CN116106436A - Method for detecting impurities in busulfan bulk drug - Google Patents
Method for detecting impurities in busulfan bulk drug Download PDFInfo
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- CN116106436A CN116106436A CN202211444052.9A CN202211444052A CN116106436A CN 116106436 A CN116106436 A CN 116106436A CN 202211444052 A CN202211444052 A CN 202211444052A CN 116106436 A CN116106436 A CN 116106436A
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- 239000012535 impurity Substances 0.000 title claims abstract description 48
- COVZYZSDYWQREU-UHFFFAOYSA-N Busulfan Chemical compound CS(=O)(=O)OCCCCOS(C)(=O)=O COVZYZSDYWQREU-UHFFFAOYSA-N 0.000 title claims abstract description 40
- 229960002092 busulfan Drugs 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 37
- 239000003814 drug Substances 0.000 title claims abstract description 31
- 229940079593 drug Drugs 0.000 title claims abstract description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 114
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 98
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims abstract description 74
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims abstract description 70
- 238000001514 detection method Methods 0.000 claims abstract description 53
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims abstract description 37
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims abstract description 35
- 238000004817 gas chromatography Methods 0.000 claims abstract description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 15
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 52
- 239000007789 gas Substances 0.000 claims description 39
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 13
- 239000002904 solvent Substances 0.000 claims description 12
- 239000002994 raw material Substances 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 7
- 239000012159 carrier gas Substances 0.000 claims description 7
- 239000002274 desiccant Substances 0.000 claims description 6
- 238000005070 sampling Methods 0.000 claims description 5
- 230000005526 G1 to G0 transition Effects 0.000 claims description 4
- -1 dimethylsiloxane Chemical class 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 3
- 229960005336 magnesium citrate Drugs 0.000 claims description 3
- 235000002538 magnesium citrate Nutrition 0.000 claims description 3
- 239000004337 magnesium citrate Substances 0.000 claims description 3
- PLSARIKBYIPYPF-UHFFFAOYSA-H trimagnesium dicitrate Chemical compound [Mg+2].[Mg+2].[Mg+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O PLSARIKBYIPYPF-UHFFFAOYSA-H 0.000 claims description 3
- 230000015556 catabolic process Effects 0.000 abstract description 4
- 238000006731 degradation reaction Methods 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 73
- 239000000523 sample Substances 0.000 description 37
- 235000019441 ethanol Nutrition 0.000 description 35
- 239000013558 reference substance Substances 0.000 description 24
- 239000012488 sample solution Substances 0.000 description 15
- 238000002347 injection Methods 0.000 description 14
- 239000007924 injection Substances 0.000 description 14
- 238000012360 testing method Methods 0.000 description 13
- 239000012085 test solution Substances 0.000 description 9
- 238000011084 recovery Methods 0.000 description 8
- 239000013557 residual solvent Substances 0.000 description 7
- 238000005303 weighing Methods 0.000 description 7
- 238000010586 diagram Methods 0.000 description 5
- 239000012088 reference solution Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 4
- 239000012490 blank solution Substances 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 238000009835 boiling Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- YGGWPDLYLUHWIQ-UHFFFAOYSA-N n,n-dimethylpyridine-2-carboxamide Chemical compound CN(C)C(=O)C1=CC=CC=N1 YGGWPDLYLUHWIQ-UHFFFAOYSA-N 0.000 description 3
- 238000013112 stability test Methods 0.000 description 3
- XVMSFILGAMDHEY-UHFFFAOYSA-N 6-(4-aminophenyl)sulfonylpyridin-3-amine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)C1=CC=C(N)C=N1 XVMSFILGAMDHEY-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 description 2
- 239000007857 degradation product Substances 0.000 description 2
- 229940098779 methanesulfonic acid Drugs 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 229940100198 alkylating agent Drugs 0.000 description 1
- 239000002168 alkylating agent Substances 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000011134 hematopoietic stem cell transplantation Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- MBABOKRGFJTBAE-UHFFFAOYSA-N methyl methanesulfonate Chemical compound COS(C)(=O)=O MBABOKRGFJTBAE-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 231100000683 possible toxicity Toxicity 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
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- 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
- G01N30/14—Preparation by elimination of some components
-
- 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
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
- G01N30/6073—Construction of the column body in open tubular form
- G01N30/6078—Capillaries
-
- 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
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8637—Peak shape
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- 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
- G01N2030/025—Gas chromatography
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- 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
- G01N2030/3076—Control of physical parameters of the fluid carrier of temperature using specially adapted T(t) profile
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- General Health & Medical Sciences (AREA)
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Abstract
The invention relates to the technical field of analysis and detection, in particular to a method for detecting impurities in busulfan bulk drug. The invention provides a method for detecting impurities in busulfan bulk drug, which comprises the following steps: removing water from a sample liquid to be detected, adopting a headspace gas phase method to sample, and carrying out gas chromatography detection to obtain a gas chromatogram of the sample to be detected, wherein the gas chromatogram comprises impurity peaks; the impurities include ethanol, acetone, pyridine and tetrahydrofuran; calculating the content of impurities in the sample to be detected according to the peak areas of the impurity peaks and a preset standard curve respectively; the standard curve is a relation curve of mass concentration and peak area of impurities. The detection method provided by the invention can be used for simultaneously detecting the process impurities (ethanol, acetone and pyridine) and the degradation impurities (tetrahydrofuran) in the busulfan bulk drug.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a method for detecting impurities in busulfan bulk drug.
Background
Busulfan (Busulfan), chemical name: 1, 4-butanediol dimethyl sulfonate is a cell cycle non-specific alkylating agent antitumor drug, can induce apoptosis, is a classical drug for pretreatment before hematopoietic stem cell transplantation, and the scheme of combining cyclophosphamide is one of clinical classical schemes. Various organic solvents may remain in the busulfan raw material medicine, wherein ethanol and acetone are used as solvents in the raw material medicine synthesis process, and pyridine is used as a catalyst in the synthesis process. Ethanol and acetone are solvents of type 3 (solvents of low potential toxicity) in ICHQ3C, pyridine is a solvent of type 2 (solvents should be restricted) in ICHQ3C, and the residual reference value (PDE) is 2.0 mg/day. Therefore, the detection of the three substances (ethanol, acetone and pyridine) is particularly important.
In addition, busulfan is easy to degrade and is decomposed into methanesulfonic acid and tetrahydrofuran when meeting water. Tetrahydrofuran is a class 2 solvent (solvent should be limited) in ICHQ3C and its PDE is 7.2 mg/day. Therefore, in consideration of the effectiveness of busulfan and the hazard of tetrahydrofuran, the tetrahydrofuran in busulfan bulk drug is controlled.
However, at present, a method for simultaneously detecting process impurities (ethanol, acetone and pyridine) and degradation impurities (tetrahydrofuran) in busulfan bulk drug is not reported.
Disclosure of Invention
In view of the above, the invention provides a method for detecting impurities in a busulfan bulk drug, which can detect process impurities (ethanol, acetone and pyridine) and degradation impurities (tetrahydrofuran) in the busulfan bulk drug at the same time.
In order to achieve the aim of the invention, the invention provides a method for detecting impurities in busulfan bulk drug, which comprises the following steps:
dissolving busulfan raw material medicine into an N, N-dimethylformamide solvent to obtain a sample liquid to be detected;
removing water from a sample liquid to be detected, adopting a headspace gas phase method to sample, and carrying out gas chromatography detection to obtain a gas chromatogram of the sample to be detected, wherein the gas chromatogram comprises impurity peaks; the impurities include ethanol, acetone, pyridine and tetrahydrofuran;
calculating the content of impurities in the sample to be detected according to the peak areas of the impurity peaks and a preset standard curve respectively; the standard curve is a relation curve of mass concentration and peak area of impurities;
the headspace equilibrium temperature of the headspace sampling by the headspace phase method is 70-75 ℃, and the equilibrium time is 10-20 min;
the conditions for the gas chromatography detection include:
the gas chromatographic column is a medium-polarity gas capillary chromatographic column; the carrier gas is nitrogen; the split ratio is 10:1-30:1; the initial temperature of the gas chromatographic column is 40 ℃; the detector is a FID detector.
Preferably, the temperature increase program in the gas chromatographic separation is as follows: the initial temperature was maintained at 40℃for 4min, then at a heating rate of 30℃per minute to 220℃and at 220℃for 5min.
Preferably, the sample injection amount of the sample injection is 1mL.
Preferably, the flow rate of the carrier gas is 1mL/min.
Preferably, the detector temperature is 250 ℃.
Preferably, the water removal mode is desiccant water removal; the water removing agent comprises one or more of anhydrous sodium sulfate, anhydrous magnesium sulfate and anhydrous magnesium citrate.
Preferably, the stationary phase of the gas chromatographic column is 6% cyanopropylphenyl-94% dimethylsiloxane.
The invention provides a method for detecting impurities in busulfan bulk drug, which comprises the following steps: dissolving busulfan raw material medicine into an N, N-dimethylformamide solvent to obtain a sample liquid to be detected; removing water from a sample liquid to be detected, adopting a headspace gas phase method to sample, and carrying out gas chromatography detection to obtain a gas chromatogram of the sample to be detected, wherein the gas chromatogram comprises impurity peaks; the impurities include ethanol, acetone, pyridine and tetrahydrofuran; calculating the content of impurities in the sample to be detected according to the peak areas of the impurity peaks and a preset standard curve respectively; the standard curve is a relation curve of mass concentration and peak area of impurities; the headspace equilibrium temperature of the headspace sampling by the headspace phase method is 70-75 ℃, and the equilibrium time is 10-20 min; the conditions for the gas chromatography detection include: the gas chromatographic column is a medium-polarity gas capillary chromatographic column; the carrier gas is nitrogen; the split ratio is 10:1-30:1; the initial temperature of the gas chromatographic column is 40 ℃; the detector is a FID detector. The invention combines the boiling point (acetone 56 ℃, tetrahydrofuran 66 ℃, ethanol 78 ℃, pyridine 115 ℃) and polarity difference (acetone 6.4, tetrahydrofuran 4.2, ethanol 4.2 and pyridine 5.3) of the object to be detected, selects a neutral polarity chromatographic column with hydrogen bond distinguishing force, and adopts temperature programming to effectively separate the four residual solvents. In addition, the water is removed from the sample liquid to be tested, the headspace balance time and temperature are controlled, the degradation of the busulfan bulk drug in the headspace balance process is effectively controlled, and the influence of the partial degradation product on the sample quality evaluation is reduced. Meanwhile, the reduction of the degradation product methanesulfonic acid is beneficial to the conversion of pyridine from an ionic state to a molecular state, and the extraction of pyridine in the sample injection process is improved, so that the pyridine is more beneficial to detection.
In addition, the detection method provided by the invention has the characteristics of convenience and quickness in operation, economy, high accuracy, high precision and high durability.
Drawings
FIG. 1 is a graph of the linear relationship of ethanol;
FIG. 2 is a graph of the linear relationship of acetone;
FIG. 3 is a linear relationship diagram of tetrahydrofuran;
FIG. 4 is a linear relationship diagram of pyridine;
FIG. 5 is a chromatogram of a blank solution;
FIG. 6 is a chromatogram of a mixed control solution;
FIG. 7 is a chromatogram of a test solution;
FIG. 8 is a chromatogram of a labeled test solution;
FIG. 9 is a chromatogram of a mixed control solution with a split ratio of 3:1 detection;
FIG. 10 is a chromatogram of a mixed control solution under detection conditions with a split ratio of 5:1;
FIG. 11 is a chromatogram of a mixed control solution under detection conditions with a split ratio of 10:1;
FIG. 12 is a chromatogram of a mixed control solution under 20:1 split detection conditions;
FIG. 13 is a chromatogram of a mixed control solution with a split ratio of 30:1 detection;
FIG. 14 is a chromatogram of a labeled test solution (1) under direct sample injection conditions;
FIG. 15 is a chromatogram of the labeled test solution (2) under headspace sampling conditions;
FIG. 16 is a chromatogram of a mixed control solution under detection conditions with an initial column temperature of 60 ℃;
FIG. 17 is a chromatogram of a mixed control solution under detection conditions with an initial column temperature of 40 ℃.
Detailed Description
The invention provides a method for detecting impurities in busulfan bulk drug, which comprises the following steps:
dissolving busulfan raw material medicine into an N, N-dimethylformamide solvent to obtain a sample liquid to be detected;
removing water from a sample liquid to be detected, adopting a headspace gas phase method to sample, and carrying out gas chromatography detection to obtain a gas chromatogram of the sample to be detected, wherein the gas chromatogram comprises impurity peaks; the impurities include ethanol, acetone, pyridine and tetrahydrofuran;
calculating the content of impurities in the sample to be detected according to the peak areas of the impurity peaks and a preset standard curve respectively; the standard curve is a relation curve of mass concentration and peak area of impurities;
in the present invention, unless otherwise specified, the reagents used are commercially available products well known to those skilled in the art.
The busulfan bulk drug is dissolved in an N, N-dimethylformamide solvent to obtain a sample liquid to be detected.
In the invention, the mass concentration of the busulfan bulk drug in the sample liquid to be detected is preferably 5-20%, more preferably 10%.
After the sample liquid to be detected is obtained, the sample liquid to be detected is subjected to water removal, and then is injected by a headspace gas phase method, and gas chromatography detection is carried out to obtain a gas chromatogram of the impurity.
In the present invention, the impurities include ethanol, acetone, pyridine and tetrahydrofuran. In the present invention, the water removal mode is drying agent water removal, and the drying agent preferably comprises one or more of anhydrous sodium sulfate, anhydrous magnesium sulfate and anhydrous magnesium citrate, and more preferably anhydrous sodium sulfate. In the invention, the water removal is particularly preferably realized by adding the sample liquid to be tested and the drying agent into the headspace bottle.
In the invention, the headspace equilibrium temperature of the headspace phase method sample injection is 70-75 ℃, and in the embodiment of the invention, the headspace equilibrium time is 10-20 min, preferably 15min.
In the invention, the gas chromatographic column for gas chromatographic detection is a middle-polarity gas capillary chromatographic column, and the stationary phase of the middle-polarity gas chromatographic column is 6% cyanopropylphenyl-94% dimethylsiloxane. In the embodiment of the invention, the gas chromatographic column is particularly preferably an AgilentDB-624 gas chromatographic column, and the specification is preferably 30m multiplied by 0.32mm multiplied by 1.8 mu m. In the invention, the carrier gas detected by the gas chromatography is nitrogen; the flow rate of the carrier gas is preferably 1mL/min. In the invention, the split ratio of the gas chromatography detection is 10:1 to 30:1, preferably 15:1 to 25:1, and more preferably 20:1. In the invention, the sample injection amount of the sample injection is preferably 1mL. In the present invention, the temperature increase program for gas chromatography detection is preferably: the initial temperature was maintained at 40℃for 4min, then at a heating rate of 30℃per minute to 220℃and at 220℃for 5min. In the present invention, the detector for gas chromatography detection is a FID detector, and the temperature of the detector is preferably 250 ℃.
After a gas chromatogram of the impurity is obtained, the content of the impurity in the sample to be detected is calculated according to the peak area in the gas chromatogram and a preset standard curve; the standard curve is a relation curve of mass concentration and peak area of impurities.
In the present invention, the method for acquiring a predetermined standard curve preferably includes:
respectively weighing ethanol reference substance, acetone reference substance, pyridine reference substance and tetrahydrofuran reference substance, and respectively preparing reference substance solutions with serial concentrations by using N, N-dimethylformamide;
detecting the reference substance solution according to the detection conditions of the gas chromatography in the detection method according to the technical scheme;
and obtaining the preset standard spectrogram by taking the peak area as an abscissa and the concentration as an ordinate.
In the present invention, the purity of the ethanol control, acetone control, tetrahydrofuran control and pyridine control is preferably analytically pure.
In the invention, the concentration of the ethanol reference solution is respectively 0.003, 0.126, 0.252, 0.505, 0.757 and 1.010mg/mL;
the concentration of the acetone control solution is 0.0006, 0.1257, 0.2515, 0.5030, 0.7544 and 1.0059mg/mL;
the concentrations of the tetrahydrofuran reference substance solutions are respectively 0.0003, 0.0181, 0.0362, 0.0723, 0.1085 and 0.1446mg/mL;
the concentrations of the pyridine reference substance solutions are respectively as follows: 1.29, 5.16, 10.32, 20.64, 30.96, 41.28. Mu.g/mL.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
1. And (3) manufacturing a standard curve:
(1) Solution configuration
(1) Blank solution: 2mLN, N-dimethylformamide was precisely measured, a 20mL headspace bottle was set, and about 1g of anhydrous sodium sulfate was added thereto, followed by sealing.
(2) Ethanol solution: about 250mg of absolute ethyl alcohol reference substance is weighed, precisely weighed, placed in a 10mL measuring flask, diluted to a scale by adding N, N-dimethylformamide, and shaken well.
(3) Acetone solution: about 250mg of acetone reference substance is weighed, precisely weighed, placed in a 10mL measuring flask, diluted to a scale by adding N, N-dimethylformamide, and shaken well.
(4) Tetrahydrofuran solution: about 180mg of tetrahydrofuran reference substance is weighed, precisely weighed, placed in a 10mL measuring flask, diluted to a scale by adding N, N-dimethylformamide, and shaken well.
(5) Pyridine solution: about 50mg of pyridine reference substance is weighed, precisely weighed, placed in a 10mL measuring flask, diluted to a scale by adding N, N-dimethylformamide, and shaken well.
(6) Mixing the control stock solution: precisely measuring 5mL of each of the ethanol solution and the acetone solution, 1mL of each of the tetrahydrofuran solution and the pyridine solution, placing the ethanol solution and the acetone solution in the same 50mL measuring flask, adding N, N-dimethylformamide to dilute to a scale, and shaking uniformly.
(7) Mixing the reference substance solution: precisely measuring 10mL of mixed reference substance stock solution, placing into a 50mL measuring flask, adding N, N-dimethylformamide for dilution to scale, shaking uniformly, precisely measuring 2mL, placing into a 20mL headspace bottle, adding anhydrous sodium sulfate about 1g, and sealing.
(8) Test solution: the busulfan crude drug is weighed about 200mg, precisely weighed, placed in a 20mL headspace bottle, added with about 1g of anhydrous sodium sulfate, precisely added with 2mL of N, N-dimethylformamide, and sealed, thus obtaining the busulfan.
(9) Adding a labeled test sample solution: weighing busulfan raw material drug about 200mg, precisely weighing, placing in a 20mL headspace bottle, adding anhydrous sodium sulfate about 1g, precisely adding mixed reference substance solution 2mL, and sealing.
(2) Determination of a Standard Curve
(1) Series concentration reference substance solutions
Taking a proper amount of ethanol solution, and adopting N, N-dimethylformamide to dilute the ethanol solution to the concentration of: 0.003mg/mL, 0.126mg/mL, 0.252mg/mL, 0.505mg/mL, 0.757mg/mL and 1.010mg/mL, thus obtaining ethanol solutions with serial concentrations;
taking a proper amount of acetone solution, and adopting N, N-dimethylformamide to dilute the acetone solution to the concentration of: 0.0006mg/mL, 0.1257mg/mL, 0.2515mg/mL, 0.5030mg/mL, 0.7544mg/mL, 1.0059mg/mL, to obtain acetone solutions of a series of concentrations;
taking a proper amount of tetrahydrofuran solution, and adopting N, N-dimethylformamide to dilute the tetrahydrofuran solution to the concentration of: 0.0003mg/mL, 0.0181mg/mL, 0.0362mg/mL, 0.0723mg/mL, 0.1085mg/mL, 0.1446mg/mL, to obtain tetrahydrofuran solutions of a series of concentrations;
taking a proper amount of pyridine solution, and adopting N, N-dimethylformamide to dilute the pyridine solution to the concentration of: 1.29. Mu.g/mL, 5.16. Mu.g/mL, 10.32. Mu.g/mL, 20.64. Mu.g/mL, 30.96. Mu.g/mL, 41.28. Mu.g/mL, give pyridine solutions of a range of concentrations.
(2) Detection of
Detecting the ethanol solution, the acetone solution, the tetrahydrofuran solution and the pyridine solution with the series of concentrations respectively according to the conditions of gas chromatography detection of two (1), taking the concentration of each reference substance as an abscissa and the peak area as an ordinate, and drawing a standard curve to obtain a standard curve regression equation, wherein the standard curve regression equation is shown in figures 1-4, and figure 1 is a linear relation diagram of ethanol; FIG. 2 is a graph of the linear relationship of acetone; FIG. 3 is a linear relationship diagram of tetrahydrofuran; fig. 4 is a linear relationship diagram of pyridine.
As can be seen from fig. 1 to 4, the peak areas and the concentrations of ethanol, acetone, tetrahydrofuran and pyridine are all linear, the linear correlation coefficients are 0.99994, and the linear relationship is good.
Example 2
Specificity test
(1) Conditions for gas chromatography detection: the gas chromatographic column is an AgilentDB-624 gas chromatographic column; the stationary phase of the gas chromatographic column is 6% cyanopropylphenyl-94% dimethylsiloxane; the temperature-raising program is as follows: the initial temperature is 40 ℃ for 4min, the temperature is increased to 220 ℃ at the heating rate of 30 ℃ per min, and the temperature is kept for 5min; the sample injection amount is 1mL; the split ratio is 20:1; the detector was a FID detector with a detector temperature of 250 ℃.
(2) Taking blank solution, mixed reference substance solution, test sample solution and standard test sample solution, injecting into gas chromatograph, detecting according to the conditions of gas chromatograph detection of two and (1), and the chromatograms are shown in fig. 5-8, wherein fig. 5 is the chromatogram of the blank solution; FIG. 6 is a chromatogram of a mixed control solution; FIG. 7 is a chromatogram of a test solution; FIG. 8 is a chromatogram of a labeled test solution. As can be seen from fig. 5 to 8: the anhydrous sodium sulfate does not interfere with the measurement of each residual solvent, the retention time of each mutagenic impurity in the standard sample solution is consistent with that of each residual solvent in the reference solution, and the separation degree of the residual solvent and the surrounding impurities is good, so that the detection method has good specificity.
Example 3
Detection limit and quantitative limit measurement
Different control solutions were diluted, tested according to "two, (1) conditions for gas chromatography detection", chromatograms were recorded and the signal to noise ratios were checked, and the results are shown in table 1 below.
TABLE 1 quantification limit and detection limit of ethanol, acetone, pyridine, tetrahydrofuran
Example 4
Repeatability test
6 parts of the labeled sample solution are taken to be detected according to the conditions of gas chromatography detection of two and 1, a chromatogram is recorded, the recovery rate is calculated, and the detection result is shown in table 2.
TABLE 2 recovery test data
From 2, it can be seen that: the recovery rates of ethanol, acetone, tetrahydrofuran and pyridine in 6 parts of the repetitive solution are respectively 101.0% -103.7%, 96.17% -101.1%, 99.22% -103.6% and 88.51% -91.75%, and the RSD% is respectively 0.9%, 1.7%, 1.6% and 1.2%.
Example 5
Solution stability test
The control solution and the standard sample solution were taken at different time points and tested according to the "conditions for gas chromatography detection" of two (1), and the chromatograms and recovery rates were recorded, and the results are shown in tables 3 to 4 below. From tables 3 to 4, it can be seen that: the recovery rate of the peak area of each residual solvent in the reference substance solution relative to the peak area at 0 hour is between 99.55 percent and 105.1 percent within 20 hours; the recovery rate of the peak area of each residual solvent in the standard sample solution is 101.2-109.6% relative to the peak area at 0 hour within 12 hours. The control solution was stable for at least 20 hours and the test solution was stable for at least 12 hours.
TABLE 3 solution stability test data
TABLE 4 solution stability test data
Example 6
Taking the mixed reference substance solution to detect according to the conditions of gas chromatography detection of II and 1, wherein the split ratio in the detection conditions is respectively replaced by 3:1, 5:1, 10:1, 20:1 and 30:1, and recording chromatograms, and the results are shown in figures 9-13, wherein figure 9 shows the chromatograms of the mixed reference solution under the detection conditions that the split ratio is 3:1;
FIG. 10 is a chromatogram of a mixed control solution under detection conditions with a split ratio of 5:1; FIG. 11 is a chromatogram of a mixed control solution under detection conditions with a split ratio of 10:1; FIG. 12 is a chromatogram of a mixed control solution under 20:1 split detection conditions; FIG. 13 is a chromatogram of a mixed control solution with a split ratio of 30:1. As can be seen from fig. 9 to 13: the split ratio reaches more than 10:1, the peak shape of each residual solvent in the mixed control solution is good, and the ethanol and the acetone can achieve baseline separation.
Example 7
Preparing and adding a standard sample solution (1), weighing about 200mg of busulfan raw material medicine, precisely weighing, placing into a 10mL measuring flask, adding ethanol, acetone, tetrahydrofuran and pyridine with the limit of 100% level, shaking for dissolving and diluting to a scale, and shaking uniformly.
Preparing a standard sample solution (2), weighing about 200mg of busulfan raw material medicine, precisely weighing, placing in a 20mL headspace bottle, adding about 1g of anhydrous sodium sulfate, precisely adding 2mL of mixed reference substance solution, and sealing.
The method comprises the steps of (1) adding a standard sample solution and (2) adding the standard sample solution, respectively carrying out sample injection in a direct sample injection mode and a headspace sample injection mode according to the conditions of gas chromatography detection of the second and the first, and detecting chromatograms shown in fig. 14-15, wherein fig. 14 is a chromatogram of the standard sample solution (1) under the direct sample injection condition; FIG. 15 is a chromatogram of the addition of the labeled test sample solution (2) under headspace sampling conditions. As can be seen from fig. 14 to 15: by adopting direct sample injection, the busulfan as a main component can be injected into a gas chromatographic column along with the solution to generate a huge interference peak around 7min, thereby interfering with the determination of pyridine. The headspace sample injection is carried out by extracting the upper air in the headspace bottle, the busulfan boiling point is about 464 ℃, the busulfan will not gasify in the headspace balance process, and the interference peak disappears.
Example 8
Detecting the mixed reference substance solution according to the conditions of gas chromatography detection (II, 1), wherein the temperature rise program is respectively replaced by 40 ℃ for 4min, the temperature is raised to 220 ℃ at the temperature rise rate of 30 ℃/min, the temperature is kept for 5min, the temperature is kept for 4min, the temperature is raised to 220 ℃ at the temperature rise rate of 30 ℃/min, the temperature is kept for 5min, the detection is carried out, the chromatograms are recorded, and the results are shown in figures 16-17, wherein figure 16 is the chromatograms of the mixed reference solution under the detection condition that the initial column temperature is 60 ℃; FIG. 17 is a chromatogram of a mixed control solution under detection conditions with an initial column temperature of 40 ℃. As can be seen from fig. 16 to 17: the initial column temperature of 60 ℃ is adopted, and the peak shape of ethanol in the mixed reference substance solution is abnormal. The initial column temperature is reduced to 40 ℃ because of the lower boiling point of the ethanol, and the peak type of the ethanol is obviously improved.
Example 9
The mixed reference solution is prepared according to the 'preparation of first solution'.
Preparing a 50% level standard test solution: the busulfan crude drug is weighed about 200mg, precisely weighed, placed in a 20mL headspace bottle, added with about 1g of anhydrous sodium sulfate, precisely added with 2mL of limited 50% concentration level ethanol, pyridine, tetrahydrofuran and pyridine N, N-dimethylformamide solution, and sealed, thus obtaining the busulfan.
Preparing 100% level standard test sample solution: the busulfan crude drug is weighed about 200mg, precisely weighed, placed in a 20mL headspace bottle, added with about 1g of anhydrous sodium sulfate, precisely added with 2mL of limited concentration 100% ethanol, pyridine, tetrahydrofuran and pyridine N, N-dimethylformamide solution, and sealed, thus obtaining the busulfan.
Preparing 150% level standard test sample solution: the busulfan crude drug is weighed about 200mg, precisely weighed, placed in a 20mL headspace bottle, added with about 1g of anhydrous sodium sulfate, precisely added with 2mL of limited concentration of 150% ethanol, pyridine, tetrahydrofuran and pyridine N, N-dimethylformamide solution, and sealed, thus obtaining the busulfan.
The mixed reference substance solution, the 50% level marked test substance solution, the 100% level marked test substance solution and the 150% level marked test substance solution are respectively replaced by a headspace condition (1) according to the conditions of gas chromatography detection of II and 1: the "headspace equilibrium temperature 80 ℃, the equilibrium time 30min" and the headspace condition (2) "headspace equilibrium temperature 75 ℃, the equilibrium time 15min" were measured respectively, and the measurement results are shown in table 5, from table 5, it can be seen that: in the test of the headspace condition (2), pyridine recovery is greatly improved, and the accuracy of the method is obviously improved.
TABLE 5 recovery of ethanol, acetone, tetrahydrofuran and pyridine under different headspace conditions
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. A detection method of impurities in busulfan bulk drug comprises the following steps:
dissolving busulfan raw material medicine into an N, N-dimethylformamide solvent to obtain a sample liquid to be detected;
removing water from a sample liquid to be detected, adopting a headspace gas phase method to sample, and carrying out gas chromatography detection to obtain a gas chromatogram of the sample to be detected, wherein the gas chromatogram comprises impurity peaks; the impurities include ethanol, acetone, pyridine and tetrahydrofuran;
calculating the content of impurities in the sample to be detected according to the peak areas of the impurity peaks and a preset standard curve respectively; the standard curve is a relation curve of mass concentration and peak area of impurities;
the headspace equilibrium temperature of the headspace sampling by the headspace phase method is 70-75 ℃, and the equilibrium time is 10-20 min;
the conditions for the gas chromatography detection include:
the gas chromatographic column is a medium-polarity gas capillary chromatographic column; the carrier gas is nitrogen; the split ratio is 10:1-30:1; the initial temperature of the gas chromatographic column is 40 ℃; the detector is a FID detector.
2. The method according to claim 1, wherein the temperature increase program for gas chromatography detection is: the initial temperature was maintained at 40℃for 4min, then at a heating rate of 30℃per minute to 220℃and at 220℃for 5min.
3. The method according to claim 1, wherein the sample amount is 1mL.
4. The method of claim 1, wherein the carrier gas has a flow rate of 1mL/min.
5. The method of claim 1, wherein the detector temperature is 250 ℃.
6. The method according to claim 1, wherein the water removal is desiccant water removal; the drying agent comprises one or more of anhydrous sodium sulfate, anhydrous magnesium sulfate and anhydrous magnesium citrate.
7. The method according to claim 1, wherein the stationary phase of the gas chromatography column is 6% cyanopropylphenyl-94% dimethylsiloxane.
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