CN114235976A - Synthesis and analysis method of intermediate product of nitrogen-containing heterocyclic organic compound - Google Patents
Synthesis and analysis method of intermediate product of nitrogen-containing heterocyclic organic compound Download PDFInfo
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- CN114235976A CN114235976A CN202111319007.6A CN202111319007A CN114235976A CN 114235976 A CN114235976 A CN 114235976A CN 202111319007 A CN202111319007 A CN 202111319007A CN 114235976 A CN114235976 A CN 114235976A
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- 239000013067 intermediate product Substances 0.000 title claims abstract description 81
- -1 nitrogen-containing heterocyclic organic compound Chemical class 0.000 title claims abstract description 48
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 10
- 238000004458 analytical method Methods 0.000 title description 8
- 238000001308 synthesis method Methods 0.000 title description 2
- 238000000034 method Methods 0.000 claims abstract description 46
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 26
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 18
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 12
- 150000002894 organic compounds Chemical class 0.000 claims abstract description 8
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 8
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 7
- 238000000926 separation method Methods 0.000 claims abstract description 5
- 238000000746 purification Methods 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 108
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 87
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 48
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 26
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- 239000012535 impurity Substances 0.000 claims description 25
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 13
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- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 6
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- RTWNYYOXLSILQN-UHFFFAOYSA-N methanediamine Chemical compound NCN RTWNYYOXLSILQN-UHFFFAOYSA-N 0.000 claims description 5
- 238000010791 quenching Methods 0.000 claims description 5
- 230000000171 quenching effect Effects 0.000 claims description 5
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 claims description 5
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- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 4
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- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 claims description 3
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 3
- 125000002490 anilino group Chemical group [H]N(*)C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- GEYXPJBPASPPLI-UHFFFAOYSA-N manganese(III) oxide Inorganic materials O=[Mn]O[Mn]=O GEYXPJBPASPPLI-UHFFFAOYSA-N 0.000 claims description 3
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- 238000002386 leaching Methods 0.000 claims description 2
- TYTHZVVGVFAQHF-UHFFFAOYSA-N manganese(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[Mn+3].[Mn+3] TYTHZVVGVFAQHF-UHFFFAOYSA-N 0.000 claims description 2
- 238000002953 preparative HPLC Methods 0.000 claims description 2
- 239000003480 eluent Substances 0.000 claims 4
- 150000003254 radicals Chemical class 0.000 claims 4
- IICCLYANAQEHCI-UHFFFAOYSA-N 4,5,6,7-tetrachloro-3',6'-dihydroxy-2',4',5',7'-tetraiodospiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound O1C(=O)C(C(=C(Cl)C(Cl)=C2Cl)Cl)=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 IICCLYANAQEHCI-UHFFFAOYSA-N 0.000 claims 3
- 239000003642 reactive oxygen metabolite Substances 0.000 claims 3
- 229930187593 rose bengal Natural products 0.000 claims 3
- 229940081623 rose bengal Drugs 0.000 claims 3
- STRXNPAVPKGJQR-UHFFFAOYSA-N rose bengal A Natural products O1C(=O)C(C(=CC=C2Cl)Cl)=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 STRXNPAVPKGJQR-UHFFFAOYSA-N 0.000 claims 3
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 claims 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims 2
- 208000017983 photosensitivity disease Diseases 0.000 claims 2
- 231100000434 photosensitization Toxicity 0.000 claims 2
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- 239000001257 hydrogen Substances 0.000 claims 1
- 229910052739 hydrogen Inorganic materials 0.000 claims 1
- DALUDRGQOYMVLD-UHFFFAOYSA-N iron manganese Chemical compound [Mn].[Fe] DALUDRGQOYMVLD-UHFFFAOYSA-N 0.000 claims 1
- NQNOMXXYKHWVKR-UHFFFAOYSA-N methylazanium;sulfate Chemical compound NC.NC.OS(O)(=O)=O NQNOMXXYKHWVKR-UHFFFAOYSA-N 0.000 claims 1
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 claims 1
- OZKCXDPUSFUPRJ-UHFFFAOYSA-N oxobismuth;hydrobromide Chemical compound Br.[Bi]=O OZKCXDPUSFUPRJ-UHFFFAOYSA-N 0.000 claims 1
- JRKICGRDRMAZLK-UHFFFAOYSA-L peroxydisulfate Chemical class [O-]S(=O)(=O)OOS([O-])(=O)=O JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 claims 1
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- 231100000820 toxicity test Toxicity 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 55
- FFGPTBGBLSHEPO-UHFFFAOYSA-N carbamazepine Chemical compound C1=CC2=CC=CC=C2N(C(=O)N)C2=CC=CC=C21 FFGPTBGBLSHEPO-UHFFFAOYSA-N 0.000 description 15
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- 239000000543 intermediate Substances 0.000 description 14
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- JLKIGFTWXXRPMT-UHFFFAOYSA-N sulphamethoxazole Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 JLKIGFTWXXRPMT-UHFFFAOYSA-N 0.000 description 14
- PJSFRIWCGOHTNF-UHFFFAOYSA-N Sulphormetoxin Chemical compound COC1=NC=NC(NS(=O)(=O)C=2C=CC(N)=CC=2)=C1OC PJSFRIWCGOHTNF-UHFFFAOYSA-N 0.000 description 7
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- 150000002391 heterocyclic compounds Chemical class 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/08—Preparation using an enricher
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The invention discloses a method for oriented synthesis, separation and purification of a nitrogenous heterocyclic organic compound intermediate product and quantitative analysis of the nitrogenous heterocyclic organic compound intermediate product. The patent innovatively provides a technical method for synthesizing and purifying the intermediate product of the organic compound containing the nitrogen heterocycle, the method can analyze and identify the intermediate product, and has important significance for carrying out toxicity experiments on the intermediate product of the organic compound containing the nitrogen heterocycle in a laboratory.
Description
Technical Field
The invention relates to the technical field of synthesis and analysis of organic compounds, in particular to a method for quantitatively synthesizing, separating and purifying a nitrogenous heterocyclic ring organic compound advanced oxidation intermediate product and realizing quantitative analysis of the nitrogenous heterocyclic ring organic compound advanced oxidation intermediate product.
Background
In recent years, nitrogen-containing heterocyclic compounds have unique biological activity, are often used as structural building blocks of medicines and pesticides, and are increasingly widely applied to other fields such as dye-sensitized solar cells. The nitrogenous heterocyclic compound is difficult to be absorbed by the body and is often discharged out of the body along with excrement and urine, so that a series of environmental behaviors occur and environmental pollution is caused. Nitrogen-containing heterocyclic compounds generally have a greater polarity and a lower n-octanol/water partition coefficient (K)ow) The water-soluble organic fertilizer has relatively high solubility, is not easy to be adsorbed by soil, is easy to exist in a water phase and is widely existed in industrial wastewater such as chemical wastewater, pharmaceutical wastewater, pesticide wastewater and the like. Novel micro-pollutants of water represented by carbamazepine have low environmental concentration but have remarkable biotoxicity of a body and a conversion product, can be exposed to human bodies and aquatic and terrestrial organisms for a long time, and poses serious threats to human health and ecological environment. Advanced oxidation technologies (AOPs) are produced by combining electricity, light radiation, catalysts and the like with common chemical oxidants in an oxidation reaction to generate high-activity free radicals which react with organic matters and are oxidized and decomposed into small molecules until the small molecules are degraded into CO2、H2O and inorganic salts. In the process, the advanced oxidation technology can remove most of the oxygenNitrogen-containing heterocyclic compounds are preferred, but their degradation profile merely illustrates the efficiency of the treatment of the contaminant material itself by the technique, whereas decomposition or elimination of the parent compound is not equivalent to toxic removal. Since the contaminants may form intermediates during degradation, they exhibit similar or even higher toxicity to the organism than the parent compound. And most intermediate products converted from the nitrogen heterocyclic ring organic compound lack a standard product, and toxicity tests and researches on the intermediate products cannot be carried out in a laboratory, so that the toxicity of the intermediate products is not clear, and the harm of the intermediate products to human and environment cannot be determined. Therefore, it is important to find a method for preparing a standard product by directionally synthesizing and purifying the intermediate product of the nitrogenous heterocyclic organic compound with strong pertinence.
The HPLC-QTOF-MS combined technology integrates the high-efficiency separation of HPLC and the high sensitivity, high specificity and high resolution of Q-TOF-MS, has obvious advantages in qualitative and quantitative analysis, better meets the requirements of modern organic compound research on an automatic and high-throughput analysis method, and becomes one of the strongest powerful analysis tools for organic compound impurity structure research. The Density Functional Theory (DFT) is a quantum chemical calculation method, can explore the specific molecular structure of a substance and predict the reactive site thereof, and has the advantages of convenient calculation, high analysis speed and the like. The specific calculation method comprises the following steps: a Fowell function, front line orbit theory, average local ionization energy, dual descriptors, electrostatic potential, etc. The structure of the intermediate product can be more accurately presumed by combining the characteristics of the two.
The semi-preparative high performance liquid chromatography has the characteristics of simplicity, practicability, economy, rapidness, easiness in amplification and the like, and is widely applied to preparation of various substance monomer standard products. HPLC-MS/MS can exclude mutual interference of chemical components with similar properties in a complex matrix, and is particularly suitable for the qualitative and quantitative determination of complex samples. And the mass spectrum detection has high sensitivity, is not limited by no chromophore of the compound, and is more suitable for measuring low-quantity components in the sample.
Disclosure of Invention
The present invention is directed to a method for synthesizing and analyzing an intermediate product of a nitrogen-containing heterocyclic organic compound, which solves the above-mentioned problems of the prior art.
In order to achieve the above purpose, the invention provides the following technical scheme:
a method for synthesizing and analyzing an intermediate product of a nitrogen-containing heterocyclic organic compound is characterized by comprising the following steps:
(1) pretreatment of the sample: mashing a sample, placing the sample into a 50mL glass centrifuge tube, adding an acetonitrile/dichloromethane mixed solution, adding anhydrous sodium sulfate after ultrasonic treatment, homogenizing the sample by a high-speed electric homogenizer, mixing the mixture by vortex, centrifuging the mixture, and carefully transferring an acetonitrile/diaminomethane layer into a 100mL chicken heart bottle. And adding the mixed acetonitrile/dichloromethane solution into the original centrifuge tube. Repeating the operation for 1-2 times, combining the two extracting solutions, and concentrating in a water bath under reduced pressure until the extracting solution is dry. And then respectively adding methanol and formic acid, uniformly mixing by vortex, transferring into a 50mL glass centrifuge tube after ultrasonic treatment, repeating the operation for 1-2 times, and combining into the centrifuge tube. And adding n-hexane, centrifuging, discarding the n-hexane layer on the upper layer, repeating the operation for 1-2 times, and adding water to dilute the lower layer. Respectively activating the solid phase extraction column with methanol and ultrapure water, adding a sample diluent, adding water and methanol for leaching, and finally eluting with methanol. Concentrating the eluate under reduced pressure to dryness, diluting with mobile phase, filtering with organic phase filter membrane, and injecting sample.
(2) Intermediate recognition of nitrogen-containing heterocyclic organic compounds: firstly, analyzing and identifying the yield change trend of an intermediate product in a target sample by using an HPLC-QTOF-MS high performance liquid chromatography time-of-flight mass spectrometer-mass spectrometer. Meanwhile, the electron cloud density of the parent compound is analyzed through a Density Functional Theory (DFT) functional, and an intermediate product possibly generated in the preliminary reaction is predicted. And then predicting the structure of the intermediate product through an HPLC-QTOF-MS secondary mass spectrum structure, and preliminarily confirming the structure and chromatographic parameters of the potential intermediate product.
(2) Intermediate synthesis of nitrogen heterocyclic organic compound: sulfate radical (SO) is generated through a designed oxidation reaction system4-), hydroxyl radical (. OH), superoxide radical (. O)2 -) And singlet oxygen: (1O2) By using the activityOxygen (ROS) quenching/capturing experiments obtain needed ROS, attack the parent body of the target nitrogen heterocyclic ring organic compound, and directionally generate a large amount of target intermediate products.
(3) And (3) separating and purifying the intermediate product of the nitrogen-containing heterocyclic organic compound: firstly, enriching and concentrating an intermediate product in a target sample by a solid-phase extraction technology, then generating a certain amount of pure target intermediate product by liquid chromatography semi-preparation, mixing a certain organic solvent, and blowing off the organic solvent by nitrogen blowing to obtain the pure target intermediate product.
(4) And (3) sample purity inspection: performing purity inspection by using HPLC-QTOF-MS, and judging whether the chromatographic peak has chloride ions, sulfate ions, heavy metals and other miscellaneous peaks; the material content of the hetero-peak, i.e. the impurity content, was quantified by known hetero-peak standards. The quality standard of control needs to be lower than 1 percent, and finally the separated and purified sample of the intermediate product of the nitrogenous heterocyclic organic compound meets the purity requirement.
(5) Quantification of intermediate products of heterocyclic organic compounds containing nitrogen: by using the prepared intermediate product standard of the nitrogen-containing heterocyclic organic compound, a standard curve can be measured by HPLC-MS/MS, and quantitative analysis of the intermediate product, the conversion product or the by-product in a target sample is realized.
Compared with the prior art, the invention has the beneficial effects that:
the patent innovatively provides a technical method for synthesizing and purifying the intermediate product of the organic compound containing the nitrogen heterocycle, the method can analyze and identify the intermediate product, and has important significance for carrying out toxicity experiments on the intermediate product of the organic compound containing the nitrogen heterocycle in a laboratory.
Drawings
FIG. 1 is a flow chart of a method of the present invention;
FIG. 2 is a table of charge profiles and Fullfun values for carbamazepine of the present invention;
FIG. 3 is a secondary mass spectrum of molecules and fragment ions of TP211 of the present invention;
FIG. 4 is a schematic diagram of the main intermediate products of the present invention after advanced oxidation of carbamazepine;
FIG. 5 is a diagram of a structure of an intermediate product to be synthesized according to the present invention;
FIG. 6 is a structural diagram of the main intermediate after the sulfamethoxazole is subjected to advanced oxidation;
FIG. 7 is a structural diagram of the main intermediate product after the sulfamethoxazole is subjected to the advanced oxidation.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
The invention provides a technical scheme that: the synthesis and analysis of intermediates of Carbamazepine (CBZ) are exemplified. The method comprises the following steps:
(1) pretreatment of the sample: smashing a sample, weighing 6g of the smashed sample, placing the smashed sample in a 50mL glass centrifuge tube, adding 30mL of acetonitrile/dichloromethane mixed solution (the volume ratio of acetonitrile to dichloromethane is 96: 5), carrying out ultrasonic treatment for 3min, adding 6g of anhydrous sodium sulfate, carrying out high-speed electric homogenizer at 12000r/min for homogenizing for 2min, carrying out vortex mixing for 1.5min, centrifuging at 4000r/min for 10min, and carefully transferring the acetonitrile/diaminomethane layer to a 100mL heart-shaped bottle. Then 30mL of the acetonitrile/dichloromethane mixed solution was added to the original centrifuge tube. Repeating the above operation for 1 time, mixing the two extractive solutions, and concentrating under reduced pressure in 55 deg.C water bath to dry. Then adding 1.5mL of methanol and 2.5mL of 1% (volume fraction, the same below) of formic acid respectively, mixing by vortex, transferring into a 50mL glass centrifuge tube after 3min of ultrasonic treatment, repeating the above operation for 1 time, and combining into the centrifuge tube. Then 10mL of n-hexane is added, the mixture is centrifuged at 3000r/min for 6min, the upper n-hexane layer is discarded, the operation is repeated for 1 time, and 7mL of water is added to the lower layer for dilution. The solid phase extraction column was activated with 4mL of methanol and 5mL of ultrapure water, followed by addition of the sample diluent, followed by addition of 5mL of water and 2mL of 5% (volume fraction) methanol for rinsing, and finally eluted with 10mL of methanol. The eluate was concentrated to dryness at 55 ℃ under reduced pressure, and the volume was determined by using 1mL of a mobile phase (methanol: 0.1% formic acid: 3: 7, volume ratio), and the sample was introduced after being filtered through a 0.22 μm organic phase filter.
(2) Identification of the carbamazepine intermediate: firstly, setting a mobile phase of acetonitrile/0.1% formic acid water as 20:80(v/v) and a sample injection volume of 15 mu L by using HPLC-QTOF-MS (high performance liquid chromatography time of flight mass spectrometry-mass spectrometer); performing mass spectrometry in a positive ion mode, wherein the scanning range is 50-500 m/z, the ion voltage is 40V, and the collision energy is set to be 50V; the temperature of the drying gas is 350 ℃, and the flow rate of the drying gas is 5.5L/min; the pressure of the atomization gas is 350 kPa; the temperature of the sheath gas is 360 ℃, and the flow rate of the sheath gas is 15L/min; capillary voltage 4200V, cone hole voltage 60V and cleavage voltage 150V. Density Functional Theory (DFT) functional analysis of the electron cloud density of carbamazepine: and establishing an optimized molecular model through Gauss View, and analyzing the molecules by Gaussian software. Then, a possible product structure is obtained through HPLC-QTOF-MS secondary mass spectrum structure prediction, and the structure and chromatographic parameters of potential intermediate products are preliminarily confirmed. FIG. 2 shows the results of analysis of a portion of the intermediates:
(3) synthesis of a carbamazepine intermediate product: PMS (Peronosulfate)/Fe by design0The (zero-valent iron) oxidation reaction system directionally generates hydroxyl free radicals (. OH); mn2O3The (manganese sesquioxide)/PMS oxidation reaction system generates sulfate radicals. ROS are purified by using a reactive oxygen quenching/capturing experiment, nitrogen-containing heterocycles of carbamazepine are attacked, and a large amount of target intermediate products are directionally generated. The main intermediate products after advanced oxidation comprise the following structures:
(4) separating and purifying a carbamazepine intermediate product: samples were extracted using a 500mg solid phase extraction column. Taking a certain volume of sample, filtering with qualitative filter paper, and then filtering with a 0.45-micron water system filter membrane; washing the solid phase extraction column twice respectively by using 5mL of methanol and 5mL of ultrapure water; the sample was then injected into the solid phase extraction column through a Teflon tube at a flow rate of 5 mL/min. The extraction column was drained and dried for 2.5h, then the column was eluted with 6mL of methanol at a flow rate of 2mL/min, and the eluate was collected in a glass tube and concentrated to 1mL using a nitrogen blower.
(5) And (3) sample purity inspection: performing purity inspection by HPLC-QTOF-MS, preparing control solutions of different impurity standard substances (chloride ions, sulfate ions, heavy metals and the like), and obtaining an impurity peak spectrogram (secondary mass spectrogram) by HPLC-QTOF-MS; obtaining a mass spectrogram of the sample through HPLC-QTOF-MS, and comparing the mass spectrogram with a mass spectrogram obtained from a reference solution to determine impurities; and calculating the content of the impurities according to the related parameters of the impurity peaks, and further estimating the purity of the sample. The quality standard of control needs to be lower than 1 percent, and the separated and purified sample of the carbamazepine intermediate product is finally judged to meet the purity requirement.
(6) Quantification of carbamazepine intermediate standards: using the prepared standard of the carbamazepine intermediate product, a standard curve can be measured by HPLC-MS/MS, the relevant conditions for HPLC-MS/MS being: the chromatographic column is C18(5 μm)250mm × 4.6mm, the mobile phase is acetonitrile and 5mmol/L ammonium formate aqueous solution with mass fraction of 0.1% formic acid, the volume flow rate is 0.6mL/min, the column temperature is set at 30 ℃, and the sample injection amount is 5 μ L. Realize the intermediate product and conversion of the carbamazepine in the target sample
The chemical products or by-products were quantitatively analyzed. Taking carbamazepine as an example, the following structures can be isolated by the above method:
example two
The invention provides a technical scheme that: taking Sulfamethoxazole (SMX) as an example, a method for synthesizing and analyzing an intermediate product of the sulfamethoxazole. The method comprises the following steps:
(1) pretreatment of the sample: smashing a sample, weighing 10g of the smashed sample, placing the smashed sample in a 50mL glass centrifuge tube, then adding 25mL of acetonitrile/dichloromethane mixed solution (the volume ratio of acetonitrile to dichloromethane is 100: 5), adding 8g of anhydrous sodium sulfate after ultrasonic treatment for 2min, homogenizing the sample for 1.5min by a high-speed electric homogenizer at 10000r/min, vortex mixing the mixture for 1.5min, centrifuging the mixture for 8min at 3000r/min, and carefully transferring an acetonitrile/diaminomethane layer to a 100mL heart-shaped bottle. Then 25mL of the acetonitrile/dichloromethane mixed solution was added to the original centrifuge tube. Repeating the above operation for 2 times, mixing the two extractive solutions, and concentrating under reduced pressure in 55 deg.C water bath to dry. Then adding 1mL of methanol and 2mL of 1% (volume fraction, the same below) formic acid respectively, mixing by vortex, transferring into a 50mL glass centrifuge tube after performing ultrasonic treatment for 3min, repeating the operation for 2 times, and combining into the centrifuge tube. Then 10mL of normal hexane is added, the mixture is centrifuged at 3000r/min for 6min, the upper normal hexane layer is discarded, the operation is repeated for 2 times, and 7mL of water is added to the lower layer for dilution. The solid phase extraction column was activated with 4mL of methanol and 5mL of ultrapure water, followed by addition of the sample diluent, followed by addition of 5mL of water and 2mL of 5% (volume fraction) methanol for rinsing, and finally eluted with 10mL of methanol. The eluate was concentrated to dryness at 55 ℃ under reduced pressure, and the volume was determined by using 1mL of a mobile phase (methanol: 0.1% formic acid: 3: 7, volume ratio), and the sample was introduced after being filtered through a 0.22 μm organic phase filter.
(2) Sulfamethoxazole intermediate recognition: firstly, setting a mobile phase of acetonitrile/0.1% formic acid water as 20:80(v/v) and a sample injection volume of 10 mu L by using HPLC-QTOF-MS (high performance liquid chromatography time of flight mass spectrometry-mass spectrometer); performing mass spectrometry in a positive ion mode, wherein the scanning range is 50-500 m/z, the ion voltage is 35V, and the collision energy is set to be 45V; the temperature of the drying gas is 350 ℃, and the flow rate of the drying gas is 5.5L/min; the pressure of the atomization gas is 350 kPa; the temperature of the sheath gas is 360 ℃, and the flow rate of the sheath gas is 15L/min; capillary voltage 3800V, cone hole voltage 65V, lysis voltage 150V. Density Functional Theory (DFT) functional analysis of electron cloud density of sulfamethoxazole: and establishing an optimized molecular model through Gauss View, and analyzing the molecules by Gaussian software. Then, a possible product structure is obtained through HPLC-QTOF-MS secondary mass spectrum structure prediction, and the structure and chromatographic parameters of potential intermediate products are preliminarily confirmed.
(3) Sulfamethoxazole intermediate synthesis: UV/TiO by design2(titanium dioxide) the oxidation reaction system directionally generates hydroxyl radicals (. OH); TiO 22The photocatalytic system generates singlet oxygen radicals. And (3) purifying ROS by using an active oxygen quenching/capturing experiment, attacking nitrogenous heterocyclic ring and aniline groups of sulfamethoxazole, and directionally generating a large amount of target intermediate products.
(4) And (3) separating and purifying sulfamethoxazole intermediate: samples were extracted using a 500mg solid phase extraction column. Taking a certain volume of sample, filtering with qualitative filter paper, and then filtering with a 0.45-micron water system filter membrane; washing the solid phase extraction column twice respectively by using 5mL of methanol and 5mL of ultrapure water; the sample was then injected into the solid phase extraction column through a Teflon tube at a flow rate of 3 mL/min. The extraction column was drained and dried for 1.5h, then the column was eluted with 6mL of methanol at a flow rate of 1mL/min, and the eluate was collected in a glass tube and concentrated to 1mL using a nitrogen blower.
(5) And (3) sample purity inspection: performing purity inspection by HPLC-QTOF-MS, preparing control solutions of different impurity standard substances (chloride ions, sulfate ions, heavy metals and the like), and obtaining an impurity peak spectrogram (secondary mass spectrogram) by HPLC-QTOF-MS; obtaining a mass spectrogram of the sample through HPLC-QTOF-MS, and comparing the mass spectrogram with a mass spectrogram obtained from a reference solution to determine impurities; and calculating the content of the impurities according to the related parameters of the impurity peaks, and further estimating the purity of the sample. The quality standard of control needs to be lower than 1 percent, and finally the sulfamethoxazole intermediate product separation and purification sample is judged to meet the purity requirement.
(6) Quantifying a sulfamethoxazole intermediate product standard product: using the prepared sulfamethoxazole intermediate standard, a standard curve can be measured by HPLC-MS/MS, and relevant conditions of HPLC-MS/MS are as follows: the chromatographic column is C18(5 μm)250mm × 4.6mm, the mobile phase is acetonitrile and 5mmol/L ammonium formate aqueous solution with mass fraction of 0.1% formic acid, the volume flow rate is 0.6mL/min, the column temperature is set to 30 ℃, and the sample injection amount is 7 μ L. Quantitative analysis on the intermediate product, the conversion product or the by-product of sulfamethoxazole in the target sample is realized.
EXAMPLE III
The invention provides a technical scheme that: taking Sulfamethazine (SIM) as an example, a method for synthesizing and analyzing an intermediate product of the sulfamethazine. The method comprises the following steps:
(1) pretreatment of the sample: the method comprises the following steps of mashing a sample, weighing 7g of the sample, placing the sample in a 50mL glass centrifuge tube, adding 25mL of acetonitrile/dichloromethane mixed solution (the volume ratio of acetonitrile to dichloromethane is 100: 5), carrying out ultrasonic treatment for 2.5min, adding 9g of anhydrous sodium sulfate, homogenizing by a high-speed electric homogenizer of 13000r/min for 1.5min, carrying out vortex mixing for 1.5min, centrifuging by 3500r/min for 9min, and carefully transferring an acetonitrile/diaminomethane layer to a 100mL heart-shaped bottle. Then 30mL of the acetonitrile/dichloromethane mixed solution was added to the original centrifuge tube. Repeating the above operation for 2 times, mixing the two extractive solutions, and concentrating under reduced pressure in 52 deg.C water bath to dry. Then adding 1.25mL of methanol and 2.25mL of 1% (volume fraction, the same below) formic acid respectively, mixing by vortex, transferring to a 50mL glass centrifuge tube after 2.5min of ultrasonic treatment, repeating the above operation for 1 time, and combining into the centrifuge tube. Adding 8.5mL of n-hexane, centrifuging at 3000r/min for 6min, discarding the upper n-hexane layer, repeating the above operation for 2 times, and adding 7mL of water to the lower layer for dilution. The solid phase extraction column was activated with 4mL of methanol and 5mL of ultrapure water, followed by addition of the sample diluent, followed by addition of 5mL of water and 2mL of 5% (volume fraction) methanol for rinsing, and finally eluted with 10mL of methanol. The eluate was concentrated to dryness at 55 ℃ under reduced pressure, and the volume was determined by using 1mL of a mobile phase (methanol: 0.1% formic acid: 3: 7, volume ratio), and the sample was introduced after being filtered through a 0.22 μm organic phase filter.
(2) Identification of sulfadoxine intermediate: firstly, setting a mobile phase of acetonitrile/0.1% formic acid water as 20:80(v/v) and a sample injection volume of 15 mu L by using HPLC-QTOF-MS (high performance liquid chromatography time of flight mass spectrometry-mass spectrometer); performing mass spectrometry in a positive ion mode, wherein the scanning range is 50-500 m/z, the ion voltage is 40V, and the collision energy is set to be 50V; the temperature of the drying gas is 350 ℃, and the flow rate of the drying gas is 4.5L/min; the atomization gas pressure is 330 kPa; the temperature of the sheath gas is 350 ℃, and the flow rate of the sheath gas is 10L/min; capillary voltage 4000V, cone hole voltage 70V and lysis voltage 130V. Density Functional Theory (DFT) functional analysis of electron cloud density of sulfadoxine: and establishing an optimized molecular model through Gauss View, and analyzing the molecules by Gaussian software. Then, a possible product structure is obtained through HPLC-QTOF-MS secondary mass spectrum structure prediction, and the structure and chromatographic parameters of potential intermediate products are preliminarily confirmed.
(3) Synthesis of sulfasolmidine intermediate: UV/TiO by design2(titanium dioxide) the oxidation reaction system directionally generates hydroxyl radicals (. OH); TiO 22The photocatalytic system generates singlet oxygen radicals. And (3) purifying ROS by using an active oxygen quenching/capturing experiment, attacking nitrogenous heterocycle and aniline groups of the sulfadiazine, and directionally generating a large amount of target intermediate products.
(4) And (3) separating and purifying a sulfadoxine intermediate product: samples were extracted using a 500mg solid phase extraction column. Taking a certain volume of sample, filtering with qualitative filter paper, and then filtering with a 0.45-micron water system filter membrane; washing the solid phase extraction column twice respectively by using 5mL of methanol and 5mL of ultrapure water; the sample was then injected into the solid phase extraction column through a Teflon tube at a flow rate of 4 mL/min. The extraction column was drained and dried for 1.5h, then the column was eluted with 6mL of methanol at a flow rate of 1.5mL/min, and the eluate was collected in a glass tube and concentrated to 1mL using a nitrogen blower.
(5) And (3) sample purity inspection: performing purity inspection by HPLC-QTOF-MS, preparing control solutions of different impurity standard substances (chloride ions, sulfate ions, heavy metals and the like), and obtaining an impurity peak spectrogram (secondary mass spectrogram) by HPLC-QTOF-MS; obtaining a mass spectrogram of the sample through HPLC-QTOF-MS, and comparing the mass spectrogram with a mass spectrogram obtained from a reference solution to determine impurities; and calculating the content of the impurities according to the related parameters of the impurity peaks, and further estimating the purity of the sample. The quality standard of control needs to be lower than 1 percent, and the separated and purified sample of the sulfadoxine intermediate product is finally judged to meet the purity requirement.
(6) Quantifying a sulfadoxine intermediate product standard: using the prepared standard substance of the sulfadoxine intermediate product, a standard curve can be measured by HPLC-MS/MS under the relevant conditions that a chromatographic column is C18(5 μm)250mm multiplied by 4.6mm, a mobile phase is acetonitrile and 5mmol/L ammonium formate aqueous solution with the mass fraction of 0.1% formic acid, the volume flow is 1.5mL/min, the column temperature is set to 40 ℃, and the sample injection amount is 10 μ L. Quantitative analysis on the intermediate product, the conversion product or the by-product of the sulfadoxine in the target sample is realized.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A method for synthesizing and analyzing an intermediate product of a nitrogen-containing heterocyclic organic compound is characterized by comprising the following steps:
(1) pretreatment of the sample: smashing a sample, placing the smashed sample in a 50mL glass centrifuge tube, adding an acetonitrile/dichloromethane mixed solution, adding anhydrous sodium sulfate after ultrasonic treatment, homogenizing the sample by a high-speed electric homogenizer, mixing the mixture by vortex, centrifuging the mixture, and carefully transferring an acetonitrile/diaminomethane layer to a 100mL chicken heart bottle; adding an acetonitrile/dichloromethane mixed solution into an original centrifuge tube, repeating the operation for 1-2 times, combining extracting solutions obtained in two times, concentrating the extracting solutions in a water bath under reduced pressure until the extracting solutions are dry, respectively adding methanol and formic acid, uniformly mixing the extracting solutions in a vortex mode, transferring the obtained mixture into a 50mL glass centrifuge tube after ultrasonic treatment, repeating the operation for 1-2 times, combining the extracting solutions into the centrifuge tube, adding n-hexane, centrifuging the obtained product, discarding an upper n-hexane layer, repeating the operation for 1-2 times, adding water to dilute a lower layer, respectively activating a solid phase extraction column with methanol and ultrapure water, adding a sample diluent, then adding water and methanol to leach, finally eluting with methanol, concentrating an eluent under reduced pressure until the obtained product is dry, fixing the volume with a mobile phase, filtering the obtained product with an organic phase filter membrane, and sampling;
(2) intermediate recognition of nitrogen-containing heterocyclic organic compounds: firstly, analyzing and identifying the yield change trend of an intermediate product in a target sample by using an HPLC-QTOF-MS high performance liquid chromatography time-of-flight mass spectrometer-mass spectrometer, meanwhile, analyzing the electron cloud density of a parent compound through a Density Functional Theory (DFT) functional, predicting the intermediate product possibly generated by a preliminary reaction, then predicting the structure of the intermediate product through an HPLC-QTOF-MS secondary mass spectrum structure, and preliminarily determining the structure and chromatographic parameters of the potential intermediate product;
(3) intermediate synthesis of nitrogen heterocyclic organic compound: sulfate radical (SO) is generated through a designed oxidation reaction system4-), hydroxyl radical (. OH), superoxideFree radical (. O)2-) and singlet oxygen ((ii)1O2) Quenching/capturing experiments by using Reactive Oxygen Species (ROS) to obtain the needed ROS, attacking a target nitrogenous heterocyclic organic compound parent and directionally generating a large amount of target intermediate products;
(4) and (3) separating and purifying the intermediate product of the nitrogen-containing heterocyclic organic compound: firstly, enriching and concentrating an intermediate product in a degraded sample by a solid-phase extraction technology, then generating a certain amount of pure target intermediate product by semi-preparative high performance liquid chromatography, mixing a certain amount of organic solvent, and then blowing off the organic solvent by nitrogen blowing to obtain the pure target intermediate product;
(5) and (3) sample purity inspection: performing purity inspection by using HPLC-QTOF-MS, and judging whether the chromatographic peak has chloride ions, sulfate ions, heavy metals and other miscellaneous peaks; the method comprises the following steps of quantifying the substance content of the miscellaneous peak, namely the impurity content, by using a known miscellaneous peak standard product, and finally judging that a separation and purification sample of the intermediate product of the organic compound containing the nitrogen heterocyclic ring meets the purity requirement when the quality standard of control needs to be lower than 1%;
(6) quantification of intermediate products of heterocyclic organic compounds containing nitrogen: by using the prepared intermediate product standard of the nitrogenous heterocyclic organic compound, a concentration standard curve can be established by HPLC-MS/MS, and quantitative analysis of the intermediate product, the conversion product or the byproduct in a target sample is realized.
2. The method according to claim 1, wherein the method comprises the steps of: the pretreatment method of the sample in the step (1) comprises the following steps of mashing the sample and weighing 5-10 g of the sample; the volume of the acetonitrile/dichloromethane mixed solution is 25-30 mL, and the volume ratio of acetonitrile to dichloromethane is 95-100: 5; then, carrying out ultrasonic treatment for 2-3 min, and adding 5-10 g of anhydrous sodium sulfate; homogenizing for 1.5-2 min by a high-speed electric homogenizer at 10000-15000 r/min; vortex mixing for 1-1.5 min, and centrifuging for 8-10 min at 3000-4000 r/min; adding 25-30 mL of acetonitrile/dichloromethane mixed solution into the original centrifuge tube, and concentrating in a water bath at 50-55 ℃ under reduced pressure until the solution is dry; and then adding 1-1.5 mL of methanol and 2-2.5 mL of 1% (volume fraction, the same below) formic acid respectively, mixing uniformly by vortex, performing ultrasonic treatment for 2-3 min, transferring into a 50mL glass centrifuge tube, repeating the operation for 1-2 times, merging into the centrifuge tube, adding 8-10 mL of n-hexane, centrifuging for 6min at 3000r/min, discarding the upper n-hexane layer, repeating the operation for 1-2 times, adding 7mL of water into the lower layer, respectively activating the HLB solid-phase extraction column by 4mL of methanol and 5mL of ultrapure water, adding a sample diluent, adding 5mL of water and 2mL of 5% (volume fraction) of methanol for leaching, finally eluting the eluent by 10mL of methanol, concentrating at 55 ℃ under reduced pressure to dryness, adding 1mL of mobile phase (methanol: 0.1% formic acid: 3: 7, volume ratio) to fix volume, injecting a sample through a 0.22 mu m organic phase filter membrane, and filtering.
3. The method according to claim 1, wherein the method comprises the steps of: the identification method of the intermediate product of the nitrogenous heterocyclic organic compound in the step (2) is characterized in that relevant conditions of the HPLC-QTOF-MS high performance liquid chromatography time-of-flight mass spectrometer are set to be that the mobile phase is acetonitrile/0.1% formic acid water (20: 80(v/v), and the sample injection volume is 10-15 mu L; the mass spectrometry is carried out in a positive ion mode, the scanning range is 50-500 m/z, the ion voltage is 30-50V, and the collision energy is set to be 45-60V; the temperature of the drying gas is 300-350 ℃, and the flow rate of the drying gas is 3.5-5.5L/min; the atomizing gas pressure is 300-350 kPa; the temperature of the sheath gas is 320-360 ℃, and the flow rate of the sheath gas is 6-15L/min; the capillary voltage is 3800-4200V, the taper hole voltage is 60-70V, and the cracking voltage is 120-150V.
4. The method according to claim 1, wherein the method comprises the steps of: and (2) identifying intermediate products of the nitrogenous heterocyclic organic compound, wherein the Density Functional Theory (DFT) functional analysis is to analyze the optimized molecular model by using software such as Gaussian, VASP, CASEP and the like.
5. The method according to claim 1, wherein the method comprises the steps of: synthesizing the intermediate product of the nitrogenous heterocyclic organic compound in the step (3), wherein the designed oxidation reaction system is PMS (Per-type methyl ammonium sulfate)sulfate)/Fe0(zero-valent iron) system, FMBO (iron manganese binary oxide)/H2O2/O3System and UV/TiO2(titanium dioxide) systems can generate hydroxyl radicals (. OH) in a directed manner; designed TAP (thermally activated persulfate) System, Mn2O3The (manganese sesquioxide)/PMS system and the BiOBr (bismuth oxybromide)/PMS system can directionally generate sulfate radical (SO)4· -); designed RB (Rose Bengal) photosensitization system and O3/H2O2The oxidation system can directionally generate singlet oxygen free radicals (1O2) (ii) a Designed TiO2The photocatalytic system can directionally generate superoxide radical (O)2-)。
6. The method according to claim 1, wherein the method comprises the steps of: synthesizing an intermediate product of the nitrogenous heterocyclic organic compound in the step (3), wherein the free radical directional attack site: PMS (Peronosulfate)/Fe0The hydroxyl free radical generated by the (zero-valent iron) system directionally attacks the nitrogen-containing heterocycle in the target object; FMBO/H2O2/O3Hydroxyl free radicals generated by the system directionally attack benzene rings in a target object or amino groups on aniline; UV/TiO2Hydroxyl radicals generated by the system directionally attack carbon-carbon double bonds or high-activity carbon-hydrogen bonds in the target object, and sulfate radicals generated by the TAP system directionally attack olefin double bonds in the target object; mn2O3Sulfate radicals generated by a PMS system directionally attack aniline groups in a target object; sulfate radicals generated by a BiOBr/PMS system directionally attack nitrogen-sulfur bonds in a target object, and singlet oxygen radicals generated by an RB (rose bengal) photosensitization system directionally attack nitrogen-containing heterocycles in the target object; o is3/H2O2The singlet oxygen free radical generated by the oxidation system directionally attacks the benzene ring and TiO in the target object2The superoxide radical generated by the photocatalytic system directionally attacks the heterocyclic double bond in the target object.
7. The method according to claim 1, wherein the method comprises the steps of: step (4) a separation and purification method of intermediate products of nitrogen heterocyclic organic compounds, wherein the solid phase extraction technology is used for concentrating the intermediate products in a target sample in an enrichment way, namely extracting the intermediate products on a solid phase extraction device, extracting the sample by using a 500mg solid phase extraction column, filtering the sample with a certain volume by using qualitative filter paper, and then filtering the sample by using a 0.45 mu m water system filter membrane; washing the solid phase extraction column twice respectively by using 5mL of methanol and 5mL of ultrapure water; then injecting the sample into a solid phase extraction column from a polytetrafluoroethylene tube at the flow rate of 3-5 mL/min, draining and drying the extraction column for 1.5-2.5 h, then performing column elution with 6mL of methanol at the flow rate of 1-2 mL/min, collecting the eluent in a glass tube, and concentrating the eluent to 1mL by using a nitrogen blower.
8. The method according to claim 1, wherein the method comprises the steps of: step (5), sample purity inspection is carried out, and the quantification of the substance content of the impurity peak through the known impurity peak standard substance means that firstly, a control solution of different impurity standard substances (chloride ions, sulfate ions, heavy metals and the like) is prepared, and an impurity peak spectrogram (secondary mass spectrogram) is obtained through HPLC-QTOF-MS; secondly, obtaining a mass spectrogram of the sample through HPLC-QTOF-MS, and comparing the mass spectrogram with a mass spectrogram obtained by a reference solution to determine impurities; and thirdly, calculating the impurity content through the impurity peak related parameters, and further estimating the purity of the sample.
9. The method according to claim 1, wherein the method comprises the steps of: the purity of the sample in the step (5) is checked, the quality standard of the control needs to be lower than 1%, and if the quality standard is lower than 1%, the next step is carried out; if not less than 1%, returning to the step (3) of claim 1 for synthesizing the intermediate product of the nitrogen-containing heterocyclic organic compound.
10. The method according to claim 1, wherein the method comprises the steps of: and (6) quantifying a standard product of the intermediate product of the nitrogenous heterocyclic organic compound, wherein in the method for measuring the standard curve by HPLC-MS/MS, a chromatographic column is C18(5 mu m)250mm multiplied by 4.6mm, a mobile phase comprises acetonitrile and an ammonium formate aqueous solution with the mass fraction of 0.1% formic acid-5 mmol/L, the volume flow is 0.6-1.5 mL/min, the column temperature is set to be 30-40 ℃, and the sample injection amount is 5-10 mu L.
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