Search

CN-121994783-A - Arginine determination method with stable color development

CN121994783ACN 121994783 ACN121994783 ACN 121994783ACN-121994783-A

Abstract

The invention provides an arginine determination method with stable color development, belonging to the technical field of analytical chemistry and biological detection. The method comprises the specific steps of preparing arginine standard solutions with different concentrations, sequentially adding alpha-naphthol and sodium hypochlorite solutions, adding ethylenediamine solution after reaction, measuring absorbance, drawing a standard curve, processing a sample to be tested by the same steps, and calculating the arginine content according to the standard curve. The method aims at the technical problems of unstable and easy fading of the color development product existing in the conventional sakaguchi reaction colorimetric method, and effectively solves the problems of narrow detection time window and poor reproducibility by introducing ethylenediamine solution into a reaction system as a reaction termination and color development stabilizer. The method has the advantages of small reagent dosage and simple and convenient operation, is particularly suitable for high-throughput and automatic detection of a 96-well plate and an enzyme-labeled instrument, and has wide application value in product quality control and scientific research in the fields of medicine, food, cosmetics, cell culture and the like.

Inventors

  • HUANG FENGJIE
  • LIAO JINLONG
  • WU XINYING

Assignees

  • 中国药科大学

Dates

Publication Date
20260508
Application Date
20260130

Claims (9)

  1. 1. A method for measuring the content of arginine with stable color development comprises the following steps: S1, preparing a standard solution containing arginine with different concentrations, sequentially adding an alpha-naphthol solution and a sodium hypochlorite solution into the standard solution, uniformly mixing, standing at room temperature, adding an ethylenediamine solution to terminate the reaction, measuring the absorbance of a reaction system, and drawing a standard curve according to the concentration of the standard solution and the corresponding absorbance; S2, sample measurement, namely adding the alpha-naphthol solution and the sodium hypochlorite solution into a sample to be measured, uniformly mixing, standing for reaction, adding the ethylenediamine solution for stopping reaction, measuring the absorbance of a reaction system, and calculating to obtain the arginine content in the sample to be measured according to the standard curve; in steps S1 and S2, the reaction conditions are the same.
  2. 2. The method for measuring the content of alpha-naphthol according to claim 1, wherein the alpha-naphthol solution is prepared by diluting a stock solution of alpha-naphthol absolute ethanol with a volume ratio of 1:4 by using a carbonate buffer solution with a pH of 10.5.
  3. 3. The method for measuring content according to claim 1 or 2, wherein the sodium hypochlorite solution is prepared by diluting a sodium hypochlorite stock solution with a carbonate buffer solution having a pH of 10.5 at a volume ratio of 1:1, wherein the sodium hypochlorite stock solution has a content of free base (calculated as NaOH) of 7.0-8.0% and a content of active chlorine (calculated as Cl) of 4.5-5.0%.
  4. 4. The method for measuring the content of ethylenediamine according to any one of claims 1 to 3, wherein the concentration of the ethylenediamine solution is 10% to 100% (v/v).
  5. 5. The method for measuring content according to claim 1 to 4, wherein the standing time at room temperature is 1 minute.
  6. 6. The method for measuring content according to any one of claims 1 to 5, wherein the absorbance is measured at a detection wavelength of 480 to 500 nm.
  7. 7. The method for measuring content according to claim 1 to 6, wherein the absorbance measurement is performed in a 96-well plate using an enzyme-labeled instrument.
  8. 8. The method according to any one of claims 1 to 7, wherein in the steps S1 and S2, the reagents are added in the volumes of 50. Mu.L of the standard solution or the sample to be measured, 50. Mu.L of the alpha-naphthol solution, 50. Mu.L of the sodium hypochlorite solution and 50. Mu.L of the ethylenediamine solution.
  9. 9. The method according to claim 1 to 8, wherein the concentration of arginine in the sample to be measured is at least 10. Mu.g/mL.

Description

Arginine determination method with stable color development Technical Field The invention belongs to the technical field of analytical chemistry and biological detection, and particularly relates to a stable arginine quantitative analysis method. Background Arginine is an important basic amino acid and has wide application in the fields of medicine, functional food, cosmetics, cell culture media and the like. The accurate and rapid determination of the content is a key link of the quality control and scientific research of related products. The arginine detection method mainly comprises an amino acid analyzer method, a liquid chromatography method, an enzyme method and a sakaguchi reagent method. The amino acid analyzer has high maintenance cost, strict operation requirement, high equipment cost of liquid chromatography, long detection period, 2-3 hours for sedimentation at 4 ℃, and low detection efficiency, and the acetone dry powder needs to be prepared by an enzymatic method, the preparation time is 40 hours. Currently, the quantitative analysis of arginine mainly adopts a colorimetric method based on sakaguchi reaction. The principle of the method is that arginine reacts with alpha-naphthol and active chlorine (usually from sodium hypochlorite) under the strong alkaline condition to generate a red product with maximum absorption near 520nm, and the absorbance and the arginine concentration are in a linear relation within a certain range, so that quantitative analysis can be realized. However, the conventional sakaguchi reaction colorimetric method has a technical disadvantage that instability of a developed product cannot be properly solved for a long time. The red complex formed by the reaction changes over time and fades in a short time, which makes it necessary to complete the detection within a very narrow time window, which is extremely demanding in terms of timing and poor in reproducibility. Therefore, the arginine content measuring method which has stable color development, simple and convenient operation, good reproducibility and wide time window detection support is developed, and has important practical application value. Disclosure of Invention The invention provides a method for measuring the content of arginine with stable color development, which aims to solve the problem of unstable color development in the process of measuring the content of arginine by a traditional sakurting reaction colorimetric method. The technical scheme adopted by the invention specifically comprises the following steps: s1, preparing standard solutions containing arginine with different concentrations, sequentially adding an alpha-naphthol solution and a sodium hypochlorite solution into the standard solutions, uniformly mixing, standing at room temperature, adding an ethylenediamine solution to terminate the reaction, measuring the absorbance of a reaction system, and drawing a standard curve according to the concentration of the standard solutions and the corresponding absorbance; S2, sample measurement, namely adding the alpha-naphthol solution and the sodium hypochlorite solution into a sample to be measured, uniformly mixing, standing at room temperature, adding the ethylenediamine solution to terminate the reaction, measuring the absorbance of a reaction system, and calculating to obtain the arginine content in the sample to be measured according to the standard curve. In the content determination method, an ethylenediamine aqueous solution is used as a reaction termination and color development stabilizer. The effective concentration thereof ranges from 10% to 100% (v/v), preferably 50%. In the content determination method, the alpha-naphthol solution is prepared by diluting 0.5% (m/v) alpha-naphthol ethanol stock solution with a carbonate buffer solution with the pH of 10.5 at 1:4, and the sodium hypochlorite solution is prepared by diluting sodium hypochlorite stock solution (free base (calculated by NaOH) of 7.0-8.0% and active chlorine (calculated by Cl) of 4.5-5.0%) with the same buffer solution at 1:1. In the content measuring method, the color reaction is completed after standing for about 1 minute at room temperature, and the absorbance is preferably detected at 480-500nm wavelength, and the optimal wavelength is 492nm. In the content determination method, the method is particularly suitable for operation of a 96-well plate and an enzyme-labeled instrument, and the addition volume of each reagent is small (50 mu L), so that the detection flux and the efficiency are greatly improved. Compared with the prior art, the invention has the following remarkable advantages: The invention solves the problems of unstable color development and serious color fading in a short time of the traditional arginine colorimetric method, solves the technical bottlenecks of easy decomposition and rapid decay of absorbance value of the traditional sakaguchi reaction product by introducing ethylenediamine solution as an e