Determination method of arginine with stable color development

Abstract

The invention provides a method for determining arginine with stable color development, and belongs to the technical field of analytical chemistry and biological detection. The method comprises the following specific steps: preparing arginine standard solutions with different concentrations, sequentially adding alpha-naphthol and a sodium hypochlorite solution, adding an ethylenediamine solution after reaction, measuring absorbance and drawing a standard curve; a sample to be detected is treated by the same steps, and the arginine content is calculated according to the standard curve. According to the method, aiming at the technical problems that a chromogenic product is unstable and easy to fade in the existing Sakaguchi reaction colorimetric method, an ethylenediamine solution is introduced into a reaction system as a reaction termination and chromogenic stabilizer, so that the problems that a detection time window is narrow and the reproducibility is poor are effectively solved. The method is small in reagent dosage and simple and convenient to operate, is particularly suitable for high-throughput and automatic detection of a 96-well plate and a microplate reader, and has wide application value in product quality control and scientific research in the fields of medicine, food, cosmetics, cell culture and the like.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry and biological detection technology, and specifically relates to a stable method for quantitative analysis of arginine. Background Technology

[0002] Arginine is an important basic amino acid with wide applications in medicine, functional foods, cosmetics, and cell culture media. Accurate and rapid determination of its content is a crucial step in quality control and scientific research for related products.

[0003] Arginine detection methods mainly include amino acid analyzer methods, liquid chromatography (LC), enzymatic methods, and the Sakaguchi reagent method. Amino acid analyzers have high maintenance costs and strict operational requirements; LC methods are expensive and have long detection cycles, requiring 2-3 hours just for sedimentation at 4℃; enzymatic methods require the preparation of acetone powder, which takes up to 40 hours, resulting in low detection efficiency. Currently, quantitative analysis of arginine mainly employs the colorimetric method based on the Sakaguchi reaction. The principle of this method is that arginine reacts with α-naphthol and active chlorine (usually derived from sodium hypochlorite) under strongly alkaline conditions to generate a red product with maximum absorption around 520 nm. The absorbance of this product is linearly related to the arginine concentration within a certain range, thus enabling quantitative analysis.

[0004] However, the traditional Sakaguchi reaction colorimetric method has a long-standing technical drawback: the instability of the colorimetric product. The red complex formed by the reaction changes over time and fades within a short period of time. This means that the detection must be completed within a very narrow time window, which places extremely stringent requirements on timing and results in poor reproducibility.

[0005] Therefore, developing a method for determining arginine content that is colorimetrically stable, easy to operate, reproducible, and supports detection over a wide time window is of significant practical value. Summary of the Invention

[0006] This invention provides a colorimetrically stable method for determining arginine content, thereby solving the problem of unstable color development in the traditional Sakaguchi reaction colorimetric method for determining arginine content.

[0007] The technical solution adopted in this invention specifically includes the following steps:

[0008] S1. Standard curve plotting: Prepare standard solutions containing different concentrations of arginine. Add α-naphthol solution and sodium hypochlorite solution to the standard solutions in sequence, mix well, and let stand at room temperature. Then add ethylenediamine solution to terminate the reaction and measure the absorbance of the reaction system. Plot the standard curve based on the concentration of the standard solution and the corresponding absorbance.

[0009] S2. Sample determination: Add the α-naphthol solution and the sodium hypochlorite solution to the sample to be tested, mix well, let stand at room temperature, then add the ethylenediamine solution to terminate the reaction, measure the absorbance of the reaction system, and calculate the content of arginine in the sample to be tested according to the standard curve.

[0010] In the above-described method for content determination, ethylenediamine aqueous solution is used as a reaction terminator and color stabilizer. Its effective concentration range is 10%-100% (v / v), preferably 50%.

[0011] In the above-mentioned method for content determination, the α-naphthol solution is prepared by diluting 0.5% (m / v) α-naphthol ethanol stock solution with a carbonate buffer at pH 10.5 at a ratio of 1:4; the sodium hypochlorite solution is prepared by diluting sodium hypochlorite stock solution (free alkali (calculated as NaOH) 7.0-8.0%; active chlorine (calculated as Cl) 4.5-5.0%) with the same buffer at a ratio of 1:1.

[0012] In the above-mentioned content determination method, the colorimetric reaction can be completed by standing at room temperature for about 1 minute; the absorbance is preferably detected at a wavelength of 480-500nm, and the optimal wavelength is 492nm.

[0013] Of the above-mentioned methods for content determination, the method is particularly suitable for operation with 96-well plates and microplate readers. The volume of each reagent added is small (50 μL), which greatly improves the detection throughput and efficiency.

[0014] Compared with the prior art, the present invention has the following significant advantages:

[0015] This invention solves the problems of unstable color development and severe fading in a short time in the traditional arginine colorimetric method; by introducing ethylenediamine solution as a highly efficient terminating stabilizer, it solves the technical bottleneck of easy decomposition of the traditional Sakaguchi reaction product and rapid decay of absorbance value; after the color development is completed, the absorbance value of the reaction system remains highly stable for 24 hours, and the results are reliable and reproducible.

[0016] The method of this invention is fully adaptable to 96-well plate operation and microplate reader detection. By miniaturizing and standardizing the volume of each reagent (50 μL each) and optimizing the reaction time (only 1 minute), parallel processing and rapid detection of large batches of samples can be achieved. The method has uniform steps and is easy to operate, significantly reducing human error and labor intensity. It can be easily integrated into automated workstations to meet the needs of high-throughput and automated detection. Attached Figure Description

[0017] Figure 1 This is a full-wavelength scan spectrum of the colorimetric reaction system in Example 1 of the present invention.

[0018] Figure 2This illustrates the effect of different concentrations of ethylenediamine on the absorbance stability of the reaction system in Example 2 of the present invention; where 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 1, and 0 represent ethylenediamine concentrations of 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, and 0%, respectively.

[0019] Figure 3 This is the arginine standard curve at different time points in Example 3 of the present invention.

[0020] Figure 4 This is the standard curve for arginine determination in Example 4 of the present invention. Detailed Implementation

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] Unless otherwise specified, all materials used in the following embodiments are commercially available.

[0023] Example 1: Optimization of colorimetric reaction conditions and detection wavelength using an ELISA reader

[0024] a. Reagent preparation

[0025] Arginine standard solution (100 μg / mL): Accurately weigh an appropriate amount of arginine hydrochloride reference standard, dissolve it in pure water and dilute to volume to prepare an arginine standard solution with a concentration of 100 μg / mL.

[0026] α-Naphthol solution (0.1%): Take 0.5% α-naphthol ethanol stock solution and dilute it 5 times with carbonate buffer solution at pH 10.5 to obtain 0.1% α-naphthol solution;

[0027] Sodium hypochlorite solution: Take sodium hypochlorite stock solution (7.0-8.0% free alkali, 4.5-5.0% active chlorine), dilute it 1 part with a carbonate buffer solution of pH 10.5, mix well and set aside.

[0028] Ethylenediamine solution (10%, v / v): Measure 10 mL of ethylenediamine, dilute it with pure water to 100 mL, and mix well.

[0029] b. Experimental methods

[0030] Add the following reagents sequentially to the 96-well plate:

[0031] Arginine standard solution (100 μg / mL): 50 μL

[0032] 0.1% α-naphthol solution: 50 μL

[0033] Sodium hypochlorite solution: 50 μL

[0034] Immediately mix using a pipette and allow the mixture to stand at room temperature for 1 minute. Then, add 50 μL of 10% ethylenediamine solution to terminate the reaction and mix again.

[0035] c. Absorbance scanning and wavelength determination

[0036] The absorbance values ​​of the reaction system were recorded by performing a full-wavelength scan (400–600 nm) using an ELISA reader. The scan results are as follows: Figure 1 As shown, the colorimetric reaction system exhibits high absorbance and a relatively stable plateau in the wavelength range of 480–500 nm, with a peak value reaching around 492 nm. This indicates that the orange-red product generated by the reaction has maximum absorption in this wavelength range. Therefore, 492 nm was selected as the optimal detection wavelength for subsequent measurements to obtain the highest detection sensitivity.

[0037] Example 2: Optimization of colorimetric reaction conditions using an enzyme-linked immunosorbent assay (ELISA) reader - ethylenediamine concentration

[0038] a. Reagent preparation

[0039] Arginine standard solution (100 μg / mL): Preparation method is the same as in Example 1;

[0040] α-Naphthol solution (0.1%), sodium hypochlorite solution: preparation method is the same as in Example 1;

[0041] Ethylenediamine gradient concentration solutions: The ethylenediamine stock solution was diluted with pure water to prepare concentrations (v / v) of 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, and 0% (pure water control).

[0042] b. Experimental methods

[0043] In a 96-well plate, add the following sequentially to each well:

[0044] Arginine standard solution (100 μg / mL): 50 μL

[0045] 0.1% α-naphthol solution: 50 μL

[0046] Sodium hypochlorite solution: 50 μL

[0047] Immediately mix the sample using a pipette, allow it to stand at room temperature for 1 minute, then add 50 μL of ethylenediamine solution of different concentrations to terminate the reaction, and mix again. Measure the absorbance using a microplate reader at the optimal detection wavelength of 492 nm determined in Example 1. Starting from the addition of the ethylenediamine solution, measure the absorbance every half hour for 12 hours, and perform an endpoint determination at 24 hours.

[0048] c. Stability and determination of ethylenediamine concentration

[0049] The absorbance of reaction systems terminated by different concentrations of ethylenediamine changes over time as follows: Figure 2 As shown in Table 1, to quantify the stability at each concentration, the percentage decrease in absorbance (R value) of each system over 24 hours was calculated, and the results are listed in Table 1.

[0050] Taking into account initial absorbance, 24-hour stability, and reagent cost:

[0051] The concentration of ethylenediamine should not be lower than 10%, otherwise the stability will not meet the requirements for quantitative analysis.

[0052] A 50% (v / v) ethylenediamine solution is recommended as a routine reaction termination stabilizer. Under these conditions, the absorbance of the reaction system retains more than 69.8% of its initial value within 24 hours after termination, and the change is gradual during the critical 0-12 hours, ensuring the accuracy and reproducibility of the measurement results.

[0053] The optimal concentration can be selected as 80% (v / v), at which the system has the highest long-term stability (78.9% retention rate over 24 hours), making it suitable for special scenarios where long-term storage is required before measurement.

[0054] Table 1. Absorbance retention rate at different ethylenediamine concentrations over 24 hours

[0055]

[0056] Example 3: Establishment and Stability Verification of Standard Curve

[0057] a. Reagent preparation

[0058] Arginine standard solution series: Accurately weigh arginine hydrochloride reference standard, dissolve it in pure water and perform serial dilutions to prepare standard solutions with concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg / mL. The 0 μg / mL solution is a blank control.

[0059] α-Naphthol solution (0.1%), sodium hypochlorite solution: preparation method is the same as in Example 1;

[0060] Ethylenediamine solution (50%, v / v): Measure 50 mL of ethylenediamine, dilute it with pure water to 100 mL, and mix well.

[0061] b. Experimental methods

[0062] In a 96-well plate, set up reaction wells for each concentration of arginine standard solution. Add the following to the wells sequentially:

[0063] Arginine standard solutions of different concentrations: 50 μL

[0064] 0.1% α-naphthol solution: 50 μL

[0065] Sodium hypochlorite solution: 50 μL

[0066] Immediately mix using a pipette and allow the mixture to stand at room temperature for 1 minute. Then, add 50 μL of 50% ethylenediamine solution to each well to terminate the reaction and mix again.

[0067] The absorbance of each well was measured immediately at 492 nm using a microplate reader, starting from the moment the ethylenediamine solution was added (0 hours). Subsequently, the 96-well plate was placed at room temperature in the dark, and the absorbance of the same batch of reaction wells was measured again at 0.5 hours, 12 hours, and 24 hours.

[0068] c. Experimental Results and Analysis

[0069] A standard curve was plotted by performing linear regression analysis with the concentration of the arginine standard solution (μg / mL) on the x-axis (X) and the measured absorbance values ​​(excluding the absorbance of the blank wells) on the y-axis (Y). The linear equations and coefficients of determination (R²) of the standard curves measured at different time points are shown. 2 As shown in Table 2, the arginine standard curves for reaction times of 0 hours, 0.5 hours, 12 hours, and 24 hours are as follows: Figure 3 As shown.

[0070] Table 2. Linear regression analysis of standard curves for arginine determination at different time points.

[0071] Measurement time (hours) Linear regression equation <![CDATA[Coefficient of determination (R 2 )]]> 0 Y = 0.002978 * X + 0.007757 0.9869 0.5 Y = 0.002776 * X + 0.01027 0.9824 12 Y = 0.002180 * X + 0.004879 0.9861 24 Y = 0.002042 * X + 0.004515 0.9839

[0072] Note: Y is the absorbance value, and X is the arginine concentration (μg / mL).

[0073] Example 4: Sample determination (spiking recovery experiment using cell culture medium as the matrix)

[0074] a. Reagent:

[0075] α-Naphthol solution (0.1%), sodium hypochlorite solution: preparation method is the same as in Example 1;

[0076] Ethylenediamine solution (50%, v / v): Preparation method is the same as in Example 3;

[0077] Standard curve solution: Arginine standard solutions with concentration gradients of 0, 20, 40, 60, 80, and 100 μg / mL were prepared using pure water as the solvent.

[0078] Spiked sample solutions: Spiked sample solutions with theoretical arginine concentrations of 10, 50, and 80 μg / mL were prepared using RPMI-1640 basal medium.

[0079] b. Experimental methods:

[0080] In a 96-well plate, add the following sequentially to each well:

[0081] Sample or standard solution: 50 μL

[0082] 0.1% α-naphthol solution: 50 μL

[0083] Sodium hypochlorite solution: 50 μL

[0084] Immediately mix the sample using a pipette and allow it to stand at room temperature for 1 minute. Then, add 50 μL of 50% ethylenediamine solution to each well to stop the reaction, mix again, and immediately measure the absorbance of each well at 492 nm using a microplate reader.

[0085] c. Result Calculation

[0086] Using the concentrations of arginine standards prepared in pure water (0, 20, 40, 60, 80, 100 μg / mL) as the x-axis and the measured absorbance values ​​as the y-axis, linear regression analysis was performed to plot a standard curve (e.g., ...). Figure 4 Substitute the absorbance value of the spiked sample into the standard curve equation to calculate the corresponding measured concentration.

[0087] Recovery rate (%) = (Measured concentration / Theoretical spiked concentration) × 100%

[0088] Relative Standard Deviation (RSD) (%) = (Standard Deviation / Average Measured Concentration) × 100%

[0089] Table 3. Spike recovery and precision results for arginine determination in 1640 medium (n=3)

[0090] Theoretical spiking concentration (μg / mL) Average measured concentration (μg / mL) Average recovery rate (%) RSD (%) 10 10.34 103.4 9.7 50 45.27 90.6 6.8 80 69.70 87.1 7.7

[0091] This spiked recovery experimental system verifies the reliability of the method of the present invention in determining the arginine content in real complex biological matrices.

[0092] Based on the above linear regression equation and recovery results, it can be seen that the spiked recovery experimental system verifies the reliability of the method of the present invention in determining the arginine content in actual complex biological matrices. The method of optimizing the colorimetric reaction and measuring absorbance using an ELISA reader is very sensitive, and rapid batch detection can be achieved using a 96-well ELISA plate, making it suitable as a quantitative detection method for arginine.

Claims

1. A colorimetrically stable method for determining the content of arginine, comprising the following steps: S1. Plotting the standard curve: Prepare standard solutions containing different concentrations of arginine. Add α-naphthol solution and sodium hypochlorite solution to the standard solutions in sequence, mix well, let stand at room temperature, add ethylenediamine solution to terminate the reaction, measure the absorbance of the reaction system, and plot the standard curve based on the concentration of the standard solution and the corresponding absorbance. S2. Sample determination: Add the α-naphthol solution and the sodium hypochlorite solution to the sample to be tested, mix well and let it stand to react, then add the ethylenediamine solution to terminate the reaction, measure the absorbance of the reaction system, and calculate the content of arginine in the sample to be tested according to the standard curve. The reaction conditions are the same in steps S1 and S2.

2. The content determination method according to claim 1, characterized in that: The α-naphthol solution was prepared by diluting a 0.5% (m / v) α-naphthol anhydrous ethanol stock solution with a carbonate buffer solution at pH 10.5 at a volume ratio of 1:

4.

3. The content determination method according to claim 1 or 2, characterized in that: The sodium hypochlorite solution is prepared by diluting the sodium hypochlorite stock solution with a carbonate buffer solution at pH 10.5 at a volume ratio of 1:1; the sodium hypochlorite stock solution contains 7.0-8.0% free alkali (calculated as NaOH) and 4.5-5.0% active chlorine (calculated as Cl).

4. The content determination method according to any one of claims 1-3, characterized in that: The concentration of the ethylenediamine solution is 10% to 100% (v / v).

5. The content determination method according to any one of claims 1-4, characterized in that: The settling time at room temperature is 1 minute.

6. The content determination method according to any one of claims 1-5, characterized in that: The absorbance was measured at a detection wavelength of 480–500 nm.

7. The method for content determination according to any one of claims 1-6, characterized in that: The absorbance was measured in a 96-well plate using an enzyme-linked immunosorbent assay (ELISA) reader.

8. The method for content determination according to any one of claims 1-7, characterized in that: In steps S1 and S2, the volumes of each reagent added are as follows: 50 μL of standard solution or sample to be tested, 50 μL of α-naphthol solution, 50 μL of sodium hypochlorite solution, and 50 μL of ethylenediamine solution.

9. The method for content determination according to any one of claims 1-8, characterized in that: The minimum concentration of arginine in the sample to be tested is 10 μg / mL.

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