阅读说明：本技术 一种羧酸衍生化试剂及其制备方法和应用 (Carboxylic acid derivatization reagent and preparation method and application thereof ) 是由 王晓 孙成龙 刘伟 耿岩玲 于 2020-05-22 设计创作，主要内容包括：本发明提供一种羧酸衍生化试剂及其制备方法和应用,属于分析化学技术领域。所述羧酸衍生化试剂具体为N,N,N-三甲基-2-(哌嗪-1-基)乙烷-1-碘化铵,其结构式如下：<Image he="170" wi="492" file="DDA0002504294820000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>本发明中的羧酸衍生化试剂可作为羧酸类代谢物质谱检测的衍生化试剂,具有良好的质谱增敏效果,因此具有良好的实际推广应用之价值。(The invention provides a carboxylic acid derivatization reagent and a preparation method and application thereof, belonging to the technical field of analytical chemistry. The carboxylic acid derivatization reagent is N, N, N-trimethyl-2- (piperazine-1-yl) ethane-1-ammonium iodide, and the structural formula is as follows: the carboxylic acid derivatization reagent can be used as a derivatization reagent for mass spectrometry detection of carboxylic acid metabolites, and has a good mass spectrum sensitization effect, so that the carboxylic acid derivatization reagent has a good value for practical popularization and application.)

1. A compound having the chemical name: n, N, N-trimethyl-2- (piperazin-1-yl) ethane-1-ammonium iodide, of the formula (I):

2. a method of synthesizing the compound of claim 1, comprising

3. The method of synthesis according to claim 2, comprising the steps of: dissolving 1- (2-dimethylaminoethyl) piperazine in ether at room temperature, adding methyl iodide, and stirring.

4. A synthesis process according to claim 3, characterized in that the molar charge ratio of 1- (2-dimethylaminoethyl) piperazine to methyl iodide is 3-10:1, preferably 5: 1; or the like, or, alternatively,

the synthesis method further comprises the following steps: and after the reaction is finished, filtering the reaction solution, washing with diethyl ether, and drying to obtain solid powder, namely the N, N, N-trimethyl-2- (piperazine-1-yl) ethane-1-ammonium iodide.

5. Use of a compound of claim 1 as a derivatizing agent; preferably, the derivatizing agent is a carboxylic acid derivatizing agent; more preferably, the application comprises the application in the mass spectrometric detection of carboxylic acid metabolites.

6. A method for mass spectrometric detection of a carboxylic acid metabolite, said method comprising: and carrying out derivatization reaction on the carboxylic acid metabolites and then carrying out mass spectrum detection.

Wherein the derivatizing agent used in the derivatizing reaction is the compound of claim 1.

7. The method of claim 6, comprising the steps of:

(1) obtaining a carboxylic acid metabolite extraction solution from a biological sample;

(2) derivatization of carboxylic acid metabolites: dissolving a derivatization reagent in a solvent, adding an excessive derivatization reagent solution into a carboxylic acid metabolite extraction solution, adding a catalyst, carrying out vortex mixing, carrying out derivatization reaction at constant temperature, and obtaining a carboxylic acid metabolite derivatization product after the reaction is finished;

(3) mass spectrum detection: and (3) performing needle pump injection mass spectrometry on the derivatization product of the carboxylic acid metabolite reference substance to obtain a corresponding accurate m/z value and ion pair information, setting mass spectrometry parameters, and analyzing the derivatization test solution of the biological sample obtained in the step (2) to obtain the information of the carboxylic acid metabolite in the biological sample.

8. The method of claim 7, wherein the carboxylic acid metabolite comprises one or more of stearic acid, oleic acid, linoleic acid, malic acid, citric acid, succinic acid, cholic acid, chenodeoxycholic acid, glycocholic acid, indoleacetic acid, abscisic acid; or the like, or, alternatively,

the biological sample used in step (1) includes an animal tissue sample or a plant tissue sample, preferably rat plasma or rice roots.

9. The method of claim 7, wherein the solvent used in step (2) is a mixed solution of acetonitrile and water, preferably acetonitrile and water in a volume ratio of 9: 1;

the concentration of the derivatization reagent is 0.5mg/mL-2mg/mL, preferably 1 mg/mL;

the catalyst comprises any one of HBOt, TEA, EDC and HATU, and HATU is preferred.

10. The method of claim 7, wherein the reaction temperature in step (2) is 20-60 ℃, preferably 40 ℃; or the like, or, alternatively,

the reaction time of the step (2) is 0.5 to 2 hours, preferably 1 hour.

Technical Field

The invention relates to the technical field of analytical chemistry, in particular to a carboxylic acid derivatization reagent and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Carboxylic acid compounds such as fatty acids, bile acids, malic acid, citric acid, succinic acid, indoleacetic acid, abscisic acid, and the like are important metabolites in the body, and they play a crucial role in cell proliferation, energy metabolism, signal transduction, and the like. The content change of these metabolites can reflect the biological state of the body. For example, abnormal regulation of tricarboxylic acid cycle-related metabolites (malate, citrate, succinate) in humans is closely associated with the development of various cancers; abnormal bile acid metabolism is an important characteristic of metabolic diseases of the body; indole acetic acid and abscisic acid are important plant hormones, and can regulate and control important life activities of plant growth and development with extremely low content. Therefore, the accurate analysis and determination of the type and content of the carboxylic acid metabolites in organisms have important significance for helping people to deeply understand the action mechanism of the acid metabolites in complex life activities.

The current determination of carboxylic acid metabolites in biological samples mainly relies on mass spectrometry technology. In the negative ion detection mode, the method of directly carrying out liquid chromatography-tandem mass spectrometry is the most common method at present, but because the content of partial carboxylic acid metabolites in organisms is extremely low, accurate determination is difficult. Therefore, amine derivatization reagents with high ionization efficiency are often used for reacting with carboxylic acid metabolites, so that the mass spectrometric detection sensitivity of the carboxylic acid metabolites is improved. The inventor finds that the currently reported carboxylic acid metabolite derivatization reagents comprise: n, N-dimethylethylenediamine, 5- (dimethylamino) pentylamine, and the like. However, these derivatization reagents have the problems of complicated derivatization operation, insufficient sensitivity, influence on the detection of the substance to be detected due to the strong ionization efficiency of the derivatization reagents, and the like.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a carboxylic acid derivatization reagent, a preparation method and an application thereof, wherein the carboxylic acid derivatization reagent is specifically N, N, N-trimethyl-2- (piperazine-1-yl) ethane-1-ammonium iodide, the carboxylic acid derivatization reagent can be used as a derivatization reagent for mass spectrometry detection of carboxylic acid metabolites, and experiments prove that the carboxylic acid derivatization reagent has a good mass spectrometry sensitization effect, so the carboxylic acid derivatization reagent has a good value in practical popularization and application.

In order to solve the technical problems, the technical scheme of the invention is as follows:

in a first aspect of the present invention, there is provided a compound which is N, N-trimethyl-2- (piperazin-1-yl) ethane-1-ammonium iodide having the formula (I):

in a second aspect of the present invention, there is provided a method for synthesizing the above compound, the method comprising:

in a third aspect of the invention, there is provided the use of a compound as described above as a derivatising agent;

specifically, the derivatization reagent is a carboxylic acid derivatization reagent;

more particularly, the application comprises the application in the mass spectrometric detection of carboxylic acid metabolites.

In a fourth aspect of the present invention, there is provided a method for mass spectrometric detection of a carboxylic acid metabolite, the method comprising: and carrying out derivatization reaction on the carboxylic acid metabolites and then carrying out mass spectrum detection.

Wherein the derivatization reagent used in the derivatization reaction is the N, N, N-trimethyl-2- (piperazine-1-yl) ethane-1-ammonium iodide.

The beneficial technical effects of one or more technical schemes are as follows:

(1) n, N, N-trimethyl-2- (piperazine-1-yl) ethane-1-ammonium iodide is used as a derivatization reagent, the derivatization condition is mild, the reaction activity is high, the reaction speed is high, the derivatization reagent has positive charges, and the ionization efficiency of a derivatization product is high.

(2) The N, N, N-trimethyl-2- (piperazine-1-yl) ethane-1-ammonium iodide is used as a derivatization reagent, so that the derivatization analysis of various carboxylic acid metabolites in a biological sample can be realized, and the method comprises the following steps: stearic acid, oleic acid, linoleic acid, malic acid, citric acid, succinic acid, cholic acid, chenodeoxycholic acid, glycocholic acid, indoleacetic acid and abscisic acid.

(3) The combined derivatization reagent and carboxylic acid metabolites have stable properties, and trimethylamine (59Da) can be stably removed to form [ M-59] + fragment ions in mass spectrum ion scanning analysis, so that the accuracy of qualitative and quantitative analysis is enhanced, and the method has good practical application value.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings according to the provided drawings without creative efforts.

FIG. 1 is a high resolution mass spectrum and a proton ion scanning mass spectrum of N, N, N-trimethyl-2- (piperazin-1-yl) ethane-1-ammonium iodide of example 1 of the present invention;

FIG. 2 is a high resolution mass spectrum of stearic acid, oleic acid, linoleic acid, malic acid, citric acid, succinic acid, cholic acid, chenodeoxycholic acid, glycocholic acid in rat plasma in example 3 of the present invention, in combination with N, N, N-trimethyl-2- (piperazin-1-yl) ethane-1-ammonium iodide derivatizing agent;

FIG. 3 is a scanning mass spectrum of a product of derivatization of oleic acid with N, N, N-trimethyl-2- (piperazin-1-yl) ethane-1-ammonium iodide in rat plasma according to example 3 of the present invention;

FIG. 4 is a high resolution mass spectrum of abscisic acid and indoleacetic acid in rice roots combined with N, N, N-trimethyl-2- (piperazin-1-yl) ethane-1-ammonium iodide derivatizing agent in example 4 of the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As mentioned above, the derivatization reagent of the currently reported carboxylic acid metabolite has the problems of complicated derivatization operation, insufficient sensitivity, influence of strong ionization efficiency of the derivatization reagent on the detection of the substance to be detected, and the like.

In view of the above, the present invention provides a compound having a chemical name of: n, N, N-trimethyl-2- (piperazin-1-yl) ethane-1-ammonium iodide, of the formula (I):

in another embodiment of the present invention, there is provided a method for synthesizing the compound of formula (I) as described above, as follows:

in another embodiment of the present invention, the synthesis method comprises the steps of: dissolving 1- (2-dimethylaminoethyl) piperazine in ether at room temperature, adding methyl iodide, and stirring.

In another embodiment of the present invention, the molar charge ratio of 1- (2-dimethylaminoethyl) piperazine to methyl iodide in the above process may be 3-10:1, preferably 5: 1.

In another embodiment of the present invention, the synthesis method further comprises: and after the reaction is finished, filtering the reaction solution, washing with diethyl ether, and drying to obtain solid powder, namely the N, N, N-trimethyl-2- (piperazine-1-yl) ethane-1-ammonium iodide.

In yet another embodiment of the present invention, there is provided the use of the above compound as a derivatizing agent; specifically, the derivatization reagent is a carboxylic acid derivatization reagent; more particularly, the application comprises the application in the mass spectrometric detection of carboxylic acid metabolites.

In another embodiment of the present invention, there is provided a method for mass spectrometric detection of a carboxylic acid metabolite, said method comprising: and carrying out derivatization reaction on the carboxylic acid metabolites and then carrying out mass spectrum detection.

Wherein the derivatization reagent used in the derivatization reaction is the N, N, N-trimethyl-2- (piperazine-1-yl) ethane-1-ammonium iodide.

In another embodiment of the present invention, a method for mass spectrometric detection of a carboxylic acid metabolite is provided, comprising the steps of:

(1) obtaining a carboxylic acid metabolite extraction solution from a biological sample;

(2) derivatization of carboxylic acid metabolites: dissolving a derivatization reagent in a solvent, adding an excessive derivatization reagent solution into a carboxylic acid metabolite extraction solution, adding a catalyst, carrying out vortex mixing, carrying out derivatization reaction at constant temperature, and obtaining a carboxylic acid metabolite derivatization product after the reaction is finished;

(3) mass spectrum detection: and (3) performing needle pump injection mass spectrometry on the derivatization product of the carboxylic acid metabolite reference substance to obtain a corresponding accurate m/z value and ion pair information, setting mass spectrometry parameters, and analyzing the derivatization test solution of the biological sample obtained in the step (2) to obtain the information of the carboxylic acid metabolite in the biological sample.

In yet another embodiment of the present invention, the carboxylic acid metabolites include one or more of stearic acid, oleic acid, linoleic acid, malic acid, citric acid, succinic acid, cholic acid, chenodeoxycholic acid, glycocholic acid, indoleacetic acid, and abscisic acid.

In still another embodiment of the present invention, the biological sample used in step (1) includes, but is not limited to, an animal tissue sample or a plant tissue sample, such as rat plasma or rice roots.

In another embodiment of the present invention, the solvent used in step (2) is a mixed solution of acetonitrile and water, preferably acetonitrile and water in a volume ratio of 9: 1.

In yet another embodiment of the present invention, the concentration of the derivatizing agent used in step (2) is from 0.5mg/mL to 2mg/mL, preferably 1 mg/mL.

In still another embodiment of the present invention, the catalyst used in step (2) comprises one of HBOt (1-hydroxybenzotriazole hydrate), TEA (triethylamine), EDC (N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide hydrochloride), HATU (O- (7-azabenzotriazol-1-yl) -N, N' -tetramethyluronium hexafluorophosphate), preferably HATU.

In still another embodiment of the present invention, the reaction temperature in step (2) is 20 to 60 ℃, preferably 40 ℃.

In still another embodiment of the present invention, the reaction time of step (2) is 0.5 to 2 hours, preferably 1 hour.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.