC-20AD, PDA detector;
chromatographic column: CHIRALPAK IG column (Daicel, 4.6 mm. Times.250 mm,5 μm); column temperature is 30 ℃; the sample feeding disc does not control the temperature; acquisition time: 20min-40min; the flow rate is 1.0ml/min; detection wavelength: 210nm; the sample was introduced in an amount of 50. Mu.l.
The experimental steps are as follows:
(1) Enantiomer stock solution (containing about 30 μg/ml of enantiomer) was prepared: about 3mg of the carfilzomib enantiomer reference substance is taken, precisely weighed, placed in a 100ml measuring flask, dissolved and diluted to a scale by acetonitrile, and uniformly shaken to obtain the carfilzomib enantiomer reference substance.
(2) Sample preparation: 1 piece of carfilzomib for injection (about 10mg containing carfilzomib) was taken, reconstituted with about 3ml of water, transferred to a 50ml measuring flask, rinsed 3 times with a 40% acetonitrile aqueous solution, transferred to the same 50ml measuring flask, precisely added with 1ml of enantiomer stock solution, diluted to scale with a 40% acetonitrile aqueous solution, and shaken well to obtain a standard sample solution (about 0.2mg/ml containing carfilzomib and about 0.6 μg/ml containing enantiomer).
(3) And (3) detection: 50 μl of the sample solution was poured into a liquid chromatograph, and the chromatogram was recorded. The results are shown in Table 1.
Table 1 flow comparative investigation results
The results show that: the volume ratio of phosphate buffer-acetonitrile in the mobile phase was 60:40-40: at 60, the separation of the carfilzomib and the enantiomer is good, and the method is suitable for detecting the enantiomer in the carfilzomib. Of these, the preferred ratio is phosphate buffer-acetonitrile (50:50), the chromatogram of which is shown in FIG. 1.
EXAMPLE 2 pH investigation of phosphate buffer in Mobile phase
Detector and chromatographic conditions
HPLC instrument: shimadzu, LC-20AD, PDA detector;
chromatographic column: CHIRALPAK IG column (Daicel, 4.6 mm. Times.250 mm,5 μm); column temperature is 30 ℃; the sample feeding disc does not control the temperature; acquisition time: 20min-30min; the flow rate is 1.0ml/min; detection wavelength: 210nm; the sample was introduced in an amount of 50. Mu.l.
The experimental steps are as follows:
(1) Sample solution formulation was the same as in example 1.
(2) And (3) detection: 50 μl of the sample solution was injected into a liquid chromatograph, and the chromatogram was recorded. The results are shown in Table 2.
TABLE 2 results of buffer pH investigation in mobile phase
The results show that: separation of carfilzomib from the enantiomer was sensitive to phosphate buffer pH. When the pH of the phosphate buffer in the mobile phase is 2.5, the separation of the carfilzomib and the enantiomer is poor, the baseline separation is not realized, and the enantiomer integral is affected; when the pH value of the phosphate buffer solution in the mobile phase is 3.0-4.5, the carfilzomib and the enantiomer are well separated, the peak shape of the enantiomer is good, and the method is suitable for detecting the enantiomer in the carfilzomib. Among these, the phosphate buffer pH in the mobile phase is preferably 3.5.
EXAMPLE 3 flow rate investigation
Detector and chromatographic conditions
HPLC instrument: shimadzu, LC-20AD, PDA detector;
chromatographic column: CHIRALPAK IG column (Daicel, 4.6 mm. Times.250 mm,5 μm); column temperature is 30 ℃; the sample feeding disc does not control the temperature; acquisition time: 20min-40min; mobile phase was 50mM phosphate buffer (ph 3.5) -acetonitrile=50:50; detection wavelength: 210nm; the sample was introduced in an amount of 50. Mu.l.
The experimental steps are as follows:
(1) Sample solution formulation was the same as in example 1.
(2) And (3) detection: 50 μl of the sample solution was injected into a liquid chromatograph, and the chromatogram was recorded. The results are shown in Table 3.
TABLE 3 flow rate investigation results
The results show that: at a flow rate of 0.8-1.2ml/min, carfilzomib is well separated from the enantiomer, the enantiomer peak shape is good, and the method is suitable for detecting the enantiomer in the carfilzomib, wherein the preferred flow rate is 1.0ml/min.
Example 4 column temperature investigation
Detector and chromatographic conditions
HPLC instrument: shimadzu, LC-20AD, PDA detector;
chromatographic column: CHIRALPAK IG column (Daicel, 4.6 mm. Times.250 mm,5 μm); the sample feeding disc does not control the temperature; acquisition time: 20min; mobile phase was 50mM phosphate buffer (ph 3.5) -acetonitrile=50:50; the flow rate is 1.0ml/min; detection wavelength: 210nm; the sample was introduced in an amount of 50. Mu.l.
The experimental steps are as follows:
(1) Sample solution formulation was the same as in example 1.
(2) And (3) detection: 50 μl of the sample solution was injected into a liquid chromatograph, and the chromatogram was recorded. The results are shown in Table 4.
Table 4 column temperature investigation results
The results show that: separation of carfilzomib from the enantiomer was sensitive to column temperature. At a column temperature of 20 ℃, carfilzomib and the enantiomer are not well separated, and the enantiomer integral is affected; when the column temperature is 25-40 ℃, the separation degree of the carfilzomib and the enantiomer is increased along with the increase of the column temperature, the peak shape of the enantiomer is sharper, and the method is suitable for detecting the enantiomer in the carfilzomib. Considering that the tolerance of the column to the column temperature is in the range of 0-40 c, it is preferable that the column temperature is 30 c in order to extend the life of the column.
EXAMPLE 5 specificity investigation
Detector and chromatographic conditions:
HPLC instrument: shimadzu, LC-20AT, LC-20AD, VWD detector;
chromatographic column: CHIRALPAK IG column (Daicel, 4.6 mm. Times.250 mm,5 μm); column temperature: 30 ℃; the sample feeding disc does not control the temperature; acquisition time: 20min; mobile phase was 50mM phosphate buffer (ph 3.5) -acetonitrile=50:50; the flow rate is 1.0ml/min; detection wavelength: 210nm; the sample was introduced in an amount of 50. Mu.l.
The experimental steps are as follows:
(1) Sample preparation:
blank auxiliary material solution: and (3) preparing a blank auxiliary material solution by using 40% acetonitrile aqueous solution according to the prescription amount by taking proper amounts of sulfobutyl beta cyclodextrin sodium, anhydrous citric acid and sodium hydroxide.
Carfilzomib drug substance acid-destruction solution: about 20mg of carfilzomib raw material medicine is taken, precisely weighed, placed in a 20ml measuring flask, added with 2ml of 4M hydrochloric acid solution, dispersed by shaking, placed at room temperature for 1h, added with 2ml of 4M sodium hydroxide solution, added with 2ml of acetonitrile, dissolved by ultrasound, diluted to scale by 40% acetonitrile aqueous solution, and evenly shaken to obtain the medicine.
(2) And (3) detection: 50 μl of each sample solution was poured into a liquid chromatograph, and the chromatogram was recorded. Typical chromatograms are shown in fig. 2-3. The results are shown in Table 5.
TABLE 5 results of specificity test
The main degradation impurities of the carfilzomib are hydrolysis impurities and chlorinated impurities, and can be generated under an acidic condition, so that the interference condition of each degradation impurity on enantiomer detection is examined by carrying out hydrochloric acid damage on the crude drug, and the specificity of the method is clarified by combining the interference condition of hollow white auxiliary materials in the preparation. The results show that: the blank auxiliary materials do not interfere with the detection of enantiomers in the carfilzomib for injection; under the condition of hydrochloric acid damage, the detection of enantiomers in the carfilzomib crude drug/injection is not interfered by carfilzomib hydrolysis impurities, chlorinated impurities and the like.
Example 6 limit of detection and limit of quantification investigation
The detector and chromatographic conditions were the same as in example 5.
(1) Sample preparation:
enantiomer control stock solution: about 4mg of enantiomer reference substance is taken, precisely weighed, placed in a 50ml measuring flask, dissolved and diluted to a scale by acetonitrile, and uniformly shaken to obtain the compound. (containing enantiomer about 80. Mu.g/ml)
Enantiomer localization solution: precisely measuring 2ml of enantiomer reference stock solution, placing into a 10ml measuring flask, diluting to scale with 40% acetonitrile water solution, and shaking; then 2ml of the solution is precisely measured, placed in a 50ml measuring flask, diluted to the scale with 40% acetonitrile water solution and shaken well. (containing enantiomer about 0.64. Mu.g/ml)
Quantitative limiting solution: 3ml of enantiomer positioning solution is precisely measured, placed in a 20ml measuring flask, diluted to scale with 40% acetonitrile aqueous solution and shaken well. (containing enantiomer about 0.1. Mu.g/ml)
Detection limit solution: 1.5ml of the enantiomer localization solution was precisely measured, placed in a 20ml measuring flask, diluted to scale with 40% acetonitrile aqueous solution, and shaken well. (containing enantiomer about 0.05. Mu.g/ml)
(2) And (3) detection: taking quantitative limiting solution and continuously sampling for 6 times, detecting limiting solution and sampling for 1 time, and recording a chromatogram.
The result shows that in the 6-needle quantitative limiting solution, the enantiomer peaks S/N are respectively 43, 47, 39, 57, 55 and 45, which are all more than 10, and the peak area RSD is 2.5 percent (which indicates that the instrument sampling precision is good); in the detection limit solution, the enantiomer peak S/N is 17 and is more than 3. The enantiomer detection limit was 0.05. Mu.g/ml and the quantification limit was 0.1. Mu.g/ml.
EXAMPLE 7 precision and accuracy investigation
The detector and chromatographic conditions were the same as in example 5.
(1) Sample preparation:
enantiomer localization solution: formulated as in example 6.
Carfilzomib control stock solution: about 3mg of carfilzomib reference substance is taken, precisely weighed, placed in a 100ml measuring flask, dissolved and diluted to a scale with acetonitrile, and shaken well. (about 30. Mu.g/ml with Carfilzomib)
Carfilzomib control solution: precisely measuring 1ml of carfilzomib reference substance stock solution, placing into a 50ml measuring flask, diluting to scale with 40% acetonitrile water solution, and shaking to obtain the final product. (about 0.6. Mu.g/ml with Carfilzomib)
Sample stock solution: taking 1 piece of carfilzomib for injection (about 10mg containing carfilzomib), adding about 3ml of water for re-dissolution, rinsing a penicillin bottle with 40% acetonitrile water solution for 3 times, transferring to the same 10ml measuring bottle, diluting to a scale with 40% acetonitrile water solution, and shaking uniformly. (3 parts in parallel) (about 1mg/ml with carfilzomib)
Blank test solution: 2ml of the sample stock solution is precisely measured, a 10ml measuring flask is placed, and diluted to a scale by using 40% acetonitrile water solution, and the sample stock solution is uniformly shaken. (3 parts in parallel) (about 0.2mg/ml with carfilzomib)
0.1% of the standard test sample solution: precisely measuring 2ml of the stock solution of the test sample, placing in a 10ml measuring flask, precisely measuring 3ml of the enantiomer positioning solution, placing in the same 10ml measuring flask, diluting to scale with 40% acetonitrile aqueous solution, and shaking uniformly. (3 parts in parallel) (about 0.2. Mu.g/ml with carfilzomib enantiomer, 0.2mg/ml with carfilzomib)
(2) And (3) detection: continuously sampling the Carfilzomib reference substance solution for 6 times, taking each sample solution, injecting the sample solution into a liquid chromatograph, and recording a chromatogram.
(3) And (3) calculating: and calculating according to a main component external standard method, and counting the recovery rate and the recovery rate RSD of 3 parts of 0.1% standard-added test sample solution.
TABLE 6 accuracy results
The result shows that the recovery rate in 3 parts of 0.1% standard-added test sample solution is 88.9% -95.6%, the average recovery rate is 92.3%, the recovery rates are all in the range of 80.0% -120.0%, the RSD (reactive species concentration) of the recovery rate is 3.6% and less than 10.0%, and the method is accurate and reliable, good in precision and suitable for detecting enantiomers in the carfilzomib crude drug/preparation.
EXAMPLE 8 diastereoisomeric Studies
The detector and chromatographic conditions were the same as in example 5.
(1) Sample preparation:
enantiomer stock solution: formulated as in example 1.
Diastereomer 1/2/3/4/5 localization solution: about 3mg of each of the Carfilzomib diastereoisomer 1/2/3/4/5 reference substances (structural formula 3-formula 7) is respectively taken, precisely weighed, put into different 50ml measuring flasks, dissolved and diluted to a scale with acetonitrile, and shaken uniformly; precisely measuring 0.5ml respectively, placing into different 50ml measuring bottles, diluting with 40% acetonitrile water solution to scale, and shaking. (containing diastereomers 1/2/3/4/5 each at about 0.6. Mu.g/ml)
Adding a labeled test sample solution: taking 1 branch of carfilzomib (about 10mg containing carfilzomib) for injection, adding about 3ml of water for re-dissolution, transferring to a 50ml measuring flask, rinsing a penicillin bottle with 40% acetonitrile aqueous solution for 3 times, transferring the rinsing solution to the same 50ml measuring flask, precisely adding 1ml of enantiomer stock solution, diluting to a scale with 40% acetonitrile aqueous solution, and shaking uniformly to obtain a standard sample solution. (containing about 0.2mg/ml of carfilzomib and about 0.6. Mu.g/ml of enantiomer)
(2) And (3) detection: taking 50 μl of each diastereomer positioning solution and the labeled test solution, injecting into a liquid chromatograph, and recording the chromatogram. Typical chromatograms are shown in fig. 1, 4-8.
TABLE 7 diastereoisomer study test results
The carfilzomib structure has 5 chiral centers, so that a plurality of chiral isomers can be introduced in the synthesis process of the bulk drug, and under the method, part of diastereoisomers are studied. The results show that: neither diastereomer 1/2/3/4/5 interferes with the peak formation of carfilzomib and enantiomer, and the method is also applicable to the determination of diastereomer 1/2/3/4/5.
Claims (10)
1. A method for detecting a carfilzomib enantiomer by reverse phase chromatography, comprising the following chromatographic conditions:
chromatographic column: taking amylose-tri (3-chloro-5-methyl phenyl carbamate) covalently bonded on the surface of silica gel as a filler,
mobile phase: 50mM phosphate buffer-acetonitrile, volume ratio of 60:40-40:60,
flow rate: 0.8-1.2ml/min,
detection wavelength: at a wavelength of 210nm,
column temperature: 25-40 ℃,
the detector is an ultraviolet detector which is used for detecting the ultraviolet radiation,
the phosphate buffer solution is potassium dihydrogen phosphate buffer solution, and the pH value of the phosphate buffer solution is 3-4.5.
2. The method of claim 1, wherein the chromatographic column is a CHIRALPAK IG column.
3. The method of claim 1, wherein the volume ratio of 50mM phosphate buffer-acetonitrile in the mobile phase is 50:50.
4. the method of claim 1, wherein the pH of the phosphate buffer in the mobile phase is 3.5.
5. The method of claim 1, wherein the flow rate is 1.0ml/min.
6. The method of claim 1, wherein the column temperature is 30 ℃.
7. The method of claim 1, wherein the chromatographic conditions are such that the sample is introduced in an amount of 50 μl.
8. The method according to any one of claims 1-7, characterized in that the method comprises the steps of:
(1) Preparation of carfilzomib control solution: taking a proper amount of carfilzomib reference substance, adding a proper amount of acetonitrile, performing ultrasonic dissolution, and preparing a reference substance solution by using a 40% acetonitrile water solution;
(2) Preparation of carfilzomib enantiomer localization solution: taking a proper amount of a carfilzomib enantiomer reference substance, adding a proper amount of acetonitrile for ultrasonic dissolution, and preparing a carfilzomib enantiomer positioning solution by using a 40% acetonitrile aqueous solution;
(3) Preparation of test solution: taking a proper amount of carfilzomib for injection, re-dissolving the carfilzomib with water, and diluting the carfilzomib with 40% acetonitrile water solution to be used as a sample solution;
or, taking a proper amount of carfilzomib raw material medicine, adding a proper amount of acetonitrile for ultrasonic dissolution, diluting with 40% acetonitrile aqueous solution, and preparing a test sample solution;
or, taking a proper amount of other carfilzomib injections except for carfilzomib for injection, and preparing a sample solution;
(4) And (3) taking a carfilzomib reference substance solution, a carfilzomib enantiomer positioning solution and a test sample solution, and injecting the solutions into a high performance liquid chromatograph for detection.
9. The method of claim 1, which is also useful for detection of carfilzomib diastereoisomers.
10. The method of claim 9, wherein the diastereomer is a compound of formula 3-formula 7:
。
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