CN116754695B - Derivatization detection method for chlorosulfonyl isocyanate - Google Patents
Derivatization detection method for chlorosulfonyl isocyanate Download PDFInfo
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- WRJWRGBVPUUDLA-UHFFFAOYSA-N chlorosulfonyl isocyanate Chemical compound ClS(=O)(=O)N=C=O WRJWRGBVPUUDLA-UHFFFAOYSA-N 0.000 title claims abstract description 76
- 238000001212 derivatisation Methods 0.000 title claims abstract description 39
- 238000001514 detection method Methods 0.000 title abstract description 28
- 239000000523 sample Substances 0.000 claims abstract description 60
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 38
- 238000012360 testing method Methods 0.000 claims abstract description 37
- 239000013558 reference substance Substances 0.000 claims abstract description 30
- PVOAHINGSUIXLS-UHFFFAOYSA-N 1-Methylpiperazine Chemical compound CN1CCNCC1 PVOAHINGSUIXLS-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000012488 sample solution Substances 0.000 claims abstract description 15
- 239000000126 substance Substances 0.000 claims abstract description 14
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 6
- 239000003960 organic solvent Substances 0.000 claims abstract description 6
- 238000010812 external standard method Methods 0.000 claims abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 111
- 239000000243 solution Substances 0.000 claims description 102
- 238000000034 method Methods 0.000 claims description 26
- NMAOAFBQWWLHQG-UHFFFAOYSA-N 4-methylpiperazine-1-sulfonamide Chemical compound CN1CCN(S(N)(=O)=O)CC1 NMAOAFBQWWLHQG-UHFFFAOYSA-N 0.000 claims description 21
- 238000005303 weighing Methods 0.000 claims description 14
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 11
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 10
- 238000010790 dilution Methods 0.000 claims description 9
- 239000012895 dilution Substances 0.000 claims description 9
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 150000002500 ions Chemical class 0.000 claims description 5
- ZMXDDKWLCZADIW-UHFFFAOYSA-N dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 11
- 238000006243 chemical reaction Methods 0.000 abstract description 9
- HCDSYEBRPDDECJ-UHFFFAOYSA-N n-methylpiperazine-1-sulfonamide Chemical compound CNS(=O)(=O)N1CCNCC1 HCDSYEBRPDDECJ-UHFFFAOYSA-N 0.000 abstract 1
- 239000011550 stock solution Substances 0.000 description 45
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 32
- 239000003795 chemical substances by application Substances 0.000 description 28
- 229910021642 ultra pure water Inorganic materials 0.000 description 26
- 239000012498 ultrapure water Substances 0.000 description 26
- 238000007865 diluting Methods 0.000 description 24
- 239000012490 blank solution Substances 0.000 description 21
- 238000002360 preparation method Methods 0.000 description 19
- 238000004128 high performance liquid chromatography Methods 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- 239000012467 final product Substances 0.000 description 10
- 239000012535 impurity Substances 0.000 description 10
- 239000012085 test solution Substances 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 9
- 239000012086 standard solution Substances 0.000 description 9
- PAMIQIKDUOTOBW-UHFFFAOYSA-N 1-methylpiperidine Chemical compound CN1CCCCC1 PAMIQIKDUOTOBW-UHFFFAOYSA-N 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- -1 chlorosulfonyl Chemical group 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 239000007791 liquid phase Substances 0.000 description 5
- 239000012088 reference solution Substances 0.000 description 5
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 4
- KMIPKYQIOVAHOP-YLGJWRNMSA-N cefditoren Chemical compound S([C@@H]1[C@@H](C(N1C=1C(O)=O)=O)NC(=O)\C(=N/OC)C=2N=C(N)SC=2)CC=1\C=C/C=1SC=NC=1C KMIPKYQIOVAHOP-YLGJWRNMSA-N 0.000 description 4
- 229960004069 cefditoren Drugs 0.000 description 4
- 229960001958 cefodizime Drugs 0.000 description 4
- XDZKBRJLTGRPSS-BGZQYGJUSA-N cefodizime Chemical compound S([C@@H]1[C@@H](C(N1C=1C(O)=O)=O)NC(=O)\C(=N/OC)C=2N=C(N)SC=2)CC=1CSC1=NC(C)=C(CC(O)=O)S1 XDZKBRJLTGRPSS-BGZQYGJUSA-N 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000004949 mass spectrometry Methods 0.000 description 4
- CURJNMSGPBXOGK-UHFFFAOYSA-N n',n'-di(propan-2-yl)ethane-1,2-diamine Chemical compound CC(C)N(C(C)C)CCN CURJNMSGPBXOGK-UHFFFAOYSA-N 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 229910052708 sodium Inorganic materials 0.000 description 4
- JSAKRLDIZOGQTN-UHFFFAOYSA-M 4-[(2-hydroxynaphthalen-1-yl)diazenyl]naphthalene-1-sulfonate Chemical compound OC1=C(C2=CC=CC=C2C=C1)N=NC1=CC=C(C2=CC=CC=C12)S(=O)(=O)[O-] JSAKRLDIZOGQTN-UHFFFAOYSA-M 0.000 description 3
- 229960003716 cilastatin sodium Drugs 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 238000012417 linear regression Methods 0.000 description 3
- 230000003252 repetitive effect Effects 0.000 description 3
- OWIKHYCFFJSOEH-UHFFFAOYSA-N Isocyanic acid Chemical group N=C=O OWIKHYCFFJSOEH-UHFFFAOYSA-N 0.000 description 2
- 241001089723 Metaphycus omega Species 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000010606 normalization Methods 0.000 description 2
- XRQDFNLINLXZLB-CKIKVBCHSA-N peramivir Chemical compound CCC(CC)[C@H](NC(C)=O)[C@@H]1[C@H](O)[C@@H](C(O)=O)C[C@H]1NC(N)=N XRQDFNLINLXZLB-CKIKVBCHSA-N 0.000 description 2
- 229960001084 peramivir Drugs 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 208000033962 Fontaine progeroid syndrome Diseases 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- UOTHZPGSFUCNBZ-UHFFFAOYSA-N [Cl].O=C=NS(=O)(=O)N=C=O Chemical compound [Cl].O=C=NS(=O)(=O)N=C=O UOTHZPGSFUCNBZ-UHFFFAOYSA-N 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000006352 cycloaddition reaction Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 201000011243 gastrointestinal stromal tumor Diseases 0.000 description 1
- 231100000024 genotoxic Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000012948 isocyanate Substances 0.000 description 1
- 150000002513 isocyanates Chemical class 0.000 description 1
- 125000001261 isocyanato group Chemical group *N=C=O 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 239000012038 nucleophile Substances 0.000 description 1
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012795 verification Methods 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
- 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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
-
- 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 provides a derivatization detection method of chlorosulfonyl isocyanate, which comprises the following steps: preparing a reference substance solution and a test substance solution; mixing a derivatization reagent with a chlorosulfonyl isocyanate solution with a set content, and reacting at room temperature to obtain a reference substance solution; mixing a derivative reagent with a sample to be tested, and reacting at room temperature to obtain a sample solution; the derivatization reagent is obtained by dissolving 1-methylpiperazine in an organic solvent; and respectively analyzing the reference substance solution and the test substance solution by adopting a liquid chromatography-mass spectrometer, recording a chromatogram, and calculating the content of the 1-methylpiperazine sulfonamide according to a peak area external standard method by combining the content of the chlorosulfonyl isocyanate in the reference substance solution so as to obtain the content of the chlorosulfonyl isocyanate in the test substance. By adopting the technical scheme of the invention, the reaction is simple and quick, the operation is convenient, the derivative product has strong response in the LC-MS, the interference can be avoided, and the sensitivity is high.
Description
Technical Field
The invention relates to the technical field of compound detection, in particular to a derivatization detection method of chlorosulfonyl isocyanate.
Background
The chemical property of chlorosulfonyl isocyanate is very active, and the compound has chlorosulfonyl and isocyanic acid groups in the molecule, and the chlorosulfonyl and the isocyanic acid groups are both very active functional groups. Furthermore, carbon and nitrogen atoms are two electrophilic sites in the molecule of the compound, and can be attacked by nucleophiles. The N=C part of isocyanate can also generate surface cycloaddition, and can be added with water, active hydrogen, alcohol, phenol, amine, amide, carboxylic acid, olefin, aldehyde ketone and ester to obtain various novel compounds, which can be used as dehydrating agents and oxidizing agents, thus having wide application in the synthesis of various medicines.
Chlorosulfonyl and isocyanato in the chlorosulfonyl isocyanate molecule are warning structures of genotoxic impurities according to ICH M7 guidelines, so that the content of chlorosulfonyl isocyanate in the drug must be strictly controlled. The development of the chlorosulfonyl isocyanate detection method with strong specificity and high sensitivity is very critical to the quality control of medicines and is also critical to the success or failure of medicine reporting.
The conventional chlorosulfonyl isocyanate detection method has a plurality of advantages and disadvantages, and the specific analysis is as follows:
(1) Derivatization spectrophotometry: the method is mainly used for detecting chlorosulfonyl isocyanate in water, has poor sensitivity and specificity, and can not meet the requirement of detection limit in medicines.
(2) Color reaction: the reaction with sodium nitrite, the solution was reddish brown, but the method was low in sensitivity.
(3) Ion chromatography: the detection sensitivity is low, and the interference of coexisting ions in the sample matrix and the limitation of instrument and equipment are large.
(4) Gas chromatography mass spectrometry: the chlorosulfonyl isocyanate reacts with alcohol substances to generate ester compounds, and then the ester compounds are measured by GCMS, but the method cannot meet the limit requirement of less than 1 ppm.
Disclosure of Invention
Aiming at the technical problems, the invention discloses a derivatization detection method of chlorosulfonyl isocyanate, which is simple, rapid in detection and high in sensitivity.
In this regard, the invention adopts the following technical scheme:
a method for detecting derivatization of chlorosulfonyl isocyanate comprises the following steps:
step S1, preparing a reference substance solution and a test substance solution;
the reference substance solution is prepared by the following steps: mixing a derivatizing reagent with a chlorosulfonyl isocyanate solution with a set content, and reacting at room temperature;
the sample solution is prepared by the following steps: mixing a derivative reagent with a test sample, and reacting at room temperature;
wherein the derivatization reagent is obtained by dissolving 1-methylpiperazine in an organic solvent;
and S2, respectively analyzing the reference substance solution and the test substance solution by adopting a liquid chromatography-mass spectrometer, recording a chromatogram, and calculating the content of the 4-methyl-1-piperazine sulfonamide according to a peak area external standard method by combining the content of the chlorosulfonyl isocyanate in the reference substance solution so as to obtain the content of the chlorosulfonyl isocyanate in the test substance.
By adopting the technical scheme, the derivatization reaction of the 1-methylpiperazine and chlorosulfonyl isocyanate by the derivatization reagent is as follows:
in the development of the present invention, a variety of derivatizing agents are employed, such asN,NWhen pure water is adopted as a derivative reagent, the response of the pure water and chlorosulfonyl isocyanate derivative products is too low to meet the sensitivity requirement of the method; when N, N-diisopropyl ethylenediamine is used as a derivative reagent, the labeled recovery rate of the derivative product is low, and a false positive result appears; when 1-methylpiperidine is used as the derivatizing agent, the derivatizing reaction product is complex and has a large number of peaks. Therefore, the 1-methylpiperazine is finally selected as the derivative reagent, the derivative product is single, the sensitivity is high, the operation is simple and convenient, the reaction is rapid, the obtained derivative product 4-methyl-1-piperazine sulfonamide has strong response in HPLC/MS, the testing method of the chlorosulfonyl isocyanate is simplified, and the detection sensitivity is improved.
As a further improvement of the present invention, in the step S1, the concentration of the 1-methylpiperazine in the derivatizing agent is 5 to 100mg/ml. Preferably, the concentration of the 1-methylpiperazine in the derivatizing agent is 20-50mg/ml.
As a further improvement of the present invention, the organic solvent comprises dimethyl sulfoxide,N,NDimethylformamide (DMA),N, NDimethylacetamide,N-at least one of methyl pyrrolidone, acetonitrile. Further preferably, the organic solvent is acetonitrile.
As a further improvement of the invention, in the step S1, the content of chlorosulfonyl isocyanate in the test sample is calculated by adopting the following formula:
，
wherein ,A i measuring the peak area of the 4-methyl-1-piperazine sulfonamide of the object to be measured in the solution of the object to be measured;
A S the average value of the peak area of the 4-methyl-1-piperazine sulfonamide of the object to be detected, which is measured in the reference substance solution;
W T the sample is the sample weighing quality of the sample liquid;
W S the mass of chlorosulfonyl isocyanate in the chlorosulfonyl isocyanate solution weighed for preparing the reference substance solution;
D T is the dilution multiple of the sample solution;
D S is the dilution multiple of the reference substance solution.
As a further improvement of the present invention, in step S2, the conditions of the liquid chromatography-mass spectrometry are: c18 column, mobile phase a:10mM ammonium formate aqueous solution, mobile phase B: acetonitrile, gradient elution. Further preferably, the C18 column is a C18-PFP, C18-AQ or EC-C18 column.
As a further improvement of the present invention, in step S2, the chromatographic column in the liquid chromatography-mass spectrometer is an ACE Excel 3 c18-PFP chromatographic column.
As a further improvement of the present invention, the conditions of the liquid chromatography-mass spectrometry in step S2 further include: the column temperature is 20-35 ℃, the flow rate is 0.2 ml/min-1 ml/min, and the sample injection amount is 1-5 mu L.
As a further improvement of the present invention, in the gradient elution, 0 to 3 min, A (%): b (%) =97:3, 4-8min, a (%): b (%) =10:90.
As a further improvement of the invention, in the liquid chromatography-mass spectrometry in the step S2, the parent ion is set to be m/z 180, the child ion is set to be m/z 101, and the collision energy is 10-20. Further preferably, the collision energy is 15.
Compared with the prior art, the invention has the beneficial effects that:
according to the technical scheme, the 4-methyl-1-piperazine sulfonamide can be quickly generated by using the derivative reagent 1-methylpiperazine and the chlorosulfonyl isocyanate under the room temperature condition, and the 4-methyl-1-piperazine sulfonamide is measured to detect the chlorosulfonyl isocyanate.
Drawings
FIG. 1 is a full LC-MS/MS scan of a control solution after addition of a derivatizing agent in an embodiment of the present invention.
FIG. 2 is a graph of a linear regression equation of the linear verification experiment of example 1 of the present invention.
Description of the embodiments
Preferred embodiments of the present invention are described in further detail below.
The method for detecting the chlorosulfonyl isocyanate derivatization specifically comprises the following steps:
instrument: an electronic balance of ten parts per million, a liquid phase mass spectrum combined instrument, a liquid phase chromatographic column and an ultrapure water instrument;
reagent: acetonitrile (HPLC), 1-methylpiperazine (98%), dichloromethane (HPLC), ammonium formate (AR), ultrapure water (resistivity. Gtoreq.18.2 M.OMEGA. Multidot.cm);
control: chlorosulfonyl isocyanate;
derivatization agent: 1-methylpiperazine, structural formula:
derivatization reaction:
1. solution preparation
Derivatizing reagent: taking 1-methylpiperazine about 1250mg to 25ml in a volumetric flask, diluting to a scale with acetonitrile, and shaking uniformly to obtain the final product.
Blank solution: transferring 50 μl acetonitrile into 5ml EP tube, adding 50 μl derivatizing agent, shaking, adding 0.9ml ultrapure water, and shaking.
Stock solution of chlorosulfonyl isocyanate: and (3) taking about 20mg of chlorosulfonyl isocyanate, precisely weighing, diluting to a scale with methylene dichloride in a 25ml volumetric flask, shaking up, transferring the solution into a 1.0ml to 100ml volumetric flask, diluting to the scale with acetonitrile, and shaking up to obtain the final product.
Control stock solution: transferring 1.0ml chlorosulfonyl isocyanate stock solution 1 to 25ml volumetric flask, diluting with acetonitrile to scale, and shaking.
Control solution: transferring 50 μl of control stock solution 3-5 ml EP tube, adding 50 μl of derivatization agent, shaking, adding 0.9ml of ultrapure water, shaking, and standing at room temperature for 5 min.
Test solution: about 20mg of the sample is taken, precisely weighed, added into a 5ml EP tube, 50 μl of acetonitrile is added, then 50 μl of derivatization agent is added, shaking is carried out, then 0.9ml of ultrapure water is added, shaking is carried out, standing is carried out at room temperature for 5min, and 2 parts of the sample are prepared in parallel.
2. The process parameters are as follows:
the LC-MS/MS full scan results obtained are shown in figure 1.
The following description is made in connection with specific examples in accordance with the above-described methods.
Example 1
And verifying the residual method of chlorosulfonyl isocyanate in cefodizime sodium.
1. Instrument, reagent, reference substance and test sample
Instrument: one ten thousandth analytical balance, liquid phase-mass spectrometry (Agilent 1260A-6470A);
chromatographic column: ACE Excel 3 c18-PFP (100 x 4.6mm,3 μm);
flow rate: 1ml/min
Solvent: acetonitrile (HPLC), 1-methylpiperazine (98%), dichloromethane (HPLC), ammonium formate (AR), ultrapure water (resistivity. Gtoreq.18.2 M.OMEGA. Multidot.cm);
control: chlorosulfonyl isocyanate;
test article: cefditoren sodium.
2. Solution preparation
Derivatizing reagent: taking 1-methylpiperazine about 1250mg to 25ml in a volumetric flask, diluting to a scale with acetonitrile, and shaking uniformly to obtain the final product.
Blank solution: transferring 50 μl acetonitrile to 5ml EP tube, adding 50 μl derivatizing agent, shaking, adding 0.9ml ultrapure water, shaking, and standing at room temperature for 5 min.
Stock solution of chlorosulfonyl isocyanate: and (3) taking about 20mg of chlorosulfonyl isocyanate, precisely weighing, diluting to a scale with methylene dichloride in a 25ml volumetric flask, shaking up, transferring the solution into a 1.0ml to 100ml volumetric flask, diluting to the scale with acetonitrile, and shaking up to obtain the final product.
Control stock solution: transferring 1.0ml chlorosulfonyl isocyanate stock solution 1 to 25ml volumetric flask, diluting with acetonitrile to scale, and shaking.
Control solution: transferring 50 μl of control stock solution 3-5 ml EP tube, adding 50 μl of derivatization agent, shaking, adding 0.9ml of ultrapure water, shaking, and standing at room temperature for 5 min.
Test solution: about 20mg of the sample is taken, precisely weighed, added into a 5ml EP tube, 50 μl of acetonitrile is added, then 50 μl of derivatization agent is added, shaking is carried out, then 0.9ml of ultrapure water is added, shaking is carried out, standing is carried out at room temperature for 5min, and 2 parts of the sample are prepared in parallel.
3. System applicability experiment
(1) Preparing a solution:
blank solution: the preparation method is the same as that of the above, 1 part is prepared;
control solution: the preparation method is the same as that of the above, 1 part is prepared;
(2) Operation of
After the system is balanced, taking 1 needle of blank solution, 6 parts of reference solution are respectively sampled by 1 needle, and recording a chromatogram. The peak area, the peak area average value and the RSD value of the to-be-detected object of 6-needle continuous sample injection of the report control solution are adopted.
(3) The results are shown in Table 1.
Table 1 system applicability results
Conclusion: the RSD of the peak area of the 6-needle 4-methyl-1-piperazine sulfonamide in the continuous sample injection of the control solution is 1.14%, which meets the requirements. The system applicability meets the detection requirement.
4. Specificity experiments
(1) Preparing a solution:
blank solution: the preparation method is the same as that of the above, 1 part is prepared;
control solution: the preparation method is the same as that of the above, 1 part is prepared;
test solution: the preparation method is the same as that of the above, 1 part is prepared;
control stock solution 3: the preparation method is the same as that of the above, 1 part is prepared;
adding a standard solution to a test sample: about 20mg of the sample is taken, precisely weighed, added into a 5ml EP tube, 50 μl of reference stock solution 3 is added, then 50 μl of derivatization agent is added, shaking is carried out, then 0.9ml of ultrapure water is added, shaking is carried out, and standing is carried out at room temperature for 5min, thus obtaining 1 part.
(2) Operation of
Taking chromatographic conditions described by the method for each solution on the premise of qualified system applicability, and recording a chromatogram after each 1 needle. Reporting retention time, peak area and separation degree of the impurity to be detected in the blank solution, the reference solution, the sample adding standard solution and the adjacent peaks.
(3) The results are shown in Table 2.
TABLE 2 specific results
Conclusion:
the blank solution has no interference to impurities to be detected in the reference substance solution and the sample solution;
the impurity peak to be detected in the reference substance solution, the test substance solution and the test substance labeling solution has no adjacent peak (namely, meets the requirement of more than or equal to 1.5);
the retention time of the impurity peak to be detected in the sample solution or the sample labeling solution is consistent with that of the reference solution;
compared with the sample solution, the peak area of each impurity to be detected in the sample labeling solution is increased, and the method has good specificity.
5. Analysis of repeatability experiments
(1) Preparing a solution:
analysis of duplicate solutions: the preparation method is used for preparing 6 parts of the sample to be tested in the special embodiment in parallel with the standard adding solution;
(2) Operation of
According to the measurement method, 6 parts of analysis repetitive solution are taken, 1 needle is injected for each sample, and the content of an object to be measured in the 6 parts of analysis repetitive solution is calculated according to the average value of the peak area of the reference substance solution in the system applicability. The peak area, single content value, mean±sd, RSD value and 95% confidence interval of the test object are reported.
(3) Results
Table 3 analysis of the repeatability results
Acceptable standards:
and 6 parts of the analysis repetitive solution have RSD less than or equal to 10.0 percent of the content of the 4-methyl-1-piperazine sulfonamide in the sample.
Conclusion: in 6 parts of the analysis repeatability solution, the RSD value of the content of the 4-methyl-1-piperazine sulfonamide is 1.41 percent (less than or equal to 10.0 percent) and meets the requirements.
6. Experiment of detection limit
(1) Preparing a solution:
blank solution: the preparation method is the same as that of the above, 1 part is prepared;
detection of the limiting stock: transferring 100 μl of control stock solution into a volumetric flask of 2-25 ml, diluting with acetonitrile to scale, and shaking.
Detection limit solution: and transferring 50 mu l of the detection limit stock solution into a 5ml EP tube, adding 50 mu l of the derivatization agent, shaking uniformly, adding 0.9ml of ultrapure water, shaking uniformly, and standing at room temperature for 5min to obtain the kit.
(2) Operation of
Taking 1 needle of blank solution sample injection under the premise of qualified system applicability, detecting 3 parts of limited solution, respectively feeding 1 needle, and recording a chromatogram. Reporting the peak area and signal to noise ratio (S/N) of the impurity to be detected of the detection limit solution; and calculating the detection limit concentration of the impurity to be detected and the concentration corresponding to the concentration percentage of the solution of the sample.
(3) The results are shown in Table 4.
TABLE 4 limit of detection results
Conclusion:
the minimum value of the signal to noise ratio of the 4-methyl-1-piperazine sulfonamide peak is 36.1 (more than or equal to 3) when the detection limit solution is continuously sampled for 3 times; the detection limit concentration level of 4-methyl-1-piperazine sulfonamide was 0.08 ppm.
7. Quantitative limit experiment
(1) Preparing a solution:
blank solution: the preparation method is the same as that of the above, 1 part is prepared;
quantitative limiting stock solution: transferring 300 μl of the control stock solution into a volumetric flask of 2-25 ml, diluting with acetonitrile to scale, and shaking.
Quantitative limiting solution: transferring 50 μl of the quantitative limiting stock solution into a 5ml EP tube, adding 50 μl of derivatization agent, shaking, adding 0.9ml of ultrapure water, shaking, and standing at room temperature for 5 min.
(2) Operation of
Taking 1 needle of blank solution sample injection under the premise of qualified system applicability, quantitatively limiting 3 parts of solution to enter 1 needle respectively, and recording a chromatogram. Reporting the peak area and the signal to noise ratio (S/N) of the impurity to be detected of the quantitative limiting solution; and calculating the quantitative limit concentration of the impurity to be detected and the concentration corresponding to the concentration percentage of the solution of the sample.
(3) The results are shown in Table 5.
TABLE 5 quantitative limit results
Acceptable standards:
1) The signal to noise ratio of the object to be detected is more than or equal to 10 after the quantitative limiting solution is continuously sampled for 3 times;
2) The RSD value of the peak area of the object to be detected is less than or equal to 15.0% after the quantitative limiting solution is continuously injected for 3 times;
conclusion:
1) The minimum value of the signal-to-noise ratio of the 4-methyl-1-piperazine sulfonamide is 107.3 (more than or equal to 10) and meets the requirements;
2) The RSD value of the peak area of the 4-methyl-1-piperazine sulfonamide is 2.42 percent (less than or equal to 15.0 percent) which meets the requirements after the quantitative solution limiting continuous sampling is carried out for 3 times; the quantitative limit of the method meets the detection requirement.
3) The quantitative limiting concentration level of 4-methyl-1-piperazine sulfonamide was 0.0047 μg/ml (0.23 ppm).
8. Linear and range experiments
(1) Preparing a solution:
blank solution, control stock solution 2, control solution: the preparation method is the same as before.
Linear solutions at each concentration: and transferring 50 mu l of linear stock solutions with different concentrations into a 5ml EP tube, adding 50 mu l of derivatization agent, shaking uniformly, adding 0.9ml of ultrapure water, shaking uniformly, standing at room temperature for 5min to obtain the product, wherein 1 part of each concentration is prepared, and specific reference is made to Table 6.
TABLE 6 Linear solution formulation
(2) Operation of
And after the applicability of the system is qualified, taking a blank solution and feeding 1 needle, taking linear solutions at all concentration levels, feeding the samples from low concentration to high concentration in sequence for 2 times, and recording a chromatogram. And (3) carrying out unitary linear regression on the concentration of each peak area average value of each object to be detected, respectively reporting a linear equation, a linear correlation coefficient r, a residual square sum, a Y-axis intercept, an absolute value of a concentration ratio of the Y-axis intercept to 100%, and a 95% confidence interval of the Y-axis intercept of the object to be detected, and drawing a linear map.
(3) The results are shown in Table 7.
TABLE 7 Linear results
From the above results, a linear analysis of fig. 2 can be obtained, in which the correlation coefficient r is 0.9995, the y-axis intercept 3505.6097 (3.93%, accounting for 100% concentration level), and the linear regression equation is y= 5833665.2834 x-3505.6097.
Acceptable standards:
1) The linear correlation coefficient r of the object to be detected is more than or equal to 0.990;
2) The ratio of the absolute value of the Y-axis intercept of the object to be detected to the peak area corresponding to the 100% limit concentration is less than or equal to 10.0%.
Conclusion:
1) The linear correlation coefficient r of the 4-methyl-1-piperazine sulfonamide is 0.9995 (more than or equal to 0.990), which meets the requirements;
2) The ratio of the absolute value of the Y-axis intercept of the 4-methyl-1-piperazine sulfonamide to the peak area corresponding to the 100% limit concentration is 3.93% (. Ltoreq.10.0%), which meets the requirements.
Overall, the present process is good in linearity for 4-methyl-1-piperazine sulfonamide at LOQ to 150% of the limit concentration.
9. Accuracy experiment
(1) Preparing a solution:
blank solution, chlorosulfonyl isocyanate stock solution, control stock solution: the preparation method is the same as before, and 1 part of each is prepared;
test solution: the preparation method is the same as before, 2 parts are prepared;
accuracy solution: about 20mg of the sample to be tested is precisely weighed and put into a 5ml EP tube, 50 μl of solutions with different concentrations (3 parts per concentration level) are respectively added according to the following table, 50 μl of derivatization agent is added, shaking is carried out, 0.9ml of ultrapure water is added, shaking is carried out, standing is carried out at room temperature for 5min, and 9 parts of solutions with the accuracy are obtained, and the details are shown in Table 8.
Table 8 accuracy solution formulation
(2) Operation of
And after the applicability of the system is qualified, taking 1 needle of blank solution, taking 2 parts of sample solution to be tested, respectively feeding 1 needle, respectively feeding 3 parts of accurate solution at each concentration level into 1 needle, and recording a chromatogram. A 95% confidence interval for the average recovery (n=9) was calculated and reported for each individual test object recovery for 9 samples.
The content and recovery rate of each object to be detected in the sample solution before and after the addition of the label are respectively calculated according to the following formula:
in the formula ：CS Adding a standard solution into the sample to obtain the concentration of stock solution of the sample to be measured, wherein mg/mL is the concentration of stock solution of the sample to be measured;
v is the volume of stock solution of the object to be detected added in each sample adding standard solution, and mL;
C S+T adding the measured residual quantity of the to-be-measured object in the standard solution to each to-be-measured object in ppm;
C i ppm as the residual amount of the object to be detected measured in the solution of the object to be detected;
the average value of the residual quantity of the to-be-detected object measured in the to-be-detected object solution is ppm;
A i the peak area of the object to be detected in the sample solution;
A S 6, aiming at the average peak area of the object to be detected in the control solution;
A S+T adding the peak area of the object to be detected in the standard solution to each object to be detected;
W T the sample is the sample weighing amount of the sample in the sample solution, mg;
W S the sample is the weighed amount of the to-be-detected object in the control solution, and mg;
W S+T adding a sample weighing amount of the test sample in the standard solution to each test sample, and mg;
D T is the dilution multiple of the sample solution;
D S is the dilution multiple of the control solution;
D S+T and adding dilution factors of the test samples in the standard solution to each test sample.
(3) The results are shown in Table 9.
TABLE 9 accuracy results
Acceptable standards: under each concentration, the recovery rate of the to-be-detected object is between 70.0 and 130.0 percent;
conclusion: under the condition that the LOQ of the limiting concentration is 100 percent and 150 percent, the single value range of the recovery rate of the 4-methyl-1-piperazine sulfonamide is 72.4 percent to 79.7 percent (between 70.0 percent and 130.0 percent) and meets the requirement; the method has good accuracy.
Example 2
And (5) measuring the residual content of chlorosulfonyl isocyanate in the cilastatin sodium intermediate.
1. Instrument, reagent, reference substance and test sample
Instrument: a liquid chromatograph-mass spectrometer, an analytical balance of one ten thousandth;
chromatographic column: agilent Proshell 120 EC-C18 (4.6 x 100mm,2.7 um);
flow rate: 0.8 ml/min
Reagent: acetonitrile (LCMS), ammonium formate (AR), 1-methylpiperazine (98%), dichloromethane (HPLC), ultrapure water (resistivity. Gtoreq.18.2M Ω. Cm);
control: chlorosulfonyl isocyanate;
test article: cilastatin sodium intermediate.
2. Solution preparation
Derivatizing reagent: taking about 1000 mg of 1-methylpiperazine, precisely weighing, putting into a 50ml volumetric flask, dissolving with acetonitrile, diluting to scale, and shaking to obtain the final product;
blank solution: transferring 100 μl of dichloromethane to a 25ml volumetric flask, adding a proper amount of derivatizing agent, shaking uniformly, adding 3.0ml of ultrapure water, diluting to scale with the derivatizing agent, shaking uniformly, and standing at room temperature for 10min to obtain the final product;
control stock solution 1: about 22mg of chlorosulfonyl isocyanate is taken, precisely weighed, put into a 50ml volumetric flask, diluted to scale by methylene dichloride and uniformly shaken to obtain the catalyst;
control stock solution 2: transferring 0.5ml of reference stock solution to a volumetric flask of 1-10 ml, diluting with dichloromethane to scale, and shaking;
control stock solution 3: transferring 1.0ml of reference stock solution to a volumetric flask of 2-10 ml, diluting with dichloromethane to scale, and shaking;
control solution: transferring 100 μl of reference stock solution to a volumetric flask of 3-25 ml, adding proper amount of derivatization agent, shaking, adding 3.0ml of ultrapure water, diluting to scale with derivatization agent, shaking, standing at room temperature for 10 min;
test solution: taking about 100mg of a sample, precisely weighing, adding 100 mu l of dichloromethane and a proper amount of derivatizing agent into a 25ml volumetric flask, shaking uniformly, adding 3.0ml of ultrapure water to dissolve the sample, diluting to a scale with the derivatizing agent, shaking uniformly, standing at room temperature for 10min, and preparing 2 parts in parallel.
3. Operation of
After the system is balanced, taking the chromatographic conditions described by the method for each solution, feeding a blank solution into 1 needle, feeding a control solution into 3 needles, feeding a test solution into 2 needles, and recording a chromatogram.
The content calculation formula:
in the formula ：Ai Measuring the peak area of the object to be measured in the solution of the object to be measured;
A S measuring the average value of the peak area of the object to be measured in the reference substance solution;
W T weighing the sample solution to be tested, and mg;
W S the mass of the measured substance is weighed for preparing a reference substance solution, and mg;
D T is the dilution multiple of the sample solution;
D S is the dilution multiple of the reference substance solution.
4. Detection result
TABLE 10 chlorosulfonyl isocyanate content detection results
5. Conclusion: the chlorosulfonyl isocyanate in the cilastatin sodium intermediate was not detected (limit 0.22ppm, limit 0.02 ppm).
Example 3
And (5) measuring the residual content of the chlorine sulfonyl isocyanate of the Para Mi Weizhong.
1. Instrument, reagent, reference substance and test sample
Instrument: a liquid chromatograph-mass spectrometer, an analytical balance of one ten thousandth;
chromatographic column: SHIMADZU Shim-pack GIST-HP C18-AQ (2.1. Times.100 mm,3 μm);
flow rate: 0.3 ml/min;
reagent: dimethyl sulfoxide (HPLC), acetonitrile, 1-methylpiperazine (99%);
control: chlorosulfonyl isocyanate.
Test article: peramivir.
2. Solution preparation
Derivatizing reagent: taking about 1250 to mg of 1-methylpiperazine, precisely weighing, putting into a 50ml volumetric flask, dissolving with DMSO, diluting to a scale, and shaking uniformly to obtain the final product;
blank solution: transferring 200 μl of dimethyl sulfoxide into 5ml EP tube, adding 200 μl of derivatizing agent, shaking, adding 0.6ml of ultrapure water, shaking, and standing at room temperature for 5 min;
control stock solution 1: taking about 20mg of chlorosulfonyl isocyanate, precisely weighing, putting into a 25ml volumetric flask, diluting to a scale with acetonitrile, and shaking uniformly to obtain the final product;
control stock solution 2: transferring 1.0ml of reference stock solution into a volumetric flask of 1-100 ml, diluting with acetonitrile to scale, and shaking;
control stock solution 3: transferring 1.0ml of reference stock solution into a volumetric flask of 1-50 ml, diluting with acetonitrile to scale, and shaking;
control solution: transferring 200 μl of control stock solution 3-5 ml EP tube, adding 200 μl of derivatizing agent, shaking, adding 0.6ml of ultrapure water, shaking, and standing at room temperature for 5 min;
test solution: taking about 200. 200mg of a test sample, precisely weighing, adding 200. Mu.l of acetonitrile into a 5ml EP tube, adding 200. Mu.l of a derivatization reagent, adding 0.6ml of ultrapure water, shaking uniformly, standing at room temperature for 5min, and preparing 2 parts in parallel.
3. Operation of
After the system is balanced, taking chromatographic conditions described by the method for each solution, feeding a blank solution into 1 needle, feeding a reference solution into 3 needles, feeding a test solution into 2 needles, recording a chromatogram, and calculating the content according to a peak area normalization method.
4. Detection result
TABLE 11 chlorosulfonyl isocyanate content detection results
5. Conclusion: no chlorosulfonyl isocyanate was detected in the peramivir sample (limit of 0.16 ppm, limit of detection of 0.015 ppm).
Comparative example 1
And (5) measuring the residual content of chlorosulfonyl isocyanate in the cefodizime sodium.
1. Instrument, reagent, reference substance and test sample
Instrument: a liquid chromatograph-mass spectrometer, an analytical balance of one ten thousandth;
reagent: dichloromethane (HPLC), acetonitrile (HPLC), N-diisopropylethylenediamine (AR);
control: chlorosulfonyl isocyanate.
Test article: cefditoren sodium.
2. Solution preparation
Derivatizing reagent: n, N-diisopropylethylenediamine: acetonitrile=1:100 (volume ratio);
blank solution: transferring 200 μl acetonitrile into 5ml EP tube, adding 200 μl derivatizing agent, shaking, adding 0.6ml ultrapure water, shaking, and standing at room temperature for 5 min;
control stock solution 1: about 20mg of chlorosulfonyl isocyanate is taken, precisely weighed, put into a 25ml volumetric flask, diluted to scale by methylene dichloride and uniformly shaken to obtain the finished product;
control stock solution 2: transferring 1.0ml of reference stock solution into a volumetric flask of 1-100 ml, diluting with acetonitrile to scale, and shaking;
control stock solution 3: transferring 1.0ml of reference stock solution into a volumetric flask with volume of 2-50 ml, diluting with acetonitrile to scale, and shaking;
control solution: transferring 200 μl of control stock solution 3-5 ml EP tube, adding 200 μl of derivatizing agent, shaking, adding 0.6ml of ultrapure water, shaking, and standing at room temperature for 5 min;
test solution: about 200mg of a sample to be tested is taken, precisely weighed, added into a 5ml EP tube, 200 mu l of acetonitrile is added, then 200 mu l of derivative reagent is added, shaking is carried out, then 0.9ml of ultrapure water is added, shaking is carried out, and standing is carried out for 5min at room temperature, thus obtaining the sample;
adding a standard solution to a test sample: about 200mg of the sample is taken, precisely weighed, added into a 5ml EP tube, 200 μl of reference stock solution 3 is added, then 200 μl of derivative reagent is added, shaking is carried out, then 0.9ml of ultrapure water is added, shaking is carried out, and standing is carried out at room temperature for 5min, thus obtaining the product.
3. Conditions of liquid phase Mass Spectrometry
4. Operation of
After the system is balanced, taking chromatographic conditions described by the method for each solution, feeding 1 needle for each of blank solution, reference solution, test solution and test standard adding solution, recording a chromatogram, and calculating the content according to a peak area normalization method.
5. Results
Chlorosulfonyl isocyanate reacts with N, N-diisopropylethylenediamine, 100% of the derivative product yields off standard (about 67%) and the sample shows false positive results. Thus, N, N-diisopropylethylenediamine is not suitable as a derivatizing reagent for detecting chlorosulfonyl isocyanate.
Comparative example 2
And (5) measuring the residual content of chlorosulfonyl isocyanate in the cefodizime sodium.
1. Instrument, reagent, reference substance and test sample
Instrument: a liquid chromatograph-mass spectrometer, an analytical balance of one ten thousandth;
reagent: acetonitrile (HPLC), ultrapure water;
control: chlorosulfonyl isocyanate.
Test article: cefditoren sodium.
2. Solution preparation
Control stock solution: taking about 50mg of chlorosulfonyl isocyanate, precisely weighing, putting into a 25ml volumetric flask, diluting to a scale with acetonitrile, and shaking uniformly to obtain the final product;
derivatization of the reaction solution: transferring 1.0ml of reference stock solution into a 5ml EP tube, adding 1.0ml of water, shaking uniformly, and standing at room temperature for 5 min.
3. Conditions of liquid phase Mass Spectrometry
4. Results:
the chlorosulfonyl isocyanate reacts with water, the response of the derivative product is too low to meet the requirement of the sensitivity of the method, and the response value of the derivative product ([ M-H ] -peak M/z 138.8) is only 180000 when the concentration of the chlorosulfonyl isocyanate is 1 mg/ml.
Comparative example 3
And (5) measuring the residual content of chlorosulfonyl isocyanate in the cefodizime sodium.
1. Instrument, reagent, reference substance and test sample
Instrument: a liquid chromatograph-mass spectrometer, an analytical balance of one ten thousandth;
reagent: acetonitrile (HPLC), 1-methylpiperidine (AR);
control: chlorosulfonyl isocyanate.
Test article: cefditoren sodium.
2. Solution preparation
Control stock solution: taking about 50mg of chlorosulfonyl isocyanate, precisely weighing, putting into a 25ml volumetric flask, diluting to a scale with acetonitrile, and shaking uniformly to obtain the final product;
derivatizing reagent: 1-methylpiperidine: acetonitrile=1:100 (volume ratio)
Derivatization of the reaction solution: transferring 1.0ml of reference stock solution into a 5ml EP tube, adding 1.0ml of derivatization reagent, shaking uniformly, and standing at room temperature for 5 min.
3. Conditions of liquid phase Mass Spectrometry
4. Results
The chlorosulfonyl isocyanate and the 1-methylpiperidine react more complicated, the derivative product is not single, and a target peak to be detected cannot be identified.
As can be seen from the experimental results of the above examples, the method for detecting chlorosulfonyl isocyanate by adopting the technical scheme of the invention has the advantages of simple and quick reaction, simple operation and high sensitivity in detecting chlorosulfonyl isocyanate.
The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.
Claims (7)
1. The method for detecting the derivatization of the chlorosulfonyl isocyanate is characterized by comprising the following steps of:
step S1, preparing a reference substance solution and a test substance solution;
the reference substance solution is prepared by the following steps: mixing a derivatizing reagent with a chlorosulfonyl isocyanate solution with a set content, and reacting at room temperature;
the sample solution is prepared by the following steps: mixing a derivative reagent with a test sample, and reacting at room temperature;
wherein the derivatization reagent is obtained by dissolving 1-methylpiperazine in an organic solvent;
s2, respectively analyzing a reference substance solution and a test substance solution by adopting a liquid chromatography-mass spectrometer, recording a chromatogram, and calculating the content of 4-methyl-1-piperazine sulfonamide according to a peak area external standard method by combining the content of chlorosulfonyl isocyanate in the reference substance solution so as to obtain the content of chlorosulfonyl isocyanate in the test substance;
in step S2, the conditions of the liquid chromatography-mass spectrometry are: c18 column, mobile phase a:10mM ammonium formate aqueous solution, mobile phase B: acetonitrile, gradient elution; in the gradient elution, 0-3 min, A (%): b (%) =97:3, 4-8min, a (%): b (%) =10:90;
in the liquid chromatography-mass spectrometry in the step S2, the parent ion is set to be m/z 180, the child ion is set to be m/z 101, and the collision energy is 10-20.
2. The method for detecting the derivatization of chlorosulfonyl isocyanate according to claim 1, wherein: in the step S1, the concentration of the 1-methylpiperazine in the derivatization reagent is 5-100mg/ml.
3. The method for detecting the derivatization of chlorosulfonyl isocyanate according to claim 2, wherein: the concentration of the 1-methylpiperazine is 20-50mg/ml.
4. The method for detecting the derivatization of chlorosulfonyl isocyanate according to claim 1, wherein: the organic solvent comprises dimethyl sulfoxide,N,NDimethylformamide (DMA),N,NDimethylacetamide,N-at least one of methyl pyrrolidone, acetonitrile.
5. The method for detecting the derivatization of chlorosulfonyl isocyanate according to claim 1, wherein: in the step S1, the content of chlorosulfonyl isocyanate in the sample is calculated by adopting the following formula:
，
wherein ,A i measuring the peak area of the 4-methyl-1-piperazine sulfonamide of the object to be measured in the solution of the object to be measured;
A S the average value of the peak area of the 4-methyl-1-piperazine sulfonamide of the object to be detected, which is measured in the reference substance solution;
W T the sample is the sample weighing quality of the sample liquid;
W S the mass of chlorosulfonyl isocyanate in the chlorosulfonyl isocyanate solution weighed for preparing the reference substance solution;
D T is the dilution multiple of the sample solution;
D S is the dilution multiple of the reference substance solution.
6. The method for detecting the derivatization of chlorosulfonyl isocyanate according to claim 1, wherein: in the step S2, the chromatographic column in the liquid chromatography-mass spectrometer is a C18-PFP, C18-AQ or EC-C18 chromatographic column.
7. The method for detecting the derivatization of chlorosulfonyl isocyanate according to claim 6, wherein: the conditions of the liquid chromatography-mass spectrometry in step S2 further include: the column temperature is 20-35 ℃, the flow rate is 0.2 ml/min-1 ml/min, and the sample injection amount is 1-5 mu L.
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