A method for determining enantiomers of polypeptide protected amino acids

Abstract

The present invention relates to a method for determining enantiomers of polypeptide protected amino acids. Compared with the prior art, a normal phase chromatographic column is not used to determine enantiomers using n-hexane and isopropanol / ethanol as a mobile phase system; instead, a reverse phase chiral chromatographic column with a surface coated with cellulose-tris(4-methylbenzoate) filler is used, and ammonium acetate solution-acetonitrile is used as a mobile phase for gradient elution; under the chromatographic conditions, enantiomers and main components can be effectively separated, and a buffer salt is used as a mobile phase, and the target peak is sharp and easy to judge and detect; and the ammonium acetate and acetonitrile used in the mobile phase system are both conventional reagents, which are easy to obtain, thereby reducing the detection cost.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for determining enantiomers of polypeptide-protected amino acids. Background Art

[0002] Cetrorelix acetate, also commonly known as cetrorelix, is an effective progesterone-releasing hormone inhibitor receptor antagonist that can control the stimulation of the ovaries, prevent premature discharge of immature follicles, and help conception; the single-dose regimen of cetrorelix can reduce the number of injections and is more convenient to use. At present, only the original drug of cetrorelix acetate for injection is on sale in China, and the competition landscape is good. It is a drug recommended by the relevant guidelines for assisted reproduction and has good clinical prospects. Cetrorelix acetate is a synthetic decapeptide, and its chemical name is: N-acetyl-3-(2-naphthyl)-D-alanyl-p-chloro-D-phenylalanyl-3-(3-pyridyl)-D-alanyl-L-seryl-L-tyrosyl-N5-carbamoyl-D-ornithine-L-leucyl-L-arginyl-L-prolyl-D-alaninamide acetate.

[0003] Its structural formula is:

[0004]

[0005] It can be seen that the peptide chain of cetrorelix acetate contains multiple chiral centers, and these chiral centers are introduced by multiple amino acid starting materials for synthesizing cetrorelix acetate. These amino acid starting materials all have the risk of enantiomeric impurities. If the chiral impurities of the starting materials are not controlled, they will be introduced into the final product, making it difficult to purify the final product, making it difficult to meet quality control standards, and increasing production costs.

[0006] Therefore, developing an analytical method that can effectively detect amino acid enantiomers has important guiding significance for the production and purification of cetrorelix acetate, and establishing strict quality control standards will have positive significance for reducing the difficulty of final product purification and improving product quality.

[0007] At present, there are few literatures on the determination and analysis methods of polypeptide protected amino acid enantiomers. Patent application CN111505161A discloses a method for detecting protected amino acid enantiomers, which uses a reverse phase chromatography method to determine the enantiomers of Fmoc protected amino acids. The chromatographic column used is a cellulose-tris (3,5-dimethylphenylcarbamate) chromatographic column, and the mobile phase is a 0.1% trifluoroacetic acid aqueous solution-acetonitrile system. The applicant tried to use this method to detect multiple amino acid starting materials of cetrorelix acetate, but found that it was not applicable. Summary of the invention

[0008] The object of the present invention is to provide a method for analyzing enantiomers of polypeptide protected amino acids, wherein the chromatographic conditions are applicable to the detection of enantiomers of a protected amino acid starting material of cetrorelix acetate, and the method can achieve effective separation of the main peak and the enantiomers, with outstanding separation effect and good specificity.

[0009] The technical solution to achieve the above invention object is:

[0010] The present invention provides a method for determining enantiomers of polypeptide protected amino acids, comprising the following steps:

[0011] (1) Sample preparation:

[0012] Preparation of test solution: accurately weigh 10 mg of the test sample, place it in a 20 ml volumetric flask, dissolve it with diluent and dilute to the scale line, shake well, and obtain;

[0013] Preparation of reference solution: accurately weigh the protected amino acid enantiomer, dissolve it with a diluent and dilute it to a concentration of 0.03 mg / ml as the reference stock solution; accurately measure 1 ml of the reference stock solution, place it in a 20 ml volumetric flask, dissolve it with a diluent and dilute it to the scale line, shake well, and obtain;

[0014] Preparation of sensitivity solution: accurately measure 2 ml of reference solution, place in a 10 ml volumetric flask, dissolve with diluent and dilute to the scale line, shake well, and obtain;

[0015] Preparation of system suitability solution: accurately weigh 10 mg of the test sample, place it in a 20 ml volumetric flask, add 1 ml of the reference stock solution, dissolve it with a diluent and dilute to the scale, shake well, and the solution is ready;

[0016] (2) Chromatographic condition setting:

[0017] Chromatographic column: The silica gel surface is coated with cellulose-tris (4-methylbenzoate) chiral stationary phase as the chromatographic column;

[0018] Mobile phase: 5 mmol / L ammonium acetate solution (pH adjusted to 4.5 with acetic acid) as mobile phase A and acetonitrile as mobile phase B for gradient elution;

[0019] Column temperature: 20-30°C;

[0020] Flow rate: 0.3~1.0ml / min;

[0021] Injection volume: 1-10 μl;

[0022] Detection wavelength: 260~270nm;

[0023] (3) Detection and calculation:

[0024] Accurately measure the system suitability solution, reference solution and test solution, inject them into the liquid chromatograph respectively, and record the chromatogram; calculate the peak area of ​​the main peak and enantiomers by the external standard method.

[0025] According to an embodiment of the present invention, the diluent is an acetonitrile solution.

[0026] According to the embodiment of the present invention, gradient elution is performed according to the following table:

[0027]

[0028] According to an embodiment of the present invention, the column temperature is 20-30°C; for example: 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, preferably 25°C.

[0029] According to an embodiment of the present invention, the flow rate is 0.3 to 1.0 ml / min; for example: 0.3 ml / min, 0.4 ml / min, 0.5 ml / min, 0.6 ml / min, 0.7 ml / min, 0.8 ml / min, 0.9 ml / min, 1.0 ml / min, preferably 1.0 ml / min.

[0030] According to an embodiment of the present invention, the injection volume is 1 to 10 μl; for example: 1 μl, 3 μl, 5 μl, 7 μl, 9 μl, 10 μl; preferably 10 μl.

[0031] According to an embodiment of the present invention, the detection wavelength is 260-270 nm; for example: 260 nm, 261 nm, 262 nm, 263 nm, 264 nm, 265 nm, 266 nm, 267 nm, 268 nm, 269 nm, 270 nm; preferably 264 nm.

[0032] According to an embodiment of the present invention, the protected amino acid is Fmoc-Tyr(tBu)-OH.

[0033] The present invention also provides a method for determining enantiomers of polypeptide protected amino acids for determining the purity of protected amino acids in cetrorelix acetate starting raw materials, wherein the protected amino acid is Fmoc-Tyr(tBu)-OH, and the content of its corresponding isomers is not higher than 0.3%.

[0034] The beneficial effects of the present invention are:

[0035] 1. The mobile phases used in the present invention are ammonium acetate and acetonitrile, which are commonly used buffer salts and polar reagents, rarely react with samples, have good elution ability, and have low pressure in the chromatographic system. At the same time, acidic additives such as trifluoroacetic acid and formic acid are not used in the mobile phase system, thereby avoiding the problem of chiral chromatographic columns and instruments being corroded due to long-term use of acidic mobile phases.

[0036] 2. The present invention locates the impurity peaks of possible degradation impurities and process impurities in a protected amino acid Fmoc-Tyr(tBu)-OH, a starting material of cetrorelix acetate, such as tyrosine, Fmoc-Tyr(tBu)-Tyr(tBu)-OH, Fmoc-β-Ala-Tyr(tBu)-OH, Fmoc-β-Ala-OH, Fmoc-Osu and H-Tyr(tBu)-OH. The present invention adopts a gradient elution method for detection. By optimizing and setting reasonable elution conditions, these impurities can be accurately located, and the separation degree with the enantiomer Fmoc-D-Tyr(tBu)-OH meets the requirements, and can be eluted quickly; it does not interfere with the detection of enantiomers in the sample, and does not affect the detection of other batches of samples in the same chromatographic system. The problem of incomplete separation of the enantiomer Fmoc-D-Tyr(tBu)-OH from other impurities in the sample and the failure to meet the requirements for the separation degree of the main peak, which affects the peak area integration and leads to inaccurate detection results, is avoided.

[0037] 3. The present invention comprehensively designs and optimizes the mobile phase ratio, reference solution and elution conditions. During the operation of the liquid chromatography system, the analytical method of the present invention has a stable baseline, and the blank solvent and other impurities have basically no interference with the detection of enantiomers in the product. The impurity separation effect is outstanding and the specificity is good. The test data proves that the detection limit can reach 0.0449 μg / ml, and the sensitivity is extremely high. The test results can provide reliable and effective information for product research and development, production and purification, lay a solid foundation for establishing the quality control standard for protecting amino acids, and play a positive role in the quality control of the final product. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the system suitability spectrum of Fmoc-Tyr(tBu)-OH and its enantiomers in Example 1.

[0039] Figure 2 It is the system suitability spectrum of Fmoc-Tyr(tBu)-OH and its enantiomers in the comparative example.

[0040] Figure 3 This is the linear trend diagram of Fmoc-D-Tyr(tBu)-OH in Example 2. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with specific examples and drawings. The following examples are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto. Unless otherwise specified, the raw materials used can be obtained from the market or homemade.

[0042] Example 1 (reverse phase) (XQRK-SM-6)

[0043] The method for detecting the enantiomer of Fmoc-Tyr(tBu)-OH, the starting material of cetrorelix acetate, specifically comprises the following steps:

[0044] (1) Chromatographic conditions: A chromatographic column with a silica gel surface coated with cellulose-tri(4-methylbenzoate) filler was used as the chromatographic column, with a column length of 15 cm, an inner diameter of 4.6 mm, and a film thickness of 5 μm; the column temperature was 25°C, the flow rate was 1.0 ml / min, the injection volume was 10 μl, the detection wavelength was 264 nm, 5 mmol / L ammonium acetate solution (pH adjusted to 4.5 with acetic acid) was used as the mobile phase A, acetonitrile was used as the mobile phase B, and gradient elution was performed according to Table 1 below.

[0045] (2) Preparation of test solution: Take about 10 mg of the product, weigh accurately, place in a 20 ml volumetric flask, add 4 ml of acetonitrile to dissolve, add solvent to dilute to the scale, shake well, and obtain;

[0046] (3) Preparation of reference solution: Take about 10 mg of Fmoc-D-Tyr(tBu)-OH reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add 4 ml of acetonitrile to dissolve it, add solvent to dilute to scale, and shake well; accurately measure an appropriate amount, add solvent to dilute to a solution containing 0.03 mg per 1 ml, as the reference stock solution; accurately measure 1 ml of the reference stock solution, place it in a 20 ml volumetric flask, dissolve it with solvent and dilute to scale, and shake well;

[0047] (4) Preparation of sensitivity solution: Accurately measure 2 ml of the reference solution, place it in a 10 ml volumetric flask, dissolve it with diluent and dilute to the scale line, shake well, and obtain;

[0048] (5) Preparation of system suitability solution: Take about 10 mg of this product, weigh accurately, place in a 20 ml volumetric flask, add 4 ml of acetonitrile to dissolve, add 1 ml of reference substance stock solution, add solvent to dilute to the scale, shake well, and obtain.

[0049] (5) Detection: Accurately measure the system suitability solution, sensitivity solution, test solution and reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. In the system suitability solution, Fmoc-D-Tyr(tBu)-OH and Fmoc-Tyr(tBu)-OH peaks appear in sequence, and the separation degree between Fmoc-D-Tyr(tBu)-OH and its adjacent peaks should meet the requirements.

[0050] (7) In the chromatogram of the test solution, if there is a chromatographic peak with the same retention time as Fmoc-D-Tyr(tBu)-OH, the peak area calculated by the external standard method shall not exceed 0.3%.

[0051] Table 1 Chromatographic conditions Gradient elution program

[0052]

[0053] Table 2 Chemical structures of Fmoc-Tyr(tBu)-OH and Fmoc-D-Tyr(tBu)-OH

[0054]

[0055] Table 3

[0056] Compound Name Fmoc-D-Tyr(tBu)-OH Fmoc-Tyr(tBu)-OH Peak time min 13.195 14.421 Peak area ratio% 0.30 99.70 Separation / 4.1

[0057] from Figure 1 From the chromatogram and the results in Table 3, it can be seen that the separation degree between the enantiomer Fmoc-D-Tyr(tBu)-OH and the main component Fmoc-Tyr(tBu)-OH is 4.1, which is greater than 1.5, indicating that the present invention can effectively separate Fmoc-Tyr(tBu)-OH and the enantiomer Fmoc-D-Tyr(tBu)-OH.

[0058] Comparative Example

[0059] Patent CN111505161A uses cellulose-tris (3,5-dimethylphenylcarbamate) as a chromatographic column, trifluoroacetic acid: water = 1:1000 as mobile phase A, column temperature 25 ° C, acetonitrile as mobile phase B, mobile phase A: mobile phase B = 80:20 (V / V) for isocratic elution for 25 minutes, and detects the enantiomers of Fmoc series protected amino acids. The present invention finds through repeated test conditions that the main component and its enantiomer can be effectively separated under the liquid phase conditions and sample detection, but the target peak shape is not sharp enough, and the sensitivity is not high, which is not easy to judge and identify; see Figure 2 The system suitability spectrum of Fmoc-Tyr(tBu)-OH and enantiomers is shown. Therefore, it is impossible to quantitatively analyze the enantiomers in the starting material and accurately detect the content in multiple batches of samples. Such chromatographic conditions are not suitable for quantitative detection of enantiomers of multiple protected amino acids in cetrorelix acetate, and further exploration of chromatographic methods is required.

[0060] Example 2

[0061] The invention adopts an external standard method to calculate the content of enantiomers, and performs a series of methodological verifications on the method, including linearity, detection limit, repeatability, accuracy, intermediate precision, solution placement stability and durability tests; the results show that the method has strong specificity and high accuracy, and can be used to detect the content of the enantiomer Fmoc-D-Tyr(tBu)-OH in the material Fmoc-Tyr(tBu)-OH.

[0062] 1. Methodological Validation:

[0063] 1. Exclusivity

[0064] Solution preparation:

[0065] 1) Blank solvent: same as diluent, acetonitrile-water (60:40).

[0066] 2) System suitability solution: Take about 10 mg of this product, accurately weigh it, and place it in a 20 ml volumetric flask. Accurately measure 1 ml of the reference substance stock solution and place it in the same volumetric flask. Add 4 ml of acetonitrile to dissolve it, then add solvent to dilute to the scale and shake well.

[0067] 3) Impurity mixed stock solution: Accurately weigh about 5 mg of each of the impurity reference substances H-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-Tyr(tBu)-OH, Fmoc-β-Ala-Tyr(tBu)-OH, Fmoc-β-Ala-OH, tyrosine, and impurity Fmoc-OSu, place them in the same 50 ml volumetric flask, add 10 ml of acetonitrile to dissolve, dilute to the mark with solvent, and shake well. (about 0.1 mg / ml)

[0068] 4) Mixed solution: weigh about 10 mg of this product accurately, place it in a 20 ml volumetric bottle, accurately measure 2 ml of impurity mixed stock solution, add solvent to dilute to the scale, and shake well. (about 0.01 mg / ml)

[0069] Table 4

[0070]

[0071] The blank solvent has no interference with the detection of enantiomers in this product; in addition, under the chromatographic conditions, the impurities tyrosine, Fmoc-Tyr(tBu)-Tyr(tBu)-OH, Fmoc-β-Ala-Tyr(tBu)-OH, Fmoc-β-Ala-OH, Fmoc-Osu, and H-Tyr(tBu)-OH have no interference with the determination of the enantiomer Fmoc-D-Tyr(tBu)-OH of this product, indicating that the method of the present invention has good specificity.

[0072] 2. Linearity and range

[0073] Preparation of linear solutions: Accurately measure the enantiomer Fmoc-D-Tyr(tBu)-OH stock solution to prepare a series of linear concentration solutions with concentrations of 0.1497, 0.4492, 0.7487, 1.4975, and 2.2462 μg / ml. Inject them into the liquid chromatograph and record the chromatogram. Perform linear regression with Fmoc-D-Tyr(tBu)-OH concentration (μg / ml) as the horizontal axis and peak area as the vertical axis.

[0074] Table 5

[0075]

[0076] From the results in Table 4, it can be concluded that the linear equation of the concentration and peak area of ​​the enantiomer Fmoc-D-Tyr(tBu)-OH is y=23.6921x+0.1271, the correlation coefficient r=0.9999, and the absolute value of the ratio of the Y-axis intercept to the 100% response value is 0.6, which all meet the test requirements. This shows that Fmoc-D-Tyr(tBu)-OH has good linearity in the range of 0.1497 to 2.2462 μg / ml.

[0077] 3. Limit of detection and limit of quantification

[0078] Accurately measure 1 ml of Fmoc-D-Tyr(tBu)-OH reference solution, place it in a 10 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain the quantification limit solution (LOQ solution); calculate the signal-to-noise ratio (S / N) of the enantiomer Fmoc-D-Tyr(tBu)-OH peak and the RSD of the peak area after 6 consecutive injections of the LOQ solution.

[0079] Accurately measure 3 ml of the quantitative limit solution, place it in a 10 ml volumetric bottle, dilute it to the mark with a diluent, and shake it well to obtain the detection limit solution (LOD solution); calculate the signal-to-noise ratio (S / N) of the enantiomer Fmoc-D-Tyr(tBu)-OH peak after injecting the LOD solution once. The results are shown in Table 6 below.

[0080] Table 6

[0081]

[0082] The results show that the S / N values ​​of the six quantitative limit solutions of Fmoc-D-Tyr(tBu)-OH are all greater than 10, with the minimum value being 49; the RSD of the six peak areas is 1.0%, which meets the requirements. The S / N value of the detection limit solution of Fmoc-D-Tyr(tBu)-OH is 17, which is greater than 3 and meets the requirements, indicating that this method has good sensitivity.

[0083] 4. Accuracy

[0084] Preparation of test solution: accurately weigh 10 mg of the test sample, place it in a 20 ml volumetric flask, dissolve it with diluent and dilute to the scale line, shake well, and obtain;

[0085] Preparation of reference solution: accurately weigh the protected amino acid enantiomer, dissolve it with a diluent and dilute it to a concentration of 0.03 mg / ml as the reference stock solution; accurately measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, dissolve it with a diluent and dilute it to the scale line, shake well, and obtain;

[0086] Quantitative limit accuracy solution: weigh about 10 mg of Fmoc-Tyr(tBu)-OH, place in a 20 ml volumetric flask, add about 5 ml of diluent, dissolve by ultrasonication, accurately add 0.1 ml of reference substance stock solution, dilute to the scale with diluent, shake well and obtain; prepare 3 portions in parallel and inject 1 needle into each.

[0087] 100% limit accuracy solution: weigh about 10 mg of Fmoc-Tyr(tBu)-OH, place in a 20 ml volumetric flask, add about 5 ml of diluent, dissolve by ultrasonication, accurately add 1.0 ml of reference substance stock solution, dilute to the scale with diluent, and shake well; prepare 3 portions in parallel and inject 1 needle into each.

[0088] 150% limit accuracy solution: weigh about 10 mg of Fmoc-Tyr(tBu)-OH, place in a 20 ml volumetric flask, add about 5 ml of diluent, dissolve by ultrasonication, accurately add 1.5 ml of reference substance stock solution, dilute to the scale with diluent, shake well and obtain; prepare 3 portions in parallel and inject 1 needle into each.

[0089] The recovery rate (%) was calculated according to the formula (AB) / C×100%, where A is the measured amount, B is the background amount, and C is the added amount; the calculation results are shown in Table 7 below.

[0090] Table 7

[0091]

[0092] The recoveries of low, medium and high concentration levels were 114.3-115.2%, 102.8-103.8% and 103.0-103.3% respectively, and the RSD of the recoveries of 9 samples of low, medium and high levels was 5.4%, which all met the requirements, indicating that the accuracy of this method was good.

[0093] 5. Repeatability

[0094] Weigh about 10 mg of Fmoc-Tyr(tBu)-OH, place in a 20 ml volumetric flask, add about 5 ml of diluent, dissolve by ultrasonication, accurately add 1.0 ml of reference substance stock solution, dilute to the scale with diluent, and shake well; prepare 6 portions in parallel and inject 1 needle into each; the results are shown in Table 8 below.

[0095] Table 8

[0096]

[0097]

[0098] The RSD of the content values ​​of 6 reproducible solutions was 0.5%, which met the requirements, indicating that this method had good repeatability.

[0099] 6. Intermediate precision

[0100] The results under the repeatability item are regarded as the experimental results of the first person.

[0101] The same batch of samples were tested by different testers on different days using different instruments with the same method. Six intermediate precision solutions were prepared according to the method under the repeatability item.

[0102] Table 9

[0103]

[0104] Table 10

[0105]

[0106] The RSD value of the content measured in 6 intermediate precision solutions was 0.3%, and the RSD value of the content measured in 12 solutions was 1.6%, both less than 10%, meeting the requirements.

[0107] 7. Solution stability

[0108] Take the reference solution and place it at room temperature. Measure the peak area of ​​the enantiomer Fmoc-D-Tyr(tBu)-OH at 0 hour, 4 hours, 12 hours, and 17 hours, respectively. Calculate the change in the peak area of ​​the enantiomer in the reference solution at each time point relative to 0 hour, and examine the stability of the solution. See Table 11.

[0109] Take the spiked test solution and place it at room temperature. Determine the peak area of ​​the enantiomer Fmoc-D-Tyr(tBu)-OH at 0 hours, 5 hours, 11 hours, and 16 hours, respectively. Calculate the enantiomer content in the spiked test solution at each time point and the change value relative to the content at 0 hours to investigate the stability of the solution. See Table 12.

[0110] Table 11

[0111]

[0112]

[0113] Table 12

[0114] Time (h) Peak area content(%) Change value (%) 0 36.366 0.285 - 5 36.419 0.286 0.4 11 36.319 0.285 0.0 16 36.361 0.285 0.0

[0115] From the table above, it can be seen that the maximum change in the peak area of ​​the reference solution at room temperature for 4h, 12h, and 17h compared with 0h is 0.5%, indicating that the reference solution is stable at room temperature for 17 hours. The maximum change in the content of the spiked test solution at room temperature for 5h, 11h, and 16h compared with 0h is 0.4%, indicating that the spiked test solution is stable at room temperature for 16 hours.

[0116] 8. Durability test

[0117] When the instrument chromatographic parameters change slightly, such as the pH value of the mobile phase buffer salt, column temperature and flow rate, examine whether the blank solvent interferes with the detection of this product. In the system suitability solution, the separation between the enantiomer and the main peak should be no less than 1.5; and after changing the chromatographic conditions, the enantiomer content in the spiked test solution should change by less than 10% compared with the value under normal chromatographic conditions; the results are shown in Table 13 below.

[0118] Table 13

[0119]

[0120] When the instrument chromatographic parameters were changed, the blank solvent did not interfere with the determination of the enantiomer Fmoc-D-Tyr(tBu)-OH under each chromatographic condition; the system suitability solution separation was between 1.5 and 2.3 under each chromatographic condition, which met the requirements. After changing the conditions, the enantiomer detection amount in the spiked test solution and the change value of the determination result under the proposed conditions were between 0.0% and 0.7%, which were all less than 10%. This shows that the method has good durability.

Claims

1. A method for determining enantiomers of protected amino acids of a polypeptide, characterized in that: The following steps are involved: (1) Sample preparation: Preparation of test solution: Accurately weigh 10 mg of the test sample, place it in a 20 ml volumetric flask, dissolve it with diluent and dilute to Graduation line, shake well, and you are done; Preparation of reference solution: Accurately weigh the protected amino acid enantiomer, dissolve it in a diluent and dilute it to a concentration of 0.03 mg / ml solution was used as the reference stock solution; Accurately measure 1 ml of the reference stock solution, place it in a 20 ml volumetric flask, dissolve it with a diluent and dilute it to the mark, shake well, and you have it. Preparation of sensitivity solution: accurately measure 2 ml of reference solution, place in a 10 ml volumetric flask, dissolve with diluent and dilute to the scale line, shake well, and obtain; Preparation of system suitability solution: accurately weigh 10 mg of the test sample, place it in a 20 ml volumetric flask, add 1 ml of the reference stock solution, dissolve it with a diluent and dilute to the scale, shake well, and the solution is ready; (2) Chromatographic condition setting: Chromatographic column: The silica gel surface is coated with cellulose-tris (4-methylbenzoate) chiral stationary phase as the chromatographic column; Mobile phase: Use 5mmol / L ammonium acetate solution (pH adjusted to 4.5 with acetic acid) as mobile phase A and acetonitrile as mobile phase B for gradient elution. Perform gradient elution according to the following table: ； Column temperature: 20~30℃; Flow rate: 0.3~1.0ml / min; Injection volume: 1~10µl; Detection wavelength: 260~270nm; (3) Detection and calculation: Accurately measure the system suitability solution, sensitivity solution, reference solution and test solution, inject them into the liquid chromatograph respectively, and record the chromatogram; calculate the peak area of ​​the main peak and enantiomers by the external standard method.

2. The measuring method according to claim 1, characterized in that The diluent is acetonitrile solution.

3. The measuring method according to claim 1, characterized in that The column temperature was 25°C.

4. The measuring method according to claim 1, characterized in that The flow rate is 1.0 ml / min.

5. The measuring method according to claim 1, characterized in that The injection volume was 10 μl.

6. The measuring method according to claim 1, characterized in that The wavelength is 264 nm.

7. The measuring method according to claim 1, characterized in that In the sensitive solution, the signal-to-noise ratio of the peak height is greater than 10.

8. The measuring method according to claim 1, characterized in that The protected amino acid is Fmoc-Tyr(tBu)-OH.

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