CN114853650B - Material for specifically capturing ammonia compounds from complex sample and preparation method and application thereof - Google Patents
Material for specifically capturing ammonia compounds from complex sample and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a material for specifically capturing ammonia compounds from complex samples. The invention combines a magnetic separation technology with an amino compound targeted analysis technology, and covalently bonds phenyl thioisocyanate (PITC) and analogues thereof on an amino material for specifically capturing the amino compound in a complex sample. The material contains disulfide bonds, the disulfide bonds can be selectively broken through reducing agents such as dithiothreitol and the like after purification is finished, the captured amino compounds are dissociated from the magnetic material into a solution, and the solution can be used for subsequent experimental analysis after simple magnetic separation and centrifugation. The material and the method have the advantages that the material can specifically capture amino compounds, and the product obtained by breaking disulfide bonds can improve the detection sensitivity of mass spectrum, thereby providing a novel technical method for high-flux separation and identification of the sub-metabolism group of the amino compounds.
Description
Technical Field
The invention relates to the technical field of metabolite separation in a complex matrix system, in particular to a material for specifically capturing ammonia compounds from a complex sample, a preparation method and application thereof.
Background
Amino metabolites such as amino acids, catecholamines, peptides and polyamines play an important role in the biosynthesis, cell growth, signal transduction and immunomodulation of cells. Amino acids are taken as an example, and not only are nutrients and provide a material basis for life activities, but also play an important role in signal transmission, gene expression and protein synthesis. In addition, amino acids, which are also called functional amino acids including arginine, cysteine, glutamine, leucine, proline, tryptophan, etc., can be involved in regulating important metabolic pathways in the body that are critical to the normal functioning of the living body, growth, repair of the body, and autoimmunity. The concentration of amino acid metabolites such as nitric oxide, polyamines, glutathione, taurine, thyroid hormone, serotonin and the like also play an important role in maintaining body function. In addition, elevated levels of amino acids and their metabolites such as ammonia, homocysteine, asymmetric dimethylarginine can cause a range of diseases such as neurological disorders, oxidative stress abnormalities, and cardiovascular disorders. Thus, stabilization of amino acid levels in the intracellular and humoral circuits appears to be critical for maintaining homeostasis. Diagnosis and prognosis of the disease can be performed by detecting the levels of amino metabolites in tissues, blood, urine, and feces.
Conventional analysis techniques for small molecules of metabolites include Nuclear Magnetic Resonance (NMR), ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) and gas chromatography-mass spectrometry (GC-MS), allowing for the parallel determination of hundreds of metabolites. Among them, mass Spectrometry (MS) is a major technique for studying complex metabolite mixtures in human samples due to its excellent detection range and sensitivity.
The components in the biological sample are complex, and besides amino compounds, a large amount of inorganic salts, organic salts, phospholipids, proteins and the like are contained to interfere the test result, so that the sample is required to be pretreated to remove or weaken the interference, and the results with high stability and good repeatability are obtained. Proteins in the sample can be removed by a method of precipitation with an organic solvent such as methanol and acetonitrile, but a large amount of water-soluble and fat-soluble impurities in the sample cannot be removed by this method. At present, the common method for amino compounds is liquid phase-mass spectrum combination, separation is carried out through a chromatographic column, and then detection is carried out through mass spectrum, so that matrix interference is reduced, particularly, the appearance of ultra-high performance liquid chromatography (UPLC) is improved, and the separation effect is further improved. However, since most amino compounds such as amino acids have strong polar groups, retention and separation of the compounds in a reverse-phase chromatographic column are not favored. Although derivatization reagents are currently used for derivatizing amino compounds to reduce the polarity of the compounds and improve the separation effect of chromatographic columns. For example, the pre-column derivatization reagent Phenyl Isothiocyanate (PITC) and 6-aminoquinolinyl-N-hydroxysuccinimidyl carbamate (AQC) are used for derivatizing amino compounds, and a liposoluble group with ultraviolet absorption is connected to a molecule, so that the subsequent liquid chromatography separation and spectral detection are facilitated. In addition, the group with the mass spectrum signal enhancement effect can be derived, so that the detection capability of the amino molecules in mass spectrum is improved. However, there is a problem in that derivatization does not remove a large amount of matrix, and excessive derivatization reagents can interfere with detection, contaminate and clog chromatographic columns, reducing instrument life.
Therefore, providing a material for specifically capturing an ammonia compound from a complex sample, which can solve the problems of matrix interference, low analysis efficiency and the like in the current direct sample injection process, is a technical problem that needs to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a material for specifically capturing ammonia compounds from complex samples, and a preparation method and application thereof, so as to solve the problems of matrix interference and low analysis efficiency in the direct sample injection process.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a material for specifically capturing an ammonia compound from a complex sample is characterized by having a structure shown in a general formula (I):
wherein, the black round dot is a magnetic core with magnetic response, X is different substituents on amino, Y is a connecting chain between two nitrogen atoms;
the side chain contains cystamine, wherein disulfide bonds are selective breaking groups; z in the side chain is isothiocyanate group combined with the parent nucleus; the tail end of the side chain contains an isothiocyanate group, which is used for specifically capturing amino compounds.
Further, the magnetic core material with magnetic response is iron, cobalt, nickel, ferroferric oxide or a coating material containing the material.
Further, the coating material is one of amino silicon dioxide, polystyrene and polyacrylic acid.
Preferably, the magnetic core material may be any material with magnetic response, and may be selected from iron powder, ferroferric oxide, nickel powder, cobalt powder and combinations thereof, and may also be a combination of the magnetic core and other materials, such as silicon dioxide coated ferroferric oxide, silicon dioxide amino coated ferroferric oxide, silicon dioxide hydroxyl coated ferroferric oxide and silicon dioxide carboxyl coated ferroferric oxide. The surface of the magnetic core contains amino and substituted amino for subsequent derivatization.
More preferably, the core is ferroferric oxide (Fe 3 O 4 @SiO 2 -NH 2 )。
Further, the linking chain Y is an acyl or aminoacyl-containing chain, including, but not limited to, the following structures:
preferably, X is one of H, CH3, CH2CH3 and CH2CH2 OH. Y is a molecule containing an amino group, a carboxyl group, an isocyanate group, an isothiocyanate group, an aldehyde group, or the like, which can be further derived. Z is a parent nucleus capable of linking isothiocyanate groups.
More preferably, X is H, Y is a compound containing an amino group or a carboxyl group, and Z is a parent nucleus containing a benzene ring.
Most preferably, Y is succinic acid.
Further, Z-NCS is a terminal isothiocyanate group, including, but not limited to, the following structures:
further, the material for specifically capturing the ammonia compound from the complex sample may be selected from the following structures:
the invention also provides a preparation method of the material for specifically capturing the ammonia compound from the complex sample, which comprises the following steps:
(1) The amino magnetic core is connected with a cystamine succinic acid chain, and the synthetic route is as follows:
the method comprises the following steps:
(1-1) preparation of a fluorenylmethoxycarbonyl cysteamine succinic acid side chain:
the cystamine dihydrochloride is dissolved in water, and sodium bicarbonate is added to continue stirring. Adding dioxane, stirring in ice bath for 10 min, adding succinic anhydride into the reaction system, and stirring at room temperature for 12 hr. After the reaction is completed, N-diisopropylethylamine is added into a reaction system in an ice bath, 9-fluorenylmethyl-N-succinimidyl carbonate is accurately weighed and dissolved in N, N-dimethylformamide, and then the solution is dropwise added into the reaction system. The reaction was returned to room temperature and stirring was continued for 2 hours. After the reaction was completed, filtration was carried out, and the filtrate was concentrated in vacuo to be viscous, after which water was added and stirring was continued, yielding a large amount of white solid in the filtrate. The white solid was collected by filtration, washed with water, and dried in vacuo to give fluorenylmethoxycarbonyl cysteamine succinate (the obtained solid was used directly in the subsequent reaction without purification;
(1-2) magnetic material attached to the fluorenylmethoxycarbonyl cysteamine succinic acid side chain:
and mixing fluorenylmethoxycarbonyl cysteamine succinic acid with 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethyl urea hexafluorophosphate, adding dimethyl sulfoxide for dissolution, adding N, N-diisopropylethylamine, uniformly mixing, adding into the magnetic beads, continuing to oscillate, and connecting Fmoc-CS. After the connection is completed, magnetically separating, removing the solvent, washing, and replacing the centrifuge tube to obtain the magnetic beads connected with Fmoc-CS;
(1-3) side chain defluorene methoxycarbonyl group of magnetic material:
adding N, N-dimethylformamide solution into a centrifuge tube containing magnetic beads, adding 1, 8-diazabicyclo [5.4.0] undec-7-ene, oscillating for 1 hour, and after the reaction is completed, washing and replacing the centrifuge tube to obtain magnetic beads containing cystamine side chains;
(2) The magnetic material is derived from isothiocyanate groups, and the synthetic route is as follows:
(2-1) preparation of 4-carboxyphenyl isothiocyanate:
weighing 4-aminobenzoic acid, adding into dichloromethane, adding triethylamine and ice bath under the magnetic stirring condition, then taking thiocarbonyldiimidazole, adding into the reaction system, continuously stirring, then taking hydrochloric acid, adding petroleum ether, mixing and oscillating, then dropwise adding into the reaction system, continuously reacting after the dropwise adding is completed, generating a large amount of white precipitate in the reaction system, filtering, washing with water to obtain light yellow solid, and evaporating water to obtain white solid, namely 4-carboxyphenyl isothiocyanate;
(2-2) magnetic material derived isocyanate groups:
mixing 4-carboxyphenyl isothiocyanate with 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethyl urea hexafluorophosphate, adding dimethyl sulfoxide for dissolution, adding N, N-diisopropylethylamine, uniformly mixing, adding into the magnetic material containing the cystamine side chain prepared in the step (1) after oscillation, and continuing to oscillate for 0.1-2. After the reaction is finished, removing the solvent by magnetic separation, washing, and replacing a centrifuge tube to obtain the material for specifically capturing the ammonia compound from the complex sample.
Further, in the step (1-1), the mass ratio of the cystamine dihydrochloride to the sodium bicarbonate is 1:1;
the volume ratio of the substance of cystamine dihydrochloride to dioxane was 10mmol:100ml;
the molar ratio of cystamine dihydrochloride to succinic anhydride is 1:1;
the volume ratio of the substance of cystamine dihydrochloride to N, N-diisopropylethylamine was 10mmol:3.3ml;
the molar ratio of cystamine dihydrochloride to 9-fluorenylmethyl-N-succinimidyl carbonate is 1:1;
the volume ratio of the substance of cystamine dihydrochloride to N, N-dimethylformamide was 10mmol:10ml;
further, the mass ratio of the fluorenylmethoxycarbonyl cysteamine succinic acid to the 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate in the step (1-2) is 1:1;
the volume ratio of the mass of the fluorenylmethoxycarbonyl cysteamine succinic acid to the dimethyl sulfoxide is 1-10mg:1ml;
the volume ratio of the mass of the fluorenylmethoxycarbonyl cysteamine succinic acid to the N, N-diisopropylethylamine is 1-10mg:0.1-1ml;
further, the N, N-dimethylformamide solution in the step (1-3) is used in an amount of 0.5ml;
the dosage of the 1, 8-diazabicyclo [5.4.0] undec-7-ene is 0.5ml;
further, the ratio of the mass of 4-aminobenzoic acid to the volume of methylene chloride in the step (2-1) was 5g:51ml;
the dosage of triethylamine is 5.6ml;
the dosage of thiocarbonyldiimidazole is 8.45g;
the dosage of hydrochloric acid is 12ml, and the dosage of petroleum ether is 50ml;
further, the mass ratio of the 4-carboxyphenyl isothiocyanate to the 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate in the step (2-2) is 15:1-20;
the dosage of dimethyl sulfoxide is 1-10ml;
the dosage of the N, N-diisopropylethylamine is 01-1ml;
the invention also provides an application method of the material for specifically capturing the ammonia compound from the complex sample, which comprises the following steps:
1) Taking 0.1-1mg of the material, adding 0.2-1ml of dimethyl sulfoxide, 0.3ml of serum and 0.001-0.1ml of triethylamine, oscillating for 0.1-5 hours at the temperature of 0-50 ℃, removing the solvent by magnetic separation after the reaction is finished, washing with phosphate buffer solution, washing with water, washing with mixed solution of acetonitrile and water, and replacing a centrifuge tube for later use;
2) Capturing the obtained amino compound to break bonds:
suspending the magnetic material prepared in the step 1) in 0.1-1ml of acetonitrile or methanol water solution, adding 0.01ml of dithiothreitol, oscillating to break bonds for 1-24h, magnetically separating after breaking bonds, sucking the solution, diluting 5 times, centrifuging, and using for subsequent mass spectrum detection and analysis.
The beneficial effect of adopting above-mentioned further scheme lies in: the method is simpler and more convenient to operate, is not limited by a large instrument (such as a high-speed centrifugal machine), realizes thorough separation of materials and solutions, and is more beneficial to follow-up experiments.
The invention has the beneficial effects that: the invention provides a brand new magnetic material, a preparation method and an application method of the material for selectively capturing amino compounds in complex samples. The magnetic material is formed by fixing amino pre-column derivatization reagent Phenylisothiocyanate (PITC) and analogues thereof on the magnetic material through a section of connecting chain. The isothiocyanate group on the material can be selectively combined with the amino compound through an addition reaction to form covalent bonding. While components of the sample that do not react with isothiocyanate groups can be removed by simple magnetic separation and washing. In addition, disulfide bonds are contained in the connecting chain, and the amino-containing magnetic material is stable in the amino capturing and washing processes, and can be selectively broken in the presence of reducing agents such as dithiothreitol and the like, so that the captured amino-containing compound can be broken off from the magnetic material. The bond-breaking solution can be directly used for subsequent experimental analysis after simple magnetic separation. The invention realizes rapid and efficient selective capture, purification and release of amino compounds in complex samples. The captured amino compound is derivative-connected with a section of fat-soluble fragment, so that the separation effect of the amino compound in a reverse chromatographic column is improved; meanwhile, the cleavage fragments have strong ultraviolet absorption, are compatible with an ultraviolet detector, and provide a multi-dimensional detection scheme. The magnetic material has the advantages of mature technical route, high efficiency in the amino capturing process, simplicity in operation and low cost, can realize high-flux operation, and has good application prospect.
Compared with the traditional amino analysis method, the method provided by the invention has the advantages of mild enrichment condition, low-cost and easily obtained materials, easy preparation, high capture specificity and remarkable effect. The method provided by the invention can be used for capturing amino groups, can remove a large amount of matrix interferences such as phospholipids, various salts and the like, is beneficial to subsequent mass spectrometry, can more truly represent the types and concentration levels of amino compounds in samples by taking isotopically-labeled metabolites as internal standards, and find out new amino compounds, thereby having important significance for biomarker screening research, early warning of serious diseases, disease development, prognosis evaluation and the like; provides a new thought and a new method for detecting amino small molecules in complex samples, screening related tumor markers and the like.
Drawings
FIG. 1 is a scanning electron microscope photograph of a silica amino magnetic core coated with ferroferric oxide in 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.
In the embodiment of the invention, the following steps are included: pipette tips were purchased from Axygenscientific.
Example 1
The preparation method of the material for specifically capturing the ammonia compound from the complex sample comprises the following steps:
(1) Amino core linked cysteamine succinic acid (cystamine succinic acid) chain:
(1-1) preparation of Fmoc-cystamine succinate (Fmoc-CS) side chain:
cystamine dihydrochloride (2.25 g,10 mmol) was dissolved in 10mL of water, and sodium bicarbonate (2.25 g,30 mmol) was added and stirring continued. 100mL dioxane was then added and the ice bath was cooled for 10 minutes. Succinic anhydride (1.00 g,10 mmol) was added to the above reaction system, and the resulting reaction system was stirred at room temperature for a further 12 hours. After the completion of the reaction, 3.3mL of N, N-diisopropylethylamine was added to the reaction system in an ice bath, 9-fluorenylmethyl-N-succinimidyl carbonate (3.37 g,10 mmol) was accurately weighed and dissolved in 10mL of N, N-dimethylformamide, followed by dropwise addition to the reaction system. The reaction was returned to room temperature and stirring was continued for 2 hours. After completion of the reaction, filtration was carried out, and the filtrate was concentrated in vacuo to be viscous, after which 200mL of water was added and stirring was continued, yielding a large amount of white solid in the filtrate. The white solid was collected by filtration, washed with water and dried under vacuum to give 2.23g of fluorenylmethoxycarbonyl cysteamine succinic acid (47%). The resulting solid was used directly in the subsequent reaction without purification.
(1-2) magnetic material attached to the fluorenylmethoxycarbonyl cysteamine succinic acid side chain:
fluorenylmethoxycarbonyl cysteamine succinic acid (5 mg) and 5mg of 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate were mixed, 1.0mL of dimethyl sulfoxide was added for dissolution, and then 0.5mL of LN, N-diisopropylethylamine was added, and the mixture was uniformly mixed, added to the above magnetic material, and continued shaking was performed. After the reaction is completed, the magnetic material with the fluorenylmethoxycarbonyl cysteamine succinic acid side chain connected is obtained by magnetic separation, solvent removal, washing and centrifuge tube replacement.
(1-3) side chain de-fluorenylmethoxycarbonyl cysteamine succinic acid protection of magnetic material:
to a centrifuge tube containing a magnetic material, 0.5mL of fluorenylmethoxycarbonyl cysteamine succinic acid solution was added, and 0.5mL of 1, 8-diazabicyclo [5.4.0] undec-7-ene was added. And (3) oscillating for 1 hour, washing and replacing the centrifuge tube after the reaction is completed, and obtaining the magnetic material containing the cystamine side chain.
(2) Magnetic material derived isocyanate groups:
(2-1) preparation of 4-carboxyphenyl isothiocyanate:
weighing 4-aminobenzoic acid (5.00 g,36.5 mmol) and adding into 51mL of dichloromethane, adding 5.6mL of triethylamine under the magnetic stirring condition, carrying out ice bath, then taking thiocarbonyldiimidazole (8.45 g,47.4 mmol), adding into the reaction system, continuously stirring, further taking 12mL of hydrochloric acid and adding 50mL of petroleum ether, mixing and oscillating, then dropwise adding into the reaction system, continuously reacting after the dropwise adding is completed, generating a large amount of white precipitate in the reaction system, filtering, washing with water to obtain a light yellow solid, and evaporating water to obtain a white solid, namely 4-carboxyphenyl isothiocyanate;
(2-2) 2.5mg of 4-carboxyphenyl isothiocyanate was mixed with 10mg of 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, 5mL of dimethyl sulfoxide was added for dissolution, 0.5mL of LN, N-diisopropylethylamine was added, and after mixing uniformly, the mixture was added to a magnetic material, and the resultant reaction system was continued to oscillate for 1 hour. After the reaction is completed, the solvent is removed by magnetic separation, and the centrifuge tube is replaced for standby.
Example 2 covalent capture of amino compounds in serum by magnetic material:
1) 0.5mg of the magnetic material prepared in example 1 was taken, 0.6mL of dimethyl sulfoxide was added, 0.03mL of serum was added, 0.05mL of triethylamine was added, and the mixture was shaken at 25℃for 3 hours. After the reaction is completed, the solvent is removed by magnetic separation, and the centrifuge tube is replaced for standby after washing by phosphate buffer solution, water washing and acetonitrile and water mixed solution in sequence.
2) Capturing the obtained amino compound to break bonds:
the magnetic material of step 1) was suspended in 0.5mL of acetonitrile in water, and 0.01mL of dithiothreitol (0.1M) was added thereto and the bond was broken by shaking for 12 hours. After the bond breaking is completed, the solution is magnetically separated, sucked, diluted 5 times and centrifuged for subsequent mass spectrometry detection and analysis. Ultra-high resolution mass spectrometry results show that the method can detect more than 1000 metabolism-related mass spectrum peaks. By aligning the human metabonomics database (HMDB, humanMetabolomeDatabase), more than 150 target metabolite molecules were found, with partial mass spectral results as shown in the attached table 1.
TABLE 1 results of the serum-enriched fraction of the magnetic material prepared in example 1
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (9)
1. A material for specifically capturing an ammonia compound from a complex sample is characterized by having a structure shown in a general formula (I):
wherein, the black round dot is a magnetic core with magnetic response, X is different substituents on amino, Y is
The side chain contains cystamine, wherein disulfide bonds are selective breaking groups; z in the side chain is 1, 4-phenylene; the tail end of the side chain contains an isothiocyanate group, which is used for specifically capturing amino compounds.
2. The material for specifically capturing ammonia compounds from complex samples according to claim 1, wherein the magnetic core material with magnetic response is iron, cobalt, nickel, ferroferric oxide or a coating material containing the material.
3. The material for specifically capturing ammonia compounds from complex samples according to claim 2, wherein the coating material is one of aminated silica, polystyrene and polyacrylic acid.
4. A method for preparing a material for specifically capturing an ammonia compound from a complex sample according to any one of claims 1 to 3, comprising the steps of:
(1) The amino magnetic core is connected with a cystamine succinic acid chain, and the synthetic route is as follows:
the method comprises the following steps:
(1-1) preparation of a fluorenylmethoxycarbonyl cysteamine succinic acid side chain:
dissolving cystamine dihydrochloride in water, adding sodium bicarbonate, continuously stirring, adding dioxane, stirring in an ice bath for 10 minutes, adding succinic anhydride into the reaction system, stirring the obtained reaction system at room temperature for 12 hours, adding N, N-diisopropylethylamine into the reaction system in an ice bath after the reaction is completed, accurately weighing 9-fluorenylmethyl-N-succinimidyl carbonate into N, N-dimethylformamide, dropwise adding the solution into the reaction system, recovering the reaction system to room temperature, continuously stirring for 2 hours, filtering after the reaction is completed, concentrating the filtrate in vacuum to be sticky, adding water, continuously stirring, generating a large amount of white solids in the filtrate, filtering and collecting the white solids, washing and drying in vacuum to obtain fluorenylmethoxycarbonyl cysteamine succinic acid, wherein the obtained solids are directly used for subsequent reactions without purification;
(1-2) magnetic material attached to the fluorenylmethoxycarbonyl cysteamine succinic acid side chain:
mixing fluorenylmethoxycarbonyl cysteamine succinic acid with 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethyl urea hexafluorophosphate, adding dimethyl sulfoxide for dissolution, then adding N, N-diisopropylethylamine, uniformly mixing, adding into the magnetic beads, continuing to oscillate, connecting Fmoc-CS, magnetically separating after connection is completed, removing a solvent, washing, and replacing a centrifuge tube to obtain the magnetic beads connected with Fmoc-CS;
(1-3) side chain defluorene methoxycarbonyl group of magnetic material:
adding N, N-dimethylformamide solution into a centrifuge tube containing magnetic beads, adding 1, 8-diazabicyclo [5.4.0] undec-7-ene, performing post-shaking reaction for 1 hour, and washing and replacing the centrifuge tube to obtain magnetic beads containing cystamine side chains;
(2) The magnetic material is derived from isothiocyanate groups, and the synthetic route is as follows:
(2-1) preparation of 4-carboxyphenyl isothiocyanate:
weighing 4-aminobenzoic acid, adding into dichloromethane, adding triethylamine and ice bath under the magnetic stirring condition, then taking thiocarbonyldiimidazole, adding into the reaction system, continuously stirring, then taking hydrochloric acid, adding petroleum ether, mixing and oscillating, then dropwise adding into the reaction system, continuously reacting after the dropwise adding is completed, generating a large amount of white precipitate in the reaction system, filtering, washing with water to obtain light yellow solid, and evaporating water to obtain white solid, namely 4-carboxyphenyl isothiocyanate;
(2-2) magnetic material derived isocyanate groups:
mixing 4-carboxyphenyl isothiocyanate with 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethyl urea hexafluorophosphate, adding dimethyl sulfoxide for dissolution, adding N, N-diisopropylethylamine, mixing uniformly, adding into magnetic beads after oscillation, continuing oscillation of the obtained reaction system, removing solvent by magnetic separation after reaction is completed, washing, and replacing a centrifuge tube for standby.
5. The method for preparing a material for specifically capturing ammonia compounds from complex samples according to claim 4, wherein the mass ratio of cystamine dihydrochloride to sodium bicarbonate in the step (1-1) is 1:1;
the volume ratio of the substance of cystamine dihydrochloride to dioxane was 10mmol:100ml;
the molar ratio of cystamine dihydrochloride to succinic anhydride is 1:1;
the volume ratio of the substance of cystamine dihydrochloride to N, N-diisopropylethylamine was 10mmol:3.3ml;
the molar ratio of cystamine dihydrochloride to 9-fluorenylmethyl-N-succinimidyl carbonate is 1:1;
the volume ratio of the substance of cystamine dihydrochloride to N, N-dimethylformamide was 10mmol:10ml.
6. The method for preparing a material for specifically capturing ammonia compounds from complex samples according to claim 4, wherein the mass ratio of the fluorenylmethoxycarbonyl cysteamine succinate to the 2- (7-azobenzotriazole) -N, N' -tetramethylurea hexafluorophosphate in the step (1-2) is 1:1;
the volume ratio of the mass of the fluorenylmethoxycarbonyl cysteamine succinic acid to the dimethyl sulfoxide is 1-10mg:1ml;
the volume ratio of the mass of the fluorenylmethoxycarbonyl cysteamine succinic acid to the N, N-diisopropylethylamine is 1-10mg:0.1-1ml.
7. The method for preparing a material for specifically capturing ammonia compounds from a complex sample according to claim 4, wherein the amount of said N, N-dimethylformamide solution used in the step (1-3) is 0.5ml;
the 1, 8-diazabicyclo [5.4.0] undec-7-ene was used in an amount of 0.5ml.
8. The method for preparing a material for specifically capturing ammonia compounds from a complex sample according to claim 4, wherein in the step (2-1)
The ratio of the mass of 4-aminobenzoic acid to the volume of dichloromethane was 5g:51ml;
the dosage of triethylamine is 5.6ml;
the dosage of thiocarbonyldiimidazole is 8.45g;
the dosage of hydrochloric acid is 12ml, and the dosage of petroleum ether is 50ml;
the mass ratio of the 4-carboxyphenyl isothiocyanate to the 2- (7-azobenzene triazole) -N, N, N ', N' -tetramethyl urea hexafluorophosphate in the step (2-2) is 15:1-20;
the dosage of dimethyl sulfoxide is 1-10ml;
the dosage of N, N-diisopropylethylamine is 0.1-1ml.
9. A method for using the material for specifically capturing ammonia compounds from complex samples according to any one of claims 1 to 3, comprising the steps of:
1) Taking the material, adding dimethyl sulfoxide, serum and triethylamine, oscillating, removing the solvent by magnetic separation after the reaction is completed, washing with phosphate buffer solution, washing with water, washing with mixed solution of acetonitrile and water, and replacing a centrifuge tube for later use;
2) Capturing the obtained amino compound to break bonds:
suspending the magnetic material prepared in the step 1) in acetonitrile or methanol water solution, adding dithiothreitol, oscillating to break bonds, magnetically separating after the break bonds are completed, sucking the solution, diluting, centrifuging, and using for subsequent mass spectrometry detection analysis.
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