Method for determining chiral amino acids in cetrocix acetate

Abstract

The application discloses a method for determining chiral amino acids in cetrotide acetate. Specifically relates to a method for determining the content of chiral amino acids in cetrotide acetate based on pre-column derivatization high performance liquid chromatography. Simple operation, without extraction separation of the sample after derivatization, the content of 10 kinds of chiral amino acids after hydrolysis of cetrotide acetate can be determined simultaneously, the sensitivity is high, more basis can be provided for structure confirmation, and reference can be provided for separation of chiral amino acids of polypeptide drugs.

Description

Technical Field

[0001] This invention relates to a method for determining chiral amino acids in cetrorex acetate, belonging to the field of biopharmaceutical quality control technology. Background Technology

[0002] Cetrorelix acetate, also known as cetrorelix, is a synthetic polypeptide composed of 10 amino acids. It is a representative of third-generation gonadotropin-releasing hormone antagonists and can stimulate ovulation to obtain an appropriate number of high-quality mature oocytes. Currently, only the original drug is available for injection in China, resulting in a favorable competitive landscape. It is a recommended drug in assisted reproductive technology guidelines and has promising clinical prospects. Cetrorelix is ​​a synthetic decapeptide with the chemical name: 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-alanylamide acetate.

[0003] The structure is as follows:

[0004]

[0005] The bioactivity of peptide drugs is closely related to the chirality of their amino acids. In studying the primary structure of cetrorex acetate, it is necessary to react the peptide at 6 mol·L⁻¹. -1 The reaction of cetrorelic acid with hydrochloric acid at 110°C may cause racemization of chiral amino acids. The "Technical Guidelines for Pharmaceutical Research of Chemically Synthesized Peptide Drugs (Trial Version)" issued by the Center for Drug Evaluation (CDE) in February 2023 requires control of isomers of amino acids containing chiral centers. Therefore, it is necessary to study and control the content of chiral amino acids in peptide synthesis. Currently, no pharmacopoeias of various countries include quality standards for cetrorelic acetate, and there are few reports on its quality research. Summary of the Invention

[0006] The purpose of this invention is to provide a method for determining chiral amino acids in cetrorex acetate. Specifically, it relates to a method for determining the content of chiral amino acids in cetrorex acetate based on pre-column derivatization high-performance liquid chromatography. The method is simple to operate, requires no extraction and separation of the derivatized sample, and can simultaneously determine the configuration and content of 10 chiral amino acids after hydrolysis of cetrorex acetate, exhibiting high sensitivity.

[0007] The technical solution to achieve the above-mentioned objective is:

[0008] This invention provides a method for determining chiral amino acids in cetrorex acetate, comprising the following steps:

[0009] (1) Sample preparation:

[0010] Preparation of chiral amino acid reference solution: Accurately weigh appropriate amounts of the 10 amino acid reference standards that constitute cetrorex acetate, dissolve and dilute with diluent to a concentration of 40 μmol·L⁻¹. -1 The solution;

[0011] Preparation of mixed solution of chiral amino acid control and enantiomer amino acid control: Accurately weigh appropriate amounts of the 10 amino acids constituting cetrorex acetate and their enantiomer reference standards, dissolve and dilute with diluent to a concentration of 20 μmol·L⁻¹. -1 The solution;

[0012] Preparation of test solution: Accurately weigh an appropriate amount of cetrorex acetate, and after hydrolysis, dissolve and dilute with a diluent to a concentration of 40 μmol·L⁻¹. -1 The solution;

[0013] (2) Derivatization reaction:

[0014] Take the chiral amino acid reference solution, the mixed solution of chiral amino acid reference and enantiomer amino acid reference and the test solution from step (1), and add Marfey derivatizing reagent under alkaline conditions to react and obtain the derivatized chiral amino acid reference solution, the mixed solution of derivatized chiral amino acid and enantiomer amino acid reference and the derivatized test solution.

[0015] A derivatized blank solution was obtained by adding Marfey's derivatizing reagent to an aqueous solution with a pH of approximately 7.0 under alkaline conditions.

[0016] (3) Chromatographic conditions settings:

[0017] Column: Hedera ODS-2C18 column;

[0018] Mobile phase: Gradient elution was performed using 0.065% formic acid solution as mobile phase A and acetonitrile as mobile phase B;

[0019] Column temperature: 30~45℃;

[0020] Flow rate: 0.5–1.1 ml / min;

[0021] Injection volume: 1–10 μl;

[0022] Detection wavelength: 340nm;

[0023] (4) Detection and calculation:

[0024] Accurately measure the derivatized blank solution, the derivatized chiral amino acid reference solution, and the mixed solution of derivatized chiral amino acid reference and enantiomer amino acid reference, and inject them into the liquid chromatograph. Record the chromatograms, calculate the peak area of ​​each derivatized amino acid by high performance liquid chromatography, and determine the content of 10 chiral amino acids by external standard method.

[0025] According to an embodiment of the present invention, the hydrolysis reaction in step (1) involves reacting cetrorex acetate with 6 mol·L⁻¹ water. -1 Dissolve and dilute with hydrochloric acid to prepare a solution with a concentration of 1.0 mmol·L⁻¹. -1 The solution was filled with nitrogen, sealed, and placed in a 110℃ oven for 6-8 hours to hydrolyze and obtain the aqueous solution of the free amino acids to be tested.

[0026] The 10 chiral amino acids obtained after hydrolysis of cetrirexate are: L-arginine (L-Arg), 3-(3-pyridyl)-D-alanine (D-Pal), L-tyrosine (L-Tyr), D-citrulline (D-Cit), L-serine (L-Ser), L-proline (L-Pro), D-alanine (D-Ala), L-leucine (L-Leu), D-4-chlorophenylalanine (D-Phe), and D-3-(2-naphthyl)-alanine (D-Nal).

[0027] The 10 enantiomers of cetrirexate after hydrolysis are: D-arginine (D-Arg), 3-(3-pyridyl)-L-alanine (L-Pal), D-tyrosine (D-Tyr), L-citrulline (L-Cit), D-serine (D-Ser), D-proline (D-Pro), L-alanine (L-Ala), D-leucine (D-Leu), L-4-chlorophenylalanine (L-Phe), and L-3-(2-naphthyl)-alanine (L-Nal).

[0028] This invention further investigated the effect of cetrorex acetate hydrolysis reaction time on chiral amino acids and the formation of their enantiomers. The specific process was as follows: 6 mol·L⁻¹ of cetrorex acetate was taken... -1 Hydrochloric acid solutions were heated at 110℃ for 4, 6, 8, 12, and 24 hours before measurement. Results showed that after 4 hours of hydrolysis, the peptides gradually began to hydrolyze, producing small amounts of D-Arg and D-Pro isomers, and a small amount of D-Cit degradation products. From 4 to 8 hours, the peak areas of most amino acid derivatives gradually increased with increasing hydrolysis time, reaching their maximum values. The peak areas of isomers and degradation products did not increase significantly. From 6 to 8 hours, the peak areas of most amino acid derivatives remained essentially unchanged.

[0029] According to an embodiment of the present invention, the pH of each solution is adjusted to 7.0 to 7.5 during the sample preparation process in step (1). For example, it can be 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5.

[0030] Preferably, during sample preparation, 0.1–1.0 mol·L⁻¹ are used respectively. -1 Hydrochloric acid or 0.1–1.0 mol·L -1 The pH was adjusted to around 7.0 using a sodium hydroxide solution.

[0031] According to an embodiment of the present invention, depending on the different solubilities of each chiral amino acid, the diluent in step (1) can be selected from 1 to 10 mol·L⁻¹. -1 hydrochloric acid solution or 1–10 mol·L -1 Sodium hydroxide solution.

[0032] Since most of the 10 chiral amino acids involved in this invention, except for those containing aromatic ring structures, do not contain chromophores, this invention further employs pre-column derivatization. Pre-column derivatization is a method that utilizes a derivatizing reagent to react with amino acids to form diastereomeric derivatized products. This invention selects Marfey's reagent as the derivatizing reagent, which possesses advantages such as strong UV absorption functional groups, high specificity and stability of the derivatized products, and simple derivatization operation.

[0033] According to an embodiment of the present invention, the Marfey derivative reagent in step (2) is selected from one or more of FDAA, FDVA, FDPA, FDIA, and FDLA solutions.

[0034] Specifically, the Marfey-derived reagent has the following structural formula:

[0035]

[0036] Preferably, the Marfey derivatizing reagent is selected from FDLA solution, and more preferably L-FDLA solution. The L-FDLA solution is prepared by dissolving an appropriate amount of L-FDLA in dimethyl sulfoxide to a solution containing approximately 1.0 g·L⁻¹. -1 The solution.

[0037] According to an embodiment of the present invention, the alkaline condition in step (2) is 0.1 mol·L⁻¹. -1 The derivatization reaction takes place in a sodium bicarbonate solution.

[0038] According to an embodiment of the present invention, the derivatization reaction in step (2) involves adding the reaction solution to 0.1 mol·L⁻¹. -1 Sodium bicarbonate solution with 1.0 g·L -1The L-FDLA derivatizing reagent was mixed well and heated at 35–45 °C for 0.5–1.5 h. Then, 0.1 mol·L⁻¹ was added. -1 The reaction was terminated with 10 μL of hydrochloric acid solution; the supernatant was collected after centrifugation for 10 min.

[0039] According to an embodiment of the present invention, gradient elution is performed in step (3) according to the following table:

[0040]

[0041] According to an embodiment of the present invention, in step (3), the chromatographic column has a size of 4.6 mm × 250 mm and a diameter of 5 μm; the flow rate is 1.0 mL·min. -1 Column temperature: 35℃; injection volume: 10μL; wavelength: 340nm.

[0042] According to the embodiment of the present invention, in step (4), the chiral amino acid content is calculated by taking the reference standard stock solution and preparing concentrations of 10.20, 16.33, 20.41, 22.45, and 24.49 μmol·L⁻¹, respectively. -1 The linear test solutions of a series of concentrations were derivatized according to the method in step (2). The peak area was plotted as the vertical axis and the molar concentration as the horizontal axis, and linear regression calculation was performed. The content of 10 chiral amino acids was calculated based on the peak area measured in step (4).

[0043] Beneficial effects of this invention:

[0044] 1. The method for detecting chiral amino acids disclosed in this invention is easy to operate, does not require extraction and separation of derivatized samples, and is suitable for quantitative detection and analysis of chiral amino acids after peptide hydrolysis.

[0045] 2. The L-FDLA derivatizing reagent of this invention contains a 2,4-dinitrophenyl structure and a leucine chiral center, which derivatizes amino acids. The derivatized amino acid molecules can show a signal in a UV detector. At the same time, the leucine chiral center of L-FDLA introduces a second chiral center into the chiral isomer of the amino acid, turning a pair of enantiomers into diastereomers. Since the two compounds have different affinities for silica gel, they can be separated using inexpensive and commonly used achiral chromatographic columns without the need for chiral columns. The detection of the same type of amino acid uses expensive mass spectrometry, while this invention only requires ordinary HPLC for detection. Therefore, this invention greatly reduces the analytical cost.

[0046] 3. The method used in this invention can simultaneously determine the content of 10 chiral amino acids after hydrolysis of cetrorex acetate. The method is efficient and simple, and can provide a basis for the structural confirmation of cetrorex acetate and a reference for the separation of chiral amino acids in polypeptide drugs. Attached Figure Description

[0047] Figure 1 This is the chromatogram of the derivatized blank solution.

[0048] Figure 2 This is a mixed solution of derivatized chiral amino acid control and enantiomeric amino acid control.

[0049] Figure 3 The chromatogram is for the derivatized test sample solution.

[0050] Figure 4 This is a graph showing the relationship between the hydrolysis time of cetrirexate acetate and the peak area of ​​the derivatized products.

[0051] Figure 5 This is a linear graph of arginine (Arg) in Experimental Example 2 of this invention.

[0052] Figure 6 This is a linear graph of 3-(3-pyridyl)-alanine (Pal) in Experimental Example 2 of the present invention.

[0053] Figure 7 This is a linear graph of tyrosine (Tyr) in Experimental Example 2 of this invention.

[0054] Figure 8 This is a linear graph of the amino acid citrulline (Cit) in Experimental Example 2 of this invention.

[0055] Figure 9 This is a linear graph of serine (Ser) in Experimental Example 2 of the present invention.

[0056] Figure 10 This is a linear graph of proline (Pro) in Experimental Example 2 of the present invention.

[0057] Figure 11 This is a linear graph of alanine (Ala) in Experimental Example 2 of this invention.

[0058] Figure 12 This is a linear graph of leucine (Leu) in Experimental Example 2 of the present invention.

[0059] Figure 13 This is a linear graph of 4-chlorophenylalanine (Phe) in Experimental Example 2 of this invention.

[0060] Figure 14 This is a linear graph of 3-(2-naphthyl)-alanine (Nal) in Experimental Example 2 of this invention. Detailed Implementation

[0061] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The following embodiments are only descriptive and not limiting, and should not be used to limit the scope of protection of the present invention. Unless otherwise specified, all raw materials used can be obtained commercially or in-house.

[0062] Since most of the 10 chiral amino acids involved in this invention, except for those containing aromatic ring structures, do not contain chromophores, this invention further employs pre-column derivatization reaction treatment.

[0063] Example 1:

[0064] A method for detecting the chiral amino acid content in cetrorexate acetate, specifically comprising the following steps:

[0065] (1) Chromatographic conditions settings:

[0066] Column: Hedera ODS-2C18, 4.6 mm × 250 mm, 5 μm;

[0067] Column temperature: 35℃;

[0068] Detection wavelength: 340nm;

[0069] Flow rate: 1.0 ml / min;

[0070] Injection volume: 10 μL

[0071] Mobile phase A: 0.065% formic acid solution;

[0072] Mobile phase B: Acetonitrile;

[0073] Perform gradient elution according to the table below:

[0074]

[0075] (2) Preparation of diluent: Diluent 1: 6 mol·L -1 Hydrochloric acid solution, diluent 2: 1 mol·L -1 Sodium hydroxide solution;

[0076] (3) Preparation of L-FDLA solution: Take an appropriate amount of L-FDLA, add dimethyl sulfoxide to dissolve it to prepare a solution containing approximately 1.0 g·L⁻¹ -1 The solution;

[0077] (4) Preparation of test solution: Accurately weigh an appropriate amount of cetrilac acetate and prepare it using 6 mol·L⁻¹ solution. -1 Dissolve and dilute with hydrochloric acid to prepare a solution with a concentration of 1.0 mmol·L⁻¹. -1 The solution was purged with nitrogen, sealed, and placed in a 110℃ oven for 8.0 h of hydrolysis. Afterward, it was removed, cooled, unsealed, and treated with 2 mol·L⁻¹ solution. -1 Sodium hydroxide solution and 0.1 mol·L -1 The pH of the sodium hydroxide solution was adjusted to approximately 7.0, ultimately yielding a concentration of 40 μmol·L⁻¹. -1 The solution;

[0078] (5) Preparation of chiral amino acid reference solution: Accurately weigh appropriate amounts of the 10 amino acid reference standards that constitute cetrorex acetate, dissolve them in a suitable solvent to obtain a concentration of 1.0 mmol·L⁻¹. -1 The stock solution. Using 0.1 mol·L⁻¹ -1 Hydrochloric acid or 0.1 mol·L -1 The pH of the sodium hydroxide solution was adjusted to approximately 7.0, ultimately yielding a concentration of 40 μmol·L⁻¹. -1 The solution;

[0079] (6) Preparation of a mixed solution of chiral amino acid control and enantiomer amino acid control: Accurately weigh appropriate amounts of the 10 amino acids that make up cetrorex acetate and their enantiomer reference standards, dissolve them in a suitable solvent to obtain a concentration of 0.5 mmol·L⁻¹. -1 The stock solution. Using 0.1 mol·L⁻¹ -1 Hydrochloric acid or 0.1 mol·L -1 The pH of the sodium hydroxide solution was adjusted to approximately 7.0, resulting in a final concentration of 20 μmol·L⁻¹. -1 The solution;

[0080] (7) Derivatization blank solution: Accurately measure 125 μL of an aqueous solution adjusted to approximately pH 7.0 and place it at the bottom of a 1.5 mL polypropylene tube. Add 0.1 mol·L⁻¹ -1 10 μL of sodium bicarbonate solution with 1.0 g·L⁻¹ -1 100 μL of L-FDLA derivatizing reagent was mixed well and placed in a 37℃ electric heating drying oven for 1 hour. After that, it was removed and 0.1 mol·L⁻¹ was added. -1 The reaction was terminated by adding 10 μL of hydrochloric acid solution; the reaction was then centrifuged at 12000 r·min. -1 Centrifuge for 10 min and collect the supernatant to obtain the derivatized blank solution;

[0081] (8) Derivatization procedure: Accurately measure 125 μL of amino acid reference solution and place it at the bottom of a 1.5 mL polypropylene tube, then add 0.1 mol·L⁻¹ -1 10 μL of sodium bicarbonate solution was mixed with 100 μL of 1.0 g·L⁻¹ L-FDLA derivatizing reagent, and the mixture was placed in a 37 °C electric heating drying oven for 1 h. After removal, 0.1 mol·L⁻¹ L ... -1 The reaction was terminated by adding 10 μL of hydrochloric acid solution; the reaction was then centrifuged at 12000 r·min. -1 Centrifuge for 10 min and collect the supernatant to obtain the derivatized control solution. The derivatized test solution and the mixed control solution of chiral amino acids and enantiomers of amino acids are processed using the same method.

[0082] (9) Detection: Accurately measure the derivatized blank solution, derivatized chiral amino acid reference solution, derivatized test solution and the mixed solution of derivatized chiral amino acid reference and enantiomer amino acid reference, inject them into the liquid chromatograph, record the chromatogram, calculate the peak area of ​​each derivatized amino acid by high performance liquid chromatography, and determine the content of 10 chiral amino acids by external standard method.

[0083] Test results are as follows Figure 1 As shown in Table 1, the corresponding retention times are as follows. The 10 chiral amino acids were completely separated from their enantiomers (resolution > 1.0) and were not affected by the derivatizing reagents and their byproducts.

[0084] Table 1

[0085]

[0086]

[0087] Structural confirmation via mass spectrometry:

[0088] 1. Samples are the same as those under the chromatographic conditions.

[0089] 2. Mass spectrometry conditions: Electrospray ionization (ESI) source, positive ion mode acquisition; Spray voltage: 4000 Ω; Sheath gas pressure: 35 psi; Ion sweep gas pressure: 0.5 psi; Aux gas pressure: 5 psi; Capillary temperature: 350 °C; Tube lens offset: 44 V; Skimmer offset: 0 V; Collision pressure: 0 psi.

[0090] 3. The results are shown in Table 2:

[0091] Table 2

[0092]

[0093] 4. Conclusion: Mass spectrometry analysis confirmed that the amino acid analysis results were correct.

[0094] Example 2: Selection of Derivative Conditions

[0095] Factors affecting the reaction between L-FDLA and amino acids were investigated. Reactions were performed at 30, 35, 40, 45, 50, and 60 °C for the same time, or at the same temperature for 0.5, 1.0, 1.5, and 2 h, respectively. The results showed that higher reaction temperatures resulted in larger derivative peak areas. However, above 45 °C, impurities generated by the derivatizing reagent significantly interfered with the determination of sample peaks. No significant interference was observed when reacting at approximately 35–45 °C for 0.5–1.5 h. Longer reaction times resulted in larger derivative peak areas. Considering experimental efficiency and previously reported methods, the reaction time was limited to 1.0 h. Experiments with a molar ratio of the total molar sum of 10 chiral amino acids to the amount of L-FDLA ranging from 1:1 to 1:10 showed that a molar ratio of approximately 1:5 achieved the maximum yield.

[0096] Example 3 Selection of hydrolysis conditions

[0097] Take 6 mol·L of cetrilac acetate -1 Hydrochloric acid solution was heated at 110℃ for 4, 6, 8, 12, and 24 hours, and the hydrolysis time was measured. The 8-hour time point, where most amino acids showed significant hydrolysis and a relatively stable trend, was selected as the hydrolysis endpoint. At 8 hours, the racemization degree of the 10 amino acids composing cetrilac acetate was low. The results of the hydrolysis time study are shown in […]. Figure 2 .

[0098] Example 4 Selection of chromatographic conditions

[0099] The separation effects of different mobile phase systems on amino acid derivatives produced by the hydrolysis of cetrirexate acetate were investigated on a C18 column. The results showed that when using a methanol-water system as the mobile phase, some amino acids exhibited tailing, and the resolution was less than 1.0. When using an acetonitrile-phosphate system as the mobile phase, regardless of adjustments to pH, column temperature, gradient, or column replacement, it was impossible to achieve a resolution of over 1.0 for all amino acids. Finally, the acetonitrile-formic acid-water system, with approximately 0.065% formic acid, achieved good separation of all amino acids with good peak shapes.

[0100] Precisely measured 10 μL of each of the following solutions were injected into the liquid chromatograph: derivatized blank solution, derivatized reference solution, derivatized test solution, and a mixed solution of derivatized chiral amino acid reference and enantiomer amino acid reference. Chromatograms were recorded. The results showed that the 10 chiral amino acids and their enantiomers constituting cetrilac acetate were not affected by the blank. In the derivatized reference solution, the minimum resolution between each amino acid derivative peak and its adjacent peak was 1.19. The content matching of the two derivatized reference solutions should be between 96% and 105%. After five consecutive injections of the first derivatized reference solution, the RSD of each amino acid derivative peak was no greater than 0.8%, indicating good injection precision. In the derivatized test solution, other peaks did not interfere with the determination of the target amino acid derivative peaks. The method has good specificity.

[0101] Test Example 1: Limit of Detection and Limit of Quantification

[0102] Accurately transfer the amino acid reference solution from "Example 1", serially dilute it to appropriate factors, and then perform derivatization treatment as in "Example 1" before injection. The limits of detection (LOD) and quantitation (LOQ) were defined as concentrations with a signal-to-noise ratio (S / N) of not less than 3 and 10, respectively. The results showed that the LOD and LOQ were 0.68 μmol·L⁻¹. -1 and 2.04 μmol·L -1 The derivatization solution at the limit of quantitation concentration was injected six times consecutively. The maximum RSD% of the peak area of ​​the amino acid derivative was 4.2, and the maximum RSD% of the retention time was 0.2, indicating that the precision of the limit of quantitation determination was good.

[0103] Experimental Example 2: Linearity and Range

[0104] Accurately transfer the amino acid reference stock solution from "Example 1" to prepare concentrations of 10.20, 16.33, 20.41, 22.45, and 24.49 μmol·L⁻¹ sequentially. -1 A series of linear test solutions with different concentrations were prepared. Derivatization was performed according to the method described in "Example 1". 10 μL of each solution was injected sequentially for analysis, and chromatograms were recorded. A graph was plotted with peak area on the ordinate and molar concentration on the abscissa, and linear regression calculations were performed. The results showed that the concentration ranged from 10.20 to 22.45 μmol·L⁻¹. -1 Within the concentration range, the 10 amino acid derivatives constituting cetrorex acetate showed good linearity, with r values ​​all greater than 0.991, and the Y-intercept should be within 25% of the 100% response value. Figures 5-12 As shown.

[0105] Experimental Example 3: Repeatability and Intermediate Precision Test

[0106] Following the procedures outlined in "Example 1," derivatization blank solution, derivatization reference solution, and six parallel derivatization test solutions were prepared. 10 μL of each solution was precisely injected into the liquid chromatograph to assess repeatability. The results showed that the RSD of the derivatized contents of the 10 amino acids constituting cetrorex acetate ranged from 0.41% to 0.90%, indicating good repeatability. Different analysts repeated the above experiments on different dates to assess the intermediate precision of the method. The results showed that the RSD of the derivatized contents of the 10 amino acids constituting cetrorex acetate ranged from 0.23% to 1.6%. In the precision test, the RSD of the derivatized contents of the 10 amino acids constituting cetrorex acetate ranged from 0.46% to 5.8%, indicating good intermediate precision of the method.

[0107] Experiment Example 4: Stability Test

[0108] According to "Example 1", derivatization blank solution, derivatization control solution, and derivatization test solution were prepared respectively. After being placed at room temperature for 0, 11, 14, 17, 22, and 40 hours, samples were injected for analysis. The peak area of ​​each amino acid after derivatization was recorded, and the content of each amino acid derivative in the test sample was calculated. The results showed that the RSD of the peak area of ​​the 10 amino acids constituting cetrorex acetate after derivatization in the control solution was all less than 1.8%; the RSD of the content of the 10 amino acid derivatization products in the hydrolyzed test sample solution was all less than 1.8%, indicating that the solution had good stability within 40 hours.

[0109] Test Example 5: Durability Test

[0110] Change the amount of formic acid added in the mobile phase (%) and the flow rate (mL·min) -1 The column temperature (°C) was used to investigate the effect of minor environmental changes on the method. The results showed that the detection of the 10 amino acids that make up cetrilac acetate was not interfered with under various chromatographic conditions. In the derivatized reference solution, the RSD of each amino acid correction factor was less than 2.7%, and the minimum resolution between peaks was not less than 1.0. Under all conditions, the RSD of each amino acid content in the test solution was less than 6.9%, indicating good method robustness. The specific results are shown in Table 2.

[0111] Table 3 Durability Results

[0112]

[0113]

[0114] Test Example 6 Sample Detection

[0115] Three batches of cetrorex acetate samples (batch numbers: XQRK-210801, XQRK-210901, XQRK-210902) were tested. The results showed that the contents of the 10 amino acid derivatives that make up cetrorex acetate in each batch were all within the range of 83.5% to 109.5%, as shown in Table 3.

[0116] Table 4. Detection results (%) of 10 amino acids in three batches of cetrorex acetate test samples

[0117]

Claims

1. A method for determining chiral amino acids in cetrorex acetate, characterized in that, Includes the following steps: (1) Sample preparation: Preparation of chiral amino acid reference solution: Accurately weigh appropriate amounts of the 10 amino acid reference standards that constitute cetrorex acetate, dissolve and dilute with diluent to a concentration of 40 µmol·L⁻¹. -1 The solution; Preparation of mixed solution of chiral amino acid control and enantiomer amino acid control: Accurately weigh appropriate amounts of the 10 amino acids constituting cetrorex acetate and their enantiomer reference standards, dissolve and dilute with diluent to a concentration of 20 µmol·L⁻¹. -1 The solution; Preparation of test solution: Accurately weigh an appropriate amount of cetrorex acetate, and after hydrolysis, dissolve and dilute with a diluent to a concentration of 40 µmol·L⁻¹. -1 The solution; (2) Derivatization reaction: Take the chiral amino acid reference solution, the chiral amino acid reference and enantiomer amino acid reference mixed solution and the test solution from step (1), and add Marfey derivatizing reagent under alkaline conditions to react and obtain the derivatized chiral amino acid reference solution, the derivatized chiral amino acid reference and enantiomer amino acid reference mixed solution and the derivatized test solution. A blank derivatization solution was obtained by adding Marfey derivatization reagent to an aqueous solution with a pH of approximately 7.0 under alkaline conditions. The derivatization reaction was carried out by adding 0.1 mol·L⁻¹ sodium bicarbonate solution and 1.0 g·L⁻¹ L-FDLA derivatization reagent to the reaction solution, mixing well, heating at 35–45 °C for 0.5–1.5 hours, removing the solution, adding 10 µL of 0.1 mol·L⁻¹ hydrochloric acid solution to terminate the reaction, centrifuging for 10 min, and collecting the supernatant. (3) Chromatographic conditions settings: Column: Hedera ODS-2 C18 column; Mobile phase: Gradient elution was performed using 0.065% formic acid solution as mobile phase A and acetonitrile as mobile phase B; Column temperature: 30~45℃; Flow rate: 0.5~1.1 ml / min; Injection volume: 1~10µl; Detection wavelength: 340nm; (4) Detection and calculation: Accurately measure the derivatized blank solution, derivatized chiral amino acid reference solution, derivatized test solution, and mixed solution of derivatized chiral amino acid reference and enantiomer amino acid reference, and inject them into the liquid chromatograph. Record the chromatograms, calculate the peak area of ​​each derivatized amino acid by high performance liquid chromatography, and determine the content of 10 chiral amino acids by external standard method.

2. The determination method according to claim 1, characterized in that, The hydrolysis reaction described in step (1) involves reacting cetrilac acetate with 6 mol·L⁻¹ -1 Dissolve and dilute with hydrochloric acid to prepare a solution with a concentration of 1.0 mmol·L⁻¹. -1 The solution was filled with nitrogen, sealed, and placed in an oven at 110℃ for 6-8 hours to hydrolyze and obtain the aqueous solution of the free amino acids to be tested.

3. The determination method according to claim 1, characterized in that, In step (1), the pH of each solution is adjusted to 7.0~7.5 during the sample preparation process.

4. The determination method according to claim 1, characterized in that, The L-FDLA solution was prepared by dissolving an appropriate amount of L-FDLA in dimethyl sulfoxide to a solution containing 1.0 g·L⁻¹. -1 The solution.

5. The determination method according to claim 1, characterized in that, In step (3), the column specifications are 4.6 mm × 250 mm, 5 μm; the flow rate is 1.0 mL·min. -1 Column temperature: 35℃; Injection volume: 10 μL.

6. The determination method according to claim 1, characterized in that: In step (4), the chiral amino acid content was calculated by taking the reference standard stock solution and preparing concentrations of 10.20, 16.33, 20.41, 22.45, and 24.49 µmol·L⁻¹, respectively. -1 The linear test solutions of a series of concentrations were derivatized according to the method in step (2). The peak area was plotted as the vertical axis and the molar concentration as the horizontal axis, and linear regression calculation was performed. The content of 10 chiral amino acids was calculated based on the peak area measured in step (4).

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