Method for detecting contents of L-carnosine and three amino acids in sodium hyaluronate composite solution

Abstract

The invention provides the method for determining the contents of the L-carnosine and the three amino acids in the sodium hyaluronate composite solution, and the method has the advantages of strong specificity, high sensitivity, good linear relationship, good precision, high accuracy, high solution stability, good durability and accurate and reliable determination result; and the quality, effectiveness and safety of related medical beauty products can be effectively controlled.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a method for detecting the content of L-carnosine and three amino acids in a sodium hyaluronate composite solution. Background Technology

[0002] Sodium hyaluronate is a widely used biomaterial in the field of medical aesthetics, possessing excellent water-locking ability and biocompatibility. When injected into the dermis, it effectively moisturizes, plumps tissue, and promotes collagen regeneration. Injectable sodium hyaluronate complex solutions, such as the Hyaluronic Acid Complex developed by Aimeike Technology Development Co., Ltd., and the Frozen Beauty product developed by Meiyan Space Biotechnology (Jilin) ​​Co., Ltd., often combine other active ingredients with sodium hyaluronate to enhance efficacy. These products frequently contain small-molecule active nutrients such as L-carnosine, glycine, alanine, and proline, which are crucial to the final performance of the product.

[0003] To ensure the quality, safety, and efficacy of such composite solution products, it is crucial to establish accurate and reliable content determination methods to monitor the content of key active ingredients (such as L-carnosine and amino acids). However, due to the similarity in physicochemical properties of these components (glycine, alanine, proline, and L-carnosine) and their co-existence within the complex sodium hyaluronate gel matrix, achieving efficient separation and accurate quantification of these four components presents technical challenges.

[0004] A search revealed a lack of existing high-performance liquid chromatography (HPLC) methods capable of simultaneously, rapidly, and accurately detecting the levels of glycine, alanine, proline, and L-carnosine in sodium hyaluronate composite solutions. Therefore, developing a specific, sensitive, precise, and accurate HPLC method is essential and urgent for effectively controlling the quality of related drug-device combination medical devices and ensuring their quality control. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for detecting the content of L-carnosine and three amino acids in sodium hyaluronate composite solution. This method has high specificity, high sensitivity, good linearity, good precision, high accuracy, high solution stability, good durability, and accurate and reliable measurement results.

[0006] To achieve the above-mentioned objective, this invention provides a method for detecting the content of L-carnosine and three amino acids in a sodium hyaluronate composite solution, wherein the three amino acids are glycine, alanine, and proline. The method is characterized by using high-performance liquid chromatography (HPLC) for determination. The chromatographic conditions include: a reversed-phase column packed with octadecylsilane-bonded silica gel; a mobile phase consisting of mobile phase A and mobile phase B, wherein mobile phase A is a methanol-acetonitrile mixture and mobile phase B is an acetic acid-sodium acetate buffer solution.

[0007] In some embodiments, the chromatographic column has the following specifications: 4.6 × 250 mm, 5 µm; preferably, a MORHCHEM Caprisil C18-AQ or Kromasil C18 column is selected.

[0008] In some embodiments, the volume ratio of methanol to acetonitrile in mobile phase A is 1:1.

[0009] In some embodiments, the concentration of sodium acetate in mobile phase B is 0.05~0.2 mol / L; preferably, the concentration of sodium acetate is 0.1 mol / L.

[0010] In some embodiments, the pH of mobile phase B is 5.5 to 6.5, preferably 6.0.

[0011] In some embodiments, gradient elution is used in the mobile phase.

[0012] In some embodiments, the gradient elution conditions are as follows:

[0013] Isocratic elution was performed from 0 to 25 min, with the volume fraction of mobile phase B in the mobile phase >85%.

[0014] Gradient elution is performed for 25-30 minutes, eluting until the volume fraction of mobile phase B in the mobile phase is not less than 15%.

[0015] Isocratic elution was performed for 30-40 minutes.

[0016] Gradient elution was performed for 40-42 minutes until the volume fraction of mobile phase B in the mobile phase was >85%.

[0017] Isocratic elution was performed for 42-55 minutes.

[0018] In some embodiments, the gradient elution conditions are as follows:

[0019] Time (min) Mobile phase A (%) Mobile phase B (%) 0 13 87 25 13 87 30 80 20 40 80 20 42 13 87 55 13 87

[0020] In some embodiments, the flow rate is 0.8 ml / min to 1.2 ml / min; more preferably, the flow rate is 0.9 ml / min to 1.1 ml / min; and even more preferably, the flow rate is 1.0 mL / min.

[0021] In some embodiments, the temperature is 30-40°C; more preferably, it is 32-38°C, and most preferably, it is 35°C.

[0022] In some embodiments, the injection volume is 2~10 μl; more preferably, 4~6 μl, and most preferably 5 μl.

[0023] In some embodiments, the detector is an ultraviolet detector; the detection wavelength is 250~260nm, preferably 254nm.

[0024] In some embodiments, the specific steps of the detection method are as follows: prepare blank solution, reference solution and test solution, inject blank solution, reference solution and test solution into the sample respectively, detect according to liquid chromatography conditions, record the chromatogram and calculate the content of L-carnosine, glycine, alanine and proline in the test solution by peak area according to the external standard method.

[0025] In some embodiments, the preparation of the test solution includes: reacting the sodium hyaluronate composite solution to be tested sequentially with triethylamine and phenyl isothiocyanate for derivatization, then adding n-hexane for extraction, and mixing the lower layer solution with water to obtain the test solution.

[0026] Preferably, the preparation of the test solution includes adding hydrochloric acid solution before adding the derivatizing reagents triethylamine and phenyl isothiocyanate, so that the sodium hyaluronate composite solution can be better dispersed in the system.

[0027] More preferably, in the preparation of the test solution, the concentration of hydrochloric acid is 0.1 mol / L, and the volumes of hydrochloric acid, 14% triethylamine acetonitrile solution, and 0.1 mol / L phenyl isothiocyanate acetonitrile solution added are equal.

[0028] In some embodiments, the derivatization reaction is carried out in a dark room at room temperature for a reaction time of 0.5 to 1.5 hours, preferably 1 hour.

[0029] In some embodiments, the volume ratio of the sodium hyaluronate composite solution to be tested to the n-hexane is 1:3, and the volume ratio of the lower layer solution to water is 1:4.

[0030] In some embodiments, the preparation of the reference solution includes: accurately weighing glycine, L-carnosine, alanine and proline reference standards, adding water to prepare a mixed reference stock solution, and then taking an appropriate amount of the mixed reference stock solution and processing it according to the same derivatization process as the test solution preparation method to obtain the solution.

[0031] The present invention also provides an application of the detection method described above for analyzing the content of L-carnosine and three amino acids in a sodium hyaluronate composite solution.

[0032] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0033] Compared with the prior art, the present invention has the following beneficial technical effects:

[0034] This invention establishes a method for detecting the content of L-carnosine and three amino acids in sodium hyaluronate composite solution. The resolution of two adjacent chromatographic peaks is greater than 1.5, indicating that the detection method provided by this invention can achieve baseline separation of L-carnosine and the three amino acids.

[0035] The detection method provided by this invention can simultaneously, rapidly, and accurately detect the content of four components—glycine, alanine, proline, and L-carnosine—in a sodium hyaluronate composite solution. Furthermore, this method exhibits high specificity, high sensitivity, good linearity, good precision, high accuracy, high solution stability, and good durability. The measurement results are accurate and reliable, providing a strong guarantee for effectively controlling the quality, efficacy, and safety of related medical aesthetic products, and laying a solid foundation for establishing quality control standards for related products. It also has broad prospects for industrialization and promotion. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0037] Figure 1 This is the chromatogram of the blank solution in Experimental Example 1 of the present invention;

[0038] Figure 2 This is the chromatogram of the blank preparation solution of the test sample in Experimental Example 1 of the present invention;

[0039] Figure 3 This is the chromatogram of the reference solution in Experimental Example 1 of the present invention;

[0040] Figure 4 This is the chromatogram of the test solution in Experimental Example 1 of the present invention;

[0041] Figure 5 This is the chromatogram of the test solution in Example 2 of the present invention;

[0042] Figure 6 This is the chromatogram of the reference solution in Comparative Example 2 of the present invention;

[0043] Figure 7 This is the chromatogram of the blank solution in Comparative Example 3 of the present invention;

[0044] Figure 8 This is a chromatogram of the reference solution in Comparative Example 3 of the present invention. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0046] The reagents and raw materials used in the following examples are all commercially available. Information on the main reagents and reference standards is shown in Table 1.

[0047] Table 1

[0048] name Grade / Purity (%) Anhydrous sodium acetate Analytical level Triethylamine Chromatographic grade phenyl isothiocyanate Chromatographic grade n-Hexane Chromatographic grade glycine 100.0% alanine 99.9% L-Carnosine 99.8% proline 100.0%

[0049] The following examples use an Agilent high-performance liquid chromatograph for detection and a WIGGENS Vortex 3000Elite vortex mixer for vortex dispersion of the test solution.

[0050] The test sample mentioned in the embodiments of the present invention is a sodium hyaluronate composite solution prepared by the method disclosed in Example 5 of patent CN117357698A, batch number: 20230406, and its composition and ratio are shown in Table 2.

[0051] Table 2

[0052] Components Mass-to-volume ratio (m / v) Sodium hyaluronate 5mg / ml L-Carnosine 2mg / ml glycine 0.1mg / ml alanine 0.1mg / ml proline 0.2mg / ml <![CDATA[Vitamin B2]]> 0.005mg / ml Phosphate buffer <![CDATA[3 mg / ml of NaH2PO4•H2O and 11 mg / ml of Na2HPO4•12H2O]]>

[0053] In order to better monitor the content of L-carnosine and the three amino acids in the sodium hyaluronate complex solution, the content of L-carnosine and the three amino acids in this product was determined to better monitor the quality of this product.

[0054] Example 1

[0055] 1. Chromatographic conditions

[0056] ① Chromatographic column: The column is a water-resistant reversed-phase chromatography column MORHCHEMCaprisil C18-AQ (4.6×250mm, 5µm or equivalent performance) with octadecylsilane-bonded silica gel as the packing material;

[0057] ②Mobile phase: Mobile phase A: methanol-acetonitrile (50:50); Mobile phase B: 0.1 mol / L sodium acetate (adjust pH to 6.0 with acetic acid);

[0058] ③ Gradient elution:

[0059] Time (min) Mobile phase A (%) Mobile phase B (%) 0 13 87 25 13 87 30 80 20 40 80 20 42 13 87 55 13 87

[0060] ④ Detector: Ultraviolet detector;

[0061] ⑤ Detection wavelength: 254nm;

[0062] ⑥ Column temperature: 35℃;

[0063] ⑦ Flow rate: 1.0 ml / min;

[0064] ⑧ Injection volume: 5µl.

[0065] 2. Solution preparation

[0066] ① Non-derivative reference solution

[0067] Reference stock solution: Weigh 25 mg of glycine reference standard, 500 mg of L-carnosine reference standard, 25 mg of alanine reference standard and 50 mg of proline reference standard respectively, accurately weigh them, place them in a 25 ml volumetric flask, add an appropriate amount of water, dissolve and dilute to the mark, shake well, and the solution is ready.

[0068] Underrived reference solution: Accurately measure 2.5 ml of the reference stock solution, place it in a 25 ml volumetric flask, dilute with water to the mark, and shake well to obtain the solution;

[0069] ② Test solution

[0070] Accurately measure 0.5 ml of the test solution and place it in a 10 ml centrifuge tube. Add 0.5 ml of 0.1 mol / L hydrochloric acid and vortex to mix thoroughly. Add 0.5 ml of 14% triethylamine acetonitrile solution and vortex to mix thoroughly. Then add 0.5 ml of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and vortex to mix thoroughly. Place the above solution in a dark room at room temperature for 1 hour. After removing it, add 1.5 ml of n-hexane, vortex for about 30 seconds, and allow it to separate into layers. Accurately measure 0.5 ml of the lower layer and mix it with 2 ml of water.

[0071] ③Blank solution

[0072] Accurately measure 0.5 ml of water and complete the blank solution derivatization according to step ② of the sample derivatization process;

[0073] ④ Reference solution

[0074] Accurately measure 0.5 ml of the underrivatized reference solution and derivatize the reference solution according to step ② of the test sample derivatization process.

[0075] 3. Measurement Method

[0076] Inject the above-mentioned derivatized solution under the specified chromatographic conditions, record the chromatogram, and calculate the content of each component by peak area using the external standard method.

[0077] Effect verification

[0078] The method described in Example 1 was validated according to the "Guidelines for Validation of Analytical Methods 9101" in the 2020 edition of the Chinese Pharmacopoeia, including specificity, limit of quantitation, linearity and range, precision, accuracy, solution stability, and robustness. The validation process and results of the method of this invention are shown in Experiments 1-7.

[0079] Experiment Example 1: Specificity and System Suitability

[0080] The study investigated baseline interference at the peak positions of each component using blank solution and blank formulation solution, determined the retention time of each component using positioning solution, and assessed the system suitability using six consecutive injections of reference solution.

[0081] 1. Solution preparation

[0082] Verification Sample Preparation method Reference stock solution Prepare according to the method Reference solution (system suitability solution) Prepare according to the method and complete the derivation process. Positioning solution Glycine: Accurately weigh approximately 5 mg of the reference standard, place it in a 50 ml volumetric flask, add an appropriate amount of water, dissolve and dilute to the mark, and shake well; L-Carnosine: Accurately weigh approximately 100 mg of the reference standard, place it in a 50 ml volumetric flask, add an appropriate amount of water, dissolve and dilute to the mark, and shake well; Alanine: Accurately weigh 5 mg of the reference standard, place it in a 50 ml volumetric flask, add an appropriate amount of water, dissolve and dilute to the mark, and shake well; Proline: Accurately weigh 10 mg of the reference standard, place it in a 50 ml volumetric flask, add an appropriate amount of water, dissolve and dilute to the mark, and shake well; Accurately measure 0.5 ml of each of the above solutions to complete the derivatization process. blank solution Precisely measure 0.5 ml of water to complete the derivatization process. Blank reagent solution for test sample <![CDATA[Precisely measure 0.5 ml of the blank preparation of the test substance (containing only sodium hyaluronate, vitamin B2, and phosphate buffer solution), and complete the derivatization process]]> Test solution Accurately measure 0.5 ml of the test sample to complete the derivatization process.

[0083] 2. Results of the investigation

[0084] The chromatogram of the blank solution is shown below. Figure 1 The chromatogram of the blank preparation solution of the test sample is shown in [reference needed]. Figure 2 The chromatogram of the reference solution is shown below. Figure 3 The chromatogram of the test solution is shown in [reference needed]. Figure 4 The results are shown in Table 3.

[0085] Table 3 Results of the Exclusivity and System Applicability Assessment

[0086] name Glycine retention time (min) L-Carnosine retention time (min) Alanine retention time (min) Proline retention time (min) blank solution / / / / Reference solution 11.01 13.90 17.95 20.25 RSD (%) of peak area after 6 consecutive injections of the reference standard 0.09 0.05 0.03 0.04 Blank reagent solution for test sample / / / / Test solution 11.01 13.91 17.96 20.26 glycine positioning solution 11.01 / / / L-Carnosine Localization Solution / 13.93 / / Alanine positioning solution / / 17.97 / Proline positioning solution / / / 20.25

[0087] Conclusion: Under the selected chromatographic conditions, neither the blank solution nor the blank preparation solution of the test sample showed chromatographic peaks with retention times consistent with the reference solution; the resolution of each component peak was greater than 1.5, meeting the standard requirements; in the chromatogram of the test sample solution, there were no other peaks interfering with detection, and the RSD of the peak areas of glycine, alanine, proline, and L-carnosine in the system suitability study was ≤0.1%, meeting the standard requirements. The specificity of this method meets the detection requirements, indicating that the high-performance liquid chromatography method provided by this invention for determining the content of L-carnosine and the three amino acids in sodium hyaluronate composite solution has good specificity.

[0088] Experimental Example 2 Limit of Quantitation

[0089] The limit of quantitation solution was injected six times consecutively for evaluation.

[0090] 1. Preparation method of limit of quantitation solution

[0091] Verification Sample Preparation method Quantitative limit solution Prepare 1 mg / ml stock solutions of reference standards, and measure 0.5 ml of each solution into a 10 ml volumetric flask. Perform derivatization according to the derivatization method, dilute with water to the mark, and shake well. Measure 0.1 ml of glycine, alanine, and proline into a 250 ml volumetric flask and dilute with water to the mark; measure 0.1 ml of L-carnosine into a 25 ml volumetric flask and dilute with water to the mark.

[0092] 2. Results of the investigation

[0093] The results of the limit of quantitation study are shown in Table 4.

[0094] Table 4 Results of Limit of Quantitation Study

[0095]

[0096] Conclusion: Six consecutive injections of the solution yielded peak area RSDs meeting the standard requirements. The limit of quantitation (LOQ) concentrations for glycine, L-carnosine, alanine, and proline were 0.019%, 0.011%, 0.018%, and 0.010% of the sample solution concentration, respectively, meeting the detection requirements. This indicates that the high-performance liquid chromatography (HPLC) method provided by this invention for determining the content of L-carnosine and the three amino acids in sodium hyaluronate composite solution has high sensitivity.

[0097] Experiment Example 3: Linearity and Range

[0098] 1. Linear and Range Solution Preparation

[0099] Verification Sample Preparation method Reference stock solution Use the reference stock solution under the specificity and system suitability section. blank solution Precisely measure 0.5 ml of water to complete the derivatization process. L-1（20%） Accurately measure 1 ml of the reference standard stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with water and mix well. Accurately measure 0.5 ml to complete the derivatization process. L-2（50%） Accurately measure 2.5 ml of the reference standard stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with water and mix well. Accurately measure 0.5 ml to complete the derivatization process. L-3（100%） Accurately measure 5 ml of the reference standard stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with water and mix well. Accurately measure 0.5 ml to complete the derivatization process. L-4（120%） Accurately measure 6 ml of the reference standard stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with water and mix well. Accurately measure 0.5 ml to complete the derivatization process. L-5（150%） Accurately measure 7.5 ml of the reference standard stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with water and mix well. Accurately measure 0.5 ml to complete the derivatization process.

[0100] 2. Results of the investigation

[0101] The results of the linearity and range tests are shown in Table 5.

[0102] Table 5 Results of Linearity and Range Examination

[0103]

[0104] Conclusion: Under the conditions of this detection method, glycine concentration and peak area showed a linear relationship in the range of 2.117 μg / ml to 15.876 μg / ml, and the linear relationship was good. The linear equation was y = 21.197 x – 2.065, the correlation coefficient r was 0.9999, and the absolute value of the intercept was 0.92% of the main peak area of ​​the 100% linear solution.

[0105] In the range of 40.157 μg / ml to 301.176 μg / ml, L-carnosine showed a linear relationship between concentration and peak area, and the linear relationship was good. The linear equation was y = 6.979x - 5.3383, the correlation coefficient r was 0.9999, and the absolute value of the intercept was 0.38% of the main peak area of ​​the 100% linear solution.

[0106] Within the range of 1.986 μg / ml to 14.898 μg / ml, the concentration of alanine showed a linear relationship with the peak area, and the linear relationship was good. The linear equation was y = 17.433x - 2.4903, the correlation coefficient r was 0.9999, and the absolute value of the intercept was 1.45% of the main peak area of ​​the 100% linear solution.

[0107] Within the range of 4.094 μg / ml to 30.708 μg / ml, proline concentration showed a linear relationship with peak area, and the linearity was good. The linear equation was y = 15.202x + 2.3357, the correlation coefficient r was 0.9999, and the absolute value of the intercept was 0.74% of the main peak area of ​​the 100% linear solution.

[0108] The verification results all met the standard requirements.

[0109] Experimental Example 4 Precision

[0110] Repeatability

[0111] The same sample was tested 6 times to examine the RSD of amino acid and carnosine content.

[0112] 1. Preparation of reproducible solutions

[0113] Verification Sample Preparation method Reference stock solution Prepare according to the method. Reference solution Prepare according to the method and complete the derivation process. blank solution Precisely measure 0.5 ml of water to complete the derivatization process. Test solution Prepare according to the method and complete the derivation process.

[0114] 2. Results of the investigation

[0115] The results of the repeatability test are shown in Table 6.

[0116] Table 6 Results of Repeatability Testing

[0117] Serial Number glycine L-Carnosine alanine proline 1 107.0 96.5 105.2 103.1 2 107.3 96.7 105.7 103.5 3 107.3 96.6 105.3 103.6 4 107.2 96.5 105.1 103.7 5 107.5 96.7 105.5 103.7 6 107.5 96.8 105.4 104.0 Average content (%) 107.3 96.6 105.4 103.6 RSD (%) 0.17 0.14 0.21 0.29

[0118] Conclusion: Using the external standard method to calculate the content based on peak area, under the conditions of this method, the RSD of glycine content in six parallel samples was 0.17%, L-carnosine content was 0.14%, alanine content was 0.21%, and proline content was 0.29%, which met the standard requirements, indicating that the method has good repeatability.

[0119] intermediate precision

[0120] For different times and different people, follow the same procedure for repetitive items.

[0121] 1. Results of the investigation

[0122] The results of the intermediate precision test are shown in Table 7.

[0123] Table 7. Results of Intermediate Precision Testing (Part 1)

[0124] Serial Number glycine L-Carnosine alanine proline 1 108.2 95.8 101.4 101.0 2 107.7 95.3 99.4 100.8 3 108.6 96.1 102.3 102.1 4 109.8 97.3 102.9 102.2 5 107.5 95.6 98.7 101.7 6 107.6 95.3 100.6 100.7 Average content (%) 108.2 95.9 100.9 101.4 RSD (%) 0.80 0.79 1.61 0.66

[0125] Table 7 Results of Intermediate Precision Testing (Part 2)

[0126] name RSD (%) (n=12) glycine 0.72 L-Carnosine 0.67 alanine 2.50 proline 1.20

[0127] Conclusion: Using the external standard method with peak area calculations at different times and by different personnel, the RSDs for glycine content in 6 parallel tests were 0.80%, L-carnosine content RSD was 0.79%, alanine content RSD was 1.61%, and proline content RSD was 0.66%. The results of 12 tests shown in the table above showed RSDs for glycine content (0.72%), L-carnosine content (0.67%), alanine content (2.50%), and proline content (1.20%), all meeting the standard requirements. This method demonstrates good intermediate precision.

[0128] Accuracy of Experiment Example 5

[0129] The accuracy of this method was evaluated by examining the recovery rate of 100% concentration level spiked solutions (n=6).

[0130] 1. Preparation of recovery solution

[0131] Verification Sample Preparation method Reference stock solution Prepare according to the method Reference solution Prepare according to the method and complete the derivation process. blank solution Precisely measure 0.5 ml of water to complete the derivatization process. Blank preparation solution Accurately measure 0.5 ml of the blank preparation to complete the derivatization process. Spiked recovery rate of sample solution (100%) Accurately measure 1 ml of the reference standard stock solution and place it in a 10 ml volumetric flask. Dilute to the mark with the blank preparation, mix well, and then accurately measure 0.5 ml to complete the derivatization process. Prepare 6 replicates.

[0132] 2. Results of the recovery rate study

[0133] The results of the accuracy assessment are shown in Table 8.

[0134] Table 8. Accuracy Assessment Results

[0135]

[0136] Conclusion: The recovery results were as follows: glycine was within the range of 102.0%–102.5%, with a mean of 102.2% and an RSD of 0.17% (≤8.0%); alanine was within the range of 98.7%–99.4%, with a mean of 98.9% and an RSD of 0.25% (≤8.0%); proline was within the range of 99.1%–99.4%, with a mean of 99.2% and an RSD of 0.19% (≤8.0%); and L-carnosine was within the range of 96.3%–96.7%, with a mean of 96.5% and an RSD of 0.17% (≤8.0%). These results met the standard requirements, indicating that the method has good accuracy.

[0137] Experimental Example 6 Solution Stability

[0138] 1. Preparation of stable solutions

[0139] Verification Sample Preparation method Reference stock solution Prepare according to the method Reference solution Prepare according to the method and complete the derivation process. Test solution Prepare according to the method and complete the derivation process.

[0140] 2. Results of the investigation

[0141] The results of the stability study are shown in Table 9.

[0142] Table 9. Results of Stability Study

[0143] name Peak area 0h Peak area 27h Peak area 45h Peak area 63h rate of change % STD-glycine 263588 266931 268406 269648 0.97 STD-L-Carnosine 1602012 1614187 1623858 1629469 0.74 STD-alanine 220518 215384 209760 204362 3.42 STD-proline 377475 380097 383172 384003 0.78 glycine 267954 270263 272431 275123 1.13 L-Carnosine 1561875 1567769 1581273 1585553 0.71 alanine 210194 203849 201622 195206 3.05 proline 362040 363867 369850 370600 1.17

[0144] Conclusion: The stability of the reference solution and the test solution was investigated at room temperature over 63 hours.

[0145] Experimental Example 7 Durability

[0146] The consistency of the test sample solution content determination results was examined by subjecting the chromatographic conditions, such as flow rate, column, column temperature, and mobile phase ratio, to minor variations.

[0147] 1. Preparation of durable solution

[0148] Verification Sample Preparation method Reference stock solution Prepare according to the method. Reference solution Prepare according to the method and complete the derivation process. blank solution Precisely measure 0.5 ml of water to complete the derivatization process. Test solution Prepare according to the method and complete the derivation process.

[0149] 2. Results of the investigation

[0150] The results of the durability test are shown in Table 10.

[0151] Table 10 Durability Test Results

[0152]

[0153] Conclusion: The method exhibits good robustness under the following chromatographic conditions: flow rate in the range of 0.9 ml / min to 1.1 ml / min, organic phase ratio in the range of 12% to 14%, column temperature in the range of 32℃ to 38℃, and with different brands of columns and different pH values.

[0154] Example 2

[0155] The testing process is basically the same as in Example 1, except that the test sample is Hi-body developed by Aimeike Technology Development Co., Ltd., batch number (10)PG20220510.

[0156] Investigation results

[0157] The chromatogram of the test solution is shown in [reference needed]. Figure 5 The retention times of glycine were 10.940 min, with a peak area of ​​209.42 and a content of 96.65%; the retention times of L-carnosine were 14.042 min, with a peak area of ​​1362.13 and a content of 96.28%; the retention times of alanine were 17.891 min, with a peak area of ​​164.46 and a content of 94.98%; and the retention times of proline were 20.194 min, with a peak area of ​​293.68 and a content of 102.10%. The detection results were accurate and reliable.

[0158] Comparative Example 1

[0159] 1. Chromatographic conditions

[0160] Other chromatographic conditions are the same as in Example 1, except that the mobile phase and gradient are different:

[0161] Mobile phase: Mobile phase A: acetonitrile; Mobile phase B: 0.1 mol / L sodium acetate (adjust pH to 6.5 with acetic acid); Flow rate: 0.8 ml / min.

[0162] Gradient elution:

[0163] Time (min) Mobile phase A (%) Mobile phase B (%) 0 5 95 3 5 95 38 50 50 42 50 50 43 5 95 55 5 95

[0164] 2. Results of the investigation

[0165] In the chromatogram of the reference solution, alanine had a retention time of 17.891 min and proline had a retention time of 18.024 min. Under these chromatographic conditions, the peaks of alanine and proline overlapped, and they could not be completely separated.

[0166] Comparative Example 2

[0167] 1. Chromatographic conditions

[0168] Other chromatographic conditions are the same as in Example 1, except that the mobile phase and gradient are different:

[0169] Mobile phase: Mobile phase A: acetonitrile; Mobile phase B: 0.1 mol / L sodium acetate (adjust pH to 6.5 with acetic acid); Flow rate: 0.8 ml / min.

[0170] Gradient elution:

[0171] Time (min) Mobile phase A (%) Mobile phase B (%) 0 5 95 3 5 95 15 15 85 40 50 50 41 5 95 55 5 95

[0172] Preparation of reference solutions: Accurately measure glycine, L-carnosine, alanine, and proline reference standards to prepare underrived reference solutions with a concentration of 0.1 mg / ml for each reference standard. Shake well and accurately measure 0.5 ml to complete the derivatization process.

[0173] Investigation results

[0174] The chromatogram of the reference solution is shown below. Figure 6 The retention times for glycine were 16.779 min, L-carnosine 19.033 min, alanine 20.465 min, and proline 20.829 min. Under these chromatographic conditions, the separation of alanine and proline peaks was still poor.

[0175] Comparative Example 3

[0176] 1. Chromatographic conditions

[0177] Other chromatographic conditions are the same as in Example 1, except that the mobile phase and gradient are different:

[0178] Mobile phase: Mobile phase A: methanol; Mobile phase B: 0.1 mol / L sodium acetate (pH adjusted to 6.0 with acetic acid); Flow rate: 0.8 ml / min.

[0179] Gradient elution:

[0180]

[0181] Preparation of reference solutions: Accurately measure glycine, L-carnosine, alanine, and proline reference standards to prepare underrived reference solutions with a concentration of 0.1 mg / ml for each reference standard. Shake well and accurately measure 0.5 ml to complete the derivatization process.

[0182] Investigation results

[0183] The chromatogram of the blank solution is shown below. Figure 7 The chromatogram of the reference solution is shown below. Figure 8 The retention times for glycine, L-carnosine, alanine, and proline were 22.513 min, 27.191 min, 29.242 min, and 30.788 min, respectively. The solvent peak followed L-carnosine, indicating that the separation did not meet the requirements, and the proline peak overlapped with the solvent peak.

[0184] Finally, it should be noted that in this invention, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus.

[0185] Although the invention has been disclosed above through the description of specific embodiments, it should be understood that those skilled in the art can design various modifications, improvements, or equivalents to the invention within the spirit and scope of the appended embodiments. These modifications, improvements, or equivalents should also be considered to be included within the scope of protection claimed by the invention.

Claims

1. A method for detecting the content of L-carnosine and three amino acids in a sodium hyaluronate composite solution, wherein the three amino acids are glycine, alanine, and proline, characterized in that: The determination was performed using high performance liquid chromatography (HPLC). The chromatographic conditions included: a reversed-phase column packed with octadecylsilane-bonded silica gel; and mobile phases consisting of mobile phase A and mobile phase B, where mobile phase A was a methanol-acetonitrile mixture and mobile phase B was an acetic acid-sodium acetate buffer solution.

2. The detection method according to claim 1, characterized in that, The chromatographic conditions shall satisfy at least one of the following conditions 1) to 9): 1) Chromatographic column: 4.6×250mm, 5µm; 2) The volume ratio of methanol to acetonitrile in mobile phase A is 1:1; 3) The concentration of sodium acetate in mobile phase B is 0.05~0.2 mol / L; 4) The mobile phase uses gradient elution; 5) Flow rate: 0.8 ml / min ~ 1.2 ml / min; 6) Column temperature: 30~40℃; 7) Injection volume: 2~10μl; 8) Detector: Ultraviolet detector; 9) Detection wavelength: 250~260nm.

3. The detection method according to claim 2, characterized in that, The chromatographic conditions shall satisfy at least one of the following conditions 1) to 5): 1) Chromatographic column: MORHCHEM Caprisil C18-AQ or Kromasil C18 column; 2) The concentration of sodium acetate in mobile phase B is 0.1 mol / L; 3) Flow rate: 0.9 ml / min ~ 1.1 ml / min; 4) Column temperature: 32~38℃; 5) Injection volume: 4~6μl.

4. The detection method according to claim 3, characterized in that, The chromatographic conditions must satisfy at least one of the following conditions 1) to 4): 1) The pH of mobile phase B is 5.5~6.5, preferably 6.0; 2) Flow rate: 1.0 ml / min; 3) Column temperature: 35℃; 4) Injection volume: 5 μl.

5. The detection method according to any one of claims 1-4, characterized in that, The gradient elution conditions are as follows: Isocratic elution was performed from 0 to 25 min, with the volume fraction of mobile phase B in the mobile phase >85%. Gradient elution is performed for 25-30 minutes, eluting until the volume fraction of mobile phase B in the mobile phase is not less than 15%. Isocratic elution was performed for 30-40 minutes. Gradient elution was performed for 40-42 minutes until the volume fraction of mobile phase B in the mobile phase was >85%. Isocratic elution was performed for 42-55 minutes.

6. The detection method according to claim 5, characterized in that, The gradient elution conditions are as follows:

7. The detection method according to any one of claims 1-5, characterized in that, The specific steps are as follows: Prepare blank solution, reference solution and test solution, inject blank solution, reference solution and test solution into the sample respectively, detect according to liquid chromatography conditions, record the chromatogram and calculate the content of L-carnosine, glycine, alanine and proline in the test solution by peak area according to the external standard method.

8. The detection method according to claim 7, characterized in that, The preparation of the test solution includes: reacting the sodium hyaluronate composite solution to be tested sequentially with triethylamine and phenyl isothiocyanate for derivatization, then adding n-hexane for extraction, and mixing the lower layer solution with water to obtain the test solution.

9. The detection method according to claim 7, characterized in that, The preparation of the reference solution includes: accurately weighing glycine, L-carnosine, alanine and proline reference standards, adding water to prepare a mixed reference stock solution, and then taking an appropriate amount of the mixed reference stock solution and processing it according to the same derivatization process as the test solution preparation method to obtain the solution.

10. The detection method according to any one of claims 1 to 9 is used to analyze the content of L-carnosine and three amino acids in sodium hyaluronate composite solution.

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