Method for detecting content of amino acid in sodium hyaluronate solution

Abstract

The invention relates to the technical field of amino acid detection, and particularly discloses a method for detecting the content of amino acid in a sodium hyaluronate solution, which is wide in application scene, reliable in determination result and easy to operate. The detection method comprises the following steps: preparing a plurality of standard solutions with different concentrations and containing glycine, proline, alanine, leucine, valine and lysine acetate to obtain a standard series of solutions, and deriving the standard series of solutions to obtain a standard solution to be detected; pretreating and deriving a sodium hyaluronate solution to be detected to obtain a sample to be detected; carrying out liquid chromatography detection on each amino acid in the standard to-be-detected solution and the to-be-detected sample by adopting a liquid chromatography, drawing a standard curve of each amino acid, and calculating a linear regression equation of each standard curve; and calculating the content of each amino acid according to the peak area of each amino acid in the sample and the corresponding linear regression equation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of amino acid detection, in particular to a method for detecting the content of amino acids in a sodium hyaluronate solution. BACKGROUND

[0002] Hyaluronic acid (HA) is an important component of human and animal skin, vitreous body, joint lubricating fluid and cartilage tissue, generally in the form of sodium salt, i.e. sodium hyaluronate (SH), with a molecular formula of (C 14 H 21 NO 11 ) n It is formed by the repeated connection of (1-β-4) D-glucuronic acid and (1-β-3) N-acetyl-D-glucosamine disaccharide units, and its polymer has a molecular weight ranging from 5000 to 200 million daltons in the body. It is considered to be a polysaccharide that fills space, stabilizes structure, coats cells and protects cells, and is widely used in repair surgery, eye surgery or as a cosmetic product filler. According to the use needs of consumers, sodium hyaluronate is often in the form of a composite solution, which not only includes cross-linked sodium hyaluronate, but also contains various amino acids. These amino acids are mainly used to maintain moisture when the sodium hyaluronate composite solution is used, in order to prolong the effect of the sodium hyaluronate. When the content of amino acids in the sodium hyaluronate composite solution is too high or too low, it will affect the effect of the sodium hyaluronate solution on the skin tissue. The determination of the content of amino acids in the sodium hyaluronate composite solution is of great significance for the quality control of the sodium hyaluronate composite solution.

[0003] At present, the industry mainly uses methods such as potentiometric titration, indole triketone colorimetry and derivatization high performance liquid chromatography to realize the detection of the content of amino acids in the solution. Among them, the potentiometric titration method is mainly used for the micro-determination of amino acids, and it is difficult to meet the content detection in the case of high content of amino acids, and the applicable scene is insufficient. When the indole triketone colorimetry method is used to determine the content of amino acids, the detection is unstable, and the standard curve obtained generally cannot reach R 2 <0.999, and the reliability of the detection result is poor. When the derivatization high performance liquid chromatography method is used to determine the content of amino acids, it needs to be extracted and treated, the steps are more, the operation is complex, the operation precision is not high, and the accuracy of the detection result is not high, thereby affecting the reliability of the detection result of the content of amino acids. SUMMARY

[0004] Therefore, it is necessary to provide a method for detecting the content of amino acids in a sodium hyaluronate solution, which has a wide applicable scene, reliable determination result and is easy to operate.

[0005] A method for detecting the content of amino acids in a sodium hyaluronate solution, comprising the following steps:

[0006] configuring a plurality of standard solutions with different concentrations and containing glycine, proline, alanine, leucine, valine, and lysine acetate to obtain a standard series of solutions, derivatizing the standard series of solutions to obtain standard test solutions;

[0007] pretreating and derivatizing the test sodium hyaluronate solution to obtain a test sample;

[0008] detecting each amino acid in the standard test solutions and the test sample by liquid chromatography, drawing a standard curve of each amino acid, and calculating a linear regression equation of each standard curve;

[0009] calculating the content of each amino acid according to the peak area of each amino acid in the sample and the corresponding linear regression equation.

[0010] In one of the embodiments, the configuration of the standard series of solutions comprises:

[0011] weighing and dissolving glycine, proline, alanine, leucine, valine, and lysine acetate to configure a mixed standard stock solution with a content of each amino acid of 1.0 mg / mL;

[0012] taking at least 5 portions of the mixed standard stock solution with different volumes, respectively diluting to obtain a plurality of standard solutions with different concentration ranges, thereby obtaining the standard series of solutions.

[0013] In one of the embodiments, the concentration range of each amino acid in each of the standard solutions is between 0.03 and 1 mg / mL.

[0014] In one of the embodiments, the derivatization of the standard series of solutions comprises:

[0015] taking an equal amount of the standard solutions with different concentration ranges and drying to obtain dried standards;

[0016] respectively adding a derivatization buffer and a derivatization reagent to each of the dried standards in sequence to obtain test derivatization solutions;

[0017] respectively placing each of the test derivatization solutions in a sealed container and heating for derivatization;

[0018] respectively adding an equilibration buffer to each of the derivatized solutions and filtering to obtain the standard test solutions.

[0019] In one of the embodiments, the pretreatment of the test sodium hyaluronate solution comprises:

[0020] taking an equal amount of the test sodium hyaluronate solutions and centrifuging each of the test sodium hyaluronate solutions, respectively, and taking the centrifugal filtrate of each of the test sodium hyaluronate solutions for standby use.

[0021] In one embodiment, the derivation of the sodium hyaluronate solution to be tested includes:

[0022] An equal amount of centrifugal filtrate of each sodium hyaluronate solution is taken and dried to obtain a dry sample;

[0023] The derivation buffer and the derivation reagent are sequentially added to each dry sample to obtain a sample to be derived;

[0024] Each sample to be derived is heated in a sealed container for derivation;

[0025] The equilibrium buffer is added to each derived sample and filtered to obtain a sample to be tested.

[0026] In one embodiment, the derivation buffer is a mixed solution of sodium bicarbonate and acetonitrile; the solute of the derivation reagent is fluorobenzene, and the solvent is acetonitrile; the equilibrium buffer is a mixed solution of potassium dihydrogen phosphate and sodium hydroxide, and the pH of the equilibrium buffer is 7.0.

[0027] In one embodiment, the liquid chromatography is used to detect each amino acid in the standard sample to be tested and the sample to be tested by liquid chromatography, draw a standard curve of each amino acid, and calculate the linear regression equation of each standard curve, which includes:

[0028] The liquid chromatography is set according to the preset conditions, and after the instrument is stabilized, the standard sample to be tested and the sample to be tested are sequentially injected into the chromatography for liquid chromatography detection to obtain the liquid chromatogram of each amino acid in the standard series solution and the liquid chromatogram of each amino acid in the sample;

[0029] According to the peak area-standard series solution of each amino acid content relationship of the standard series solution, the standard curve of each amino acid is drawn respectively, and the linear regression equation of each standard curve is obtained.

[0030] In one embodiment, the liquid chromatography conditions are as follows: the chromatography column for liquid chromatography uses Waters AccQ.Tag amino acid column, the mobile phase A is 0.5 mol / L sodium acetate, the mobile phase B is acetonitrile:water in a volume ratio of 3:2, the flow rate is 1.0 mL / min, the column temperature is 30℃, the ultraviolet detector temperature is 40℃, the injection amount is 10 uL, the retention time is 26 min, and the delay time is 5 min.

[0031] In one embodiment, the configuration of the mobile phase A includes: accurately weighing anhydrous sodium acetate and dissolving and filtering, sequentially adding N,N-dimethylformamide and glacial acetic acid and mixing, and the pH of the mobile phase A is 6.4;

[0032] The configuration of the mobile phase B includes: acetonitrile and water are mixed in a volume ratio of 3:2, and the water is injection water.

[0033] The detection method for the amino acid content in the sodium hyaluronate solution implementing the present application adopts a derivatizing agent to perform pre-column derivatization on the standard to-be-detected liquid and the to-be-detected sample respectively, so that the standard to-be-detected liquid and the to-be-detected sample have ultraviolet absorption, liquid chromatography is used to separate each amino acid in the standard to-be-detected liquid and the to-be-detected sample, detection is performed by an ultraviolet detector of the liquid chromatograph, a standard chromatographic curve of the standard to-be-detected liquid is drawn and a linear regression equation is calculated, and the peak area of each amino acid in the sample is quantitatively calculated according to the linear regression equation of the standard to-be-detected liquid, which is simple in operation, accurate and reliable in determination result, and can be applied to determination of the amino acid content in a solution with small content and determination of the amino acid content in a solution with large content, thereby expanding the applicable scenarios of the determination of the amino acid content. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The calibration curve of the amino acid concentration and the chromatographic peak area in the standard series solution in one embodiment of the present application;

[0035] Figure 2 The chromatogram of each amino acid in the sample in one embodiment of the present application;

[0036] Figure 3 The chromatogram of the blank control test in one embodiment of the present application. DETAILED DESCRIPTION

[0037] In order to make the above objectives, characteristics and advantages of the present application more apparent, specific embodiments of the present application are described in detail below with reference to the accompanying drawings. In the following description, a large number of specific details are set forth in order to fully understand the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the scope of the present application, so the present application is not limited to the specific embodiments disclosed below.

[0038] The present application discloses a detection method for the amino acid content in a sodium hyaluronate solution, which is widely applicable, reliable in determination result and easy to operate, and the detection method comprises the following steps:

[0039] A plurality of standard solutions with different concentrations and containing glycine, proline, alanine, leucine, valine and lysine acetate are configured to obtain a standard series solution, and the standard series solution is derivatized to obtain a standard to-be-detected liquid;

[0040] The to-be-detected sodium hyaluronate solution is pretreated and derivatized to obtain a to-be-detected sample;

[0041] Liquid chromatography is used to detect each amino acid in the standard to-be-detected liquid and the to-be-detected sample, a standard curve of each amino acid is drawn, and a linear regression equation of each standard curve is calculated;

[0042] According to the peak area of each amino acid in the sample and the corresponding linear regression equation, the content of each amino acid is calculated.

[0043] The above detection method uses a derivatizing agent to perform pre-column derivatization on the standard sample solution and the sample solution respectively, so that they have ultraviolet absorption. Each amino acid in the standard sample solution and the sample solution is separated by liquid chromatography, detected by the ultraviolet detector of the liquid chromatograph, a standard chromatogram curve of the standard sample solution is drawn, and a linear regression equation is calculated. According to the peak area of each amino acid in the sample and the linear regression equation of the standard sample solution, the quantitative calculation of each amino acid in the sample is realized. The operation is simple, the determination result is accurate and reliable, and it can be applied not only to the determination of small amount of amino acids in solution, but also to the determination of large amount of amino acids in solution, thereby expanding the applicable scenarios of the determination of amino acid content.

[0044] In each embodiment of the present application, injection water is used for dissolving and diluting each medicament. The injection water in the present embodiment is obtained by distillation of purified water with a residual salt content controlled below 0.1 mg / L and a conductivity of 0.1 μs / cm at a water temperature of 25℃.

[0045] In the present embodiment, the configuration of the standard series solution includes:

[0046] Glycine, proline, alanine, leucine, valine and lysine acetate are weighed and dissolved to configure a mixed standard stock solution with a content of each amino acid of 1.0 mg / mL, that is, the content of each amino acid in the mixed standard stock solution is the same. The weighing of each amino acid can use an electronic balance or other analytical balance with a precision of 0.001 g. In other embodiments, the content of glycine, proline, alanine, leucine, valine and lysine acetate in the mixed standard stock solution can also be different. After the mixed standard stock solution with different concentrations of each amino acid is configured, derivatized and detected by liquid chromatography, only the concentration of each amino acid in the standard solution needs to be converted.

[0047] Solutions of different concentrations are obtained by diluting the mixed standard stock solution of different volumes. In this embodiment, the mixed standard stock solution is prepared in advance and can be stored for a long time. The mixed standard stock solution of the same concentration is taken according to the requirement and diluted to obtain the standard solution of the preset concentration range. Then, the standard series solution composed of the standard solutions of different concentration ranges is detected by liquid chromatography, and the standard curve is drawn according to the chromatogram. After the sample to be detected is detected by liquid chromatography, the detection result is substituted into the linear regression equation corresponding to the appropriate standard curve for calculation, so as to improve the accuracy and reliability of the detection of the amino acid content in the sodium hyaluronate solution. Preferably, the concentration range of each amino acid in each standard solution in this embodiment is between 0.03 and 1 mg / mL, that is, the concentration range of each amino acid in the same standard solution is the same, and the concentration is between 0.03 mg / mL and 1 mg / mL.

[0048] Derivation of the standard series solution includes:

[0049] The standard solutions of different concentration ranges are taken in equal amounts and dried to obtain dry standards;

[0050] The derivation buffer and the derivation reagent are sequentially added to each dry standard to obtain a solution to be derived;

[0051] Each solution to be derived is heated in a sealed container for derivation;

[0052] The balanced buffer is added to each derived solution and filtered to obtain a standard test solution.

[0053] Further, the pretreatment of the sodium hyaluronate solution to be detected includes:

[0054] The sodium hyaluronate solutions to be detected are taken in equal amounts and centrifuged, and the centrifugal filtrate of each sodium hyaluronate solution is taken for standby use. In this embodiment, the sodium hyaluronate solution to be detected is centrifuged in an ultrafiltration centrifuge tube by using a high-speed centrifuge. Since the ultrafiltration membrane is arranged in the ultrafiltration centrifuge tube, the macromolecular substances in the sodium hyaluronate solution to be detected are intercepted on one side of the ultrafiltration membrane after the solution in the ultrafiltration centrifuge tube is centrifuged, so that the small molecular solution passing through the ultrafiltration membrane enters the lower layer of the ultrafiltration centrifuge tube and is used as a sample filtrate to be derived, so as to avoid the interference of the macromolecular substances in the sodium hyaluronate solution on the determination of the content of various amino acids.

[0055] In this embodiment, the derivation of the sodium hyaluronate solution to be detected includes:

[0056] The centrifugal filtrate of each sodium hyaluronate solution is taken in equal amounts and dried to obtain a dry sample;

[0057] The derivative buffer and the derivative reagent are sequentially added to each dry sample to obtain a sample to be derivatized;

[0058] Each sample to be derivatized is heated in a sealed container to obtain a sample after derivatization.

[0059] The equilibrium buffer is added to each sample after derivatization, and the sample is filtered to obtain a sample to be measured.

[0060] It should be noted that in this embodiment, the derivatization step of the sodium hyaluronate solution to be measured is the same as that of the standard series solution, and the derivative buffer, the derivative reagent, and the equilibrium buffer used are the same. Specifically, in this embodiment, the derivative buffer used by the sodium hyaluronate solution to be measured and the standard series solution is a mixed solution of sodium bicarbonate and acetonitrile. The solute of the derivative reagent is fluorobenzene, and the solvent is acetonitrile, that is, the derivative reagent is obtained by dissolving fluorobenzene in acetonitrile. The equilibrium buffer is a mixed solution of potassium dihydrogen phosphate and sodium hydroxide, and the pH of the equilibrium buffer is 7.0. In addition, during the derivatization of the sodium hyaluronate solution to be measured and the standard series solution, an electric heating incubator, an electric heating thermostat, or a constant temperature water bath is used for heating to promote the derivatization of the sodium hyaluronate solution to be measured and the standard series solution.

[0061] In this embodiment, liquid chromatography is used to detect each amino acid in the standard sample and the sample to be measured by liquid chromatography, draw a standard curve of each amino acid, and calculate the linear regression equation of each standard curve, including:

[0062] The liquid chromatograph is set according to the preset conditions, and after the instrument is stabilized, the standard sample and the sample to be measured are sequentially injected into the chromatograph for liquid chromatography detection to obtain the liquid chromatogram of each amino acid in the standard series solution and the liquid chromatogram of each amino acid in the sample.

[0063] According to the peak area-standard series solution of each amino acid content relationship of the standard series solution, the standard curve of each amino acid is drawn, and the linear regression equation of each standard curve is calculated.

[0064] In this embodiment, both the standard series solution and the sample solution use gradient elution, which sets two mobile phases, mobile phase A and mobile phase B. In a single analysis period (liquid chromatography detection of the standard series solution or liquid chromatography detection of the sample solution), the concentration ratio of the mobile phase A and the mobile phase B is constantly changed to a certain extent to change the polarity of the mobile phase, so that each eluted amino acid component has a suitable capacity factor k, and all components in the sample can be separated optimally in the shortest time.

[0065] Further, the liquid chromatography conditions are as follows: the liquid chromatography column is a Waters AccQ.Tag amino acid column, the mobile phase A is 0.5 mol / L sodium acetate, the mobile phase B is acetonitrile: water in a volume ratio of 3:2, the flow rate is 1.0 mL / min, the column temperature is 30°C, the ultraviolet detector temperature is 40°C, the injection amount is 10 uL, the retention time is 26 min, and the delay time is 5 min. In other embodiments, the liquid chromatography column can also be a C18 chromatographic column, which can remove hydrophobic compounds in the sample to avoid interference of the hydrophobic compounds in the sample with the detection of the content of each amino acid. The configuration of the mobile phase A includes: accurately weighing anhydrous sodium acetate and dissolving and filtering, sequentially adding N,N-dimethylformamide and glacial acetic acid and mixing, and the pH of the mobile phase A is 6.4; the configuration of the mobile phase B includes: mixing acetonitrile and water in a volume ratio of 3:2, and the water is injection water. In addition, in this embodiment, when the standard sample solution and the sample solution are injected into the chromatograph, a microsyringe or an automatic sampler can be used to suck the solution and inject it into the chromatograph to realize the injection of the chromatographic column.

[0066] In addition, in this embodiment, the peak area of the amino acid in the chromatograph is obtained by integrating the curve of the amino acid chromatographic detection spectrum, and the peak area is calculated by the area integral formula in calculus. The linear regression equation can be calculated by using the operation software equipped with the liquid chromatograph or manually calculated. The linear regression equation is obtained by linear regression according to the least square method according to the relationship between the concentration of the amino acid in the standard series solution and the corresponding amino acid chromatographic detection peak area. The linear regression equation can be calculated manually or by using the operation software equipped with the liquid chromatograph.

[0067] In this embodiment, after selecting appropriate chromatographic columns and mobile phases, the liquid chromatograph is opened, the chromatographic column of the liquid chromatograph is flushed, and after the chromatographic column reaches equilibrium and the baseline is flat, a microsyringe is used to first inject the standard series solution into the sample inlet of the liquid chromatograph. The standard series solution is introduced into the chromatographic column for separation, and when each amino acid component dissolved in the mobile phase passes through the stationary phase in the chromatographic column, the retention time of each amino acid component in the stationary phase is different due to the different sizes and strengths of the interaction (adsorption, distribution, ion attraction, exclusion, affinity) with the stationary phase, so that each amino acid component is eluted from the stationary phase in turn, and the eluted amino acid components flow into the flow cell of the ultraviolet detector in turn. The ultraviolet detector converts the concentration signal of the detected amino acid component into an electrical signal, which is amplified and recorded by a recorder to obtain a chromatogram. In this way, a standard curve can be drawn according to the chromatogram of each amino acid and its concentration relationship to obtain the concentration of each amino acid and the chromatographic peak area relationship, which is used as a standard for detecting the concentration of amino acids in the sample. After detection, the amino acid components and the eluent are discharged into the effluent collector. The liquid chromatography detection of the sample solution is the same as the liquid chromatography detection of the standard series solution, and the specific steps can be referred to the aforementioned liquid chromatography detection steps of the standard series solution.

[0068] It should be noted that each standard solution in the standard series of solutions was subjected to liquid chromatography detection after derivatization. The liquid chromatography detection process of each standard solution was the same as the liquid chromatography detection process of the above-mentioned standard series solutions. The aforementioned liquid chromatography detection process of the standard series solutions is used to overall illustrate how each standard solution is subjected to liquid chromatography detection. The liquid chromatography detection of various amino acids will not be described one by one here.

[0069] It should also be noted that when calculating the content of each amino acid in the sample solution, it can be calculated according to the following equation:

[0070] ρa / Sa=ρa / Sa, where ρa refers to the concentration of an amino acid in a standard solution of a certain concentration in the standard series, Sa refers to the peak area of ​​the chromatographic peak of the amino acid in the standard solution of that concentration after liquid chromatography, ρa refers to the concentration of the corresponding amino acid in the sample, and Sa refers to the peak area of ​​the chromatographic peak of the amino acid in the sample after liquid chromatography. The amino acid types in the standard solution of the standard series used in the same calculation formula are the same as the amino acid types in the sample. For example, when calculating the concentration of glycine in a sodium hyaluronate sample, ρGly / SGly=ρGly / SGly, where ρGly refers to the concentration of glycine in a standard solution of a certain concentration in the standard series, SGly refers to the peak area of ​​the chromatographic peak of glycine in the standard solution of that concentration after liquid chromatography, ρGly refers to the concentration of the corresponding glycine in the sample, and SGly refers to the peak area of ​​the chromatographic peak of glycine in the sample after liquid chromatography. The calculation of the concentrations of the remaining amino acids can refer to the above formula.

[0071] The following describes the specific process for detecting the amino acid content in the sodium hyaluronate solution of the present invention with reference to specific examples. It should be noted that in this example, to improve the reliability of the test method, while simultaneously determining the amino acid content in the sodium hyaluronate solution to be tested, a control experiment was also performed to verify the detection method of the present invention.

[0072] Prepare experimental equipment and reagents according to the following table

[0073]

[0074] Solution preparation:

[0075] Accurately weigh 200 g of sodium hydroxide using an electronic balance, dissolve it in 200 mL of water, cool it to room temperature, dilute it to 1000 mL with water and mix it to obtain a sodium hydroxide solution with a concentration of 5 mol / L.

[0076] Accurately pipette 20 mL of glacial acetic acid into a 100 mL volumetric flask, and dilute to the mark on the top of the flask with distilled water to obtain a 20% glacial acetic acid solution.

[0077] Accurately weigh 3.4 g of potassium dihydrogen phosphate, and add to 145.5 mL of 0.1 mol / L sodium hydroxide solution, and dilute to 500 mL with distilled water, mix well and filter to obtain a pH 7.0 buffer solution, which is stored in a brown bottle. The 0.1 mol / L sodium hydroxide solution is obtained by diluting a 5 mol / L sodium hydroxide solution.

[0078] Accurately weigh 2 g of fluorobenzene, and dissolve and dilute to 100 mL with acetonitrile, and shake well to obtain a 2% derivative reagent.

[0079] Accurately weigh 21.0 g of sodium bicarbonate, and dissolve in 470 mL of water, filter, and add the filtrate and 30 mL of acetonitrile to a 500 mL reagent flask to obtain a derivative buffer solution with a pH of 9.0.

[0080] Weigh 4.6 g of anhydrous sodium acetate, and dilute to 1000 mL in a volumetric flask, filter with a 0.45 um filter membrane to obtain about 800 mL of filtrate, transfer the filtrate to a 1000 mL volumetric flask, and add 100 mL of N,N-dimethylformamide, 20% glacial acetic acid (about 0.4 mL), and distilled water to 1000 mL, and ultrasonically dissolve for 30 min to mix well for use, to obtain mobile phase A with a pH of about 6.4 and a concentration of 0.05 mol / L.

[0081] Filter about 1000 mL of acetonitrile and distilled water, take 200 mL of filtered distilled water into a 1000 mL volumetric flask, then add 600 mL of filtered acetonitrile and shake well, and when the temperature is 25℃±2℃, dilute to 1000 mL with filtered distilled water, and ultrasonically dissolve for 30 min for use, to obtain mobile phase B with a volume ratio of acetonitrile to water of 3:2.

[0082] Preparation of standard solution:

[0083] Weigh 0.5 g of each of glycine, proline, alanine, leucine, valine, and acetic acid lysine, accurately count, and place each amino acid in the same 50 mL volumetric flask, dissolve and dilute to volume with pure water, shake well for use, to obtain a mixed standard stock solution with a concentration of 1.0 mg / mL for each amino acid to be tested. Commercially available mixed stock standard solution of amino acids can also be used.

[0084] Take 1.5 mL, 2.5 mL, 5.0 mL, 25 mL, 50 mL mixed standard stock solution respectively, and place them in different 50 ml volumetric flasks. Add pure water to each volumetric flask to make up the volume, shake well, and prepare for use. Five standard solutions with the content of each amino acid of 0.030 mg / mL, 0.050 mg / mL, 0.10 mg / mL, 0.50 mg / mL, and 1.0 mg / mL are obtained to obtain a standard series solution.

[0085] Standard solution derivation:

[0086] Take 100 ul of standard solution of different concentrations respectively (5 different concentrations are prepared here), and place them in 5 small test tubes. Dry them under vacuum, and accurately add 200 ul of derivatization buffer to each test tube. Mix the derivatization buffer with the dry material, add 200 ul of derivatization reagent, and mix well. Seal each test tube with a sealing film. Place all the sealed test tubes in an electric incubator or thermostat at 60°C for 30 min. After derivatization, take out the test tubes and air dry them to room temperature. Add 1600 ul of buffer equilibration solution to each test tube. Filter the solution in each test tube with a filter head with a pore size of 0.22 um. Collect the filtrate in the corresponding sample injection bottle for use.

[0087] Sample processing and derivatization:

[0088] Take 3 samples to be tested respectively, and push them into 10 kDa ultrafiltration centrifuge tubes. Centrifuge at 10000 r / min for 10 min. Take 100 ul of the lower layer filtrate of each sample into the corresponding small test tubes and dry them under vacuum. Accurately add 200 ul of derivatization buffer to each test tube. Mix the derivatization buffer with the dry material, add 200 ul of derivatization reagent, and mix well. Seal each test tube with a sealing film. Place all the sealed test tubes in an electric incubator or thermostat at 60°C for 30 min. After derivatization, take out the test tubes and air dry them to room temperature. Add 1600 ul of buffer equilibration solution to each test tube. Filter the solution in each test tube with a filter head with a pore size of 0.22 um. Collect the filtrate in the corresponding sample injection bottle for use.

[0089] Control sample processing and derivatization:

[0090] Specifically, take one sample to be tested, and completely transfer it to a 200 mL volumetric flask. Add 50 mL of mixed standard stock solution, and add pure water to make up the volume. Shake well and prepare for use. The control solution is obtained. The actual amount of each amino acid added in the control solution is 0.25 mg / ml. Take about 5 mL of the control solution and place it in an ultrafiltration centrifuge tube. Centrifuge at 10000 r / min for 30 min, and take the lower layer solution for use.

[0091] Take 100 ul of the above control solution and place it in a small test tube for vacuum drying. After drying, accurately add 200 ul of the derivatization buffer to the test tube, mix well, then add 200 ul of the derivatization reagent and mix well. Seal the test tube with a sealing film and place the sealed test tube in an electric heating air drying oven at 60°C for 30 minutes. After derivatization, take out the test tube and place it at room temperature. Then add 1600 ul of the buffer equilibration solution to the test tube, filter with a 0.22 um filter head, and pour into the control solution sample bottle as a control for future use.

[0092] In addition, experimental water is used instead of the sample, and is treated in the same way as the sample to serve as a blank control test. The experimental water is deionized water with a resistivity of ≥18 MΩ·cm at 25°C, obtained by filtering pure water with a vacuum filtration device.

[0093] Verification of detection method: accuracy (recovery rate / correction coefficient) test

[0094] The liquid chromatograph is set, and the chromatographic conditions include selecting an amino acid column (Waters AccQ.Tag), using 0.5 mol / L sodium acetate as the mobile phase A, using acetonitrile:water = 3:2 as the mobile phase B, a flow rate of 1.0 mL / min, a column temperature of 30°C, a detector temperature of 40°C, a sample injection amount of 10 uL, a retention time of 26 min, and a delay of 5 min.

[0095] In this embodiment, the gradient elution conditions for liquid chromatography detection of standard series solutions, sample solutions, and controls are shown in the following table:

[0096] Time (min) Flow rate (mL / min) Mobile phase A (%) Mobile phase B (%) / 1.00 84.0 16.0 1.00 1.00 84.0 16.0 4.00 1.00 75.0 25.0 8.00 1.00 75.0 25.0 9.00 1.00 62.0 38.0 15.00 1.00 62.0 38.0 16.00 1.00 35.0 65.0 21.00 1.00 35.0 65.0 22.00 1.00 0.0 100.0 26.00 1.00 84.0 16.0

[0097] After the liquid chromatograph is set and the instrument is stable, the standard sample solution, the sample solution, and the control are injected into the chromatograph in turn, with a sample injection amount of 10 uL. According to the peak area-standard series solution content relationship of each amino acid in the standard series solution, a standard curve for each amino acid is drawn, and a linear regression equation is calculated by the least squares method.

[0098] Result calculation:

[0099] According to the peak area of each amino acid in the sample solution, the content of each amino acid in the sample (mg / mL) is calculated by substituting it into the linear regression equation.

[0100] After the liquid chromatography is stable, under the chromatographic conditions of the liquid chromatography, 5 standard test solutions are injected into the chromatogram in sequence, with an injection volume of 10uL. According to the peak area of ​​each amino acid in the standard series solution and the content of each amino acid in the standard series solution, a linear regression equation of each amino acid is obtained by linear fitting through the origin. After experiments, the regression coefficient R of the linear regression equation of each amino acid is 2 All ≥0.999, calibration curve as Figure 1 shown.

[0101] Then, the sample solution and the reference solution were taken and injected into the chromatograph respectively. The injection volume was 10 μL. The peak area of ​​each amino acid was substituted into the linear regression equation of the amino acid in the standard solution. The content of each amino acid in the two solutions was calculated respectively. The content of each amino acid in the reference solution minus the content of the amino acid in the sample solution was the content of each amino acid detected. The recovery rate of each amino acid was obtained by dividing the calculated content of each amino acid actually added. The specific calculation formula is as follows (1):

[0102] Recovery rate α i =S i / S i0 ×100%(1)

[0103] Where: α i is the recovery rate of target i, unit %; S i is the detection value of target i, in mg; S i0 is the amount of target substance i added, in mg.

[0104] After testing and calculation, the recovery rate of each amino acid in the sample was between 90% and 108%, the separation of each chromatographic peak was greater than 1.8, and the tailing coefficient was less than 2.0. The chromatograms of each amino acid in the sample were as follows: Figure 2 shown.

[0105] At the same time, the blank control solution was injected into the chromatogram, and its chromatogram was as follows: Figure 3 Time (min) Flow rate (mL / min) Mobile phase A (%) Mobile phase B (%) As shown, the blank control test results showed that there was no interfering chromatographic peak.

[0106] Repeatability test

[0107] Set up multiple reference substances with the same concentration and perform tests. Based on the test results of the multiple reference substances, calculate the relative standard deviation (RSD) of the test results of the amino acid concentration in the sample calculated using each reference substance according to the following formulas (2) and (3).

[0108]

[0109]

[0110] Where S is the standard deviation, in mg;

[0111] x i Detection value of target i, unit: mg;

[0112] Addition amount of target i, unit: mg;

[0113] n is the parallel test number of the sample.

[0114] Through experiments, the repeatability standard deviation RSD of the results of multiple experiments is less than 3%, proving that the method is reliable.

[0115] Each technical feature of the above-described embodiments can be combined arbitrarily. In order to make the description simple, each technical feature in the above-described embodiments is not described in all possible combinations, however, as long as the combination of the technical features does not exist contradictory, it should be considered as the scope of the description.

[0116] The above-described embodiments only express several embodiments of the present application, the description is more specific and detailed, but it cannot be understood as the limitation of the scope of the patent. It should be pointed out that for ordinary skilled in the art, without departing from the concept of the present application, a number of variations and improvements can be made, which belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for detecting the content of an amino acid in a sodium hyaluronate solution, characterized by, The method comprises the following steps: configuring a plurality of standard solutions with different concentrations and containing glycine, proline, alanine, leucine, valine and lysine acetate to obtain a standard series solution, and deriving the standard series solution to obtain a standard test solution; pretreating and deriving a test sodium hyaluronate solution to obtain a test sample; detecting each amino acid in the standard test solution and the test sample by liquid chromatography, drawing a standard curve of each amino acid, and calculating a linear regression equation of each standard curve; calculating the content of each amino acid according to the peak area of each amino acid in the sample and the corresponding linear regression equation.

2. The method for detecting the content of amino acids in a sodium hyaluronate solution according to claim 1, characterized by, The configuration of the standard series solution comprises: weighing and dissolving glycine, proline, alanine, leucine, valine and lysine acetate to configure a mixed standard stock solution with a content of each amino acid of 1.0 mg / mL; pumping at least 5 portions of the mixed standard stock solution with different volumes and diluting them respectively to obtain a plurality of standard solutions with different concentration ranges, thereby obtaining the standard series solution.

3. The method for detecting the content of amino acids in a sodium hyaluronate solution according to claim 2, characterized by, The concentration of each amino acid in each standard solution ranges from 0.03 mg / mL to 1 mg / mL.

4. The method for detecting the content of amino acids in a sodium hyaluronate solution according to claim 2, characterized by, The derivation of the standard series solution comprises: pumping an equal amount of the standard solutions with different concentration ranges and drying them to obtain dried standards; sequentially adding a derivation buffer and a derivation reagent to each dried standard to obtain a to-be-derived solution; heating each to-be-derived solution in a sealed container to derive it; adding an equilibrium buffer to each derived solution and filtering it to obtain the standard test solution.

5. The method for detecting the content of amino acids in a sodium hyaluronate solution according to claim 4, characterized by, The pretreatment of the test sodium hyaluronate solution comprises: pumping an equal amount of a plurality of test sodium hyaluronate solutions and centrifuging them respectively, and taking the centrifugal filtrate of each sodium hyaluronate solution for standby use.

6. The method for detecting the content of amino acids in a sodium hyaluronate solution according to claim 5, characterized by, The derivation of the test sodium hyaluronate solution comprises: pumping an equal amount of the centrifugal filtrate of each sodium hyaluronate solution and drying it to obtain a dried sample; sequentially adding a derivation buffer and a derivation reagent to each dried sample to obtain a to-be-derived sample; heating each to-be-derived sample in a sealed container to derive it; adding an equilibrium buffer to each derived sample and filtering it to obtain the test sample.

7. The method for detecting the content of amino acids in a sodium hyaluronate solution according to any one of claims 4 to 6, characterized in that, The derivation buffer is a mixed solution of sodium bicarbonate and acetonitrile; the derivation reagent has fluorobenzene as a solute and acetonitrile as a solvent; the equilibrium buffer is a mixed solution of potassium dihydrogen phosphate and sodium hydroxide, and the pH of the equilibrium buffer is 7.

0.

8. The method of claim 1, wherein the sodium hyaluronate solution is a pharmaceutical composition for injection. The detection of each amino acid in the standard test solution and the test sample by liquid chromatography, the drawing of a standard curve of each amino acid, and the calculation of a linear regression equation of each standard curve comprise: setting the liquid chromatograph according to preset conditions, injecting the standard test solution and the test sample into the chromatograph in sequence for liquid chromatography detection after the instrument is stabilized, and obtaining the liquid chromatogram of each amino acid in the standard series solution and the liquid chromatogram of each amino acid in the sample; drawing a standard curve of each amino acid according to the peak area-standard content relationship of each amino acid in the standard series solution, and calculating a linear regression equation of each standard curve.

9. The method for detecting the content of amino acids in a sodium hyaluronate solution according to claim 8, characterized by, The liquid chromatography conditions are as follows: the liquid chromatography column is Waters AccQ.Tag amino acid column, the mobile phase A is 0.5 mol / L sodium acetate, the mobile phase B is acetonitrile:water in a volume ratio of 3:2, the flow rate is 1.0 mL / min, the column temperature is 30 DEG C, the ultraviolet detector temperature is 40 DEG C, the injection amount is 10 uL, the retention is 26 min, and the delay is 5 min.

10. The method for detecting the content of amino acids in a sodium hyaluronate solution according to claim 9, characterized by, The configuration of the mobile phase A comprises: accurately weighing anhydrous sodium acetate and dissolving and filtering, sequentially adding N,N-dimethylformamide and glacial acetic acid and mixing, and the PH of the mobile phase A is 6.4; The configuration of the mobile phase B comprises: mixing acetonitrile and water in a volume ratio of 3:2, and the water is injection water.

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