CN115389661B - Method for detecting genotoxic impurities in linezolid glucose injection - Google Patents
Method for detecting genotoxic impurities in linezolid glucose injection Download PDFInfo
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- CN115389661B CN115389661B CN202211016056.7A CN202211016056A CN115389661B CN 115389661 B CN115389661 B CN 115389661B CN 202211016056 A CN202211016056 A CN 202211016056A CN 115389661 B CN115389661 B CN 115389661B
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- 239000012535 impurity Substances 0.000 title claims abstract description 79
- 231100000024 genotoxic Toxicity 0.000 title claims abstract description 74
- 230000001738 genotoxic effect Effects 0.000 title claims abstract description 74
- 238000000034 method Methods 0.000 title claims abstract description 35
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 title claims abstract description 34
- 229940093181 glucose injection Drugs 0.000 title claims abstract description 34
- TYZROVQLWOKYKF-ZDUSSCGKSA-N linezolid Chemical compound O=C1O[C@@H](CNC(=O)C)CN1C(C=C1F)=CC=C1N1CCOCC1 TYZROVQLWOKYKF-ZDUSSCGKSA-N 0.000 title claims abstract description 34
- 229960003907 linezolid Drugs 0.000 title claims abstract description 34
- 238000001514 detection method Methods 0.000 claims abstract description 58
- GOJJPJCUSDMTAT-SSDOTTSWSA-N [(2s)-1-acetamido-3-chloropropan-2-yl] acetate Chemical compound CC(=O)NC[C@@H](CCl)OC(C)=O GOJJPJCUSDMTAT-SSDOTTSWSA-N 0.000 claims abstract description 23
- ZEQCFSSBZPZEFJ-UHFFFAOYSA-N 4-(2-fluoro-4-nitrophenyl)morpholine Chemical compound FC1=CC([N+](=O)[O-])=CC=C1N1CCOCC1 ZEQCFSSBZPZEFJ-UHFFFAOYSA-N 0.000 claims abstract description 20
- FQGIBHQUVCGEAC-UHFFFAOYSA-N 3-Fluoro-4-morpholinoaniline Chemical compound FC1=CC(N)=CC=C1N1CCOCC1 FQGIBHQUVCGEAC-UHFFFAOYSA-N 0.000 claims abstract description 18
- 150000002500 ions Chemical class 0.000 claims abstract description 16
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims abstract description 16
- -1 compound ions Chemical class 0.000 claims abstract description 10
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 10
- 238000012544 monitoring process Methods 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims description 62
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 239000013558 reference substance Substances 0.000 claims description 21
- 239000012071 phase Substances 0.000 claims description 18
- 239000011550 stock solution Substances 0.000 claims description 17
- 238000001228 spectrum Methods 0.000 claims description 14
- 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 13
- 235000019253 formic acid Nutrition 0.000 claims description 13
- 238000010828 elution Methods 0.000 claims description 11
- 238000007865 diluting Methods 0.000 claims description 8
- 239000007791 liquid phase Substances 0.000 claims description 8
- 239000007921 spray Substances 0.000 claims description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 238000001819 mass spectrum Methods 0.000 claims description 6
- 238000010812 external standard method Methods 0.000 claims description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 4
- 238000005507 spraying Methods 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 230000002452 interceptive effect Effects 0.000 abstract description 2
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 30
- 238000011084 recovery Methods 0.000 description 14
- 239000007789 gas Substances 0.000 description 13
- 239000000463 material Substances 0.000 description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000012467 final product Substances 0.000 description 3
- 238000002552 multiple reaction monitoring Methods 0.000 description 3
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 2
- 229960004543 anhydrous citric acid Drugs 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 229960001031 glucose Drugs 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229960003085 meticillin Drugs 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000008215 water for injection Substances 0.000 description 2
- 208000031729 Bacteremia Diseases 0.000 description 1
- 241000194031 Enterococcus faecium Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
Classifications
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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
-
- 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
-
- 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
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
-
- 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
- G01N2030/065—Preparation using different phases to separate parts of sample
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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)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention belongs to the technical field of analysis and detection, and provides a method for detecting genotoxic impurities in linezolid glucose injection. According to the invention, three genotoxic impurities (S) -N- (2-acetoxyl-3-chloropropyl) acetamide, 4- (2-fluoro-4-nitrophenyl) morpholine and 3-fluoro-4- (4-morpholinyl) aniline in linezolid glucose injection are quantitatively detected by a triple quadrupole liquid chromatography-mass spectrometry method, a multi-reaction monitoring (MRM) mode is adopted for mass spectrometry detection, and a large amount of interfering ions are removed through selection of compound ions on two-stage ions, so that the chemical background of mass spectrometry is reduced, and the signal to noise ratio of a target detection object is obviously improved, thereby realizing high specificity and high sensitivity of detection.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a method for detecting genotoxic impurities in linezolid glucose injection.
Background
Linezolid glucose injection may be used to treat the following infections caused by a particular microbiologically sensitive strain: infections caused by vancomycin-resistant enterococcus faecium, including concurrent bacteremia; the pathogenic bacteria are staphylococcus aureus (methicillin-sensitive or methicillin-resistant strains) or streptococcus pneumoniae (including multi-drug resistant strains [ MDRSP ])). However, linezolid glucose injection has genotoxic impurities such as: (S) -N- (2-acetoxy-3-chloropropyl) acetamide, 4- (2-fluoro-4-nitrophenyl) morpholine and 3-fluoro-4- (4-morpholinyl) aniline; can influence the use effect of the linezolid glucose injection. Therefore, quantitative studies on three genotoxic impurities in linezolid glucose injection are needed. The commonly used analysis method is high performance liquid chromatography, but the liquid chromatography has low sensitivity and cannot meet the detection requirement.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for detecting genotoxic impurities in linezolid glucose injection. The detection method provided by the invention has high sensitivity and meets the detection requirement.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for detecting genotoxic impurities in linezolid glucose injection, which comprises the following steps:
performing triple quadrupole liquid chromatography-mass spectrometry detection on the linezolid glucose injection to be detected to obtain a chromatogram of genotoxic impurities in the linezolid glucose injection to be detected;
comparing the chromatogram of the genotoxic impurity in the linezolid glucose injection to be detected with a standard chromatogram of a preset genotoxic impurity, and calculating by an external standard method to obtain the content of the genotoxic impurity in the sample to be detected;
the genotoxic impurities include: (S) -N- (2-acetoxy-3-chloropropyl) acetamide, 4- (2-fluoro-4-nitrophenyl) morpholine and 3-fluoro-4- (4-morpholinyl) aniline;
the triple quadrupole rod liquid chromatography-mass spectrometry detection comprises liquid phase detection and mass spectrometry detection;
the parameters of the mass spectrum detection include:
the ion source type is ESI + ；
The detection mode is positive ions;
the monitoring mode is a multi-reaction monitoring mode;
the air pressure of the air curtain is 30-35 psi;
the collision air pressure is 9-11 psi;
the spraying voltage is 5500V;
the temperature is 650 ℃;
the spray gas pressure is 55-70 psi;
the pressure of the drying air is 50-70 psi;
the valve events are shown in table 1:
table 1 valve event
Compound ion pair information is shown in table 2:
table 2 compound ion pair information
Preferably, the parameters of the liquid phase detection include:
the filler of the chromatographic column is octadecylsilane chemically bonded silica gel;
the mobile phase system comprises a mobile phase A and a mobile phase B;
the mobile phase A is an aqueous solution with the formic acid volume fraction of 0.09-0.11%;
the mobile phase B is methanol solution with the volume fraction of formic acid of 0.09-0.11%;
the elution mode is gradient elution;
the procedure for the gradient elution is shown in table 3:
TABLE 3 gradient elution procedure
The flow rate is 0.8mL/min;
the sample injection amount is 1 mu L;
column temperature is 40 ℃;
the temperature of the sample chamber was 15 ℃.
Preferably, the standard spectrogram of the predetermined genotoxic impurity is a linear curve of genotoxic impurity reference substance concentration-chromatographic information.
Preferably, the method for obtaining a standard spectrum of the predetermined genotoxic impurity comprises the steps of:
dissolving the genotoxic impurity reference substance to obtain a genotoxic impurity reference substance stock solution;
diluting the genotoxic impurity control stock solution to prepare a series of linear solutions of 5ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 150ng/mL, 200ng/mL, 250ng/mL, 300ng/mL and 400 ng/mL;
carrying out the triple quadrupole rod liquid chromatography-mass spectrometry detection on the series of linear solutions to obtain chromatographic information of the series of linear solutions;
and establishing a linear standard curve based on the concentration and chromatographic information of the series of linear solutions to obtain a standard spectrogram of the predetermined genotoxic impurities.
Preferably, the chromatographic information comprises chromatographic peak areas.
The invention provides a method for detecting genotoxic impurities in linezolid glucose injection, which comprises the following steps: performing triple quadrupole liquid chromatography-mass spectrometry detection on the linezolid glucose injection to be detected to obtain a chromatogram of genotoxic impurities in the linezolid glucose injection to be detected; comparing the chromatogram of the genotoxic impurity in the linezolid glucose injection to be detected with a standard chromatogram of a preset genotoxic impurity, and calculating by an external standard method to obtain the content of the genotoxic impurity in the sample to be detected; the genotoxic impurities include: (S) -N- (2-acetoxy-3-chloropropyl) acetamide and 4- (2-fluoro-4-nitrophenyl) morpholineAnd 3-fluoro-4- (4-morpholinyl) aniline; the triple quadrupole rod liquid chromatography-mass spectrometry detection comprises liquid phase detection and mass spectrometry detection; the parameters of the mass spectrum detection include: the ion source type is ESI + The method comprises the steps of carrying out a first treatment on the surface of the The detection mode is positive ions; monitoring mode: a multiple reaction monitoring mode; the air pressure of the air curtain is 30-35 psi; the collision air pressure is 9-11 psi; the spraying voltage is 5500V; the temperature is 650 ℃; the spray gas pressure is 55-70 psi; the pressure of the drying air is 50-70 psi; the valve events are shown in table 1: the compound ion pair information is shown in table 2.
According to the invention, three genotoxic impurities (S) -N- (2-acetoxyl-3-chloropropyl) acetamide, 4- (2-fluoro-4-nitrophenyl) morpholine and 3-fluoro-4- (4-morpholinyl) aniline in linezolid glucose injection are quantitatively detected by a triple quadrupole liquid chromatography-mass spectrometry method, a multi-reaction monitoring (MRM) mode is adopted for mass spectrometry detection, and a large amount of interfering ions are removed through selection of compound ions on two-stage ions, so that the chemical background of mass spectrometry is reduced, and the signal to noise ratio of a target detection object is obviously improved, thereby realizing high specificity and high sensitivity of detection.
Drawings
FIG. 1 is a standard spectrum of the predetermined genotoxic impurity (S) -N- (2-acetoxy-3-chloropropyl) acetamide;
FIG. 2 is a standard spectrum of a predetermined genotoxic impurity (4- (2-fluoro-4-nitrophenyl) morpholine;
FIG. 3 is a standard spectrum of 3-fluoro-4- (4-morpholinyl) aniline, a predetermined genotoxic impurity.
Detailed Description
The invention provides a method for detecting genotoxic impurities in linezolid glucose injection, which comprises the following steps:
performing triple quadrupole liquid chromatography-mass spectrometry detection on the linezolid glucose injection to be detected to obtain a chromatogram of genotoxic impurities in the linezolid glucose injection to be detected;
comparing the chromatogram of the genotoxic impurity in the linezolid glucose injection to be detected with a standard chromatogram of a preset genotoxic impurity, and calculating by an external standard method to obtain the content of the genotoxic impurity in the sample to be detected.
In the present invention, the materials used in the present invention are preferably commercially available products unless otherwise specified.
According to the invention, triple quadrupole liquid chromatography-mass spectrometry detection is carried out on the linezolid glucose injection to be detected, and a chromatogram of genotoxic impurities in the linezolid glucose injection to be detected is obtained.
In the invention, the triple quadrupole liquid chromatography-mass spectrometry detection comprises liquid phase detection and mass spectrometry detection.
In the present invention, the parameters of mass spectrometry detection include: the ion source type is ESI + The method comprises the steps of carrying out a first treatment on the surface of the The detection mode is positive ions; monitoring mode: a multiple reaction monitoring mode; the air curtain air pressure is 30-35 psi, particularly preferably 30psi, and the air curtain air is preferably nitrogen; the collision gas pressure is 9-11 psi, preferably 10psi, and the collision gas is preferably nitrogen; the spraying voltage is 5500V; the temperature is 650 ℃; the spray gas pressure is 55-70 psi, preferably 60psi; the spray gas is preferably nitrogen; the drying air pressure is 50-70 psi, preferably 60psi; the drying gas is preferably nitrogen; the valve events are shown in table 1; the compound ion pair information is shown in table 2.
In the present invention, the parameters of the liquid phase detection include: the packing of the chromatographic column is preferably octadecylsilane chemically bonded silica, and the particle size of the packing is preferably 3 μm; the chromatography column is preferably of ACE EXCEL3C18 type. In the present invention, the size of the column is preferably 4.6X100 mm.
In the present invention, the mobile phase system of the liquid phase detection preferably includes a mobile phase a and a mobile phase B; the mobile phase A is preferably an aqueous solution with the volume fraction of formic acid of 0.09-0.11%, and is further preferably an aqueous solution with the volume fraction of formic acid of 0.1%; the mobile phase B is preferably methanol solution with the volume fraction of formic acid of 0.09-0.11%, and more preferably methanol solution with the volume fraction of formic acid of 0.1%; the elution mode is preferably gradient elution; the procedure for the gradient elution is preferably as shown in table 3: the flow rate is preferably 0.8mL/min; the sample injection amount is preferably 1 mu L; the column temperature is preferably 40 ℃; the temperature of the sample chamber is preferably 15 ℃.
In the present invention, the genotoxic impurities include: (S) -N- (2-acetoxy-3-chloropropyl) acetamide, 4- (2-fluoro-4-nitrophenyl) morpholine and 3-fluoro-4- (4-morpholinyl) aniline.
After obtaining a chromatogram of genotoxic impurities in linezolid glucose injection to be detected, comparing the chromatogram of the genotoxic impurities in the linezolid glucose injection to be detected with a standard chromatogram of preset genotoxic impurities, and calculating by an external standard method to obtain the content of the genotoxic impurities in a sample to be detected.
In the present invention, the standard spectrum of the predetermined genotoxic impurity is preferably a linear curve of genotoxic impurity control concentration-chromatographic information.
In the present invention, the method for obtaining a standard spectrum of the predetermined genotoxic impurity preferably comprises the steps of:
dissolving the genotoxic impurity reference substance to obtain a genotoxic impurity reference substance stock solution;
diluting the genotoxic impurity control stock solution to prepare a series of linear solutions of 5ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 150ng/mL, 200ng/mL, 250ng/mL, 300ng/mL and 400 ng/mL;
carrying out the triple quadrupole rod liquid chromatography-mass spectrometry detection on the series of linear solutions to obtain chromatographic information of the series of linear solutions;
and establishing a linear standard curve based on the concentration and chromatographic information of the series of linear solutions to obtain a standard spectrogram of the predetermined genotoxic impurities.
According to the invention, the genotoxic impurity reference substance is dissolved to obtain the genotoxic impurity reference substance stock solution. In the invention, when the genotoxic impurity reference substances are (S) -N- (2-acetoxy-3-chloropropyl) acetamide reference substance and 3-fluoro-4- (4-morpholinyl) aniline reference substance, the dissolved reagent is preferably an aqueous solution with the volume fraction of acetonitrile of 10%. In the present invention, when the genotoxic impurity reference substance is 4- (2-fluoro-4-nitrophenyl) morpholine reference substance, the dissolved reagent is preferably an aqueous solution with a volume fraction of 50% acetonitrile. In the invention, the concentration of the genotoxic impurity control stock solution is preferably 0.05mg/mL.
After the genotoxic impurity control stock solution is obtained, the genotoxic impurity control stock solution is diluted into a series of linear solutions of 5ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 150ng/mL, 200ng/mL, 250ng/mL, 300ng/mL and 400 ng/mL. In the present invention, the diluted reagent is preferably a blank adjuvant solution. In the present invention, the preparation method of the blank auxiliary material solution preferably includes: precisely weighing 0.164g of sodium citrate, 0.085g of anhydrous citric acid, 4.57g of anhydrous glucose, adding 100mL of water for injection, and sterilizing at 121 ℃ for 12 minutes to obtain the blank auxiliary material solution.
After obtaining the linear solution with a series of concentration, the invention carries out the triple quadrupole liquid chromatography-mass spectrometry detection on the series of linear solution to obtain the chromatographic information of the series of linear solution. In the present invention, the parameters of the triple quadrupole liquid chromatography-mass spectrometry detection are preferably consistent with the above technical solutions, and will not be described herein. In the present invention, the chromatographic information is preferably a chromatographic peak area.
After obtaining the chromatographic information of the series of linear solutions, the invention establishes a linear standard curve based on the concentration and the chromatographic information of the series of linear solutions to obtain the standard spectrogram of the predetermined genotoxic impurities. The parameters for establishing the linear standard curve are not particularly limited, and conventional technical means well known to those skilled in the art are adopted.
The method for detecting genotoxic impurities in linezolid glucose injection provided by the present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
Examples
(1) Instrument and reagent
Instrument: TRIPLE quadrupole liquid chromatography (model TRIPLE QUAD 4500); ACE EXCEL3C18-AR 3 μm, 4.6X100 mm column; an electronic analytical balance.
Reagent: formic acid (mass spectrum grade), acetonitrile (mass spectrum grade or chromatographic purity), methanol (mass spectrum grade or chromatographic purity), purified water, impurity (S) -N- (2-acetoxy-3-chloropropyl) acetamide, 4- (2-fluoro-4-nitrophenyl) morpholine and 3-fluoro-4- (4-morpholinyl) aniline controls.
(2) Conditions for chromatographic detection
Chromatographic column: octadecylsilane chemically bonded silica as filler (ACE EXCEL3C18-AR 3 μm, 4.6X100 mm);
mobile phase a: water (0.1% formic acid) (0.1% formic acid in water by volume);
mobile phase B: methanol (0.1% formic acid) (methanol solution with 0.1% formic acid volume fraction);
mobile phase gradient procedure is shown in table 3;
flow rate: 0.8mL/min;
sample injection amount: 1 μl;
column temperature: 40 ℃;
sample chamber: 15 ℃;
(3) Conditions for mass spectrometry detection
Ion source type: ESI (electronic service provider interface) + ；
Detection mode: a positive ion;
monitoring mode: multiple Reaction Monitoring (MRM) mode;
curtain Gas (curtaingas) pressure: 30psi;
collision Gas (Collision Gas) pressure: 10psi;
spray voltage (ionspray voltage): 5500V;
temperature (Temperature): 650 ℃;
spray Gas (Ion Source Gas 1) pressure: 60psi;
drying Gas (Ion Source Gas 2) pressure: 60psi;
the valve event table is shown in table 1; the compound ion pair information is shown in table 2.
(4) Solution preparation
10% acetonitrile solution: v (V) Acetonitrile ：V Water and its preparation method ＝10：90；
50% acetonitrile solution: v (V) Acetonitrile ：V Water and its preparation method ＝50：50；
Blank auxiliary material solution: precisely weighing 0.164g of sodium citrate, 0.085g of anhydrous citric acid and 4.57g of anhydrous glucose, adding 100mL of water for injection, and sterilizing at 121 ℃ for 12 minutes.
(S) -N- (2-acetoxy-3-chloropropyl) acetamide control stock: precisely weighing 2.5mg of (S) -N- (2-acetoxy-3-chloropropyl) acetamide reference substance, placing into a 50mL measuring flask, adding 10% acetonitrile solution, ultrasonically dissolving, diluting to scale, and shaking uniformly to obtain the final product.
4- (2-fluoro-4-nitrophenyl) morpholine control stock solution: precisely weighing 2.5mg of 4- (2-fluoro-4-nitrophenyl) morpholine reference substance, placing into a 50mL measuring flask, adding 50% acetonitrile solution, ultrasonically dissolving, diluting to scale, and shaking uniformly to obtain the final product.
3-fluoro-4- (4-morpholino) aniline control stock solution: precisely weighing 2.5mg of 3-fluoro-4- (4-morpholinyl) aniline reference substance, placing into a 50mL measuring flask, adding 10% acetonitrile solution, ultrasonically dissolving, diluting to scale, and shaking to obtain the final product.
(5) Specialization of
And (3) accurately measuring the reference substance stock solution under the item "(4)" respectively, diluting with blank auxiliary materials to prepare an impurity positioning solution, a mixed reference substance solution and a standard test substance solution which respectively contain 200ng per 1mL, taking 1 mu L of each solution respectively, and measuring according to the methods under the items "(2) and (3)".
The results show that the determination of the blank auxiliary material solution and the blank solvent does not interfere with the determination of the impurities (S) -N- (2-acetoxy-3-chloropropyl) acetamide, 4- (2-fluoro-4-nitrophenyl) morpholine and 3-fluoro-4- (4-morpholinyl) aniline, and the method has better specificity.
(6) Quantitative limit and detection limit
And (3) accurately measuring the storage solutions of the reference substances under the item (4), diluting with blank auxiliary materials to prepare quantitative limiting solutions with the signal to noise ratio of about 10 and detection limiting solutions with the signal to noise ratio of 3, taking 1 mu L of each quantitative limiting solution and each detection limiting solution, and measuring according to the methods under the items (2) and (3).
The result shows that the quantitative limit concentration of the (S) -N- (2-acetoxy-3-chloropropyl) acetamide is 0.5048ng/mL, and the detection limit concentration is 0.1514ng/mL; the quantitative limit concentration of 4- (2-fluoro-4-nitrophenyl) morpholine is 0.9970ng/mL, and the detection limit concentration is 0.2991ng/mL; the quantitative limit concentration of the 3-fluoro-4- (4-morpholinyl) aniline is 0.3959ng/mL, and the detection limit concentration is 0.1188ng/mL; the method is high in detection sensitivity.
(7) Linearity and range
The control stock solutions under the item "(4)" are respectively measured precisely, and the stock solutions are diluted by blank auxiliary materials to prepare linear solutions with target concentrations of 5ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 150ng/mL, 200ng/mL, 250ng/mL, 300ng/mL and 400ng/mL, and 1 mu L of each linear solution is respectively taken and sampled for measurement according to the methods under the items "(2) and (3)". The results are shown in FIGS. 1 to 3.
FIG. 1 is a standard spectrum of the predetermined genotoxic impurity (S) -N- (2-acetoxy-3-chloropropyl) acetamide; calculated concentrations were obtained using the standard spectra shown in fig. 1, as shown in table 4.
TABLE 4 actual and calculated concentrations of (S) -N- (2-acetoxy-3-chloropropyl) acetamide Linear solution
| Actual concentration/ng/mL | Number of sample injections | Calculate concentration/ng/mL | Accuracy% |
| 4.82 | 1 | 4.75 | 98.47 |
| 19.3 | 1 | 20.29 | 105.14 |
| 48.2 | 1 | 49.48 | 102.65 |
| 96.4 | 1 | 94.77 | 98.30 |
| 145 | 1 | 153.37 | 105.77 |
| 193 | 1 | 187.04 | 96.91 |
| 241 | 1 | 236.01 | 97.93 |
| 289 | 1 | 284.7 | 98.51 |
| 386 | 1 | 369.25 | 95.66 |
As can be seen from fig. 1 and table 4: the linear equation of (S) -N- (2-acetoxy-3-chloropropyl) acetamide is y=7.42e+003x+93 (r= 0.9992), indicating that (S) -N- (2-acetoxy-3-chloropropyl) acetamide has good linearity in the concentration range of 4.82ng/mL to 386 ng/mL.
FIG. 2 is a standard spectrum of a predetermined genotoxic impurity (4- (2-fluoro-4-nitrophenyl) morpholine) and calculated concentrations using the standard spectrum shown in FIG. 2 are shown in Table 5.
TABLE 5 actual and calculated concentrations of (S) -N- (2-acetoxy-3-chloropropyl) acetamide Linear solution
As can be seen from fig. 2 and table 5: the linear equation of 4- (2-fluoro-4-nitrophenyl) morpholine was y=7.64e+003x+1.8e+003 (r=0.9999), indicating that 4- (2-fluoro-4-nitrophenyl) morpholine was well linear over the concentration range of 5.08ng/mL to 406 ng/mL.
FIG. 3 is a standard spectrum of a predetermined genotoxic impurity 3-fluoro-4- (4-morpholinyl) aniline; calculated concentrations were obtained using the standard spectrum shown in fig. 3, as shown in table 6.
TABLE 63 actual and calculated concentrations of linear solution of fluoro-4- (4-morpholino) aniline
| Actual concentration/ng/mL | Number of sample injections | Calculate concentration/ng/mL | Accuracy% |
| 5.12 | 1 | 5.09 | 99.50 |
| 20.5 | 1 | 20.87 | 101.81 |
| 51.2 | 1 | 51.97 | 101.51 |
| 102 | 1 | 100.49 | 98.52 |
| 154 | 1 | 153.10 | 99.42 |
| 205 | 1 | 202.70 | 98.88 |
| 256 | 1 | 255.95 | 99.98 |
| 307 | 1 | 304.59 | 99.22 |
| 410 | 1 | 414.21 | 101.03 |
As can be seen from fig. 3 and table 6: the linear equation of 3-fluoro-4- (4-morpholinyl) aniline is y=8.74 e+003x-2.33e+003 (r=0.9999), indicating that 3-fluoro-4- (4-morpholinyl) aniline is well linear in the concentration range of 5.12ng/ml to 410 ng/ml.
(8) Accuracy of
The control stock solutions under the item "(4)" are respectively measured precisely, the solutions with the impurity concentrations of 100ng/mL, 200ng/mL and 300ng/mL are prepared by using the linezolid glucose injection, 1 μl of each solution is respectively taken and sampled, and the solutions are measured according to the methods under the items "(2) and (3)".
The results show that: the recovery rate of the (S) -N- (2-acetoxy-3-chloropropyl) acetamide is 73.33-81.82%, and the RSD is 4.0%; the recovery rate of 4- (2-fluoro-4-nitrophenyl) morpholine is between 100.6% and 103.1%, and RSD is 0.94%; the recovery rate of the 3-fluoro-4- (4-morpholinyl) aniline is between 78.17% and 86.22%, and the RSD is 3.5%. The method is shown to have good accuracy.
(9) Precision of
Different analysts respectively measure the reference substance stock solutions under the item (4) precisely on different dates, 12 parts of 100% recovery rate solution with the impurity concentration of 200ng/mL is prepared by using linezolid glucose injection, 1 mu L of each solution is taken and sampled, and the solutions are measured according to the methods under the items (2) and (3).
The results show that: the recovery rate of the 6 repeated solutions of the (S) -N- (2-acetoxy-3-chloropropyl) acetamide is between 77.01 and 81.82 percent, and the RSD of the recovery rate is 2.2 percent; the recovery of 6 intermediate precision solutions of (S) -N- (2-acetoxy-3-chloropropyl) acetamide was between 74.26% and 82.67%, with 6 parts RSD 4.9% and 12 parts recovery calculated for repeatability with RSD 3.7%. The recovery rate of the 6 repeated solutions of the 4- (2-fluoro-4-nitrophenyl) morpholine is between 101.0 and 104.6 percent, and the RSD of the recovery rate is 1.4 percent; the recovery rate of the 6 intermediate precision solutions of 4- (2-fluoro-4-nitrophenyl) morpholine is between 99.50% and 101.5%, the RSD of 6 parts is 0.68%, and the RSD of 12 parts calculated by repeatability is 1.8%. The recovery rate of the 6 repeated solutions of the 3-fluoro-4- (4-morpholino) aniline is between 81.63% and 86.22%, and the RSD of the recovery rate is 2.0%; the recovery rate of the 6 intermediate precision solutions of the 3-fluoro-4- (4-morpholino) aniline is between 77.23% and 80.69%, the RSD of 6 parts is 2.1%, and the RSD of 12 parts calculated by repeatability is 3.8%. The method has better precision.
(10) Sample measurement
And detecting 3 batches of produced samples by the established method, wherein the results of (S) -N- (2-acetoxyl-3-chloropropyl) acetamide and 4- (2-fluoro-4-nitrophenyl) morpholine are lower than the detection limit, and the detection result of 3-fluoro-4- (4-morpholinyl) aniline is in a linear range.
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 (4)
1. The method for detecting the genotoxic impurities in the linezolid glucose injection is characterized by comprising the following steps of:
performing triple quadrupole liquid chromatography-mass spectrometry detection on the linezolid glucose injection to be detected to obtain a chromatogram of genotoxic impurities in the linezolid glucose injection to be detected;
comparing the chromatogram of the genotoxic impurity in the linezolid glucose injection to be detected with a standard chromatogram of a preset genotoxic impurity, and calculating by an external standard method to obtain the content of the genotoxic impurity in the sample to be detected;
the genotoxic impurities include: (S) -N- (2-acetoxy-3-chloropropyl) acetamide, 4- (2-fluoro-4-nitrophenyl) morpholine and 3-fluoro-4- (4-morpholinyl) aniline;
the triple quadrupole rod liquid chromatography-mass spectrometry detection comprises liquid phase detection and mass spectrometry detection;
the parameters of the mass spectrum detection include:
the ion source type is ESI + ；
The detection mode is positive ions;
the monitoring mode is a multi-reaction monitoring mode;
the air pressure of the air curtain is 30-35 psi;
the collision air pressure is 9-11 psi;
the spraying voltage is 5500V;
the temperature is 650 ℃;
the spray gas pressure is 55-70 psi;
the pressure of the drying air is 50-70 psi;
the valve events are shown in table 1:
table 1 valve event
Compound ion pair information is shown in table 2:
table 2 compound ion pair information
The parameters of the liquid phase detection include:
the filler of the chromatographic column is octadecylsilane chemically bonded silica gel;
the mobile phase system comprises a mobile phase A and a mobile phase B;
the mobile phase A is an aqueous solution with the formic acid volume fraction of 0.09-0.11%; the mobile phase B is methanol solution with the volume fraction of formic acid of 0.09-0.11%; the elution mode is gradient elution;
the procedure for the gradient elution is shown in table 3:
TABLE 3 gradient elution procedure
The flow rate is 0.8mL/min;
the sample injection amount is 1 mu L;
column temperature is 40 ℃;
the temperature of the sample chamber was 15 ℃.
2. The method according to claim 1, wherein the standard spectrum of the predetermined genotoxic impurity is a linear curve of genotoxic impurity control concentration-chromatographic information.
3. The detection method according to claim 1 or 2, wherein the method for obtaining a standard spectrum of the predetermined genotoxic impurity comprises the steps of:
dissolving the genotoxic impurity reference substance to obtain a genotoxic impurity reference substance stock solution;
diluting the genotoxic impurity control stock solution to prepare a series of linear solutions of 5ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 150ng/mL, 200ng/mL, 250ng/mL, 300ng/mL and 400 ng/mL;
carrying out the triple quadrupole rod liquid chromatography-mass spectrometry detection on the series of linear solutions to obtain chromatographic information of the series of linear solutions;
and establishing a linear standard curve based on the concentration and chromatographic information of the series of linear solutions to obtain a standard spectrogram of the predetermined genotoxic impurities.
4. The method of claim 3, wherein the chromatographic information comprises chromatographic peak area.
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| Design, synthesis and activity against Staphylococcus epidermidis of 5-chloro-2- or 5-chloro-4-methyl-9H-xanthen-9-one and some of its derivatives;Gabriela Mazur等;Chem Biol Drug Des;全文 * |
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| UPLC-MS/MS 法测定利奈唑胺及其中间体（S）-N-（2- 乙酰氧基-3- 氯丙基）乙酰胺中的遗传毒性杂质（S）-1- 氨基-3-氯-2- 丙醇;刘峰 等;中南药学;第19卷(第7期);第1439-1443页 * |
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