CN117030867A - Method for detecting various genotoxic impurities in flunarizine hydrochloride capsules - Google Patents
Method for detecting various genotoxic impurities in flunarizine hydrochloride capsules Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 36
- 231100000024 genotoxic Toxicity 0.000 title claims abstract description 29
- 230000001738 genotoxic effect Effects 0.000 title claims abstract description 29
- RXKMOPXNWTYEHI-RDRKJGRWSA-N Flunarizine hydrochloride Chemical compound Cl.Cl.C1=CC(F)=CC=C1C(C=1C=CC(F)=CC=1)N1CCN(C\C=C\C=2C=CC=CC=2)CC1 RXKMOPXNWTYEHI-RDRKJGRWSA-N 0.000 title claims abstract description 20
- 229960002807 flunarizine hydrochloride Drugs 0.000 title claims abstract description 20
- 239000002775 capsule Substances 0.000 title claims abstract description 16
- 238000001514 detection method Methods 0.000 claims abstract description 24
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- 239000000243 solution Substances 0.000 claims description 31
- 239000000843 powder Substances 0.000 claims description 17
- 239000013558 reference substance Substances 0.000 claims description 17
- 238000005303 weighing Methods 0.000 claims description 17
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 claims description 15
- 239000003085 diluting agent Substances 0.000 claims description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- 239000012086 standard solution Substances 0.000 claims description 12
- 238000007865 diluting Methods 0.000 claims description 11
- 239000000706 filtrate Substances 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 9
- 238000000227 grinding Methods 0.000 claims description 9
- 150000002500 ions Chemical class 0.000 claims description 8
- 238000004090 dissolution Methods 0.000 claims description 7
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 5
- 239000005695 Ammonium acetate Substances 0.000 claims description 5
- 235000019257 ammonium acetate Nutrition 0.000 claims description 5
- 229940043376 ammonium acetate Drugs 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 claims description 5
- 238000004949 mass spectrometry Methods 0.000 claims description 4
- 238000001819 mass spectrum Methods 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 239000011550 stock solution Substances 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 239000007921 spray Substances 0.000 claims description 3
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- 230000005526 G1 to G0 transition Effects 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims description 2
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 2
- 238000005173 quadrupole mass spectroscopy Methods 0.000 claims description 2
- 238000009210 therapy by ultrasound Methods 0.000 claims description 2
- 229960000326 flunarizine Drugs 0.000 claims 5
- SMANXXCATUTDDT-QPJJXVBHSA-N flunarizine Chemical compound C1=CC(F)=CC=C1C(C=1C=CC(F)=CC=1)N1CCN(C\C=C\C=2C=CC=CC=2)CC1 SMANXXCATUTDDT-QPJJXVBHSA-N 0.000 claims 5
- 239000003814 drug Substances 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
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- 238000012795 verification Methods 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 2
- 238000003908 quality control method Methods 0.000 abstract description 2
- 239000012488 sample solution Substances 0.000 description 13
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- 239000007789 gas Substances 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 238000002156 mixing Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
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- MWUXSHHQAYIFBG-UHFFFAOYSA-N nitrogen oxide Inorganic materials O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- UTOPLTQLBVDCNX-REZTVBANSA-N 1-[2-(4-fluorophenyl)-2-phenylethyl]-4-[(E)-3-phenylprop-1-enyl]piperazine Chemical compound C1CN(CCN1CC(C2=CC=CC=C2)C3=CC=C(C=C3)F)/C=C/CC4=CC=CC=C4 UTOPLTQLBVDCNX-REZTVBANSA-N 0.000 description 2
- YBPMDGWKXCGXEY-XRVIQIRUSA-N 1-[bis(4-fluorophenyl)methyl]-4-[(Z)-3-phenylprop-1-enyl]piperazine Chemical compound C1CN(CCN1/C=C\CC2=CC=CC=C2)C(C3=CC=C(C=C3)F)C4=CC=C(C=C4)F YBPMDGWKXCGXEY-XRVIQIRUSA-N 0.000 description 2
- TTXIFFYPVGWLSE-UHFFFAOYSA-N 1-[bis(4-fluorophenyl)methyl]piperazine Chemical compound C1=CC(F)=CC=C1C(C=1C=CC(F)=CC=1)N1CCNCC1 TTXIFFYPVGWLSE-UHFFFAOYSA-N 0.000 description 2
- 206010008111 Cerebral haemorrhage Diseases 0.000 description 2
- 230000002490 cerebral effect Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000012088 reference solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- WGEIOMTZIIOUMA-QPJJXVBHSA-N 1-[(e)-3-phenylprop-2-enyl]piperazine Chemical compound C1CNCCN1C\C=C\C1=CC=CC=C1 WGEIOMTZIIOUMA-QPJJXVBHSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 229940127291 Calcium channel antagonist Drugs 0.000 description 1
- 206010065559 Cerebral arteriosclerosis Diseases 0.000 description 1
- 206010008088 Cerebral artery embolism Diseases 0.000 description 1
- 206010008132 Cerebral thrombosis Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 201000001429 Intracranial Thrombosis Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000000576 arachnoid Anatomy 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 239000000480 calcium channel blocker Substances 0.000 description 1
- 230000001964 calcium overload Effects 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000007787 electrohydrodynamic spraying Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 201000005851 intracranial arteriosclerosis Diseases 0.000 description 1
- 201000010849 intracranial embolism Diseases 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-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
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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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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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- 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
- G01N2030/062—Preparation extracting sample from raw material
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8872—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample impurities
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Abstract
The invention relates to a method for detecting various genotoxic impurities in flunarizine hydrochloride capsules, which is characterized in that the genotoxic impurities A, B and C are quantitatively detected simultaneously by a liquid chromatography-mass spectrometry technical means, and the detection method provided by the invention has the characteristics of sensitivity, accuracy and better repeatability through the verification of methodology such as specificity and sensitivity, thereby providing a reliable method for quality control of products and being beneficial to improving the medication safety of patients.
Description
Technical Field
The invention belongs to the field of medicine analysis and detection, and particularly relates to a method for detecting various genotoxic impurities in flunarizine hydrochloride capsules.
Background
Flunarizine hydrochloride is a calcium channel blocker and can prevent cell damage caused by pathological calcium overload in cells due to ischemia and the like. Is suitable for cerebral arteriosclerosis, cerebral thrombosis, cerebral embolism, cerebral circulatory disturbance caused by hypertension, cerebral hemorrhage, cerebral circulatory disturbance caused by arachnoid, cerebral hemorrhage, etc.
The prior art uses HPLC for detection of substances related to flunarizine hydrochloride formulations, for example CN110967421A discloses the separation and detection of substances related to 1- [ bis- (4-fluorophenyl) methyl ] piperazine, 1- [ (4-fluorophenyl) phenylethyl ] -4- [ (2E) -3-phenylpropenyl ] piperazine, 1- [ bis- (4-fluorophenyl) methyl ] -4- [ (2Z) -3-phenylpropenyl ] piperazine, trans-1-cinnamylpiperazine, 4-difluorobenzophenone, 4-difluorobenzhydrol and nitrogen oxides by HPLC.
At present, no clear control is carried out on genotoxic impurity A (structural formula A), genotoxic impurity B (structural formula B) and genotoxic impurity C (structural formula C), and no method for simultaneously detecting the genotoxic impurity A, the genotoxic impurity B and the genotoxic impurity C is available.
A is a kind of
B (B)
C (C)
Therefore, it is important to study a quantitative detection method of the above gene impurities.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a method for detecting various genotoxic impurities in flunarizine hydrochloride capsules, which can realize effective separation and quantitative detection of various genotoxic impurities.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a method for detecting various genotoxic impurities in flunarizine hydrochloride capsules, wherein the various genoviral impurities are genotoxic impurity A, genotoxic impurity B and genotoxic impurity C, and the method comprises the following steps:
s1 preparation of sample to be tested
Grinding the content of the sample into fine powder, precisely measuring a proper amount of the fine powder, adding a solvent, swirling, performing ultrasonic treatment, fixing the volume, centrifuging, filtering, and diluting the subsequent filtrate to obtain a sample to be measured;
s2 standard curve preparation
Dissolving the genotoxic impurity A, B, C reference substance to obtain a genotoxic impurity reference substance stock solution;
diluting stock solution into serial standard curve solutions with different concentrations, respectively adopting the method of the step S3 to detect, recording peak areas, and establishing a linear standard curve;
s3 liquid chromatography-mass spectrometry combined detection
And (3) performing high performance liquid chromatography-mass spectrometry detection on the to-be-detected product obtained in the step (S1), wherein a stationary phase of the high performance liquid chromatography-mass spectrometry detection method is a C18 chromatographic column, a mobile phase A is an aqueous solution containing 8-12 mM ammonium acetate, a mobile phase B is acetonitrile, gradient elution is adopted, the flow rate is 0.2-0.4 mL/min, the column temperature is 20-40 ℃, the mass spectrometry adopts a positive ion mode, an electrospray ion source is used for multi-reaction monitoring, and a detector is a triple quadrupole mass spectrometry detector.
As some preferred embodiments of the present invention, the step S1 is: grinding the content of the product into fine powder, taking the fine powder which is approximately equal to 20mg of flunarizine hydrochloride, precisely weighing a proper amount of the fine powder, placing the fine powder into a 10ml measuring flask, adding 5ml of methanol for dissolution, swirling for 5 minutes, shaking for 10 minutes, diluting to a scale with methanol, shaking uniformly, centrifuging, filtering, adding a diluent into 5ml of the subsequent filtrate to 10ml, and shaking uniformly to obtain a sample to be detected.
As some preferred embodiments of the present invention, the concentration of the series of standard solutions in step S2 is about 150ng/ml, 100ng/ml, 75ng/ml, 50ng/ml, 7.5ng/ml, 3ng/ml, respectively.
As some preferred embodiments of the invention, the chromatographic column in step S3 is Agilent Poroshell EC c18.
As some preferred embodiments of the present invention, the elution procedure of step S3 is as follows:
as some preferred embodiments of the present invention, in the step S3, the mass spectrometry condition is that the temperature of the dry gas is 350 ℃; the air curtain air pressure is 15psi; the spray gas pressure was 35psi; the spraying voltage is 5500V; the drying gas pressure was 35psi.
As some preferred embodiments of the present invention, in the step S3, the multiple reaction monitoring parameters are as follows
The beneficial effects of adopting above-mentioned technical scheme to produce lie in:
the invention provides the detection method of the three genotoxic impurities for the first time, and the condition parameters of the detection method are designed, so that the three genoviral impurities can be thoroughly separated and sensitively detected.
The detection method provided by the invention can realize effective separation of various genotoxic impurities, and proved by methodological verification such as specificity, sensitivity and the like, the method provided by the invention is sensitive, accurate and good in reproducibility.
The method can more simply and conveniently realize quantitative detection of more impurities in the flunarizine hydrochloride capsule, provides a reliable method for quality control of products, and is beneficial to improving medication safety of patients.
Drawings
FIG. 1 is a standard graph of 1- [ bis- (4-fluorophenyl) methyl ] piperazine;
FIG. 2 is a standard graph of 1- [ (4-fluorophenyl) phenylethyl ] -4- [ (2E) -3-phenylpropenyl ] piperazine;
FIG. 3 is a standard graph of 1- [ bis- (4-fluorophenyl) methyl ] -4- [ (2Z) -3-phenylpropenyl ] piperazine;
fig. 4 is an MRM mass spectrum of a sample to be tested.
Detailed Description
The present invention provides various embodiments for making the objects, technical solutions and advantages of the present invention more clear. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, well known methods, procedures, means, equipment and steps have not been described in detail so as not to obscure the present invention. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Without being specifically illustrated, the reagents, solvents, and reagents used in this example section were all commercially available, and the apparatus, materials, and reagents used in this example section were as follows:
high performance liquid chromatography-mass spectrometry (Shimadzu liquid LC-20AT, mass Spectrometry AB-6500);
flunarizine hydrochloride capsule (Shijia city Hua Xin pharmaceutical industry Limited liability company), lot number: 190302
Impurity a control: the source is as follows: TLC, lot number: 3883-087A4, content: 95.0%.
Impurity B control: the source is as follows: ECO, lot number: CB-62413-05, content: 98.5%.
Impurity C control: the source is as follows: reno medicine, lot number: RN1037-21-1, content: 95.0%.
Methanol, water and formic acid are all LC-MS grade.
Example 1
1 preparation of solution
1.1 preparation of samples to be tested
Taking 20 grains of the product, precisely weighing the total weight, pouring out the content, grinding and uniformly mixing, precisely weighing a proper amount of fine powder (approximately equivalent to 20mg of flunarizine hydrochloride), placing into a 10ml measuring flask, adding 5ml of methanol for dissolution, swirling for 5 minutes, shaking for 10 minutes, diluting to a scale with methanol, shaking uniformly, centrifuging, filtering, taking 5ml of the subsequent filtrate, adding a diluent to 10ml, shaking uniformly, and taking the filtrate as a sample solution to be measured.
1.2 preparation of control solution
1.21 control solution (1): accurately weighing about 10mg of impurity A, B, C reference substance, placing into a 10ml measuring flask, adding methanol for dissolving, fixing volume to scale, and shaking; and precisely weighing the mixture into a measuring flask with 0.1ml to 100ml, adding a diluent to dilute the mixture to a scale, and shaking the mixture uniformly.
1.22 Control solution (2): precisely measuring the reference substance solution (1) in a measuring flask with 5ml to 50ml, adding a diluent to dilute to a scale, and shaking uniformly.
1.23 series of standard solutions: precisely measuring 1.5ml of the reference substance solution (1), respectively placing 1ml into 10ml measuring flask, adding diluent to dilute to scale, and shaking to obtain serial standard solutions (1) and (2). Precisely measuring 7.5ml, 5ml, 0.75ml and 0.3ml of the reference substance solution (2), respectively placing into 10ml measuring bottles, adding diluent to dilute to scale, and shaking to obtain serial standard solutions (3), (4), (5) and (6). (the concentrations of the series of standard solutions are about 150ng/ml, 100ng/ml, 75ng/ml, 50ng/ml, 7.5ng/ml, 3ng/ml, respectively).
1.3 adding a labeled test sample solution: taking 20 grains of the product, precisely weighing the total weight, pouring out the content, grinding and uniformly mixing, precisely weighing a proper amount of fine powder (approximately equivalent to 20mg of flunarizine hydrochloride), placing into a 10ml measuring flask, adding 5ml of methanol for dissolution, swirling for 5 minutes, shaking for 10 minutes, diluting to a scale with methanol, shaking uniformly, centrifuging, filtering, precisely measuring 5ml of subsequent filtrate, precisely adding 0.75ml of reference solution A, adding a diluent to 10ml, shaking uniformly, and taking the mixture as a standard sample solution.
2 methodological verification
2.1 chromatographic conditions
Chromatographic column: agilent Poroshell 120 EC C18.0X105 mm, 2.7. Mu.m; mobile phase: acetonitrile-10 mmol/L ammonium acetate solution gradient elution (elution program see table), flow rate: 0.3ml/min; column temperature: 30 ℃.
| Time | Acetonitrile | 10mmol/L ammonium acetate solution |
| 0 | 20 | 80 |
| 3 | 25 | 75 |
| 5 | 40 | 60 |
| 10 | 95 | 5 |
| 20 | 95 | 5 |
| 26 | 20 | 80 |
Mass spectrum detection conditions
Adopting a triple quaternary rod mass spectrum detector, electrospraying a positive ion mode (ESI+), and performing multi-reaction monitoring (MRM), wherein the temperature of the dry gas is 350 ℃; the air curtain air pressure is 15psi; the spray gas pressure was 35psi; the spraying voltage is 5500V; the drying gas pressure was 35psi.
MRM parameters
| Names of Compounds | Parent ion (m/z) | Sub-ions (m/z) | DP（V） | CE（V） |
| * Impurity A | 437.3 | 203.2 | 86 | 21 |
| Impurity A | 437.3 | 117.1 | 86 | 21 |
| * Impurity C | 152.1 | 115.1 | 30 | 16 |
| Impurity C | 152.1 | 91.1 | 30 | 17 |
| * Impurity B | 283.1 | 111.1 | 35 | 15 |
| Impurity B | 283.1 | 205.2 | 35 | 18 |
Note that: * To quantify ion pairs
2.2 specificity
And (3) respectively injecting a series of standard solutions (3), a blank solution (diluent), a test sample solution and a labeled test sample solution into a liquid chromatograph-mass spectrometer, detecting according to a 2.1 method, and recording a chromatogram. The result shows that the internal standard and the reference substance have no interference at the peak position, and the blank solvent has no peak at the peak position, thus the method is suitable for measuring the impurity A, B, C in the flunarizine hydrochloride capsule.
2.3 quantitative limit and detection limit
2.3.1 taking a series of standard solutions (6) as quantitative limiting solutions.
2.3.2 precisely measuring 1ml of quantitative limiting solution, adding 2ml of diluent, and shaking uniformly to obtain the detection limiting solution.
The test results showed that the test results were as follows according to the method 2.1: when the concentration of the impurity A reference substance is 3.02ng/ml respectively, the signal to noise ratio s/n is not less than 10, namely the quantitative limiting solution. When the concentration of the reference substance is 1.01ng/ml respectively, the signal to noise ratio s/n is not less than 3, namely the detection limit solution. When the concentration of the impurity B reference substance is 3.00ng/ml respectively, the signal to noise ratio s/n is not less than 10, namely the quantitative limiting solution. When the concentration of the reference substance is 1.00ng/ml respectively, the signal to noise ratio s/n is not less than 3, namely the detection limit solution. When the concentration of the impurity C reference substance is 3.01ng/ml respectively, the signal to noise ratio s/n is not less than 10, namely the quantitative limiting solution. When the concentration of the reference substance is 1.00ng/ml respectively, the signal to noise ratio s/n is not less than 3, namely the detection limit solution.
2.4 Linear sum Range
Taking the standard curve solution prepared under the 1.2 item, detecting by adopting a 2.1 method, recording peak areas, and establishing a linear standard curve: impurity a has a standard curve of y= 1589.1x-1385.1, r, r= 0.9983, as shown in fig. 1; the standard curve for impurity B is y=y= 1273.9x-1434, r= 0.9988, as shown in fig. 2; the standard curve of the impurity C is y= 1426.2 x-1443.9, R is=0.9987, and the linear relation among the impurity A, the impurity B and the impurity C in the range of 3-150 ng/ml is good as shown in figure 3.
2.5 recovery rate
Preparation of 50% recovery solution: taking 20 grains of the product, precisely weighing the total weight, pouring out the content, grinding and uniformly mixing, precisely weighing a proper amount of fine powder (approximately equivalent to 20mg of flunarizine hydrochloride), placing into a 10ml measuring flask, adding 5ml of methanol for dissolution, swirling for 5 minutes, shaking for 10 minutes, diluting to a scale with methanol, shaking uniformly, centrifuging, filtering, precisely weighing 5ml of subsequent filtrate, precisely adding 0.375ml of reference solution (1), adding 10ml of diluent, shaking uniformly, and preparing three parts in parallel.
Preparation of 100% recovery solution: taking 20 grains of the product, precisely weighing the total weight, pouring out the content, grinding and uniformly mixing, precisely weighing a proper amount of fine powder (approximately equivalent to 20mg of flunarizine hydrochloride), placing into a 10ml measuring flask, adding 5ml of methanol for dissolution, swirling for 5 minutes, shaking for 10 minutes, diluting to a scale with methanol, shaking uniformly, centrifuging, filtering, precisely weighing 5ml of the subsequent filtrate, precisely adding 0.75ml of reference substance solution (1), adding a diluting agent to 10ml, and shaking uniformly. Triplicate were prepared.
Preparation of 150% recovery solution: taking 20 grains of the product, precisely weighing the total weight, pouring out the content, grinding and uniformly mixing, precisely weighing a proper amount of fine powder (approximately equivalent to 20mg of flunarizine hydrochloride), placing into a 10ml measuring flask, adding 5ml of methanol for dissolution, swirling for 5 minutes, shaking for 10 minutes, diluting to a scale with methanol, shaking uniformly, centrifuging, filtering, precisely weighing 5ml of the subsequent filtrate, precisely adding 1.125ml of reference substance solution (1), adding a diluting agent to 10ml, and shaking uniformly. Triplicate were prepared.
The results were obtained by detecting according to the method 2.1: the recovery rate ranges of the impurity A and the impurity B are respectively 98.0% -109.7%, 99.7% -108.2% and 98.3% -105.3%, respectively, and the recovery rate is good.
2.6 stability investigation
Precisely measuring a series of standard solutions (3) and test sample solutions, placing the solutions at room temperature for 0, 1, 2, 4, 10 and 16 hours for investigation, and detecting the investigation result according to a 2.1 method: the serial standard solution (3) and the sample solution are placed for 16 hours at room temperature, the peak area RSD of the impurity A in the serial standard solution (3) is 4.1 percent, the peak area RSD of the impurity A in the sample solution is 3.0 percent, the peak area RSD of the impurity B in the sample solution is 4.7 percent, and the peak area RSD of the impurity C in the sample solution is not detected. The series of standard solutions (3) and the test solutions were shown to be stable over 16 hours.
2.7 Precision of
Precisely measuring the sample solution and the labeled sample solution, respectively injecting into a liquid chromatograph-mass spectrometer, detecting according to 2.1 method, and recording the chromatogram. Wherein, the sample solution to be tested is added with the standard and is repeatedly sampled for 6 times, the RSD is calculated, and the precision of the method is examined. From the test results, it can be seen that: the area RSD of the peak of the impurity A is 2.2%, the area RSD of the peak of the impurity B is 1.8%, the area RSD of the peak of the impurity C is 2.5%, and the sample injection precision is good.
2.8 sample detection
The flunarizine hydrochloride capsules of different batches were tested by the method of this example, and the results of the sample measurement are shown in table 1.
TABLE 1 detection results of samples to be tested
| Lot number | Results | A | B | C |
| 712190602 | Content (ppm) | Not detected | Not detected | Not detected |
| 712190505 | Content (ppm) | Not detected | Not detected | Not detected |
| 712190508 | Content (ppm) | Not detected | Not detected | Not detected |
| 712190510 | Content (ppm) | Not detected | Not detected | Not detected |
| 712190511 | Content (ppm) | Not detected | Not detected | Not detected |
Although the invention has been described in detail with reference to the foregoing embodiments, those skilled in the art may modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (7)
1. The method for detecting various genotoxic impurities in the flunarizine hydrochloride capsule is characterized in that the various genoviral impurities are impurity A, impurity B and impurity C, and the method comprises the following steps:
s1 preparation of sample to be tested
Grinding the content of the sample into fine powder, precisely measuring a proper amount of the fine powder, adding a solvent, swirling, performing ultrasonic treatment, fixing the volume, centrifuging, filtering, and diluting the subsequent filtrate to obtain a sample to be measured;
s2 standard curve preparation
Dissolving the genotoxic impurity A, B, C reference substance to obtain a genotoxic impurity reference substance stock solution;
diluting stock solution into serial standard curve solutions with different concentrations, respectively adopting the method of the step S3 to detect, recording peak areas, and establishing a linear standard curve;
s3 liquid chromatography-mass spectrometry combined detection
And (3) performing high performance liquid chromatography-mass spectrometry detection on the to-be-detected product obtained in the step (S1), wherein a stationary phase of the high performance liquid chromatography-mass spectrometry detection method is a C18 chromatographic column, a mobile phase A is an aqueous solution containing 8-12 mM ammonium acetate, a mobile phase B is acetonitrile, gradient elution is adopted, the flow rate is 0.2-0.4 mL/min, the column temperature is 20-40 ℃, the mass spectrometry adopts a positive ion mode, an electrospray ion source is used for multi-reaction monitoring, and a detector is a triple quadrupole mass spectrometry detector.
2. The method for detecting various genotoxic impurities in flunarizine dihydrochloride capsules according to claim 1, wherein the step S1 is: grinding the content of the product into fine powder, taking the fine powder which is approximately equal to 20mg of flunarizine hydrochloride, precisely weighing a proper amount of the fine powder, placing the fine powder into a 10ml measuring flask, adding 5ml of methanol for dissolution, swirling for 5 minutes, shaking for 10 minutes, diluting to a scale with methanol, shaking uniformly, centrifuging, filtering, adding a diluent into 5ml of the subsequent filtrate to 10ml, and shaking uniformly to obtain a sample to be detected.
3. The method according to claim 1, wherein the concentration of the standard solutions in the series of step S2 is about 150ng/ml, 100ng/ml, 75ng/ml, 50ng/ml, 7.5ng/ml, 3ng/ml, respectively.
4. The method for detecting various genotoxic impurities in flunarizine dihydrochloride capsules according to claim 1, wherein the chromatographic column in the step S3 is Agilent Poroshell EC c18.
5. The method for detecting various genotoxic impurities in flunarizine dihydrochloride capsules according to claim 1, wherein the elution procedure of step S3 is as follows:
。
6. The method for detecting various genotoxic impurities in flunarizine dihydrochloride capsules according to claim 1, wherein in the step S3, the mass spectrum condition is that the temperature of the dry gas is 350 ℃; the air curtain air pressure is 15psi; the spray gas pressure was 35psi; the spraying voltage is 5500V; the drying gas pressure was 35psi.
7. The method for detecting multiple genotoxic impurities in flunarizine dihydrochloride capsule according to claim 1, wherein in the step S3, multiple reaction monitoring parameters are as follows
。
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