CN114689751A - Non-target screening method for alkylating genotoxic impurities - Google Patents
Non-target screening method for alkylating genotoxic impurities Download PDFInfo
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- CN114689751A CN114689751A CN202210326645.9A CN202210326645A CN114689751A CN 114689751 A CN114689751 A CN 114689751A CN 202210326645 A CN202210326645 A CN 202210326645A CN 114689751 A CN114689751 A CN 114689751A
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- 239000012535 impurity Substances 0.000 title claims abstract description 85
- 231100000024 genotoxic Toxicity 0.000 title claims abstract description 73
- 230000001738 genotoxic effect Effects 0.000 title claims abstract description 73
- 238000012216 screening Methods 0.000 title claims abstract description 47
- 230000002152 alkylating effect Effects 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 33
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 68
- 238000001212 derivatisation Methods 0.000 claims abstract description 32
- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
- -1 amino compound Chemical class 0.000 claims abstract description 23
- 238000001819 mass spectrum Methods 0.000 claims abstract description 14
- 238000005804 alkylation reaction Methods 0.000 claims abstract description 13
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 13
- 230000029936 alkylation Effects 0.000 claims abstract description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 54
- 150000002500 ions Chemical group 0.000 claims description 29
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 24
- 239000002904 solvent Substances 0.000 claims description 18
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 16
- FEZGQPNGBGISAY-UHFFFAOYSA-N 3-amino-n-(4-methoxyphenyl)propanamide Chemical compound COC1=CC=C(NC(=O)CCN)C=C1 FEZGQPNGBGISAY-UHFFFAOYSA-N 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- BHAAPTBBJKJZER-UHFFFAOYSA-N p-anisidine Chemical compound COC1=CC=C(N)C=C1 BHAAPTBBJKJZER-UHFFFAOYSA-N 0.000 claims description 6
- WHQSYGRFZMUQGQ-UHFFFAOYSA-N n,n-dimethylformamide;hydrate Chemical compound O.CN(C)C=O WHQSYGRFZMUQGQ-UHFFFAOYSA-N 0.000 claims description 5
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- 150000001412 amines Chemical class 0.000 claims description 4
- 150000002118 epoxides Chemical class 0.000 claims description 4
- 235000021317 phosphate Nutrition 0.000 claims description 4
- 150000003013 phosphoric acid derivatives Chemical class 0.000 claims description 4
- 150000003871 sulfonates Chemical class 0.000 claims description 4
- 238000004896 high resolution mass spectrometry Methods 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 238000001195 ultra high performance liquid chromatography Methods 0.000 claims description 2
- 238000013051 Liquid chromatography–high-resolution mass spectrometry Methods 0.000 abstract description 6
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- 239000000243 solution Substances 0.000 description 40
- 239000000047 product Substances 0.000 description 28
- 238000001514 detection method Methods 0.000 description 14
- MBABOKRGFJTBAE-UHFFFAOYSA-N methyl methanesulfonate Chemical compound COS(C)(=O)=O MBABOKRGFJTBAE-UHFFFAOYSA-N 0.000 description 14
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- 229960002620 cefuroxime axetil Drugs 0.000 description 5
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 4
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 description 4
- 239000012295 chemical reaction liquid Substances 0.000 description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 4
- 239000010931 gold Substances 0.000 description 4
- 229910052737 gold Inorganic materials 0.000 description 4
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- 230000011987 methylation Effects 0.000 description 4
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- 108090000623 proteins and genes Proteins 0.000 description 4
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 4
- FQYUMYWMJTYZTK-VIFPVBQESA-N (2r)-2-(phenoxymethyl)oxirane Chemical compound C([C@@H]1OC1)OC1=CC=CC=C1 FQYUMYWMJTYZTK-VIFPVBQESA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PLUBXMRUUVWRLT-UHFFFAOYSA-N Ethyl methanesulfonate Chemical compound CCOS(C)(=O)=O PLUBXMRUUVWRLT-UHFFFAOYSA-N 0.000 description 3
- 239000007810 chemical reaction solvent Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- PQXKWPLDPFFDJP-UHFFFAOYSA-N 2,3-dimethyloxirane Chemical compound CC1OC1C PQXKWPLDPFFDJP-UHFFFAOYSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 2
- RQBBFKINEJYDOB-UHFFFAOYSA-N acetic acid;acetonitrile Chemical compound CC#N.CC(O)=O RQBBFKINEJYDOB-UHFFFAOYSA-N 0.000 description 2
- LFLBHTZRLVHUQC-UHFFFAOYSA-N butyl methanesulfonate Chemical compound CCCCOS(C)(=O)=O LFLBHTZRLVHUQC-UHFFFAOYSA-N 0.000 description 2
- AJEHNBIPLQJTNU-UHFFFAOYSA-N cyanomethyl acetate Chemical compound CC(=O)OCC#N AJEHNBIPLQJTNU-UHFFFAOYSA-N 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 230000001149 cognitive effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229940098779 methanesulfonic acid Drugs 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- NQHXCOAXSHGTIA-SKXNDZRYSA-N nelfinavir mesylate Chemical compound CS(O)(=O)=O.CC1=C(O)C=CC=C1C(=O)N[C@H]([C@H](O)CN1[C@@H](C[C@@H]2CCCC[C@@H]2C1)C(=O)NC(C)(C)C)CSC1=CC=CC=C1 NQHXCOAXSHGTIA-SKXNDZRYSA-N 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000013557 residual solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Landscapes
- 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 non-targeted screening method of alkylation genotoxic impurities, which comprises the following steps: (1) and (3) taking an amino compound as a derivatization agent, carrying out alkylation reaction on the amino compound and alkylated genotoxic impurities, and then separating to generate a derivative product with the structure of the derivatization agent. (2) And (3) fragmenting the derivative product under the condition of mass spectrum to generate the same ion fragments, and screening unknown alkylated genotoxic impurities by ion fragment screening. The invention provides a derivatization-ultra-high performance liquid chromatography-high resolution mass spectrometry non-targeted method for screening alkylating genotoxic impurities, which is characterized in that based on the characteristic of reaction of amine compounds and alkylating genotoxic impurities, the amine compounds are selected as derivative reagents, and the derivative reagent structures contained in the derivative products are cracked under the mass spectrometry condition to generate the characteristic of specific ion fragments, so that the specific ion fragments are screened, the screening of unknown alkylating genotoxic impurities is realized, and the result shows that the specificity of the method provided by the invention is good.
Description
Technical Field
The invention relates to the technical field of genotoxic impurity detection, in particular to a non-targeted screening method for alkylating genotoxic impurities.
Background
The information disclosed in this background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
The genetic material of the human body is DNA, which mainly contains four bases, adenine, guanine, cytosine and thymine. The nitroxides on both the pyrimidine and purine bases are electron rich and are susceptible to substitution and other reactions with electrophilic alkylating impurities, resulting in mutations in the gene triggered by the modified bases of these genotoxic impurities. Alkylating reagents are reagents commonly used in drug synthesis, and trace residues may exist in drugs; alkylated impurities can be generated in the production and storage processes of the medicine, and in 2007, alkylated genotoxic impurity ethyl methanesulfonate is found in nelfinavir methanesulfonate, and the impurities are generated by the reaction of methanesulfonic acid in the medicine and residual solvent ethanol in the storage process.
Due to the cognitive limitation, the alkylated genotoxic impurities in the medicine have certain unpredictability, and the alkylated genotoxic impurities have poor stability and multiple types, so that no screening method for the alkylated genotoxic impurities is known at present.
Disclosure of Invention
Aiming at the problems, the invention provides a non-targeted screening method for the alkylated genotoxic impurities, which can realize the screening of unknown alkylated genotoxic impurities, has good stability of the generated derivative product and solves the problems of poor temperature property and difficult direct detection of the alkylated genotoxic impurities. Specifically, to achieve the above object, the technical solution of the present invention is as follows:
in a first aspect of the invention, the use of an amino compound as a derivatizing agent in the detection of alkylated genotoxic impurities is disclosed.
Further, the amino compound comprises any one of 3-amino-N- (4-methoxyphenyl) propionamide, p-anisidine and the like as an amine derivative reagent.
Further, the alkylation genotoxic impurities are impurities which generate alkylation reaction; alternatively, the alkylated genotoxic impurities include sulfonates, epoxides, phosphates, and the like.
In a second aspect of the present invention, a non-targeted screening method for alkylated genotoxic impurities is disclosed, comprising the steps of:
(1) and (3) taking an amino compound as a derivatization agent, carrying out alkylation reaction on the amino compound and alkylated genotoxic impurities, and then separating out a derivative product with the structure of the derivatization agent for later use.
(2) And fragmenting the derivative product under the condition of mass spectrum to generate the same ion fragments, and screening unknown alkylated genotoxic impurities by ion fragment screening.
Further, in the step (1), the amino compound is used after being dissolved in a solvent to form a derivatization reagent solution. Alternatively, the solvent includes any one of acetonitrile, dimethyl sulfoxide (DMSO), water, N-Dimethylformamide (DMF), and the like.
Further, in the step (1), the alkylated genotoxic impurities are dissolved in a solvent to form an alkylating reagent solution, and then the solution is used. Preferably, the solvent includes any one of acetonitrile, dimethyl sulfoxide (DMSO), water, N-Dimethylformamide (DMF), and the like.
Further, in the step (1), the amino compound comprises any one of 3-amino-N- (4-methoxyphenyl) propionamide, p-anisidine and the like as an amine derivative reagent.
Further, in the step (1), the alkylating genotoxic impurities are impurities for alkylation reaction; alternatively, the alkylated genotoxic impurities include sulfonates, epoxides, phosphates, and the like.
Further, in the step (1), the reaction temperature is 20-80 ℃, and the reaction time is 1 hour. The reaction is preferably carried out at 40 ℃ for 1 hour.
Further, in the step (2), derivative products in the reaction system are separated by an ultra-high performance liquid chromatography technology.
Further, in step (2), the derivatized product is fragmented using high resolution mass spectrometry to produce identical ion fragments.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a derivatization-ultra-high performance liquid chromatography-high resolution mass spectrometry non-targeted screening method for alkylating genotoxic impurities, which is characterized in that based on the characteristic of reaction of amine compounds and alkylating genotoxic impurities, the amine compounds are selected as derivative reagents, and the derivative reagent structures contained in the derivative products are cracked under the mass spectrometry condition to generate the characteristic of specific ion fragments, and the specific ion fragments are screened, so that the screening of unknown alkylating genotoxic impurities is realized, and the result shows that the specificity of the method provided by the invention is good.
(2) The derivatization-ultra-high performance liquid chromatography-high resolution mass spectrometry non-targeted screening method for the alkylated genotoxic impurities, which is provided by the invention, takes an amino compound as a derivatization agent, and the derivatization product generated by the reaction of the amino compound and the alkylated genotoxic impurities has good stability, so that the problems of poor temperature property and difficulty in direct detection of the alkylated genotoxic impurities are solved.
(3) Because the alkylation gene toxic impurities are various in types and structures, the alkylation gene toxic impurities have no structural characteristics for screening. The invention uses amino compound as derivative agent to react with alkylated genotoxic impurity to generate derivative product with derivative reagent structure, and realizes the screening of unknown alkylated genotoxic impurity through the characteristic fragment ion relative to the derivative reagent generated by the derivative product under mass spectrum condition.
Detailed Description
In the following description, further specific details of the invention are set forth in order to provide a thorough understanding of the invention. The terminology used in the description of the invention herein is for the purpose of describing particular advantages and features of the invention only and is not intended to be limiting of the invention.
Unless defined otherwise, all 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. Unless otherwise indicated, the drugs or agents used in the present invention are used according to the instructions of the product or by the conventional methods in the art. The technical solution of the present invention will now be further explained based on the specific embodiments.
First embodiment
A non-targeted screening method for alkylating genotoxic impurities, which is referred to as reaction scheme 1 and comprises the following steps:
(1) selecting 3-amino-N- (4-methoxyphenyl) propionamide as a derivatization reagent, placing about 100mg of the derivatization reagent in a measuring flask with the volume of 10ml, respectively dissolving the derivatization reagent by taking acetonitrile, DMSO, water and DMF as solvents, and diluting to scale to obtain four derivatization reagent solutions with different concentrations of 10mg/ml, wherein the derivatization reagent solutions obtained by taking the acetonitrile, the DMSO, the water and the DMF as solvents are respectively named as A1, A2, A3 and A4 for later use.
(2) Taking 10mg of methyl methanesulfonate, placing the methyl methanesulfonate in a measuring flask with the volume of 10ml, respectively dissolving the methyl methanesulfonate by using acetonitrile, DMSO, water and DMF as solvents, diluting the methyl methanesulfonate to a scale, and diluting the solution in a gradient manner to obtain 10 mu g/ml of alkylating reagent solution, thereby obtaining four different alkylating reagent solutions, wherein the deriving reagent solutions obtained by using the acetonitrile, the DMSO, the water and the DMF as solvents are respectively named as B1, B2, B3 and B4 for later use.
(3) Measuring 1ml of the derivative reagent solution prepared in the step (1), adding 1ml of the derivative reagent solution prepared in the step (2), uniformly mixing (mixing, namely mixing the derivative reagent solution with the same solvent with the alkylating reagent solution, namely mixing A1 with B1, mixing A2 with B3, mixing A3 with B3 and mixing A4 with B4), reacting for 1h at 40 ℃, and precisely measuring 1 mu l of reaction liquid to be injected into a liquid chromatograph-mass spectrometer (namely an ultra-high performance liquid chromatography-high resolution mass spectrometry combined method) to analyze the generation amount of reaction products: firstly, a reaction product and a derivative reagent which does not participate in the reaction are eluted and separated by a chromatographic method, then the product is fragmented under the condition of mass spectrum to generate the same ion fragment, and the screening of unknown alkylated genotoxic impurities is realized by the ion fragment screening. Wherein, the conditions of the liquid chromatography-mass spectrometry are as follows: the liquid chromatography conditions were: the chromatographic column is Thermo Hypersil Gold C18(2.1X100mm, 3 μm); the column temperature was 40 ℃; the mobile phase A is 0.1% acetic acid water solution; the mobile phase B is 0.1 percent acetonitrile acetate solution; gradient elution: 0-3min, 2% B, 3-10mi, 2% → 98% B, 10-11min, 98% B, 11.1-14min, 2% B; the flow rate is 0.3 mL/h; the sample injection amount is 1 mu L; mass spectrum conditions: ESI positive ion detection mode, Full MS-ddMS2 detection was performed.
Reaction scheme 1
The amount of the derivative product generated by different reaction solutions is shown in table 1, and it can be seen that four reaction solvents, namely acetonitrile, DMSO, water and DMF, can effectively react, wherein the reaction efficiency of acetonitrile as the solvent is the highest, and the reaction solvent can be selected according to the solubility of the substance to be detected.
TABLE 1
| Different reaction solvents | Peak area of methylation derivative product EIC (m/z 209.1289) |
| Acetonitrile | 4567543 |
| DMSO | 4367542 |
| Water (I) | 3890654 |
| DMF | 4209872 |
Second embodiment
A non-target screening method for alkylating genotoxic impurities comprises the following steps:
(1) selecting 3-amino-N- (4-methoxyphenyl) propionamide as a derivatization reagent, placing about 100mg of the derivatization reagent into a measuring flask with the volume of 10ml, adding acetonitrile as a solvent to dissolve the derivatization reagent, and diluting to scale to obtain a derivatization reagent solution with the concentration of 10mg/ml for later use.
(2) Taking 10mg of methyl methanesulfonate, placing the methyl methanesulfonate into a measuring flask with the volume of 10ml, dissolving the methyl methanesulfonate by using acetonitrile as a solvent, diluting the methyl methanesulfonate to a scale, and carrying out gradient dilution on the solution to obtain an alkylating reagent solution with the concentration of 10 mu g/ml for later use.
(3) Measuring 1ml of the derivative reagent solution prepared in the step (1), adding 1ml of the alkylating reagent solution prepared in the step (2), uniformly mixing to obtain a mixed solution, taking four parts of the mixed solution, respectively heating and reacting at 20 ℃, 40 ℃, 60 ℃ and 80 ℃ for 1h, precisely measuring 1 mu l of each reaction to obtain a reaction solution after the reaction is finished, and respectively analyzing the generation amount of reaction products by using a liquid chromatograph-mass spectrometer (namely an ultra-high performance liquid chromatography-high resolution mass spectrometry combination method): firstly, a reaction product and a derivative reagent which does not participate in the reaction are eluted and separated by a chromatographic method, then the product is fragmented under the condition of mass spectrum to generate the same ion fragment, and the screening of unknown alkylated genotoxic impurities is realized by the ion fragment screening. Wherein, the LC-MS conditions are as follows: the liquid chromatography conditions were: the chromatographic column is Thermo Hypersil Gold C18(2.1X100mm, 3 μm); the column temperature was 40 ℃; the mobile phase A is 0.1% acetic acid water solution; the mobile phase B is 0.1 percent acetic acid acetonitrile solution; gradient elution: 0-3min, 2% B, 3-10mi, 2% → 98% B, 10-11min, 98% B, 11.1-14min, 2% B; the flow rate is 0.3 mL/h; the sample injection amount is 1 mu L; mass spectrum conditions: ESI positive ion detection mode, Full MS-ddMS2 detection was performed.
The amounts of the derived products produced at different reaction temperatures are shown in Table 2, and it can be seen that the target reaction product can be obtained at all four temperatures, but the reaction efficiency is higher at 40 ℃ to 80 ℃, and the environment in which genotoxic impurities act is a human physiological environment, so 40 ℃ is preferred as the reaction temperature.
TABLE 2
| Reaction temperature | Peak area of methylation derivative product EIC (m/z 209.1289) |
| 20℃ | 1368528 |
| 40℃ | 4265734 |
| 60℃ | 43765432 |
| 80℃ | 42976665 |
Third embodiment
A non-target screening method for alkylating genotoxic impurities comprises the following steps:
(1) selecting 3-amino-N- (4-methoxyphenyl) propionamide as a derivatization reagent, placing about 100mg of the derivatization reagent into a measuring flask with the volume of 10ml, adding acetonitrile as a solvent to dissolve the derivatization reagent, and diluting to scale to obtain a derivatization reagent solution with the concentration of 10mg/ml for later use.
(2) Taking 10mg of methyl methanesulfonate, placing the methyl methanesulfonate into a measuring flask with the volume of 10ml, dissolving the methyl methanesulfonate by using acetonitrile as a solvent, diluting the methyl methanesulfonate to a scale, and carrying out gradient dilution on the solution to obtain an alkylating reagent solution with the concentration of 10 mu g/ml for later use.
(3) Measuring 1ml of the derivative reagent solution prepared in the step (1), adding 1ml of the alkylating reagent solution prepared in the step (2), uniformly mixing, heating at 40 ℃ for 1h, precisely measuring 1 microliter of each reaction to obtain reaction liquid after the reaction is finished, and analyzing the generation amount of reaction products by using a liquid mass spectrometer (namely an ultra-high performance liquid chromatography-high resolution mass spectrometry combination method) respectively: firstly, a reaction product and a derivative reagent which does not participate in the reaction are eluted and separated by a chromatographic method, then the product is fragmented under the condition of mass spectrum to generate the same ion fragment, and the screening of unknown alkylated genotoxic impurities is realized by the ion fragment screening. Wherein, the LC-MS conditions are as follows: the liquid chromatography conditions were: the chromatographic column is Thermo Hypersil Gold C18(2.1X100mm, 3 μm); the column temperature was 40 ℃; the mobile phase A is 0.1% acetic acid water solution; the mobile phase B is 0.1 percent acetonitrile acetate solution; gradient elution conditions 1: 0-3min, 2% B; 3-10mi, 2% → 98% B; 10-11min, 98% B; 11.1-14min, 2% B; gradient elution conditions 2: 0-3min, 5% B; 3-10mi, 5% → 98% B; 10-11min, 98% B; 11.1-14min, 5% B; gradient elution conditions 3: 0-3min, 10% B; 3-10mi, 10% → 98% B; 10-11min, 98% B; 11.1-14min, 10% B. The flow rate is 0.3 mL/h; the sample injection amount is 1 mu L; mass spectrum conditions: ESI positive ion detection mode, FullMS-ddMS2 detection was performed.
The separation results of the derivative reagent and the derivative reagent methylated product under different gradient elution conditions are shown in table 3, the separation effect of the derivative reagent and the derivative reagent methylated product is poor under the gradient elution conditions 2 and 3, the retention time of the derivative reagent under the gradient elution condition 1 is 2.01min, the retention time of the derivative reagent methylated product is 3.88min, and the separation effect of the derivative reagent and the derivative reagent methylated product is good. The C18 chromatographic column separates the compound according to the polarity of the compound, the smaller the polarity of the compound, the larger the retention time, the less polar the other alkylated forms of the derivatization reagent have polarity than the methylation product of the derivatization reagent, and the retention time is longer than the methylation product of the derivatization reagent, so that the alkylation product of the derivatization reagent can be effectively separated from the derivatization reagent.
TABLE 3
| Derivatization reagent retention time (min) | Derivatized product retention time (min) | |
| Gradient elution conditions 1 | 2.01 | 3.88 |
| Gradient elution conditions 2 | 1.89 | 2.32 |
| Gradient elution Condition 3 | 1.80 | 1.94 |
Fourth embodiment
A non-target screening method for alkylating genotoxic impurities comprises the following steps:
1. methyl methanesulfonate, ethyl methanesulfonate, butyl methanesulfonate, methyl propylene oxide and (R) -phenoxymethyl ethylene oxide are respectively added into cefuroxime axetil to prepare five samples with 100 mu g/g of cefuroxime axetil as the alkylating genotoxic impurity for later use.
2. Taking 100mg of 3-amino-N- (4-methoxyphenyl) propionamide, placing the 3-amino-N- (4-methoxyphenyl) propionamide into a measuring flask with the volume of 10ml, adding acetonitrile to dissolve and dilute the 3-amino-N- (4-methoxyphenyl) propionamide to a scale, and obtaining a derivatization reagent solution with the concentration of 10 mg/ml. Respectively taking 1g of cefuroxime axetil sample added with different impurities, placing the sample into a measuring flask with the volume of 10ml, and then adding acetonitrile to dissolve the sample to obtain sample solution.
3. Measuring 1ml of the derivatization reagent solution, adding 1ml of the sample solution into the derivatization reagent solution, uniformly mixing the mixture, and reacting the mixture for 1 hour at 40 ℃. Meanwhile, after acetonitrile is added into cefuroxime axetil without the alkylated genotoxic impurity in the step (1), the cefuroxime axetil is used as a blank sample solution to react with the derivatization reagent solution at 40 ℃ for 1h to be used as a blank group.
4. And (3) after the reaction in the step (3) is finished, precisely measuring 1 mu l of reaction liquid, and injecting the reaction liquid into a liquid chromatograph-mass spectrometer to analyze the product. Wherein, the conditions of LC-MS are as follows: the liquid chromatography conditions were: the chromatographic column is Thermo Hypersil Gold C18(2.1X100mm, 3 μm); the column temperature was 40 ℃; the mobile phase A is 0.1% acetic acid water solution; the mobile phase B is 0.1 percent acetic acid acetonitrile solution; gradient elution: 0-3min, 2% B, 3-10mi, 2% → 98% B, 10-11min, 98% B, 11.1-14min, 2% B; the flow rate is 0.3 mL/h; the sample injection amount is 1 mu L; mass spectrum conditions: ESI positive ion detection mode, Full MS-ddMS2 detection was performed.
5. Fragment ion screening is carried out on the Mass spectrum data obtained in the step 4 by using Mass spectrometer Mass Frontier, and the results are shown in Table 4. The results show that: the derivative products of the alkylated impurities produced common ionic fragments m/z of 124.0757, 166.0863. Whether the alkylated genotoxic impurity exists can be conveniently judged by checking whether chromatographic peaks with m/z of 124.0757 and/or 166.0863 exist in the obtained ion fragments. The blank group did not detect the ion fragments m/z124.0757, m/z 166.0863. The specificity of the method for screening the alkylating genotoxic impurities by using the amino compound as the derivative reagent is good. When the alkylated genotoxic impurities exist in the detected sample, corresponding ion fragments can be generated under the method of the embodiment, so that the alkylated genotoxic impurities exist in the detected sample can be judged, and the specific alkylated genotoxic impurities do not need to be known at the moment, and the screening of the unknown alkylated genotoxic impurities can be realized as long as the alkylated genotoxic impurities exist in the detected sample are known.
TABLE 4
Fifth embodiment
A non-targeted screening method for alkylating genotoxic impurities, which is the same as the fourth embodiment except that p-anisidine is used as a derivative reagent to replace the 3-amino-N- (4-methoxyphenyl) propionamide. The obtained Mass spectrum data were subjected to fragment ion screening using Mass spectrometer, Mass Frontier, and the results are shown in table 5. The results show that: the p-anisidine is used as a derivative reagent, a derivative product of the alkylated impurity generates a common ion fragment m/z of 123.0680, and whether the alkylated genotoxic impurity exists can be conveniently judged by checking whether a chromatographic peak with the m/z of 123.0680 exists in the obtained ion fragment. The blank group does not detect characteristic ion fragments, which shows that the method for screening genotoxic impurities by selecting amino compounds as derivative reagents has good specificity and can realize the screening of unknown alkylated genotoxic impurities.
TABLE 5
Sixth embodiment
A non-targeted screening method for alkylating genotoxic impurities, which is different from the fourth embodiment in that: replacing the 3-amino-N- (4-methoxyphenyl) propanamide with 4-dimethylaminopyridine as derivatizing agent. The obtained Mass spectrum data were subjected to fragment ion screening using Mass spectrometer, Mass Frontier, and the results are shown in table 6. The results show that: only the (R) -phenoxymethyl oxirane derivative was detected, and no other derivatives were detected. This shows that the use of 4-dimethylaminopyridine as a derivatizing reagent obviously causes problems such as undetectable detection and missed detection.
TABLE 6
| Addition of genotoxic impurities | Retention time (min) | Derivatizing characteristic fragment ions of reagent structures |
| Blank group (not added) | Undetected | Not detected out |
| Methanesulfonic acid methyl ester | Not detected out | |
| Methanesulfonic acid ethyl ester | Not detected out | |
| Methanesulfonic acid butyl ester | Not detected out | |
| Methyl propylene oxide | Not detected out | |
| (R) -phenoxymethyl oxirane | 4.27 | 123.0917 |
The above description is only illustrative of several embodiments of the present invention and should not be taken as limiting the scope of the invention. It should be noted that other persons skilled in the art can make modifications, substitutions, improvements and the like without departing from the spirit and scope of the present invention, and all of them belong to the protection scope of the present invention. Therefore, the scope of the invention should be determined from the description and claims.
Claims (10)
1. Application of amino compound as derivative agent in detecting alkylating genotoxic impurity.
2. The use according to claim 1, wherein the amino compound comprises amine derivatizing agent selected from 3-amino-N- (4-methoxyphenyl) propionamide and p-anisidine.
3. The use according to claim 1 or 2, wherein the alkylating genotoxic impurity is an impurity that undergoes an alkylation reaction; preferably, the alkylating genotoxic impurities include any one of sulfonates, epoxides, and phosphates.
4. A non-targeted screening method for alkylating genotoxic impurities is characterized by comprising the following steps:
(1) taking an amino compound as a derivatization agent, carrying out alkylation reaction on the amino compound and alkylation genotoxic impurities, and then separating out a derivative product with the structure of the derivatization agent for later use;
(2) and fragmenting the derivative product under the condition of mass spectrum to generate the same ion fragments, and screening unknown alkylated genotoxic impurities by ion fragment screening.
5. The method for non-targeted screening of alkylated genotoxic impurities according to claim 4, wherein the step (1) comprises dissolving the amino compound in a solvent to form a derivative reagent solution; preferably, the solvent includes any one of acetonitrile, dimethyl sulfoxide, water, and N, N-dimethylformamide.
6. The method for non-targeted screening of alkylated genotoxic impurities according to claim 5, wherein in step (1), the alkylated genotoxic impurities are dissolved in a solvent to form an alkylating reagent solution; preferably, the solvent comprises any one of acetonitrile, dimethyl sulfoxide, water and N, N-dimethylformamide.
7. The non-targeted screening method for alkylated genotoxic impurities according to claim 4, wherein in the step (1), the amino compound comprises any one of 3-amino-N- (4-methoxyphenyl) propionamide and p-anisidine as amine derivative reagent.
8. The method for non-targeted screening of alkylating genotoxic impurities according to claim 4, wherein in step (1), the alkylating genotoxic impurities are impurities that undergo alkylation reaction; preferably, the alkylating genotoxic impurities include any one of sulfonates, epoxides, and phosphates.
9. The non-targeted screening method for alkylated genotoxic impurities according to any one of claims 4 to 8, wherein in the step (1), the reaction temperature is 20 to 80 ℃, and the reaction time is 1 hour; the reaction is preferably carried out at 40 ℃ for 1 hour.
10. The non-targeted screening method for alkylated genotoxic impurities according to any one of claims 4-8, wherein in the step (2), derivative products in the reaction system are separated by ultra-high performance liquid chromatography;
preferably, in step (2), the derivatized product is fragmented using high resolution mass spectrometry to produce identical ion fragments.
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