TW202227814A - Method and kit for detecting biogenic amine - Google Patents

Abstract

A method for detecting biogenic amine is used to solve the problem that the conventional method is not suitable for detecting biogenic amine. The method for detecting biogenic amine includes providing a sample with biogenic amine. The sample, a derivatization reagent (coumarin-3-carboxylic acid, C-3-CA), a coupling reagent and an alkaline working solution are mixed to form a mixture, while an amidation reaction between the biogenic amines in the sample and the derivatization reagent in the presence of the coupling reagent, forming a derivative of biogenic amine. The derivative of biogenic amine in a derivatization solution is then detected to obtain a value of biogenic amine. A kit for detecting biogenic amine is also disclosed.

Description

Detection method and detection kit of biogenic amines

The present invention relates to a detection method, especially a detection method of biogenic amines; the present invention also relates to a detection kit for implementing the detection method of biogenic amines.

Biogenic amine is a biological substance with one or more amine groups, which is a basic nitrogenous compound, mainly through the decarboxylation of amino acids ( decarboxylation) or by the amination and transamination of aldehydes and ketones.

The human body contains a variety of endogenous biogenic amines (endogenous biogenic amine), which can be produced from different tissues, such as epinephrine (epinephrine), norepinephrine (norepinephrine), dopamine (dopamine), serotonin (serotonin) , 5-hydroxyindole-3-acetic acid (5-HIAA), metanephrine (metanephrine), normetanephrine (normetanephrine) and 3-methoxytyramine (3-methoxytyramine) , referred to as 3-MT) and so on. The amount of the aforementioned endogenous biogenic amines in the body may be helpful for the diagnosis of diseases; for example, free metanephrine in plasma or urine can be used for the diagnosis of pheochromocytoma (phaeochromocytoma); platelet-rich Tryptophan (tryptophan), 5-hydroxytryptophan (5-HTP), serotonin and 5-hydroxyindoleacetic acid in plasma can be used for neuroendocrine tumor (NET) Catecholamine (catecholamine), serotonin and 5-hydroxyindoleacetic acid in cerebrospinal fluid or urine can be used for the diagnosis of neuroblastoma.

In addition, biogenic amines exist in a variety of foods (especially in fermented foods such as cheese, wine, beer, rice wine, meat, etc.), and the accumulation of biogenic amines in food is usually produced by the decarboxylation of free amino acids by bacterial-derived enzymes Therefore, biogenic amines are recognized as indicators that can be used to judge the freshness of food, and biogenic amines are also potentially toxic to the human body. Therefore, many countries in the world have formulated limit standards for the content of biogenic amines in food. For example, the United States stipulates that the histamine content in aquatic products shall not exceed 50 mg/kg, the European Union stipulates that the histamine content in food shall not exceed 100 mg/kg, and the tyramine content shall not exceed 100-800 mg/kg; some European countries There are also recommended upper limits for biogenic amines in wine.

However, since most biogenic amines are highly polar substances, they do not have specific ultraviolet luminophores, and they also lack fluorescent properties, making biogenic amines unsuitable for conventional detection methods. In view of this, it is still necessary to improve a biogenic amine detection method and detection kit.

In order to solve the above problems, the purpose of the present invention is to provide a method for detecting biogenic amines, which can effectively detect biogenic amines in a sample to be tested.

Another object of the present invention is to provide a method for detecting biogenic amines, which can shorten the time course of detection.

Another object of the present invention is to provide a biogenic amine detection kit, which is used to detect whether the sample to be tested contains biogenic amines.

The method for detecting biogenic amines of the present invention may include: providing a sample to be tested, the sample to be tested contains biogenic amines; using coumarin-3-carboxylic acid as a derivatizing reagent, mixing the sample to be tested, the derivatizing reagent, A coupling reagent and an alkaline working solution are used to obtain a mixed solution to be reacted, and the derivatization reagent and the biogenic amine in the sample to be tested are subjected to an amidation reaction with the aid of the coupling reagent to obtain a solution dissolved in a derivatized amine. A biogenic amine derivative in the solution, the biogenic amine derivative is a labeled biogenic amine substituted on the amine group; and the derivative solution is used as a solution to be tested, and the biogenic amine derivative in the solution to be tested is detected. Obtain a biogenic amine strength value.

Accordingly, by making the amine group of the biogenic amine in the test sample and the carboxyl group of coumarin-3-carboxylic acid to carry out the amidation reaction with the aid of the coupling reagent, the amine group can be obtained Displaced labeled biogenic amine derivatives, the presence of which can be detected by various conventional methods (e.g., after separation by liquid chromatography, analysis by UV spectroscopy, fluorescence spectroscopy, or mass spectrometry) Therefore, the method for detecting biogenic amines of the present invention has good sensitivity, precision and accuracy, and can reduce the usage amount of the sample to be tested, which is the effect of the present invention.

The method for detecting biogenic amines of the present invention, wherein the coupling reagent can be (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinocarbonium Hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate or 2-(7-benzotriazole oxide)-N,N, N',N'-tetramethylurea hexafluorophosphate; in this way, the coupling reagent can assist the formation of the amide bond to promote the amide amination reaction.

The detection method of biogenic amine of the present invention, wherein, the alkaline working solution is formed by dissolving an organic base compound in a working solvent, and the organic base compound can be 4-dimethylaminopyridine, N-methylmorpholine, imidazole, or triethylamine, and the working solvent can be acetonitrile; in this way, the organic base compound can convert the carboxyl group of coumarin-3-carboxylic acid into a carboxylate anion, so that the coupling reagent can assist the amide progress of the chemical reaction.

The method for detecting biogenic amines of the present invention, wherein the mixed solution to be reacted further comprises an extraction solvent and a salt solution, and after the amidation reaction, the derivative solution is layered to form an upper layer solution and a lower layer solidified product , continue to use the upper layer solution as the solution to be tested, detect the biogenic amine derivatives in the solution to be tested, the extraction solvent can be acetonitrile, dimethyl carbonate, ethyl acetate or dichloromethane, and the salt solution can be It is an aqueous solution of disodium hydrogen phosphate; in this way, the extraction solvent and the salt solution can work together to remove the derivatizing reagent (coumarin-3-carboxylic acid) or the coupling reagent added in excess in the derivatizing solution, and remove the The by-products generated by the amidation reaction are further enriched in the biogenic amine derivative, so that the biogenic amine derivative can be more easily detected to obtain the biogenic amine intensity value.

Based on the same technical concept, the biogenic amine detection kit of the present invention may include: a derivatizing reagent, the derivatizing reagent is coumarin-3-carboxylic acid, and is used for the detection of the biogenic amine in the sample to be tested. An amidation reaction to form a biogenic amine derivative, the biogenic amine derivative is a labeled biogenic amine substituted on the amine group; a coupling reagent for promoting the amidation reaction; and a basic working The solution is used to form a mixed solution to be reacted together with the sample to be tested, the coupling reagent and the derivatizing reagent, and the alkaline working solution contains a working solvent that can dissolve the derivatizing reagent, the coupling reagent and the biogenic amine.

Accordingly, the detection kit can be applied to implement the aforementioned detection method of biogenic amines, and can effectively form biogenic amine derivatives with good stability together with biogenic amines. Therefore, workers can select various conventional methods for detection according to their needs. The biogenic amine derivatives (eg, separated by liquid chromatography and detected by ultraviolet spectroscopy, fluorescence spectroscopy, or mass spectrometry) are the effects of the present invention.

The biogenic amine detection kit of the present invention, wherein, the coupling reagent can be (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinocarbon Onium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate or 2-(7-benzotriazole oxide)-N,N ,N',N'-tetramethylurea hexafluorophosphate; in this way, the coupling reagent can assist the formation between the amine group of the biogenic amine in the test sample and the carboxyl group of the coumarin-3-carboxylic acid The amide bond to promote the amide amination reaction.

The biogenic amine detection kit of the present invention, wherein, the alkaline working solution is formed by dissolving an organic base compound in the working solvent, and is used to activate the carboxyl group of the derivatizing reagent, and the organic base compound can be 4-dimethyl aminopyridine, N-methylmorpholine, imidazole, or triethylamine, and the working solvent can be acetonitrile; thus, the organic base compound can convert the carboxyl group of coumarin-3-carboxylic acid into a carboxylate anion , so that the coupling reagent assists the amidation reaction.

The biogenic amine detection kit of the present invention further comprises: an extraction solvent, which is an aprotic solvent, and is used to dissolve the biogenic amine derivative; and a salt solution, which is immiscible with the extraction solvent and used to form Salting out effect, the extraction solvent can be acetonitrile, dimethyl carbonate, ethyl acetate or dichloromethane, and the salt solution can be an aqueous solution of disodium hydrogen phosphate; in this way, the extraction solvent and the salt solution can act together to Remove the derivatizing reagent (coumarin-3-carboxylic acid) or the coupling reagent added in excess in the derivatizing solution, and remove the by-products generated by the amidation reaction, and then enrich the biogenic amine derivative, which can Make the biogenic amine derivative easier to detect to obtain the biogenic amine strength value.

In order to make the above-mentioned and other objects, features and advantages of the present invention more obvious and easy to understand, the preferred embodiments of the present invention are exemplified below, and are described in detail as follows in conjunction with the accompanying drawings:

Referring to FIG. 1 , the method for detecting biogenic amines according to the first embodiment of the present invention may include: a sample providing step S1 , a derivatization step S2 and an analysis step S3 .

Specifically, in the sample providing step S1, a sample to be tested containing biogenic amine is provided. For example, the sample to be tested can be from a food sample or from a biological sample (eg, a blood sample, a urine sample, or a cerebrospinal fluid sample). When the sample to be tested is the food sample from a liquid state, the derivatization step S2 can be directly performed on the sample to be tested, and when the sample to be tested is the food sample from a solid state, the food sample can be added to water, Ultrasonic shock was used to dissolve the biogenic amines in the food sample in water, and a supernatant was obtained after centrifugation (14,800 rpm, 1 minute), and the supernatant was used as the sample to be tested in the derivatization step S2.

It is worth noting that, due to the complex composition of the biological sample, the protein in the biological sample must be removed before the derivatization step S2, so as to prevent the protein from interfering with the subsequent derivatization step S2 and affecting the monoamidation The benefit of the amidation reaction; for example, the worker can first mix the biological sample with a protein precipitant, which can be acetone or acetonitrile (ACN), so that The protein in the blood sample forms a protein precipitate, and the protein precipitate is removed by centrifugation (14,800 rpm, 1 min) to obtain a supernatant, which can be used as the subsequent derivatization step S2 sample to be tested.

In the derivatization step S2, the worker can use coumarin-3-carboxylic acid (C-3-CA) as a derivatization reagent, and dissolve the derivatization reagent in a Alkaline working solution, and adding the sample to be tested in it, then a mixed solution to be reacted can be prepared. In this way, the derivatization reagent and the biogenic amine in the sample to be tested are carried out as shown in Figure 2 The amidation reaction shown, makes an amide bond (amide bond) to form a carboxyl group (-COOH) of the derivatizing reagent and an amino group (amino group, ie, -COOH) of the biological amine in the test sample. ie -NH ₂ ) to obtain a biogenic amine derivative, the biogenic amine derivative is a labeled biogenic amine substituted on the amine group. For example, in the mixture to be reacted, the concentration of the derivatizing reagent (coumarin-3-carboxylic acid, C-3-CA) can be 25-100 mM; in this embodiment, the concentration of the derivatizing reagent to 75 mM.

The alkaline working solution can be formed by dissolving an organic base compound in a working solvent, the organic base compound can convert the carboxyl group of the derivatizing reagent into a carboxylate anion (-COO) ^- ), so that the coupling reagent can assist the amidation reaction. For example, the working solvent can be acetonitrile, acetone, dimethyl sulfoxide (DMSO for short) or dimethyl carbonate (DMC for short), and the organic base compound can be 4-dimethylamino Pyridine (4-dimethylaminopyridine, referred to as DMAP), N-Methylmorpholine ( N -Methylmorpholine, referred to as NMM), imidazole (imidazole) or triethylamine (triethylamine, referred to as TEA). The concentration of the base compound can be 50-180 mM; in this embodiment, the working solvent is acetonitrile, the organic base compound is 4-dimethylaminopyridine (DMAP), and the concentration of the organic base compound is 140 mM.

Since the formation of the amide bond between the carboxyl group and the amine group is slow, the worker can add a coupling reagent to assist the amide amination reaction when preparing the mixture to be reacted. For example, the coupling reagent may be (1-cyano-2-ethoxy-2-oxoethyleneamine) capable of forming an activated ester-mediated coupling reaction Oxy) dimethylamino morpholinocarbonium hexafluorophosphate ((1-cyano-2-ethoxy-2-oxoethylidenaminooxy) dimethylamino-morpholino-carbenium hexafluorophosphate, referred to as COMU), O-benzotriazole-N ,N,N',N'-tetramethylurea tetrafluoroborate (O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, referred to as TBTU) or 2-(7 -benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate (1-[bis(dimethylamino)methylene]-1H-1,2,3- triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, referred to as HATU), and the concentration of the coupling reagent in the mixture to be reacted can be 50-150 mM; in this embodiment, the coupling reagent is (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinocarbonium hexafluorophosphate (COMU), and the concentration of the coupling reagent was 125 mM.

In order to accelerate the progress of the amidation reaction, the worker can apply an energy to the mixture to be reacted. For example, the worker can heat the mixture to be reacted in a dry bath at 30-70° C. for 1-20 minutes to promote the formation of the biogenic amine derivative; in this embodiment, the Heat in a dry bath at 30°C for 3 minutes.

Next, in the analysis step S3, the worker can use the derivative solution as a solution to be tested, and use various conventional methods to detect the biogenic amine derivatives in the solution to be tested to obtain a biogenic amine intensity value, and The biogenic amine intensity value is used to evaluate whether the test sample contains biogenic amines, or even the content of biogenic amines in the test sample. For example, workers can detect biogenic amine derivatives in the solution to be tested by UV spectroscopy, fluorescence spectroscopy or mass spectrometry after separation by liquid chromatography; for example, by combining UV light Detection is performed by capillary high performance liquid chromatography combined with UV detector (CapLC-UV for short), or by nanoscale liquid chromatography coupled to mass spectrometry (nanoscale liquid chromatography coupled to tandem mass spectrometry, referred to as nano LC-MS/MS) for detection.

In addition, please refer to Figure 3, because when the analysis step S3 is performed, the solution to be tested (the derivatization solution) may contain excessively added derivatization reagent (coumarin-3-carboxylic acid) or the coupling reagents, and the amidation reaction may also produce byproducts. The method for detecting biogenic amines in the second embodiment of the present invention may include: the sample providing step S1, a derivatization extraction step S2' and the analysis step S3.

In the derivatization extraction step S2', in addition to the sample to be tested, the derivatization reagent, the alkaline working solution and the coupling reagent, the mixed solution to be reacted may also include an extraction solvent and a salt solution ( salt solution), the extraction solvent is an aprotic solvent that can dissolve the biogenic amine derivative, which can assist the derivatization reaction to form the biogenic amine derivative, and can be extracted from the sample to be tested Extraction of biogenic amine derivatives; the salt solution is a solution immiscible with the extraction solvent, which can form a salting-out effect, so that excess derivatization reagents, by-products, etc. can dissolve with the biogenic amine derivative of the extraction solvent. For example, the extraction solvent can be acetonitrile, dimethyl carbonate, ethyl acetate (EA) or dichloromethane (DCM), and the salt solution can be a solution containing disodium hydrogen phosphate (disodium phosphate). hydrogen phosphate, Na ₂ HPO ₄ ) solution, and the concentration of the salt solution in the mixed solution to be reacted can be 100-1000 mM; in this embodiment, the extraction solvent is dimethyl carbonate, and the salt solution is Aqueous solution of disodium hydrogen phosphate at a concentration of 1000 mM.

After mixing the sample to be tested, the derivatization reagent, the alkaline working solution, the coupling reagent, the extraction solvent and the salt solution to obtain the mixed solution to be reacted, the worker can apply the mixed solution to the mixed solution to be reacted. After the energy is given, the salt solution undergoes a phase change, transforming from a liquid phase to a solid phase to form a solidified product, which is easier to dissolve with the salt solution. Extraction solvent separation of biogenic amine derivatives. For example, the worker can heat the to-be-reacted mixture in a dry bath at 30-70°C for 1-20 minutes, then take the to-be-reacted mixture in an ice bath, and then take the upper layer solution as the to-be-reacted mixture. The solution is measured, and the analysis step S3 is performed.

According to the foregoing, based on the same technical concept, the biogenic amine detection kit according to an embodiment of the present invention may include: the derivatizing reagent, the coupling reagent and the alkaline working solution, and the derivatizing reagent is coumarin-3 -Carboxylic acid, which is used for the amidation reaction with biogenic amine to form the biogenic amine derivative together, and the coupling reagent is used to promote the amidation reaction, such as (1-cyano- 2-Ethoxy-2-oxoethyleneaminooxy) dimethylaminomorpholinocarbonium hexafluorophosphate, O-benzotriazole-N,N,N',N'- Tetramethylurea tetrafluoroborate or 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate, the alkaline working fluid is the organic The base compound is formed by dissolving the working solvent. The organic base compound is used to convert the carboxyl group of the derivatizing reagent into a carboxylate anion, so that the coupling reagent can assist the amidation reaction. The working solvent is soluble Acetonitrile of the derivatizing reagent, the coupling reagent, the biogenic amine and the organic base compound.

The biogenic amine detection kit may further comprise: the extraction solvent and the salt solution, the extraction solvent is an aprotic solvent capable of dissolving the biogenic amine derivative, which can assist in the derivatization reaction. The biogenic amine derivative is formed, and the biogenic amine derivative can be extracted from the sample to be tested; the salt solution is a solution immiscible with the extraction solvent, which can form a salting-out (salting-out) effect, so that excessive Derivatization reagents, by-products, etc. can be layered with the extraction solvent in which the biogenic amine derivative is dissolved. For example, the extraction solvent can be acetonitrile, dimethyl carbonate, ethyl acetate or dichloromethane; the salt solution can be an aqueous solution of disodium hydrogen phosphate.

In order to confirm that the detection method of biogenic amines can be used to detect the content of biogenic amines in the sample to be tested, the following tests were carried out:

Preparation of biogenic amine standard solution: respectively mix histamine (His), putrescine (Put), spermidine (Spd), cadaverine dihdrochloride (Cad), spermine Biogenic amines such as spermine (Spm) and tyramine (Tyr) were dissolved in water to form a biogenic amine standard solution with a concentration of 400 μM.

Preparation of derivatization reagent solution: Dissolve organic base compounds such as 4-dimethylaminopyridine (DMAP), N-methylmorpholine (NMM), imidazole (imidazole) and triethylamine (TEA) in the working solvent ( acetonitrile) to form an alkaline working solution with a concentration of 140 mM, and then the derivatizing reagent (coumarin-3-carboxylic acid, C-3-CA) was dissolved in the aforementioned alkaline working solution to form a concentration of 75 mM mM derivatization reagent solution.

The preparation of the coupling reagent solution: (1-cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylaminomorpholinocarbonium hexafluorophosphate (COMU), O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU) and 2-(7-benzotriazole)-N,N,N' Coupling reagents such as ,N'-tetramethylurea hexafluorophosphate hexafluorophosphate (HATU) were dissolved in the working solvent (acetonitrile) to form a coupling reagent solution with a concentration of 125 mM.

Preparation of salt solution: Dissolve disodium hydrogen phosphate (Na ₂ HPO ₄ ) in water to form a 1 M salt solution.

(A) Selection of derivatizing reagents

Please refer to Table 1. In this test, coumarin-3-carboxylic acid (C-3-CA, Group A1), chromone-2-carboxylic acid (chromone-2-carboxylic acid, Group A2), Mefenamic acid (group A3), tert -cinnamic acid (group A4), 3-indoleacrylic acid (group A5), nalidixic acid , Group A6) and 1-naphthoic acid (1-naphthoic acid, Group A7) were used as the derivatizing reagents of each group, and the derivatizing reagents of each group were dissolved in the alkaline working solution (DMAP) to form a concentration of 75 mM derivatization reagent solution, take the derivatization reagent solution (75 μL), mix the coupling reagent solution (COMU, 75 μL) and biogenic amine standard solution (100 μL), then add the extraction solvent (DMC, 100 μL) and The salt solution (Na ₂ HPO _4(aq) , 400 μL) was shaken for 15 seconds, centrifuged at 14,800 rpm for 2 minutes, then reacted in a dry bath at 30°C for 3 minutes, and then subjected to an ice bath, and the upper layer solution was taken. As the solution to be tested, use thin layer chromatography (TLC) to observe whether each group can observe the generation of the biogenic amine derivative under the ultraviolet wavelength of 302 nm, and then use ImageJ software to quantify the results. Obtain this biogenic amine strength value.

Table 1, the formula of the mixed solution to be reacted in each group of this experiment group Derivatization reagent (dissolved in alkaline working solution) Coupling reagents extraction solvent salt solution A1 Coumarin-3-carboxylic acid (4-dimethylaminopyridine) (1-Cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinocarbonium hexafluorophosphate (COMU) Dimethyl Carbonate (DMC) Disodium hydrogen phosphate aqueous solution (Na ₂ HPO _4(aq) ) A2 Chromone 2-carboxylic acid (4-dimethylaminopyridine) A3 Mefenamic acid (4-dimethylaminopyridine) A4 Tertiary cinnamic acid (4-dimethylaminopyridine) A5 3-Indoleacrylic acid (4-dimethylaminopyridine) A6 Picolinic acid (4-dimethylaminopyridine) A7 1-Naphthoic acid (4-dimethylaminopyridine)

Please refer to Table 2. For the 6 biogenic amines tested, only coumarin-3-carboxylic acid (C-3-CA) of group A1 can successfully form the biogenic amine derivative, and Mefenamic acid of group A3 can only successfully form derivatives of histamine (His), spermidine (Spd), spermine (Spm) and tyramine (Tyr), while the color of group A2 chromone-2-carboxylic acid, tert-cinnamic acid of group A4, 3-indoleacrylic acid of group A5, pyridone of group A6 Neither nalidixic acid nor 1-naphthoic acid of Group A7 could form this biogenic amine derivative.

Table 2, the strength values of biogenic amines in each group of solutions to be tested in this test group Derivatization reagent Intensity (Crest Area) Histamine (His) Putrescine (Put) Spermidine (Spd) Cadaverine (Cad) Spermine (Spm) Tyramine (Tyr) A1 coumarin-3-carboxylic acid 804.39 1895.01 1228.39 866.49 559.50 487.35 A2 Chromone 2-carboxylic acid ─ ─ ─ ─ ─ ─ A3 mefenamic acid 64.43 0 114.14 0 125.44 117.34 A4 tertiary cinnamic acid ─ ─ ─ ─ ─ ─ A5 3-Indoleacrylic acid ─ ─ ─ ─ ─ ─ A6 pyridoxic acid ─ ─ ─ ─ ─ ─ A7 1-Naphthoic acid ─ ─ ─ ─ ─ ─

(B) Selection of organic base compounds

Please refer to Table 3. In this test, 4-dimethylaminopyridine (DMAP, Group B1), N-methylmorpholine (NMM, Group B2), imidazole (imidazole, Group B3), Or organic base compounds such as triethylamine (TEA, group B4), dissolved in the working solvent (acetonitrile) to form an alkaline working solution with a concentration of 140 mM, and then the derivatizing reagent (coumarin-3- Carboxylic acid, C-3-CA) was dissolved in the alkaline working solution of each group to form the derivatization reagent solution, take the derivatization reagent solution (75 μL), mix the coupling reagent solution (COMU, 75 μL) and biogenic amine Standard solution (100 μL), then the extraction solvent (DMC, 100 μL) and the salt solution (Na ₂ HPO ₄ , 400 μL) were added, after shaking for 15 seconds, centrifuged at 14,800 rpm for 2 minutes, and then at 30 The reaction was carried out in a dry bath at ℃ for 3 minutes, followed by an ice bath, and the upper layer solution was taken as the solution to be tested, and was quantified by capillary liquid chromatography (CapLC) to obtain the biogenic amine strength value.

Table 3, the formula of the mixed solution to be reacted in each group of this experiment group Derivatization reagent (dissolved in alkaline working solution) Coupling reagents extraction solvent salt solution B1 Coumarin-3-carboxylic acid (4-dimethylaminopyridine) (1-Cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinocarbonium hexafluorophosphate (COMU) Dimethyl Carbonate (DMC) Disodium hydrogen phosphate aqueous solution (Na ₂ HPO _4(aq) ) B2 Coumarin-3-carboxylic acid (N-methylmorpholine) B3 Coumarin-3-carboxylic acid (imidazole) B4 Coumarin-3-carboxylic acid (triethylamine)

The stationary phase used in capillary liquid chromatography was a C18 column (Waters XBridge BEH C18 XP, 2.1 mm × 100 mm; 2.5 μm), and the mobile phase contained solvent A (solvent A, 0.1 μm). % formic acid in water) and solvent B (solvent B, acetonitrile), gradient elution was performed under the conditions shown in Table 4, the detection wavelength was 294 nm, and the flow rate was 180 μL/min.

Table 4. Conditions for gradient elution used in capillary liquid chromatography time (min) Solvent A Solvent B 0 95 5 3 95 5 4 40 60 13 40 60 14 0 100 20 0 100

Please refer to Figure 4, whether it is 4-Dimethylaminopyridine (DMAP) from Group B1, N-Methylmorpholine (NMM) from Group B2, imidazole from Group B3, or Group B4 Triethylamine (TEA, group B4) of all groups can successfully form the biogenic amine derivatives, among which 4-dimethylaminopyridine (DMAP) of group B1 has the best effect.

(C) Choice of coupling reagents

Please refer to Table 5. In this test, (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinocarbonium hexafluorophosphate ( COMU, group C1), O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU, group C2), or 2-(7-benzooxybenzoate) Triazole)-N,N,N',N'-tetramethylurea hexafluorophosphate hexafluorophosphate (HATU, Group C3) and other coupling reagents to form a coupling reagent solution with a concentration of 125 mM. The derivatization reagent solution (coumarin-3-carboxylic acid, C-3-CA, dissolved in a basic working solution containing 4-dimethylaminopyridine, 75 μL), was mixed with the coupling reagent solutions of the previous groups (75 μL) and biogenic amine standard solution (100 μL), then add the extraction solvent (DMC, 100 μL) and the salt solution (Na ₂ HPO ₄ , 400 μL), shake for 15 seconds, and rotate at 14,800 rpm Centrifuge for 2 minutes, then react in a dry bath at 30°C for 3 minutes, then take an ice bath, take the upper layer solution as the solution to be tested, and quantify by capillary liquid chromatography (CapLC) to obtain the biogenic amine strength value.

Table 5, the formula of the mixed solution to be reacted in each group of this experiment group Derivatization reagent (dissolved in alkaline working solution) Coupling reagents extraction solvent salt solution C1 Coumarin-3-carboxylic acid (4-dimethylaminopyridine) (1-Cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinocarbonium hexafluorophosphate (COMU) Dimethyl Carbonate (DMC) Disodium hydrogen phosphate aqueous solution (Na ₂ HPO _4(aq) ) C2 O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU) C3 2-(7-Benzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate Hexafluorophosphate (HATU)

Please refer to Figure 5, whether it is Group C1 (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinocarbonium hexafluorophosphate Salt (COMU), O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU) of group C2 or 2-(7-oxidation of group C3 Benzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate hexafluorophosphate (HATU) can successfully form the biogenic amine derivatives, in which the group C1 ( 1-Cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylaminomorpholinocarbonium hexafluorophosphate (COMU) works best.

(D) Choice of extraction solvent

Please refer to Table 6. In this test, acetonitrile (ACN, Group D1), dimethyl carbonate (DMC, Group D2), ethyl acetate (EA, Group D3) or dichloromethane (DCM, Group D4) etc. as the extraction solvent, take the derivatization reagent solution (coumarin-3-carboxylic acid, C-3-CA, dissolved in the basic working solution containing 4-dimethylaminopyridine, 75 μL ), mixed the coupling reagent solution (COMU, 75 μL) and the biogenic amine standard solution (100 μL), and then added the extraction solvent (100 μL) of the previous groups and the salt solution (Na ₂ HPO ₄ , 400 μL) After shaking for 15 seconds, centrifuge at 14,800 rpm for 2 minutes, then react in a dry bath at 30 °C for 3 minutes, and then take an ice bath. ) were quantified to obtain this biogenic amine intensity value.

Table 6, the formula of the mixed solution to be reacted in each group of this experiment group Derivatization reagent (dissolved in alkaline working solution) Coupling reagents extraction solvent salt solution D1 Coumarin-3-carboxylic acid (4-dimethylaminopyridine) (1-Cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinocarbonium hexafluorophosphate (COMU) Acetonitrile (ACN) Disodium hydrogen phosphate aqueous solution (Na ₂ HPO _4(aq) ) D2 Dimethyl Carbonate (DMC) D3 Ethyl acetate (EA) D4 Dichloromethane (DCM)

Please refer to Figure 6, whether it is acetonitrile (ACN) in group D1, dimethyl carbonate (DMC) in group D2, ethyl acetate (EA) in group D3 or dichloromethane in group D4 ( DCM) can successfully form the biogenic amine derivatives, among which the effect of group D2 dimethyl carbonate (DMC) is the best.

(E) Choice of salt solution

Please refer to Table 7. In this test, salts such as disodium hydrogen phosphate (Na ₂ HPO ₄ , Group E1) or sodium sulfate (Na ₂ SO ₄ , Group E2) are dissolved in water to form a concentration of 1000 mM salt solution, take the derivatization reagent solution (coumarin-3-carboxylic acid, C-3-CA, dissolved in a basic working solution containing 4-dimethylaminopyridine, 75 μL), mix the couple Combine reagent solution (COMU, 75 μL) and biogenic amine standard solution (100 μL), then add the extraction solvent (dichloromethane, 100 μL) and the salt solutions (400 μL) of the previous groups, and after shaking for 15 seconds, Centrifuge at 14,800 rpm for 2 minutes, then react in a dry bath at 30°C for 3 minutes, and then take an ice bath. The upper layer solution was taken as the solution to be tested and analyzed by capillary liquid chromatography (CapLC).

Table 7, the formula of the mixed solution to be reacted in each group of this experiment group Derivatization reagent (dissolved in alkaline working solution) Coupling reagents extraction solvent salt solution E1 Coumarin-3-carboxylic acid (4-dimethylaminopyridine) (1-Cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinocarbonium hexafluorophosphate (COMU) Dimethyl Carbonate (DMC) Disodium hydrogen phosphate aqueous solution (Na ₂ HPO _4(aq) ) E2 Aqueous sodium sulfate solution (Na ₂ SO _4(aq) )

Please refer to Figure 7. Disodium hydrogen phosphate (Na ₂ HPO ₄ ) in group E1 can form histamine (His), putrescine (Put), spermidine (Spd), cadaverine (Cad), Derivatives of biogenic amines such as spermine (Spm) and tyramine (Tyr), the peak positions of which are shown in 1 to 6; while the sodium sulfate (Na ₂ SO ₄ ) of group E2 is retained in the 12th to 14th minutes. Time interferes, so the individual biogenic amine derivatives cannot be effectively separated.

(F) Comparison of the first embodiment and the second embodiment

Please refer to Table 8. In this experiment, the derivatization reagent solution (coumarin-3-carboxylic acid, C-3-CA, was dissolved in an alkaline working solution containing 4-dimethylaminopyridine, 75 μL ), mix the coupling reagent solution (COMU, 75 μL) and water (100 μL), then add the extraction solvent (dichloromethane, 100 μL) and the salt solution (disodium hydrogen phosphate, 400 μL), shake for 15 After 2 seconds, centrifuge at 14,800 rpm for 2 minutes, then react in a dry bath at 30 °C for 3 minutes, and then take an ice bath. ) for analysis.

The derivatizing reagent solution (coumarin-3-carboxylic acid, C-3-CA, dissolved in a basic working solution containing 4-dimethylaminopyridine, 75 μL), mixed with the coupling reagent solution (COMU , 75 μL) and biogenic amine standard solution (100 μL), then add the extraction solvent (dichloromethane, 100 μL) and the salt solution (disodium hydrogen phosphate, 400 μL), after shaking for 15 seconds, at 14,800 rpm Centrifuge at 30°C for 2 minutes, then react in a dry bath at 30 °C for 3 minutes, and then take an ice bath. The upper layer solution was taken as the solution to be tested in Group F1 and analyzed by capillary liquid chromatography (CapLC).

and the derivatization reagent solution (coumarin-3-carboxylic acid, C-3-CA, dissolved in an alkaline working solution containing 4-dimethylaminopyridine, 75 μL), mixed with the coupling reagent solution ( COMU, 75 μL) and biogenic amine standard solution (100 μL), shake for 15 seconds, centrifuge at 14,800 rpm for 2 minutes, then react in a dry bath at 30°C for 3 minutes as the test solution for group F2 , and analyzed by capillary liquid chromatography (CapLC).

Table 8, the formula of the mixed solution to be reacted in each group of this experiment group Sample to be tested Derivatization reagent (dissolved in alkaline working solution) Coupling reagents extraction solvent salt solution F0 water Coumarin-3-carboxylic acid (4-dimethylaminopyridine) (1-Cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinocarbonium hexafluorophosphate (COMU) Dimethyl Carbonate (DMC) Disodium hydrogen phosphate aqueous solution (Na ₂ HPO _4(aq) ) F1 Biogenic amine standard Dimethyl Carbonate (DMC) Disodium hydrogen phosphate aqueous solution (Na ₂ HPO _4(aq) ) F2 - -

Please refer to FIG. 8 , compared with the F2 group without the extraction solvent and the salt solution (ie, the method for detecting biogenic amines in the first embodiment), the F1 group with the extraction solvent and the salt solution added The group (ie, the method for detecting biogenic amines in the second embodiment) can relatively clearly separate histamine (His), putrescine (Put), spermidine (Spd), cadaverine (Cad), spermine (Spm) And tyramine (Tyr) and other biogenic amine derivatives, the peak positions are shown in 1-6.

(G) Mass spectrometry identification of biogenic amine derivatives

Please refer to Table 9. In this test, histamine (His, group G1), putrescine (Put, group G2), spermidine (Spd, group G3), cadaverine (Cad, group G4) Group), spermine (Spm, Group G5), or tyramine (Tyr, Group G6) and other biogenic amines were dissolved in water to form a 400 μM biogenic amine standard solution, and the derivatizing reagent solution (Coumarin -3-Carboxylic acid, C-3-CA, dissolved in a basic working solution containing 4-dimethylaminopyridine, 75 μL), mix the coupling reagent solution (COMU, 75 μL) and biogenic amine standard solution (100 μL), then the extraction solvent (dichloromethane, 100 μL) and the salt solution (disodium hydrogen phosphate, 400 μL) were added, after shaking for 15 seconds, centrifuged at 14,800 rpm for 2 minutes, and then at 30 The reaction was performed in a dry bath at ℃ for 3 minutes, followed by an ice bath, and the upper layer solution was taken as the solution to be tested. Nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoscale liquid chromatography coupled to tandem mass spectrometry, referred to as nano LC- MS/MS) for detection.

The ninth table, the formula of the mixed solution to be reacted in each group of this test group Sample to be tested Derivatization reagent (dissolved in alkaline working solution) Coupling reagents extraction solvent salt solution G1 Histamine (His) Coumarin-3-carboxylic acid (4-dimethylaminopyridine) (1-Cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinocarbonium hexafluorophosphate (COMU) Dimethyl Carbonate (DMC) Disodium hydrogen phosphate aqueous solution (Na ₂ HPO _4(aq) ) G2 Putrescine (Put) G3 Spermidine (Spd) G4 Cadaverine (Cad) G5 Spermine (Spm) G6 Tyramine (Tyr)

The stationary phase used in the nano-liquid chromatography tandem mass spectrometry method is a C18 column (Waters ACQUITY UPLC BEH C18, 75 mm × 50 mm; 1.7 μm), and the mobile phase contains a solvent A (solvent A, 0.1% formic acid in water) and solvent B (solvent B, 0.1% formic acid in acetonitrile) were subjected to gradient elution under the conditions shown in Table 3 at a flow rate of 300 nL/min.

Table 10. Gradient elution conditions for nano-liquid chromatography tandem mass spectrometry time (min) Solvent A Solvent B 0 99 1 1 88 12 5 0 100 45 0 100

As shown in the precursor ion spectrum and fragment ion spectrum of the histamine derivative shown in Figures 9a and 9b, the peaks of the histamine derivative obtained by scanning with MS-1 The mass-to-charge ratio ( m/z ) of ([M+H] ⁺ ) is 284.1033, and the mass-to-charge ratio ( m/z ) of the peak of the fragment ion of the histamine derivative obtained by MS2 scanning in MRM mode is 95 and 266.

As shown in the precursor ion spectrum and fragment ion spectrum of the putrescine derivative shown in Figures 10a and 10b, the mass of the peak ([M+H] ⁺ ) of the putrescine derivative obtained by scanning with MS-1 The charge ratio ( m/z ) was 433.1400, and the mass-to-charge ratios ( m/z ) of the peaks of the fragment ions of the putrescine derivatives obtained by MS2 scanning in the MRM mode were 173, 244 and 415.

As shown in the precursor ion spectrum and fragment ion spectrum of spermidine derivatives shown in Figures 11a and 11b, the peaks ([M+H] ⁺ ) of the spermidine derivatives obtained by scanning with MS-1 The mass-to-charge ratio ( m/z ) of 662.2159, and the mass-to-charge ratios ( m/z ) of the peaks of the fragment ions of spermidine derivatives obtained by MS2 scanning in MRM mode were 230, 244 and 472.

As shown in the precursor ion spectrum and fragment ion spectrum of the cadaverine derivative shown in Figures 12a and 12b, the mass of the peak ([M+H] ⁺ ) of the cadaverine derivative obtained by scanning with MS-1 The charge ratio ( m/z ) was 447.1559, and the mass-to-charge ratios ( m/z ) of the peaks of the fragment ions of the spermidine derivative obtained by MS2 scanning in the MRM mode were 201, 258 and 429.

As shown in the precursor ion spectrum and fragment ion spectrum of the spermine derivative shown in Figures 13a and 13b, the mass of the peak ([M+H] ⁺ ) of the spermine derivative obtained by scanning with MS-1 The charge ratio ( m/z ) was 891.2883, and the mass-to-charge ratios ( m/z ) of the peaks of the fragment ions of spermine derivatives obtained by MS2 scanning in MRM mode were 473 and 701.

As shown in the precursor ion spectrum and fragment ion spectrum of the tyramine derivative shown in Figures 14a and 14b, the mass of the peak ([M+H] ⁺ ) of the tyramine derivative obtained by scanning with MS-1 The charge ratio ( m/z ) was 482.1237, and the mass-to-charge ratios ( m/z ) of the peaks of the fragment ions of the tyramide derivative obtained by MS2 scanning in the MRM mode were 173, 292 and 464.

(H) Analysis validation (validation)

In this test, biogenic amines with concentrations ranging from 3 to 400 μM are used for testing. After the derivatization and extraction step S2', capillary liquid chromatography (CapLC) is used for analysis. The concentration of the biogenic amine derivatives As the X-axis and the peak area ratio of the biogenic amine derivative to the internal standard (internal control) as the Y-axis, the formula for the calibration curve was obtained by linear regression analysis and the coefficient of determination (r ² for short).

0.992 腐胺（Put） 3～400

0.995 亞精胺（Spd） 3～400

0.997 屍胺（Cad） 3～400

0.997 精胺（Spm） 5～400

0.995 酪胺（Tyr） 3～400

0.994 Table 11, the calibration line analysis results of the same batch of biogenic amine standard products in this test Biogenic amine standard Linear relationship (μM) Calibration curve formula (n=6) decisive factor Histamine (His) 3～400

0.992 Putrescine (Put) 3～400

0.995 Spermidine (Spd) 3～400

0.997 Cadaverine (Cad) 3～400

0.997 Spermine (Spm) 5～400

0.995 Tyramine (Tyr) 3～400

0.994

0.992 腐胺（Put） 3～400

0.992 亞精胺（Spd） 3～400

0.996 屍胺（Cad） 3～400

0.995 精胺（Spm） 5～400

0.993 酪胺（Tyr） 3～400

0.996 Table 12, the calibration line analysis results of different batches of biogenic amine standard products in this experiment Biogenic amine standard Linear relationship (μM) Calibration curve formula (n=6) decisive factor Histamine (His) 3～400

0.992 Putrescine (Put) 3～400

0.992 Spermidine (Spd) 3～400

0.996 Cadaverine (Cad) 3～400

0.995 Spermine (Spm) 5～400

0.993 Tyramine (Tyr) 3～400

0.996

As shown in Tables 15a, 15b and 11, 12, the biogenic amines were assayed at concentrations of 3 to 400 μM, either within an intrabatch assy or an interbatch assay. In the range, its concentration has a good linear relationship with the peak area.

Next, with low concentration (10 μM), medium concentration (150 μM) and high concentration (350 μM) biogenic amine standards, the precision and accuracy within the same batch and between different batches were carried out. ) analysis.

Table 13, the test results of the precision and accuracy of each biogenic amine standard in this experiment Biogenic amine standard Concentration (μM) Within the same batch (n=6) Different batches (n=6) Relative Standard Deviation (RSD, %) Relative error (RE, %) Relative Standard Deviation (RSD, %) Relative error (RE, %) Histamine (His) 10 3.75 -2.80 5.20 2.05 150 2.99 2.10 2.54 0.07 350 5.31 2.15 1.64 0.68 Putrescine (Put) 10 2.76 4.04 5.27 1.50 150 0.89 2.68 1.82 1.24 350 1.09 -0.22 2.63 0.64 Spermidine (Spd) 10 1.69 2.19 0.87 2.41 150 1.75 3.30 4.11 2.12 350 1.03 1.04 8.35 0.66 Cadaverine (Cad) 10 1.61 1.34 2.41 0.41 150 0.84 2.21 2.12 2.82 350 0.60 1.94 0.66 -0.69 Spermine (Spm) 10 1.67 -4.50 7.87 -3.69 150 1.32 2.92 6.34 -0.90 350 0.45 0.89 5.35 -2.59 Tyramine (Tyr) 10 4.31 -4.38 7.64 4.39 150 4.73 2.43 4.35 2.62 350 5.24 2.38 1.10 -0.19

As shown in Table 13, the relative standard deviation (RSD) and the relative error (RE) were both less than 10% in the analysis within the same batch or between different batches, indicating that the The detection method of biogenic amines has good accuracy and precision.

(I) Analysis of liquid food samples

In this experiment, soy sauce samples (groups I1-1 to I1-10), fish sauce samples (groups I2-1 to I2-6) or rye juice samples (groups I3-1 to I3-9) were selected for testing. , and analyzed by capillary liquid chromatography (CapLC) after the derivatization extraction step S2'.

Table 14. Analysis results of biogenic amine content of 10 kinds of commercial soy sauce Content (μg/g) ﹝ Relative standard deviation (%) ﹞ (n=3) group Histamine (His) Putrescine (Put) Spermidine (Spd) Cadaverine (Cad) Spermine (Spm) Tyramine (Tyr) I1-1 368.78﹝4.95﹞ 494.80﹝3.74﹞ 62.11 ﹝3.07﹞ 519.04 ﹝5.06﹞ 75.80﹝2.30﹞ 794.01 ﹝0.92﹞ I1-2 99.21﹝4.84﹞ 20.21﹝4.80﹞ 43.99﹝2.12﹞ 56.37﹝3.40﹞ ND 89.60﹝3.65﹞ I1-3 75.07﹝5.55﹞ 147.67﹝4.07﹞ 94.82﹝3.91﹞ 113.94﹝5.97﹞ <LOQ 385.70 ﹝0.34﹞ I1-4 232.38 ﹝4.10﹞ 307.50﹝4.49﹞ 73.99﹝2.94﹞ 64.93﹝5.52﹞ <LOQ 199.58 ﹝6.37﹞ I1-5 34.28﹝0.11﹞ 25.21﹝0.49﹞ 63.52 ﹝1.21﹞ 64.87﹝2.39﹞ <LOQ 124.59 ﹝0.91﹞ I1-6 149.76 ﹝2.79﹞ 126.36 ﹝0.71﹞ 51.28﹝2.50﹞ 172.55﹝1.98﹞ <LOQ 343.52 ﹝4.67﹞ I1-7 204.37 ﹝2.24﹞ 321.30 ﹝3.13﹞ 64.33﹝1.36﹞ 135.24﹝3.29﹞ <LOQ 756.00 ﹝3.71﹞ I1-8 92.28﹝1.90﹞ 41.48﹝3.65﹞ 56.11 ﹝2.25﹞ 174.72 ﹝5.39﹞ <LOQ 74.48﹝4.50﹞ I1-9 255.91 ﹝2.11﹞ 118.12 ﹝4.72﹞ 41.63﹝9.22﹞ 73.76 ﹝5.51﹞ ND 495.70﹝7.91﹞ I1-10 68.10﹝1.59﹞ 55.98﹝1.32﹞ 112.57﹝3.48﹞ 237.56﹝1.50﹞ 42.14﹝1.34﹞ 235.14 ﹝2.36﹞

Table 15. Analysis results of biogenic amine content of 9 commercially available fish sauces Content (μg/g) ﹝ Relative standard deviation (%) ﹞ (n=3) group Histamine (His) Putrescine (Put) Spermidine (Spd) Cadaverine (Cad) Spermine (Spm) Tyramine (Tyr) I2-1 95.02 ﹝6.67﹞ 172.22﹝2.90﹞ 41.59﹝3.37﹞ 455.02 ﹝2.30﹞ 19.09﹝9.34﹞ 198.41 ﹝4.75﹞ I2-2 48.07﹝2.49﹞ 39.60﹝6.69﹞ 25.12﹝5.32﹞ 121.40﹝2.69﹞ ND 61.69﹝3.39﹞ I2-3 247.34﹝3.07﹞ 284.94﹝4.23﹞ 138.69 ﹝6.72﹞ 759.10﹝4.75﹞ 249.63﹝3.70﹞ 426.22﹝7.48﹞ I2-4 237.43﹝4.54﹞ 176.30﹝7.00﹞ 175.39 ﹝4.89﹞ 512.25﹝3.32﹞ 117.29﹝2.20﹞ 391.44 ﹝4.24﹞ I2-5 129.26 ﹝3.29﹞ 121.77 ﹝4.77﹞ 128.21 ﹝0.77﹞ 360.74﹝2.54﹞ 99.55﹝2.01﹞ 326.24﹝3.25﹞ I2-6 95.33﹝1.31﹞ 89.92﹝0.93﹞ 91.11﹝3.11﹞ 273.79﹝2.08﹞ 58.35﹝1.41﹞ 120.50﹝3.43﹞ I2-7 76.68﹝1.64﹞ 64.12 ﹝1.51﹞ 72.14﹝2.12﹞ 141.80 ﹝2.17﹞ <LOQ 70.55﹝2.36﹞ I2-8 119.65 ﹝1.16﹞ 101.68 ﹝0.92﹞ 40.60﹝0.60﹞ 308.27﹝2.08﹞ 42.34﹝1.87﹞ 134.92 ﹝6.10﹞ I2-9 131.45﹝1.84﹞ 127.91 ﹝2.46﹞ 38.86﹝5.80﹞ 403.04﹝2.97﹞ 59.66﹝3.89﹞ 145.62 ﹝3.62﹞

Table 16, Analysis results of biogenic amine content of 9 kinds of commercially available rye juice Content (μg/g) ﹝ Relative standard deviation (%) ﹞ (n=3) group Histamine (His) Putrescine (Put) Spermidine (Spd) Cadaverine (Cad) Spermine (Spm) Tyramine (Tyr) I3-1 2.47﹝7.89﹞ 39.48﹝3.97﹞ 2.58﹝5.53﹞ 40.58﹝7.33﹞ 3.63﹝2.40﹞ 31.40﹝3.70﹞ I3-2 3.12 ﹝5.99﹞ 3.33﹝3.31﹞ 2.94﹝4.82﹞ 3.89﹝3.93﹞ 2.28﹝2.37﹞ 2.37﹝7.73﹞ I3-3 ND 6.36﹝0.53﹞ 27.39 ﹝0.71﹞ 4.83﹝1.26﹞ <LOQ 3.52﹝0.48﹞ I3-4 ND 41.86﹝2.82﹞ 2.54﹝4.41﹞ 41.04﹝4.35﹞ <LOQ 15.75﹝5.31﹞ I3-5 ND 4.20﹝5.30﹞ 2.70﹝5.15﹞ 4.28﹝2.66﹞ <LOQ 2.15﹝2.84﹞ I3-6 1.40﹝3.96﹞ 2.85﹝0.92﹞ 2.42﹝3.74﹞ 2.29﹝1.33﹞ <LOQ 1.94﹝5.82﹞ I3-7 3.07﹝1.31﹞ 3.18﹝0.84﹞ 3.59 ﹝2.12﹞ 2.69﹝0.95﹞ 2.48﹝1.95﹞ 3.65﹝0.44﹞ I3-8 1.00﹝1.20﹞ 0.90﹝5.90﹞ 1.79﹝8.63﹞ 1.43﹝8.96﹞ <LOQ 2.68﹝6.59﹞ I3-9 1.31 ﹝6.41﹞ 0.65﹝4.05﹞ 2.17﹝5.15﹞ 1.45﹝7.27﹞ ND <LOQ

As shown in Tables 14 to 16 and Figures 16a to 16c, whether it is commercially available soy sauce, commercially available fish sauce or commercially available rye juice, the presence of biogenic amines can be detected by this biogenic amine detection method.

(J) Analysis results of solid food samples

In this experiment, a miso sample is selected for testing. The miso sample is dissolved in water, shaken by ultrasonic for 15 minutes, and centrifuged at 14,800 rpm for 5 minutes. , and analyzed by capillary liquid chromatography (CapLC), and the results are shown in Figure 17. The presence of biogenic amines can be detected by this biogenic amine detection method.

(K) Analysis results of biological samples

In this experiment, plasma samples were selected for testing. The plasma samples were mixed with acetonitrile and centrifuged at 14,800 rpm for 5 minutes. CapLC) analysis, the results are shown in Figure 18, the presence of biogenic amines can also be detected by this biogenic amine detection method.

To sum up, by making the amine group of the biogenic amine in the test sample and the carboxyl group of coumarin-3-carboxylic acid to carry out the amidation reaction with the aid of the coupling reagent, the amine can be obtained A labeled biogenic amine derivative substituted on the base, the presence of which can be detected by various conventional methods (for example, after separation by liquid chromatography, using UV spectroscopy, fluorescence spectroscopy, or detection by mass spectrometry), therefore, the method for detecting biogenic amines of the present invention has good sensitivity, precision and accuracy, and can reduce the amount of the sample to be tested, which is the effect of the present invention.

Furthermore, the detection kit can be applied to implement the above-mentioned detection method of biogenic amines, and can effectively form biogenic amine derivatives with good stability together with biogenic amines. Therefore, workers can select various conventional methods for detection according to their needs. The biogenic amine derivatives (eg, separated by liquid chromatography and detected by ultraviolet spectroscopy, fluorescence spectroscopy, or mass spectrometry) are the effects of the present invention.

Although the present invention has been disclosed by the above-mentioned preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes and modifications relative to the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the patent application attached hereto.

﹝this invention﹞ S1: Sample providing step S2: Derivation step S2': Derivative extraction step S3: Detection step

[Fig. 1] A flowchart of the method for detecting biogenic amines according to the first embodiment of the present invention. [Picture 2] The chemical equation for the derivatization of biogenic amines with coumarin-3-carboxylic acid. [Fig. 3] A flow chart of the method for detecting biogenic amines according to the second embodiment of the present invention. [Figure 4] In test (B), the bar graph of the biogenic amine strength values of the solutions to be tested in groups B1 to B4. [Figure 5] In test (C), the bar graph of the biogenic amine strength values of the solutions to be tested in groups C1 to C3. [Picture 6] In the test (D), the bar graph of the biogenic amine strength values of the solutions to be tested in groups D1 to D4. [Figure 7] In test (E), the capillary liquid chromatograms of the solutions to be tested in groups E1 to E2, wherein peaks 1 to 6 are histamine derivatives, putrescine derivatives, and spermidine derivatives, respectively , cadaverine derivatives, spermine derivatives and tyramine derivatives formed peaks. [Figure 8] In test (F), the capillary liquid chromatograms of the solutions to be tested in groups F0 to F2, where peaks 1 to 6 are histamine derivatives, putrescine derivatives, and spermidine derivatives, respectively , cadaverine derivatives, spermine derivatives and tyramine derivatives formed peaks. [Figure 9a] In test (G), the precursor ion spectrum of the solution to be tested in Group G1. [Figure 9b] In the test (G), the fragment ion spectrum of the solution to be tested in Group G1. [Figure 10a] In test (G), the precursor ion spectrum of the solution to be tested in Group G2. [Figure 10b] In test (G), the fragment ion spectrum of the solution to be tested in Group G2. [Figure 11a] In test (G), the precursor ion spectrum of the solution to be tested in Group G3. [Figure 11b] In test (G), the fragment ion spectrum of the solution to be tested in Group G3. [Figure 12a] In the test (G), the precursor ion spectrum of the solution to be tested in Group G4. [Figure 12b] In test (G), the fragment ion spectrum of the solution to be tested in Group G4. [Figure 13a] In test (G), the precursor ion spectrum of the solution to be tested in Group G5. [Figure 13b] In test (G), the fragment ion spectrum of the solution to be tested in Group G5. [Figure 14a] In test (G), the precursor ion spectrum of the solution to be tested in Group G6. [Figure 14b] In test (G), the fragment ion spectrum of the solution to be tested in Group G6. [Figure 15a] In test (H), the calibration line analysis results of the same batch of biogenic amine standards. [Figure 15b] In test (H), the calibration curve analysis results of different batches of biogenic amine standard products. [Fig. 16a] In test (I), the capillary liquid chromatogram of the soy sauce samples in group I1-1, in which peaks 1 to 6 are histamine derivatives, putrescine derivatives, spermidine derivatives, Peaks formed by cadaverine derivatives, spermine derivatives and tyramine derivatives. [Fig. 16b] In test (I), the capillary liquid chromatogram of the fish sauce sample of group I2-1, wherein the peaks 1 to 6 are histamine derivatives, putrescine derivatives and spermidine derivatives, respectively , cadaverine derivatives, spermine derivatives and tyramine derivatives formed peaks. [Fig. 16c] In test (I), the capillary liquid chromatogram of the rye juice sample of group I3-1, in which peaks 1 to 6 are histamine derivatives, putrescine derivatives, spermidine derivatives, respectively The peaks formed by compounds, cadaverine derivatives, spermine derivatives and tyramine derivatives. [Figure 17] In test (J), the capillary liquid chromatogram of the miso sample, in which peaks 1 to 6 are histamine derivatives, putrescine derivatives, spermidine derivatives, cadaverine derivatives, Peaks formed by spermine derivatives and tyramine derivatives. [Fig. 18] In test (K), the capillary liquid chromatogram of the plasma sample, in which peaks 1 to 6 are histamine derivatives, putrescine derivatives, spermidine derivatives, cadaverine derivatives, Peaks formed by spermine derivatives and tyramine derivatives.

S1: Sample providing step

S2: Derivation step

S3: Detection step

Claims (16)

A detection method for biogenic amines, comprising: Provide a sample to be tested, the sample to be tested contains biogenic amines; Using coumarin-3-carboxylic acid as a derivatizing reagent, mix the sample to be tested, the derivatizing reagent, a coupling reagent and an alkaline working solution to obtain a mixed solution to be reacted, so that the derivatizing reagent and the The biogenic amine in the sample is subjected to an amidation reaction with the aid of the coupling reagent to obtain a biogenic amine derivative dissolved in a derivatizing solution, and the biogenic amine derivative is a labeled biogenic amine substituted on the amine group; and Using the derivative solution as a test solution, the biogenic amine derivative in the test solution is detected to obtain a biogenic amine intensity value. The method for detecting biological amines according to claim 1, wherein the coupling reagent is (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbon Onium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate or 2-(7-benzotriazole oxide)-N,N ,N',N'-tetramethylurea hexafluorophosphate. The method for detecting biogenic amines according to claim 1, wherein the alkaline working solution is formed by dissolving an organic base compound in a working solvent. The method for detecting biogenic amines according to claim 3, wherein the organic base compound is 4-dimethylaminopyridine, N-methylmorpholine, imidazole, or triethylamine. The method for detecting biogenic amines according to claim 3, wherein the working solvent is acetonitrile. The method for detecting biogenic amines according to claim 1, wherein the mixed solution to be reacted further comprises an extraction solvent and a salt solution, and after the amidation reaction, the derivative solution is layered to form an upper layer solution and a lower layer solution For the solidified product, the upper layer solution is used as the solution to be tested, and the biogenic amine derivatives in the solution to be tested are detected. The method for detecting biogenic amines according to claim 6, wherein the extraction solvent is acetonitrile, dimethyl carbonate, ethyl acetate or dichloromethane. The method for detecting biogenic amines according to claim 6, wherein the salt solution is disodium hydrogen phosphate. A biogenic amine detection kit, comprising: A derivatizing reagent, the derivatizing reagent is coumarin-3-carboxylic acid, and is used for monoamidation reaction with the biogenic amine in the test sample to form a biogenic amine derivative, and the biogenic amine derivative is an amine Substitute a labeled biogenic amine on the base; a coupling reagent for promoting the amidation reaction; and an alkaline working solution for forming a mixed solution to be reacted together with the sample to be tested, the coupling reagent and the derivatizing reagent, and the alkaline working solution contains a solution that can dissolve the derivatizing reagent, the coupling reagent and the biogenic amine A working solvent. The detection kit for biogenic amines according to claim 9, wherein the coupling reagent is (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholino Carbonium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate or 2-(7-oxybenzotriazole)-N, N,N',N'-tetramethylurea hexafluorophosphate. The biogenic amine detection kit according to claim 9, wherein the alkaline working solution is formed by dissolving an organic base compound in the working solvent, and is used to activate the carboxyl group of the derivatizing reagent. The biogenic amine detection kit according to claim 11, wherein the organic base compound is 4-dimethylaminopyridine, N-methylmorpholine, imidazole or triethylamine. The detection kit for biogenic amines according to claim 11, wherein the working solvent is acetonitrile. As claimed in claim 9, the biogenic amine detection kit also includes: an extraction solvent, which is an aprotic solvent for dissolving the biogenic amine derivative; and A salt solution, immiscible with the extraction solvent, and used to create a salting-out effect. The detection kit for biogenic amines according to claim 14, wherein the extraction solvent is acetonitrile, dimethyl carbonate, ethyl acetate or dichloromethane. The detection kit for biogenic amines according to claim 14, wherein the salt solution is disodium hydrogen phosphate.

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