CN114235976B - 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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- -1 nitrogen-containing heterocyclic organic compound Chemical class 0.000 title claims abstract description 73
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- 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
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Abstract
The invention discloses a method for directional synthesis, separation and purification of a nitrogen-containing heterocyclic organic compound intermediate product and quantitative analysis of the nitrogen-containing heterocyclic organic compound intermediate product, which comprises pretreatment of a sample, recognition of the nitrogen-containing heterocyclic organic compound intermediate product, synthesis of the nitrogen-containing heterocyclic organic compound intermediate product, separation and purification of the nitrogen-containing heterocyclic organic compound intermediate product, purity inspection of the sample and quantitative analysis of the nitrogen-containing heterocyclic organic compound intermediate product by using a standard substance. The patent innovatively provides a technical method for synthesizing and purifying the intermediate product of the nitrogen-containing heterocyclic organic compound, and the method can analyze and identify the intermediate product and has important significance for toxicity experiments of the intermediate product of the nitrogen-containing heterocyclic organic compound 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 nitrogen-containing heterocyclic organic compound advanced oxidation intermediate product and realizing quantitative analysis thereof.
Background
In recent years, because the nitrogen-containing heterocyclic compound has unique biological activity, the nitrogen-containing heterocyclic compound is often used as a structural constituent unit of medicines and pesticides, and is also increasingly widely applied to other fields such as dye-sensitized solar cells. The nitrogenous heterocyclic compound is difficult to be absorbed by the organism, often discharged out of the body along with excrement and urine, and then a series of environmental behaviors occur, and environmental pollution is caused. The nitrogen-containing heterocyclic compounds generally have a greater polarity and a lower n-octanol/water partition coefficient (K ow ) The water-soluble organic compound has relatively high solubility, is not easy to be adsorbed by soil and is easy to exist in water phase, and widely exists in industrial wastewater such as chemical wastewater, pharmaceutical wastewater, pesticide wastewater and the like. The novel micro-pollutants of the water body represented by carbamazepine have low environmental concentration but obvious biological toxicity of the body and the conversion products, can be exposed to human bodies and aquatic and terrestrial organisms for a long time, and cause serious threat to human health and ecological environment. Advanced oxidation techniques (AOPs) are those in which, in an oxidation reaction, electricity, optical radiation, catalysts, etc. are combined with a common chemical oxidant to produce highly reactive free radicals which react with organics to oxidize and decompose them into small molecules until they degrade into CO 2 、H 2 O and inorganic salts. In this process, advanced oxidation techniques, while removing most of the nitrogen-containing heterocyclic compounds, their degradation is merely illustrative of the processing efficiency of the technique on the contaminant itself, whereas decomposition or elimination of the parent compound is not equivalent to removal of toxicity. Because contaminants may form intermediates during degradation, they may exhibit even higher toxicity to the organism than the parent compound. Most intermediate products converted from nitrogen-containing heterocyclic organic compounds lack standard products, and toxicity tests and researches on the intermediate products cannot be carried out in a laboratory, so that toxicity of the intermediate products is not clear, and harm to human beings 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 nitrogen-containing heterocyclic organic compound with high pertinence.
The high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometer combined technology is a combined technology integrating high sensitivity, high specificity and high resolution of High Performance Liquid Chromatography (HPLC) and Q-TOF-MS, has obvious advantages in qualitative and quantitative analysis, well 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 (Density functional theory, DFT) is a quantum chemical calculation method, which can explore the specific molecular structure of a substance and forecast the reactive sites of the substance, and has the advantages of convenient calculation, high analysis speed and the like. The specific calculation method comprises the following steps: fullwell function, front trajectory theory, average localized ionization energy, dual descriptors, electrostatic potential, etc. The structure of the intermediate product can be estimated more accurately by combining the characteristics of the two.
The semi-preparative high performance liquid chromatography has the characteristics of simplicity, easiness in implementation, economy, rapidness, easiness in amplification and the like, and is widely applied to the preparation of various material monomer standard substances. The high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry combined instrument can eliminate mutual interference of chemical components with similar properties in a complex matrix, and is particularly suitable for qualitative and quantitative analysis of complex samples. The sensitivity of mass spectrum detection is high, the method is not limited by compounds without chromophores, and the method is more suitable for measuring low-content components in samples.
Disclosure of Invention
The invention aims to provide a synthesis and analysis method of a nitrogenous heterocyclic organic compound intermediate product, which aims to solve the problems in the prior art.
In order to achieve the above object, the present invention provides the following technical solutions:
a method for synthesizing and analyzing a nitrogen-containing heterocyclic organic compound intermediate, comprising the steps of:
(1) Pretreatment of a sample: (a) After the sample is smashed, the mixture is placed in a 50mL glass centrifuge tube (1), acetonitrile/dichloromethane mixed solution is added, anhydrous sodium sulfate is added after ultrasonic treatment, a high-speed electric homogenizer is used for homogenizing, vortex mixing and centrifugation are carried out, and an acetonitrile/dichloromethane layer is carefully transferred into a 100mL heart-shaped bottle. (b) Then, acetonitrile/dichloromethane mixed solution was added to the glass centrifuge tube (1). (c) Repeating the operation of (b) 1-2 times, combining the extracting solutions of (a) and (b), and concentrating in water bath under reduced pressure until dry. (d) Respectively adding methanol and formic acid, mixing by vortex, and transferring to a 50mL glass centrifuge tube (2) after ultrasonic treatment; (e) Repeating the operation (d) for 1-2 times, and combining the two operations into a glass centrifuge tube (2). (f) Adding n-hexane, centrifuging, and discarding the upper n-hexane layer; (g) Repeating the operation in the step (f) for 1-2 times, and adding water to dilute the lower layer. The solid phase extraction column is activated by methanol and ultrapure water respectively, then the sample diluent is added, then water and methanol are added for leaching, and finally methanol is used for eluting. Concentrating the eluent under reduced pressure to dryness, fixing volume by using a mobile phase, filtering by using an organic phase filter membrane, and then injecting samples.
(2) Recognition of nitrogen-containing heterocyclic organic compound intermediates: firstly, analyzing and identifying the yield change trend of the intermediate product in the target sample by using a high performance liquid chromatography time-of-flight mass spectrometer. Meanwhile, electron cloud density of the parent compound is analyzed through the generalized function of density functional theory, and intermediate products possibly generated by the preliminary reaction are predicted. And then predicting the structure of the intermediate product through a secondary mass spectrum structure of a high performance liquid chromatography time-of-flight mass spectrum-mass spectrometer, and preliminarily confirming the structure and chromatographic parameters of the potential intermediate product.
(2) Synthesis of intermediate products of nitrogen-containing heterocyclic organic compounds: through a designed oxidation reaction system, sulfate radical, hydroxyl radical, superoxide radical and singlet oxygen radical are generated, and active oxygen quenching/capturing experiments are utilized to obtain the required active oxygen, so that a target nitrogenous heterocyclic organic compound matrix is attacked, and a large amount of target intermediate products are directionally generated.
(3) Separating and purifying intermediate products of the nitrogen-containing heterocyclic organic compounds: firstly, enriching and concentrating intermediate products in a target sample by a solid phase extraction technology, then preparing and generating a certain amount of pure products of the target intermediate products by liquid chromatography, mixing a certain amount of organic solvents, and blowing off the organic solvents by nitrogen blowing to obtain the pure products of the target intermediate products.
(4) Sample purity test: adopting a high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry combined instrument to carry out purity inspection, and judging whether a chromatographic peak has chloride ions, sulfate ions and heavy metal impurity peaks; the substance content of the impurity peak, i.e. the impurity content, is quantified by the known impurity peak standard. The quality standard of control needs to be lower than 1 percent, and finally, the separation and purification sample of the intermediate product of the nitrogen-containing heterocyclic organic compound is judged to meet the purity requirement.
(5) Quantification of intermediate standards for nitrogen-containing heterocyclic organic compounds: the prepared intermediate product standard of the nitrogen-containing heterocyclic organic compound can be used for measuring a standard curve through a high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry combined instrument and realizing quantitative analysis of the intermediate product, conversion product or by-product in a target sample.
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 nitrogen-containing heterocyclic organic compound, and the method can analyze and identify the intermediate product and has important significance for toxicity experiments of the intermediate product of the nitrogen-containing heterocyclic organic compound in a laboratory.
Drawings
FIG. 1 is a flow chart of the method of the present invention;
FIG. 2 is a chart of the charge distribution and Fujingpi function values of carbamazepine according to the present invention;
FIG. 3 is a second-order mass spectrum of the molecules and fragment ions of TP211 of the present invention;
FIG. 4 is a block diagram of the major intermediate product of carbamazepine of the present invention after advanced oxidation;
FIG. 5 is a schematic diagram of an intermediate product synthesized in accordance with the present invention;
FIG. 6 is a diagram showing the structure of the main intermediate product after advanced oxidation of sulfamethoxazole in accordance with the present invention;
FIG. 7 is a diagram showing the structure of the main intermediate product after advanced oxidation of sulfamethoxazole in accordance with the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The invention provides a technical scheme that: taking Carbamazepine (CBZ) as an example, methods for the synthesis and analysis of intermediate products. The method comprises the following steps:
(1) Pretreatment of a sample: (a) After the sample is smashed, 6g is weighed and placed in a 50mL glass centrifuge tube (1), then 30mL of mixed solution of acetonitrile and dichloromethane with the volume ratio of 96:5 is added, after 3min of ultrasonic treatment, 6g of anhydrous sodium sulfate is added, a high-speed electric homogenizer 12000r/min is homogenized for 2min, 1.5min is mixed by vortex, 4000r/min is centrifuged for 10min, and an acetonitrile/dichloromethane layer is carefully transferred into a 100mL chicken heart bottle. (b) To the glass centrifuge tube (1) was then added 30mL of acetonitrile/dichloromethane mixture. (c) Repeating the operation in the step (b) for 1 time, combining the extracting solutions in the step (a) and the step (b), and concentrating the extracting solutions in a water bath at 55 ℃ under reduced pressure until the extracting solutions are dried. (d) 1.5mL of methanol and 2.5mL of 1% formic acid by volume were added separately, mixed by vortexing, sonicated for 3min and transferred to a 50mL glass centrifuge tube (2). (e) Repeating the operation in the step (d) for 1 time, and combining the operation with the glass centrifuge tube (2). (f) 10mL of n-hexane was added thereto, and the mixture was centrifuged at 3000r/min for 6min, and the upper n-hexane layer was discarded. (g) The above operation (f) was repeated 1 time, and the lower layer was diluted with 7mL of water. The solid phase extraction column was activated with 4mL of methanol and 5mL of ultrapure water, respectively, and then added with the sample dilution, followed by 5mL of water and 2mL of 5% volume fraction methanol, and finally eluted with 10mL of methanol. The eluate was concentrated to dryness under reduced pressure at 55℃and was subjected to filtration through a 0.22 μm organic phase filter to give a mobile phase volume of 1mL of methanol and 0.1% formic acid in a volume ratio of 3:7.
(2) Carbamazepine intermediate identification: firstly, setting the volume ratio of acetonitrile to 0.1% formic acid water as a mobile phase to be 20:80 and the sample injection volume to be 15 mu L by a high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry combined instrument; the mass spectrometry is carried out in a positive ion mode, 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 atomization air pressure is 350kPa; sheath gas temperature is 360 ℃, and sheath gas flow rate is 15L/min; capillary voltage 4200V, cone voltage 60V, lysis voltage 150V. Density functional theory generalized function analysis of carbamazepine's electron cloud density: an optimized molecular model is built through Gauss View, and the molecules are analyzed through Gaussian software. And then, predicting and obtaining a possible product structure through a secondary mass spectrum structure of a high performance liquid chromatography time-of-flight mass spectrum-mass spectrometer, and preliminarily confirming the structure and chromatographic parameters of the potential intermediate product. Fig. 2 shows the analysis of a portion of the intermediates:
(3) Carbamazepine intermediate synthesis: generating hydroxyl free radicals directionally through a designed persulfate/zero-valent iron oxidation reaction system; the manganese trioxide/persulfate oxidation reaction system generates sulfate radicals. Active oxygen is purified by an active oxygen quenching/capturing experiment, and a nitrogen-containing heterocycle of carbamazepine is attacked to directionally produce a large amount of target intermediate products. The main intermediate products after advanced oxidation include the following structures:
(4) Separating and purifying carbamazepine intermediate products: samples were extracted using a 500mg solid phase extraction column. Taking a certain volume of sample, filtering with qualitative filter paper, and filtering with 0.45 μm water-based filter membrane; washing the solid phase extraction column twice with 5mL of methanol and 5mL of ultrapure water respectively; the sample was then injected into the solid phase extraction column from a polytetrafluoroethylene tube at a flow rate of 5 mL/min. The column was drained and dried for 2.5h, then the column was eluted with 6mL of methanol at a flow rate of 2mL/min, the eluate was collected in a glass tube and concentrated to 1mL using a nitrogen blower.
(5) Sample purity test: adopting a high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry to perform purity inspection, preparing control solutions of different impurity standards (chloride ions, sulfate ions and heavy metals), and obtaining an impurity peak spectrogram (a secondary mass spectrogram) through the high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry; obtaining a mass spectrum of a sample through a high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry combined instrument, and comparing an impurity peak with the mass spectrum obtained by the control liquid to determine impurities; and calculating the impurity content through the impurity peak related parameters, and further estimating the purity of the sample. The quality standard of control needs to be lower than 1 percent, and the final judgment of the purity requirement of the carbamazepine intermediate product separation and purification sample is achieved.
(6) Quantification of carbamazepine intermediate standards: the standard curve can be measured by using the prepared carbamazepine intermediate product standard substance through a high performance liquid chromatography time-of-flight mass spectrometer, and the relevant conditions of the high performance liquid chromatography time-of-flight mass spectrometer are as follows: the chromatographic column is a C18 column with the specification of 250mm multiplied by 4.6mm and the diameter of a packing material in the column of 5 mu m, the mobile phase is acetonitrile and an ammonium formate aqueous solution with the mass fraction of 0.1% of 5mmol/L of formic acid, the volume flow is 0.6mL/min, the column temperature is set to 30 ℃, and the sample injection amount is 5 mu L. The quantitative analysis of the intermediate product, the conversion product or the by-product of carbamazepine in the target sample is realized. Taking carbamazepine as an example, the following structure can be isolated by the above method:
example two
The invention provides a technical scheme that: taking Sulfamethoxazole (SMX) as an example, the synthesis and analysis method of the intermediate product. The method comprises the following steps:
(1) Pretreatment of a sample: (a) 10g of the sample is weighed after mashing, the sample is placed in a 50mL glass centrifuge tube (1), 25mL of a mixed solution of acetonitrile and dichloromethane with the volume ratio of 100:5 is added, 8g of anhydrous sodium sulfate is added after ultrasonic treatment for 2min, 10000r/min of a high-speed electric homogenizer is homogenized for 1.5min, 1.5min is mixed by vortex, 3000r/min is centrifuged for 8min, and an acetonitrile/dichloromethane layer is carefully transferred into a 100mL chicken heart bottle. (b) To the glass centrifuge tube (1) was then added 25mL of acetonitrile/dichloromethane mixture. (c) Repeating the operation in the step (b) for 2 times, combining the extracting solutions in the step (a) and the step (b), and concentrating the extracting solutions in a water bath at 55 ℃ under reduced pressure until the extracting solutions are dried. (d) Then 1mL of methanol and 2mL of 1% volume fraction formic acid were added respectively, mixed well by vortexing, sonicated for 3min and transferred to a 50mL glass centrifuge tube (2). (e) Repeating the operation in the step (d) for 2 times, and combining the operation with the glass centrifuge tube (2). (f) 10mL of n-hexane was added thereto, and the mixture was centrifuged at 3000r/min for 6min, and the upper n-hexane layer was discarded. (g) The above operation (f) was repeated 2 times, and the lower layer was diluted with 7mL of water. The solid phase extraction column was activated with 4mL of methanol and 5mL of ultrapure water, respectively, and then added with the sample dilution, followed by 5mL of water and 2mL of 5% volume fraction methanol, and finally eluted with 10mL of methanol. The eluate was concentrated to dryness under reduced pressure at 55℃and was subjected to filtration through a 0.22 μm organic phase filter to give a mobile phase volume of 1mL of methanol and 0.1% formic acid in a volume ratio of 3:7.
(2) Identification of sulfamethoxazole intermediate: firstly, setting the volume ratio of acetonitrile to 0.1% formic acid water as a mobile phase to be 20:80 and the sample injection volume to be 10 mu L by a high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry combined instrument; the mass spectrometry is carried out in a positive ion mode, 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 atomization air pressure is 350kPa; sheath gas temperature is 360 ℃, and sheath gas flow rate is 15L/min; capillary voltage 3800V, cone voltage 65V, lysis voltage 150V. The electron cloud density of sulfamethoxazole is analyzed by the general function of the density functional theory: an optimized molecular model is built through Gauss View, and the molecules are analyzed through Gaussian software. And then, predicting and obtaining a possible product structure through a secondary mass spectrum structure of a high performance liquid chromatography time-of-flight mass spectrum-mass spectrometer, and preliminarily confirming the structure and chromatographic parameters of the potential intermediate product.
(3) Synthesis of sulfamethoxazole intermediate: generating hydroxyl free radicals directionally through a designed ultraviolet/titanium dioxide oxidation reaction system; the titanium dioxide photocatalytic system generates singlet oxygen radicals. Active oxygen is purified by an active oxygen quenching/capturing experiment, nitrogen-containing heterocycle and anilino groups of sulfamethoxazole are attacked, and a large amount of target intermediate products are directionally produced.
(4) Separating and purifying the sulfamethoxazole intermediate: samples were extracted using a 500mg solid phase extraction column. Taking a certain volume of sample, filtering with qualitative filter paper, and filtering with 0.45 μm water-based filter membrane; washing the solid phase extraction column twice with 5mL of methanol and 5mL of ultrapure water respectively; the sample was then injected into the solid phase extraction column from a polytetrafluoroethylene 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 methanol at a flow rate of 1mL/min, the eluate was collected in a glass tube and concentrated to 1mL using a nitrogen blower.
(5) Sample purity test: adopting a high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry to perform purity inspection, preparing control solutions of different impurity standards (chloride ions, sulfate ions and heavy metals), and obtaining an impurity peak spectrogram (a secondary mass spectrogram) through the high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry; obtaining a mass spectrum of a sample through a high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry combined instrument, and comparing an impurity peak with the mass spectrum obtained by the control liquid to determine impurities; and calculating the impurity content through the impurity peak related parameters, and further estimating the purity of the sample. The quality standard of control is required to be lower than 1 percent, and the separation and purification sample of the sulfamethoxazole intermediate product is finally judged to meet the purity requirement.
(6) Quantification of sulfamethoxazole intermediate standard: the standard curve can be measured by using the prepared sulfamethoxazole intermediate product standard substance through a high performance liquid chromatography time-of-flight mass spectrometer, and the relevant conditions of the high performance liquid chromatography time-of-flight mass spectrometer are as follows: the chromatographic column is a C18 column with the specification of 250mm multiplied by 4.6mm and the diameter of the packing material in the column of 5 mu m, the mobile phase is acetonitrile and ammonium formate aqueous solution with the mass fraction of 0.1% formic acid of 5mmol/L, the volume flow is 0.6mL/min, the column temperature is set to 30 ℃, and the sample injection amount is 7 mu L. The quantitative analysis of the intermediate product, the conversion product or the byproducts of the sulfamethoxazole in the target sample is realized.
Example III
The invention provides a technical scheme that: taking Sulfadiazine (SIM) as an example, and a method for synthesizing and analyzing an intermediate product. The method comprises the following steps:
(1) Pretreatment of a sample: (a) After the sample is smashed, 7g is weighed and placed in a 50mL glass centrifuge tube (1), then 25mL of mixed solution of acetonitrile and dichloromethane with the volume ratio of 100:5 is added, 9g of anhydrous sodium sulfate is added after ultrasonic treatment for 2.5min, a high-speed electric homogenizer 13000r/min is used for homogenizing for 1.5min, vortex mixing is carried out for 1.5min, centrifugation is carried out for 9min at 3500r/min, and an acetonitrile/dichloromethane layer is carefully transferred into a 100mL chicken heart bottle. (b) To the glass centrifuge tube (1) was then added 30mL of acetonitrile/dichloromethane mixture. (c) Repeating the operation in the step (b) for 2 times, combining the extracting solutions in the step (a) and the step (b), and concentrating the extracting solutions in a water bath at 52 ℃ under reduced pressure until the extracting solutions are dried. (d) 1.25mL of methanol and 2.25mL of 1% formic acid by volume fraction were added separately, mixed by vortexing, sonicated for 2.5min and transferred to a 50mL glass centrifuge tube (2). (e) Repeating the operation in the step (d) for 1 time, and combining the operation with the glass centrifuge tube (2). (f) 8.5mL of n-hexane was added thereto, and the mixture was centrifuged at 3000r/min for 6min, and the upper n-hexane layer was discarded. (g) The above operation (f) was repeated 2 times, and the lower layer was diluted with 7mL of water. The solid phase extraction column was activated with 4mL of methanol and 5mL of ultrapure water, respectively, and then added with the sample dilution, followed by 5mL of water and 2mL of 5% volume fraction methanol, and finally eluted with 10mL of methanol. The eluate was concentrated to dryness under reduced pressure at 55℃and was subjected to filtration through a 0.22 μm organic phase filter to give a mobile phase volume of 1mL of methanol and 0.1% formic acid in a volume ratio of 3:7.
(2) Sulfadiazine intermediate identification: firstly, setting the volume ratio of acetonitrile to 0.1% formic acid water as a mobile phase to be 20:80 and the sample injection volume to be 15 mu L by a high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry combined instrument; the mass spectrometry is carried out in a positive ion mode, 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 air pressure is 330kPa; the temperature of the sheath gas is 350 ℃, and the flow rate of the sheath gas is 10L/min; capillary voltage 4000V, cone voltage 70V, lysis voltage 130V. Density Functional Theory (DFT) functional analysis of electronic cloud density of sulfadiazine: an optimized molecular model is built through Gauss View, and the molecules are analyzed through Gaussian software. And then, predicting and obtaining a possible product structure through a secondary mass spectrum structure of a high performance liquid chromatography time-of-flight mass spectrum-mass spectrometer, and preliminarily confirming the structure and chromatographic parameters of the potential intermediate product.
(3) Sulfadiazine intermediate product synthesis: generating hydroxyl free radicals directionally through a designed ultraviolet/titanium dioxide oxidation reaction system; the titanium dioxide photocatalytic system generates singlet oxygen radicals. Active oxygen is purified by an active oxygen quenching/capturing experiment, nitrogen-containing heterocycle and anilino groups of the sulfamethoxazole are attacked, and a large amount of target intermediate products are directionally produced.
(4) Separating and purifying a sulfadiazine intermediate product: samples were extracted using a 500mg solid phase extraction column. Taking a certain volume of sample, filtering with qualitative filter paper, and filtering with 0.45 μm water-based filter membrane; washing the solid phase extraction column twice with 5mL of methanol and 5mL of ultrapure water respectively; the sample was then injected into the solid phase extraction column from a polytetrafluoroethylene tube at a flow rate of 4 mL/min. The column was drained and dried for 1.5h, then column eluted with 6mL methanol at a flow rate of 1.5mL/min, the eluate was collected in a glass tube and concentrated to 1mL using a nitrogen blower.
(5) Sample purity test: adopting a high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry to perform purity inspection, preparing control solutions of different impurity standards (chloride ions, sulfate ions and heavy metals), and obtaining an impurity peak spectrogram (a secondary mass spectrogram) through the high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry; obtaining a mass spectrum of a sample through a high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry combined instrument, and comparing an impurity peak with the mass spectrum obtained by the control liquid to determine impurities; and calculating the impurity content through the impurity peak related parameters, and further estimating the purity of the sample. The quality standard of control needs to be lower than 1 percent, and the separation and purification sample of the sulfadiazine intermediate product is finally judged to meet the purity requirement.
(6) Quantification of sulfadiazine intermediate standard: the standard curve can be measured by using the prepared sulfadiazine intermediate product standard substance through a high performance liquid chromatography time-of-flight mass spectrometer, and the related conditions of the high performance liquid chromatography time-of-flight mass spectrometer are that a chromatographic column is a C18 column with the specification of 250mm multiplied by 4.6mm and the diameter of a packing material in the column of 5 mu m, a mobile phase is acetonitrile and ammonium formate aqueous solution with the mass fraction of 0.1% formic acid of 5mmol/L, the volume flow is 1.5mL/min, the column temperature is set to 40 ℃, and the sample injection amount is 10 mu L. The quantitative analysis of the intermediate product, the conversion product or the byproducts of the sulfadiazine in the target sample is realized.
It is noted that relational terms such as first and second, and the like are 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. Moreover, 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 understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein 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 a nitrogen-containing heterocyclic organic compound intermediate, comprising the steps of:
(1) Pretreatment of a sample: (a) Mashing a sample, placing the sample into a 50mL glass centrifuge tube (1), then adding acetonitrile/dichloromethane mixed solution, adding anhydrous sodium sulfate after ultrasonic treatment, homogenizing by a high-speed electric homogenizer, uniformly mixing by vortex, centrifuging, and carefully transferring an acetonitrile/dichloromethane layer into a 100mL heart bottle; (b) Adding acetonitrile/dichloromethane mixed solution into the glass centrifuge tube (1); (c) Repeating the operation in the step (b) for 1 to 2 times, combining the extracting solutions in the step (a) and the step (b), and concentrating the extracting solutions in a water bath under reduced pressure until the extracting solutions are dried; (d) Respectively adding methanol and formic acid, mixing by vortex, and transferring to a 50mL glass centrifuge tube (2) after ultrasonic treatment; (e) Repeating the operation in the step (d) for 1-2 times, and combining the operation with the glass centrifuge tube (2); (f) Adding n-hexane, centrifuging, and discarding the upper n-hexane layer; (g) Repeating the operation in the step (f) for 1-2 times, adding water to dilute the lower layer, respectively using methanol and ultrapure water to activate the solid phase extraction column, adding sample diluent, adding water and methanol for leaching, eluting with methanol, concentrating the eluent under reduced pressure to dryness, using a mobile phase to fix the volume, filtering by an organic phase filter membrane, and then introducing samples;
(2) Recognition of nitrogen-containing heterocyclic organic compound intermediates: firstly, analyzing and identifying the yield change trend of an intermediate product in a target sample by using a high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry combined instrument, simultaneously, analyzing the electron cloud density of a parent compound by using a density functional theory overtime function, predicting an intermediate product possibly generated by a preliminary reaction, and then predicting the structure of the intermediate product by using a secondary mass spectrometry structure of the high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry combined instrument, and preliminarily confirming the structure and chromatographic parameters of the potential intermediate product;
(3) Synthesis of intermediate products of nitrogen-containing heterocyclic organic compounds: generating sulfate radical, hydroxyl radical, superoxide radical and singlet oxygen radical through a designed oxidation reaction system, obtaining required active oxygen by utilizing an active oxygen quenching/capturing experiment, attacking a target nitrogen-containing heterocyclic organic compound matrix, and directionally generating a large amount of target intermediate products;
(4) Separating and purifying intermediate products of the nitrogen-containing heterocyclic organic compounds: firstly, enriching and concentrating intermediate products in a degradation sample by a solid phase extraction technology, then generating a certain amount of target intermediate product pure products by semi-preparative high performance liquid chromatography, mixing a certain amount of organic solvent, and blowing off the organic solvent by nitrogen blowing to obtain the target intermediate product pure products;
(5) Sample purity test: adopting a high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry combined instrument to carry out purity inspection, and judging whether a chromatographic peak has chloride ions, sulfate ions and heavy metal impurity peaks; the impurity content, namely the impurity content, of the impurity peak is quantified by the known impurity peak standard substance, and the quality standard is controlled to be lower than 1 percent, so that the separation and purification sample of the intermediate product of the nitrogen-containing heterocyclic organic compound is finally judged to meet the purity requirement;
(6) Quantification of intermediate standards for nitrogen-containing heterocyclic organic compounds: the prepared intermediate product standard of the nitrogen-containing heterocyclic organic compound can be used for establishing a concentration standard curve through a high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry combined instrument, and realizing quantitative analysis of intermediate products, conversion products or byproducts in a target sample.
2. The method for synthesizing and analyzing a nitrogen-containing heterocyclic organic compound intermediate according to claim 1, wherein: the pretreatment method of the sample in the step (1) comprises the steps of (a) mashing the sample and weighing 5-10 g; 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 adding 5-10 g anhydrous sodium sulfate for 2-3 min by ultrasonic treatment; homogenizing for 1.5-2 min by 10000-15000 r/min of a high-speed electric homogenizer; vortex mixing for 1-1.5 min, and centrifuging for 8-10 min at 3000-4000 r/min; (b) Then adding 25-30 mL of acetonitrile/dichloromethane mixed solution into the glass centrifuge tube (1); (c) Repeating the operation in the step (b) for 1 to 2 times, combining the extracting solutions in the step (a) and the step (b), and concentrating the extracting solutions in a water bath at 50 to 55 ℃ under reduced pressure until the extracting solutions are dried; (d) Respectively adding 1-1.5 mL of methanol and 2-2.5 mL of formic acid with the volume fraction of 1%, mixing uniformly by vortex, carrying out ultrasonic treatment for 2-3 min, and transferring into a 50mL glass centrifuge tube (2); (e) Repeating the operation in the step (d) for 1-2 times, and combining the operation with the glass centrifuge tube (2); (f) Adding 8-10 mL of n-hexane, centrifuging for 6min at 3000r/min, and discarding an upper n-hexane layer; (g) Repeating the operation (f) for 1-2 times, adding 7mL of water into the lower layer for dilution, respectively using 4mL of methanol and 5mL of ultrapure water for activating treatment, adding sample diluent, adding 5mL of water and 2mL of 5% volume fraction methanol for leaching, eluting with 10mL of methanol, concentrating the eluent under reduced pressure at 55 ℃ to dryness, using 1mL of mobile phase for constant volume, wherein the mobile phase consists of 0.1% volume fraction of methanol and formic acid, the volume ratio of 0.1% of methanol to formic acid is 3:7, and filtering by a 0.22 mu m organic phase filter membrane for sample injection.
3. The method for synthesizing and analyzing a nitrogen-containing heterocyclic organic compound intermediate according to claim 1, wherein: the method for identifying the intermediate product of the nitrogen-containing heterocyclic organic compound in the step (2) comprises the steps that the related conditions of the high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry combined instrument are set to be that the mobile phase is acetonitrile and the volume ratio of 0.1% formic acid water is 20:80, 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 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 atomization air pressure is 300-350 kPa; the sheath gas temperature is 320-360 ℃, and the sheath gas flow rate is 6-15L/min; capillary voltage 3800-4200V, taper hole voltage 60-70V, cracking voltage 120-150V.
4. The method for synthesizing and analyzing a nitrogen-containing heterocyclic organic compound intermediate according to claim 1, wherein: and (2) identifying the intermediate product of the nitrogen-containing heterocyclic organic compound, wherein the Density Functional Theory (DFT) functional analysis is carried out on the optimized molecular model through Gaussian, VASP and CASTEP software.
5. The method for synthesizing and analyzing a nitrogen-containing heterocyclic organic compound intermediate according to claim 1, wherein: synthesizing the intermediate product of the nitrogen-containing heterocyclic organic compound in the step (3), wherein the designed oxidation reaction system is a persulfate/zero-valent iron system, a ferro-manganese binary oxide/hydrogen peroxide/ozone system and an ultraviolet/titanium dioxide system, and hydroxyl free radicals can be directionally generated; the designed heat-activated persulfate system, the manganese trioxide/persulfate system and the bismuth oxybromide/persulfate system can directionally generate sulfate radicals; the designed rose red light sensitization system and the ozone/hydrogen peroxide oxidation system can directionally generate singlet oxygen free radicals; the designed titanium dioxide photocatalysis system can directionally generate superoxide radical.
6. The method for synthesizing and analyzing a nitrogen-containing heterocyclic organic compound intermediate according to claim 1, wherein: step (3) synthesis of intermediate products of nitrogen-containing heterocyclic organic compounds, wherein the hydroxyl radicals, sulfate radicals, superoxide radicals and singlet oxygen radicals are targeted to attack sites: hydroxyl radicals generated by the peroxymonosulfate/zero-valent iron system directionally attack nitrogen-containing heterocycles in the target; hydroxyl radical generated by the ferro-manganese binary oxide/hydrogen peroxide/ozone system directionally attacks amino on benzene ring or aniline in the target object; hydroxyl radicals generated by the ultraviolet/titanium dioxide system directionally attack carbon-carbon double bonds or high-activity carbon-hydrogen bonds in the target object, and sulfate radicals generated by the heat-activated persulfate system directionally attack olefin double bonds in the target object; the sulfate radical generated by the manganese trioxide/persulfate system directionally attacks the anilino group in the target; the sulfate radical generated by the bismuth oxybromide/persulfate system directionally attacks the nitrogen-sulfur bond in the target, and the singlet oxygen radical generated by the rose bengal photosensitization system directionally attacks the nitrogen-containing heterocycle in the target; the singlet oxygen free radical generated by the ozone/hydrogen peroxide oxidation system directionally attacks the benzene ring in the target object, and the superoxide free radical generated by the titanium dioxide photocatalysis system directionally attacks the heterocyclic double bond in the target object.
7. The method for synthesizing and analyzing a nitrogen-containing heterocyclic organic compound intermediate according to claim 1, wherein: the method for separating and purifying the intermediate product of the nitrogen-containing heterocyclic organic compound in the step (4), wherein the solid phase extraction technology is used for enriching and concentrating the intermediate product in a target sample, namely extracting the intermediate product on a solid phase extraction device, extracting the sample by using a 500mg solid phase extraction column, taking a certain volume of the sample, filtering the sample by qualitative filter paper, and filtering the sample by using a 0.45 mu m water system filter membrane; washing the solid phase extraction column twice with 5mL of methanol and 5mL of ultrapure water respectively; then the sample is injected into a solid phase extraction column from a polytetrafluoroethylene tube at a flow rate of 3-5 mL/min, the extraction column is drained and dried for 1.5-2.5 h, then 6mL of methanol is used for column elution at a flow rate of 1-2 mL/min, the eluent is collected in a glass tube, and then the eluent is concentrated to 1mL by a nitrogen blower.
8. The method for synthesizing and analyzing a nitrogen-containing heterocyclic organic compound intermediate according to claim 1, wherein: the sample purity test of the step (5), the quantitative impurity peak substance content by the known impurity peak standard substance means (1) preparing different impurity standard substances, chloride ion, sulfate ion and heavy metal reference solution, obtaining impurity peak secondary mass spectrum by high performance liquid chromatography time-of-flight mass spectrum-mass spectrum combined instrument; (2) obtaining a mass spectrum of a sample through a high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry combined instrument, and comparing an impurity peak with the mass spectrum obtained by the control liquid to determine impurities; (3) and calculating the impurity content through the impurity peak related parameters, and further estimating the purity of the sample.
9. The method for synthesizing and analyzing a nitrogen-containing heterocyclic organic compound intermediate according to claim 1, wherein: step (5) sample purity test, wherein the quality standard of the control needs to be lower than 1%, and if the quality standard is lower than 1%, the step (6) nitrogen-containing heterocyclic organic compound intermediate product standard quantification is carried out; and if the ratio is not less than 1%, returning to the step (3) for synthesizing the intermediate product of the nitrogen-containing heterocyclic organic compound.
10. The method for synthesizing and analyzing a nitrogen-containing heterocyclic organic compound intermediate according to claim 1, wherein: in the method for measuring a standard curve by using a high performance liquid chromatography time-of-flight mass spectrometry-mass spectrometry combined instrument, a chromatographic column is a C18 column with the specification of 250mm multiplied by 4.6mm and the diameter of a packing material in the column of 5 mu m, a mobile phase is acetonitrile and 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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