CN117805265A - Loxoprofen sodium and analysis method of genotoxic impurities in preparation thereof - Google Patents
Loxoprofen sodium and analysis method of genotoxic impurities in preparation thereof Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 76
- 229960002373 loxoprofen Drugs 0.000 title claims abstract description 26
- 231100000024 genotoxic Toxicity 0.000 title claims abstract description 20
- 230000001738 genotoxic effect Effects 0.000 title claims abstract description 20
- YMBXTVYHTMGZDW-UHFFFAOYSA-N loxoprofen Chemical compound C1=CC(C(C(O)=O)C)=CC=C1CC1C(=O)CCC1 YMBXTVYHTMGZDW-UHFFFAOYSA-N 0.000 title claims abstract 5
- 238000002360 preparation method Methods 0.000 title abstract description 11
- 238000004458 analytical method Methods 0.000 title description 9
- 238000000034 method Methods 0.000 claims abstract description 38
- 238000001514 detection method Methods 0.000 claims abstract description 34
- 239000012488 sample solution Substances 0.000 claims abstract description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 45
- 239000000243 solution Substances 0.000 claims description 32
- 239000000523 sample Substances 0.000 claims description 27
- 238000012360 testing method Methods 0.000 claims description 24
- 150000002500 ions Chemical class 0.000 claims description 22
- 239000011550 stock solution Substances 0.000 claims description 21
- 239000013558 reference substance Substances 0.000 claims description 19
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 15
- 239000003085 diluting agent Substances 0.000 claims description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 238000001819 mass spectrum Methods 0.000 claims description 10
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- 239000007789 gas Substances 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 4
- 239000008186 active pharmaceutical agent Substances 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 229940088679 drug related substance Drugs 0.000 claims description 4
- 235000019253 formic acid Nutrition 0.000 claims description 4
- 229910001873 dinitrogen Inorganic materials 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 239000000945 filler Substances 0.000 claims description 3
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 3
- 239000007921 spray Substances 0.000 claims description 3
- 238000010812 external standard method Methods 0.000 claims description 2
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 claims description 2
- 238000010829 isocratic elution Methods 0.000 claims description 2
- 238000004811 liquid chromatography Methods 0.000 claims description 2
- 239000000825 pharmaceutical preparation Substances 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 7
- 238000001212 derivatisation Methods 0.000 abstract description 3
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 abstract description 3
- 239000012088 reference solution Substances 0.000 abstract 1
- BAZQYVYVKYOAGO-UHFFFAOYSA-M loxoprofen sodium hydrate Chemical compound O.O.[Na+].C1=CC(C(C([O-])=O)C)=CC=C1CC1C(=O)CCC1 BAZQYVYVKYOAGO-UHFFFAOYSA-M 0.000 description 22
- 238000007865 diluting Methods 0.000 description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 238000011084 recovery Methods 0.000 description 8
- 238000005303 weighing Methods 0.000 description 8
- 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 description 7
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 239000012490 blank solution Substances 0.000 description 5
- 238000002552 multiple reaction monitoring Methods 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000004807 localization Effects 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- OXFCILAOEGBINA-UHFFFAOYSA-N methyl 2-[4-(bromomethyl)phenyl]propanoate Chemical compound COC(=O)C(C)C1=CC=C(CBr)C=C1 OXFCILAOEGBINA-UHFFFAOYSA-N 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- QQXBRVQJMKBAOZ-UHFFFAOYSA-N 2-[4-(bromomethyl)phenyl]propanoic acid Chemical compound OC(=O)C(C)C1=CC=C(CBr)C=C1 QQXBRVQJMKBAOZ-UHFFFAOYSA-N 0.000 description 1
- 208000008035 Back Pain Diseases 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- 208000008930 Low Back Pain Diseases 0.000 description 1
- 206010034464 Periarthritis Diseases 0.000 description 1
- AUALQMFGWLZREY-UHFFFAOYSA-N acetonitrile;methanol Chemical compound OC.CC#N AUALQMFGWLZREY-UHFFFAOYSA-N 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 229940035676 analgesics Drugs 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 229940124599 anti-inflammatory drug Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000007012 clinical effect Effects 0.000 description 1
- 238000011217 control strategy Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- 150000008282 halocarbons Chemical class 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 238000012502 risk assessment Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 210000000707 wrist 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
- G01N30/04—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
- G01N30/26—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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a method for analyzing genotoxic impurities in loxoprofen sodium and a preparation thereof, which adopts a liquid chromatography-mass spectrometry method, adopts conventional reagents to dissolve and dilute a sample solution and a reference solution, does not need to detect after derivatization, has simple operation, has the detection limit of two genotoxic impurities below 0.5ppm and the quantitative limit below 2ppm, and can realize the accurate detection of the genotoxic impurities.
Description
Technical Field
The invention belongs to the technical field of chemical analysis, and particularly relates to loxoprofen sodium and a method for analyzing genotoxic impurities in a preparation thereof.
Background
Loxoprofen sodium, chemical name of 2- [4- (2-oxo-cyclopentane-1-ylmethyl) phenyl]Sodium propionate, CAS number 80382-23-6, molecular formula C 15 H 17 O 3 ·2H 2 O, molecular weight 304.32. Loxoprofen sodium and its clinical congenersCompared with the medicine, the medicine is characterized in that: has good clinical effect and small side effect. The other kind of medicine has the features of wide application, and may be used clinically in treating rheumatoid arthritis, lumbago, scapulohumeral periarthritis, neck, shoulder and wrist syndrome, etc.
At present, the synthetic method of loxoprofen sodium is more, wherein a more common synthetic route is as follows:
in this synthetic route: 2- (4- (bromomethyl) phenyl) propionic acid (SM 1) and 2- (4-bromomethyl phenyl) propionic acid methyl ester (LSL-1) are respectively a starting material and an intermediate 1 for synthesizing loxoprofen sodium bulk drug, both contain halogenated hydrocarbon warning structures, and are potential genotoxic impurities. The risk assessment of potentially genotoxic impurities according to ICH M7 guidelines can be attributed to class 3 (i.e., containing a warning structure, but not a mutability dataset, independent of the structure of the drug substance), and is applicable to TTC (toxicological concern threshold) algorithms to formulate acceptable limits. Loxoprofen sodium is an analgesic and anti-inflammatory drug, the maximum daily dosage is 180 mg/day, the daily intake is calculated according to 1.5 mug/day, and the control limits of the impurity SM1 and the impurity LSL-1 are both 0.00083 percent.
The existing impurity detection method of loxoprofen sodium mainly comprises two methods of high performance liquid chromatography detection and liquid chromatography-mass spectrometry detection. For example, in the publication No. CN115236255B, "a method for detecting related substances of loxoprofen sodium", a high performance liquid phase method is adopted, wherein a mobile phase of the method consists of an aqueous solution of phosphoric acid and acetonitrile, the pH value of which is regulated to be 2.3-2.7 by adding phosphoric acid, and gradient elution is carried out; from the test results, the detection limit of the impurity M1 (intermediate 1 in the above synthetic route) was 0.02504. Mu.g/mL (equivalent to 0.005%), and the detection results did not meet the requirements of the genotoxic impurity standard. In another example, in publication CN110940744a, "a method for detecting genotoxic impurities in loxoprofen or its sodium salt", a method using liquid chromatography-mass spectrometry is adopted, but piperidine is adopted as a derivatization reagent in the method, and impurity a (corresponding to SM1 in the above synthetic route) and impurity B are derivatized first, but the reaction of piperidine and impurity a or B is not single, and the amino group in piperidine may react with the carboxyl group in impurity a or B or may also react with the halogen in impurity a or B, so the derivative may not be unique. Furthermore, because the impurity A is bromide and has higher activity than the impurity B, the impurity A is used as a reference in the whole analysis process, and the total amount of the impurity A and the impurity B is detected, so that the impurity control strategy is very low in precision; moreover, because the activity of the impurity B is low, the impurity B can not be derived or can not be completely derived, and whether the impurity A and the impurity B show peaks in a chromatogram at the same time or not and whether the impurity B can be detected are unknown, which directly influence the reliability of data and the accuracy of results. Also used in this patent is a SIM mode, m/z=248, which is a single ion detection, not strongly specific, and in particular for samples after derivatization, other substances of this molecular weight are likely to interfere with the detection; the feasibility of the method and the accuracy of the data in this patent are thus both to be examined. From the selection of the solvent in the patent, DMSO is used as the solvent, has strong toxicity, is not friendly to the environment and operators, is difficult to gasify because of easy solidification at low temperature, and has different degrees of damage to chromatographic columns and liquid-mass spectrometry equipment, so that the sensitivity of the DMSO is reduced, and the use of the DMSO is not recommended. From the detection results of the patent; the detection limit of the impurity A in the detection method described in the patent is 7.3ppm, the limit (limit of quantification) of the impurity A which can be accurately quantified is estimated to be about 24ppm, and the detection requirement of 8.3ppm (generally, the limit of quantification is required to be less than 30% of the limit) of the impurity A is not met, so that the method can only qualitatively examine the impurity and can not accurately quantify the result. In summary, in order to ensure the safety and quality controllability of loxoprofen sodium, it is necessary to develop a method for detecting loxoprofen sodium genotoxic impurities with a simple method and a low detection limit.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide the loxoprofen sodium and the analysis method of the genotoxic impurities in the preparation thereof, wherein the test sample solution and the reference substance solution are dissolved and diluted by adopting conventional reagents, complex pretreatment is not needed, the operation is simple, the detection limit of the two genotoxic impurities is less than 0.5ppm, the quantitative limit is less than 2ppm, and the method verification test shows that the accurate detection of the genotoxic impurities can be realized.
The invention provides a method for analyzing genotoxic impurities in loxoprofen sodium and a preparation thereof, which comprises the following steps:
s1, preparing a sample solution: preparing a test sample solution by using a diluent, wherein the diluent consists of acetonitrile and methanol;
s2, preparing a reference substance solution: firstly, acetonitrile is adopted to prepare a reference substance stock solution, and then the diluent in the step S1 is further adopted to dilute the reference substance stock solution to the required concentration to be used as a reference substance solution;
s3, detecting: performing high performance liquid chromatography-mass spectrometry tandem detection on the sample solution in the step S1 and the reference substance solution in the step S2 respectively, and recording a related spectrogram;
wherein: the scanning mode in the mass spectrum in step S3 adopts the MRM mode.
According to the mass spectrum provided by the invention, the mass spectrum adopts the MRM mode, and a pair of ions are respectively adopted to identify substances, so that the specificity is stronger, and the qualitative accuracy is improved.
Preferably, in the step S1, the sample is loxoprofen sodium drug substance or a pharmaceutical preparation containing loxoprofen sodium.
Preferably, in the step S1, the volume ratio of acetonitrile to methanol is (75-85): 20-25.
Preferably, in the step S2, the genotoxic impurity is one or both of impurity SM1 and impurity LSL-1; the specific structural formula is as follows:
。
preferably, in the step S3, the test conditions of the liquid chromatography are as follows: the chromatographic column adopts octadecylsilane chemically bonded silica gel as filler or equivalent chromatographic column; the mobile phase is acetonitrile- (0.25-0.35)% formic acid aqueous solution, the volume ratio is (40-60)% (40-60), and the flow rate is 0.3-0.5 ml/min; the column temperature is 35-45 ℃; the elution mode adopts isocratic elution, the running time is about 26-30 min, and the valve is switched to mass spectrum detection according to the peak time of the impurity SM1 and the impurity LSL-1.
Preferably, in the step S3, the test conditions of the mass spectrum are as follows: ionization mode: electrospray positive ion mode; ion spray voltage: 4.0KV; atomizer: nitrogen, 50psi; collision gas: nitrogen gas; drying gas: the flow rate of nitrogen is 12-14L/min.
Preferably, in the step S3, the detection conditions of the mass spectrum are: impurity SM1: the parent ion is 243.1 and the child ion is 197.0; impurity LSL-1: the parent ion is 256.9, and the child ion is 197.0;
collision voltage: m/z 243.1/197.0 is 111V; m/z 256.1/197.0 is 105V;
collision Energy (CE): m/z 243.1/197.0 is 10V; m/z 256.9/197.0 is 6V.
Preferably, the method calculates the content of genotoxic impurities in the test sample according to an external standard method.
The invention has the beneficial effects that:
1) In the method, loxoprofen sodium and a blank solution do not interfere with detection of impurities, and the two impurities SM1 and LSL-1 can be effectively separated and are not interfered, so that the specificity is good.
2) In the method, the acquisition time is reasonable, and the overlong balance time and the acquisition time are not needed.
3) In the method, the mobile phase is only acetonitrile and acetic acid aqueous solution, the reagent is simple and easy to obtain, the mobile phase is simple to prepare, the sample pretreatment operation list is not needed to be derived, no special reagent is needed to be used, the selected reagent is a conventional reagent, the environment is protected, the specification of the selected chromatographic column is common, and the cost performance is high.
4) The method of the invention has higher sensitivity from the test result, and can accurately carry out quantitative detection on the impurities SM1 and LSL-1 (the quantitative limit concentration of the impurities SM1 is 24.895ng/ml, which is equivalent to 1.660ppm of the concentration of the test sample, and the detection limit concentration is 7.469ng/ml, which is equivalent to 0.498ppm of the concentration of the test sample). The quantitative limit concentration of the impurity LSL-1 is 5.054ng/ml, which corresponds to 0.337ppm of the concentration of the test sample, the detection limit concentration is 1.516ng/ml, which corresponds to 0.101ppm of the concentration of the test sample), and the standard detection requirement can be met.
5) In the method, the mass spectrum adopts the MRM mode, and a pair of ions are respectively adopted to identify the substances, so that compared with single ion detection in the SIM mode, the specificity is stronger, and the qualitative accuracy is improved.
Drawings
FIG. 1 is a map of SM1 localization solution in example 1 of the present invention.
FIG. 2 is a map of LSL-1 localization solution in example 1 of the present invention.
FIG. 3 is a graph of the sample+control solution in example 1 of the present invention.
FIG. 4 is a diagram showing the detection limit of impurity SM1 and impurity LSL-1 in example 2 of the present invention.
FIG. 5 is a quantitative limit diagram of impurity SM1 and impurity LSL-1 in example 2 of the present invention.
FIG. 6 is a linear relationship diagram of impurity SM1 and impurity LSL-1 in example 3 of the present invention.
Detailed Description
The advantages of the invention will now be further described by the following examples, which are given for illustrative purposes only and do not limit the scope of the invention, while variations and modifications apparent to those skilled in the art in light of the present disclosure are included within the scope of the invention.
The loxoprofen sodium drug substance sample used in the present invention is provided by the company of Hunan Jiujingzhu drug Co., ltd.
Example 1: specialization of
Test conditions
Liquid phase conditions: octadecylsilane chemically bonded silica is used as filler (Agilent ZORBAX SB-C18, 4.6mm.times.150mm, 5 μm or column with equivalent performance); acetonitrile-0.3% formic acid water solution (50:50) is taken as a mobile phase, and the flow rate is 0.4ml per minute; column temperature is 40 ℃; the detection wavelength is 222nm; the sample volume was 10. Mu.l.
Mass spectrometry conditions:
ionization mode: electrospray positive ion mode; ion spray voltage: 4.0KV;
atomizer: nitrogen, 50psi; collision gas: nitrogen gas;
drying gas: nitrogen, flow rate 13L/min, temperature 300 ℃;
scanning mode: multiple Reaction Monitoring (MRM);
impurity SM1: the parent ion is 243.1 and the child ion is 197.0;
impurity LSL-1: the parent ion is 256.9, and the child ion is 197.0;
collision voltage: m/z 243.1/197.0 is 111V; m/z 256.1/197.0 is 105V
Collision Energy (CE): m/z 243.1/197.0 is 10V; m/z 256.9/197.0 is 6V
Cell accelerator Voltage：4
Solution preparation
Blank solution (diluent): acetonitrile-methanol (80:20).
Test solution (about 15 mg/ml): weighing about 750mg of the product, precisely weighing, placing into a 50ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking to obtain the final product.
SM1 stock solution # 1 (about 311.25 μg/ml): weighing about 24.9mg of SM1 reference substance, precisely weighing, placing into a 20ml measuring flask, adding acetonitrile for dissolving and diluting to scale, and shaking; precisely measuring 5ml, placing into a 20ml measuring flask, diluting with acetonitrile to scale, and shaking.
SM1 stock solution # 2 (about 3112.5 ng/ml): precisely measuring 1# 1ml of SM1 stock solution, placing into a 100ml measuring flask, diluting to scale with acetonitrile, and shaking.
SM1 positioning solution (about 124.5 ng/ml): precisely measuring 2# 2ml of SM1 stock solution, placing into a 50ml measuring flask, diluting to scale with diluent, and shaking.
LSL-1 stock solution # 1 (about 311.25 μg/ml): weighing about 24.9mg of LSL-1 reference substance, precisely weighing, placing into a 20ml measuring flask, adding acetonitrile for dissolving and diluting to scale, and shaking uniformly; precisely weighing 5ml, placing in a 20ml measuring flask, diluting to scale with acetonitrile, and shaking.
LSL-1 stock solution # 2 (about 3112.5 ng/ml): precisely measuring 1ml, placing in a 100ml measuring flask, diluting with acetonitrile to scale, and shaking.
LSL-1 localization solution (about 124.5 ng/ml): precisely measuring 2# 2ml of LSL-1 stock solution, placing into a 50ml measuring flask, diluting to scale with diluent, and shaking.
Control stock solution: precisely weighing SM1 stock solution 1#1 ml and LSL-1 stock solution 1#1 ml, placing into a 100ml measuring flask, diluting with acetonitrile to scale, and shaking. (about 3112.5 ng/ml)
Control solution: taking 2ml of reference stock solution, placing into a 50ml measuring flask, diluting to scale with diluent, and shaking to obtain the final product. (about 124.5 ng/ml)
Test article + control article solution: and taking a proper amount of loxoprofen sodium, the impurities SM1 and the impurities LSL-1, adding a diluent to dissolve and dilute the loxoprofen sodium and the impurities into a solution containing about 15mg of loxoprofen sodium in each 1ml and 0.1245 mug of each impurity.
Precisely measuring 10 μl of each of the blank solution, each impurity positioning solution, test sample solution, reference substance solution, and test sample+reference substance solution, respectively, and injecting into chromatograph for testing, and recording chromatogram.
From the results of fig. 1 to 3 and table 1, it is shown that: the blank solution does not interfere detection of the impurities SM1 and LSL-1, the impurities SM1 and LSL-1 can be effectively separated, and the method has good specificity.
Example 2: limit of detection and limit of quantification
Precisely measuring a proper amount of the reference substance solution in the embodiment 1, and gradually diluting to obtain the detection limit when the S/N is about 3; when the S/N is about 10, the limit of the amount is defined. The solutions were analyzed separately and chromatograms were recorded, and the results are shown in tables 2 and 3.
From the results of fig. 4 to 5 and tables 2 to 3, it is shown that: in the method, the quantitative limit concentration of the impurity SM1 is 24.895ng/ml, which is equivalent to 1.660ppm of the concentration of the test sample, the detection limit concentration is 7.469ng/ml, which is equivalent to 0.498ppm of the concentration of the test sample; the quantitative limit concentration of the impurity LSL-1 was 5.054ng/ml, which corresponds to 0.337ppm of the concentration of the sample, and the detection limit concentration was 1.516ng/ml, which corresponds to 0.101ppm of the concentration of the sample.
Example 3: linearity and range
The control stock solution of example 1 was diluted to a suitable concentration, and a quantitative Limit (LOQ) solution, 25%, 50%, 100%, 150%, 200% linear solution were sampled for analysis (2 needles each), and a chromatogram was recorded. Linear regression was performed with the concentration on the abscissa (C) and the average peak area on the ordinate (a), and a linear equation was found. The experimental results are shown in table 4 and fig. 6.
Example 4: precision of
(1) Precision of sample injection
Taking control solution, continuously injecting for 6 needles, recording a chromatogram, and calculating a result.
The results show that: continuously injecting a control solution for 6 needles, wherein the retention time RSD of the impurity SM1 is 0.22%, and the peak area RSD is 1.63%; the retention time RSD of the impurity LSL-1 was 0.18% and the peak area RSD was 3.23%; the method has better sample injection precision.
(2) Repeatability of
6 parts of sample solution of the test sample is prepared for sample injection analysis, a chromatogram is recorded, and the result is calculated.
The results show that: the impurities SM1 and LSL-1 in the 6 parts of sample solutions are not detected, and the method has good repeatability.
(3) Intermediate precision
6 samples of the sample solution to be tested are prepared respectively at different days for sample injection analysis, a chromatogram is recorded, and a result is calculated.
The results show that: no impurity SM1 and no impurity LSL-1 are detected in 12 sample solutions, and the method has better intermediate precision.
Example 5: accuracy of
Control stock solution: preparation of control stock solution as in example 1
50% recovery solution: about 750mg of the sample is taken, precisely weighed, placed in a 50ml measuring flask, precisely measured with 1ml of the reference substance stock solution, added with the diluent for dissolution and dilution to the scale, and shaken well. Parallel preparation of 3 parts
100% recovery solution: about 750mg of the sample is taken, precisely weighed, placed in a 50ml measuring flask, precisely measured with 2ml of the reference substance stock solution, added with the diluent for dissolution and dilution to the scale, and shaken well. Parallel preparation of 3 parts
150% recovery solution: about 750mg of the sample is taken, precisely weighed, placed in a 50ml measuring flask, precisely measured with 3ml of the reference substance stock solution, added with the diluent for dissolution and dilution to the scale, and shaken well. Parallel preparation of 3 parts
Sample analysis was performed on the control solution of example 1 and the recovery solutions of each concentration level (3 parts were prepared in parallel), and chromatograms were recorded.
The results show that: in the concentration range of 50% -150%, the recovery rate of the impurity SM1 is 82.09% -95.20%, the average recovery rate is 87.81%, and the RSD is 5.53%; the recovery rate of the impurity LSL-1 is 97.68% -101.11%, the average recovery rate is 99.19%, and the RSD is 1.19%, so that the method is accurate and reliable.
Example 6: durability of
The degree of tolerance of the measurement results, which is not affected when there is a slight variation in the measurement condition parameters, is evaluated by changing the column temperature, the proportion of formic acid in the mobile phase, and the different chromatographic columns. The blank solution, the control solution and the test solution were analyzed, and chromatograms were recorded, and the results are shown in tables 10 and 11.
Remarks: the chromatographic columns 1 and 2 differ only by their numbers.
The results show that: the column temperature (+ -5 ℃) and the formic acid proportion (+ -0.05%) are respectively regulated, and different chromatographic columns are used for detecting the sample, the experimental result is consistent with the repeatability test, so that the measurement result is not affected when the measurement condition parameters slightly change, namely the method has better durability.
The foregoing is a further detailed description of the present application in connection with the specific embodiments, and it is not intended that the practice of the present application be limited to such descriptions. It should be understood that those skilled in the art to which the present application pertains may make several simple deductions or substitutions without departing from the spirit of the present application, and all such deductions or substitutions should be considered to be within the scope of the present application.
Claims (8)
1. A method for analyzing genotoxic impurities in loxoprofen sodium and its formulations, comprising the steps of:
s1, preparing a sample solution: preparing a test sample solution by using a diluent, wherein the diluent consists of acetonitrile and methanol;
s2, preparing a reference substance solution: firstly, acetonitrile is adopted to prepare a reference substance stock solution, and then the diluent in the step S1 is further adopted to dilute the reference substance stock solution to the required concentration to be used as a reference substance solution;
s3, detecting: performing high performance liquid chromatography-mass spectrometry tandem detection on the sample solution in the step S1 and the reference substance solution in the step S2 respectively, and recording a related spectrogram;
wherein: the scanning mode in the mass spectrum in step S3 adopts the MRM mode.
2. The method according to claim 1, wherein in the step S1, the sample is loxoprofen sodium drug substance or a pharmaceutical preparation containing loxoprofen sodium.
3. The method according to claim 1, wherein in the step S1, the volume ratio of acetonitrile to methanol is (75-85): 20-25.
4. The method according to claim 1, wherein in the step S2, the genotoxic impurity is one or both of impurity SM1 and impurity LSL-1; the specific structural formula is as follows:
。
5. the method according to claim 1, wherein, preferably, in the step S3, the test conditions of the liquid chromatography are: the chromatographic column adopts octadecylsilane chemically bonded silica gel as filler or equivalent chromatographic column; the mobile phase is acetonitrile- (0.25-0.35)% formic acid aqueous solution, the volume ratio is (40-60)% (40-60), and the flow rate is 0.3-0.5 ml/min; the column temperature is 35-45 ℃; the elution mode adopts isocratic elution.
6. The method according to claim 1, wherein in the step S3, the test conditions of the mass spectrum are: ionization mode: electrospray positive ion mode; ion spray voltage: 4.0KV; atomizer: nitrogen, 50psi; collision gas: nitrogen gas; drying gas: the flow rate of nitrogen is 12-14L/min.
7. The method according to claim 6, wherein in the step S3, the detection conditions of the mass spectrum are: impurity SM1: the parent ion is 243.1 and the child ion is 197.0; impurity LSL-1: the parent ion is 256.9, and the child ion is 197.0;
collision voltage: m/z 243.1/197.0 is 111V; m/z 256.1/197.0 is 105V;
collision Energy (CE): m/z 243.1/197.0 is 10V; m/z 256.9/197.0 is 6V.
8. The method according to claim 1, wherein the content of genotoxic impurities in the sample is calculated by an external standard method.
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