CN116609453A - Detection method of formylacetate in moxifloxacin hydrochloride - Google Patents
Detection method of formylacetate in moxifloxacin hydrochloride Download PDFInfo
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- 229960005112 moxifloxacin hydrochloride Drugs 0.000 title claims abstract description 86
- IDIIJJHBXUESQI-DFIJPDEKSA-N moxifloxacin hydrochloride Chemical compound Cl.COC1=C(N2C[C@H]3NCCC[C@H]3C2)C(F)=CC(C(C(C(O)=O)=C2)=O)=C1N2C1CC1 IDIIJJHBXUESQI-DFIJPDEKSA-N 0.000 title claims abstract description 86
- 238000001514 detection method Methods 0.000 title claims abstract description 36
- OAKURXIZZOAYBC-UHFFFAOYSA-M 3-oxopropanoate Chemical compound [O-]C(=O)CC=O OAKURXIZZOAYBC-UHFFFAOYSA-M 0.000 title description 17
- SYFFHRPDTQNMQB-UHFFFAOYSA-N ethyl 3-oxopropanoate Chemical compound CCOC(=O)CC=O SYFFHRPDTQNMQB-UHFFFAOYSA-N 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 27
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 claims abstract description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 57
- 239000002904 solvent Substances 0.000 claims description 42
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 14
- 150000002500 ions Chemical class 0.000 claims description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 8
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 7
- 239000000945 filler Substances 0.000 claims description 6
- 238000001819 mass spectrum Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 5
- 239000000377 silicon dioxide Substances 0.000 claims description 5
- 238000000132 electrospray ionisation Methods 0.000 claims description 4
- 238000004949 mass spectrometry Methods 0.000 claims description 4
- 238000012544 monitoring process Methods 0.000 claims description 4
- 239000007921 spray Substances 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 7
- 239000000243 solution Substances 0.000 description 53
- 238000007865 diluting Methods 0.000 description 24
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- 238000004090 dissolution Methods 0.000 description 12
- 239000000523 sample Substances 0.000 description 12
- 238000012360 testing method Methods 0.000 description 12
- 229960003702 moxifloxacin Drugs 0.000 description 10
- FABPRXSRWADJSP-MEDUHNTESA-N moxifloxacin Chemical compound COC1=C(N2C[C@H]3NCCC[C@H]3C2)C(F)=CC(C(C(C(O)=O)=C2)=O)=C1N2C1CC1 FABPRXSRWADJSP-MEDUHNTESA-N 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 9
- 239000013558 reference substance Substances 0.000 description 9
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 239000012467 final product Substances 0.000 description 8
- 239000012488 sample solution Substances 0.000 description 8
- 239000012085 test solution Substances 0.000 description 5
- 238000012795 verification Methods 0.000 description 4
- 206010059866 Drug resistance Diseases 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 229940124350 antibacterial drug Drugs 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 108020000946 Bacterial DNA Proteins 0.000 description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Natural products CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 2
- 241000192125 Firmicutes Species 0.000 description 2
- 102000004310 Ion Channels Human genes 0.000 description 2
- -1 aldehyde compound Chemical class 0.000 description 2
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- 231100000024 genotoxic Toxicity 0.000 description 2
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- 239000000047 product Substances 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- FGBGEKHVEJJJLT-LLVKDONJSA-N 1-cyclopropyl-7-[(3r)-3-ethylpyrrolidin-1-yl]-6-fluoro-8-methoxy-4-oxoquinoline-3-carboxylic acid Chemical compound C1[C@H](CC)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN(C3CC3)C2=C1OC FGBGEKHVEJJJLT-LLVKDONJSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 241000606161 Chlamydia Species 0.000 description 1
- 102000003915 DNA Topoisomerases Human genes 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 206010034972 Photosensitivity reaction Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
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- 238000010812 external standard method Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
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- 238000011835 investigation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 208000007578 phototoxic dermatitis Diseases 0.000 description 1
- 231100000018 phototoxicity Toxicity 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 201000009890 sinusitis Diseases 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000002229 urogenital system Anatomy 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
- G01N30/26—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
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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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
- G01N2030/027—Liquid chromatography
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- 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
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Abstract
The invention provides a detection method of formylacetic acid ethyl ester in moxifloxacin hydrochloride, belonging to the technical field of medicine quality detection. The invention provides a detection method of formylacetic acid ethyl ester in moxifloxacin hydrochloride, which comprises the following steps: providing moxifloxacin hydrochloride solution to be tested; performing high performance liquid chromatography-mass spectrometry on the moxifloxacin hydrochloride solution to be detected to obtain a chromatogram of moxifloxacin hydrochloride to be detected; and obtaining the content of the formylacetic acid ethyl ester in the moxifloxacin hydrochloride to be detected according to the standard curve of the formylacetic acid ethyl ester and the chromatogram of the moxifloxacin hydrochloride to be detected. The method provided by the invention can effectively detect the ethyl formylacetate in the moxifloxacin hydrochloride, and is simple to operate.
Description
Technical Field
The invention relates to the technical field of medicine quality detection, in particular to a detection method of formylacetic acid ethyl ester in moxifloxacin hydrochloride.
Background
Moxifloxacin is a fourth-generation broad-spectrum 8-methoxy fluoroquinolone antibacterial drug, belongs to a novel variety in the synthesis of antibacterial drugs, and achieves the antibacterial purpose by inhibiting bacterial DNA topoisomerase and repairing, copying, recombining and transcribing bacterial DNA. Because methoxy is introduced into the 8 th carbon atom of moxifloxacin, the combination capability and the cell membrane penetrating power of the medicine and bacteria are enhanced, and the drug resistance, phototoxicity and cytotoxicity of gram-positive bacteria are reduced, so that the moxifloxacin has the pharmacokinetic characteristics of wide antibacterial spectrum, low drug resistance, good oral absorption, high absolute bioavailability and the like. Moxifloxacin can effectively inhibit skin inflammation, has very good anti-inflammatory and anti-infective effects, has good antibacterial effects on gram-negative bacteria, gram-positive bacteria, mycoplasma, chlamydia and the like, and is often orally taken in clinic. Moxifloxacin hydrochloride is clinically applied to treat community-acquired pneumonia, acute bacterial sinusitis, genitourinary system infection and the like. The moxifloxacin venous preparation and the oral preparation have almost the same pharmacokinetic parameters in human bodies, and are particularly suitable for sequential treatment of antibacterial drugs. The medicine is taken for 1 time every day, has good compliance and is helpful for psychological rehabilitation of patients. In clinical application, the moxifloxacin has the characteristics of less adverse reaction, low drug resistance, reduced corresponding hospitalization time, reduced economic burden and the like, and is worthy of popularization and application.
The formylacetate is a byproduct generated in the moxifloxacin hydrochloride synthesis process, is an aldehyde compound and is a potential genotoxic impurity, but related reports of a detection method of the formylacetate in moxifloxacin hydrochloride are not yet seen at present.
Disclosure of Invention
The invention aims to provide a method for detecting ethyl formylacetate in moxifloxacin hydrochloride, which can effectively detect ethyl formylacetate in moxifloxacin hydrochloride.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a detection method of formylacetic acid ethyl ester in moxifloxacin hydrochloride, which comprises the following steps:
providing moxifloxacin hydrochloride solution to be tested;
performing high performance liquid chromatography-mass spectrometry on the moxifloxacin hydrochloride solution to be detected to obtain a chromatogram of moxifloxacin hydrochloride to be detected;
obtaining the content of the formylacetic acid ethyl ester in the moxifloxacin hydrochloride to be detected according to the standard curve of the formylacetic acid ethyl ester and the chromatogram of the moxifloxacin hydrochloride to be detected;
the high performance liquid chromatography-mass spectrometry conditions include:
the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an ammonia water solution with the volume fraction of 0.1%, and the mobile phase B is methanol; gradient elution procedure: 0 to 6.00min, the volume fraction of the mobile phase A is reduced from 65% to 5% at a constant speed; 6.00-7.00 min, and the volume fraction of the mobile phase A is kept 5%; 7.00-7.01 min, the volume fraction of the mobile phase A is increased from 5% to 65%; 7.01-10.00 min, the volume fraction of the mobile phase A is kept 65%.
Preferably, the filler in the chromatographic column used for the high performance liquid chromatography is octadecylsilane chemically bonded silica, and the granularity of the filler is 2.7 mu m; the specification of the chromatographic column is 3.0mm×100mm.
Preferably, the column temperature of the high performance liquid chromatography is 30 ℃.
Preferably, the mobile phase flow rate of the high performance liquid chromatography is 0.5mL/min.
Preferably, the sample injection volume of the high performance liquid chromatography is 5 μl.
Preferably, the mass spectrometry conditions of the high performance liquid chromatography-mass spectrometry include: an electrospray ionization source; the detection mode is negative ion detection; the source temperature was 550 ℃.
Preferably, the ion spray voltage of the mass spectrum is-4500V.
Preferably, the mass spectrum has an atomizing gas 1 flow rate of 50psi, an atomizing gas 2 flow rate of 55psi, and a curtain gas flow rate of 40psi.
Preferably, the scanning mode of the mass spectrum is a multi-reaction ion monitoring mode.
Preferably, the solvent in the moxifloxacin hydrochloride solution to be detected is methanol.
The invention provides a detection method of formylacetic acid ethyl ester in moxifloxacin hydrochloride, which comprises the following steps: providing moxifloxacin hydrochloride solution to be tested; performing high performance liquid chromatography-mass spectrometry on the moxifloxacin hydrochloride solution to be detected to obtain a chromatogram of moxifloxacin hydrochloride to be detected; obtaining the content of the formylacetic acid ethyl ester in the moxifloxacin hydrochloride to be detected according to the standard curve of the formylacetic acid ethyl ester and the chromatogram of the moxifloxacin hydrochloride to be detected; the high performance liquid chromatography-mass spectrometry conditions include: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an ammonia water solution with the volume fraction of 0.1%, and the mobile phase B is methanol; gradient elution procedure: 0 to 6.00min, the volume fraction of the mobile phase A is reduced from 65% to 5% at a constant speed; 6.00-7.00 min, and the volume fraction of the mobile phase A is kept 5%; 7.00-7.01 min, the volume fraction of the mobile phase A is increased from 5% to 65%; 7.01-10.00 min, the volume fraction of the mobile phase A is kept 65%. The method provided by the invention can effectively detect the ethyl formylacetate in the moxifloxacin hydrochloride, and is simple to operate. The results of the examples show that the specificity, sensitivity, linear range, accuracy and repeatability of the detection method are verified through methodology verification, whether residual ethyl formylacetate is contained in moxifloxacin hydrochloride can be effectively detected, the content of the residual ethyl formylacetate can be accurately measured, the quality of moxifloxacin hydrochloride products is effectively controlled through the method, and the medication safety of medicines is improved.
Drawings
FIG. 1 is a typical HPLC chromatogram of a sample addition solution;
FIG. 2 is a typical HPLC chromatogram of a blank solvent;
FIG. 3 is a typical HPLC chromatogram of a control solution;
FIG. 4 is a typical HPLC chromatogram of a test solution;
fig. 5 is a typical graph of linear range.
Detailed Description
The invention provides a detection method of formylacetic acid ethyl ester in moxifloxacin hydrochloride, which comprises the following steps:
providing moxifloxacin hydrochloride solution to be tested;
performing high performance liquid chromatography-mass spectrometry on the moxifloxacin hydrochloride solution to be detected to obtain a chromatogram of moxifloxacin hydrochloride to be detected;
obtaining the content of the formylacetic acid ethyl ester in the moxifloxacin hydrochloride to be detected according to the standard curve of the formylacetic acid ethyl ester and the chromatogram of the moxifloxacin hydrochloride to be detected;
the high performance liquid chromatography-mass spectrometry conditions include:
the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an ammonia water solution with the volume fraction of 0.1%, and the mobile phase B is methanol; gradient elution procedure: 0 to 6.00min, the volume fraction of the mobile phase A is reduced from 65% to 5% at a constant speed; 6.00-7.00 min, and the volume fraction of the mobile phase A is kept 5%; 7.00-7.01 min, the volume fraction of the mobile phase A is increased from 5% to 65%; 7.01-10.00 min, the volume fraction of the mobile phase A is kept 65%.
In the present invention, the raw materials used are commercially available products well known to those skilled in the art unless specified otherwise.
The invention provides a moxifloxacin hydrochloride solution to be tested. In the invention, the solvent in the moxifloxacin hydrochloride solution to be detected is preferably methanol; the preparation method of the moxifloxacin hydrochloride solution to be detected is not particularly limited, and moxifloxacin hydrochloride to be detected is dissolved in methanol and diluted to the required concentration by the methanol. In the embodiment of the invention, the moxifloxacin hydrochloride solution to be detected is specifically a sample solution, the concentration of the sample solution is not particularly limited, and the concentration of the moxifloxacin hydrochloride solution to be detected in the sample solution is preferably 0.4mg/mL according to the principle that the concentration of the moxifloxacin hydrochloride solution to be detected can be a solution with a conventional concentration in the field so as to facilitate detection.
After the moxifloxacin hydrochloride solution to be detected is obtained, the high performance liquid chromatography-mass spectrometry analysis is carried out on the moxifloxacin hydrochloride solution to be detected, so that a chromatogram of the moxifloxacin hydrochloride to be detected is obtained. In the present invention, the hplc conditions for hplc-ms analysis include: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an ammonia water solution with the volume fraction of 0.1 percent (the preparation method comprises the steps of taking 1mL of ammonia water with the mass concentration of 25-28 percent, adding water to 1000 mL), and the mobile phase B is methanol; gradient elution procedure: 0 to 6.00min, the volume fraction of the mobile phase A is reduced from 65% to 5% at a constant speed; 6.00-7.00 min, and the volume fraction of the mobile phase A is kept 5%; 7.00-7.01 min, the volume fraction of the mobile phase A is increased from 5% to 65%; 7.01-10.00 min, the volume fraction of the mobile phase A is kept 65%.
In the present invention, the high performance liquid chromatography conditions preferably further include: the filler in the chromatographic column is preferably octadecylsilane chemically bonded silica, the particle size of the filler is preferably 2.7 μm, and the specification of the chromatographic column is preferably 3.0mm×100mm; in the present invention, the column is preferably an octadecylsilane chemically bonded silica reversed phase column; in the examples of the present invention, welchEX-C18 column (3.0 mm. Times.100 mm,2.7 μm) was used in particular.
In the present invention, the high performance liquid chromatography conditions preferably further include: the column temperature is preferably 30 ℃, and the flow rate of the mobile phase is preferably 0.5mL/min; the sample volume is preferably 5. Mu.L.
In the present invention, the mass spectrometry conditions of the high performance liquid chromatography-mass spectrometry preferably include: an electrospray ionization source; the detection mode is negative ion detection; the source temperature was 550 ℃; the ion spray voltage is-4500V; the flow rate of the atomizing gas 1 is 50psi, the flow rate of the atomizing gas 2 is 55psi, and the flow rate of the air curtain gas is 40psi; the scanning mode is a multi-reaction ion monitoring mode.
After obtaining a chromatogram of moxifloxacin hydrochloride to be detected, the content of the ethyl formyl acetate in the moxifloxacin hydrochloride to be detected is obtained according to a standard curve of the ethyl formyl acetate and the chromatogram of the moxifloxacin hydrochloride to be detected. In the present invention, the standard curve of the ethyl formylacetate is preferably a linear regression equation of the concentration of the standard solution and the chromatographic peak area of the standard solution, specifically, the standard curve of the ethyl formylacetate uses the chromatographic peak area corresponding to the ethyl formylacetate in the standard solution as the ordinate and uses the concentration of the ethyl formylacetate as the abscissa. In the present invention, the solvent of the standard solution is preferably methanol; in embodiments of the invention, the concentration of ethyl formylacetate in the standard solution is preferably 1ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL. The invention carries out high performance liquid chromatography-mass spectrometry analysis on the standard solution to obtain chromatographic peak areas corresponding to the ethyl formylacetate in the standard solution; the conditions for high performance liquid chromatography-mass spectrometry are identical to those for high performance liquid chromatography-mass spectrometry of the moxifloxacin hydrochloride solution to be tested, and are not described in detail herein. According to the invention, the content of the formylacetate in moxifloxacin hydrochloride can be obtained according to the standard curve of the formylacetate and the chromatogram of the moxifloxacin hydrochloride to be detected, so that the content of the formylacetate in the moxifloxacin hydrochloride solution to be detected can be obtained.
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. Instrument and HPLC-MS conditions
High performance liquid chromatography-mass spectrometry (LC-MC/MC): shimadzu, LCMS-8045; MSA6.6S-0CE parts per million on the day (Sartorius company, germany); model AB265-S analytical balance (METTER TOLEDO Co., USA).
High performance liquid chromatography conditions: the column was packed with octadecylsilane chemically bonded silica (Welch EXT-C18,3.0 mm. Times.100 mm,2.7 μm); the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an ammonia water solution with the volume fraction of 0.1 percent (the preparation method comprises the steps of taking 1mL of ammonia water with the mass concentration of 25-28 percent, adding water to 1000 mL), and performing gradient elution with the mobile phase B being methanol, wherein the specific gradient elution program is shown in the table 1, and the total operation is 10min; the flow rate is 0.5mL/min; the column temperature is 30 ℃; the sample volume was 5. Mu.L.
TABLE 1 gradient elution procedure
| Time (min) | Mobile phase a (volume fraction,%) | Mobile phase B (volume fraction,%) |
| 0.00 | 65 | 35 |
| 6.00 | 5 | 95 |
| 7.00 | 5 | 95 |
| 7.01 | 65 | 35 |
| 10.00 | 65 | 35 |
Mass spectrometry conditions: electrospray ionization source (ESI source); the detection mode is as follows: detecting negative ions; the source temperature was 550 ℃; the ion spray voltage is-4500V; the air flow rates of the atomizing air 1, the atomizing air 2 and the air curtain are respectively 50psi, 55psi and 40psi; scanning mode: multiple reactive ion monitoring mode (MRM); other parameters of the target substance and the ion channel of the object to be measured are shown in table 2.
TABLE 2 other parameters of target substance and ion channel of the object to be measured
2. Material
Formyl ethyl acetate control (source: sinco Pharmachem, lot number: 20-08-0905); moxifloxacin hydrochloride (source: jiangxi morning sun pharmaceutical Co., ltd., lot numbers 20190801, 20200405, 20210101); methanol (source: thermo Fisher, lot number: 218273); ammonia (source: national medicine, lot number: 20201020).
3. Solution preparation and detection of genotoxic impurity formylacetate in moxifloxacin hydrochloride
Preparing a reference substance solution: accurately weighing a proper amount of formylacetic acid ethyl ester reference substance, adding methanol to dissolve and dilute the reference substance into a solution containing about 0.1 mug of formylacetic acid ethyl ester in 1mL, and taking the solution as the reference substance solution.
Sample solution preparation: taking a proper amount of moxifloxacin hydrochloride, precisely weighing, adding methanol for dissolving and quantitatively diluting to prepare a solution containing about 0.4mg of moxifloxacin hydrochloride in each 1mL of the solution, and taking the solution as a test sample solution.
Precisely measuring 5 mu L of each solution, respectively injecting into a high performance liquid chromatograph-mass spectrometer, and recording a chromatogram. The ethyl formylacetate content of moxifloxacin hydrochloride was measured by an external standard method, and the results are shown in table 3.
TABLE 3 determination of ethyl formylacetate in moxifloxacin hydrochloride
| Sample of | Content of |
| 20190801 | Not detected |
| 20200405 | Not detected |
| 20210101 | Not detected |
4. Methodological verification
4.1 specificity (methanol as solvent)
Control stock solution (1): taking 10mg of formylacetic acid ethyl ester reference substance, precisely weighing, placing into a 10mL measuring flask, adding a solvent for dissolution, diluting to a scale, and shaking uniformly to obtain the final product.
Control stock solution (2): precisely measuring the reference stock solution (1 mL, placing in a 100mL measuring flask, diluting to scale with solvent, shaking, precisely measuring 1mL, placing in a 100mL measuring flask, diluting to scale with solvent, and shaking to obtain the final product.
Control solution: precisely measuring the reference stock solution (2:1 mL, placing into a 10mL measuring flask, diluting with solvent to scale, and shaking.
Test solution: taking about 40mg of moxifloxacin hydrochloride, precisely weighing, placing into a 10mL measuring flask, adding a solvent for dissolution and dilution to a scale, shaking uniformly, precisely weighing 1mL, placing into the 10mL measuring flask, diluting to the scale with the solvent, and shaking uniformly to obtain the moxifloxacin hydrochloride.
Adding a standard solution to a test sample: taking about 40mg of moxifloxacin hydrochloride, precisely weighing, placing into a 10mL measuring flask, adding a solvent for dissolution and dilution to a scale, shaking uniformly, precisely weighing 1mL, placing into the 10mL measuring flask, precisely adding a reference stock solution (2) 1mL, diluting with the solvent to the scale, and shaking uniformly to obtain the moxifloxacin hydrochloride.
Precisely measuring 5 mu L of each solution, respectively injecting into a high performance liquid chromatograph-mass spectrometer, and recording a chromatogram. Typical patterns are shown in FIGS. 1-4, and specific results are shown in Table 4. From this, it was found that the solvent and the sample solution did not interfere with the detection of ethyl formylacetate.
TABLE 4 detection methodology of formylacetate in moxifloxacin hydrochloride-specific results
4.2 quantitative limit and detection limit
The detection Limit (LOD) and the quantification Limit (LOQ) are determined according to the signal-to-noise ratio method. The stock solution of the formylacetate with known concentration is diluted, the concentration at S/N is about 10 is taken as a quantitative limit concentration, and the concentration at S/N is about 3 is taken as a detection limit concentration.
According to the chromatographic conditions, precisely measuring 5 mu L of each of the quantitative limit solution and the detection limit solution, respectively injecting into a high performance liquid chromatography-mass spectrometry combined instrument, and recording the chromatograms, wherein the results are shown in tables 5-6. As can be seen from tables 5 to 6, the minimum S/N of the three-needle detection limit is 3.0, the concentration is 0.20ng/mL, and the concentration corresponds to 3.0% of the limit concentration; the minimum S/N=10.1 in the six-needle quantitative limit is larger than 10, the concentration is 0.66ng/mL, which is equivalent to 10.0% of the limit concentration, and the RSD=2.9% of the peak area of the six continuous needles in the quantitative limit is smaller than 20.0%, which indicates that the sensitivity of the method is higher.
TABLE 5 detection methodology of formylacetate in moxifloxacin hydrochloride-quantitative limit test results
TABLE 6 detection methodology of formylacetate in moxifloxacin hydrochloride-detection limit test results
4.3 linearity and Range
The concentration of the test solution is 0.4mg/mL, the limit of the ethyl formylacetate is 25ppm, and the limit concentration is 10ng/mL; the linear investigation range was set to a quantitative limit of 500% of the limit concentration.
The solvent used to prepare the following solutions was methanol:
control stock solution (1): taking 10mg of formylacetic acid ethyl ester reference substance, precisely weighing, placing into a 10mL measuring flask, adding a solvent for dissolution, diluting to a scale, and shaking uniformly to obtain the final product.
Control stock solution (2): precisely measuring the reference stock solution (1 mL, placing in a 100mL measuring flask, diluting to scale with solvent, shaking, precisely measuring 1mL, placing in a 100mL measuring flask, diluting to scale with solvent, and shaking to obtain the final product.
Preparation of a linear solution: a suitable amount of the control stock solution (2) was precisely measured according to Table 7, placed in a suitable measuring flask, diluted to a scale with a solvent, and shaken to give a linear solution, as shown in Table 7.
TABLE 7 preparation of linear solutions of formylacetate
Taking 5 mu L of each linear solution, respectively injecting into a high performance liquid chromatography-mass spectrometer, and recording a chromatogram. The linear relationship is plotted as a function of measured peak area versus concentration, and linear regression is performed with the concentration being on the abscissa (x) and the peak area being on the ordinate (y). Specifically, table 8 and fig. 5 show the results. From this, it was found that ethyl formylacetate was linear in the range of 0.6604 to 33.02ng/mL, and the linear equation was y=1.234×10 4 x+3.493×10 3 Correlation coefficient r=0.9995, r 2 =0.9990, sum of squares of residuals is 99987891.
TABLE 8 results of the linear test of formylacetate
4.4 accuracy (methanol as solvent)
Control stock solution (1): taking 10mg of formylacetic acid ethyl ester reference substance, precisely weighing, placing into a 10mL measuring flask, adding a solvent for dissolution, diluting to a scale, and shaking uniformly to obtain the final product.
Control stock solution (2): precisely measuring the reference stock solution (1 mL, placing in a 100mL measuring flask, diluting to scale with solvent, shaking, precisely measuring 1mL, placing in a 100mL measuring flask, diluting to scale with solvent, and shaking to obtain the final product.
Control solution: precisely measuring the reference stock solution (2:1 mL, placing into a 10mL measuring flask, diluting with solvent to scale, and shaking.
Test solution: taking about 40mg of moxifloxacin hydrochloride, precisely weighing, placing into a 10mL measuring flask, adding a solvent for dissolution and dilution to a scale, shaking uniformly, precisely weighing 1mL, placing into the 10mL measuring flask, diluting to the scale with the solvent, and shaking uniformly to obtain the moxifloxacin hydrochloride.
50% of test sample adding standard solution: taking about 40mg of moxifloxacin hydrochloride, precisely weighing, placing into a 10mL measuring flask, adding a solvent for dissolution and dilution to a scale, shaking uniformly, precisely weighing 1mL, placing into the 10mL measuring flask, precisely adding a reference stock solution (2) (0.5 mL), diluting with the solvent to the scale, and shaking uniformly to obtain the moxifloxacin hydrochloride. 3 parts of solution was prepared in the same manner.
100% of test sample adding standard solution: taking about 40mg of moxifloxacin hydrochloride, precisely weighing, placing into a 10mL measuring flask, adding a solvent for dissolution and dilution to a scale, shaking uniformly, precisely weighing 1mL, placing into the 10mL measuring flask, precisely adding a reference stock solution (2) 1mL, diluting with the solvent to the scale, and shaking uniformly to obtain the moxifloxacin hydrochloride. 3 parts of solution was prepared in the same manner.
150% of test sample adding standard solution: taking about 40mg of moxifloxacin hydrochloride, precisely weighing, placing into a 10mL measuring flask, adding a solvent for dissolution and dilution to a scale, shaking uniformly, precisely weighing 1mL, placing into the 10mL measuring flask, precisely adding a reference stock solution (2) (1.5 mL), diluting with the solvent to the scale, and shaking uniformly to obtain the moxifloxacin hydrochloride. 3 parts of solution was prepared in the same manner.
Respectively precisely measuring 5 mu L of each of the reference substance solution and the sample adding standard solution, respectively injecting into a high performance liquid chromatograph-mass spectrometer, and recording the chromatograms, wherein the specific results are shown in tables 9-10. As shown in tables 9-10, the recovery rate of 9 parts of the solution with the standard sample is 98.3-104.3%, the recovery rate is in the range of 90-110%, the RSD value is less than 10%, and the verification requirement is met.
TABLE 9 detection methodology of formylacetate in moxifloxacin hydrochloride-accuracy test results
Table 10 results of formylacetate detection methodology-accuracy test in moxifloxacin hydrochloride
4.5 repeatability (methanol as solvent)
Control stock solution (1): taking 10mg of formylacetic acid ethyl ester reference substance, precisely weighing, placing into a 10mL measuring flask, adding a solvent for dissolution, diluting to a scale, and shaking uniformly to obtain the final product.
Control stock solution (2): precisely measuring the reference stock solution (1 mL, placing in a 100mL measuring flask, diluting to scale with solvent, shaking, precisely measuring 1mL, placing in a 100mL measuring flask, diluting to scale with solvent, and shaking to obtain the final product.
Control solution: precisely measuring the reference stock solution (2:1 mL, placing into a 10mL measuring flask, diluting with solvent to scale, and shaking.
Test solution: taking about 40mg of moxifloxacin hydrochloride, precisely weighing, placing into a 10mL measuring flask, adding a solvent for dissolution and dilution to a scale, shaking uniformly, precisely weighing 1mL, placing into the 10mL measuring flask, diluting to the scale with the solvent, and shaking uniformly to obtain the moxifloxacin hydrochloride.
Adding a standard solution to a test sample: taking about 40mg of moxifloxacin hydrochloride, precisely weighing, placing into a 10mL measuring flask, adding a solvent for dissolution and dilution to a scale, shaking uniformly, precisely weighing 1mL, placing into the 10mL measuring flask, precisely adding a reference stock solution (2) 1mL, diluting with the solvent to the scale, and shaking uniformly to obtain the moxifloxacin hydrochloride. 6 parts of solution was prepared in the same manner.
Precisely measuring 5 μl of each of the reference solution and the sample solution, respectively, and injecting into high performance liquid chromatography-mass spectrometry instrument to record the chromatograms. The specific results are shown in Table 11. As shown in Table 11, the RSD of the measurement result of the 6-part labeled sample solution is less than 10%, which meets the verification requirement.
TABLE 11 detection methodology of formylacetate in moxifloxacin hydrochloride-repeatability test results
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 (10)
1. A detection method of formylacetic acid ethyl ester in moxifloxacin hydrochloride comprises the following steps:
providing moxifloxacin hydrochloride solution to be tested;
performing high performance liquid chromatography-mass spectrometry on the moxifloxacin hydrochloride solution to be detected to obtain a chromatogram of moxifloxacin hydrochloride to be detected;
obtaining the content of the formylacetic acid ethyl ester in the moxifloxacin hydrochloride to be detected according to the standard curve of the formylacetic acid ethyl ester and the chromatogram of the moxifloxacin hydrochloride to be detected;
the high performance liquid chromatography-mass spectrometry conditions include:
the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an ammonia water solution with the volume fraction of 0.1%, and the mobile phase B is methanol; gradient elution procedure: 0 to 6.00min, the volume fraction of the mobile phase A is reduced from 65% to 5% at a constant speed; 6.00-7.00 min, and the volume fraction of the mobile phase A is kept 5%; 7.00-7.01 min, the volume fraction of the mobile phase A is increased from 5% to 65%; 7.01-10.00 min, the volume fraction of the mobile phase A is kept 65%.
2. The method according to claim 1, wherein the filler in the column for high performance liquid chromatography is octadecylsilane chemically bonded silica, and the particle size of the filler is 2.7 μm; the specification of the chromatographic column is 3.0mm×100mm.
3. The method according to claim 1, wherein the column temperature of the high performance liquid chromatography is 30 ℃.
4. The method according to claim 1, wherein the mobile phase flow rate of the high performance liquid chromatography is 0.5mL/min.
5. The method according to claim 1, wherein the sample volume of the high performance liquid chromatograph is 5 μl.
6. The method of claim 1, wherein the mass spectrometry conditions of the high performance liquid chromatography-mass spectrometry comprise: an electrospray ionization source; the detection mode is negative ion detection; the source temperature was 550 ℃.
7. The method of claim 6, wherein the ion spray voltage of the mass spectrum is-4500V.
8. The method of claim 6, wherein the mass spectrum has an atomizing gas 1 flow rate of 50psi, an atomizing gas 2 flow rate of 55psi, and a curtain gas flow rate of 40psi.
9. The method of claim 6, wherein the mass spectrum is scanned in a multi-reactive ion monitoring mode.
10. The detection method according to claim 1, wherein the solvent in the moxifloxacin hydrochloride solution to be detected is methanol.
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