Pre-column derivatization kit containing 2, 4-dinitrofluorobenzene and application of pre-column derivatization kit

Abstract

The invention relates to a pre-column derivatization kit containing 2, 4-dinitrofluorobenzene and application of the pre-column derivatization kit, and belongs to the technical field of amino acid detection. The kit comprises an amino acid releasing agent, the amino acid releasing agent comprises a sulfosalicylic acid solution, an EDTA (Ethylene Diamine Tetraacetic Acid) solution and ethanol, the concentration of the sulfosalicylic acid solution is 3-8wt%, and the concentration of the EDTA solution is 0.5-1.5 wt%; the derivatization reagent comprises a sodium carbonate-sodium bicarbonate buffer solution, sorbitan monopalmitate and a 2, 4-dinitrofluorobenzene solution, the pH range of the sodium carbonate-sodium bicarbonate buffer solution is 8.0-10.0, the concentration of the 2, 4-dinitrofluorobenzene solution is 0.5-2wt%, and the solvent is acetonitrile; the pH (Potential of Hydrogen) of the phosphate buffer solution is 6.5 to 7.5. According to the kit, the reagent stability is guaranteed, the operation steps can be reduced, the reaction time is shortened, the derivatization effect on amino acid is also obviously improved, and the accuracy of amino acid detection through an HPLC method can be improved.

Description

Technical Field

[0001] The present application relates to a pre-column derivatization kit containing 2,4-dinitrofluorobenzene and application thereof, belonging to the technical field of amino acid detection. Background Art

[0002] Amino acids are not only the basic structural units of peptides and proteins, but also the material basis of life metabolism, playing a vital role in the life activities of organisms. The accurate analysis of amino acids plays a wide and important role in biomedical research, food safety testing, agricultural science research, environmental protection, pharmaceutical industry, clinical disease diagnosis, metabolomics and other fields. Since amino acids are a class of biologically active substances with similar chemical properties, the sensitivity of the detection method plays a very important role in the accuracy of the analysis during the analysis process. However, the existing amino acid detection technologies have their own shortcomings, such as high analysis cost, high detection limit and slow test speed, so the development of new amino acid detection technologies is imminent.

[0003] Reversed-phase high performance liquid chromatography (HPLC) is widely used due to its high sensitivity and high selectivity. However, most amino acids have no UV absorption and are very polar, so they must be derivatized in UV detection mode. The most commonly used derivatization methods include: o-phthalaldehyde (OPA) method, fluorenyl methyl chloroformate (FMOC) method, dansyl chloride (Dansyl-Cl) method and phenyl isothiocyanate (PITC) method. Among them, the OPA method, FMOC method and Dansyl-Cl method all require the use of fluorescence detectors, which are relatively expensive; the PITC derivatization method has cumbersome steps and will reduce the life of the chromatographic column.

[0004] 2,4-Dinitrofluorobenzene, as an amino acid derivatization reagent, has attracted much attention from domestic and foreign researchers due to its high efficiency, stability, and simple operation. In previous studies by the inventors of this application, see "2,4-Dinitrofluorobenzene Pre-column Derivatization HPLC Method for Determination of 17 Amino Acids in Fertilizers" (Yan Xuewei, Lu Qingya, Su Hongli, et al., Henan Chemical Industry, Issue 3, 2019), which has confirmed the method and established a high-performance liquid chromatography method for determining amino acids in fertilizers using 2,4-Dinitrofluorobenzene pre-column derivatization. The 2,4-Dinitrofluorobenzene (2,4-Dinitrofluorobenzene) pre-column derivatization method has the characteristics of simple operation of the derivatization agent and stable derivatives, and is suitable for quantitative determination.

[0005] However, during the research process, the experimenters found that the derivatization ability of the derivatization reagent still needs to be improved, and the application steps of the derivatization reagent are cumbersome. Therefore, it is necessary to provide a pre-column derivatization kit containing 2,4-dinitrofluorobenzene to solve the problems of long reaction time of the derivatization product in the existing technology and poor derivatization effect for high-concentration samples. Summary of the invention

[0006] In order to solve the above problems, a pre-column derivatization kit containing 2,4-dinitrofluorobenzene and a derivatization method are provided. The kit of the present application provides an amino acid releaser premix and a derivatization organic phase premix, which ensure the stability of the reagents while reducing the operation steps. The ethanol in the amino acid releaser is combined with EDTA and sulfosalicylic acid to improve the extraction efficiency of amino acids in amino acid fertilizers, and the addition of a light stabilizer can better improve the stability of the reagents. The derivatization organic premix in the derivatization reagent, ethanol, sorbitan monopalmitate and 2,4-dinitrofluorobenzene can improve the contact efficiency between amino acids and 2,4-dinitrofluorobenzene, and promote the rapid absorption of the generated HF by the sodium carbonate-sodium bicarbonate buffer solution, which can not only reduce the derivatization reaction time, but also significantly improve the derivatization effect of 2,4-dinitrofluorobenzene on amino acids.

[0007] According to one aspect of the present application, a pre-column derivatization kit containing 2,4-dinitrofluorobenzene is provided, the kit comprising:

[0008] An amino acid releaser, the amino acid releaser comprising a sulfosalicylic acid solution, an EDTA solution and ethanol, the concentration of the sulfosalicylic acid solution being 3-8wt%, and the concentration of the EDTA solution being 0.5-1.5wt%;

[0009] A derivatization reagent, wherein the derivatization reagent comprises a sodium carbonate-sodium bicarbonate buffer solution, sorbitan monopalmitate and a 2,4-dinitrofluorobenzene solution, wherein the pH range of the sodium carbonate-sodium bicarbonate buffer solution is 8.0 to 10.0, the concentration of the 2,4-dinitrofluorobenzene solution is 0.5 to 2 wt %, and the solvent in the 2,4-dinitrofluorobenzene solution is acetonitrile;

[0010] Phosphate buffer, wherein the pH value of the phosphate buffer is 6.5 to 7.5.

[0011] In the present application, ethanol in the amino acid releaser is combined with EDTA and sulfosalicylic acid to improve the extraction efficiency of amino acids in the amino acid fertilizer. The derivatization organic premix in the derivatization reagent, sorbitan monopalmitate and 2,4-dinitrofluorobenzene can improve the contact efficiency between amino acids and 2,4-dinitrofluorobenzene, and promote the rapid absorption of the generated HF by the sodium carbonate-sodium bicarbonate buffer solution, which can not only reduce the derivatization reaction time, but also significantly improve the derivatization effect of 2,4-dinitrofluorobenzene on amino acids.

[0012] Optionally, the amino acid releaser further includes a light stabilizer, and the amino acid releaser is a premixed liquid, and the premixed liquid is obtained by mixing the light stabilizer, sulfosalicylic acid solution, EDTA solution and ethanol.

[0013] The stability of the reagent can be improved by adding a light stabilizer. In addition, the kit of the present application provides an amino acid releaser as a premixed solution, which ensures the stability of the reagent and can be directly added to the sample solution for extraction, thereby significantly reducing the number of operating steps and improving operating efficiency.

[0014] Optionally, the light stabilizer is selected from one or more of benzophenones, benzotriazoles, triazines, salicylates and acrylonitrile derivatives.

[0015] Optionally, the light stabilizer is BAD or UV-9.

[0016] Optionally, the ethanol concentration in the amino acid releaser is 5 to 15 vol%; and / or,

[0017] The concentration of the light stabilizer in the amino acid releaser is 0.1-0.5 wt %.

[0018] Since sulfosalicylic acid is sensitive to light, even if it is placed in a light-shielding reagent cabinet, it often needs to be used or taken out repeatedly and is easily decomposed into salicylic acid when exposed to light. In order to solve the problem of stable storage of amino acid releasers, the experimenters compared a variety of different light stabilizers and found that BAD or UV-9 has better compatibility with ethanol and sulfosalicylic acid. BAD or UV-9 can better protect sulfosalicylic acid. After 168 hours, the sulfosalicylic acid in the amino acid releaser still maintains a good effect.

[0019] Optionally, the derivatization reagent further includes ethanol, and the derivatization reagent includes a sodium carbonate-sodium bicarbonate buffer solution and a derivatization organic premix solution, and the derivatization organic premix solution is obtained by mixing ethanol, dehydrated sorbitan monopalmitate and 2,4-dinitrofluorobenzene solution.

[0020] The derivatization reagent also includes ethanol. The combination of ethanol and sorbitan monopalmitate with 2,4-dinitrofluorobenzene can improve the contact efficiency between amino acids and 2,4-dinitrofluorobenzene. The presence of ethanol can further promote the rapid absorption of the generated HF by the sodium carbonate-sodium bicarbonate buffer solution, thereby reducing the derivatization reaction time and significantly improving the derivatization effect.

[0021] Optionally, the ethanol concentration in the derivatized organic premix is ​​3-8 vol%.

[0022] According to another aspect of the present application, there is provided an application of the above-mentioned pre-column derivatization kit containing 2,4-dinitrofluorobenzene in the pre-column derivatization step when detecting amino acids in amino acid fertilizers by HPLC, the application comprising the following steps:

[0023] 1) Weighing an amino acid fertilizer sample, dissolving it in water, and filtering it through a microporous filter membrane to obtain an amino acid fertilizer liquid;

[0024] 2) adding an amino acid releaser to the amino acid fertilizer liquid, mixing well, letting the mixture stand, and filtering with a microporous membrane to obtain an intermediate filtrate;

[0025] 3) adding a derivatization reagent to the intermediate filtrate, mixing well, and then heating to react;

[0026] 4) After cooling, adding phosphate buffer solution and mixing well to obtain a test sample solution, which is used for HPLC analysis to detect amino acids in the amino acid fertilizer.

[0027] Optionally, the microporous filter membrane in steps 1) and 2) has a pore size ranging from 0.40 to 0.50 μm.

[0028] Optionally, the heating reaction in step 3) is carried out at a temperature of 50 to 70° C. for 20 to 30 minutes.

[0029] Optionally, in step 2), the amino acid releasing agent is added to the amino acid fertilizer liquid and then allowed to stand for no less than 40 minutes.

[0030] In the case of no premixing, the following steps are included:

[0031] 1) Weighing an amino acid fertilizer sample, dissolving it in water, and filtering it through a microporous filter membrane to obtain an amino acid fertilizer liquid;

[0032] 2) adding sulfosalicylic acid solution to the amino acid fertilizer solution and letting it stand, then adding ethanol and EDTA solution, mixing and letting it stand, filtering with a microporous membrane to obtain an intermediate filtrate;

[0033] 3) adding sodium carbonate-sodium bicarbonate buffer solution, 2,4-dinitrofluorobenzene solution and sorbitan monopalmitate to the intermediate filtrate, mixing well and heating to react;

[0034] 4) After cooling, adding phosphate buffer solution and mixing well to obtain a test sample solution, which is used for HPLC analysis to detect amino acids in the amino acid fertilizer.

[0035] Optionally, in step 2), the sulfosalicylic acid solution and the ethanol and EDTA solutions are added separately, wherein the sulfosalicylic acid solution is first added to the amino acid fertilizer solution and then allowed to stand for no less than 30 minutes, and then the ethanol and EDTA solutions are added, mixed, and allowed to stand for no less than 10 minutes.

[0036] The beneficial effects of this application include but are not limited to:

[0037] 1. The pre-column derivatization kit containing 2,4-dinitrofluorobenzene and its application according to the present application have the advantages of simple operation, few steps and short reaction time. The derivatization reaction can be completed in 20 to 30 minutes, and the derivatization efficiency is high and the effect is better, which can improve the accuracy of HPLC detection of amino acid content in amino acid fertilizers.

[0038] 2. According to the pre-column derivatization kit containing 2,4-dinitrofluorobenzene and its application in the present application, two premixed solutions are provided, including an amino acid releaser and a derivatization organic premixed solution, which can significantly reduce the operating steps in the detection process, and the premixed solution remains stable for at least 168 hours and has a good amino acid derivatization effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0040] Figure 1 The research technology roadmap involved in this application;

[0041] Figure 2 This is a reaction schematic diagram of the amino acid derivatization of 2,4-dinitrofluorobenzene involved in the examples of the present application. DETAILED DESCRIPTION

[0042] The present application is described in detail below in conjunction with examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and reagents in the examples of the present application are purchased through commercial channels.

[0043] like Figure 1 The figure shows the technical roadmap of the research scheme of this application. After exploring and studying the derivatization technology, the experimenters also studied and explored the derivatization products in the derivatization technology process, especially the derivatization kit, in which the derivatization reagents A, B and C correspond to the amino acid releaser, the derivatization reagent and the phosphate buffer solution, respectively.

[0044] Among them, the amino acid releaser is a premixed liquid obtained by mixing a light stabilizer, a sulfosalicylic acid solution, an EDTA solution and ethanol. The addition of ethanol can promote protein denaturation, and EDTA can be used to precipitate the protein, and the amino acid monomers adsorbed or bound therein can be fully extracted. The experimenters found that even if the amino acid releaser is placed in a light-shielded reagent cabinet, the prepared solution needs to be taken out repeatedly, so it is still difficult to ensure the stability of the amino acid releaser, especially the sulfosalicylic acid component therein. Therefore, by adding a light stabilizer and ethanol, a better protective effect on sulfosalicylic acid can be achieved, thereby ensuring the stability of the amino acid releaser and the stability of its effect.

[0045] The derivatization organic premix in the derivatization reagent is a premix, which is obtained by mixing ethanol, sorbitan monopalmitate and 2,4-dinitrofluorobenzene solution. The presence of sorbitan monopalmitate and ethanol can promote the contact and reaction effect between 2,4-dinitrofluorobenzene and amino acids. The ethanol can promote the transfer of the generated HF to the sodium carbonate-sodium bicarbonate buffer solution, thereby promoting the reaction degree of amino acids and 2,4-dinitrofluorobenzene. Even when treating high-concentration amino acid fertilizer samples, the amino acids can be fully derivatized, thereby ensuring the accuracy of HPLC detection. In addition, the derivatization reaction time is also significantly reduced. A better amino acid derivatization effect can be achieved by heating at 60°C for 20 to 30 minutes. In addition, the added ethanol component also has a certain promoting effect on the stability of the premix.

[0046] Among them, the sodium carbonate-sodium bicarbonate buffer solution and the phosphate buffer solution are prepared as needed or prepared in advance, and the two buffer solutions are stored separately. The sodium carbonate-sodium bicarbonate buffer solution mainly provides an alkaline environment for the amino acid derivatization reaction, and the phosphate buffer solution is added according to the requirements of HPLC injection.

[0047] The present application scheme is described below through specific embodiments.

[0048] Example 1

[0049] This embodiment provides a pre-column derivatization kit containing 2,4-dinitrofluorobenzene, comprising:

[0050] a) an amino acid releaser, the amino acid releaser comprising a sulfosalicylic acid solution, an EDTA solution, a light stabilizer and ethanol, the amino acid releaser being a premixed solution obtained by uniformly mixing the light stabilizer, the sulfosalicylic acid solution, the EDTA solution and the ethanol, wherein the sulfosalicylic acid concentration is 5wt%, the EDTA concentration is 1.0wt%, the light stabilizer is BAD, the BAD concentration is 0.3wt%, and the ethanol concentration is 10vol%;

[0051] b) a derivatization reagent, the derivatization reagent comprising a sodium carbonate-sodium bicarbonate buffer solution and a derivatization organic premix solution, wherein the derivatization organic premix solution is obtained by mixing ethanol, sorbitan monopalmitate and 2,4-dinitrofluorobenzene solution, the pH of the sodium carbonate-sodium bicarbonate buffer solution is 9.0, the concentration of 2,4-dinitrofluorobenzene in the derivatization organic premix solution is 1.5wt%, the concentration of ethanol is 6vol%, the concentration of sorbitan monopalmitate is 2.0wt%, the solvent is acetonitrile, and the sodium carbonate-sodium bicarbonate buffer solution and the derivatization organic premix solution are stored separately;

[0052] c) Phosphate buffer, the pH of the phosphate buffer is 7.0.

[0053] Example 2

[0054] This embodiment provides a pre-column derivatization kit containing 2,4-dinitrofluorobenzene, comprising:

[0055] a) an amino acid releaser, the amino acid releaser comprising a sulfosalicylic acid solution, an EDTA solution, a light stabilizer and ethanol, the amino acid releaser being a premixed solution obtained by uniformly mixing the light stabilizer, the sulfosalicylic acid solution, the EDTA solution and the ethanol, wherein the sulfosalicylic acid concentration is 8wt%, the EDTA concentration is 1.5wt%, the light stabilizer is BAD, the BAD concentration is 0.5wt%, and the ethanol concentration is 5vol%;

[0056] b) a derivatization reagent, the derivatization reagent comprising a sodium carbonate-sodium bicarbonate buffer solution and a derivatization organic premix solution, wherein the derivatization organic premix solution is obtained by mixing ethanol, sorbitan monopalmitate and 2,4-dinitrofluorobenzene solution, the pH of the sodium carbonate-sodium bicarbonate buffer solution is 10.0, the concentration of 2,4-dinitrofluorobenzene in the derivatization organic premix solution is 2wt%, the concentration of ethanol is 8vol%, the concentration of sorbitan monopalmitate is 3.0wt%, the solvent is acetonitrile, and the sodium carbonate-sodium bicarbonate buffer solution and the derivatization organic premix solution are stored separately;

[0057] c) Phosphate buffer, the pH of the phosphate buffer is 7.5.

[0058] Example 3

[0059] This embodiment provides a pre-column derivatization kit containing 2,4-dinitrofluorobenzene, comprising:

[0060] a) an amino acid releaser, the amino acid releaser comprising a sulfosalicylic acid solution, an EDTA solution, a light stabilizer and ethanol, the amino acid releaser being a premixed solution obtained by uniformly mixing the light stabilizer, the sulfosalicylic acid solution, the EDTA solution and the ethanol, wherein the sulfosalicylic acid concentration is 3wt%, the EDTA concentration is 0.5wt%, the light stabilizer is UV-9, the UV-9 concentration is 0.1wt%, and the ethanol concentration is 15vol%;

[0061] b) a derivatization reagent, the derivatization reagent comprising a sodium carbonate-sodium bicarbonate buffer solution and a derivatization organic premix solution, wherein the derivatization organic premix solution is obtained by mixing ethanol, sorbitan monopalmitate and 2,4-dinitrofluorobenzene solution, the pH of the sodium carbonate-sodium bicarbonate buffer solution is 8.0, the concentration of 2,4-dinitrofluorobenzene in the derivatization organic premix solution is 0.5wt%, the concentration of ethanol is 3vol%, the concentration of sorbitan monopalmitate is 0.1wt%, the solvent is acetonitrile, and the sodium carbonate-sodium bicarbonate buffer solution and the derivatization organic premix solution are stored separately;

[0062] c) Phosphate buffer, the pH of the phosphate buffer is 6.5.

[0063] Example 4 (No light stabilizer)

[0064] This embodiment provides a pre-column derivatization kit containing 2,4-dinitrofluorobenzene, comprising:

[0065] a) an amino acid releaser, the amino acid releaser comprising a sulfosalicylic acid solution, an EDTA solution and ethanol, the amino acid releaser being a premixed solution obtained by uniformly mixing a light stabilizer, a sulfosalicylic acid solution, an EDTA solution and ethanol, wherein the sulfosalicylic acid concentration is 3wt%, the EDTA concentration is 0.5wt%, and the ethanol concentration is 10vol%;

[0066] b) a derivatization reagent, the derivatization reagent comprising a sodium carbonate-sodium bicarbonate buffer solution and a derivatization organic premix solution, wherein the derivatization organic premix solution is obtained by mixing ethanol, sorbitan monopalmitate and 2,4-dinitrofluorobenzene solution, the pH of the sodium carbonate-sodium bicarbonate buffer solution is 8.0, the concentration of 2,4-dinitrofluorobenzene in the derivatization organic premix solution is 0.5wt%, the concentration of ethanol is 6vol%, the concentration of sorbitan monopalmitate is 1.0wt%, the solvent is acetonitrile, and the sodium carbonate-sodium bicarbonate buffer solution and the derivatization organic premix solution are stored separately;

[0067] c) Phosphate buffer, the pH of the phosphate buffer is 6.5.

[0068] Example 5 (Light stabilizer is other types of benzophenone)

[0069] This embodiment is substantially the same as embodiment 1, except that the light stabilizer is replaced with an equal amount of UV-531.

[0070] Example 6 (Light stabilizer is benzotriazole)

[0071] This example is substantially the same as Example 1, except that the light stabilizer is replaced with an equal amount of UV-329.

[0072] Example 7 (Light stabilizer is triazine)

[0073] This embodiment is substantially the same as embodiment 1, except that the light stabilizer is replaced with an equal amount of UV-1577.

[0074] Example 8 (Light stabilizer is acrylonitrile derivative)

[0075] This example is substantially the same as Example 1, except that the light stabilizer is replaced with an equal amount of N-539.

[0076] Example 9 (derivatization reagent does not contain ethanol)

[0077] This embodiment is substantially the same as embodiment 1, except that the derivatized organic premix solution does not contain ethanol.

[0078] Comparative Example 1

[0079] This comparative example provides a pre-column derivatization kit containing 2,4-dinitrofluorobenzene, comprising:

[0080] a) an amino acid releaser, the amino acid releaser comprising a sulfosalicylic acid solution and an EDTA solution, wherein the concentration of the sulfosalicylic acid solution is 5wt%, the concentration of the EDTA solution is 1.0wt%, and the sulfosalicylic acid solution and the EDTA solution are stored separately;

[0081] b) a derivatization reagent, wherein the derivatization reagent comprises a sodium carbonate-sodium bicarbonate buffer solution and a 2,4-dinitrofluorobenzene solution, wherein the pH of the sodium carbonate-sodium bicarbonate buffer solution is 9.0, the concentration of the 2,4-dinitrofluorobenzene solution is 1.5 wt %, the solvent is acetonitrile, and the sodium carbonate-sodium bicarbonate buffer solution and the 2,4-dinitrofluorobenzene solution are stored separately;

[0082] c) Phosphate buffer, the pH of the phosphate buffer is 7.0.

[0083] Comparative Example 2

[0084] This comparative example is substantially the same as Example 1, except that the amino acid releaser does not contain ethanol.

[0085] Comparative Example 3

[0086] This comparative example is substantially the same as Example 1, except that the derivatized organic premix does not contain sorbitan monopalmitate.

[0087] Example 10 (Amino Acid Fertilizer Derivatization Treatment)

[0088] This example provides a method for using the pre-column derivatization kit containing 2,4-dinitrofluorobenzene in Examples 1 to 9 and Comparative Examples 1 to 3, i.e., a method for derivatizing amino acid fertilizers, wherein the general process in the prior art includes the following steps:

[0089] 1) Weighing an amino acid fertilizer sample, dissolving it in water, and filtering it through a microporous filter membrane to obtain an amino acid fertilizer liquid;

[0090] 2) adding the sulfosalicylic acid solution to the amino acid fertilizer solution and letting it stand, then adding the EDTA solution, mixing well, letting it stand, and filtering with a microporous membrane to obtain an intermediate filtrate;

[0091] 3) adding sodium carbonate-sodium bicarbonate buffer solution and 2,4-dinitrofluorobenzene solution to the intermediate filtrate, mixing well and heating to react;

[0092] 4) After cooling, phosphate buffer is added and mixed to obtain a test sample solution, which can be used for HPLC analysis to detect amino acids in the amino acid fertilizer.

[0093] The method for using the kit of the present invention comprises the following steps:

[0094] 1) Weighing an amino acid fertilizer sample, dissolving it in water, and filtering it through a microporous filter membrane to obtain an amino acid fertilizer liquid;

[0095] 2) adding an amino acid releaser to the amino acid fertilizer liquid, mixing well, letting it stand, and filtering with a microporous membrane to obtain an intermediate filtrate;

[0096] 3) adding sodium carbonate-sodium bicarbonate buffer solution and derivatized organic premix solution to the intermediate filtrate, mixing well and heating to react;

[0097] 4) After cooling, phosphate buffer is added and mixed to obtain a test sample solution, which can be used for HPLC analysis to detect amino acids in the amino acid fertilizer.

[0098] The method for using the kit without premix in this application includes the following steps:

[0099] 1) Weighing an amino acid fertilizer sample, dissolving it in water, and filtering it through a microporous filter membrane to obtain an amino acid fertilizer liquid;

[0100] 2) adding sulfosalicylic acid solution to the amino acid fertilizer solution and letting it stand, then adding ethanol and EDTA solution, mixing and letting it stand, filtering with a microporous membrane to obtain an intermediate filtrate;

[0101] 3) adding sodium carbonate-sodium bicarbonate buffer solution, 2,4-dinitrofluorobenzene solution and sorbitan monopalmitate to the intermediate filtrate, mixing well and heating to react;

[0102] 4) After cooling, phosphate buffer is added and mixed to obtain a test sample solution, which is used for HPLC analysis to detect amino acids in the amino acid fertilizer.

[0103] Wherein, the microporous filter membrane in steps 1) and 2) has a pore size range of 0.40 to 0.50 μm, for example, specifically 0.45 μm, and the heating reaction condition in step 3) is 50 to 70°C for 20 to 30 minutes, for example, in a specific embodiment, 60°C for 25 minutes. In step 2), the sulfosalicylic acid solution is added to the amino acid fertilizer solution and then allowed to stand for no less than 30 minutes, and the ethanol and EDTA solution are added and mixed and then allowed to stand for no less than 10 minutes. For example, in a specific embodiment, the sulfosalicylic acid solution is added to the amino acid fertilizer solution and then allowed to stand for 30 minutes, and the ethanol and EDTA solution are added and mixed and then allowed to stand for 10 minutes. For the kit using a premixed solution, the amino acid releaser is added to the amino acid fertilizer solution and then allowed to stand for 40 minutes.

[0104] It should be noted that for the scheme of the present application without premixing, those skilled in the art can determine it according to the steps of amino acid derivatization of 2,4-dinitrofluorobenzene in the prior art and the scheme and steps recorded in the present application. For example, the existing order of addition of ethanol and EDTA can be adjusted, for example, the order of addition of 2,4-dinitrofluorobenzene solution and sorbitan monopalmitate can also be adjusted. For the case of containing other ingredients, such as two or three, those skilled in the art can also determine the order of addition according to the content of the scheme recorded in the present application, as long as the effect of amino acid derivatization of 2,4-dinitrofluorobenzene recorded in the prior art can be met.

[0105] like Figure 2 As shown in FIG. 1 , the reaction principle of amino acid derivatization by 2,4-dinitrofluorobenzene is shown. In the present application scheme, the amino acid fertilizer is treated with an amino acid releaser to extract and release the amino acid to be detected, and the 2,4-dinitrofluorobenzene in the derivatization reagent can react with the amino acid as follows: Figure 2 The derivatization reaction shown is performed to label the amino acids, and then the amino acids labeled with 2,4-dinitrofluorobenzene are detected by HPLC, so that the amino acid components in the amino acid fertilizer can be qualitatively or quantitatively analyzed. For the present application, HPLC is mainly used for quantitative analysis, so the derivatization level of the amino acids is crucial to the accuracy of the quantitative analysis.

[0106] Test Example 1 (Amino Acid Extraction Rate Test)

[0107] In order to test the effect of amino acid releaser and derivatization reagent, 5.0 mg / L, 25 mg / L, and 50 mg / L mixed amino acid standard solutions were used for derivatization reaction, and the derivatization ratio of amino acids was calculated by separating the derivatized amino acid products, so as to evaluate the extraction effect of amino acids and the derivatization effect of 2,4-dinitrofluorobenzene. The mixed amino acids were mixed standards of 17 amino acids, including aspartic acid, glutamic acid, serine, glycine, arginine, threonine, proline, alanine, valine, methionine, cysteine, isoleucine, leucine, phenylalanine, histidine, lysine, and tyrosine. The 5.0 mg / L, 25 mg / L, and 50 mg / L mixed amino acid standard solutions were different in total amount of amino acids, while the amount of each amino acid in each mixed amino acid standard solution was the same, that is, the content ratio of any two amino acids was 1:1. In addition, in order to conform to the environment in the actual detection process of amino acid fertilizers as much as possible, 10 wt% protein and copper, manganese, and ferrous ions were additionally added to simulate the amino acid fertilizers in actual applications.

[0108] The amino acid derivatization test of the mixed amino acid standard solution was carried out using the kits of Examples 1 to 3 and Comparative Examples 1 to 3. The amino acid derivatization ratio was obtained by detecting the content of 2,4-dinitrofluorobenzene-derivatized amino acids and calculating their ratio to the total amount of initial amino acids.

[0109] Except for the different components of the kit used, the amount of active ingredient added to each reagent is controlled to be consistent, and the derivatization operating conditions are also the same. Specifically, the microporous filter membrane has a pore size range of 0.45 μm, and the heating condition for the reaction in step 3) is to react at 60°C for 25 minutes. Other conditions not described in detail are also controlled to be consistent. The calculation results of the amino acid derivatization ratio are shown in Table 1 below.

[0110] Table 1 Amino acid derivatization ratio

[0111]

[0112]

[0113] According to the results in Table 1, the kit provided in the present application can not only fully extract and derivatize the amino acids in low-concentration amino acid fertilizers, but also efficiently extract and derivatize higher-concentration amino acid fertilizers. This can improve the detection accuracy of amino acids in amino acid fertilizers, make the detection results more representative, and the application scenarios of the kit are more universal.

[0114] Test Example 2 (Stability Test)

[0115] Since the amino acid derivatization process involves a large number of added reagents, in order to reduce the complexity of the operations in the derivatization process, some reagents are premixed to prepare a premix solution for storage, and the premix solution is directly added for processing when used, including the amino acid releaser and the derivatization organic premix solution. The effect of the premix solution has been verified in Test Example 1, but the storage stability of the premix solution still needs to be tested and determined, so as to guide the solution configuration and storage in the actual process.

[0116] During the test, after the relevant reagents in the example kit were configured, they were placed in a laboratory reagent light-shielding cabinet for storage and observed after 168 hours. The effect of the derivatization kit after 168 hours was tested using the same method as in Test Example 1. In addition, in order to be able to distinguish the stability effects of different kits as much as possible, a derivatization reaction was carried out on a 50 mg / L mixed amino acid standard solution. The results are shown in Table 2 below.

[0117] Table 2 Stability test results

[0118]

[0119]

[0120] According to the results in Table 2, the addition of light stabilizers has a significant effect on the stability of the reagents in the kit of the present application, especially the derivatization effect, and BAD or UV-9 has a significantly better effect than other light stabilizers. In addition, the addition of ethanol to the derivatized organic premix can also improve the stability of the derivatized organic premix.

[0121] More importantly, the results in Table 2 show that the premixed liquid kit provided in the present application, especially the amino acid releaser and the derivatized organic premixed liquid therein, can significantly reduce the number of operating steps in the detection process, and the premixed liquid can remain stable for at least 168 hours, and the amino acid derivatization effect remains stable.

[0122] The above is only the embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

1. A pre-column derivatization kit containing 2,4-dinitrofluorobenzene, characterized in that: The kit comprises: An amino acid releaser, the amino acid releaser comprising a sulfosalicylic acid solution, an EDTA solution and ethanol, the concentration of the sulfosalicylic acid solution being 3-8wt%, and the concentration of the EDTA solution being 0.5-1.5wt%; A derivatization reagent, wherein the derivatization reagent comprises a sodium carbonate-sodium bicarbonate buffer solution, sorbitan monopalmitate and a 2,4-dinitrofluorobenzene solution, wherein the pH range of the sodium carbonate-sodium bicarbonate buffer solution is 8.0 to 10.0, the concentration of the 2,4-dinitrofluorobenzene solution is 0.5 to 2 wt %, and the solvent in the 2,4-dinitrofluorobenzene solution is acetonitrile; Phosphate buffer, wherein the pH value of the phosphate buffer is 6.5 to 7.

5.

2. The pre-column derivatization kit containing 2,4-dinitrofluorobenzene according to claim 1, characterized in that: The amino acid releaser also includes a light stabilizer, and the amino acid releaser is a premixed liquid, which is obtained by mixing the light stabilizer, sulfosalicylic acid solution, EDTA solution and ethanol.

3. The pre-column derivatization kit containing 2,4-dinitrofluorobenzene according to claim 2, characterized in that: The light stabilizer is selected from one or more of benzophenones, benzotriazoles, triazines, salicylates and acrylonitrile derivatives; Optionally, the light stabilizer is BAD or UV-9.

4. The pre-column derivatization kit containing 2,4-dinitrofluorobenzene according to claim 3, characterized in that: The ethanol concentration in the amino acid releaser is 5 to 15 vol%; and / or, The concentration of the light stabilizer in the amino acid releaser is 0.1-0.5 wt %.

5. The pre-column derivatization kit containing 2,4-dinitrofluorobenzene according to claim 1, characterized in that: The derivatization reagent also includes ethanol, and the derivatization reagent includes a sodium carbonate-sodium bicarbonate buffer solution and a derivatization organic premix solution, and the derivatization organic premix solution is obtained by mixing ethanol, dehydrated sorbitan monopalmitate and 2,4-dinitrofluorobenzene solution.

6. The pre-column derivatization kit containing 2,4-dinitrofluorobenzene according to claim 1, characterized in that: The ethanol concentration in the derivatized organic premix is ​​3-8 vol%.

7. Use of the pre-column derivatization kit containing 2,4-dinitrofluorobenzene as claimed in any one of claims 1 to 6 in the pre-column derivatization step when detecting amino acids in amino acid fertilizers by HPLC, characterized in that: The application comprises the following steps: 1) Weighing an amino acid fertilizer sample, dissolving it in water, and filtering it through a microporous filter membrane to obtain an amino acid fertilizer liquid; 2) adding an amino acid releaser to the amino acid fertilizer liquid, mixing well, letting the mixture stand, and filtering with a microporous membrane to obtain an intermediate filtrate; 3) adding a derivatization reagent to the intermediate filtrate, mixing well, and then heating to react; 4) After cooling, adding phosphate buffer solution and mixing well to obtain a test sample solution, which is used for HPLC analysis to detect amino acids in the amino acid fertilizer.

8. The use according to claim 7, characterized in that: The microporous filter membrane in steps 1) and 2) has a pore size ranging from 0.40 to 0.50 μm.

9. The use according to claim 7, characterized in that: The heating reaction condition in the step 3) is 50-70° C. for 20-30 min.

10. The use according to claim 7, characterized in that: In the step 2), the amino acid releasing agent is added to the amino acid fertilizer solution and then allowed to stand for no less than 40 minutes.

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