Method for determining ratio of amino acids in desmopressin acetate

Abstract

The invention belongs to the technical field of pharmaceutical testing methods, and particularly relates to a method for determining the ratio of amino acids in desmopressin acetate. The method comprises the following steps: respectively carrying out acid hydrolysis treatment and oxidation hydrolysis treatment on a test sample, respectively determining a reference substance solution, an acid hydrolysis test sample solution and an oxidation hydrolysis test sample solution by adopting a liquid chromatography method, respectively calculating the mole number of each amino acid by peak area according to an external standard method, and then calculating the relative ratio of each amino acid. The method provided by the invention solves the problem of determination deviation of the amino acid ratio of desmopressin acetate in the prior art, solves the problems existing in detection of the amino acid ratio of desmopressin acetate in the existing pharmacopeia standard, improves the accuracy of a drug detection result, and ensures the drug quality.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical testing methods, and in particular relates to a method for determining the amino acid ratio in desmopressin acetate. Background Art

[0002] In the quality standard of desmopressin acetate in Part II of the 2020 edition of the Chinese Pharmacopoeia, the amino acid ratio inspection method is: subject the sample to acid hydrolysis and use an appropriate amino acid analysis method for determination. However, the structural formula of desmopressin acetate contains cysteine. Due to the unstable chemical properties of cysteine, it will be partially destroyed during the acid hydrolysis process, which will make the cysteine ​​determination result low.

[0003] The method for checking the amino acid ratio in the quality standard of octreotide acetate in Part II of the 2020 edition of the Chinese Pharmacopoeia is as follows: the test sample is first oxidized and then acid hydrolyzed. The cysteine ​​in the test sample is oxidized to cysteic acid in equal molar amounts and determined using an appropriate amino acid analysis method. The molar number of cysteine ​​is calculated as cysteic acid. However, the structural formula of desmopressin acetate also contains tyrosine. Due to the unstable chemical properties of tyrosine, it will be partially destroyed during the oxidation process, which will make the tyrosine determination result low. Summary of the invention

[0004] In order to solve the problems existing in the above-mentioned prior art, the present invention provides a method for determining the amino acid ratio in desmopressin acetate. In order to ensure that both cysteine ​​and tyrosine can be accurately determined, the present invention performs acid hydrolysis and oxidative hydrolysis on the test sample respectively, and adopts a suitable amino acid analysis method for determination.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A method for determining the ratio of amino acids in desmopressin acetate comprises the steps of subjecting a test sample to acid hydrolysis treatment and oxidative hydrolysis treatment, and using liquid chromatography to determine the reference solution, the acid hydrolysis test sample solution, and the oxidative hydrolysis test sample solution, and calculating the molar number of each amino acid by peak area according to the external standard method, and then calculating the relative ratio of each amino acid.

[0007] The determination method is specifically:

[0008] Prepare reference solution, acid hydrolysis test solution, and oxidative hydrolysis test solution respectively;

[0009] Liquid chromatography was used for determination. The molar numbers of aspartic acid, glutamic acid, proline, tyrosine, and phenylalanine in the acid hydrolysis test solution and the molar numbers of glycine and cysteic acid in the oxidative hydrolysis test solution were calculated by peak area according to the external standard method.

[0010] Taking one sixth of the total molar number of aspartic acid, glutamic acid, proline, glycine, phenylalanine and arginine as 1, the relative ratio of each amino acid is calculated. The relative ratios of aspartic acid, glutamic acid, proline, glycine, phenylalanine and arginine should all be 0.90-1.10, tyrosine should be 0.70-1.05, and cysteine ​​should be 0.80-1.10 calculated as cysteic acid.

[0011] A method for determining the amino acid ratio in desmopressin acetate specifically comprises the following steps:

[0012] (1) Preparation of reference solution: Take appropriate amount of tyrosine, phenylalanine, glutamic acid, aspartic acid, cysteic acid, proline, arginine and glycine reference substances respectively, weigh accurately, dissolve in 0.02 mol / L hydrochloric acid solution and quantitatively dilute to prepare a solution containing about 0.1 μmol of each of tyrosine, phenylalanine, glutamic acid, aspartic acid, cysteic acid, proline, arginine and glycine per 1 ml;

[0013] (2) Preparation of acid hydrolysis test solution: Take 10 mg of the test sample, place it in a glass bottle, add 4 ml of 6 mol / L hydrochloric acid solution, fill it with nitrogen and seal it, place it at 110°C for hydrolysis for 22 hours, cool it, take 0.1 ml, add water to 1 ml, dry it under reduced pressure, add 2 ml of 0.02 mol / L hydrochloric acid solution to dissolve it, filter it, and take the filtrate;

[0014] (3) Preparation of oxidative hydrolysis test solution: Take 10 mg of the test sample, add 1 ml of performic acid solution (mix 30% hydrogen peroxide and formic acid in a ratio of 1:9 and place at room temperature for 2 hours), place in an ice-water bath for 6 hours, take 0.1 ml, add water to 1 ml, dry under reduced pressure until dry, add 0.4 ml of 6 mol / L hydrochloric acid solution to dissolve, seal after nitrogen filling, place at 110°C for hydrolysis for 22 hours, cool, take 0.1 ml, add water to 1 ml, dry under reduced pressure until dry, add 2 ml of 0.02 mol / L hydrochloric acid solution to dissolve, filter, and take the filtrate;

[0015] (4) Determination: Accurately measure 20 μl of each of the reference solution, acid hydrolysis test solution and oxidative hydrolysis test solution, and inject them into the amino acid analyzer respectively. Record the chromatograms and calculate the molar numbers of tyrosine, phenylalanine, glutamic acid, aspartic acid, proline and arginine in the acid hydrolysis test solution and the molar numbers of cysteic acid and glycine in the oxidative hydrolysis test solution by the external standard method based on the peak areas;

[0016] (5) Calculation of relative ratios of amino acids: Taking one sixth of the total molar number of phenylalanine, glutamic acid, aspartic acid, proline, glycine and arginine as 1, the relative ratios of each amino acid should be calculated. The relative ratios of aspartic acid, glutamic acid, proline, glycine, phenylalanine and arginine should all be 0.90-1.10; the relative ratios of tyrosine should be 0.70-1.05; and the relative ratios of cysteine, calculated as cysteic acid, should be 0.80-1.10.

[0017] Beneficial Effects

[0018] The invention discloses a method for determining the amino acid ratio in desmopressin acetate, which solves the problem of the deviation of the amino acid ratio determination of desmopressin acetate in the prior art, solves the problem existing in the inspection of the amino acid ratio in desmopressin acetate in the current pharmacopoeia standard, improves the accuracy of drug detection results, and ensures the quality of drugs.

[0019] The method of the present invention has been verified, and it is proved that the method has good specificity, instrument precision, intermediate precision, repeatability and linearity, and the method has good accuracy and good durability. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Cysteic Acid Linear Relationship Diagram;

[0021] Figure 2 : Linear relationship diagram of aspartic acid;

[0022] Figure 3 : Glutamate linear relationship diagram;

[0023] Figure 4 : Linear relationship diagram of proline;

[0024] Figure 5 : Linear relationship diagram of glycine;

[0025] Figure 6 : Tyrosine linear relationship diagram;

[0026] Figure 7 : Phenylalanine linear relationship diagram;

[0027] Figure 8 : Arginine linear relationship diagram;

[0028] Fig. 9 :Chromatographic results of acid hydrolysis samples;

[0029] Fig.10 :Chromatographic results of oxidative hydrolysis samples;

[0030] Fig.11 : Chromatographic results of mixed reference substance. DETAILED DESCRIPTION

[0031] Hereinafter, the present invention will be described in detail. Before describing, it should be understood that the terms used in this specification and the appended claims should not be interpreted as being limited to the general meaning and dictionary meaning, but should be interpreted according to the meaning and concept corresponding to the technical aspects of the present invention on the basis of the principle that the inventor is allowed to appropriately define the terms for the best interpretation. Therefore, the descriptions presented here are only preferred examples for illustrative purposes and are not intended to limit the scope of the present invention, so that it should be understood that other equivalents or improvements can be obtained therefrom without departing from the spirit and scope of the present invention.

[0032] The following examples are only listed as examples of embodiments of the present invention and do not constitute any limitation to the present invention. It can be understood by those skilled in the art that modifications within the scope of the essence and concept of the present invention fall within the scope of protection of the present invention. Unless otherwise specified, the reagents and instruments used in the following examples are all commercially available products.

[0033] Example 1 Determination of amino acid ratios in desmopressin acetate:

[0034] 1. Reagents and test solutions

[0035] Formic acid (analytical grade), 30% hydrogen peroxide (analytical grade), hydrochloric acid (analytical grade), purified water.

[0036] 2. Reference Material Information

[0037] Table 1. Reference material information

[0038]

[0039] 3. Instruments and Equipment

[0040] Table 2. Instruments and equipment

[0041] Instrument Name Instrument Model Instrument Manufacturer Amino Acid Analyzer LA8080 Hitachi Corporation Freeze drying machine LABCONCO Beijing Zhaoshengxing Instrument Equipment Co., Ltd. Drying box DGG-9070B Shanghai Senxin Experimental Instrument Co., Ltd. Electronic balance XSE205 METTLER TOLEDO

[0042] 4. Inspection method: Liquid chromatography

[0043] (1) Analysis conditions

[0044] B1: pH-1

[0045] B2: pH-2

[0046] B3: pH-3

[0047] B4: pH-4

[0048] B5: H2O

[0049] B6: pH-RG

[0050] R1: Ninhydrin

[0051] R2: Ninhydrin buffer solution

[0052] R3: 5% ethanol The above reagents are the buffer and color developing solution of Hitachi amino acid analyzer.

[0053] Table 3. Liquid chromatography conditions

[0054]

[0055] Chromatographic column: Hitachi 2622PH-4.6×60mm

[0056] Column temperature: 57°C

[0057] Injection volume: 20 μl

[0058] Detection wavelength: 570nm, 440nm

[0059] (2) Solution preparation

[0060] Preparation of reference solution: Take appropriate amount of tyrosine, phenylalanine, glutamic acid, aspartic acid, cysteic acid, proline, arginine and glycine reference substances respectively, weigh accurately, dissolve them with 0.02 mol / L hydrochloric acid solution and quantitatively dilute them to make a solution containing about 0.1 μmol each of tyrosine, phenylalanine, glutamic acid, aspartic acid, cysteic acid, proline, arginine and glycine per 1 ml.

[0061] Preparation of acid hydrolysis test solution: Take 10 mg of the test sample, place it in a glass bottle, add 4 ml of 6 mol / L hydrochloric acid solution, seal it after filling with nitrogen, and hydrolyze it at 110°C for 22 hours. Cool it, take 0.1 ml, add water to 1 ml, dry it under reduced pressure to dryness, add 2 ml of 0.02 mol / L hydrochloric acid solution to dissolve it, filter it, and take the filtrate.

[0062] Preparation of oxidative hydrolysis test solution: Take 10 mg of the test sample, add 1 ml of performic acid solution (mix 30% hydrogen peroxide and formic acid in a ratio of 1:9 and leave at room temperature for 2 hours), place in an ice-water bath for 6 hours, take 0.1 ml, add water to 1 ml, dry under reduced pressure to dry, add 0.4 ml of 6 mol / L hydrochloric acid solution to dissolve, seal after nitrogen filling, place at 110°C for hydrolysis for 22 hours, cool, take 0.1 ml, add water to 1 ml, dry under reduced pressure to dry, add 2 ml of 0.02 mol / L hydrochloric acid solution to dissolve, filter, and take the filtrate.

[0063] (3) Determination

[0064] Accurately measure 20 μl of each of the reference solution, acid hydrolysis test solution and oxidative hydrolysis test solution, and inject them into the amino acid analyzer respectively. Record the chromatograms and calculate the molar numbers of tyrosine, phenylalanine, glutamic acid, aspartic acid, proline and arginine in the acid hydrolysis test solution and the molar numbers of cysteic acid and glycine in the oxidative hydrolysis test solution by peak areas according to the external standard method.

[0065] 5. Standard regulations

[0066] Taking one-sixth of the total molar number of phenylalanine, glutamic acid, aspartic acid, proline, glycine and arginine as 1, the relative ratio of each amino acid is calculated. The relative ratios of aspartic acid, glutamic acid, proline, glycine, phenylalanine and arginine should all be 0.90-1.10, tyrosine should be 0.70-1.05, and cysteine ​​should be 0.80-1.10 as cysteic acid.

[0067] Example 2 Validation of the analytical method

[0068] In accordance with the "Technical Guidelines for Validation of Analytical Methods for Quality Control of Chemical Drugs", "Technical Guidelines for Standardized Processes for Establishing Quality Standards for Chemical Drugs", "Technical Guidelines for Impurity Research of Chemical Drugs", and the Validation Guidelines for Analytical Methods 9101 in Part IV of the 2020 Edition of the Chinese Pharmacopoeia, the amino acid ratio inspection method in desmopressin acetate was methodologically validated.

[0069] Table 4. Summary of samples used for analysis and verification

[0070]

[0071] 2.1 Amino acid ratio check

[0072] 2.1.1 Summary of the validation of the amino acid ratio test methodology

[0073] Table 5. Summary results of amino acid ratio test method validation

[0074]

[0075]

[0076] 2.1.1 Test method selection

[0077] The amino acid ratio inspection method in the 2020 edition of the Chinese Pharmacopoeia, Part II, Desmopressin Acetate Quality Standard, was used to acid hydrolyze the sample and determine it using an appropriate amino acid analysis method. The desmopressin acetate structure contains cysteine. Due to the unstable chemical properties of cysteine, it will be partially destroyed during the acid hydrolysis process, which will make the cysteine ​​determination result low. Therefore, the cysteine ​​determination method refers to the amino acid ratio inspection method in the 2020 edition of the Chinese Pharmacopoeia, Part II, Octreotide Acetate Quality Standard, and the sample was first oxidized and then acid hydrolyzed. In the hydrolysis treatment, the cysteine ​​in the test sample is oxidized to cysteic acid in equal molar amounts and determined by a suitable amino acid analysis method. The molar number of cysteine ​​is calculated as cysteic acid, but the desmopressin acetate structural formula also contains tyrosine. Due to the unstable chemical properties of tyrosine, it will be partially destroyed during the oxidation process, which will make the tyrosine determination result low. Therefore, in order to ensure that both cysteine ​​and tyrosine can be accurately determined, the test sample is subjected to acid hydrolysis treatment and oxidative hydrolysis treatment respectively, and is determined by a suitable amino acid analysis method.

[0078] 2.1.2 Specificity test

[0079] The test samples were subjected to acid hydrolysis and oxidative hydrolysis treatments, respectively, and analyzed using the amino acid analyzer protein hydrolysis analysis method.

[0080] Preparation of amino acid mixed reference solution: Take 1 ml of amino acid mixed solution standard substance (17 kinds of amino acids), place it in a 10 ml volumetric flask, dilute to the scale with 0.02 mol / L hydrochloric acid solution, and shake well.

[0081] Preparation of cysteic acid reference solution: Take 20 mg of cysteic acid, place it in a 50 ml volumetric flask, add 0.02 mol / L hydrochloric acid solution to dissolve and dilute to the scale, shake well, accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the scale with 0.02 mol / L hydrochloric acid solution, and shake well.

[0082] Preparation of cysteine ​​reference oxidation solution: Take 10 mg of cysteine ​​hydrochloride, add 1 ml of performic acid, place in an ice-water bath for 6 hours, take 0.1 ml, add water to 1 ml, dry under reduced pressure, and add 10 ml of 0.02 mol / L hydrochloric acid solution to dissolve.

[0083] Preparation of acid hydrolysis test solution: Take 10 mg of the test sample, place it in a glass bottle, add 4 ml of 6 mol / L hydrochloric acid solution, seal it after filling with nitrogen, and hydrolyze it at 110°C for 22 hours. Cool it, take 0.1 ml, add water to 1 ml, dry it under reduced pressure to dryness, add 2 ml of 0.02 mol / L hydrochloric acid solution to dissolve it, filter it, and take the filtrate.

[0084] Preparation of oxidative hydrolysis test solution: Take 10 mg of the test sample, add 1 ml of performic acid solution (mix 30% hydrogen peroxide and formic acid in a ratio of 1:9 and leave at room temperature for 2 hours), place in an ice-water bath for 6 hours, take 0.1 ml, add water to 1 ml, dry under reduced pressure to dry, add 0.4 ml of 6 mol / L hydrochloric acid solution, seal after nitrogen filling, place at 110°C for hydrolysis for 22 hours, cool, take 0.1 ml, add water to 1 ml, dry under reduced pressure to dry, add 2 ml of 0.02 mol / L hydrochloric acid solution to dissolve, filter, and take the filtrate.

[0085] Accurately measure 20 μl of each of the above solutions, inject them into the liquid chromatograph, record the chromatogram, and the test results are shown in Table 6.

[0086] Table 6. Results of specificity test for amino acid ratio inspection methodology validation

[0087]

[0088] Conclusion: The amino acids in the test solution were located and analyzed using the amino acid mixed reference solution. The results showed that the separation between the chromatographic peaks of the amino acids in the acid hydrolysis test solution and the oxidative hydrolysis test solution met the requirements, indicating that the method has good specificity.

[0089] The retention time of the chromatographic peak after cysteine ​​oxidation is consistent with that of the cysteic acid peak. Therefore, the amount of cysteic acid in the oxidative hydrolysis test solution can be used to characterize the amount of cysteine. In the chromatogram of the acid hydrolysis test solution, the baseline at 10-11 min is elevated, affecting the integration of the glycine peak. However, in the chromatogram of the oxidative hydrolysis test solution, the baseline at 10-11 min is normal, and the peak shape of glycine is also normal. The reason may be that during the acid hydrolysis treatment of the test sample, the degradation products produced after the cysteine ​​is destroyed, as well as cysteine ​​itself, raise the baseline at 10-11 min. During the oxidative hydrolysis treatment of the test sample, all cysteine ​​is converted into cysteic acid, making the baseline at 10-11 min normal. Therefore, the amount of glycine in the test sample can be calculated based on the amount of glycine in the oxidative hydrolysis test solution.

[0090] 2.1.3 Instrument precision test

[0091] Accurately measure 20 μl of the reference solution, inject it into the amino acid analyzer, record the chromatogram, inject it 6 times continuously, and calculate the RSD values ​​of the retention time (T) and peak area (A) 6 times to examine the instrument precision of this method. The test results are shown in Table 7.

[0092] Table 7. Results of the instrument precision test for amino acid ratio inspection methodology verification

[0093]

[0094]

[0095] Verification requirements: The RSD of retention time shall not exceed 1.5%, and the RSD of peak area shall not exceed 2.0%.

[0096] Conclusion: From the above test results, it can be seen that the reference solution was injected 6 times continuously, and the RSD results of the retention time and peak area of ​​cysteic acid, aspartic acid, glutamic acid, proline, glycine, tyrosine, phenylalanine and arginine all met the validation requirements, indicating that the instrument precision of this method is good.

[0097] 2.1.5 Linear relationship test

[0098] Preparation of reference stock solution: Take about 13 mg of L-aspartic acid, about 15 mg of glutamic acid, about 10 mg of glycine, about 17 mg of arginine, about 12 mg of proline, about 18 mg of tyrosine, about 17 mg of phenylalanine, and about 17 mg of cysteic acid, accurately weigh them, place them in a 50 ml volumetric flask, add 0.02 mol / L hydrochloric acid solution to dissolve and dilute to the scale, and shake well.

[0099] Preparation of linear series solutions: Accurately measure 0.1, 0.4, 0.8, 1.0, 1.2, 1.5, and 2.0 ml of the reference stock solution, place them in 20 ml volumetric flasks respectively, dilute to the scale with 0.02 mol / L hydrochloric acid solution, and shake well.

[0100] Accurately measure 20 μl of each linear series solution and inject it into the amino acid analyzer. Record the chromatogram and perform linear regression with the reference substance concentration (C, μg / ml) as the horizontal axis and the peak area (A) as the vertical axis. The test results are shown in Table 8 and the spectrum is attached. Figure 1-8 .

[0101] Table 8. Results of linear relationship test for amino acid ratio inspection methodology verification

[0102]

[0103]

[0104] Verification requirements: The correlation coefficient R shall not be less than 0.99.

[0105] Conclusion: The concentration ranges of cysteic acid, glutamic acid, proline, tyrosine, phenylalanine, arginine and cysteic acid were 1.69-33.85 μg / ml, 1.33-26.65 μg / ml, 1.59-31.71 μg / ml, 1.29-25.84 μg / ml, 1.09-21.82 μg / ml, 1.89-37.78 μg / ml, 1.77-35.34 μg / ml and 1.73-34.58 μg / ml, respectively, and the correlation coefficients were all 1.000, which met the validation requirements, indicating that the method had a good linear relationship.

[0106] 2.1.6 Repeatability test

[0107] Take this product according to the preparation method of acid hydrolysis test solution and oxidation hydrolysis test solution, prepare 6 portions respectively in the same way as the repeatability test test solution, accurately measure 20μl respectively, inject into the amino acid analyzer, record the chromatogram, calculate the molar amount of each amino acid in the test sample by peak area according to the external standard method, take one sixth of the total molar number of aspartic acid, glutamic acid, proline, glycine, phenylalanine and arginine as 1, calculate the relative ratio of each amino acid, and count the RSD values ​​of the 6 determination results to examine the repeatability of this method. The test results are shown in Tables 9 and 10.

[0108] Table 9. Results of repeatability test for amino acid ratio inspection methodology validation (amino acid content)

[0109]

[0110] Verification requirements: The RSD of the measurement results shall not exceed 3%.

[0111] Table 10. Results of repeatability test for amino acid ratio inspection methodology validation (amino acid ratio)

[0112]

[0113] Conclusion: From the above test results, it can be seen that the RSD of the contents of cysteine, aspartic acid, glutamic acid, proline, glycine, tyrosine, phenylalanine, and arginine and the amino acid ratios measured after repeated determination for 6 times all meet the verification requirements, indicating that the method has good repeatability.

[0114] 2.1.8 Intermediate precision test

[0115] Different analysts and different dates took this product according to the preparation method of acid hydrolysis test solution and oxidation hydrolysis test solution, and prepared 6 portions respectively as the intermediate precision test test solution. 20μl was accurately measured and injected into the amino acid analyzer, and the chromatogram was recorded. The molar amount of each amino acid in the test sample was calculated by the peak area according to the external standard method. The relative ratio of each amino acid was calculated by taking one sixth of the total molar number of aspartic acid, glutamic acid, proline, glycine, phenylalanine and arginine as 1. Combined with the repeatability test, the RSD values ​​of 12 determination results were counted to investigate the intermediate precision of this method. The test results are shown in Tables 11 and 12.

[0116] Table 11. Results of repeatability test for amino acid ratio inspection methodology validation (amino acid content)

[0117]

[0118] Verification requirements: The RSD of the measurement results shall not exceed 6%.

[0119] Table 12. Results of repeatability test for amino acid ratio inspection methodology validation (amino acid ratio)

[0120]

[0121]

[0122] Verification requirements: The RSD of the measurement results shall not exceed 6%.

[0123] Conclusion: From the above test results, it can be seen that different analysts, on different dates, repeated the determination 6 times for a total of 12 times, and the RSDs of cysteine, aspartic acid, glutamic acid, proline, glycine, tyrosine, phenylalanine and arginine contents measured all met the validation requirements, indicating that the intermediate precision of this method is good.

[0124] 2.1.7 Accuracy test

[0125] Prepare spiked test solution by adding different concentrations (low, medium, and high) of amino acid mixed reference substances to the test sample, and then treat it in the same way as the test sample solution preparation method. Prepare 3 copies of each concentration in the same way.

[0126] Preparation of reference solution: Take about 13 mg of L-aspartic acid, about 15 mg of glutamic acid, about 10 mg of glycine, about 17 mg of arginine, about 12 mg of proline, about 18 mg of tyrosine, about 17 mg of phenylalanine, and about 17 mg of cysteic acid, respectively, accurately weigh, place in a 50 ml volumetric flask, add 0.02 mol / L hydrochloric acid solution to dissolve and dilute to the scale, shake well, accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to the scale with 0.02 mol / L hydrochloric acid solution, and shake well.

[0127] Preparation of reference substance stock solution I: Take about 13 mg of L-aspartic acid, about 15 mg of glutamic acid, about 17 mg of arginine, about 12 mg of proline, about 18 mg of tyrosine, and about 17 mg of phenylalanine, accurately weigh them, place them in a 10 ml volumetric flask, add 6 mol / L hydrochloric acid solution to dissolve and dilute to the scale, and shake well.

[0128] Preparation of reference substance stock solution II: Take about 16 mg of cysteine ​​hydrochloride and about 10 mg of glycine, accurately weigh them, place them in a 10 ml volumetric flask, add performic acid solution to dissolve and dilute to the scale, and shake well.

[0129] Preparation of acid hydrolysis spiked test solution: Take about 10 mg of the test sample, a total of 3 portions, put in glass bottles, add 0.64 ml, 0.80 ml, and 0.96 ml of reference substance stock solution I respectively, then add 6 mol / L hydrochloric acid solution to 4 ml respectively, seal after nitrogen filling, hydrolyze at 110℃ for 22 hours, cool, take 0.1 ml, add water to 1 ml, dry under reduced pressure to dry, add 4 ml of 0.02 mol / L hydrochloric acid solution to dissolve, filter, and take the filtrate.

[0130] Preparation of oxidative hydrolysis spiked test solution: Take about 10 mg of the test sample, a total of 3 portions, add 0.64 ml, 0.80 ml, and 0.96 ml of reference substance stock solution II respectively, then add performic acid solution to 1 ml, place in an ice-water bath for 6 hours, take 0.1 ml, add water to 1 ml, dry under reduced pressure to dry, add 0.4 ml of 6 mol / L hydrochloric acid solution to dissolve, seal after nitrogen filling, place at 110°C for hydrolysis for 22 hours, cool, take 0.1 ml, add water to 1 ml, dry under reduced pressure to dry, add 4 ml of 0.02 mol / L hydrochloric acid solution to dissolve, filter, and take the filtrate.

[0131] Accurately measure 20 μl of each of the reference solution, the acid hydrolysis spiked test solution, and the oxidative hydrolysis spiked test solution, and inject them into the amino acid analyzer respectively, record the chromatogram, and calculate the content of aspartic acid, glutamic acid, arginine, proline, tyrosine, and phenylalanine in the acid hydrolysis spiked test solution and the content of glycine and cysteic acid in the oxidative hydrolysis spiked test solution by the external standard method using the peak area, and calculate the recovery rate by the ratio of the measured amount to the added amount to investigate the accuracy of the method. The test results are shown in Tables 13 to 20.

[0132] Table 13. Results of the accuracy test of the amino acid ratio test methodology validation (aspartic acid)

[0133]

[0134] Verification requirements: The recovery rate should be between 90% and 110%, and the RSD should not exceed 5%.

[0135] Table 14. Results of the accuracy test of the amino acid ratio inspection methodology validation (glutamic acid)

[0136]

[0137] Verification requirements: The recovery rate should be between 90% and 110%, and the RSD should not exceed 5%.

[0138] Table 15. Results of the accuracy test of the amino acid ratio inspection methodology validation (proline)

[0139]

[0140] Verification requirements: The recovery rate should be between 90% and 110%, and the RSD should not exceed 5%.

[0141] Table 16. Results of the accuracy test of the amino acid ratio inspection methodology validation (tyrosine)

[0142]

[0143] Verification requirements: The recovery rate should be between 90% and 110%, and the RSD should not exceed 5%.

[0144] Table 17. Results of the accuracy test of the amino acid ratio test methodology validation (phenylalanine)

[0145]

[0146]

[0147] Verification requirements: The recovery rate should be between 90% and 110%, and the RSD should not exceed 5%.

[0148] Table 18. Results of the accuracy test of the amino acid ratio test methodology validation (arginine)

[0149]

[0150] Verification requirements: The recovery rate should be between 90% and 110%, and the RSD should not exceed 5%.

[0151] Table 19. Results of the accuracy test of the amino acid ratio test methodology validation (cysteine)

[0152]

[0153] Verification requirements: The recovery rate should be between 90% and 110%, and the RSD should not exceed 5%.

[0154] Table 20. Results of the accuracy test of the amino acid ratio test methodology validation (glycine)

[0155]

[0156]

[0157] Verification requirements: The recovery rate should be between 90% and 110%, and the RSD should not exceed 5%.

[0158] Conclusion: From the above test results, it can be seen that by adding different concentrations of various amino acid reference substances to the test samples, the recovery rates of aspartic acid, glutamic acid, proline, tyrosine, phenylalanine, cysteine, glycine, and arginine were measured between 94.8% and 104.6%, 93.3% and 101.3%, 101.9% and 104.9%, 101.6% and 109.8%, 104.8% and 109.3%, respectively. The recovery rates are between 90% and 110%, and the RSDs are all less than 5%, which meet the validation requirements, indicating that the method has good accuracy.

[0159] 2.1.9 Durability test

[0160] This method uses the protein hydrolysis analysis method of the amino acid analyzer. The mobile phase and color developing solution are the original imported reagents of the Hitachi amino acid analyzer. By fine-tuning the preparation conditions of the test solution, such as the acid hydrolysis time and the oxidation time, the molar amount of each amino acid in the test sample is determined under different test conditions. The RSD values ​​of the results measured under different test conditions are statistically analyzed to investigate the durability of this method. The test results are shown in Table 21.

[0161] Table 21. Results of the robustness test for amino acid ratio inspection methodology validation

[0162]

[0163]

[0164] Verification requirements: The RSD of the measurement results under different test conditions is no more than 3%, which is considered to be good durability.

[0165] Conclusion: From the above test results, it can be seen that by changing the acid hydrolysis time and oxidation time, the RSD of the results measured under different test conditions meets the verification requirements and has little effect on the measurement results, indicating that this method has good durability.

[0166] 3. Sample inspection

[0167] Referring to the test conditions of Example 1 and the preparation methods of the reference solution, the acid hydrolysis test solution, and the oxidative hydrolysis test solution, the liquid chromatography method was used for determination, and the molar number of aspartic acid, glutamic acid, proline, tyrosine, and phenylalanine in the acid hydrolysis test solution and the molar number of glycine and cysteic acid in the oxidative hydrolysis test solution were calculated by peak area according to the external standard method. Taking one sixth of the total molar number of aspartic acid, glutamic acid, proline, glycine, phenylalanine, and arginine as 1, the relative ratio of each amino acid was calculated, and aspartic acid, glutamic acid, proline, glycine, phenylalanine, and arginine should all be 0.90 to 1.10, tyrosine should be 0.70 to 1.05, and cysteine ​​and cysteic acid should all be 0.80 to 1.10.

[0168] The test results are shown in Table 22 and the chromatogram results are shown in Attachment Figure 9-11 .

[0169] Table 22 Amino Acid Ratio Test Sample Test Results

[0170]

[0171] Conclusion: The amino acid ratios of aspartic acid, glutamic acid, proline, glycine, phenylalanine, arginine, tyrosine and cysteine ​​in the three batches of test samples were in compliance with the regulations.

[0172] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A method for determining the amino acid ratio in desmopressin acetate, characterized in that: The test samples were subjected to acid hydrolysis and oxidative hydrolysis treatments, respectively. The reference solution, acid hydrolysis test sample solution, and oxidative hydrolysis test sample solution were determined by liquid chromatography, and the molar number of each amino acid was calculated by peak area using the external standard method, and then the relative ratio of each amino acid was calculated.

2. The method for determining the amino acid ratio in desmopressin acetate according to claim 1, characterized in that: The amino acids measured in the acid hydrolysis test solution include aspartic acid, glutamic acid, proline, tyrosine, and phenylalanine; the amino acids measured in the oxidative hydrolysis test solution include glycine and cysteic acid.

3. The method for determining the amino acid ratio in desmopressin acetate according to claim 1, characterized in that: The method specifically comprises the following steps: (1) Preparation of reference solution: Take appropriate amount of tyrosine, phenylalanine, glutamic acid, aspartic acid, cysteic acid, proline, arginine and glycine reference substances respectively, weigh accurately, dissolve in 0.02 mol / L hydrochloric acid solution and quantitatively dilute to prepare a solution containing 0.1 μmol each of tyrosine, phenylalanine, glutamic acid, aspartic acid, cysteic acid, proline, arginine and glycine per 1 ml; (2) Preparation of acid hydrolysis test solution; (3) Preparation of oxidative hydrolysis test solution; (4) Determination: Accurately measure the reference solution, the acid hydrolysis test solution and the oxidative hydrolysis test solution, inject them into the amino acid analyzer respectively, record the chromatograms, and calculate the molar number of tyrosine, phenylalanine, glutamic acid, aspartic acid, proline, and arginine in the acid hydrolysis test solution and the molar number of cysteic acid and glycine in the oxidative hydrolysis test solution by the external standard method based on the peak area; (5) Calculation of relative ratios of amino acids: Taking one sixth of the total molar number of phenylalanine, glutamic acid, aspartic acid, proline, glycine and arginine as 1, the relative ratios of each amino acid should be 0.90-1.10 for aspartic acid, glutamic acid, proline, glycine, phenylalanine and arginine; tyrosine should be 0.70-1.05; and cysteine ​​should be 0.80-1.10 in terms of cysteic acid.

4. The method for determining the amino acid ratio in desmopressin acetate according to claim 3, characterized in that: The reference substances in step (1) include L-aspartic acid, glutamic acid, glycine, arginine, proline, tyrosine, phenylalanine, cysteic acid, and cysteine ​​hydrochloride.

5. The method for determining the amino acid ratio in desmopressin acetate according to claim 3, characterized in that: The preparation of the acid hydrolysis test solution in step (2) is specifically as follows: take the test sample, place it in a glass bottle, add 6 mol / L hydrochloric acid solution, seal it after nitrogen filling, place it at 110°C for hydrolysis, cool it, take a portion, add water, dry it to dryness under reduced pressure, add 0.02 mol / L hydrochloric acid solution to dissolve it, filter it, and take the filtrate.

6. The method for determining the amino acid ratio in desmopressin acetate according to claim 3, characterized in that: The preparation of the oxidative hydrolysis test solution described in step (3) is specifically as follows: take the test sample, add formic acid solution, place in an ice water bath, take a portion and dilute with water, dry under reduced pressure to dryness, add 6 mol / L hydrochloric acid solution to dissolve, seal after nitrogen filling, place at 110°C for hydrolysis, cool, take a portion and dilute with water, dry under reduced pressure to dryness, add 0.02 mol / L hydrochloric acid solution to dissolve, filter, and take the filtrate.

7. The method for determining the amino acid ratio in desmopressin acetate according to claim 6, characterized in that: The performic acid solution in step (3) is prepared by mixing 30% hydrogen peroxide and formic acid in a ratio of 1:9 and leaving the mixture at room temperature for 2 hours.

8. The method for determining the amino acid ratio in desmopressin acetate according to claim 3, characterized in that: The liquid chromatography analysis conditions in step (4) are: B 1: pH-1; B2: pH-2; B3: pH-3; B4: pH-4; B5: H2O; B6: pH-RG; R1: ninhydrin; R2: ninhydrin buffer solution; R3: 5% ethanol; chromatographic column: Hitachi 2622PH-4.6×60mm; column temperature: 57°C; injection volume: 20μl; detection wavelength: 570nm, 440nm.

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