CN114062557B - Detection method for degradation impurities in mizoribine bulk drug - Google Patents
Detection method for degradation impurities in mizoribine bulk drug Download PDFInfo
- Publication number
- CN114062557B CN114062557B CN202111392140.4A CN202111392140A CN114062557B CN 114062557 B CN114062557 B CN 114062557B CN 202111392140 A CN202111392140 A CN 202111392140A CN 114062557 B CN114062557 B CN 114062557B
- Authority
- CN
- China
- Prior art keywords
- mobile phase
- mizoribine
- column
- bulk drug
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000012535 impurity Substances 0.000 title claims abstract description 53
- HZQDCMWJEBCWBR-UUOKFMHZSA-N Mizoribine Chemical compound OC1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 HZQDCMWJEBCWBR-UUOKFMHZSA-N 0.000 title claims abstract description 44
- 229950000844 mizoribine Drugs 0.000 title claims abstract description 44
- 239000003814 drug Substances 0.000 title claims abstract description 32
- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 229940079593 drug Drugs 0.000 title claims abstract description 27
- 230000015556 catabolic process Effects 0.000 title claims abstract description 24
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 24
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 14
- 238000005259 measurement Methods 0.000 claims abstract description 10
- 239000007791 liquid phase Substances 0.000 claims abstract description 7
- 238000010612 desalination reaction Methods 0.000 claims abstract description 6
- 239000002994 raw material Substances 0.000 claims abstract description 5
- 239000012071 phase Substances 0.000 claims description 60
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 32
- 239000000523 sample Substances 0.000 claims description 25
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 13
- 239000007789 gas Substances 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 11
- 238000004811 liquid chromatography Methods 0.000 claims description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 238000001819 mass spectrum Methods 0.000 claims description 9
- 239000012488 sample solution Substances 0.000 claims description 9
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 8
- 238000002347 injection Methods 0.000 claims description 8
- 239000007924 injection Substances 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000013558 reference substance Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000008213 purified water Substances 0.000 claims description 5
- 229910052786 argon Inorganic materials 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 4
- 238000010812 external standard method Methods 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 239000012085 test solution Substances 0.000 claims description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 3
- 239000003085 diluting agent Substances 0.000 claims description 3
- 235000019253 formic acid Nutrition 0.000 claims description 3
- 239000012088 reference solution Substances 0.000 claims description 3
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 abstract description 6
- 238000000926 separation method Methods 0.000 abstract description 5
- 230000000593 degrading effect Effects 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 150000002500 ions Chemical class 0.000 description 8
- 238000012360 testing method Methods 0.000 description 7
- 239000008186 active pharmaceutical agent Substances 0.000 description 5
- 229940088679 drug related substance Drugs 0.000 description 5
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- -1 imidazole nucleoside Chemical class 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000005777 Lupus Nephritis Diseases 0.000 description 1
- 206010029164 Nephrotic syndrome Diseases 0.000 description 1
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- MRWXACSTFXYYMV-FDDDBJFASA-N nebularine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC=C2N=C1 MRWXACSTFXYYMV-FDDDBJFASA-N 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002212 purine nucleoside Substances 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 238000012795 verification 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
-
- 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
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/38—Flow patterns
- G01N30/46—Flow patterns using more than one column
- G01N30/461—Flow patterns using more than one column with serial coupling of separation columns
- G01N30/463—Flow patterns using more than one column with serial coupling of separation columns for multidimensional 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/78—Detectors specially adapted therefor using more than one detector
-
- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8682—Group type analysis, e.g. of components having structural properties in common
-
- 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
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
- G01N2030/3007—Control of physical parameters of the fluid carrier of temperature same temperature for whole column
-
- 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
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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)
- Spectroscopy & Molecular Physics (AREA)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a detection method for degrading impurities in mizoribine bulk drug, which belongs to the technical field of drug analysis, adopts liquid-mass spectrometry to determine the content and structure of the degrading impurities in mizoribine bulk drug, and comprises the following steps: (1) Adopting two-dimensional liquid phase to carry out content measurement, trapping and desalination on degradation impurities in the mizoribine bulk drug; (2) And (3) adopting mass spectrometry to carry out structural measurement on the degradation impurities trapped in the step (1). The detection method is simple, convenient and reliable, has good impurity separation degree, is beneficial to protecting the chromatographic column, and can accurately position specific degradation impurities in the chromatogram under the condition of liquid phase of the mizoribine raw material medicine and identify the impurity structure.
Description
Technical Field
The invention relates to a detection method for degradation impurities in mizoribine bulk drug, belonging to the technical field of drug analysis.
Background
Mizoribine (mizoribine MZR) is an imidazole nucleoside immunosuppressant separated from a culture solution of mould Eupenicillum brefeldianum in 1971, and specifically inhibits purine nucleoside synthesis and inhibits the growth rate of lymphocytes. In recent years, the composition is clinically used for inhibiting rejection reaction during kidney transplantation, and can also be used for treating autoimmune diseases such as lupus nephritis, rheumatoid arthritis and nephrotic syndrome. Imidazole standThe guest is white or slightly yellow crystal powder, the chemical name is 5-hydroxy-1-beta-D-ribofuranose-1H-imidazole-4-carboxamide, and the molecular formula is C 9 H 13 N 3 O 6 Molecular weight 259.22, readily soluble in water or dimethylsulfoxide, practically insoluble in methanol, ethanol or chloroform, melting point about 198 ℃, structural formula as follows:
according to the requirements of four 9001 bulk drugs and preparation stability test guidelines of Chinese pharmacopoeia 2020, performing an influence factor test, placing a sample to be tested in an open weighing bottle, spreading the sample into a thin layer with the thickness of 5mm, respectively placing the sample for 0 and 10 days at the temperature of 60 ℃, the relative humidity of 45% and the relative humidity of 25 ℃, detecting that one impurity is obviously increased, and performing structural identification according to the requirements of the guidelines. At present, only the detection method of the mizoribine related substances is described in the Japanese pharmacopoeia, because of the limit of the solubility of the mizoribine, the Japanese pharmacopoeia adopts a phosphoric acid aqueous solution as a mobile phase, the column pressure is high in the detection process, the chromatographic column is easy to damage, the separation degree of impurity peaks before the main peak is poor, as shown in FIG. 10, all peaks of 2.568, 3.279 and 3.813 minutes cannot be separated, and the condition of detecting an unknown impurity structure by liquid chromatography-mass spectrometry cannot be met.
Disclosure of Invention
The invention aims to provide a detection method for degrading impurities in mizoribine bulk drug, which has good impurity separation degree, and can accurately position specific impurities in a chromatogram under a liquid phase condition and identify impurity structures.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the detection method of the degradation impurity in the mizoribine bulk drug adopts liquid-mass spectrometry to determine the content and the structure of the degradation impurity in the mizoribine bulk drug, and comprises the following steps:
(1) Adopting two-dimensional liquid phase to carry out content measurement, trapping and desalination on degradation impurities in the mizoribine bulk drug;
(2) And (3) adopting mass spectrometry to carry out structural measurement on the degradation impurities trapped in the step (1).
The technical scheme of the invention is further improved as follows: the step (1) specifically comprises the following steps:
s1: preparing a test solution: taking a mobile phase as a solvent, dissolving and fixing the volume of the mizoribine raw material medicine to obtain a sample solution;
s2: preparing a reference substance liquid: diluting the sample solution with mobile phase as diluent to obtain reference solution;
s3: and (3) measuring: precisely measuring the solution of the sample and the solution of the reference substance respectively, injecting into a two-dimensional liquid chromatograph, and recording the chromatograms under the following specific conditions:
1 st -D liquid chromatography conditions: chromatographic column: c18 column (4.6 mm. Times.250 mm,5 μm), mobile phase: phosphoric acid in water-methanol (98:2), flow rate: 0.8ml/min, column temperature: 20 ℃, detection wavelength: 220nm, sample injection amount: 20. Mu.L;
2 nd -D liquid chromatography conditions: chromatographic column: c18 column (2.1 mm. Times.50 mm,2 μm), detection wavelength: 220nm, column temperature: 20 ℃, flow rate: sample injection amount of 0.3 ml/min: 20 μl, mobile phase a:0.1% formic acid, mobile phase B: acetonitrile, gradient elution procedure: 0-3.00 minutes, 95% mobile phase A and 5% mobile phase B; 3.00-8.00 minutes, the mobile phase A gradually changes from 95% to 70%, and the mobile phase B gradually changes from 5% to 30%;8.00-10.00 minutes, 70% mobile phase A and 30% mobile phase B; 10.00-10.10 minutes, the mobile phase A gradually changes from 70% to 95%, and the mobile phase B gradually changes from 30% to 5%; the mobile phase A is 95% and the mobile phase B is 5% in 10.10-12.00 minutes.
The technical scheme of the invention is further improved as follows: the mass spectrum conditions of the step (2) are as follows: ion source: ESI (+/-), interface voltage: 4.5kV, scanning range: primary mass spectrometry: m/z is 200-1500; automatic multistage: m/z is 100-1000; atomizing gas: nitrogen 1.5L/min, dry gas: nitrogen 10L/min, collision gas: argon, desolventizing tube temperature: 200 ℃, heating module temperature: 200 ℃, detector voltage: 1.65kV, calibration method: and (5) automatically tuning the optimized voltage and calibrating the mass number by an external standard method.
The technical scheme of the invention is further improved as follows: the preparation process of the phosphoric acid aqueous solution of the mobile phase is to dissolve 1ml of phosphoric acid in 1500ml of purified water.
The technical scheme of the invention is further improved as follows: said 1 st the-D liquid chromatography column is Venusil MP C18 chromatography column, 2 nd the-D liquid chromatographic column is InertSustatin TM C18 chromatographic column
By adopting the technical scheme, the invention has the following technical effects:
the detection method has good impurity separation degree, is beneficial to protecting a chromatographic column, and can accurately position specific degradation impurities in a chromatogram under the condition of liquid phase of the mizoribine bulk drug and identify an impurity structure.
The invention completes the desalination process of the mobile phase while rapidly analyzing the target peak, and the detection method is simple, convenient and reliable.
The invention is described in 1 st And methanol is added into the mobile phase D, so that the solubility of a sample is ensured under the corresponding liquid chromatography condition, the impurity separation degree is good, the detection result is accurate, and the protection of a chromatographic column is facilitated.
Drawings
FIG. 1 is sample 1 of the mizoribine drug substance of the present invention tested for influence factor for 0 day st D, a chromatogram;
FIG. 2 is sample 1 of the mizoribine drug substance of the present invention for 10 days st D, a chromatogram;
FIG. 3 is the invention 1 st D, verifying a specificity result diagram through methodology;
FIG. 4 is the invention 1 st D, verifying a detection limit result diagram through methodology;
FIG. 5 is a primary mass spectrum of the degradation impurity positive ion mode of the mizoribine bulk drug of the invention;
FIG. 6 is a secondary mass spectrum of the degraded impurity positive ion mode of the mizoribine bulk drug of the invention;
FIG. 7 is a primary mass spectrum of the degradation impurity anion mode of the mizoribine bulk drug of the invention;
FIG. 8 is a secondary mass spectrum of the degradation impurity anion mode of the mizoribine bulk drug of the invention;
FIG. 9 is sample 1 of comparative example 1 mizoribine drug substance influence factor test for 10 days st D, a chromatogram;
FIG. 10 is a sample 1stD chromatogram of comparative example 2 mizoribine drug substance influence factor test for 10 days.
Detailed Description
The invention is further described in detail below with reference to the attached drawings and specific examples:
the detection method of the degradation impurity in the mizoribine bulk drug adopts liquid-mass spectrometry to determine the content and the structure of the degradation impurity in the mizoribine bulk drug, and comprises the following steps:
(1) The method adopts two-dimensional liquid phase to carry out content measurement, trapping and desalination on degradation impurities in the mizoribine bulk drug, and specifically comprises the following steps:
s1: preparing a test solution: taking a mobile phase as a solvent, dissolving and fixing the volume of the mizoribine raw material medicine to obtain a sample solution;
s2: preparing a reference substance liquid: diluting the sample solution with mobile phase as diluent to obtain reference solution;
s3: and (3) measuring: precisely measuring the solution of the sample and the solution of the reference substance respectively, injecting into a two-dimensional liquid chromatograph, and recording the chromatograms under the following specific conditions:
1 st -D liquid chromatography conditions: chromatographic column: venusil MP C18 column (4.6 mm. Times.250 mm,5 μm), mobile phase: phosphoric acid aqueous solution-methanol (98:2) prepared by dissolving 1ml phosphoric acid in 1500ml purified water at a flow rate: 0.8ml/min, column temperature: 20 ℃, detection wavelength: 220nm, sample injection amount: 20. Mu.L;
2 nd -D liquid chromatography conditions: inertSustain TM C18 column (2.1 mm. Times.50 mm,2 μm), detection wavelength: 220nm, column temperature: 20 ℃, flow rate: sample injection amount of 0.3 ml/min: 20 μl, mobile phase a:0.1% formic acid, mobile phase B: acetonitrile, gradient elution procedure: 0-3.00 minutes, 95% mobile phase A and 5% mobile phase B; 3.00-8.00 minutes, the mobile phase A gradually changes from 95% to 70%,mobile phase B was graded from 5% to 30%;8.00-10.00 minutes, 70% mobile phase A and 30% mobile phase B; 10.00-10.10 minutes, the mobile phase A gradually changes from 70% to 95%, and the mobile phase B gradually changes from 30% to 5%; the mobile phase A is 95% and the mobile phase B is 5% in 10.10-12.00 minutes.
(2) And (3) adopting mass spectrum to carry out structural measurement on the degradation impurities trapped in the step (1), wherein the mass spectrum conditions are as follows: ion source: ESI (+/-), interface voltage: 4.5kV, scanning range: primary mass spectrometry: m/z is 200-1500; automatic multistage: m/z is 100-1000; atomizing gas: nitrogen 1.5L/min, dry gas: nitrogen 10L/min, collision gas: argon, desolventizing tube temperature: 200 ℃, heating module temperature: 200 ℃, detector voltage: 1.65kV, calibration method: and (5) automatically tuning the optimized voltage and calibrating the mass number by an external standard method.
Example 1
1. Instrument and reagent
Shimadzu 2D-LC-IT-TOF-MS/MS,
mizoribine (lot numbers 1906002, 20190901),
phosphoric acid (national instruments for medicine, analytical grade), methanol (Fisher, USA), purified water was prepared in the laboratory.
2. Chromatographic conditions
1 st -D liquid chromatography conditions: venusil MP C18 column (4.6 mm. Times.250 mm,5 μm); taking phosphoric acid aqueous solution-methanol (98:2) as a mobile phase, wherein the preparation process of the phosphoric acid aqueous solution comprises the steps of dissolving 1ml of phosphoric acid into 1500ml of purified water; the flow rate is 0.8ml/min, the column temperature is 20 ℃, the detection wavelength is 220nm, and the sample injection amount is as follows: 20. Mu.L.
2 nd -D liquid chromatography conditions: inertSustain TM C18 column (2.1 mm. Times.50 mm,2 μm), detection wavelength: 220nm, column temperature: 20 ℃, flow rate: 0.3ml/min, sample injection amount of 20 μl, mobile phase: 0.1% formic acid-acetonitrile, gradient elution, elution procedure see table below (valve switch flow path into mass spectrum after 1.5 min):
| time (min) | 0 | 3.00 | 8.00 | 10.00 | 10.10 | 12.00 |
| Mobile phase a (%) | 95 | 95 | 70 | 70 | 95 | 95 |
| Mobile phase B (%) | 5 | 5 | 30 | 30 | 5 | 5 |
Mass spectrometry conditions: ion source: ESI (+/-), interface voltage: 4.5kV, scanning range: primary mass spectrometry: m/z is 200-1500; automatic multistage: m/z is 100-1000; atomizing gas: nitrogen 1.5L/min, dry gas: nitrogen 10L/min, collision gas: argon, desolventizing tube temperature: 200 ℃, heating module temperature: 200 ℃, detector voltage: 1.65kV, calibration method: and (5) automatically tuning the optimized voltage and calibrating the mass number by an external standard method.
3. Measurement
Preparation of test solution: taking 50mg of mizoribine bulk drug, precisely weighing, placing into a 25mL volumetric flask, adding a mobile phase (phosphoric acid aqueous solution-methanol (98:2)) to fix the volume and dissolving to prepare a solution containing about 2mg of mizoribine per 1mL, and taking the solution as a sample solution.
Preparation of control solution: precisely measuring 1ml of the sample solution, placing in a 100ml volumetric flask, diluting to scale with mobile phase, and shaking; then, 5ml of the diluted solution was precisely removed and placed in a 50ml volumetric flask to prepare a solution containing about 2. Mu.g per 1ml as a control solution.
And (3) measuring: the samples were measured in an amount of 25. Mu.l, and the samples were injected into a liquid chromatograph to record a chromatogram. The relative main peak retention time of the chromatogram of the sample solution is 0.34, and the impurity peak area is not larger than the main peak area (0.1%) of the control solution, if the impurity peak area is larger than the main peak area of the control solution, the structure identification is needed.
4. Methodological verification
4.1 specificity
Taking the mobile phase as a blank sample, precisely measuring 25 μl, injecting into a liquid chromatograph, and recording the chromatogram. As can be seen from fig. 3, the blank solvent has no interference to the sample measurement.
4.2 limit of detection
The control solution was measured precisely, diluted gradually with mobile phase, measured precisely at 25. Mu.l, injected into a liquid chromatograph, and the sample concentration at 3 XSNR was the detection limit, which was 100ng/ml, as shown in FIG. 4.
5. Analysis of results
5.1 1 st D-LC analysis results
1 st The main purpose of D analysis is to determine the retention time of target impurities to facilitate entrapment into LOOP for two-dimensional desalination analysis. Sample 1 of mizoribine drug substance influence factor test for 0 day and 10 days st D chromatograms are shown in fig. 1 and 2, and can be seen by comparing fig. 1 and 2: the relative retention time of degraded impurities was 0.34 (i.e., the peak with peak time 2.993min in fig. 2).
The test samples of the influence factors of two different existing factories are detected, and the results are shown in the following table:
| sample of | Specific impurity (%) | Other maximum mono-impurity (%) | Impurity sum (%) |
| 1906002 (0 days) | Not detected | 0.05 | 0.19 |
| 20190901 (0 days) | Not detected | 0.06 | 0.21 |
| 1906002 (10 days) | 0.16 | 0.06 | 0.39 |
| 20190901 (10 days) | 0.17 | 0.07 | 0.42 |
Among them, the specific impurity results of 1906002 batches of samples for 10 days are shown in the following table:
| sequence number | Retention time (min) | Area of | Area (%) |
| 1 | 2.568 | 1244 | 0.03 |
| 2 | 2.678 | 2149 | 0.05 |
| 3 | 2.993 | 7526 | 0.16 |
| 4 | 3.265 | 536 | 0.02 |
| 5 | 3.372 | 489 | 0.03 |
| 6 | 3.721 | 2066 | 0.04 |
| 7 | 3.899 | 3005 | 0.06 |
5.2 2 nd D-LC and Mass Spectrometry analysis results
Degradation impurity (r.t. = 2.993 min) was switched into LOOP 1, pass 2 nd D-LC and mass spectrometry to obtain multi-stage mass spectrograms under positive ion and negative ion modes.
As shown in FIGS. 5-8, the molecular ion peaks are identical in positive and negative ion modes, the cracking rule is identical, and the degradation impurity has a relative molecular weight of 277.0878 and a possible molecular formula C 9 H 15 N 3 O 7 The structural formula is presumed to be as follows:
comparative example 1
Detecting mizoribine in sample (1906002 batches) of mizoribine bulk drug effect factor test for 10 days by adopting a mizoribine detection method reported in literature (journal of Chinese clinical pharmacology, volume 36, 15) for measuring mizoribine concentration in plasma of patients after kidney transplantation by using high performance liquid chromatography-tandem mass spectrometry, 1 st The D chromatogram is shown in FIG. 9, and no impurity peak was detected.
Comparative example 2
1, detecting a sample (1906002 batches) of mizoribine raw material drug influence factor test for 10 days by adopting a detection method of mizoribine related substances recorded in Japanese pharmacopoeia, wherein a mobile phase is a phosphoric acid aqueous solution and a pure phosphoric acid aqueous solution system is adopted as a mobile phase, and 1 st The D chromatogram is shown in FIG. 10, 2.568, 3.279 andthe peak at 3.813 minutes cannot be effectively separated, and a plurality of impurities are possibly combined to form a peak, so that the single impurity amount is high, the upper limit specified in Japanese pharmacopoeia is reached, and the specific impurity results are shown in the following table:
Claims (2)
1. a detection method for degradation impurities in mizoribine bulk drug is characterized by comprising the following steps: the content and structure of degraded impurities in the mizoribine bulk drug are measured by adopting liquid-mass spectrometry, and the method comprises the following steps:
(1) Adopting two-dimensional liquid phase to carry out content measurement, trapping and desalination on degradation impurities in the mizoribine bulk drug;
(2) Carrying out structure measurement on the degradation impurities trapped in the step (1) by adopting mass spectrometry;
the step (1) specifically comprises the following steps:
s1: preparing a test solution: taking a mobile phase as a solvent, dissolving and fixing the volume of the mizoribine raw material medicine to obtain a sample solution;
s2: preparing a reference substance liquid: diluting the sample solution with mobile phase as diluent to obtain reference solution;
s3: and (3) measuring: precisely measuring the solution of the sample and the solution of the reference substance respectively, injecting into a two-dimensional liquid chromatograph, and recording the chromatograms under the following specific conditions:
1 st -D liquid chromatography conditions: chromatographic column: c18 column, 4.6mm X250 mm,5 μm mobile phase: phosphoric acid aqueous solution-methanol, volume ratio of 98:2, flow rate: 0.8ml/min, column temperature: 20 ℃, detection wavelength: 220nm, sample injection amount: 20. Mu.L; the preparation process of the phosphoric acid aqueous solution of the mobile phase comprises the steps of dissolving 1ml of phosphoric acid in 1500ml of purified water;
2 nd -D liquid chromatography conditions: chromatographic column: c18 column, 2.1 mm. Times.50 mm,2 μm, detection wavelength: 220nm, column temperature: 20 ℃, flow rate: sample injection amount of 0.3 ml/min: 20 μl, mobile phase a:0.1% formic acid, mobile phase B: acetonitrile, gradient washThe procedure for removing is as follows: 0-3.00 minutes, 95% mobile phase A and 5% mobile phase B; 3.00-8.00 minutes, the mobile phase A gradually changes from 95% to 70%, and the mobile phase B gradually changes from 5% to 30%;8.00-10.00 minutes, 70% mobile phase A and 30% mobile phase B; 10.00-10.10 minutes, the mobile phase A gradually changes from 70% to 95%, and the mobile phase B gradually changes from 30% to 5%;10.10-12.00 minutes with 95% mobile phase A and 5% mobile phase B;
the mass spectrum conditions of the step (2) are as follows: ion source: esi+/-, interface voltage: 4.5kV, scanning range: primary mass spectrometry: m/z is 200-1500; automatic multistage: m/z is 100-1000; atomizing gas: nitrogen 1.5L/min, dry gas: nitrogen 10L/min, collision gas: argon, desolventizing tube temperature: 200 ℃, heating module temperature: 200 ℃, detector voltage: 1.65kV, calibration method: automatically tuning the optimized voltage and calibrating the mass number by an external standard method;
the relative molecular weight of the degraded impurity is 277.0878, and the molecular formula is C 9 H 15 N 3 O 7 The structure is as follows:
2. the method for detecting degradation impurities in mizoribine bulk drug according to claim 1, which is characterized in that: 1 st the-D liquid chromatography column is Venusil MP C18 chromatography column, 2 nd the-D liquid chromatographic column is InertSustatin TM C18 column.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111392140.4A CN114062557B (en) | 2021-11-19 | 2021-11-19 | Detection method for degradation impurities in mizoribine bulk drug |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111392140.4A CN114062557B (en) | 2021-11-19 | 2021-11-19 | Detection method for degradation impurities in mizoribine bulk drug |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114062557A CN114062557A (en) | 2022-02-18 |
| CN114062557B true CN114062557B (en) | 2023-07-25 |
Family
ID=80279244
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111392140.4A Active CN114062557B (en) | 2021-11-19 | 2021-11-19 | Detection method for degradation impurities in mizoribine bulk drug |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114062557B (en) |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100414293C (en) * | 2006-01-16 | 2008-08-27 | 复旦大学附属华山医院 | Method for determining blood drug level of mizoribine |
| CN101226172A (en) * | 2007-01-17 | 2008-07-23 | 中山大学附属第一医院 | Method for rapidly determining mizoribine blood concentration |
| JP5602349B2 (en) * | 2007-11-20 | 2014-10-08 | 旭化成ファーマ株式会社 | Method for measuring mizoribine and / or ribavirin |
-
2021
- 2021-11-19 CN CN202111392140.4A patent/CN114062557B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN114062557A (en) | 2022-02-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109406650A (en) | Kit and detection method for four kinds of immunosuppressant drug concentrations in Accurate Determining people's whole blood | |
| CN109187839A (en) | The kit and detection method of four kinds of immunosuppressant drug concentrations in Accurate Determining people's whole blood | |
| CN111766317B (en) | Method for measuring GS-441524 content in preparation by using polyethylene glycol and water | |
| CN112067723A (en) | UPLC-MS detection method of p-toluenesulfonate in medicine | |
| CN116773693A (en) | Method for detecting 11 antihypertensive drugs and 1 metabolite in blood by liquid chromatography-tandem mass spectrometry and application of method | |
| CN107991415B (en) | Method for simultaneously separating and measuring pyroglutamic acid and methionine sulfoxide impurities in compound amino acid injection 18AA by liquid chromatography | |
| CN114509526A (en) | LC-MS/MS-based pure solvent type whole blood immunosuppressant drug concentration joint inspection kit and detection method | |
| CN108152411B (en) | Method for detecting impurities in 3-amino-2-methyl crotonate | |
| CN114062557B (en) | Detection method for degradation impurities in mizoribine bulk drug | |
| CN114689737B (en) | Analysis method of S-o-chlorophenylglycine methyl tartrate related substances | |
| CN113237973B (en) | Method for detecting content of impurities in eldecalcitol soft capsules | |
| CN110836930A (en) | Method for measuring content of dichlorobutane in levetiracetam by gas chromatography-mass spectrometry | |
| CN112611813B (en) | Method for testing genotoxic impurities of Sacubitril valsartan sodium starting material | |
| CN115015416A (en) | Method for synchronously measuring content of five antioxidants in medicinal rubber plug | |
| CN107703221A (en) | It is a kind of while determine the method for RABEPRAZOLE SODIUM and its metabolin in Beagle dog plasmas | |
| CN114166960A (en) | Detection method of brexpiprazole related substance | |
| CN110412164B (en) | Method for detecting related substances of mexiletine hydrochloride | |
| CN109298081B (en) | Method for determining impurity A biological sample in Cetilistat | |
| CN110806447B (en) | Screening method and content determination method for bordetella pertussis tracheal cytotoxin | |
| CN112710752A (en) | Detection method of residual solvent of meclofenoxate sodium | |
| CN112321562A (en) | Preparation method and detection method of impurities in omeprazole sodium bicarbonate dry suspension | |
| CN104833752B (en) | Measure the kounidine in elegant jessamine and the method for koumine | |
| CN111323493A (en) | Method for detecting enantiomer of posaconazole starting material | |
| CN109324139A (en) | Ribosylzeatin liquid-liquid extraction-liquid chromatography-tandem mass spectrometry measuring method in a kind of tobacco leaf | |
| CN115856144B (en) | Method for detecting trimethyl sulfoxide iodide impurity in fluconazole bulk drug and injection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| OL01 | Intention to license declared | ||
| OL01 | Intention to license declared | ||
| EE01 | Entry into force of recordation of patent licensing contract | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20220218 Assignee: SHIJIAZHUANG HUAXIN PHARMACEUTICAL Co.,Ltd. Assignor: Hebei Institute for drug and medical device inspection (Hebei cosmetic inspection and Research Center) Contract record no.: X2024980011076 Denomination of invention: A detection method for degradation impurities in imidacloprid raw materials Granted publication date: 20230725 License type: Open License Record date: 20240802 |