Fluorescence analysis method and application of plasmalogens

Abstract

The present invention provides a method for fluorescently labeling plasmalogens, which method comprises: contacting a compound represented by formula (I) or a stereoisomer, tautomer or salt thereof with a substrate to be labeled, wherein the substrate to be labeled comprises at least one vinyl ether bond. The method of the present invention can selectively label plasmalogens without interference from other lipids. This fluorescent labeling method can be combined with simple instrument devices such as high performance liquid chromatography and fluorescence detector as an accurate fluorescence analysis method for plasmalogens, and is used for rapid analysis and screening of plasmalogens in blood lipids. This method has high accuracy and low cost in detecting plasmalogens in blood lipids.

Description

Technical Field

[0001] The present invention belongs to the technical field of spectral analysis and detection. Specifically, it relates to a fluorescence analysis method and application of plasmalogens, and more specifically, to a fluorescent small molecule, a method for fluorescently labeling plasmalogens, a fluorescently labeled plasmalogen, a method for detecting plasmalogens, and the use of the fluorescent small molecule and the fluorescently labeled plasmalogen in the preparation of a kit. Background Art

[0002] Plasmalogens are a special type of glycerol ether phospholipids. Compared with diacylglycerol phospholipids linked by ester bonds and other glycerol ether phospholipids linked by saturated ether bonds, their main structural feature is that an aliphatic chain is linked through an enol ether bond at the sn-1 site of the glycerol backbone ( Figure 1 ). In mammals, according to the different head groups, plasmalogens are mainly divided into phosphatidylethanolamine plasmalogens (PE-Pls) and phosphatidylcholine plasmalogens (PC-Pls). In recent years, studies have reported a high correlation between plasmalogens and diseases, and significant decreases in the levels of specific plasmalogen species have been observed in patients with Alzheimer's disease, cardiovascular diseases, and peroxisome-related diseases, etc. However, there is currently no simple and feasible analysis method for plasmalogens. Therefore, developing a simple and accurate labeling and analysis method is of great significance for the diagnosis of plasmalogen-related diseases.

[0003] Fluorescence analysis technology has the advantages of high sensitivity, simple equipment, easy modification, and good biocompatibility, and has been widely used in the field of lipid analysis through methods such as fluorescent lipid analogs, lipid-binding protein-fluorophore conjugates, and fluorescent labeling. However, due to the difficulty in selectively recognizing and labeling plasmalogens, there is currently no accurate fluorescence analysis method for plasmalogens. In addition, in order to exclude the interference of saturated ether bonds in glycerol ether phospholipid isomers on the detection of plasmalogens, it is necessary to analyze them using complex mass spectrometry instruments, resulting in a significant increase in detection costs.

[0004] Therefore, there is an urgent need in the art to design a method for highly selectively fluorescently labeling plasmalogens in order to improve the detection precision and reduce the detection cost. Summary of the Invention

[0005] This application is based on the inventor's discovery and recognition of the following facts and problems:

[0006] Currently, the detection of plasmalogens in blood lipids is easily interfered by other lipids. In order to exclude the interference of other lipids, it is necessary to perform mass spectrometry analysis on the sample using precise and complex instruments, which results in a high cost and is difficult to apply clinically. In order to be able to highly selectively label plasmalogens, the inventor labeled plasmalogens by synthesizing fluorescent small molecules, and then detected the labeled products using high performance liquid chromatography-fluorescence analysis. Realize the rapid analysis and screening of plasmalogens in blood lipids with high selectivity and low cost.

[0007] In the first aspect of the present invention, there is provided the use of a compound represented by formula (I), or a stereoisomer, tautomer or salt thereof of the compound represented by formula (I) in labeling plasmalogens.

[0008]

[0009] Wherein, R1, R2, R3, R4 and R5 are each independently selected from H, D, F, Cl, Br, I, OH, NH2, NO2, CN, N3, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 hydroxyalkyl, C 1-6 alkylamino, C 3-12 carbocyclic group, heterocyclic group composed of 3-12 atoms, C 6-10 aryl or heteroaryl composed of 5-12 atoms, wherein each of R1, R2, R3, R4 and R5 is independently optionally substituted by one or more R 6 substituents.

[0010] Said R 6 is independently H, D, F, Cl, Br, I, =O, OH, NH2, NO2, CN, N3, C 1-4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C 1-4 alkoxy, C 1-4 alkylamino, C 1-4 haloalkyl, C 1-4 haloalkoxy, C 1-4 hydroxyalkyl or C 1-4 haloalkylamino.

[0011] According to an embodiment of the present invention, the compound or its stereoisomer, tautomer and its salt can be used for highly selectively labeling plasmalogens.

[0012] According to an embodiment of the present invention, the above-mentioned compound or its stereoisomer, tautomer and its salt may further include at least one of the following additional technical features:

[0013] According to an embodiment of the present invention, R1, R2, R3, R4 and R5 are each independently selected from H.

[0014] In a second aspect of the present invention, the present invention provides a method for fluorescently labeling a substrate. According to an embodiment of the present invention, the method includes: contacting a compound represented by formula (I) or a stereoisomer, tautomer or salt thereof with a substrate to be labeled, wherein the substrate to be labeled includes at least one vinyl ether bond.

[0015]

[0016] Wherein, the substrate includes at least one vinyl ether bond;

[0017] R1, R2, R3, R4 and R5 are each independently selected from H, D, F, Cl, Br, I, OH, NH2, NO2, CN, N3, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 hydroxyalkyl, C 1-6 alkylamino, C 3-12 carbocyclic group, heterocyclic group composed of 3-12 atoms, C 6-10 aryl or heteroaryl composed of 5-12 atoms, wherein each of R1, R2, R3, R4 and R5 is independently optionally substituted by one or more R 6 ;

[0018] The R 6 is independently H, D, F, Cl, Br, I, =O, OH, NH2, NO2, CN, N3, C 1-4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C 1-4 alkoxy, C 1-4 alkylamino, C 1-4 haloalkyl, C 1-4 haloalkoxy, C 1-4 hydroxyalkyl or C 1-4 haloalkylamino.

[0019] The inventors found that the method can highly selectively label substrates containing at least one vinyl ether bond, such as plasmalogens, without interference from other lipids. This fluorescence labeling method can be combined with simple instrument devices such as high-performance liquid chromatography and fluorescence detectors as an accurate fluorescence analysis method for substrates such as plasmalogens, which can be used for rapid analysis and screening of plasmalogens in test samples. Furthermore, the screened plasmalogens can be further scientifically analyzed, such as analyzing the content or structure of plasmalogens, and can also be used to guide clinical diagnosis or treatment through the content of plasmalogens in test samples such as blood. Analyzing and screening plasmalogens in blood lipids using this method can significantly save detection costs and has more practical significance in actual hospital detections.

[0020] According to an embodiment of the present invention, the method for fluorescently labeling plasmalogens further includes at least one of the following additional technical features:

[0021] According to an embodiment of the present invention, the contact is carried out in a first solvent, and the concentration of the compound represented by formula (I) or a stereoisomer, tautomer or salt thereof of the compound represented by formula (I) in the first solvent is 100 μM - 100 mM.

[0022] According to an embodiment of the present invention, the first solvent includes a buffer solution and an organic solvent.

[0023] According to an embodiment of the present invention, the buffer solution includes at least one of phosphate, HEPES, MOPS, and MES buffer solutions. It should be noted that the phosphate buffer solution includes PBS buffer solution.

[0024] According to an embodiment of the present invention, the pH of the buffer solution is 4.0 - 9.0. The inventors found that at a high buffer solution pH, the compound represented by formula (I) or a stereoisomer, tautomer or salt thereof of the compound represented by formula (I) is prone to generating by-products. At a low buffer solution pH, the vinyl ether bond in the substrate is prone to hydrolysis.

[0025] According to an embodiment of the present invention, the pH of the buffer solution is 6.0. According to an embodiment of the present invention, selecting a buffer solution with a pH of 6.0 can maximize the cycloaddition reaction.

[0026] According to an embodiment of the present invention, the organic solvent includes acetonitrile.

[0027] According to an embodiment of the present invention, the volume ratio of the buffer solution to the organic solvent is 1:9 to 9:1. The inventors found that when the reaction solvent is only an organic solvent, the compound represented by formula (I) or a stereoisomer, tautomer or salt thereof of the compound represented by formula (I) is not easily dehydrated, resulting in the inaccessibility of the cycloaddition reaction with the vinyl ether bond. When the reaction solvent is only a buffer solution, the solubility of the substrate is poor.

[0028] According to an embodiment of the present invention, the volume ratio of the buffer solution to the organic solvent is 7:3. The inventors have found that selecting a volume ratio of buffer solution to organic solvent of 7:3 has the advantage of maximizing the cycloaddition reaction.

[0029] According to an embodiment of the present invention, the substrate is pre-acylated.

[0030] According to an embodiment of the present invention, the substrate includes plasmalogen.

[0031] According to an embodiment of the present invention, the acylation treatment is carried out in the presence of an acylating reagent and a catalyst. It should be noted that the acylating reagent and the acylation reagent have the same representative meaning.

[0032] According to an embodiment of the present invention, the acylating reagent includes at least one selected from N-hydroxysuccinimide esters and their common derivatives.

[0033] According to an embodiment of the present invention, the acylating reagent is N-hydroxysuccinimide acetate.

[0034] According to an embodiment of the present invention, the concentration of N-hydroxysuccinimide acetate in the acylation system is 500 μM - 50 mM.

[0035] According to an embodiment of the present invention, the catalyst is an organic base.

[0036] According to an embodiment of the present invention, the organic base is selected from at least one of 4-dimethylaminopyridine and triethylamine.

[0037] According to an embodiment of the present invention, the plasmalogen includes a carbon chain at the sn-1 position, a polar group, and optionally an acyl chain at the sn-2 position. According to an embodiment of the present invention, the type of plasmalogen that can be labeled is determined by the carbon chain at the sn-1 position, the acyl chain at the sn-2 position, and the type of polar group on its glycerol backbone.

[0038] According to an embodiment of the present invention, the carbon chain at the sn-1 position includes at least one selected from C16:0, C18:0, C18:1, C18:2, C20:0, C20:1, C20:2, C22:0, C22:1, C22:2.

[0039] According to an embodiment of the present invention, the acyl chain at the sn-2 position includes at least one of C16:0, C18:0, C18:1, C18:2, C18:3, C20:3, C20:4, C20:5, C22:4, C22:5, C22:6, C22:7, C24:8.

[0040] According to an embodiment of the present invention, the polar group includes at least one of phosphoric acid, ethanolamine phosphate, choline phosphate, serine phosphate, glycerol phosphate, and inositol phosphate.

[0041] In a third aspect of the present invention, the present invention provides a fluorescently labeled plasmalogen. According to an embodiment of the present invention, the fluorescently labeled plasmalogen is obtained by labeling using the method described in the second aspect of the present invention.

[0042] According to an embodiment of the present invention, the fluorescently labeled plasmalogen can be used for a sample to be tested, such as the analysis and screening of plasmalogens in blood lipids.

[0043] In a fourth aspect of the present invention, the present invention provides a method for detecting plasmalogens. According to an embodiment of the present invention, the method includes: performing a labeling treatment on plasmalogens according to the method described in the second aspect of the present invention, detecting the labeled plasmalogens using high performance liquid chromatography to obtain a liquid chromatogram, and determining plasmalogens based on the obtained liquid chromatogram.

[0044] The inventors found that after labeling plasmalogens with the above-mentioned compound and then detecting them using high performance liquid chromatography, interference caused by its isomers, such as glycerol ether phospholipids connected by saturated ether bonds, to the detection results can be excluded. Moreover, this method has higher detection accuracy, lower cost, and wider applicability compared to the mass spectrometry analysis of a single plasmalogen sample.

[0045] According to an embodiment of the present invention, the method for detecting plasmalogens further includes at least one of the following additional technical features:

[0046] According to an embodiment of the present invention, the mobile phase used in the liquid chromatography is selected from at least one of water, methanol, acetonitrile, isopropanol, n-hexane, acetic acid, formic acid, ammonium acetate, and ammonium formate.

[0047] According to an embodiment of the present invention, the chromatographic column used in the liquid chromatography is selected from at least one of a C18 column, a C8 column, an amino column, and a silica gel column.

[0048] According to an embodiment of the present invention, the detection wavelength of the liquid chromatography is 300 - 350 nm, and the emission wavelength is 350 - 430 nm.

[0049] According to an embodiment of the present invention, the plasmalogens are derived from a blood sample.

[0050] According to an embodiment of the present invention, the blood sample is selected from at least one of whole blood, serum, plasma, red blood cells, white blood cells, and platelets.

[0051] In the fifth aspect of the present invention, there is provided the use of a compound represented by formula (I) or a stereoisomer, tautomer or salt thereof of the compound represented by formula (I), or the fluorescently labeled plasmalogen described in the third aspect of the present invention in the preparation of a kit. According to an embodiment of the present invention, the kit is used for detecting plasmalogen in blood lipids or plasmalogen-related diseases,

[0052]

[0053] wherein R1, R2, R3, R4 and R5 are each independently selected from H, D, F, Cl, Br, I, OH, NH2, NO2, CN, N3, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 hydroxyalkyl, C 1-6 alkylamino, C 3-12 carbocyclic group, heterocyclic group composed of 3-12 atoms, C 6-10 aryl or heteroaryl composed of 5-12 atoms, wherein each of R1, R2, R3, R4 and R5 is independently optionally substituted by one or more R 6 ;

[0054] Said R 6 is independently H, D, F, Cl, Br, I, =O, OH, NH2, NO2, CN, N3, C 1-4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C 1-4 alkoxy, C 1-4 alkylamino, C 1-4 haloalkyl, C 1-4 haloalkoxy, C 1-4 hydroxyalkyl or C 1-4 haloalkylamino.

[0055] According to an embodiment of the present invention, the use further includes at least one of the following additional technical features:

[0056] According to an embodiment of the present invention, the plasmalogen-related diseases include at least one of Alzheimer's disease, cardiovascular diseases and peroxisome-related diseases.

[0057] The additional aspects and advantages of the present invention will be partly given in the following description, partly will become apparent from the following description, or will be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0059] Figure 1 is a schematic diagram of the plasmalogen structure according to an embodiment of the present invention;

[0060] Figure 2 is a schematic diagram of the reaction of the fluorescent small molecule QQMP with ethanolamine plasmalogen (PE-Pls) and choline plasmalogen (PC-Pls) according to an embodiment of the present invention;

[0061] Figure 3 is the structural formula of QQMP according to Embodiment 1 of the present invention;

[0062] Figure 4 is a schematic diagram of the plasmalogen and lipid structure without vinyl ether bond according to Embodiment 2 of the present invention;

[0063] Figure 5 is a high performance liquid chromatography (HPLC) diagram of the screening results of the buffer pH value and the ratio of buffer to organic solvent in the cycloaddition reaction according to Embodiment 2 of the present invention;

[0064] Figure 6 is a high performance liquid chromatography (HPLC) diagram after the reaction of QQMP with lipid standards according to Embodiment 3 of the present invention;

[0065] Figure 7 is a secondary mass spectrometry diagram of the labeled plasmalogen case according to Embodiment 3 of the present invention;

[0066] Figure 8 is a high performance liquid chromatography (HPLC) diagram after the reaction of QQMP with blood lipids according to Embodiment 4 of the present invention. Detailed Embodiments

[0067] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the drawings. The embodiments described below by referring to the drawings are exemplary and are intended to explain the present invention, and should not be construed as a limitation to the present invention.

[0068] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include at least one of such features. In the description of the present invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0069] The endpoints and any values in the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0070] In one aspect of the present invention, the present invention provides the use of a compound represented by formula (I) or a stereoisomer, tautomer or salt thereof of the compound represented by formula (I) in labeling a substrate,

[0071]

[0072] wherein the substrate comprises at least one vinyl ether bond;

[0073] R1, R2, R3, R4 and R5 are each independently selected from H, D, F, Cl, Br, I, OH, NH2, NO2, CN, N3, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 hydroxyalkyl, C 1-6 alkylamino, C 3-12 carbocyclic group, heterocyclic group composed of 3 - 12 atoms, C 6-10 aryl or heteroaryl composed of 5 - 12 atoms, wherein each of R1, R2, R3, R4 and R5 is independently optionally substituted by one or more R 6 ;

[0074] The R 6 is independently H, D, F, Cl, Br, I, =O, OH, NH2, NO2, CN, N3, C 1-4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C 1-4 alkoxy, C 1-4 alkylamino, C 1-4 haloalkyl, C 1-4 haloalkoxy, C 1-4 hydroxyalkyl or C 1-4 haloalkylamino.

[0075] Generally, the term "substituted" means that one or more hydrogen atoms in a given structure or group are replaced by specific substituents. Unless otherwise indicated, a substituent can be substituted at each reasonable position where substitution is possible in the group. When more than one position in a given structural formula can be substituted by one or more specific substituents selected therefrom, the substituents can be the same or different and can be substituted at each reasonable position in the structural formula. The substituents described in the present invention include, but are not limited to, -NH-, -O-, -COO-, C 1-6 alkylene, C 2-6 alkenylene, C 2-6 alkynylene, C 1-6 haloalkylene, C 1-6 alkoxyalkylene, C 1-6 haloalkoxyalkylene, C 1-6 alkanimino, C 1-6 hydroxy-substituted alkylene, (C 1-6 alkylene)-C(=O)-, (C 1-6 alkoxy)-C(=O)-, (C 1-6 alkanimino)-C(=O)-, cycloalkyl, heterocyclic group, aryl or heteroaryl, and the like.

[0076] The term "stereoisomer" refers to compounds having the same chemical constitution but different arrangements of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans isomers), atropisomers, and the like.

[0077] The stereochemical definitions and rules used herein generally follow S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S, “Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc, New York, 1994. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. When describing optically active compounds, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule with respect to one or more chiral centers. The prefixes d and l or (+) and (-) are symbols used to specify the rotation of plane-polarized light caused by the compound, where (-) or l indicates that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. A specific stereoisomer is an enantiomer, and a mixture of such isomers is called a racemic mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process.

[0078] Any mixture of the resulting stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, diastereomers, e.g., by chromatography and / or fractional crystallization, based on differences in the physicochemical properties of the components.

[0079] The racemate of any resulting end product or intermediate can be resolved into the optical enantiomers by known methods familiar to those skilled in the art, e.g., by separation of the diastereomeric salts obtained thereof. The racemic product can also be separated by chiral chromatography, e.g., high performance liquid chromatography (HPLC) using a chiral adsorbent. In particular, the enantiomers can be prepared by asymmetric synthesis, for example, see Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2nd Ed. Robert E. Gawley, Jeffrey Aube, Elsevier, Oxford, UK, 2012); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S. H. Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972); Chiral Separation Techniques: A Practical Approach (Subramanian, G. Ed., Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2007).

[0080] The terms “tautomer” or “tautomeric form” refer to structural isomers of different energies that can interconvert via a low energy barrier. If tautomerism is possible (e.g., in solution), a chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (also called prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization.

[0081] The "salts" used in the present invention refer to the organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well-known in the art, as described in the literature: S.M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. The salts formed from pharmaceutically acceptable non-toxic acids include, but are not limited to, inorganic acid salts formed by reacting with amino groups such as hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, and organic acid salts such as acetate, oxalate, maleate, tartrate, citrate, succinate, malonate, or obtained by other methods described in books and literature such as ion exchange method to obtain these salts. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, mesylate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and so on. The salts obtained by appropriate bases include salts of alkali metals, alkaline earth metals, ammonium, and N+(C1-4 alkyl)4. The present invention also contemplates quaternary ammonium salts formed from any compound containing an N group. Water-soluble, oil-soluble, or dispersible products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and so on. Pharmaceutically acceptable salts further include appropriate, non-toxic ammonium, quaternary ammonium salts, and amine cations formed with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C1-8 sulfonates, and aromatic sulfonates.

[0082] In yet another aspect of the present invention, the present invention provides a method for fluorescently labeling a substrate. According to an embodiment of the present invention, the method includes: contacting a compound represented by formula (I) or a stereoisomer, tautomer, or salt thereof with a substrate to be labeled, wherein the substrate to be labeled includes at least one vinyl ether bond.

[0083]

[0084] Wherein, R1, R2, R3, R4 and R5 are each independently selected from H, D, F, Cl, Br, I, OH, NH2, NO2, CN, N3, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 hydroxyalkyl, C 1-6 alkylamino, C 3-12 carbocyclic group, heterocyclic group composed of 3 - 12 atoms, C 6-10 aryl or heteroaryl composed of 5 - 12 atoms, wherein each of R1, R2, R3, R4 and R5 is independently and optionally substituted by one or more R 6 .

[0085] Said R 6 is independently H, D, F, Cl, Br, I, =O, OH, NH2, NO2, CN, N3, C 1-4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C 1-4 alkoxy, C 1-4 alkylamino, C 1-4 haloalkyl, C 1-4 haloalkoxy, C 1-4 hydroxyalkyl or C 1-4 haloalkylamino.

[0086] It should be noted that the substrate of the fluorescent label is a lipid after acylation treatment, and the compound shown in the foregoing formula (I) or a stereoisomer, tautomer or salt of the compound shown in formula (I) undergoes a cycloaddition reaction with the vinyl ether bond of plasmalogen in the lipid to achieve fluorescent labeling of the substrate ( Figure 2 ).

[0087] According to an embodiment of the present invention, the contact is carried out in a first solvent, and the concentration of the compound shown in formula (I) or a stereoisomer, tautomer or salt of the compound shown in formula (I) in the first solvent is 100 μM - 100 mM.

[0088] According to an embodiment of the present invention, the first solvent includes a buffer solution and an organic solvent.

[0089] According to an embodiment of the present invention, the buffer solution includes at least one of phosphate, HEPES, MOPS, and MES buffer solutions. It should be noted that the phosphate buffer solution includes PBS buffer solution.

[0090] According to an embodiment of the present invention, the pH of the buffer solution is 4.0 - 9.0.

[0091] According to an embodiment of the present invention, the pH of the buffer is 6.0.

[0092] According to an embodiment of the present invention, the organic solvent includes acetonitrile.

[0093] According to an embodiment of the present invention, the volume ratio of the buffer to the organic solvent is 1:9 to 9:1.

[0094] According to an embodiment of the present invention, the volume ratio of the buffer to the organic solvent is 7:3.

[0095] According to an embodiment of the present invention, the substrate is pre-acylated.

[0096] According to an embodiment of the present invention, the substrate includes plasmalogen.

[0097] According to an embodiment of the present invention, the acylation treatment is carried out in the presence of an acylating reagent and a catalyst. It should be noted that the acylating reagent and the acylating agent have the same representative meaning.

[0098] According to an embodiment of the present invention, the acylating reagent includes at least one selected from N-hydroxysuccinimide esters and their common derivatives.

[0099] According to an embodiment of the present invention, the acylating reagent is N-hydroxysuccinimide acetate.

[0100] According to an embodiment of the present invention, the concentration of N-hydroxysuccinimide acetate in the acylation system is 500 μM - 50 mM.

[0101] According to an embodiment of the present invention, the catalyst is an organic base.

[0102] According to an embodiment of the present invention, the organic base is selected from at least one of 4-dimethylaminopyridine and triethylamine.

[0103] According to an embodiment of the present invention, the plasmalogen includes a carbon chain at the sn-1 position, a polar group, and optionally an acyl chain at the sn-2 position. According to an embodiment of the present invention, the types of plasmalogens that can be labeled are determined by the carbon chain at the sn-1 position, the acyl chain at the sn-2 position, and the types of polar groups on its glycerol backbone.

[0104] According to an embodiment of the present invention, the carbon chain at the sn-1 position includes at least one selected from C16:0, C18:0, C18:1, C18:2, C20:0, C20:1, C20:2, C22:0, C22:1, C22:2.

[0105] According to an embodiment of the present invention, the acyl chain at the sn-2 position includes at least one of C16:0, C18:0, C18:1, C18:2, C18:3, C20:3, C20:4, C20:5, C22:4, C22:5, C22:6, C22:7, C24:8.

[0106] In this article, the structure of glycerophospholipid has glycerol (glycerin) as the structural center, and three carbons of glycerol are respectively connected to R x1 , R x2 , phosphate group (other groups can also be connected behind the phosphate), R x1 , R x2 is a hydrocarbon, and the carbon atoms to which R x1 , R x2 are connected are called the sn-1 and sn-2 positions of the phospholipid. There are many connection methods at the sn-1 and sn-2 positions. In addition to being connected by an ester bond, they can also be connected by an ether bond.

[0107] It should be noted that in this article, taking C16:0 as an example (sn-1 position), among them, 16 in C16:0 represents the number of C atoms in the carbon chain, 0 represents the number of double bonds, and it contains an enol ether bond. Taking C18:1 as an example (sn-1 position), 18 in C18:1 represents the number of C atoms in the carbon chain, 1 represents the presence of one double bond, and it contains an enol ether bond. The sn-2 position may not contain a fatty acyl chain.

[0108] Specific introductions to the embodiments will be given below. For those not specifying specific technologies or conditions in the embodiments, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product instructions. For those reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchases.

[0109] Example 1: Synthesis of 7-hydroxy-8-hydroxymethylquinoline (QQMP)

[0110] According to an embodiment of the present invention, the specific steps for preparing the fluorescent small molecule QQMP (7-hydroxy-8-hydroxymethylquinoline) are as follows:

[0111] 1) Dissolve 435 mg (3 mmol) of 7-hydroxyquinoline in 1.8 mL of 2 M aqueous sodium hydroxide solution, and then add 2.5 mL of 37% aqueous formaldehyde solution to obtain a mixed solution;

[0112] 2) After stirring the mixed solution in step 1) at room temperature for 1.5 h, adjust the pH of the reaction system to 2-3 with 5 M hydrochloric acid, and continue the reaction for 3 min;

[0113] 3) Alkalize the reaction system in step 2) with saturated sodium bicarbonate solution, then extract with a mixture of isopropanol and chloroform (volume ratio 1:5) of 40 ml, repeat three times to obtain the organic phase.

[0114] 4) Combine the organic phases obtained by extraction, wash once with saturated sodium chloride solution, then dry and concentrate with anhydrous sodium sulfate to obtain the concentrated crude product.

[0115] 5) Purify the concentrated crude product by silica gel column chromatography: first elute the by-products with low polarity with petroleum ether and ethyl acetate (volume ratio 5:1), then elute the target product with petroleum ether and ethyl acetate (volume ratio 3:2), concentrate the target product to remove the solvent to obtain 7-hydroxy-8-hydroxymethylquinoline.

[0116] The results showed that a total of 295 mg of 7-hydroxy-8-hydroxymethylquinoline (pale yellow solid) was obtained, with a yield of 56.2%. The target product was verified by NMR and mass spectrometry. The NMR results: 1 H NMR (methanol-d4, 400 MHz), δ (ppm): 5.31 (s, 2H), 7.19 (d, J = 8.0 Hz, 1H), 7.28 (m, 1H), 7.69 (d, J = 8.0 Hz, 1H), 8.15 (m, 1H), 8.73 (m, 1H). 13 C NMR (methanol-d4, 100 MHz), δ (ppm): 56.18, 118.07, 118.23, 119.00, 123.17, 128.37, 136.65, 147.86, 149.29, 156.63; the mass spectrometry results: C 10 H9NO2 [M-H]-theoretical value 174.0555, actual value 174.0589. The above results indicate that the prepared 7-hydroxy-8-hydroxymethylquinoline product has a high purity ( Figure 3 )

[0117] Example 2: Labeling plasmalogen with QQMP fluorescence

[0118] According to the embodiment of the present invention, the specific steps for labeling plasmalogen with QQMP fluorescence are as follows:

[0119] 1. Acetylate and block the primary amino group of the lipid with N-hydroxysuccinimide acetate

[0120] Mix 200 μL of acetonitrile, 20 μL of 2 mM lipid standards (PE-Pls 18:0 / 18:1, PE-Pls 18:0 / 20:4, PC-Pls 18:0 / 18:1, DOPE, DOPC, PS 16:0, PA18:1, PG 18:1, Sph d18:1, SM, Chol, TAG)( Figure 4 ), 20 μL of 100 mM N-hydroxysuccinimide acetate, and 4 μL of 100 mM 4-dimethylaminopyridine, and react with shaking at 37 °C for 1 h to obtain an acetylated and blocked lipid standard reaction solution.

[0121] 2. The heteroatom Diels-Alder reaction (cycloaddition reaction) occurs between QQMP and the vinyl ether bond of plasmalogen

[0122] 2.1 pH screening process of PBS buffer in the cycloaddition reaction (pH 4.0 - 9.0, optimal 6.0)

[0123] Use 1-butene ethyl ether containing a vinyl ether bond instead of plasmalogen to screen the pH value of the buffer and the ratio of buffer to organic solvent in the cycloaddition reaction. Since 1-butene ethyl ether is a mixture of cis and trans isomers, there are also two isomers of the cycloaddition product. Add QQMP and 1-butene ethyl ether to the mixed solution of 1×PBS buffer (pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0) and acetonitrile, react with shaking at 37 °C for 24 h, collect the reaction product, and perform high performance liquid chromatography detection. According to the high performance liquid chromatography results, when the pH value of the buffer solution is 6.0, the yield of the cycloaddition reaction is the highest ( Figure 5 left).

[0124] 2.2 Screening process of the ratio of buffer to organic solvent in the cycloaddition reaction (optimal 7:3)

[0125] Add QQMP and 1-butene ethyl ether to the mixed solution of 1×PBS buffer and acetonitrile (volume ratio 1:9, 3:7, 5:5, 7:3, 9:1), react with shaking at 37 °C for 24 h, collect the reaction product, and perform high performance liquid chromatography detection. According to the high performance liquid chromatography results, when the volume ratio of the buffer solution to acetonitrile is 7:3, the yield of the cycloaddition reaction is the highest ( Figure 5 right).

[0126] 2.3 Perform the cycloaddition reaction between QQMP and plasmalogen according to the optimal pH and the optimal ratio of buffer to organic solvent screened in 2.1 and 2.2

[0127] Add 200 μL of acetonitrile, 1.4 mL of 1×PBS buffer (pH 6.0), and 200 μL of QQMP (concentration: 20 mM) to the acetylated and blocked lipid standard reaction solution obtained in Step 1. React with shaking at 37 °C for 24 h, collect the reaction product, and perform high-performance liquid chromatography detection.

[0128] Example 3: Detection of Labeled Products by High-Performance Liquid Chromatography-Fluorescence Analysis

[0129] According to the embodiments of the present invention, in order to determine that QQMP only undergoes a fluorescence labeling reaction with plasmalogens and does not react with other lipids; the labeled products of plasmalogens are analyzed by tandem mass spectrometry to further verify the cycloaddition reaction of QQMP with plasmalogens at the vinyl ether bond site. The specific detection steps are as follows:

[0130] 1. Reaction Sample Treatment

[0131] Place the reaction product obtained in Example 2 in an argon stream to remove the organic solvent, and then extract it three times with 0.7 ml of a dichloromethane and methanol mixture (volume ratio 10:1). Combine the extracted organic phases, remove the solvent in an argon stream, and then redissolve it with 100 μL of a dichloromethane and methanol mixture (volume ratio 2:1). The injection volume is 10 μL for high-performance liquid chromatography-fluorescence analysis.

[0132] 2. High-Performance Liquid Chromatography-Fluorescence Analysis

[0133] Detect the lipid standard according to the following chromatographic conditions:

[0134] Chromatographic column: C18, length 250 mm, inner diameter 4.6 mm, particle size 5 μm;

[0135] Mobile phase A: Dissolve 0.77 g of ammonium acetate in 400 mL of ultrapure water, and then add 600 mL of HPLC-grade acetonitrile;

[0136] Mobile phase B: Mix 100 mL of HPLC-grade acetonitrile with 900 mL of HPLC-grade isopropanol;

[0137] Column temperature: 35 °C; Flow rate: 1 mL / min;

[0138] Fluorescence detector: Excitation wavelength 330 nm, emission wavelength 370 nm;

[0139] The gradient elution program is shown in Table 1.

[0140] Table 1: Gradient Elution Program

[0141] Time (min) A(％) B(％) 0 63 37 3 63 37 8 55 45 10 48 52 16 42 58 22 34 66 28 30 70 36 25 75 40 2 98 44 2 98 44.2 63 37 50 63 37

[0142] The results showed that for the reaction samples of the above lipid standards ethanolamine plasmalogen (PE-Pls) 18:0 / 18:1, ethanolamine plasmalogen (PE-Pls) 18:0 / 20:4, choline plasmalogen (PC-Pls) 18:0 / 18:1, dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylcholine (DOPC), phosphatidylserine (PS) 16:0, phosphatidic acid (PA) 18:1, phosphatidylglycerol (PG) 18:1, sphingosine (Sph) d18:1, sphingomyelin (SM), cholesterol (Chol), triglyceride (TAG) and QQMP, high performance liquid chromatography-fluorescence analysis was carried out. Only the samples containing plasmalogen standards PE-Pls 18:0 / 18:1, PE-Pls 18:0 / 20:4 and PC-Pls 18:0 / 18:1 had strong chromatographic peaks, and the samples of other lipids without vinyl ether bonds had no obvious signals( Figure 6 ).

[0143] 3. Tandem mass spectrometry analysis

[0144] Tandem mass spectrometry analysis was carried out on the chromatographic peak eluates of the above samples containing plasmalogen standards PE-Pls 18:0 / 18:1, PE-Pls 18:0 / 20:4 and PC-Pls 18:0 / 18:1 respectively( Figure 7 ). The results showed that in the positive ion mode, the characteristic fragment ion peak F1 of the derivatized carbon chain at the sn-1 site of the glycerol backbone was observed; in the negative ion mode, the characteristic fragment ion peak F2 of the acyl chain at the sn-2 site of the glycerol backbone was observed. Through tandem mass spectrometry analysis, it was determined that QQMP underwent a cycloaddition reaction with the vinyl ether bond of plasmalogen.

[0145] Example 4: Fluorescent labeling method combined with high performance liquid chromatography-fluorescence analysis technology for the analysis and screening of plasmalogen in blood lipids

[0146] According to the embodiments of the present invention, the specific analysis and screening steps are as follows:[[]]

[0147] 1. Lipid extraction

[0148] 800 μL of a dichloromethane and methanol mixture (volume ratio 2:1) was added to 200 μL of human plasma, vortexed and left to stand, repeated 3 times. After sufficient extraction, centrifugation (4 °C, 10,000 rpm, 20 min) was carried out to achieve solution stratification. The lower organic phase was taken out and the solvent was dried in an argon stream. Then 50 μL of a dichloromethane and methanol mixture (volume ratio 2:1) was added for reconstitution to obtain a human plasma lipid extract.

[0149] 2. Acetylation blocking of primary amino groups in lipids

[0150] Take 50 μL of the human plasma lipid extract obtained in step 1, mix it with 200 μL of acetonitrile, 30 μL of 100 mM N-hydroxysuccinimide acetate, and 4 μL of 100 mM 4-dimethylaminopyridine, and react with shaking at 37 °C for 1 h to obtain an acetylation reaction solution.

[0151] 3. Use QQMP fluorescence labeling of plasmalogens in blood lipids

[0152] Mix the acetylation reaction solution obtained in step 2 with 1.4 mL of 1×PBS buffer (pH 6.0) and 400 μL of 40 mM QQMP, and react with shaking at 37 °C for 24 h for labeling, and collect the reaction products.

[0153] 4. Reaction sample treatment

[0154] Place the reaction products obtained in step 3 in an argon stream to remove the organic solvents, then extract with 0.7 ml of a dichloromethane and methanol mixture (volume ratio 10:1) three times. Combine the extracted organic phases, remove the solvents in an argon stream, and then redissolve with 100 μL of a dichloromethane and methanol mixture (volume ratio 2:1). The injection volume is 10 μL for high performance liquid chromatography-fluorescence analysis.

[0155] 5. High performance liquid chromatography-fluorescence analysis

[0156] Detect the above blood lipid samples according to the following chromatographic conditions:

[0157] Chromatographic column: C18, length 250 mm, inner diameter 4.6 mm, particle size 5 μm;

[0158] Mobile phase A: Dissolve 0.77 g of ammonium acetate in 400 mL of ultrapure water, and then add 600 mL of HPLC grade acetonitrile;

[0159] Mobile phase B: Mix 100 mL of HPLC grade acetonitrile with 900 mL of HPLC grade isopropanol;

[0160] Column temperature: 35 °C; Flow rate: 1 mL / min;

[0161] Fluorescence detector: Excitation wavelength 330 nm, emission wavelength 370 nm;

[0162] The gradient elution program is shown in Table 1.

[0163] Table 1: Gradient elution program

[0164] Time (min) A(％) B(％) 0 63 37 3 63 37 8 55 45 10 48 52 16 42 58 22 34 66 28 30 70 36 25 75 40 2 98 44 2 98 44.2 63 37 50 63 37

[0165] The results show that there are multiple obvious chromatographic signal peaks in the blood lipid samples after reaction with QQMP, and these signal peaks can be used for the analysis and screening of plasmalogens in blood lipids (Figure 8 )。

[0166] 6. High performance liquid chromatography - tandem mass spectrometry analysis

[0167] The reacted blood lipid samples were analyzed by high performance liquid chromatography - tandem mass spectrometry. Using F1 (the characteristic fragment ion peak of the derivatized carbon chain at the sn - 1 position of the glycerol backbone) and F2 (the characteristic fragment ion peak of the acyl chain at the sn - 2 position of the glycerol backbone) as characteristic fragment ions, 60 labeled plasmalogens were found, including 29 plasmalogens with ethanolamine as the polar head and 31 plasmalogens with choline as the polar head, verifying that the cycloaddition reaction of QQMP with the vinyl ether bond is applicable to the plasmalogens in fluorescently labeled blood lipids.

[0168] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0169] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

Use of a compound represented by formula (I) or a stereoisomer, tautomer or salt thereof of the compound represented by formula (I) in a labeled substrate, Among them, wherein the substrate comprises at least one vinyl ether bond; R1, R2, R3, R4, and R5 are each independently selected from H, D, F, Cl, Br, I, OH, NH2, NO2, CN, N3, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 hydroxyalkyl, C 1-6 alkylamino, C 3-12 carbocyclic group, heterocyclic group consisting of 3 to 12 atoms, C 6-10 aryl or heteroaryl consisting of 5 to 12 atoms, wherein each of R1, R2, R3, R4, and R5 is independently and optionally substituted by one or more R 6 substituted, Said R 6 is independently H, D, F, Cl, Br, I, =O, OH, NH2, NO2, CN, N3, C 1-4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C 1-4 alkoxy, C 1-4 alkylamino, C 1-4 haloalkyl, C 1-4 haloalkoxy, C 1-4 hydroxyalkyl or C 1-4 haloalkylamino.

2. The use according to claim 1, wherein R1, R2, R3, R4 and R5 are each independently selected from H.

3. A method for fluorescently labeling a substrate, characterized in that, Comprising: contacting the compound represented by formula (I) or a stereoisomer, tautomer or salt thereof of the compound represented by formula (I) with a substrate to be labeled, wherein the substrate to be labeled comprises at least one vinyl ether bond, wherein, R1, R2, R3, R4 and R5 are each independently selected from H, D, F, Cl, Br, I, OH, NH2, NO2, CN, N3, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 hydroxyalkyl, C 1-6 alkylamino, C 3-12 carbocyclic group, heterocyclic group composed of 3-12 atoms, C 6-10 aryl or heteroaryl composed of 5-12 atoms, wherein each of R1, R2, R3, R4 and R5 is independently and optionally substituted by one or more R 6 substituted, Said R 6 is independently H, D, F, Cl, Br, I, =O, OH, NH2, NO2, CN, N3, C 1-4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C 1-4 alkoxy, C 1-4 alkylamino, C 1-4 haloalkyl, C 1-4 haloalkoxy, C 1-4 hydroxyalkyl or C 1-4 haloalkylamino.

4. The method according to claim 3, wherein the contacting is carried out in a first solvent, and the concentration of the compound represented by formula (I) or a stereoisomer, tautomer or salt thereof of the compound represented by formula (I) in the first solvent is 100 μM - 100 mM.

5. The method according to claim 4, characterized in that, The first solvent comprises a buffer solution and an organic solvent.

6. The method according to claim 5, wherein The buffer solution comprises at least one of phosphate, HEPES, MOPS, MES buffer solutions.

7. The method according to claim 5, characterized in that, The pH of the buffer solution is 4.0 - 9.

0.

8. The method according to claim 7, wherein The pH of the buffer solution is 6.

0.

9. The method according to claim 5, wherein The organic solvent comprises acetonitrile.

10. The method according to claim 5, wherein The volume ratio of the buffer solution to the organic solvent is 1:9 to 9:

1.

11. The method according to claim 10, wherein The volume ratio of the buffer solution to the organic solvent is 7:

3.

12. The method according to claim 3, wherein The substrate is pre-acylated.

13. The method according to claim 12, wherein The substrate comprises plasmalogen.

14. The method according to claim 12, wherein The acylation treatment is carried out in the presence of an acylating agent and a catalyst.

15. The method according to claim 14, wherein The acylating agent comprises at least one selected from N-hydroxysuccinimide esters and their common derivatives.

16. The method according to claim 15, wherein The acylating agent is N-hydroxysuccinimide acetate.

17. The method according to claim 16, wherein In the acylation treatment, the concentration of N-hydroxysuccinimide acetate in the acylation system is 500 μM - 50 mM.

18. The method according to claim 14, wherein The catalyst is an organic base.

19. The method according to claim 18, wherein The organic base is selected from at least one of 4-dimethylaminopyridine and triethylamine.

20. The method according to claim 13, wherein The plasmalogen comprises an sn-1 site carbon chain, a polar group and optionally an sn-2 site acyl chain.

21. The method according to claim 20, wherein The sn-1 site carbon chain comprises at least one selected from C16:0, C18:0, C18:1, C18:2, C20:0, C20:1, C20:2, C22:0, C22:1, C22:

2.

22. The method according to claim 20, wherein The sn-2 site acyl chain comprises at least one of C16:0, C18:0, C18:1, C18:2, C18:3, C20:3, C20:4, C20:5, C22:4, C22:5, C22:6, C22:7, C24:

8.

23. The method according to claim 20, wherein The polar group comprises at least one of phosphoric acid, phosphoethanolamine, phosphocholine, phosphoserine, phosphoglycerol, phosphoinositol.

24. A fluorescently labeled plasmalogen, characterized in that, The fluorescently labeled plasmalogen is obtained by labeling according to the method of any one of claims 3 - 23.

25. A method for detecting plasmalogen, characterized in that, Comprising: labeling plasmalogen according to the method of any one of claims 3 - 23; detecting the labeled plasmalogen by high performance liquid chromatography to obtain a liquid chromatogram; determining plasmalogen based on the obtained liquid chromatogram.

26. The method according to claim 25, characterized in that, The mobile phase used in the high performance liquid chromatography is selected from at least one of water, methanol, acetonitrile, isopropanol, n-hexane, acetic acid, formic acid, ammonium acetate, ammonium formate; Optionally, the chromatographic column used in the liquid chromatography is selected from at least one of a C18 column, a C8 column, an amino column, and a silica gel column; Optionally, the detection wavelength of the liquid chromatography is 300 - 350 nm, and the emission wavelength is 350 - 430 nm.

27. The method according to claim 25, wherein The plasmalogen is derived from a blood sample; Optionally, the blood sample is selected from at least one of whole blood, serum, plasma, red blood cells, white blood cells, and platelets. Use of the compound represented by formula (I) or a stereoisomer, tautomer, or salt thereof of the compound represented by formula (I) or the fluorescently labeled plasmalogen according to claim 24 in the preparation of a kit for detecting plasmalogen or plasmalogen-related diseases in blood lipids, Among them, R1, R2, R3, R4 and R5 are each independently selected from H, D, F, Cl, Br, I, OH, NH2, NO2, CN, N3, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 hydroxyalkyl, C 1-6 alkylamino, C 3-12 carbocyclic group, heterocyclic group composed of 3 - 12 atoms, C 6-10 aryl or heteroaryl composed of 5 - 12 atoms, wherein each of R1, R2, R3, R4 and R5 is independently optionally substituted by one or more R 6 ; Said R 6 is independently H, D, F, Cl, Br, I, =O, OH, NH2, NO2, CN, N3, C 1-4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C 1-4 alkoxy, C 1-4 alkylamino, C 1-4 haloalkyl, C 1-4 haloalkoxy, C 1-4 hydroxyalkyl or C 1-4 haloalkylamino.

29. The use according to claim 28, wherein, The plasmalogen-related diseases include at least one of Alzheimer's disease, cardiovascular diseases, and peroxisome-related diseases.

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