Qualitative and quantitative detection method of 3-hydroxy-L-proline

Abstract

The invention discloses a qualitative and quantitative analysis method of 3-hydroxy-L-proline. The method comprises the following steps: S1, establishing a concentration-absorbance standard curve of the 3-hydroxy-L-proline; s2, the solution to be detected is subjected to an oxidation reaction with hydrogen peroxide and copper sulfate under the alkaline condition, and an oxidation solution is obtained; s3, the oxidation solution reacts with p-dimethylaminobenzaldehyde to obtain a color developing solution, and if the solution to be detected contains 3-hydroxy-L-proline, the color developing solution is purplish red; and S4, determining the absorbance of the color developing solution at 560 nm, and obtaining the concentration of the 3-hydroxy-L-proline in the solution to be detected according to the concentration-absorbance standard curve of the 3-hydroxy-L-proline. Through a mild reaction mode, rapid detection and quantification of 3-hydroxy-L-proline in a solution are realized, and large-scale detection can be realized by matching with a deep hole plate and a multi-channel photometer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a qualitative and quantitative detection method for 3-hydroxy-L-proline. Background Technology

[0002] 3-Hyp is an amino acid with a hydroxyl group introduced at the C-3 position of the proline ring. Its molecular formula is C5H9NO3, and its molecular weight is 131.13. This modification significantly enhances molecular polarity and hydrogen bonding ability, improving the tertiary structural stability and solubility of collagen peptides and other peptide chains. It can also improve cell adhesion, promote wound healing, and aid in tissue recovery. In cosmetics and nutritional products, it can be used in anti-aging masks, functional beverages, and joint health formulas to support skin hydration and joint health. Furthermore, 3-Hyp can also serve as an important chiral intermediate in the synthesis of pharmaceuticals, biodegradable polymers, and smart hydrogels.

[0003] Because 3-hydroxy-L-proline lacks strong ultraviolet absorption or fluorescence, direct detection has very low sensitivity. In existing technologies, the quantitative detection of 3-hydroxy-L-proline in reaction solutions largely relies on high-performance liquid chromatography (HPLC) or mass spectrometry (MS). While these methods offer high accuracy, they are expensive, require complex pretreatment, and have long detection cycles, making them unsuitable for large-scale sample screening.

[0004] Furthermore, the position of the hydroxyl group on the proline ring (C-3 or C-4) has a decisive influence on its chemical properties and detection methods. Currently widely used rapid detection methods such as the chloramine T method are mainly for 4-hydroxy-proline because its reaction mechanism depends on a specific oxidative ring-opening process, which is ineffective for 3-hydroxy-proline and may even lead to its degradation. Therefore, it cannot be used for the detection of 3-hydroxy-L-proline.

[0005] Therefore, it is necessary to provide a new qualitative and quantitative detection method for 3-hydroxy-L-proline to solve the above problems. Summary of the Invention

[0006] The purpose of this invention is to provide a qualitative and quantitative detection method for 3-hydroxy-L-proline, which can achieve rapid identification and quantitative detection of 3-hydroxy-L-proline in solution, and can effectively distinguish 3-hydroxy-L-proline from its positional isomer 4-hydroxy-L-proline.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A qualitative and quantitative analysis method for 3-hydroxy-L-proline includes the following steps:

[0009] S1: Establish a concentration-absorbance standard curve for 3-hydroxy-L-proline;

[0010] S2: The test solution undergoes an oxidation reaction with hydrogen peroxide and copper sulfate under alkaline conditions to obtain an oxidized solution;

[0011] S3: The oxidizing solution is reacted with p-dimethylaminobenzaldehyde (p-DAB) to obtain a colorimetric solution. If the test solution contains 3-hydroxy-L-proline, the colorimetric solution will be purple-red.

[0012] S4: Measure the absorbance of the colorimetric solution at 560 nm, and obtain the concentration of 3-hydroxy-L-proline in the test solution according to the concentration-absorbance standard curve of 3-hydroxy-L-proline.

[0013] As a further improved technical solution of the present invention, step S1 includes: diluting 3-hydroxy-L-proline with water to prepare standard solutions of multiple concentrations, performing colorimetric reactions and measuring the absorbance at 560 nm, and establishing a concentration-absorbance standard curve of 3-hydroxy-L-proline based on the concentration and absorbance of the standard solutions.

[0014] As a further improvement to the present invention, step S2 includes:

[0015] S21: Add 50 mmol / L copper sulfate pentahydrate solution, 2.5 mmol / L sodium hydroxide solution and 3% hydrogen peroxide solution to the test solution in sequence to form an oxidation reaction system, mix well and let stand;

[0016] S22: The oxidation reaction system is heated in a water bath at 70°C and mixed to obtain an oxidation solution.

[0017] As a further improvement of the present invention, in step S21, the oxidation reaction system is sealed and then mixed.

[0018] In step S22, the oxidation reaction system is heated in a 70°C water bath under sealed conditions, and after mixing, it is heated in a 70°C water bath under unsealed conditions.

[0019] As a further improvement to the present invention, step S3 includes:

[0020] S31: Cool the oxidizing solution;

[0021] S32: Add 4 mmol / L sulfuric acid solution and 5% p-DAB ethanol solution to the cooled oxidized solution to form a colorimetric reaction system. After mixing, heat in a water bath at 70°C to complete the color development and obtain the colorimetric solution.

[0022] As a further improvement of the present invention, the volume ratio of the test solution, 50 mmol / L copper sulfate pentahydrate solution, 2.5 mmol / L sodium hydroxide solution and 3% hydrogen peroxide solution is 100:20:50:50.

[0023] As a further improvement of the present invention, the volume ratio of the test solution, the 4 mmol / L sulfuric acid solution and the 5% p-DAB ethanol solution is 100:80:250.

[0024] As a further improvement of the present invention, steps S2 and S3 are performed on a deep-hole plate, and step S4 uses a multi-channel photometer to measure absorbance.

[0025] Compared to existing technologies, the qualitative and quantitative analysis method for 3-hydroxy-L-proline of this invention offers the following advantages: Through a milder reaction mechanism and precise control of oxidation conditions, 3-hydroxy-L-proline undergoes a specific oxidative ring-opening reaction to generate an intermediate that reacts with p-DAB for color development, enabling rapid detection and quantification of 3-hydroxy-L-proline in solution. 4-hydroxy-proline does not undergo a similar reaction under these conditions, thus achieving specific detection. This method can be used in conjunction with deep-well plates and multi-channel spectrophotometers for large-scale detection, enabling rapid identification and preliminary quantification of 3-hydroxy-L-proline in solution. It can also be used for large-scale screening of proline hydroxylase mutant libraries. Attached Figure Description

[0026] Figure 1 This is a colorimetric diagram of 3-hydroxy-L-proline and 4-hydroxy-L-proline or proline in a specific embodiment of the present invention;

[0027] Figure 2 This is a schematic diagram of the concentration-absorbance standard curve of 3-hydroxy-L-proline in a specific embodiment of the present invention;

[0028] Figure 3 This is a schematic diagram illustrating the preliminary screening of a proline hydroxylase mutant library using a 96-well plate according to a specific embodiment of the present invention. Detailed Implementation

[0029] The exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. If several embodiments exist, features in these embodiments may be combined with each other without conflict. When the description refers to the drawings, unless otherwise stated, the same numbers in different drawings represent the same or similar elements. The descriptions in the following exemplary embodiments do not represent all embodiments consistent with the present invention; rather, they are merely examples of apparatuses, products, and / or methods consistent with some aspects of the present invention as set forth in the claims.

[0030] The terminology used in this invention is for the purpose of describing particular embodiments only and is not intended to limit the scope of protection of this invention. The singular forms “a,” “the,” or “the” as used in the specification and claims of this invention are also intended to include the plural forms unless the context clearly indicates otherwise.

[0031] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0032] 3-Hydroxy-L-proline is not directly encoded by the genetic code, but is produced from proline during post-translational modification under the action of prolyl hydroxylase. In some cases, if the reaction conditions are harsh (e.g., high temperature, strong acid), a small amount of the trans isomer may be converted to the cis isomer. 4-Hydroxy-L-proline can be rapidly detected using the chloramine-T labeling method, but this method is ineffective for proline hydroxylated at the 3-position because the reaction is too vigorous, causing the 3-hydroxy-L-proline to undergo ring-opening.

[0033] Based on this, the present invention uses a relatively mild reaction method that can effectively and rapidly detect 3-hydroxy-L-proline while avoiding a response to 4-hydroxy-proline, thus ensuring the specificity of the detection.

[0034] This invention discloses a qualitative and quantitative analysis method for 3-hydroxy-L-proline, comprising the following steps:

[0035] S1: Establish a concentration-absorbance standard curve for 3-hydroxy-L-proline;

[0036] S2: The test solution undergoes an oxidation reaction with hydrogen peroxide and copper sulfate under alkaline conditions to obtain an oxidized solution;

[0037] S3: The oxidizing solution is reacted with p-dimethylaminobenzaldehyde (p-DAB) to obtain a colorimetric solution. If the test solution contains 3-hydroxy-L-proline, the colorimetric solution will be purple-red.

[0038] S4: Measure the absorbance of the colorimetric solution at 560 nm, and obtain the concentration of 3-hydroxy-L-proline in the test solution according to the concentration-absorbance standard curve of 3-hydroxy-L-proline.

[0039] One of the key aspects of this invention lies in the precise control of the oxidation step. The steric hindrance and electronic effects of the C-3 hydroxyl group differ from those of the C-4 hydroxyl group, resulting in differences in their reactivity with the oxidizing agent. This invention utilizes a specific concentration of Cu... 2+ The weakly alkaline H₂O₂ system selectively oxidizes 3-hydroxy-proline to open its ring, generating an aldehyde intermediate, which then reacts with p-DAB to produce a characteristic color reaction (purple-red). 4-hydroxy-proline remains stable under these conditions and does not react with p-DAB, thus enabling specific recognition of the target analyte.

[0040] In this embodiment of the invention, a concentration-absorbance standard curve for 3-hydroxy-L-proline is first established. Then, the test solution is oxidized and colored through a mild reaction. Finally, its absorbance is measured, and the concentration can be obtained by referring to the concentration-absorbance standard curve. The operation is simple and fast, and the detection cost is low.

[0041] Step S1 includes: diluting 3-hydroxy-L-proline with pure water to prepare multiple standard solutions of different concentrations, performing colorimetric reactions and measuring the absorbance at 560 nm, and establishing a concentration-absorbance standard curve of 3-hydroxy-L-proline based on the concentration and absorbance of the standard solutions.

[0042] According to the Lambert-Beer Law, at a specific wavelength, the absorbance of a solution is directly proportional to its concentration within a certain range. By measuring the absorbance of a standard with a known concentration, a linear relationship curve between concentration and absorbance can be plotted. When measuring an unknown sample, simply measure its absorbance and substitute it into the formula of the standard curve to calculate its accurate concentration.

[0043] By establishing an effective standard curve, the optimal detection concentration range can be determined, thus avoiding the blind use of excessively high concentration solutions in subsequent experiments, which would otherwise lead to waste. Furthermore, once a stable and reliable standard curve is established, it is not necessary to bring a complete set of standards for each batch of samples in subsequent batch testing; only a few quality control points are needed for verification, which greatly improves the detection efficiency.

[0044] It is understandable that after preparing standard solutions of different concentrations, the standard solutions and the test solutions are subsequently subjected to oxidation and colorimetric reactions under the same conditions to ensure the accuracy of the test results.

[0045] Further, step S2 includes:

[0046] S21: Add 50 mmol / L copper sulfate pentahydrate (CuSO4·5H2O) solution, 2.5 mmol / L sodium hydroxide (NaOH) solution and 3% hydrogen peroxide (H2O2) solution to the test solution in sequence to form an oxidation reaction system, mix well and let stand;

[0047] S22: The oxidation reaction system is heated in a water bath at 70°C and mixed to obtain an oxidation solution.

[0048] The 2.5 mmol / L NaOH solution provides a weakly alkaline environment. At this pH, copper ions can form complexes and exist stably in the solution, avoiding the formation of copper hydroxide precipitate and deactivation under strongly alkaline conditions. Furthermore, the weakly alkaline conditions of this system result in less corrosiveness, making it safer for equipment and operators. 50 mmol / L Cu 2+ Both 3% and H2O2 are medium concentrations. This ratio ensures an effective oxidation reaction without the uncontrollable, excessively vigorous reaction seen in high-concentration systems, reducing the risk of ineffective decomposition and violent boiling of hydrogen peroxide. Copper sulfate, sodium hydroxide, and hydrogen peroxide are all common, basic chemicals that are inexpensive and readily available.

[0049] Further, step S3 includes:

[0050] S31: Cool the oxidizing solution;

[0051] S32: Add 4 mmol / L sulfuric acid (H2SO4) solution and 5% p-DAB ethanol solution to the cooled oxidizing solution to form a colorimetric reaction system. After mixing, heat in a water bath at 70°C to complete the color development and obtain the colorimetric solution.

[0052] In this embodiment, the reaction of the oxidizing solution with H₂SO₄ solution and p-DAB ethanol solution produces a dye molecule with a large conjugated system, exhibiting strong absorption in the visible light region. Even at very low concentrations in the test solution, it produces a very vivid color visible to the naked eye. p-DAB possesses relatively specific reaction characteristics, effectively indicating the presence of the target compound and reducing interference from other substances. H₂SO₄ can shift the reaction equilibrium towards the formation of the colored product. Furthermore, both H₂SO₄ and p-DAB are common, basic chemicals, inexpensive, and readily available, and the reaction procedure is simple.

[0053] The Lambert-Beer Law and standard curves hold true only when all other conditions (including temperature and pH) are identical, except for the concentration. If some standard solutions or test solutions are at higher temperatures and others at lower temperatures, their reaction rates will differ from the moment the colorimetric reagent is added. Even if all solutions are cooled to room temperature before measurement, the rapid reaction phase experienced by the hot solution in the initial stage may result in a deeper color, leading to a poorer linearity of the standard curve. This can cause different test results for samples of the same concentration, ultimately resulting in a loss of reliability and comparability of the entire experimental data.

[0054] Therefore, in this embodiment, all standard solutions and test solutions are cooled before the colorimetric reagent is added, and the colorimetric reaction only begins to accelerate when actively heated, ensuring that the initial and process conditions of the reaction are the same, thereby ensuring the accuracy of data measurement.

[0055] In some embodiments, in step S4, the absorbance is measured after the colorimetric solution has been cooled.

[0056] One of the prerequisites for the Lambert-Beer Law to hold is that the composition and properties of the solution are stable during the measurement process. Temperature changes directly affect the properties of the solution, thus affecting its actual concentration and potentially the completeness of the colorimetric reaction. Therefore, in this embodiment, all test solutions and standard solutions are cooled and stabilized at the same temperature after the colorimetric reaction is complete. This ensures that measurements and comparisons are performed on the same baseline, guaranteeing the comparability and accuracy of the established standard curve and the measured data for unknown samples.

[0057] In some embodiments, in step S21, the oxidation reaction system is sealed and then mixed; in step S22, the oxidation reaction system is heated in a 70°C water bath under a sealed state, mixed, and then heated in a 70°C water bath under an unsealed state; in step S32, the colorimetric reaction system is sealed and then mixed and heated in a 70°C water bath.

[0058] Thus, in the oxidation reaction process, creating a closed and stable reaction environment first prevents the loss of volatile reactants, ensures that the initial concentration of each component in the oxidation reaction system remains unchanged, allows the reaction to proceed fully as designed, avoids interference from gases such as oxygen and carbon dioxide in the air, reduces unnecessary side reactions, and accelerates the reaction rate and shortens the reaction time. After the reaction is complete, there may still be unreacted reagents in the system, such as hydrogen peroxide. The remaining hydrogen peroxide can interfere with the subsequent colorimetric reaction. Therefore, unsealing and heating can decompose the remaining hydrogen peroxide into water and oxygen and volatilize it to eliminate interference. The oxidation reaction may also generate volatile byproducts, which may inhibit the forward reaction. Therefore, unsealing and heating can remove these byproducts and promote a more complete oxidation reaction.

[0059] Colorimetric reactions typically occur within a specific volume. If heated in an open container, the solvent evaporates, reducing the solution volume and increasing the concentration of the test solution and the colorimetric product. This volume-induced concentration change can lead to a significantly higher measured absorbance value. If the standard solution and the test solution evaporate at different rates, the established standard curve will be incorrect, resulting in inaccurate concentration data for unknown samples. Furthermore, oxygen in the air may oxidize the colorimetric reagent or reaction products, causing the solution to fade or producing byproducts.

[0060] In some embodiments, the volume ratio of the test solution, 50 mmol / L CuSO4·5H2O solution, 2.5 mmol / L NaOH solution, and 3% H2O2 solution is 100:20:50:50.

[0061] In some implementations, the volume ratio of the test solution, the 4 mmol / L H2SO4 solution, and the 5% p-DAB ethanol solution is 100:80:250.

[0062] In some embodiments, steps S2 and S3 are performed on a deep-hole plate, and step S4 uses a multi-channel photometer to measure absorbance.

[0063] This embodiment achieves rapid detection and quantification of 3-hydroxy-L-proline in solution through a relatively mild reaction method, and can be combined with deep-well plates and multi-channel photometers to achieve large-scale detection, enabling rapid identification and preliminary quantification of 3-hydroxy-L-proline in solution.

[0064] The present invention will be further explained and illustrated below with examples. These examples are only used to illustrate the method and are not intended to limit the scope of application of the present invention.

[0065] Example 1

[0066] Establishment of a colorimetric reaction system.

[0067] Add 100 μL of the test solution (containing 3-hydroxy-L-proline, 4-hydroxy-L-proline, or proline) to a 1.5 mL EP tube, then add 20 μL of 50 mmol / L CuSO4·5H2O solution, 50 μL of 2.5 mmol / L NaOH solution, and 50 μL of 3% H2O2 solution in sequence. Seal the tube, mix well, and let stand for 5 min. Place the reaction system in a 70 °C water bath and heat for 5 min. Vortex the mixture again, then open the lid and continue heating for 5 min.

[0068] The reaction system was cooled on ice, and then 80 μL of 4 mmol / L H2SO4 solution and 250 μL of 5.0% p-DAB ethanol solution were added sequentially. After sealing and mixing, the mixture was heated in a 70℃ water bath for 5 min to complete the color development.

[0069] If the test solution contains 3-hydroxy-L-proline, a distinct purple-red color will appear, and the result can be quantified by measuring the absorbance at 560 nm.

[0070] like Figure 1As shown, the test solution on the left contains 3-hydroxy-L-proline, and the solution turns purple-red after color development; the test solution on the right shows no obvious color change (close to colorless), and contains only 4-hydroxy-L-proline or proline. This result proves that the method of the present invention has high selectivity for 3-hydroxy-L-proline, and can quantify 3-hydroxy-L-proline by further measuring the absorbance at 560 nm.

[0071] Example 2

[0072] Establish a concentration-absorbance standard curve for 3-hydroxy-L-proline.

[0073] 3-hydroxy-L-proline was diluted with pure water to different concentrations, and the colorimetric reaction was performed as in Example 1. The absorbance at 560 nm was measured and a concentration-absorbance standard curve was established.

[0074] As a control, parallel experiments were conducted using a 4-hydroxy-L-proline solution of the same concentration, and the absorbance values ​​were close to those of the blank control, further confirming the specificity of this method.

[0075] like Figure 2 As shown, when the concentration of 3-hydroxy-L-proline is higher than 1 mmol / L, its concentration is positively correlated with absorbance, which can be used as a basis for qualitative or preliminary quantification of 3-hydroxy-L-proline in the sample.

[0076] Example 3

[0077] Large-scale screening application of proline hydroxylase mutant libraries.

[0078] The supernatant of the reaction catalyzed by different proline hydroxylase mutants was aspirated into a 96-well plate. 20 μL of 50 mmol / L CuSO4·5H2O solution, 50 μL of 2.5 mmol / L NaOH solution, and 50 μL of 3% H2O2 solution were added sequentially. The plate was sealed with an aluminum film, vortexed, and allowed to stand for 5 min. The reaction system was then heated in a 70℃ water bath for 5 min, vortexed again, the aluminum film was removed, and heating continued for another 5 min.

[0079] The colorimetric reaction system was cooled on ice for 5 min, and then 80 μL of 4 mmol / L H2SO4 solution and 250 μL of 5.0% p-DAB ethanol solution were added sequentially. The deep well plate was then resealed with an aluminum film, mixed well, and heated in a 70℃ water bath for 5 min to obtain the colorimetric solution.

[0080] Pipette 100 μL of the colorimetric reaction solution into the corresponding well of the 96-well microplate and measure the absorbance at 560 nm.

[0081] like Figure 3As shown, the absorbance value is positively correlated with the concentration of 3-hydroxy-L-proline, which can be used as a basis for the improvement of enzyme activity.

[0082] In summary, the qualitative and quantitative analysis method for 3-hydroxy-L-proline of the present invention enables rapid detection and quantification of 3-hydroxy-L-proline in solution. This system is characterized by its rapid and batch operation capability, and can be used as a preliminary screening scheme for proline hydroxylase mutant libraries. Combined with deep-well plates and multi-channel spectrophotometers, it enables large-scale detection, achieving rapid identification and preliminary quantification of 3-hydroxy-L-proline in solution, greatly shortening the screening time of mutant libraries and reducing workload.

[0083] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. The understanding of this specification should be based on those skilled in the art. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still make modifications or equivalent substitutions to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A qualitative and quantitative analysis method for 3-hydroxy-L-proline, characterized in that, Includes the following steps: S1: Establish a concentration-absorbance standard curve for 3-hydroxy-L-proline; S2: The test solution undergoes an oxidation reaction with hydrogen peroxide and copper sulfate under alkaline conditions to obtain an oxidized solution; S3: The oxidizing solution is reacted with p-dimethylaminobenzaldehyde (p-DAB) to obtain a colorimetric solution. If the test solution contains 3-hydroxy-L-proline, the colorimetric solution will be purple-red. S4: Measure the absorbance of the colorimetric solution at 560 nm, and obtain the concentration of 3-hydroxy-L-proline in the test solution according to the concentration-absorbance standard curve of 3-hydroxy-L-proline.

2. The qualitative and quantitative analysis method for 3-hydroxy-L-proline according to claim 1, characterized in that, Step S1 includes: diluting 3-hydroxy-L-proline with water to prepare standard solutions of multiple concentrations, performing colorimetric reactions and measuring the absorbance at 560 nm, and establishing a concentration-absorbance standard curve of 3-hydroxy-L-proline based on the concentration and absorbance of the standard solutions.

3. The qualitative and quantitative analysis method for 3-hydroxy-L-proline according to claim 1, characterized in that, Step S2 includes: S21: Add 50 mmol / L copper sulfate pentahydrate solution, 2.5 mmol / L sodium hydroxide solution and 3% hydrogen peroxide solution to the test solution in sequence to form an oxidation reaction system, mix well and let stand; S22: The oxidation reaction system is heated in a water bath at 70°C and mixed to obtain an oxidation solution.

4. The qualitative and quantitative analysis method for 3-hydroxy-L-proline according to claim 3, characterized in that: In step S21, the oxidation reaction system is sealed and then mixed. In step S22, the oxidation reaction system is heated in a 70°C water bath under a sealed condition, and after mixing, it is heated in a 70°C water bath under an unsealed condition.

5. The qualitative and quantitative analysis method for 3-hydroxy-L-proline according to claim 1, characterized in that, Step S3 includes: S31: Cool the oxidizing solution; S32: Add 4 mmol / L sulfuric acid solution and 5% p-DAB ethanol solution to the cooled oxidized solution to form a colorimetric reaction system. After mixing, heat in a water bath at 70°C to complete the color development and obtain the colorimetric solution.

6. The qualitative and quantitative analysis method for 3-hydroxy-L-proline according to claim 3, characterized in that: The volume ratio of the test solution, 50 mmol / L copper sulfate pentahydrate solution, 2.5 mmol / L sodium hydroxide solution, and 3% hydrogen peroxide solution is 100:20:50:

50.

7. The qualitative and quantitative analysis method for 3-hydroxy-L-proline according to claim 5, characterized in that: The volume ratio of the test solution, 4 mmol / L sulfuric acid solution, and 5% p-DAB ethanol solution was 100:80:

250.

8. The qualitative and quantitative analysis method for 3-hydroxy-L-proline according to any one of claims 1 to 7, characterized in that: Steps S2 and S3 are performed on a deep well plate, and step S4 uses a multi-channel photometer to measure absorbance.

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