Amino acid derivatization method based on transesterification and application of amino acid derivatization method in measurement of bacterial amino acid stable carbon isotope

Abstract

The invention discloses an amino acid derivatization method based on ester exchange and application of the amino acid derivatization method in bacterial amino acid stable carbon isotope measurement. According to the method, an alcoholic solution of boron trifluoride serves as a catalyst, carboxyl of amino acid reacts with ester groups in carboxylic ester, the ester groups are transferred to the amino acid, amino-acid ester is generated, and esterification of the carboxyl is achieved; and then carrying out acylation reaction on the amino-acid ester, triethylamine and acetic anhydride to realize acylation of amino groups. According to the method, chemical reagents causing damage to instruments do not need to be used, and generation of water in the reaction process can be avoided, so that the water removal step is omitted, the drying time is shortened, and meanwhile, side reactions and generation of by-products can be reduced. The method disclosed by the invention has good suitability with a stable isotope analysis instrument, can be used for rapidly and accurately determining the stable carbon isotope of the bacterial amino acid in an environmental sample, and has a wide application prospect in the aspects of establishing a bacterial amino acid monomer stable carbon isotope fingerprint database and widening an environmental microorganism traceability technical means.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of environmental microorganisms and amino acid stable isotope analysis, and in particular to an amino acid derivatization method based on ester exchange and its application in bacterial amino acid stable carbon isotope measurement. BACKGROUND

[0002] Amino acids, as the basic building blocks of proteins, account for more than 70% of organic nitrogen and nearly 50% of total carbon in organisms, and are key carriers of carbon and nitrogen cycles in marine and terrestrial ecosystems. The conservation of amino acid molecular structure, the diversity of metabolic pathways, and the universality in organisms make amino acids ideal biomarkers for analyzing biogeochemical processes. Amino acid isotope fingerprinting (AA-δ 13 C, AA-δ 15 N) is also widely used in ecological tracing (such as animal migration tracking and food web construction) and food origin identification.

[0003] Currently, gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is the main method for amino acid isotope analysis. However, due to the presence of both amino (-NH2) and carboxyl (-COOH) groups, amino acids are strongly polar and not easily volatile. Direct gas chromatography separation often results in peak tailing and low separation efficiency, so derivatization is necessary to generate derivatives with high thermal stability and strong volatility to meet detection requirements.

[0004] Existing derivatization methods have significant limitations: silane derivatization and trifluoroacetic acid acylation are commonly used pretreatment techniques for gas chromatography, but the silane derivatives or fluorine-containing compounds formed in the GC-C-IRMS system combustion tube can produce SiO2 or fluorinated deposits, reducing the efficiency of the oxidation reactor and possibly damaging the capillary column; Pv / iPr (pivaloyl isopropyl esterification) and NAIP (N-acetyl isopropyl esterification) methods commonly used for nitrogen isotope analysis, as well as NACME (N-acetyl methyl ester) derivatization method used for carbon and hydrogen isotope analysis, all require complex esterification and acylation steps. These methods usually use acetyl chloride or thionyl chloride to acidify the alcohol solution (which requires ice bath treatment for exothermic reaction), followed by reaction at 110°C for 1-2 h, with a total processing time of 6-7 h. The water generated as a byproduct not only hinders the forward progress of the reversible reaction, but also increases the generation of subsequent byproducts, so long-term nitrogen blowing drying is required to remove water and excess reagents. At the same time, the reagents used in existing methods, such as acetyl chloride, thionyl chloride, pivaloyl chloride, and methyl chloroformate, have strong acute toxicity and pose potential risks to the health of experimental personnel.

[0005] Therefore, it is necessary to develop a new amino acid derivatization method which is good in instrument adaptability, anhydrous esterification, low-toxic reagent and simple and rapid, to provide an efficient pretreatment process for microbial amino acid isotope analysis using a GC-C-IRMS system, so as to improve the analysis efficiency and promote the development of the technology in the field of environmental microorganisms. SUMMARY

[0006] In order to overcome the above-mentioned defects and deficiencies in the prior art, the present application provides an amino acid derivatization method based on ester exchange and application thereof in measurement of stable carbon isotope of bacterial amino acid.

[0007] A first object of the present application is to provide an amino acid derivatization method.

[0008] A second object of the present application is to provide a method for measuring stable carbon isotope of bacterial amino acid.

[0009] The present application claims the following: An amino acid derivatization method, comprising the following steps: S11. mixing an amino acid sample and a carboxylic acid ester to perform an ester exchange reaction with an alcohol solution of boron trifluoride as a catalyst, and obtaining an ester exchange product after drying; wherein a general structure of the carboxylic acid ester is R-COO-R1, a general structure of alcohol in the alcohol solution of boron trifluoride is R2-OH, and R1 and R2 are alkyl groups with the same structure; The purpose of this step is to react -COOH of the amino acid with the ester group on the carboxylic acid ester, transfer the ester group on the carboxylic acid ester to the amino acid, generate acid and new amino acid ester, and realize esterification of the carboxyl group; S12. resolubilizing the ester exchange product in anhydrous alcohol solution, and obtaining amino acid ester after drying; wherein a general structure of alcohol in the anhydrous alcohol solution is R3-OH, and R3 is an alkyl group with the same structure as R1 and R2 in step S1; The purpose of this step is to remove residual solvent (mainly the alcohol solution of boron trifluoride); S13. dissolving the amino acid ester in a mixed buffer solvent, adding triethylamine and acetic anhydride to perform acylation reaction, and obtaining amino acid derivative crude product; The mixed buffer solvent is obtained by mixing anhydrous acetonitrile and anhydrous 1,4 dioxane; S14. adding an extraction solvent to the amino acid derivative crude product, and separating organic phase to obtain the amino acid derivative; The extraction solvent is obtained by mixing chloroform and sodium bicarbonate solution.

[0010] Preferably, in step S11, the transesterification product is obtained after drying, and the transesterification product is dissolved in the alcohol solution of boron trifluoride again and then dried. This step can further promote and strengthen the esterification effect, and also remove residual low-boiling carboxylic acid esters.

[0011] Preferably, in step S11, the carboxylic acid ester includes methyl acetate, propyl acetate, isopropyl acetate or butyl acetate.

[0012] Preferably, in step S11, the alcohol solution of boron trifluoride includes methanol solution of boron trifluoride, propanol solution of boron trifluoride, isopropanol solution of boron trifluoride or butanol solution of boron trifluoride.

[0013] Preferably, in step S12, the anhydrous alcohol solution includes anhydrous methanol, anhydrous propanol, anhydrous isopropanol or anhydrous butanol.

[0014] Preferably, in step S11, the volume ratio of the carboxylic acid ester and the alcohol solution of boron trifluoride is 5-8:2.5-3.

[0015] More preferably, in step S11, the volume ratio of the carboxylic acid ester and the alcohol solution of boron trifluoride is 5:2.5.

[0016] Preferably, in step S11, the esterification reaction is performed by heating at 70-110°C for 25-120 min.

[0017] More preferably, in step S11, the esterification reaction is performed by heating at 75°C for 30 min.

[0018] Preferably, in step S13, the volume ratio of the anhydrous acetonitrile and the anhydrous 1,4 dioxane in the mixed buffer solvent is (2-4):1.

[0019] More preferably, in step S13, the volume ratio of the anhydrous acetonitrile and the anhydrous 1,4 dioxane in the mixed buffer solvent is 2:1.

[0020] Preferably, in step S13, the volume ratio of the triethylamine and the acetic anhydride is 2-3:1.2.

[0021] More preferably, in step S13, the volume ratio of the triethylamine and the acetic anhydride is 2:1.2.

[0022] Preferably, in step S13, the acylation reaction is performed by heating at 40-60°C for 10-35 min.

[0023] More preferably, the acylation reaction is performed by heating at 40°C for 30 min.

[0024] Preferably, in step S14, the volume ratio of chloroform to sodium bicarbonate solution in the extraction solvent is 1: (1.5-2.5), and the concentration of the sodium bicarbonate solution is (0.001-0.1) M.

[0025] More preferably, in step S14, the volume ratio of chloroform to sodium bicarbonate solution in the extraction solvent is 1:2, and the concentration of the sodium bicarbonate solution is 0.001 M.

[0026] Preferably, in steps S11-S12, the drying method is nitrogen blowing.

[0027] Application of any of the above amino acid derivatization methods in bacterial amino acid stable carbon isotope measurement.

[0028] A method for bacterial amino acid stable carbon isotope measurement, comprising the following steps: S21. Amplifying and culturing bacteria using a culture medium without amino acids and collecting the bacterial culture; This step is to make the bacteria "synthesize" amino acids from scratch; Then, the bacterial culture is digested with hydrochloric acid, and after digestion, the solvent is removed, n-hexane and dichloromethane are added to remove impurities, and after drying, an amino acid sample is obtained; S22. Derivatizing the amino acid sample obtained in step S21 using any of the above amino acid derivatization methods to obtain an amino acid derivative; S23. Detecting the amino acid derivative on a GC-MS and / or GC-C-IRMS machine; Wherein, the n-hexane and dichloromethane are used to wash and remove hydrophobic impurities such as lipids.

[0029] Preferably, the volume ratio of n-hexane to dichloromethane is (3-6): (1-5).

[0030] More preferably, the volume ratio of n-hexane to dichloromethane is 6:5.

[0031] Preferably, the bacteria are Escherichia coli, Enterococcus faecalis, Salmonella or Enterococcus faecium.

[0032] Compared with the prior art, the present application has the following beneficial effects: The present application discloses an amino acid derivatization method based on ester exchange and its application in bacterial amino acid stable carbon isotope measurement, which has the following advantages: (1) The derivatization reagent used in the present application does not contain silicon, fluorine and other toxic components which are harmful to the instrument system, and has good compatibility with stable isotope analysis instruments; (2) The amino acid derivatization method of the present application includes ester exchange and acetylation, wherein the ester exchange process avoids the generation of water in principle, thereby eliminating the water removal step, significantly shortening the sample nitrogen blow drying time, and at the same time, reducing the generation of side reactions and by-products. (3) The present application can quickly and accurately determine the stable carbon isotope of bacterial amino acid in the environmental sample, and has wide application prospects in establishing the stable carbon isotope fingerprint library of bacterial amino acid monomers of different environmental sources and different types and widening the environmental microbial tracing technology means. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The chromatogram of the E. coli amino acid derivative of Example 1 on GC-MS (top) and GC-C-IRMS (bottom).

[0034] Figure 2 The GC-MS chromatogram of the amino acid derivative of Example 11.

[0035] Figure 3 The NIST library comparison structure diagram of the amino acid derivative of Example 11; A: Ala, B: Gly, C: Val, D: L-Nva, E: Leu, F: Ile, G: Ser, H: Asp, I: Pro, J: Glu, K: Met, L: Phe, M: Lys.

[0036] Figure 4 The consistency results of the carbon isotope values of the amino acid derivative prepared in Example 11 and the carbon isotope values of the amino acid powder directly determined by EA-IRMS.

[0037] Figure 5 The GC-MS chromatogram of the amino acid derivative of Comparative Example 1, A: glutamic acid, B: leucine, C: expected effect.

[0038] Figure 6 The GC-MS results of the amino acid derivative of Comparative Example 2, A: chromatogram, B: mass spectrum.

[0039] Figure 7 The GC-MS chromatogram of the amino acid derivative of Comparative Example 3.

[0040] Figure 8 The NIST library comparison structure diagram of the amino acid derivative of Comparative Example 3; A: Gly, B: L-Vla, C: L-Cys, D: Phe, E: L-Nva, F: Ile, G: L-Met, H: L-Thr, I: L-Lys•HCl, J: L-Trp, K: L-Nle.

[0041] Figure 9 GC-MS chromatogram of the amino acid derivative of Comparative Example 4. DETAILED DESCRIPTION

[0042] The application will be further described in conjunction with specific examples, which in no way represent any form of limitation to the application. Unless otherwise specified, the reagents, methods and apparatus used in the present application are those conventional in the art.

[0043] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0044] Example 1: An ester exchange-based amino acid derivatization method The present example provides an ester exchange-based amino acid derivatization method, the specific steps of which are as follows: S1. Use a loop to pick up E. coli and inoculate into a modified Arabidopsis thaliana glucose acid liquid medium without amino acids for culture. After reaching the logarithmic growth phase, centrifuge 5-15 mL of bacterial solution at 8000 g for 5 min, discard the supernatant after centrifugation, wash twice with normal saline, and centrifuge again at 8000 g for 5 min and discard the supernatant. Resuspend the bacterial pellet in 5 mL of 6 mol / L hydrochloric acid solution, then transfer to a digestion tube, add L-norvaline (L-Nva) as an internal standard, seal the digestion tube after filling with N2, and digest at 110°C for 24 h. After the sample is digested and cooled to room temperature, dry it with nitrogen, and redissolve it in 1 mL of 0.1 mol / L dilute hydrochloric acid solution. Add 0.5 mL of mixed solution (V 正己烷 :V 二氯甲烷 =6:5), vortex to mix, discard the organic phase after removing the hydrophobic components, repeat this step 3 times, then dry the aqueous phase with nitrogen to obtain the purified amino acid sample; S2. Add 500 μL of methyl acetate and 250 μL of boron trifluoride methanol solution to the purified amino acid sample obtained in step S1. Tighten the lid, and place the sealed tube in a metal dry bath at 75°C for 30 min. After the sample is cooled to room temperature, dry it with high-purity nitrogen to remove the solvent and reagent; S3. Redissolve the dried sample in 500 μL of boron trifluoride methanol solution, and dry it with a gentle stream of nitrogen; S4. Redissolve the dried sample in 300 μL of anhydrous methanol, and dry it with a gentle stream of nitrogen, repeat this step 2 times to obtain the amino acid ester; S5. Dissolve the amino acid ester in the buffer solvent mixed from 200 μL anhydrous acetonitrile and 100 μL anhydrous 1,4 dioxane, and transfer the solution to a new centrifuge tube. Add 200 μL triethylamine, mix well, and then add 120 μL fresh anhydrous acetic anhydride. After sealing, heat at 40 °C for 30 min for acylation reaction; S6. After the reaction is completed and cooled, add 400 μL CHCl3to the cooled solution, then add 800 μL of 0.001 mol / L NaHCO3solution (add in two times, 400 μL each time), shake and extract the derivative into the CHCl3phase. Discard the upper aqueous phase, add molecular sieves to the organic phase for drying, and obtain the amino acid derivative.

[0045] Example 2 An amino acid derivatization method based on ester exchange This example provides an amino acid derivatization method based on ester exchange, which is performed according to Example 1, with the difference that in step S1, a mixed solution (V 正己烷 :V 二氯甲烷 = 3:1) is used to replace the mixed solution (V 正己烷 :V 二氯甲烷 = 6:5).

[0046] Example 3 An amino acid derivatization method based on ester exchange This example provides an amino acid derivatization method based on ester exchange, which is performed according to Example 1, with the difference that in step S2, 800 μL of isopropyl acetate is used to replace 500 μL of methyl acetate, and isopropyl alcohol solution of boron trifluoride is used to replace the methanol solution of boron trifluoride; in step S3, isopropyl alcohol solution of boron trifluoride is used to replace the methanol solution of boron trifluoride; in step S4, anhydrous isopropyl alcohol is used to replace anhydrous methanol.

[0047] Example 4 An amino acid derivatization method based on ester exchange This example provides an amino acid derivatization method based on ester exchange, which is performed according to Example 1, with the difference that in step S2, butyl acetate is used to replace methyl acetate, and 300 μL of butanol solution of boron trifluoride is used to replace 250 μL of methanol solution of boron trifluoride; in step S3, 600 μL of butanol solution of boron trifluoride is used to replace 500 μL of methanol solution of boron trifluoride; in step S4, anhydrous butanol is used to replace anhydrous methanol.

[0048] Example 5 An amino acid derivatization method based on ester exchange This example provides a method for amino acid derivatization based on ester exchange, which is performed according to Example 1, except that in step S2, isopropyl acetate is used to replace methyl acetate, and boron trifluoride isopropyl alcohol solution is used to replace boron trifluoride methanol solution, and the reaction temperature is 110°C; in step S3, 600 μL of boron trifluoride isopropyl alcohol solution is used to replace 500 μL of boron trifluoride methanol solution; and in step S4, anhydrous isopropyl alcohol is used to replace anhydrous methanol.

[0049] Example 6 A method for amino acid derivatization based on ester exchange This example provides a method for amino acid derivatization based on ester exchange, which is performed according to Example 1, except that in step S5, the volume of anhydrous acetonitrile is 400 μL.

[0050] Example 7 A method for amino acid derivatization based on ester exchange This example provides a method for amino acid derivatization based on ester exchange, which is performed according to Example 1, except that in step S5, the volume of triethylamine is 300 μL.

[0051] Example 8 A method for amino acid derivatization based on ester exchange This example provides a method for amino acid derivatization based on ester exchange, which is performed according to Example 1, except that in step S5, the acylation reaction is performed at 60°C for 30 min.

[0052] Example 9 A method for amino acid derivatization based on ester exchange This example provides a method for amino acid derivatization based on ester exchange, which is performed according to Example 1, except that in step S6, the concentration of NaHCO3 solution is 0.1 mol / L.

[0053] Example 10 A method for amino acid derivatization based on ester exchange This example provides a method for amino acid derivatization based on ester exchange, and the specific steps are as follows: Collecting an environmental atmospheric sample: using an Anderson single-stage sampler and a micro air pump, atmospheric samples are collected on a mixed cellulose membrane at a flow rate of 28.3 L / min for 30 min. After sampling, the membrane sample is placed in a 250 mL conical flask, 80 mL of PBS buffer is added, and oscillation is performed at 37°C at a speed of 140 rpm for 30 min. After taking out the conical flask, stand still, use a pipette to suck 200 μL of supernatant, and spread it on a MacConkey plate culture medium, and incubate at 37°C for 24 h, and then perform streak isolation to obtain a single bacterial colony.

[0054] Using an inoculating loop to pick the single bacterial colony, and preparing the amino acid derivative according to steps S1-S6 of Example 1.

[0055] Experimental Example 1 GC-MS analysis and monomer compound stable isotope analysis I. Experimental method The amino acid derivatives prepared in Examples 1-10 were transferred to GC sample bottles respectively, and subjected to GC-MS and GC-C-IRMS machine analysis.

[0056] II. Experimental results The δ 13 C isotope values of the E. coli amino acid derivatives prepared in Examples 1-10 are shown in Table 1, and the chromatograms of the E. coli amino acid derivative in Example 1 on GC-MS (gas chromatograph-mass spectrometer) and GC-C-IRMS (gas chromatograph-combustion-isotope mass spectrometer) are shown in Figure 1 Figure 1 It can be seen that the δ 13 C isotope values of each amino acid derivative appear successively at 500-1800 s. The above results show that the amino acid derivatization methods of Examples 1-10 can be used for efficient determination of stable carbon isotope ratios of bacterial amino acids.

[0057] Table 1 δ 13 C isotope values of amino acid derivatives

[0058] Example 11 An amino acid derivatization method based on ester exchange I. Experimental method This example provides an amino acid derivatization method based on ester exchange, which is performed according to Example 1, with the difference that step S1 is omitted, and the amino acid standard (Ala, Gly, Val, L-Nva, Leu, Ile, Ser, Asp, Pro, Glu, Met, Phe, Lys) is weighed, dissolved in 0.1 mol / L HCl solution, mixed, and made up to 100 mL in a volumetric flask to prepare an amino acid standard mixed solution, so that the final concentration of each amino acid standard is 0.0025-0.005 mol / L. 550 μL of the amino acid standard mixed solution is taken with a 1000 μL microsyringe and placed in a 5 mL cryotube. It is blown dry with gentle nitrogen gas at 60°C to obtain an amino acid sample, and then the amino acid derivative is prepared according to steps S2-S6.

[0059] The amino acid derivative is transferred to a GC sample bottle and subjected to GC-MS and GC-C-IRMS machine analysis.

[0060] II. Experimental results The results are shown in Figure 2 The chromatographic peaks of each amino acid component are clearly distinguishable, and the expected derivative can be generated​Figure 2 and Figure 3 ). The carbon isotope values of each amino acid derivative obtained from GC-C-IRMS measurement were corrected by subtracting the isotope values of the carbon atoms added by the derivatization reagents, and matched the carbon isotope values of the amino acid powders directly measured by EA-IRMS ( Figure 4 ).

[0061] Comparative Example 1 I. Experimental Method This comparative example used the NAIP method (N-acetyl-i-propyl (NAIP) esters) for the derivatization of amino acids, and the specific steps were as follows: S1. Weigh 0.5 mg of leucine powder and 0.5 mg of glutamic acid powder, and add 1 mL of acidified isopropanol solution (V 异丙醇 :V 乙酰氯 = 5 mL: 800 μL) at 110°C for 1 h of propyl esterification; S2. Put the reaction tube in the refrigerator to terminate the reaction, then remove the residual isopropanol under a gentle nitrogen stream at 40°C. Add 0.25 mL of dichloromethane, and evaporate at room temperature to remove excess isopropanol and water, repeat this step once; S3. Add 1 mL of mixed solution (V 乙酸酐 :V 三乙胺 :V 丙酮 = 1:2:5), and acylate at 60°C for 10 min, then dry at room temperature with a gentle nitrogen stream; S4. Add 2 mL of ethyl acetate and 1 mL of saturated NaCl solution, vortex to promote phase separation, and dry the organic phase at room temperature with a gentle nitrogen stream. Then add 500 μL of dichloromethane under ice bath conditions, remove the residual reagent under a gentle nitrogen stream, repeat this step 2 times to obtain the amino acid derivative.

[0062] Use a micro-injection needle to aspirate 500 μL of ethyl acetate to dissolve the amino acid derivative, then transfer it to a GC injection vial for GC-MS analysis.

[0063] II. Experimental Results The results are shown in Figure 5 , the carboxyl group on glutamic acid was not esterified (A in Figure 5 ), the carboxyl group on leucine formed an ethyl ester (B in Figure 5 ), indicating that the esterification did not achieve the desired effect (theoretically the carboxyl group should be isopropyl esterified, and the amino group should be acetylated, Figure 5 C).

[0064] Comparative Example 2 I. Experimental method This comparative example uses the NAIP method (N-acetyl-i-propyl (NAIP) esters) for the derivatization of amino acids, which is performed according to Comparative Example 1, with the difference that the specific steps of step S1 are as follows: Take 100 μL of the amino acid mixed standard (brand: Tanmoji, product number: 81987, containing 21 kinds of amino acids, each amino acid concentration is 1 mmol / L) into a 5 mL cryotube, dry it with gentle nitrogen blowing at 60°C, then add 1 mL of acidified isopropanol solution (V 异丙醇 :V 乙酰氯 = 5 mL: 800 μL), and propyl esterify at 110°C for 1 h.

[0065] II. Experimental results The results are shown in Figure 6 , only Phe (A in Figure 6 ) can be clearly identified and matched in the chromatogram, but from the figure it can be seen that the amino group (-NH2) of Phe is acetylated, but the carboxyl group is not esterified (B in Figure 6 ), so the expected esterification effect is not achieved.

[0066] Comparative Example 3 I. Experimental method This comparative example uses the NAIP method (N-acetyl-i-propyl (NAIP) esters) for the derivatization of amino acids, which is performed according to Comparative Example 1, with the difference that the specific steps of step S1 are as follows: Weigh Gly, L-Val, L-Cys, Phe, L-Nva, Ile, L-Met, L-Thr, L-Lys•HCl, L-Trp, L-His, L-Nle standard powder, respectively, to prepare 5000 mg / L of amino acid single standard solution. Take 100 μL of each amino acid single standard solution and place it in a 5 mL cryotube, dry it with gentle nitrogen blowing at 60°C, then add 1 mL of acidified isopropanol solution (V 异丙醇 :V 乙酰氯 = 5 mL: 800 μL), and propyl esterify at 110°C for 1 h.

[0067] II. Experimental results The results of the GC-MS chromatogram of each amino acid are shown in Figure 7 , the NIST library comparison structure diagram is shown in Figure 8 , and the Figure 7 , Figure 8As shown in Table 2, derivatization using the NAIP method is prone to poor esterification results (such as the formation of ethyl ester, methyl ester, or amino acid tandem with other substances instead of isopropyl ester).

[0068] Table 2 Analysis of amino acid derivatization

[0069] Comparative Example 4 I. Experimental Methods This comparative example uses the NAIP method (N-acetyl-i-propyl (NAIP)esters) for amino acid derivatization, following the procedure of Comparative Example 1, except that the specific steps of step S1 are as follows: Weigh out 0.5 mg of amino acid powder (Ile, L-Leu, L-Lys•HCl, Phe, L-Thr, L-Val, L-Nva) to prepare an amino acid mixed sample, then add 1 mL of acidified isopropanol solution (V... 异丙醇 V 乙酰氯 =5 mL:800 μL), propyl esterification was performed at 110 °C for 1 h.

[0070] II. Experimental Results The results are as follows Figure 9 As shown, after derivatization, chromatographic peak sticking is likely to occur. This is because the carboxyl groups of amino acids are not derivatized (isopropyl esterified), and therefore still have high polarity and poor volatility, resulting in poor chromatographic separation and making it impossible to continue monomeric stable isotope analysis.

[0071] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method of amino acid derivatization, characterized by, The method comprises the following steps: S11. mixing the amino acid sample and the carboxylic acid ester to perform an ester exchange reaction with an alcohol solution of boron trifluoride as a catalyst, and drying to obtain an ester exchange reaction product; wherein the carboxylic acid ester has a general structure of R-COO-R1, the alcohol in the alcohol solution of boron trifluoride has a general structure of R2-OH, and R1 and R2 are alkyl groups with the same structure; S12. re-dissolving the ester exchange product in anhydrous alcohol solution, and drying to obtain an amino acid ester; wherein the alcohol in the anhydrous alcohol solution has a general structure of R3-OH, and R3 is an alkyl group with the same structure as R1 and R2 in step S1; S13. dissolving the amino acid ester in a mixed buffer solvent, adding triethylamine and acetic anhydride to perform an acylation reaction, and obtaining an amino acid derivative crude product; the mixed buffer solvent is obtained by mixing anhydrous acetonitrile and anhydrous 1,4 dioxane; S14. adding an extraction solvent to the amino acid derivative crude product, and separating an organic phase to obtain an amino acid derivative; the extraction solvent is obtained by mixing chloroform and sodium bicarbonate solution.

2. The amino acid derivatization method of claim 1, wherein, In step S11, the ester exchange reaction product is re-dissolved in an alcohol solution of boron trifluoride and then dried.

3. The amino acid derivatization method of claim 1, wherein, In step S11, the carboxylic acid ester includes methyl acetate, propyl acetate, isopropyl acetate or butyl acetate.

4. The amino acid derivatization method of claim 1, wherein, In step S11, the alcohol solution of boron trifluoride includes a methanol solution of boron trifluoride, a propanol solution of boron trifluoride, an isopropanol solution of boron trifluoride or a butanol solution of boron trifluoride.

5. The amino acid derivatization method of claim 1, wherein, In step S12, the anhydrous alcohol solution includes anhydrous methanol, anhydrous propanol, anhydrous isopropanol or anhydrous butanol.

6. The amino acid derivatization method of claim 1, wherein, In step S11, the volume ratio of the carboxylic acid ester to the alcohol solution of boron trifluoride is 5-8:2.5-3.

7. The amino acid derivatization method of claim 1, wherein, In step S11, the ester exchange reaction is performed under the condition of heating at 70-110°C for 25-120 min.

8. The amino acid derivatization method of claim 1, wherein, In step S13, the volume ratio of the triethylamine to the acetic anhydride is 2-3:1.

2.

9. The amino acid derivatization method of claim 1, wherein, In step S13, the acylation reaction is performed under the condition of heating at 40-60°C for 10-35 min.

10. A method for bacterial amino acid stable carbon isotope measurement, characterized by, The method comprises the following steps: S21. amplifying and culturing bacteria using a culture medium without amino acid, collecting the bacterial culture, and then digesting the bacterial culture with hydrochloric acid, removing the solvent after digestion, adding n-hexane and dichloromethane to remove impurities, and drying to obtain an amino acid sample; S22. performing a derivatization reaction on the amino acid sample obtained in step S21 using the amino acid derivatization method according to any one of claims 1-9 to obtain an amino acid derivative; S23. performing GC-MS and / or GC-C-IRMS detection on the amino acid derivative.

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