Use of a secondary amine reagent in the detection and analysis of carboxylic acids

Abstract

The application discloses application of a secondary amine reagent in carboxylic acid analysis and / or separation, and the structural formula of the secondary amine reagent is one of the following structures: (PMBA), (5-PMBA). d 5-PMBA). The PMBA and 5-PMBA provided by the application can be used for analysis and detection of carboxylic acid. d 5-PMBA can be used for analysis and detection of carboxylic acid, and by N -(piperidin-4-ylmethyl)benzamide (PMBA) and stable isotope forms thereof d 5-PMBA is used alone or in combination as a pair of isotope-labeled reagents, and a condensation reaction occurs with carboxylic acid; the obtained derivative can effectively detect and separate the carboxylic acid, and an easy ionizable group is introduced, so that the mass spectrum response in the ESI positive ion mode is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of carboxylic acid detection technology, specifically to the application of a secondary amine reagent in the detection and analysis of carboxylic acids. Background Technology

[0002] The carboxylic acid family is one of the busiest signaling molecules in organisms: from energy factories to interspecies communication, their concentration fluctuations are like a real-time updated "health check report." The carboxylic acid family is vast, and common examples include short-, medium-, and long-chain fatty acids, tricarboxylic acid cycle intermediates, aromatic / phenyl carboxylic acids, and plant hormones.

[0003] Changes in the levels of these carboxylic acids can reflect the biological state of an organism. For example, the tricarboxylic acid cycle intermediate—the mitochondrial "gear"—is also an epigenetic-immune messenger; an imbalance often indicates that a tumor has reprogrammed its energy pathways. Fatty acids—essential biomolecules for all organisms and one of the body's main energy sources; aromatic / phenyl carboxylic acids—a cross-border language between microorganisms and plants, possessing both stress resistance and neural activity.

[0004] Currently, the determination of carboxylic acid metabolites in biological samples mainly relies on mass spectrometry. In negative ion detection mode, direct liquid chromatography-tandem mass spectrometry (LC-MS / MS) is the most commonly used method. However, because some carboxylic acid metabolites are present in extremely low concentrations in organisms, accurate determination is difficult. Therefore, amine derivatization reagents with high ionization efficiency are often used to react with carboxylic acid metabolites, thereby improving the mass spectrometric detection sensitivity of carboxylic acid metabolites.

[0005] In summary, developing novel derivatization reagents is of great significance for reducing detection costs, improving the sensitivity of carboxylic acids in mass spectrometry, and enabling quantitative detection. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to improve the sensitivity of carboxylic acid detection and how to separate carboxylic acids and their isomers.

[0007] The present invention solves the above-mentioned technical problems through the following technical means:

[0008] The application of a secondary amine reagent in the analysis and / or separation of carboxylic acids, wherein the secondary amine reagent has one of the following structural formulas: .

[0009] Preferably, the preparation method of the secondary amine reagent includes the following steps: S1. Reaction of benzoic acid or deuterated benzoic acid and oxaloyl chloride in a solvent; S2. The product obtained in S1 is reacted with 1-tert-butoxycarbonyl-4-aminomethylpiperidine in the presence of a base and a solvent. S3. The product obtained in S2 is reacted with trifluoroacetic acid in a solvent to obtain the secondary amine reagent.

[0010] Preferably, in S1, the ratio of benzoic acid or deuterated benzoic acid to oxaloyl chloride is 500 mg: 0.42 mL; the mass ratio of 1-tert-butoxycarbonyl-4-aminomethylpiperidine used in S2 to benzoic acid or deuterated benzoic acid used in S1 is 1:1; and in S3, the ratio of the product obtained in S2 to trifluoroacetic acid is 500 mg: 1.6 mL.

[0011] Preferably, in S1, the reaction process includes reacting under ice bath conditions for half an hour and then reacting at room temperature for 4 hours; in S2, the reaction includes reacting under ice bath conditions for half an hour and then reacting at room temperature for 1 hour; in S3, the reaction includes reacting at room temperature for 1 hour; in S1, the solvent is dichloromethane; in S2, the solvent is dichloromethane and the base is triethylamine; in S3, the solvent is dichloromethane.

[0012] Preferably, the carboxylic acid is one or a mixture of carboxylic acid metabolites from the tricarboxylic acid cycle, short-chain fatty acids, hydroxy acids, and keto acids.

[0013] Preferably, the carboxylic acid is one or a mixture of succinic acid (SA), fumaric acid (FA), malic acid (MA), 2-ketobutyric acid (2-KA), pyruvic acid (PYR), lactic acid (LA), methoxyacetic acid (MAA), 2-hydroxybutyric acid (2-HBA), 3-hydroxybutyric acid (3-HBA), n-butyric acid (BA), n-valeric acid (VA), propionic acid (PA), hexanoic acid (CA), valproic acid (VPA), 4-pentenoic acid (4-PA), and phenyllactic acid (PLA).

[0014] Preferably, the carboxylic acid is a mixture of succinic acid (SA), fumaric acid (FA), malic acid (MA), 2-ketobutyric acid (2-KA), pyruvic acid (PYR), lactic acid (LA), methoxyacetic acid (MAA), 2-hydroxybutyric acid (2-HBA), 3-hydroxybutyric acid (3-HBA), n-butyric acid (BA), n-valeric acid (VA), propionic acid (PA), hexanoic acid (CA), valproic acid (VPA), 4-pentenoic acid (4-PA), and phenyllactic acid (PLA).

[0015] This invention also proposes a method for analyzing and / or separating carboxylic acids using a secondary amine reagent, comprising the following steps: reacting a secondary amine reagent as a labeling reagent with a carboxylic acid to obtain a derivative; and then detecting the derivative using LC-MS; wherein the secondary amine reagent is... , One or two of them.

[0016] Preferably, a secondary amine reagent is used. or As a labeling reagent, it reacts with carboxylic acids to yield derivatives; the derivatives are then detected using LC-MS; or, a secondary amine reagent is used. As a labeling reagent, it reacts with carboxylic acids to give derivative A, which is then used as a secondary amine reagent. As a labeling reagent, it reacts with carboxylic acid to obtain derivative B, which is used as an internal standard; derivative A and derivative B are mixed and then detected by LC-MS.

[0017] Preferably, the reaction occurs with a carboxylic acid in the presence of a catalyst.

[0018] Preferably, the carboxylic acid solution is mixed with a catalyst and a secondary amine reagent, and the reaction is carried out at 30-55°C for 30-60 minutes to complete the labeling and obtain the derivative.

[0019] Preferably, the catalyst is one or more of the following: a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt); a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxy-7-azabenzotriazole (HOAt); and a mixture of triphenylphosphine (TPP) and 2,2'-dithiopyridine (DPDS).

[0020] Preferably, in the mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt), the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to 1-hydroxybenzotriazole (HOBt) is 1:1; in the mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxy-7-azabenzotriazole (HOAt), the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to N-hydroxy-7-azabenzotriazole (HOAt) is 1:1; and in the mixture of triphenylphosphine (TPP) and 2,2'-dithiodipyridine (DPDS), the molar ratio of triphenylphosphine (TPP) to 2,2'-dithiodipyridine (DPDS) is 1:1.

[0021] Preferably, the solution of carboxylic acid is reacted with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N The derivative is obtained by mixing 1-hydroxy-7-azabenzotriazole (HOAt) and a secondary amine reagent and reacting at 30-55℃ for 30-60 minutes.

[0022] Preferably, in the LC-MS detection process, the chromatographic column is a SHIMADZU shim pack GIST C18, with dimensions of 2.1 × 100 mm and 2.0 μm. The column temperature is 35℃. The mobile phase consists of mobile phase A and mobile phase B. A 0.1% (v / v) formic acid aqueous solution and acetonitrile are used as mobile phase A and mobile phase B, respectively, for chromatographic detection. The chromatographic gradient is as follows: 0-3 min, 5% (v / v) mobile phase B; 3-8 min, 5-15% (v / v) mobile phase B; 8-15 min, 15-25% (v / v) mobile phase B; 15-20 min, 25-60% (v / v) mobile phase B; 20-24 min, 60-90% (v / v) mobile phase B; 24-26 min, 90% (v / v) mobile phase B; 26-28 min, 90%-5% (v / v) mobile phase B; 28-30 min... The mobile phase B has a volume fraction of 5% and a flow rate of 0.35 mL / min.

[0023] The advantages of this invention are: (1) The PMBA and provided by this invention d 5-PMBA can be used for the analytical detection of carboxylic acids, by... N 3-(piperidin-4-ylmethyl)benzamide (PMBA) and its stable isotopic forms d 5-PMBA, used alone or in combination as a pair of isotope labeling reagents, undergoes a condensation reaction with carboxylic acids to obtain derivatives that introduce easily ionized groups, greatly improving their mass spectrometry response in ESI positive ion mode.

[0024] (2) This invention not only greatly improves the detection sensitivity of carboxylic acids, but also successfully separates two pairs of isomers (LA / MAA and 2-HBA / 3-HBA) in chromatography.

[0025] (3) This invention provides a method for preparing PMBA and d The 5-PMBA method is simple and easy to operate. Attached Figure Description

[0026] Figure 1 The NMR spectrum of the PMBA prepared in Example 1 of this invention; Figure 2 The sample prepared in Example 1 of this invention d 5-PMBA NMR; Figure 3 Different carboxylic acids (SA, 2-KA, VPA) in Example 2 of this invention were subjected to PMBA and d Secondary mass spectrum of the 5-PMBA labeled product; Figure 4 Different carboxylic acids (2-HBA, 3-HBA, PA) in Example 2 of this invention were subjected to PMBA and d Secondary mass spectrum of the 5-PMBA labeled product; Figure 5 Different carboxylic acids (4-PA, LA, MAA) in Example 2 of this invention were subjected to PMBA and d Secondary mass spectrum of the 5-PMBA labeled product; Figure 6 Different carboxylic acids (FA, MA, PYR) in Example 2 of this invention were subjected to PMBA and d Secondary mass spectrum of the 5-PMBA labeled product; Figure 7 Different carboxylic acids (BA, VA, CA) in Example 2 of this invention were subjected to PMBA and d Secondary mass spectrum of the 5-PMBA labeled product; Figure 8 The carboxylic acid (PLA) in Example 2 of this invention was processed with PMBA and d Secondary mass spectrum of the 5-PMBA labeled product; Figure 9 These are chromatograms of different carboxylic acids before labeling in Example 3 of the present invention; Figure 10 The chromatograms are of different carboxylic acids labeled with PMBA in Example 3 of this invention; Figure 11 PMBA and in Embodiment 3 of the present invention d Chromatogram of the labeled product after 5-PMBA labeling of carboxylic acid isomers (red indicates PMBA labeling, blue indicates...). d 5-PMBA marker); Figure 12 PMBA and in Embodiment 3 of the present invention d 5-PMBA-labeled carboxylic acid isomers, chromatograms after changing chromatographic conditions (red indicates PMBA labeling, blue indicates...). d 5-PMBA marker); Figure 13 PMBA and in Embodiment 3 of the present invention d Chromatogram of 5-PMBA simultaneously labeling multiple carboxylic acids (red indicates PMBA labeling, blue indicates...). d 5-PMBA marker); Figure 14 This is a comparison of the mass spectrometry responses of the labeled products (SA, 2-KA, BA, PA, CA, 4-PA, VA, PYR) using different catalysts in the PMBA-labeled carboxylic acid reaction of Example 4 of the present invention. Figure 15This is a comparison of the mass spectrometry responses of the labeled products (LA, MAA, 3-HBA, 2-HBA, VPA, MA, PLA, FA) using different catalysts in the PMBA-labeled carboxylic acid reaction of Example 4 of the present invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0029] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0030] It should be noted that the chemical reagents used in this invention are commercially available reagents. The LC-MS analysis of this invention was performed on a Shimadzu MS-8050 mass spectrometer (Shimadzu, Japan), equipped with an electrospray ionization source (ESI) (Turbo Ion Spray) and a Shimadzu LC-30AD UPLC system. The chromatographic column was a SHIMADZU shim pack GIST C18, with dimensions of 2.1 × 100 mm and a diameter of 2.0 μm.

[0031] The carboxylic acids in the embodiments of the present invention include succinic acid (SA), fumaric acid (FA), malic acid (MA), 2-ketobutyric acid (2-KA), pyruvic acid (PYR), lactic acid (LA), methoxyacetic acid (MAA), 2-hydroxybutyric acid (2-HBA), 3-hydroxybutyric acid (3-HBA), n-butyric acid (BA), n-valeric acid (VA), propionic acid (PA), hexanoic acid (CA), valproic acid (VPA), 4-pentenoic acid (4-PA), and phenyllactic acid (PLA) mixed in equal mass proportions.

[0032] Example 1 The reagent PMBA and the present invention d Synthesis of 5-PMBA Benzoic acid (500 mg) was dissolved in dichloromethane (5 mL), and oxaloyl chloride (0.42 mL) was slowly added dropwise. After reacting in an ice bath for half an hour, the mixture was transferred to room temperature and reacted for 4 hours. After the reaction was completed, the solution was evaporated to dryness, dissolved in dichloromethane (3 mL), and 1-tert-butoxycarbonyl-4-aminomethylpiperidine (500 mg) was added. Then, triethylamine (0.7 mL) was slowly added dropwise. After the addition was completed, the mixture was reacted in an ice bath for half an hour, and then transferred to room temperature and reacted for 1 hour. After the reaction was completed, 1 mL of water was added to quench the reaction. Dichloromethane and saturated brine (1:1 v / v) were added for extraction, and the organic layer was dried with anhydrous sodium sulfate. The intermediate was purified by column chromatography (petroleum ether / ethyl acetate, 2:1 v / v) to obtain a pale yellow intermediate.

[0033] The obtained intermediate (500 mg) was dissolved in 10 mL of dichloromethane, and then trifluoroacetic acid (1.6 mL) was slowly added dropwise. The reaction was carried out at room temperature for 1 h. After the reaction was completed, the solution was evaporated to dryness and extracted with dichloromethane. The product PMBA was obtained by purification using a chromatography column (dichloromethane / methanol / triethylamine, v / v ratio 95:4:1). Its NMR spectrum is as follows Figure 1 As shown; By replacing the initial substrate in the above steps with deuterated benzoic acid, the following preparation was obtained. d 5-PMBA Its NMR spectrum is as follows Figure 2 As shown.

[0034] Example 2 PMBA / d Chemical labeling of 5-PMBA Take an acetonitrile ACN solution of the above carboxylic acids (each carboxylic acid concentration in the solution is 10 ug / mL, 5 μL), and sequentially add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 100 mmol / L, 12 μL) and N-hydroxy-7-azabenzotriazole (HOAt, 100 mmol / L, 12 μL) dissolved in acetonitrile, PMBA or d Add 5-PMBA (100 mmol / L, 12 μL), then add acetonitrile to make the total volume of the labeling reaction 200 μL, and react at 35 °C for 30 minutes to obtain the carboxylic acid derivative.

[0035] The labeling reaction formula is shown below:

[0036] Figure 3-8 The mixture of carboxylic acids was subjected to PMBA and dThe secondary mass spectra of the corresponding labeled products after effective labeling with 5-PMBA show that different labeled products can produce characteristic secondary fragments, which can help with qualitative analysis.

[0037] Example 3 chromatographic behavior In Example 2, the secondary amine PMBA and d LC-ESI-MS analysis of 5-PMBA-labeled carboxylic acid derivatives and underrivatized carboxylic acids was performed on a Shimadzu MS-8050 mass spectrometer (Shimadzu, Japan), equipped with an electrospray ionization source and a Shimadzu LC-30AD UPLC system. Mass spectrometry parameters for carboxylic acid derivatives and underrivatized carboxylic acids were optimized in both positive and negative ion modes to achieve optimal analytical performance, using the MRM mode. Direct injection was employed to optimize the MRM parameters for best analytical performance. A SHIMADZU shim pack GIST C18 column (2.1 × 100 mm, 2.0 μm) was used, and the column temperature was 35 °C. Chromatographic analysis was performed using a 0.1% (v / v) formic acid aqueous solution (phase A) and acetonitrile (phase B) as mobile phases. The optimized chromatographic gradient was as follows: 0–3 min, 5% (v / v) mobile phase B; 3–8 min, 5–15% (v / v) mobile phase B; 8–15 min, 15–25% (v / v) mobile phase B; 15–20 min, 25–60% (v / v) mobile phase B; 20–24 min, 60–90% (v / v) mobile phase B; 24–26 min, 90% (v / v) mobile phase B; 26–28 min, 90%–5% (v / v) mobile phase B; 28–30 min, 5% (v / v) mobile phase B; the flow rate was 0.35 mL / min. Figure 9 The chromatogram of underrivatized carboxylic acids is given by... Figure 9 It can be seen that the carboxylic acid before labeling has weaker retention and the peak time is concentrated in the first minute; Figure 10 The chromatograms are of different carboxylic acids labeled with PMBA. Figure 10 It can be seen that the retention time of different carboxylic acids is significantly increased in reverse chromatography, which enables better separation of carboxylic acids.

[0038] The secondary amine PMBA in Example 2 and d 5-PMBA-labeled carboxylic acid derivatives were mixed in a 1:1 molar ratio and detected under the chromatographic conditions described above. Figure 13 For PMBA (shown in red) and d The chromatogram of the labeled product after 5-PMBA (shown in blue) labeling shows that the chromatographic behavior of the lightly and heavily labeled products is basically the same.

[0039] Figure 11The chromatogram for the separation of carboxylic acids from PMBA (shown in red) is shown. Figure 11 As can be seen from this, the two pairs of isomers, LA and MAA, and 2-HBA and 3-HBA, were effectively separated with good resolution. d 5-PMBA (shown in blue) has the same physicochemical properties as PMBA, and its separation spectrum is completely identical to that of PMBA. However, if the chromatographic conditions are changed, the separation effect is not ideal, such as... Figure 12 As shown, the chromatographic conditions were as follows: The column was a Thermo Scientific™ Accucore™ C18 (2.1 × 150 mm, 2.6 μm); the column temperature was 35 ℃; and the mobile phases were 0.1% formic acid aqueous solution (phase A) and acetonitrile (phase B). The chromatographic gradient was as follows: 0–3 min, 5% mobile phase B; 3–8 min, 5–10% mobile phase B; 8–13 min, 10–50% mobile phase B; 13–20 min, 50–65% mobile phase B; 20–24 min, 65–90% mobile phase B; 24–26 min, 90% mobile phase B; 26–28 min, 90%–5% mobile phase B; 28–30 min, 5% mobile phase B; and the flow rate was 0.35 m / s. mL / min.

[0040] Example 4 Catalyst type optimization The only difference from Example 2 is that N,N'-dicyclohexylcarbodiimide (DCC), or 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt), or triphenylphosphine (TPP) and 2,2'-dithiodipyridine (DPDS) are used instead of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxy-7-azabenzotriazole (HOAt) as catalysts, and the concentration of each substance in the system is 6.0 mmol / L.

[0041] Figure 14-15To compare the mass spectrometric responses of different catalysts used in the PMBA-labeled carboxylic acid reaction, four different catalyst systems were compared: N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxy-7-azabenzotriazole (HOAt), and triphenylphosphine (TPP) and 2,2'-dithiopyridine (DPDS). The figures show that the labeling efficiency of the EDC / HOAt group and the TPP / DPDS group in Example 4 is higher than that of the DCC group and the EDC / HOBt group. Considering all factors, EDC / HOAt was selected as the preferred catalyst for the catalytic reaction.

[0042] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of a secondary amine reagent in the analysis and / or separation of carboxylic acids for non-disease diagnosis or treatment, wherein the secondary amine reagent has a structural formula of one of the following: 、 ； During the analysis, the carboxylic acid is one or a mixture of succinic acid, fumaric acid, malic acid, 2-ketobutyric acid, pyruvic acid, lactic acid, methoxyacetic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, n-butyric acid, n-valeric acid, propionic acid, hexanoic acid, valproic acid, 4-pentenoic acid, and phenyllactic acid. During the separation process, the carboxylic acid is a mixture of multiple substances selected from lactic acid, hexanoic acid, valproic acid, 4-pentenoic acid, succinic acid, 2-hydroxybutyric acid, 2-ketobutyric acid, and 3-hydroxybutyric acid; or, the carboxylic acid is a mixture of multiple substances selected from lactic acid, hexanoic acid, valproic acid, 4-pentenoic acid, fumaric acid, 2-hydroxybutyric acid, n-pentanoic acid, and 3-hydroxybutyric acid; or, the carboxylic acid is a mixture of multiple substances selected from lactic acid, hexanoic acid, valproic acid, 4-pentenoic acid, succinic acid, 2-hydroxybutyric acid, phenyllactic acid, and 3-hydroxybutyric acid; or, the carboxylic acid is a mixture of multiple substances selected from lactic acid, hexanoic acid, valproic acid, 4-pentenoic acid, succinic acid, malic acid, phenyllactic acid, and pyruvic acid; or, the carboxylic acid is a mixture of lactic acid, hexanoic acid, and valproic acid. A mixture of multiple acids including 4-pentenoic acid, fumaric acid, n-butyric acid, 2-ketobutyric acid, and propionic acid; or, a mixture of multiple acids including lactic acid, hexanoic acid, valproic acid, 4-pentenoic acid, succinic acid, malic acid, 2-ketobutyric acid, and methoxyacetic acid; or, a mixture of multiple acids including lactic acid, hexanoic acid, valproic acid, 4-pentenoic acid, fumaric acid, n-butyric acid, n-valproic acid, and methoxyacetic acid; or, a mixture of multiple acids including lactic acid, hexanoic acid, valproic acid, 4-pentenoic acid, succinic acid, 2-hydroxybutyric acid, phenyllactic acid, and methoxyacetic acid; or, a mixture of lactic acid and methoxyacetic acid; or, a mixture of 2-hydroxybutyric acid and 3-hydroxybutyric acid.

2. Use of a secondary amine reagent according to claim 1 in the analysis and / or isolation of carboxylic acids in the diagnosis or treatment of non-diseases, characterized in that: The preparation method of the secondary amine reagent includes the following steps: S1. Reaction of benzoic acid or deuterated benzoic acid and oxaloyl chloride in a solvent; S2. The product obtained in S1 is reacted with 1-tert-butoxycarbonyl-4-aminomethylpiperidine in the presence of a base and a solvent. S3. The product obtained in S2 is reacted with trifluoroacetic acid in a solvent to obtain the secondary amine reagent.

3. The application of the secondary amine reagent according to claim 2 in the analysis and / or separation of carboxylic acids for non-disease diagnosis or treatment, characterized in that: In S1, the ratio of benzoic acid or deuterated benzoic acid to oxaloyl chloride is 500 mg: 0.42 mL; in S2, the mass ratio of 1-tert-butoxycarbonyl-4-aminomethylpiperidine used to benzoic acid or deuterated benzoic acid used in S1 is 1:1; in S3, the ratio of the product obtained in S2 to trifluoroacetic acid is 500 mg: 1.6 mL.

4. The application of the secondary amine reagent according to claim 2 in the analysis and / or separation of carboxylic acids for non-disease diagnosis or treatment, characterized in that: In S1, the reaction process includes reacting under ice bath conditions for half an hour, followed by reacting at room temperature for 4 hours; in S2, the reaction includes reacting under ice bath conditions for half an hour, followed by reacting at room temperature for 1 hour; in S3, the reaction includes reacting at room temperature for 1 hour; in S1, the solvent is dichloromethane; in S2, the solvent is dichloromethane, and the base is triethylamine; in S3, the solvent is dichloromethane.

5. A method for the analysis and / or separation of carboxylic acids for non-disease diagnosis or treatment using secondary amine reagents, characterized in that: Includes the following steps: A secondary amine reagent is used as a labeling reagent to react with a carboxylic acid to obtain a derivative; the derivative is then detected by LC-MS; the secondary amine reagent is... , One or two of them; During the analysis, the carboxylic acid is one or a mixture of succinic acid, fumaric acid, malic acid, 2-ketobutyric acid, pyruvic acid, lactic acid, methoxyacetic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, n-butyric acid, n-valeric acid, propionic acid, hexanoic acid, valproic acid, 4-pentenoic acid, and phenyllactic acid. During the separation process, the carboxylic acid is a mixture of multiple substances selected from lactic acid, hexanoic acid, valproic acid, 4-pentenoic acid, succinic acid, 2-hydroxybutyric acid, 2-ketobutyric acid, and 3-hydroxybutyric acid; or, the carboxylic acid is a mixture of multiple substances selected from lactic acid, hexanoic acid, valproic acid, 4-pentenoic acid, fumaric acid, 2-hydroxybutyric acid, n-pentanoic acid, and 3-hydroxybutyric acid; or, the carboxylic acid is a mixture of multiple substances selected from lactic acid, hexanoic acid, valproic acid, 4-pentenoic acid, succinic acid, 2-hydroxybutyric acid, phenyllactic acid, and 3-hydroxybutyric acid; or, the carboxylic acid is a mixture of multiple substances selected from lactic acid, hexanoic acid, valproic acid, 4-pentenoic acid, succinic acid, malic acid, phenyllactic acid, and pyruvic acid; or, the carboxylic acid is a mixture of lactic acid, hexanoic acid, and valproic acid. A mixture of multiple acids including 4-pentenoic acid, fumaric acid, n-butyric acid, 2-ketobutyric acid, and propionic acid; or, a mixture of multiple acids including lactic acid, hexanoic acid, valproic acid, 4-pentenoic acid, succinic acid, malic acid, 2-ketobutyric acid, and methoxyacetic acid; or, a mixture of multiple acids including lactic acid, hexanoic acid, valproic acid, 4-pentenoic acid, fumaric acid, n-butyric acid, n-valproic acid, and methoxyacetic acid; or, a mixture of multiple acids including lactic acid, hexanoic acid, valproic acid, 4-pentenoic acid, succinic acid, 2-hydroxybutyric acid, phenyllactic acid, and methoxyacetic acid; or, a mixture of lactic acid and methoxyacetic acid; or, a mixture of 2-hydroxybutyric acid and 3-hydroxybutyric acid. The catalyst used in the reaction is one or more of the following: a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC and 1-hydroxybenzotriazole HOBt; a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxy-7-azabenzotriazole HOAt; or a mixture of triphenylphosphine TPP and 2,2'-dithiopyridine DPDS.

6. The method for analyzing and / or separating carboxylic acids for non-disease diagnosis or treatment using secondary amine reagents according to claim 5, characterized in that: secondary amine reagent or As a labeling reagent, it reacts with carboxylic acids to yield derivatives; the derivatives are then detected using LC-MS; or, a secondary amine reagent is used. As a labeling reagent, it reacts with carboxylic acids to give derivative A, which is then used as a secondary amine reagent. As a labeling reagent, it reacts with carboxylic acid to obtain derivative B, which is used as an internal standard; derivative A and derivative B are mixed and then detected by LC-MS.

7. The method for analyzing and / or separating carboxylic acids for non-disease diagnosis or treatment using secondary amine reagents according to claim 5, characterized in that: The carboxylic acid solution is mixed with a catalyst and a secondary amine reagent, and the reaction is carried out at 30-55°C for 30-60 minutes to complete the labeling and obtain the derivative.

8. The method for analyzing and / or separating carboxylic acids for the diagnosis or treatment of non-diseases using secondary amine reagents according to any one of claims 5-7, characterized in that: In the LC-MS detection process, the chromatographic column was a SHIMADZU shim pack GIST C18, with dimensions of 2.1 × 100 mm and 2.0 μm. The column temperature was 35℃. The mobile phase consisted of mobile phase A and mobile phase B. 0.1% formic acid aqueous solution and acetonitrile were used as mobile phase A and mobile phase B, respectively, for chromatographic detection. The chromatographic gradient was as follows: 0–3 min, 5% mobile phase B; 3–8 min, 5–15% mobile phase B; 8–15 min, 15–25% mobile phase B; 15–20 min, 25–60% mobile phase B; 20–24 min, 60–90% mobile phase B; 24–26 min, 90% mobile phase B; 26–28 min, 90%–5% mobile phase B; 28–30 min… The mobile phase B has a volume fraction of 5% and a flow rate of 0.35 mL / min.

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