CN113552231A - Method for detecting residual content of genotoxic impurity 4-dimethylaminopyridine in zopiclone - Google Patents
Method for detecting residual content of genotoxic impurity 4-dimethylaminopyridine in zopiclone Download PDFInfo
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- CN113552231A CN113552231A CN202010334868.0A CN202010334868A CN113552231A CN 113552231 A CN113552231 A CN 113552231A CN 202010334868 A CN202010334868 A CN 202010334868A CN 113552231 A CN113552231 A CN 113552231A
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- dimethylaminopyridine
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- zopiclone
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- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 title claims abstract description 120
- GBBSUAFBMRNDJC-MRXNPFEDSA-N (5R)-zopiclone Chemical compound C1CN(C)CCN1C(=O)O[C@@H]1C2=NC=CN=C2C(=O)N1C1=CC=C(Cl)C=N1 GBBSUAFBMRNDJC-MRXNPFEDSA-N 0.000 title claims abstract description 55
- 229960000820 zopiclone Drugs 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 35
- 239000012535 impurity Substances 0.000 title claims abstract description 25
- 231100000024 genotoxic Toxicity 0.000 title claims abstract description 17
- 230000001738 genotoxic effect Effects 0.000 title claims abstract description 17
- 239000013558 reference substance Substances 0.000 claims abstract description 55
- 239000003085 diluting agent Substances 0.000 claims abstract description 53
- 239000000243 solution Substances 0.000 claims abstract description 52
- 239000012085 test solution Substances 0.000 claims abstract description 24
- 238000005303 weighing Methods 0.000 claims abstract description 18
- 238000012360 testing method Methods 0.000 claims abstract description 13
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims abstract description 11
- 238000010812 external standard method Methods 0.000 claims abstract description 6
- 238000007865 diluting Methods 0.000 claims abstract description 5
- 239000012088 reference solution Substances 0.000 claims abstract description 5
- 239000011550 stock solution Substances 0.000 claims description 50
- 239000000523 sample Substances 0.000 claims description 26
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- GBBSUAFBMRNDJC-INIZCTEOSA-N eszopiclone Chemical compound C1CN(C)CCN1C(=O)O[C@H]1C2=NC=CN=C2C(=O)N1C1=CC=C(Cl)C=N1 GBBSUAFBMRNDJC-INIZCTEOSA-N 0.000 claims description 12
- 229960001578 eszopiclone Drugs 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 9
- 238000001819 mass spectrum Methods 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 239000012488 sample solution Substances 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 239000007921 spray Substances 0.000 claims description 3
- 238000004364 calculation method Methods 0.000 claims description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims 2
- 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 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims 1
- 235000019253 formic acid Nutrition 0.000 claims 1
- 150000002500 ions Chemical class 0.000 claims 1
- 239000003643 water by type Substances 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 12
- 238000001228 spectrum Methods 0.000 description 8
- 238000012546 transfer Methods 0.000 description 6
- 238000011084 recovery Methods 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- BJEPYKJPYRNKOW-UWTATZPHSA-N (R)-malic acid Chemical compound OC(=O)[C@H](O)CC(O)=O BJEPYKJPYRNKOW-UWTATZPHSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 102100024239 Sphingosine-1-phosphate lyase 1 Human genes 0.000 description 3
- 101710122496 Sphingosine-1-phosphate lyase 1 Proteins 0.000 description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 3
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 208000019116 sleep disease Diseases 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- KAESVJOAVNADME-UHFFFAOYSA-N 1H-pyrrole Natural products C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 1
- SVUOLADPCWQTTE-UHFFFAOYSA-N 1h-1,2-benzodiazepine Chemical compound N1N=CC=CC2=CC=CC=C12 SVUOLADPCWQTTE-UHFFFAOYSA-N 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 1
- BPIHPZPPEKKBPL-UHFFFAOYSA-N [6-(5-chloropyridin-2-yl)-5-oxo-7H-pyrrolo[3,4-b]pyrazin-7-yl] (4-methylpiperazin-1-yl) carbonate Chemical compound CN1CCN(CC1)OC(=O)OC2C3=NC=CN=C3C(=O)N2C4=NC=C(C=C4)Cl BPIHPZPPEKKBPL-UHFFFAOYSA-N 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940049706 benzodiazepine Drugs 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003326 hypnotic agent Substances 0.000 description 1
- 230000000147 hypnotic effect Effects 0.000 description 1
- 206010022437 insomnia Diseases 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- PSHKMPUSSFXUIA-UHFFFAOYSA-N n,n-dimethylpyridin-2-amine Chemical compound CN(C)C1=CC=CC=N1 PSHKMPUSSFXUIA-UHFFFAOYSA-N 0.000 description 1
- 238000009522 phase III clinical trial Methods 0.000 description 1
- -1 pyrrole cyclic ketone compounds Chemical class 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000007430 reference method Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004799 sedative–hypnotic effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- GBBSUAFBMRNDJC-UHFFFAOYSA-N zopiclone Chemical compound C1CN(C)CCN1C(=O)OC1C2=NC=CN=C2C(=O)N1C1=CC=C(Cl)C=N1 GBBSUAFBMRNDJC-UHFFFAOYSA-N 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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
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Abstract
The invention discloses a method for detecting the residual content of genotoxic impurity 4-dimethylaminopyridine in zopiclone, which comprises the following steps: control solution: taking a proper amount of 4-dimethylaminopyridine as a reference substance, precisely weighing, placing into a brown volumetric flask, dissolving with a diluent, and sequentially diluting to prepare a reference substance solution; test solution: taking a proper amount of zopiclone to be tested, precisely weighing, placing in a volumetric flask, and dissolving with a diluent to a constant volume to prepare a test solution; the determination method comprises the following steps: respectively taking a test solution and a reference solution, precisely measuring and injecting the sample, recording a chromatogram by adopting a liquid chromatography-mass spectrometry method, and calculating the peak area according to an external standard method to obtain the residual content of the genotoxic impurity 4-dimethylaminopyridine in the zopiclone. The method adopts LC-MS detection, uses an external standard method to measure the sample and analyzes the residual content of the 4-dimethylaminopyridine, and can ensure the accuracy and reliability of the test result.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to the field of medicine impurity detection, and particularly relates to a method for detecting the residual content of a genotoxic impurity, namely 4-dimethylaminopyridine in zopiclone.
Background
Zopiclone (ZOP), which is chemically named 6- (5-chloro-2-pyridyl) -7- [ (4-methylpiperazin-1-yl) carboxyloxy ] -5, 6-dihydropyrrolo [3, 4-b ] pyrazin-5-one, belongs to pyrrole cyclic ketone compounds, is used as a novel clinically used non-benzodiazepine sedative hypnotic drug, and can be effectively used for short-term treatment of insomnia. Racemic zopiclone was marketed by Rhone-Poulenc Rorer, Inc. under the trade names IMOVANE and AMOBAN in over 80 countries, Europe and the like, from the mid-80 s for the treatment of sleep disorders. Reminiscent of the Return to dream (IMOVANE) in 1999 is sold in the order of $ 1 million and $ 6 million, and is a leading drug for treating sleep disorders outside the U.S. market and has not been filed for FDA. The company Sepracor believes that the racemic short-acting hypnotic effect may be due to its (+) isomer, so it was possible to develop Eszopiclone from 1998, to date successful in completing phase III clinical trials, and the US FDA approved the drug for marketing in the US 12 months 2004.
The eszopiclone is an (S) -configuration enantiomer of zopiclone and is prepared by the following steps: the zopiclone is split by D- (+) -malic acid to obtain an intermediate, namely, eszopiclone D- (+) -malate, then the D- (+) -malic acid is removed by alkalization to obtain a crude eszopiclone product, and finally the finished eszopiclone product is prepared by refining. The preparation process of the eszopiclone only splits the zopiclone, does not involve the change of a molecular structure, and does not introduce special organic impurities except chiral isomers (R-zopiclone), D- (+) -malic acid and ethyl acetate. The inorganic reagent used in the process can be effectively removed.
Besides different stereo configurations, the chemical structures of the zopiclone and the eszopiclone are consistent, impurities contained in the zopiclone can be introduced into the eszopiclone, and the synthetic process route of the zopiclone is as follows:
according to the production process of the zopiclone, the possibility that genotoxic impurities possibly contained in the zopiclone, namely 4-dimethylamino pyridine, are introduced into the eszopiclone exists, so that the research on the residual situation of the zopiclone has important significance for the quality control of the zopiclone and the eszopiclone.
However, in the existing legal standards of zopiclone, a method for checking the residual content of 4-dimethylaminopyridine is not carried, the standard is lacked, no relevant literature reports exist at home and abroad, the medicine quality cannot be evaluated, and potential safety hazards exist.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for detecting the residual content of the genotoxic impurity 4-dimethylaminopyridine in zopiclone, the method adopts LC-MS (liquid chromatography-mass spectrometry) detection, an external standard method is used for determining a sample, the residual content of the 4-dimethylaminopyridine in the sample is analyzed, and the method can ensure the accuracy and the reliability of a test result.
The invention is realized by the following technical scheme:
the method for detecting the residual content of the genotoxic impurity 4-dimethylaminopyridine in zopiclone comprises the following steps: control solution: taking a proper amount of 4-dimethylaminopyridine as a reference substance, precisely weighing, placing into a brown volumetric flask, dissolving with a diluent, and sequentially diluting to prepare a reference substance solution;
test solution: taking a proper amount of zopiclone to be tested, precisely weighing, placing in a volumetric flask, and dissolving with a diluent to a constant volume to prepare a test solution;
the determination method comprises the following steps: respectively taking a test solution and a reference solution, precisely measuring and injecting samples, recording a chromatogram by adopting a liquid chromatography-mass spectrometry method, and calculating the peak area according to an external standard method to obtain the residual content of the genotoxic impurity 4-dimethylaminopyridine in the zopiclone;
wherein, the diluent is acetonitrile and water is 1:9(V: V).
The invention further improves the scheme as follows:
the preparation process of the reference substance solution comprises the following steps:
precisely weighing 0.01005g of 4-dimethylaminopyridine in a 10mL volumetric flask, adding a diluent to dissolve and dilute the mixture to a scale mark, and obtaining a reference substance stock solution I.
And (3) transferring 0.5mL of the reference substance stock solution I into a 10mL volumetric flask by using a 0.5mL pipette, adding a diluent to a constant volume, shaking up, and marking as a reference substance stock solution II.
And (3) transferring 1.0mL of the reference substance stock solution II into a 10mL volumetric flask by using a 1mL pipette, adding a diluent, fixing the volume and shaking up to obtain a reference substance stock solution III.
And (3) transferring 1.0mL of the reference substance stock solution III into a 100mL volumetric flask by using a 1mL pipette, adding a diluent, fixing the volume and shaking up to obtain a reference substance solution with the concentration of 50 ng/mL.
Further, the preparation process of the test solution is as follows: weighing 10mg of the sample of the eszopiclone precisely, placing the sample in a 100mL volumetric flask, and dissolving the sample with a diluent to a constant volume to prepare a sample solution with the concentration of 0.1 mg/mL.
Further, the mass spectrum condition of the liquid chromatography-mass spectrometry is
| Spray type | ESI source |
| Scanning type | SIM |
| Fragmentor | 70V |
| Pressure of atomized gas | 35psi |
| Temperature of |
350℃ |
| Drying gas flowSpeed measuring device | 12L/min |
| Polarity | Positive |
| Capillary voltage | 3000V |
| SIMIon | 123.00 |
。
Further, the mass spectrum condition of the liquid chromatography-mass spectrometry is
Furthermore, according to the requirements of ICH three-party coordination guidance principle on impurities in new raw material medicines, the content of 4-dimethylaminopyridine in a test sample is not more than 500 ppm.
The calculation formula of the content of the 4-dimethylaminopyridine in the test solution is as follows:
wherein:
ASPLmeans the peak area of 4-dimethylaminopyridine in the sample solution
WSPLThe sample weighing (mg) of the sample
VSPLVolume of test solution (mL)
ASTDRefers to the peak area of 4-dimethylaminopyridine in a control solution
WSTDThe weight of 4-dimethylaminopyridine (mg) in the control solution is referred to
VSTDRefers to the volume (mL) of the control solution.
The invention has the beneficial effects that:
the method provided by the invention adopts a liquid chromatography-mass spectrometry method for separation and detection, has high equipment popularization rate and low use cost, and thus, the application occasion and the use range of the method can be effectively enlarged.
In the detection method provided by the invention, all the used reagents are pollution-free, the treatment processes of the reference substance and the test sample are simple, the use amount of the reference substance and the test sample is small, and the reference substance solution, the test sample solution and the self reference solution can be conveniently and quickly prepared.
Under the chromatographic condition provided by the invention, the separation degree between impurities and main components is good, the system stability and repeatability are good, the accuracy is high, the peak-off time is short, the analysis time is short, and the experiment cost is saved.
The method provided by the invention can quickly, effectively and simply measure the amount of genotoxic impurities, namely 4-dimethylaminopyridine impurities, which may be contained in zopiclone, can be used for evaluating or evaluating the components of the medicine, avoids potential safety hazards, and can provide reliable data monitoring for the quality safety of the raw material medicine.
Tests are used for verifying the detection method provided by the invention, and the results show that the method has strong specificity, good precision and high accuracy, and can quickly determine the amount of 4-dimethylaminopyridine in zopiclone.
Drawings
FIG. 1 spectrum of blank solvent;
FIG. 2 is a spectrum of the control of example 1;
FIG. 3 is a spectrum of test article 851411009 of example 1;
FIG. 4 is a spectrum of the control of example 2;
FIG. 5 is a spectrum of test article 18000491 of example 2;
FIG. 6 is a spectrum of the control of example 3;
FIG. 7 is a spectrum of test article 851409007 of example 3;
FIG. 8 is a standard graph of 4-dimethylaminopyridine.
Due to the limited space, the spectrum of the validation part of the method is not described in the specification.
Detailed Description
Instrument, chromatographic column, reagent and reference substance instrument
Chromatographic column
Reagent and reference substance
Examples 1 to 3
Diluent
Acetonitrile water 1:9(V: V)
Blank solvent
Acetonitrile water 1:9(V: V)
Control solution (50ng/mL 4-dimethylaminopyridine solution)
Precisely weighing 0.01005g of 4-dimethylaminopyridine in a 10mL volumetric flask, adding a diluent to dissolve and dilute the mixture to a scale mark, and obtaining a reference substance stock solution I.
And (3) transferring 0.5mL of the reference substance stock solution I into a 10mL volumetric flask by using a 0.5mL pipette, adding a diluent to a constant volume, and shaking up to obtain a reference substance stock solution II.
And (3) transferring 1.0mL of the reference substance stock solution II into a 10mL volumetric flask by using a 1mL pipette, adding a diluent, fixing the volume and shaking up to obtain a reference substance stock solution III.
Using a 1mL pipette to transfer 1.0mL of the reference substance stock solution III into a 100mL volumetric flask, adding a diluent to a constant volume and shaking up to obtain a reference substance solution.
Test solution (0.1mg/mL zopiclone)
Precisely weighing about 10mg of samples of different batches respectively, transferring the samples into a 100mL volumetric flask, diluting the samples to a scale with a diluent, shaking up the samples to be used as a test solution of zopiclone.
Respectively taking the test solution and the reference solution, precisely measuring 2 mu L of sample injection, recording the chromatogram, and calculating according to the peak area by an external standard method to obtain the content of the 4-dimethylaminopyridine in the test solution.
The mass spectrum conditions are
| Spray type | ESI source |
| Scanning type | SIM |
| Fragmentor | 70V |
| Pressure of atomized gas | 35psi |
| Temperature of drying |
350℃ |
| Dry air flow rate | 12L/min |
| Polarity | Positive |
| Capillary voltage | 3000V |
| SIMIon | 123.00 |
The mass spectrum conditions are
Calculating the formula:
wherein:
ASPLmeans the peak area of 4-dimethylaminopyridine in the sample solution
WSPLThe sample weighing (mg) of the sample
VSPLVolume of test solution (mL)
ASTDRefers to the peak area of 4-dimethylaminopyridine in a control solution
WSTDThe weight of 4-dimethylaminopyridine (mg) in the control solution is referred to
VSTDRefers to the volume (mL) of the control solution.
The limit of quantitation (LOQ) of the method is 50pmm, the limit of detection (LOD) is 15 ppm; the sample should not contain more than 500ppm of 4-dimethylaminopyridine.
The chromatograms are shown in fig. 1 to 7, and the detection results are shown in table 1.
TABLE 1 test results
The experimental results show that 4-dimethylaminopyridine is not detected in three batches of samples.
Analytical method validation
1. System adaptability
Diluent
Acetonitrile water 1:9(V: V)
Positioning solution (50ng/mL 4-dimethylaminopyridine solution)
Precisely weighing 0.01011g of 4-dimethylaminopyridine in a 10mL volumetric flask, adding a diluent to dissolve and dilute the mixture to a scale mark, and obtaining a reference substance stock solution I.
And (3) transferring 0.5mL of the reference substance stock solution I into a 10mL volumetric flask by using a 0.5mL pipette, adding a diluent to a constant volume, and shaking up to obtain a reference substance stock solution II.
And (3) transferring 1.0mL of the reference substance stock solution II into a 10mL volumetric flask by using a 1mL pipette, adding a diluent, fixing the volume and shaking up to obtain a reference substance stock solution III.
1.0mL of the control stock solution III was pipetted into a 100mL volumetric flask with a 1mL pipette, and the volume was adjusted by adding diluent and shaking up, and the volume was labeled STD-1.
Another control solution, designated STD-2 (0.01000 g of 4-dimethylaminopyridine) was prepared following the same formulation procedure described above.
Test solution (0.1mg/mL zopiclone)
0.01003g of zopiclone was precisely weighed, transferred to a 100mL volumetric flask, made to volume with diluent, shaken well, designated SPL, as a test solution of zopiclone.
Sample adding solution
0.01001g of zopiclone was precisely weighed, transferred to a 100mL volumetric flask, added with 1mL of control stock solution III, made to volume with diluent, shaken well, designated SPL-STD, as a test sample labeling solution for zopiclone.
According to the chromatographic and mass spectrum conditions of the invention, STD-1 was measured 6 times, and the system adaptability results are shown in Table 2
TABLE 2 System Adaptation results
2. Specificity
According to the chromatographic and mass spectrometric conditions of the present invention, the STD, SPL and SPL-STD samples were measured, respectively, and the results of specificity are shown in Table 3.
TABLE 3 specificity results
The blank has no interference, the separation degree between the 4-dimethylamino pyridine and the adjacent chromatographic peak is more than 1.5, and the specificity meets the requirement.
3. Limit of quantification
Quantitative limiting solution
0.01011g, 0.01000g, 0.01007g, 0.01008g, 0.01008g and 0.01004g of 4-dimethylaminopyridine are precisely weighed and respectively placed in 6 10mL volumetric flasks, and a diluent is added to dissolve and dilute the 4-dimethylaminopyridine to a scale, so that reference substance stock solutions I-1-I-6 are obtained.
Precisely transferring 0.5mL of the reference substance stock solution I into 6 10mL volumetric flasks, adding a diluent to a constant volume, shaking up, and marking as reference substance stock solutions II-1-II-6.
Precisely transferring 1.0mL of the reference substance stock solution II into 6 10mL volumetric flasks respectively, adding a diluent, fixing the volume and shaking up to obtain reference substance stock solutions III-1 to III-6.
Precisely transferring 1.0mL of the reference substance stock solution III into 6 10mL volumetric flasks respectively, adding a diluent to a constant volume, and shaking up to obtain reference substance stock solutions IV-1-IV-6.
Precisely transferring 1.0mL of reference substance stock solution into 6 volumetric flasks of 100mL, adding diluent to constant volume, shaking up, and respectively marking as LOQ-1-LOQ-6.
Test solution
0.00999g and 0.01003g of zopiclone are precisely weighed, respectively transferred into 2 100mL volumetric flasks, subjected to constant volume by using a diluent, and uniformly shaken, and respectively marked as SPL-1 and SPL-2.
Quantitative limit recovery rate solution
0.01000g, 0.01002g, 0.00999g, 0.01003g, 0.00995g and 0.00995g of zopiclone are precisely weighed, respectively transferred into 6 volumetric flasks of 100mL, respectively added with IV-11.0 mL of reference stock solution, and subjected to constant volume with a diluent and shaking up, wherein the volumes are respectively marked as SPL-LOQ-1-SPL-LOQ-6.
According to the chromatographic and mass spectrum conditions of the invention, the solutions were subjected to sample injection detection, and the quantitative limit recovery rate solutions were analyzed, respectively, with the results shown in tables 4 and 5.
TABLE 4 quantitative limit results
TABLE 5 recovery in quantitative limits
The experimental results show that: the signal-to-noise ratio of each solution response under 6 parts of quantitative concentration is more than 10, the RSD of the peak area response factor is less than 8%, and the quantitative limit repeatability meets the requirement. The recovery rate under the quantitative limit concentration is between 80% and 115%, the accuracy of the quantitative limit is good, and the quantitative limit is 50 ppm.
4. Detection limit
A3 mL pipette is used to remove 3mL of LOQ-2 solution under the LOQ repeatability term into a 10m volumetric flask, the volume is determined by using a diluent, the solution is marked as LOD, and 2 parts are prepared in parallel and marked as LOD-1 and LOD-2.
The solutions were analyzed by sample injection, and the S/N in the LOD solution was calculated, the results are shown in Table 6.
TABLE 6 detection limit results
The experimental results show that: the signal-to-noise ratio of 4-dimethylaminopyridine in the LOD solution is not less than 3, and the detection limit is 15 ppm.
5. Linearity and range
Solution preparation
L-200. transfer the control stock solution III-3 to 100mL volumetric flasks under the quantitation limit of 2.0mL with a 2mL pipette, dilute to the mark with diluent, shake up, and mark as L-200.
L-150. transfer the control stock solution III-3 to 100mL volumetric flasks under the quantitation limit of 1.5mL with a 2mL pipette, dilute to the mark with diluent, shake up, and mark as L-150.
L-100. transfer the control stock solution III-3 to 100mL volumetric flasks under the quantitative limit of 1.0mL with a 1mL pipette, dilute to the mark with diluent, shake up, and mark as L-100.
L-80. transfer the control stock solution III-3 to 100mL volumetric flasks under the quantitative limit of 0.8mL with a 1mL pipette, dilute to the mark with diluent, shake up, and mark as L-80.
L-50. transfer the control stock solution III-3 to 100mL volumetric flasks under the quantitative limit of 0.5mL with a 0.5mL pipette, dilute to the mark with diluent, shake up, and mark as L-50.
LOQ: formulation of LOQ in reference method.
The solutions were analyzed by injection, and the results are shown in Table 7 and FIG. 8.
TABLE 7 Linear and Range results
The experimental result shows that the linear relation between the 4-dimethylamino pyridine and the peak area is good in the concentration range of 5.035 ng/mL-100.700 ng/mL, the correlation coefficient is 0.9997, the Y-axis intercept is within 25% of the 100% response value, and the linear range meets the determination requirement.
6. Accuracy of
Solution preparation
Test solution (0.1mg/mL zopiclone)
0.01001g and 0.01007g of zopiclone are precisely weighed, respectively transferred into 2 100mL volumetric flasks, subjected to constant volume by using a diluent, shaken up and respectively marked as SPL-3-SPL-4.
Zopiclone sample accuracy
Solutions of 80%, 100%, 120% limiting concentration were prepared in 3 parts, 6 parts, and 3 parts, respectively, and the accuracy was determined.
SPL-80% STD solution: 0.01008g, 0.01007g and 0.01006g of zopiclone are precisely weighed, respectively transferred into 3 100mL volumetric flasks, added with 0.8mL of stock solution III-1 of a reference substance under the special item, diluted to the scale with a diluent and shaken uniformly. Respectively denoted as SPL-80% STD-1 to SPL-80% STD-3.
SPL-100% STD solution: precisely weighing 0.01001g, 0.01001g, 0.01004g, 0.00996g, 0.00999g and 0.01004g of zopiclone, respectively transferring the zopiclone to 6 100mL volumetric flasks, adding 1.0mL of stock solution III-1 of a reference substance under the special item, diluting the stock solution to the scale with a diluent, and shaking up. Respectively denoted as SPL-100% STD-1 to SPL-100% STD-6.
SPL-120% STD solution: 0.01002g, 0.01000g and 0.01007g of zopiclone are precisely weighed, respectively transferred into 3 100mL volumetric flasks, added with 1.2mL of stock solution III-1 of a reference substance under the special item, diluted to the scale with a diluent and shaken uniformly. 3 parts of the mixture are prepared in parallel and are respectively marked as SPL-120 percent STD-1 to SPL-120 percent STD-3.
The results are shown in Table 8.
TABLE 84 dimethylaminopyridine accuracy
The experimental result shows that the recovery rate of the 4-dimethylaminopyridine is between 85 and 110 percent under the limit concentration of 80 percent, the limit concentration of 100 percent and the limit concentration of 120 percent, and the requirement is met. The accuracy of the method is good.
7. Repeatability of
Preparation of the solution
Results for 6 100% limiting concentrations of spiked samples in zopiclone accuracy were taken as reproducibility results.
The 4-dimethylaminopyridine content of 6 parts of each 100% STD-SPL solution was calculated and the results are shown in Table 10.
The results of the verification are shown in tables 9 and 10.
8. Intermediate precision
Solution preparation
Precisely weighing 0.01002g of 4-dimethylaminopyridine in a 10mL volumetric flask, adding a diluent to dissolve and dilute to a scale mark to obtain a reference substance stock solution I.
And (3) transferring 0.5mL of the reference substance stock solution I into a 10mL volumetric flask by using a 0.5mL pipette, adding a diluent to a constant volume, shaking up, and marking as a reference substance stock solution II.
And (3) transferring 1.0mL of the reference substance stock solution II into a 10mL volumetric flask by using a 1mL pipette, adding a diluent, fixing the volume and shaking up to obtain a reference substance stock solution III.
1.0mL of the control stock solution III was pipetted into a 100mL volumetric flask with a 1mL pipette, and the volume was adjusted by adding diluent and shaking up, and the volume was labeled STD-1.
Preparation of intermediate precision solution
SPL-100% STD-1-B to SPL-100% STD-6-B solutions: 0.00992g, 0.01003g, 0.01001g, 0.00997g, 0.01008g and 0.01005g of zopiclone are precisely weighed, respectively transferred into 6 100mL volumetric flasks, added with 1.0mL of reference stock solution III-1, diluted to the scale with diluent and shaken uniformly. Respectively denoted as SPL-100% STD-1-B to SPL-100% STD-6-B.
Analyzing the above solutions by injecting sample respectively
The 4-dimethylaminopyridine content of 6 parts of the STD-SPL solution was calculated and the results are entered in tables 9, 10.
RSD% of 4-dimethylaminopyridine content in 12 parts of STD-SPL was calculated and the results are shown in tables 9 and 10.
TABLE 9 intermediate precision System suitability
TABLE 10 precision
The experimental result shows that the RSD of the 4-dimethylamino pyridine is less than or equal to 6 percent when the RSD is continuously measured for 6 times under the limit concentration of 100 percent, and the RSD of the 4-dimethylamino pyridine is less than or equal to 11 percent when the RSD is continuously measured for 12 times under the limit concentration of 100 percent, so the repeatability and the intermediate precision of the method meet the requirements.
Claims (6)
1. The method for detecting the residual content of the genotoxic impurity 4-dimethylaminopyridine in zopiclone is characterized by comprising the following steps of:
control solution: taking a proper amount of 4-dimethylaminopyridine as a reference substance, precisely weighing, placing into a brown volumetric flask, dissolving with a diluent, and sequentially diluting to prepare a reference substance solution;
test solution: taking a proper amount of zopiclone to be tested, precisely weighing, placing in a volumetric flask, and dissolving with a diluent to a constant volume to prepare a test solution;
the determination method comprises the following steps: respectively taking a test solution and a reference solution, precisely measuring and injecting samples, recording a chromatogram by adopting a liquid chromatography-mass spectrometry method, and calculating the peak area according to an external standard method to obtain the residual content of the genotoxic impurity 4-dimethylaminopyridine in the zopiclone;
wherein, the diluent is acetonitrile and water is 1:9(V: V).
2. The method of claim 1 for detecting the residual content of the genotoxic impurity, 4-dimethylaminopyridine, in zopiclone, wherein: the preparation process of the reference substance solution comprises the following steps:
precisely weighing 0.01005g of 4-dimethylaminopyridine in a 10mL volumetric flask, adding a diluent to dissolve and dilute the mixture to a scale mark, and obtaining a reference substance stock solution I.
And (3) transferring 0.5mL of the reference substance stock solution I into a 10mL volumetric flask by using a 0.5mL pipette, adding a diluent to a constant volume, shaking up, and marking as a reference substance stock solution II.
And (3) transferring 1.0mL of the reference substance stock solution II into a 10mL volumetric flask by using a 1mL pipette, adding a diluent, fixing the volume and shaking up to obtain a reference substance stock solution III.
And (3) transferring 1.0mL of the reference substance stock solution III into a 100mL volumetric flask by using a 1mL pipette, adding a diluent, fixing the volume and shaking up to obtain a reference substance solution with the concentration of 50 ng/mL.
3. The method of claim 1 for detecting the residual content of the genotoxic impurity, 4-dimethylaminopyridine, in zopiclone, wherein: the preparation process of the test solution comprises the following steps: weighing 10mg of the sample of the eszopiclone precisely, placing the sample in a 100mL volumetric flask, and dissolving the sample with a diluent to a constant volume to prepare a sample solution with the concentration of 0.1 mg/mL.
4. The method of claim 1 for detecting the residual content of the genotoxic impurity, 4-dimethylaminopyridine, in zopiclone, wherein: the mass spectrum conditions of the liquid chromatography-mass spectrometry are as follows:
spray type ESI sources;
scanning the type SIM;
Fragmentor 70V；
atomizing gas pressure 35 psi;
the temperature of the drying gas is 350 ℃;
the flow rate of the drying gas is 12L/min;
Polarity Positive；
capillary voltage 3000V;
SIM Ion 123.00。
5. the method of claim 1 for detecting the residual content of the genotoxic impurity, 4-dimethylaminopyridine, in zopiclone, wherein: the chromatographic conditions of the LC-MS are as follows:
mobile phase a 10 mmol/ammonium formate-0.05% formic acid (vol/vol) in water;
mobile phase B acetonitrile;
column Waters XSelect HST 3(150 mm. times.4.6 mm, 3.5 μm);
the column flow rate is 1 mL/min;
the column temperature is 35 ℃;
the sample injection volume is 2 mu L;
blank solvent acetonitrile: 1:9 of water;
PostRun Time 5min。
6. the method of claim 1 for detecting the residual content of the genotoxic impurity, 4-dimethylaminopyridine, in zopiclone, wherein: according to the requirements of an ICH three-party coordination guiding principle on impurities in a new raw material medicament, if a test product contains 4-dimethylaminopyridine, the content of the impurities does not exceed 500 ppm;
the calculation formula of the content of the 4-dimethylaminopyridine in the test solution is as follows:
wherein:
ASPLmeans the peak area of 4-dimethylaminopyridine in the sample solution
WSPLThe sample weighing (mg) of the sample
VSPLVolume of test solution (mL)
ASTDRefers to the peak area of 4-dimethylaminopyridine in a control solution
WSTDThe weight of 4-dimethylaminopyridine (mg) in the control solution is referred to
VSTDRefers to the volume (mL) of the control solution.
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