A method for separating etelcalcetide hydrochloride and impurities and application thereof

Abstract

The application discloses a separation method and application of etelcalcetide hydrochloride and impurities. The separation method comprises the following steps: detecting a to-be-detected sample by high performance liquid chromatography; wherein the to-be-detected sample is an aqueous solution containing etelcalcetide hydrochloride; the high performance liquid chromatography adopts a gradient elution mode; a mobile phase A is an ammonium sulfate buffer solution, and the pH value is 1.5-1.7; a mobile phase B is a water phase-organic phase system, the type and the pH value of the water phase are consistent with the type and the pH value of the mobile phase A, the organic phase is acetonitrile, and the volume ratio of the water phase to the organic phase is 1:(0.82-1.22); in the high performance liquid chromatography, the column temperature of a chromatographic column is 40-45 DEG C, and the particle size of the filler in the chromatographic column is 2.7-3.5 mu m. The method has the characteristics of strong specificity, good separation degree, fast analysis speed, high sensitivity and the like, and can be used for process quality control in etelcalcetide hydrochloride bulk drug synthesis process and quality evaluation of a final product.

Description

Technical Field

[0001] This invention relates to a method and application for separating etakatide hydrochloride and its impurities. Background Technology

[0002] Itcatide hydrochloride, as a synthetically produced octapeptide compound, possesses a unique stereochemical structure. Its molecular formula is N-acetyl-D-cysteyl-S-(L-cysteine ​​disulfide bridge)-D-alanyl-D-arginyl-D-arginyl-D-arginyl-D-alanyl-D-arginamide hydrochloride, represented by the chemical formula C1. 38 H 73 N 21 O 10 S2 • xHCl (4 ≤ x ≤ 5), with a theoretical molecular weight of 1047.5·xHCl. This drug has been approved for the clinical treatment of secondary hyperparathyroidism in patients with chronic kidney disease undergoing hemodialysis. Its efficacy is closely related to the stability of the formulation quality.

[0003] In industrial production, itlactide hydrochloride is prepared using a solid-phase peptide synthesis process. However, due to the complexity of peptide molecular structures, process impurities such as amino acid deletions, oxidation products, and residual side-chain protecting groups are easily generated during synthesis. Furthermore, the formulation may undergo degradation reactions such as disulfide bond breakage and amide hydrolysis during long-term storage, generating various structural analogs. The main process impurities and potential degradation impurities are shown in Table 1.

[0004] Table 1. 12 potential impurities in etakatide hydrochloride

[0005]

[0006] The presence of these impurities can affect drug efficacy and safety, but currently, comprehensive impurity profiling methods have not been established in pharmacopoeias and publicly available literature. Therefore, developing high-resolution analytical methods and establishing systematic property control strategies covering process impurities and degradation products have become core technical problems that urgently need to be solved in this field. Summary of the Invention

[0007] The technical problem to be solved by this invention is to overcome the shortcomings of existing technologies that cannot simultaneously separate eticatrix hydrochloride and multiple impurities and perform content detection, and to provide a method and application for separating eticatrix hydrochloride and impurities. This method has the characteristics of high specificity, good separation, fast analysis speed, and high sensitivity, and can be used for process quality control and final product quality evaluation in the synthesis process of eticatrix hydrochloride raw material.

[0008] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0009] This invention provides a method for separating etakatide hydrochloride and impurities, which includes the following steps: detecting the sample to be tested by high performance liquid chromatography;

[0010] The sample to be tested is an aqueous solution containing itcatide hydrochloride;

[0011] The high-performance liquid chromatography method employs gradient elution; mobile phase A is ammonium sulfate buffer with a pH of 1.5-1.7; mobile phase B is an aqueous-organic phase system, with the type and pH of the aqueous phase being consistent with those of mobile phase A, and the organic phase being acetonitrile, with a volume ratio of aqueous phase to organic phase of 1:(0.82-1.22).

[0012] The gradient elution conditions for mobile phase A and mobile phase B are as follows:

[0013]

[0014] The percentages mentioned above are the ratio of the volume of mobile phase A or mobile phase B to the total volume of "mobile phase A and mobile phase B";

[0015] In the high-performance liquid chromatography method, the column temperature is 40~45℃;

[0016] In the high-performance liquid chromatography method, the particle size of the packing material in the chromatographic column is 2.7~3.5μm.

[0017] In this invention, the concentration of itcatide hydrochloride in the sample to be tested can be 0.001~5 mg / mL, preferably 0.01~2 mg / mL, for example 1 mg / mL.

[0018] In this invention, the concentration of a single impurity in the sample to be tested can be 0.0001~2 mg / mL, preferably 0.001~0.1 mg / mL, for example 0.05 mg / mL.

[0019] In this invention, the impurities can be "impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, impurity 10, impurity 11 and impurity 12";

[0020] Impurity 1 is cystine, impurity 2 is cysteine ​​hydrochloride, and impurity 3 is [D-Orn]. 3 [Itecaide, wherein impurity 4 is [D-Orn] 7 [Ittca peptide, wherein impurity 5 is [For-D-Cys] 1 [Itecaide, wherein impurity 6 is [β-Ala] 2 Itacreptide, wherein impurity 7 is [D-Arg] 7[-OH] eptacinide, wherein impurity 8 is eptacinide dimer-II, and impurity 9 is [β-Ala] 2' Impurity 10 is an oxidized impurity of etacopeptide, impurity 11 is etacopeptide dimer-I and impurity 12 is etacopeptide dimer-III.

[0021] In this invention, impurity 1 is cystine with the sequence Cys-Cys.

[0022] Impurity 2 is cysteine ​​hydrochloride with the sequence Cys•HCl.

[0023] The impurity 3 is [D-Orn] 3 Itca peptide, with the sequence Ac-D-Cys(Cys)-D-Ala-D-Orn-D-Arg-D-Arg-D-Ala-D-Arg-NH2.

[0024] The impurity 4 is [D-Orn] 7 Itca peptide, with the sequence Ac-D-Cys(Cys)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Orn-NH2.

[0025] The impurity 5 is [For-D-Cys] 1 Itca peptide, with the sequence For-D-Cys(Cys)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2.

[0026] The impurity 6 is [β-Ala] 2 Itca peptide, with the sequence Ac-D-Cys(Cys)-β-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2.

[0027] The impurity 7 is [D-Arg] 7 [-OH] Itca peptide, with the sequence Ac-D-Cys(Cys)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-OH.

[0028] Impurity 8 is itcatide dimer-II, with the sequence Ac-D-Cys. 1 (HD-Cys 1’ -D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2.

[0029] The impurity 9 is [β-Ala]2' Itca peptide, with the sequence Ac-D-Cys(Cys)-D-Ala-β-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2.

[0030] Impurity 10 is an oxidized impurity of itcatide, with the sequence Ac-D-Cys[Cys(O)]-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2.

[0031] Impurity 11 is itcatide dimer-I, with the sequence Ac-D-Cys. 1 (Ac-D-Cys 1’ -D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2.

[0032] Impurity 12 is etkatapeptide dimer-III, with the sequence Ac-D-Cys. 1 (Ac-D-Cys 1’ -D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-OH)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2.

[0033] In this invention, the impurities may further include unknown impurities. The unknown impurities are unidentified impurities in the mixed solution with unknown sequences.

[0034] In this invention, the injection volume of the sample to be tested can be 5~100 μL, for example 10 μL.

[0035] In this invention, the concentration of the ammonium sulfate buffer in the mobile phase A can be 10~50 mmol / L, for example 20 mmol / L.

[0036] In this invention, the pH value of the ammonium sulfate buffer solution in the mobile phase A is preferably 1.5 or 1.7.

[0037] In this invention, the volume ratio of the aqueous phase to the organic phase in the mobile phase B can be 1:(0.90~1.15), for example 1:1.

[0038] In a preferred embodiment, the gradient elution conditions for mobile phase A and mobile phase B are as follows:

[0039] .

[0041] In this invention, the total flow rate of the mobile phase A and the mobile phase B in the high performance liquid chromatography method can be 0.5~1.0 mL / min, for example 0.7 mL / min.

[0042] In this invention, the chromatographic column used in the high-performance liquid chromatography method can be an octadecylsilane-bonded silica column, such as a Welch Xtimate C column. 18 Column or Welch Ultimate XB C 18 Chromatographic column.

[0043] In this invention, the particle size of the packing material in the chromatographic column is preferably 3 μm in the high performance liquid chromatography method.

[0044] In this invention, the inner diameter × column length of the chromatographic column is preferably 4.6 mm × 150 mm in the high performance liquid chromatography method.

[0045] In this invention, the column temperature of the chromatographic column in the high-performance liquid chromatography method is preferably 40°C or 45°C.

[0046] In this invention, the detector used in the high-performance liquid chromatography method can be an ultraviolet detector.

[0047] In this invention, the detection wavelength in the high-performance liquid chromatography method can be 205~215 nm, preferably 210 nm.

[0048] In this invention, preferably, the separation method includes the following steps:

[0049] a. Take itatitra hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, impurity 10, impurity 11 and impurity 12, add mobile phase A to prepare 13 stock solutions with a concentration of 1-3 mg / mL.

[0050] b. Measure 0.5-1.5 mL of itcatide hydrochloride and each impurity stock solution, and add mobile phase A to prepare 13 positioning solutions with a concentration of 40-60 µg / mL.

[0051] c. Measure 0.5-1.5 mL of each of the stock solutions of etakatide hydrochloride and each impurity, add mobile phase A to prepare a solution containing 40-60 µg of etakatide hydrochloride and each impurity per 1 mL, which is the mixed solution;

[0052] d. Take 10 µL each of the etakatide hydrochloride localization solution, each impurity localization solution, and the mixed solution, and detect them by high performance liquid chromatography. Record the retention times of etakatide hydrochloride and each impurity, and observe the separation of etakatide hydrochloride and each impurity in the mixed solution, as well as the separation between each impurity.

[0053] In a preferred embodiment, the separation method includes the following steps:

[0054] a. Weigh appropriate amounts of itcatide hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, impurity 10, impurity 11 and impurity 12 respectively, dissolve and dilute them with mobile phase A to prepare 13 solutions with a concentration of 1 mg / mL as stock solutions.

[0055] b. Measure 1 mL each of itcatide hydrochloride and each impurity stock solution, place them in different 20 mL volumetric flasks, dilute and bring to volume with mobile phase A, shake well, and obtain 13 positioning solutions with a concentration of 50 µg / mL.

[0056] c. Measure 1 mL each of itacapeptide hydrochloride and each impurity stock solution, place them in the same 20 mL volumetric flask, dilute with mobile phase A and make up to volume, shake well, and obtain a solution containing 50 µg of itacapeptide hydrochloride and each impurity per 1 mL, which is the mixed solution.

[0057] d. Take 10 µL of each of the following solutions: etakatide hydrochloride localization solution, localization solutions of each impurity, and mixed solution. Detect them by high performance liquid chromatography (HPLC). Record the retention times of etakatide hydrochloride and each impurity. Observe the separation of etakatide hydrochloride from each impurity in the mixed solution, and the separation between each impurity.

[0058] The present invention also provides the application of the separation method of etakatide hydrochloride and impurities in the analysis, detection or content determination of etakatide hydrochloride and impurities.

[0059] In this invention, the high performance liquid chromatography method is used to analyze and detect etakatide hydrochloride and impurities or to determine their content.

[0060] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0061] The reagents and raw materials used in this invention are all commercially available.

[0062] The positive and progressive effects of this invention are as follows:

[0063] This invention provides a high-performance liquid chromatography (HPLC) detection method that can simultaneously and effectively separate eticatrix hydrochloride and 13 other impurities. It features high specificity, good separation, fast analysis speed, and high sensitivity. This method can be used to detect impurities in eticatrix hydrochloride raw materials, solving the technical problem of the lack of methods for separating eticatrix hydrochloride and impurities in published literature and pharmacopoeias. It enables precise control of process impurities and potential degradation impurities in eticatrix hydrochloride, thereby improving the quality of eticatrix hydrochloride products. Attached Figure Description

[0064] Figure 1 This is the HPLC chromatogram of the mixed solution detected in Example 1.

[0065] Figure 2 This is the HPLC chromatogram of the mixed solution detected in Example 2.

[0066] Figure 3 This is the HPLC chromatogram of the mixed solution detected in Example 3.

[0067] Figure 4 This is the HPLC chromatogram of the mixed solution detected in Example 4.

[0068] Figure 5 This is the HPLC chromatogram of the mixed solution detected in Example 5.

[0069] Figure 6 This is a blank HPLC chromatogram of the 800241105 batch of itlactoide hydrochloride in Example 6.

[0070] Figure 7 This is the HPLC chromatogram of the control solution for detecting itlactin hydrochloride in batch 800241105 of Example 6.

[0071] Figure 8 The image shows the HPLC chromatogram of the test sample from batch 800241105 of itlactoide hydrochloride in Example 6.

[0072] Figure 9 The image shows the HPLC chromatogram of the mixed solution in Comparative Example 1.

[0073] Figure 10 This is the HPLC chromatogram of the mixed solution in Comparative Example 2.

[0074] Figure 11 This is the HPLC chromatogram of the mixed solution in Comparative Example 3.

[0075] Figure 12 This is the HPLC chromatogram of the mixed solution in Comparative Example 4.

[0076] Figure 13 This is the HPLC chromatogram of the mixed solution in Comparative Example 5.

[0077] Figure 14 This is the HPLC chromatogram of the mixed solution in Comparative Example 6.

[0078] Figure 15 This is the HPLC chromatogram of the mixed solution in Comparative Example 7.

[0079] Figure 16 This is the HPLC chromatogram of the mixed solution in Comparative Example 8.

[0080] Figure 17This is the HPLC chromatogram of the mixed solution in Comparative Example 9. Detailed Implementation

[0081] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0082] In the following embodiments, the product's source and model are as follows:

[0083] Acetonitrile, HPLC grade, purchased from Fisher Scientific;

[0084] Ammonium sulfate was AR grade, and phosphoric acid was HPLC grade, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0085] Three batches of etakatide hydrochloride raw material (batch numbers 2023082901, 2023101701 and 2023102701), impurity 3 (batch number: IMP21-230214-01), impurity 4 (batch number: IMP24-230214-01), impurity 5 (batch number: IMP30-230220-01), impurity 6 (batch number: IMP31-230506-01), and impurity 7 (batch number: IM) were found to contain impurities 3, 4, 5, 6, 6, and 7 respectively. P25-230506-01), impurity 8 (batch number: IMP39-230804-01), impurity 9 (batch number: IMP34-230220-01), impurity 10 (batch number: IMP26-20240402), impurity 11 (batch number: IMP27-230602-01), and impurity 12 (batch number: IMP40-230804-01) were purchased from Beijing PunoWangKang Pharmaceutical Technology Co., Ltd.

[0086] Impurity 1 (batch number: 140632-202204) and impurity 2 (batch number: 140699-202105) were purchased from the National Institutes for Food and Drug Control.

[0087] The etakatide hydrochloride raw material (batch number 800241105) was prepared by Shanghai Pharmaceutical First Biochemical Pharmaceutical Co., Ltd.

[0088] The instrument models and sources used in the following embodiments are as follows:

[0089] Infinity 1260 High Performance Liquid Chromatograph (Agilent Technologies, USA);

[0090] Welch Ultimate XB C 18 Chromatographic column (3 μm, 4.6 × 150 mm);

[0091] Welch Ultimate C 18 Chromatographic column (3 μm, 4.6 × 150 mm);

[0092] Xbridge C 18 Chromatographic column (5 μm, 4.6 × 150 mm);

[0093] XPR56 / A microbalance (Mettler-Toledo, Switzerland);

[0094] AL204 analytical balance (Mettler-Toledo, Switzerland);

[0095] S210 pH meter (Switzerland, Mettler-Toledo).

[0096] Example 1

[0097] This example illustrates the separation of etakatide hydrochloride and 13 impurities.

[0098] (1) Sample preparation

[0099] Take appropriate amounts of etakatide hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, impurity 10, impurity 11 and impurity 12 respectively, dissolve and dilute them with mobile phase A to prepare 13 solutions with a concentration of approximately 1 mg / mL, as stock solutions.

[0100] Measure 1 mL each of itcatide hydrochloride and each impurity stock solution, place them in different 20 mL volumetric flasks, dilute and bring to volume with mobile phase A, shake well, and obtain 13 positioning solutions with a concentration of 50 µg / mL.

[0101] Take 1 mL each of etakatide hydrochloride and each impurity stock solution, place them in the same 20 mL volumetric flask, dilute with mobile phase A and make up to volume, shake well, and obtain a solution containing approximately 50 µg of etakatide hydrochloride and each impurity per 1 mL, which is the mixed solution.

[0102] (2) Sample testing

[0103] Take 10 µL each of itlactoide hydrochloride and each impurity localization solution, and perform high performance liquid chromatography under the following experimental conditions. Record the retention times of itlactoide hydrochloride and each impurity.

[0104] Take 10 μL of the mixed solution and perform high-performance liquid chromatography (HPLC) under the following experimental conditions to obtain the chromatogram shown below. Figure 1 As shown.

[0105] The experimental conditions are as follows:

[0106] Column: Welch Ultimate XB C 18 Chromatographic column (3 μm, 4.6 × 150 mm)

[0107] Column temperature: 45℃

[0108] Mobile phase: Mobile phase A is 20 mmol / L ammonium sulfate buffer (pH=1.5), and mobile phase B is a mixed solution of mobile phase A and acetonitrile (volume ratio 50:50).

[0109] Total flow rate of mobile phase: 0.7 mL / min.

[0110] The gradient elution procedure is shown in Table 2:

[0111] Table 2

[0112]

[0113] Injection volume: 10 μL.

[0114] Detector: Ultraviolet detector.

[0115] Detection wavelength: 210nm.

[0116] (3) Experimental results

[0117] Figure 1 The HPLC chromatogram for the mixed solution detection is shown. Based on the retention times of etlactam hydrochloride and various impurities, the peak with a retention time of 11.162 min is the etlactam hydrochloride peak. The remaining 13 peaks are those of known and unknown impurities, as shown in Table 3. All 12 known impurities and 1 unknown impurity were detected. Etilactam hydrochloride and impurities, as well as other impurities, were effectively separated, with a minimum resolution of 1.2. The chromatographic method in this embodiment has a short analysis time of 46 min and can detect 13 potential impurities in etlactam hydrochloride, demonstrating strong specificity.

[0118] Table 3

[0119]

[0120] Example 2

[0121] This example illustrates the separation of etakatide hydrochloride and 13 impurities.

[0122] (1) Sample preparation

[0123] Same as Example 1.

[0124] (2) Sample testing

[0125] This example uses Welch Xtimate C 18The chromatographic column (3 μm, 4.6 × 150 mm) was used, and other conditions were the same as in Example 1. The resulting chromatogram is shown below. Figure 2 As shown.

[0126] (3) Experimental results

[0127] Figure 2 The HPLC chromatogram for the mixed solution detection is shown. Based on the retention times of etlactam hydrochloride and various impurities, the peak with a retention time of 9.941 min is the etlactam hydrochloride peak. The remaining 13 peaks are those of known and unknown impurities, as shown in Table 4. All 12 known impurities and 1 unknown impurity were detected. Etilactam hydrochloride and impurities, as well as other impurities, were effectively separated, with a minimum resolution of 1.5. The chromatographic method in this embodiment has a short analysis time of 46 min and can detect 13 potential impurities in etlactam hydrochloride, demonstrating strong specificity.

[0128] Table 4

[0129]

[0130] Example 3

[0131] This example illustrates the separation of etakatide hydrochloride and 13 impurities.

[0132] (1) Sample preparation

[0133] Same as Example 1.

[0134] (2) Sample testing

[0135] In this example, mobile phase A was 20 mmol / L ammonium sulfate buffer (pH=1.7), and other conditions were the same as in Example 2. The resulting chromatogram is shown below. Figure 3 As shown.

[0136] (3) Experimental results

[0137] Figure 3 The HPLC chromatogram for the mixed solution detection is shown. Based on the retention times of etlactam hydrochloride and various impurities, the peak with a retention time of 9.605 min is the etlactam hydrochloride peak. The remaining 13 peaks are those of known and unknown impurities, as shown in Table 5. All 12 known impurities and 1 unknown impurity were detected. Etilactam hydrochloride and impurities, as well as other impurities, were effectively separated, with a minimum resolution of 2.5. This embodiment demonstrates a short chromatographic analysis time of 46 min, capable of detecting 13 potential impurities in etlactam hydrochloride, exhibiting strong specificity.

[0138] Table 5

[0139]

[0140] Example 4

[0141] This example illustrates the separation of etakatide hydrochloride and 13 impurities.

[0142] (1) Sample preparation

[0143] Same as Example 1.

[0144] (2) Sample testing

[0145] In this embodiment, the column temperature was 40°C, and other conditions were the same as in Example 1. The resulting chromatogram is shown below. Figure 4 As shown.

[0146] (3) Experimental results

[0147] Figure 4 The HPLC chromatogram for the mixed solution detection is shown. Based on the retention times of etlactam hydrochloride and various impurities, the peak with a retention time of 11.990 min is the etlactam hydrochloride peak. The remaining 13 peaks are those of known and unknown impurities, as shown in Table 6. All 12 known impurities and 1 unknown impurity were detected. Etilactam hydrochloride and impurities, as well as other impurities, were effectively separated, with a minimum resolution of 1.7. The chromatographic method in this embodiment has a short analysis time of 46 min and can detect 13 potential impurities in etlactam hydrochloride, demonstrating strong specificity.

[0148] Table 6

[0149]

[0150] Example 5

[0151] This example illustrates the separation of etakatide hydrochloride and 13 impurities.

[0152] (1) Sample preparation

[0153] Same as Example 1.

[0154] (2) Sample testing

[0155] This embodiment uses a gradient elution program as shown in Table 7, with other conditions the same as in Example 1. The resulting chromatogram is shown below. Figure 5 As shown.

[0156] Table 7

[0157]

[0158] (3) Experimental results

[0159] Figure 5The HPLC chromatogram for the mixed solution detection is shown. Based on the retention times of etlactam hydrochloride and various impurities, the peak with a retention time of 11.942 min is the etlactam hydrochloride peak. The remaining 13 peaks are those of known and unknown impurities, as shown in Table 8. All 12 known impurities and 1 unknown impurity were detected. Etilactam hydrochloride and impurities, as well as other impurities, were effectively separated, with a minimum resolution of 1.8. This embodiment demonstrates a short chromatographic analysis time of 46 min, capable of detecting 13 potential impurities in etlactam hydrochloride, exhibiting strong specificity.

[0160] Table 8

[0161]

[0162] Example 6

[0163] This example illustrates the detection of related substances in itcatide hydrochloride.

[0164] Using the same experimental conditions as in Example 1, the related substances of four batches of itcatide hydrochloride were determined.

[0165] a. Test solution: Accurately weigh an appropriate amount of etakatide hydrochloride, dissolve and dilute it in mobile phase A to prepare a solution with a concentration of approximately 1 mg / mL. Prepare one solution for each batch. Take 10 µL of the test solution and perform high-performance liquid chromatography (HPLC) under the experimental conditions of Example 1. Record the retention time and peak area of ​​the main peak and each impurity.

[0166] b. Control solution: Accurately measure 1 mL of the test solution and place it in a 200 mL volumetric flask. Dilute to the mark with mobile phase A and shake well. Prepare one set for each batch. Take 10 µL of the control solution and perform high-performance liquid chromatography (HPLC) under the experimental conditions of Example 1. Record the peak area of ​​the main peak.

[0167] c. Positioning solutions: Prepared in the same manner as Comparative Example 1. Take 10 µL each of the positioning solutions for etakatide hydrochloride and each impurity, and perform high-performance liquid chromatography (HPLC) under the experimental conditions of Example 1. Record the retention times of etakatide hydrochloride and each impurity.

[0168] The types of impurities in the test sample were determined based on the retention times of etakatide hydrochloride and other impurities in the positioning solution. Impurities 7 and 11 were detected in all four batches of etakatide hydrochloride. The content of related substances in etakatide hydrochloride was calculated using a self-comparison method, and the calculation formula is as follows:

[0169]

[0170]

[0171] Among them, A XThe peak area of ​​a single impurity in the test solution;

[0172] A R The peak area of ​​the main peak in the control solution;

[0173] A 总 The total peak area in the test solution;

[0174] A 主 This represents the peak area of ​​the main peak in the test sample solution.

[0175] The results are summarized in Table 10. Taking batch 800241105 of itlactide hydrochloride as an example, Figure 6 For blank, Figure 7 For control solution, Figure 8 The HPLC chromatogram of the test sample shows that impurity 7 has a retention time of 13.689 min and impurity 11 has a retention time of 23.518 min.

[0176] Table 10

[0177]

[0178] Example 7

[0179] This example demonstrates a sensitivity test for itcatide hydrochloride and impurities.

[0180] The experiment was conducted under the same experimental conditions as in Example 1.

[0181] Weigh appropriate amounts of etakatide hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 9 and impurity 11 respectively, dissolve and dilute them with mobile phase A to prepare 10 solutions with a concentration of approximately 1 mg / mL as stock solutions; measure the etakatide hydrochloride and each impurity stock solution and dilute them stepwise with mobile phase A to obtain the limit of detection and limit of quantitation solutions.

[0182] Under the experimental conditions of Example 1, high-performance liquid chromatography (HPLC) was used for detection. In the obtained chromatograms, the peak height of etakatide hydrochloride or impurities was approximately 3 to 8 times higher than the baseline noise; the concentration of the solution at this point was the limit of detection (LOD). In the obtained chromatograms, the peak height of etakatide hydrochloride or impurities was approximately 10 to 20 times higher than the baseline noise; the concentration of the solution at this point was the limit of quantitation (LOQ). The LODs and LOQs of etakatide hydrochloride and the nine impurities are shown in Table 9. All LOQs reached the ng level, indicating that this method has high sensitivity.

[0183] Table 9

[0184]

[0185] Comparative Example 1

[0186] (1) Sample preparation

[0187] Same as Example 1.

[0188] (2) Sample testing

[0189] This comparative example used mobile phase A of 20 mmol / L ammonium sulfate buffer (pH=1.3), and all other conditions were the same as in Example 2. The resulting chromatogram is shown below. Figure 9 As shown.

[0190] (3) Experimental results

[0191] Figure 9 The HPLC chromatogram for the mixed solution is shown. Based on the retention times of eticatriptyline hydrochloride and each impurity, the peak with a retention time of 8.152 min is the eticatriptyline hydrochloride peak. The remaining 11 peaks are those of known and unknown impurities, as shown in Table 11. Etecatriptyline hydrochloride can be effectively separated from 13 impurities. However, not all of the 13 impurities can be separated. Specifically, the chromatograms of the unknown impurity and impurity 6 overlap, as do the chromatograms of impurities 7 and 8.

[0192] Table 11

[0193]

[0194] Comparative Example 2

[0195] (1) Sample preparation

[0196] Same as Example 1.

[0197] (2) Sample testing

[0198] This comparative example used mobile phase A of 20 mmol / L ammonium sulfate buffer (pH=4.1), and all other conditions were the same as in Example 2. The resulting chromatogram is shown below. Figure 10 As shown.

[0199] (3) Experimental results

[0200] Figure 10 The HPLC chromatogram for the mixed solution is shown. Based on the retention times of eticatriptyline hydrochloride and each impurity, the peak with a retention time of 11.191 min is the eticatriptyline hydrochloride peak. The remaining 12 peaks are those of known and unknown impurities, as shown in Table 12. Etecatriptyline hydrochloride can be effectively separated from 13 impurities, but not all of the 13 impurities can be separated. Among them, the chromatographic peaks of impurities 8 and 9 overlap.

[0201] Table 12

[0202]

[0203] Comparative Example 3

[0204] (1) Sample preparation

[0205] Same as Example 1.

[0206] (2) Sample testing

[0207] This comparative example used mobile phase A as an aqueous phosphoric acid solution (pH=1.5), and all other conditions were the same as in Example 1. The resulting chromatogram is shown below. Figure 11 As shown.

[0208] (3) Experimental results

[0209] Figure 11 The HPLC chromatogram for the mixed solution detection is shown. Based on the retention times of eticarapeptide hydrochloride and each impurity, the peak with a retention time of 7.141 min is a coincidence peak of eticarapeptide hydrochloride and impurity 6. The remaining 10 peaks are those of known and unknown impurities, as shown in Table 13. Etecarapeptide hydrochloride cannot be completely separated from the 13 impurities, nor can all of the 13 impurities be separated from each other. Specifically, the chromatograms of impurity 4 and impurity 11 coincide, the chromatograms of eticarapeptide hydrochloride and impurity 6 coincide, the chromatograms of the unknown impurity and impurity 8 coincide, and the resolution of impurity 3 with its adjacent chromatogram peak is less than 1.2.

[0210] Table 13

[0211]

[0212] Comparative Example 4

[0213] (1) Sample preparation

[0214] Same as Example 1.

[0215] (2) Sample testing

[0216] The column temperature for this comparative example was 35°C, and other conditions were the same as in Example 1. The resulting chromatogram is shown below. Figure 12 As shown.

[0217] (3) Experimental results

[0218] Figure 12 The HPLC chromatogram for the mixed solution is shown. Based on the retention times of eticatriptyline hydrochloride and each impurity, the peak with a retention time of 12.918 min is the eticatriptyline hydrochloride peak. The remaining 12 peaks are those of known and unknown impurities, as shown in Table 14. Eticaptyline hydrochloride can be effectively separated from 13 impurities. However, not all of the 13 impurities can be separated. Specifically, the peaks of impurity 5 and the unknown impurity overlap, and the resolution between impurities 10 and 11 is less than 1.2.

[0219] Table 14

[0220]

[0221] Comparative Example 5

[0222] (1) Sample preparation

[0223] Same as Example 1.

[0224] (2) Sample testing

[0225] The column temperature used in this comparative example was 50°C, and other conditions were the same as in Example 1. The resulting chromatogram is shown below. Figure 13 As shown.

[0226] (3) Experimental results

[0227] Figure 13 The HPLC chromatogram for the mixed solution is shown. Based on the retention times of eticatriptyline hydrochloride and each impurity, the peak with a retention time of 9.694 min is the eticatriptyline hydrochloride peak. The remaining 12 peaks are those of known and unknown impurities, as shown in Table 15. Etecatriptyline hydrochloride can be effectively separated from 13 impurities, but not all of the 13 impurities can be separated. Among them, the unknown impurity and impurity 6 have overlapping chromatographic peaks.

[0228] Table 15

[0229]

[0230] Comparative Example 6

[0231] (1) Sample preparation

[0232] Same as Example 1.

[0233] (2) Sample testing

[0234] This comparative example uses Xbridge C 18 The chromatographic column (5 μm, 4.6 × 150 mm) was used, and other conditions were the same as in Example 1. The resulting chromatogram is shown below. Figure 14 As shown.

[0235] (3) Experimental results

[0236] Figure 14 The HPLC chromatogram for the mixed solution is shown. Based on the retention times of eticatriptyline hydrochloride and each impurity, the peak with a retention time of 8.573 min is the eticatriptyline hydrochloride peak. The remaining 12 peaks are those of known and unknown impurities, as shown in Table 16. Eticaptyline hydrochloride can be effectively separated from 13 impurities, but not all of the 13 impurities can be separated. Among them, the chromatographic peaks of impurity 5 and the unknown impurity overlap.

[0237] Table 16

[0238]

[0239] Comparative Example 7

[0240] (1) Sample preparation

[0241] Same as Example 1.

[0242] (2) Sample testing

[0243] This comparative example used mobile phase B, which was a mixture of mobile phase A and acetonitrile (volume ratio 25:75), and all other conditions were the same as in Example 1. The resulting chromatogram is shown below. Figure 15 As shown.

[0244] (3) Experimental results

[0245] Figure 15 The HPLC chromatogram for the mixed solution is shown. Based on the retention times of eticatriptyline hydrochloride and each impurity, the peak with a retention time of 9.887 min is the eticatriptyline hydrochloride peak. The remaining 11 peaks are those of known and unknown impurities, as shown in Table 17. Etecatriptyline hydrochloride can be effectively separated from 13 impurities. However, not all of the 13 impurities can be separated. Specifically, the chromatograms of the unknown impurity and impurity 6 overlap, as do the chromatograms of impurities 7 and 8.

[0246] Table 17

[0247]

[0248] Comparative Example 8

[0249] (1) Sample preparation

[0250] Same as Example 1.

[0251] (2) Sample testing

[0252] This comparative example used a gradient elution program as shown in Table 18, with other conditions identical to those in Example 1. The resulting chromatogram is shown below. Figure 16 As shown.

[0253] Table 18

[0254]

[0255] (3) Experimental results

[0256] Figure 16The HPLC chromatogram for the mixed solution is shown. Based on the retention times of eticatriptyline hydrochloride and each impurity, the peak with a retention time of 14.294 min is the eticatriptyline hydrochloride peak. The remaining 10 peaks are those of known and unknown impurities, as shown in Table 19. Etecatriptyline hydrochloride can be effectively separated from 13 impurities. However, not all of the 13 impurities can be separated. Specifically, the chromatographic peaks of impurity 5 and the unknown impurity overlap, as do the chromatographic peaks of impurities 10, 11, and 12.

[0257] Table 19

[0258]

[0259] Comparative Example 9

[0260] (1) Sample preparation

[0261] Same as Example 1.

[0262] (2) Sample testing

[0263] This comparative example used a gradient elution program as shown in Table 20, with other conditions identical to those in Example 1. The resulting chromatogram is shown below. Figure 17 As shown.

[0264] Table 20

[0265]

[0266] (3) Experimental results

[0267] Figure 17 The HPLC chromatogram for the mixed solution is shown. Based on the retention times of eticatriptyline hydrochloride and each impurity, the peak with a retention time of 6.807 min is the eticatriptyline hydrochloride peak. The remaining 10 peaks are those of known and unknown impurities, as shown in Table 21. Etecatriptyline hydrochloride can be effectively separated from 13 impurities. However, not all of the 13 impurities can be separated. Specifically, the peaks of impurities 3 and 4 overlap, as do the peaks of impurities 6 and 8, and the peaks of impurities 10 and 12.

[0268] Table 21

[0269]

[0270] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but all such changes and modifications fall within the scope of protection of the present invention.

Claims

1. A method for separating etakatide hydrochloride and impurities, characterized in that, It includes the following steps: the sample to be tested is detected by high performance liquid chromatography; The sample to be tested is an aqueous solution containing itcatide hydrochloride; The high-performance liquid chromatography method employs gradient elution; mobile phase A is ammonium sulfate buffer with a pH of 1.5-1.7; mobile phase B is an aqueous-organic phase system, with the type and pH of the aqueous phase being consistent with those of mobile phase A, and the organic phase being acetonitrile, with a volume ratio of aqueous phase to organic phase of 1:(0.82-1.22). The gradient elution conditions for mobile phase A and mobile phase B are as follows: The percentages mentioned above are the ratio of the volume of mobile phase A or mobile phase B to the total volume of "mobile phase A and mobile phase B"; In the high-performance liquid chromatography method, the column temperature is 40~45℃, and the particle size of the packing material in the column is 2.7~3.5μm.

2. The method for separating etakatide hydrochloride and impurities as described in claim 1, characterized in that, In the sample to be tested, the concentration of itcatide hydrochloride is 0.001~5 mg / mL, preferably 0.01~2 mg / mL, for example 1 mg / mL; And / or, in the sample to be tested, the concentration of a single impurity is 0.0001~2 mg / mL, preferably 0.001~0.1 mg / mL, for example 0.05 mg / mL; And / or, in the sample to be tested, the impurities are "impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, impurity 10, impurity 11, and impurity 12"; wherein, impurity 1 is cystine, impurity 2 is cysteine ​​hydrochloride, and impurity 3 is [D-Orn] 3 [Itecaide, wherein impurity 4 is [D-Orn] 7 [Ittca peptide, wherein impurity 5 is [For-D-Cys] 1 [Itecaide, wherein impurity 6 is [β-Ala] 2 Itacreptide, wherein impurity 7 is [D-Arg] 7 [-OH] eptacinide, wherein impurity 8 is eptacinide dimer-II, and impurity 9 is [β-Ala] 2' Impurity 10 is an oxidized impurity of etacopeptide, impurity 11 is etacopeptide dimer-I and impurity 12 is etacopeptide dimer-III; Preferably, the impurities also include unknown impurities, which are unidentified impurities in the mixed solution; And / or, the injection volume of the sample to be tested is 5~100 μL, for example 10 μL.

3. The method for separating etakatide hydrochloride and impurities as described in claim 1, characterized in that, In the mobile phase A, the concentration of the ammonium sulfate buffer is 10~50 mmol / L, for example 20 mmol / L; And / or, in the mobile phase A, the pH of the ammonium sulfate buffer is 1.5 or 1.

7.

4. The method for separating etakatide hydrochloride and impurities as described in claim 1, characterized in that, In the mobile phase B, the volume ratio of the aqueous phase to the organic phase is 1:(0.90~1.15), for example, 1:

1.

5. The method for separating etakatide hydrochloride and impurities as described in claim 1, characterized in that, The gradient elution conditions for mobile phase A and mobile phase B are as follows: 。 6. The method for separating etakatide hydrochloride and impurities as described in claim 1, characterized in that, In the high-performance liquid chromatography method, the total flow rate of mobile phase A and mobile phase B is 0.5~1.0 mL / min, for example 0.7 mL / min.

7. The method for separating etakatide hydrochloride and impurities as described in claim 1, characterized in that, In the high-performance liquid chromatography method described above, the chromatographic column is an octadecylsilane-bonded silica column, such as a Welch Xtimate C column. 18 chromatographic column or Welch Ultimate XB C 18 Chromatographic column; And / or, in the high performance liquid chromatography method, the particle size of the packing material in the chromatographic column is 3 μm; And / or, in the high performance liquid chromatography method, the inner diameter × column length of the chromatographic column is 4.6 mm × 150 mm; And / or, in the high performance liquid chromatography method, the column temperature is 40°C or 45°C.

8. The method for separating etakatide hydrochloride and impurities as described in claim 1, characterized in that, In the high-performance liquid chromatography method, the detector used is an ultraviolet detector; And / or, in the high performance liquid chromatography method, the detection wavelength is 205~215 nm, preferably 210 nm.

9. The method for separating etakatide hydrochloride and impurities as described in claim 1, characterized in that, The separation method includes the following steps: a. Take itatitra hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, impurity 10, impurity 11 and impurity 12, add mobile phase A to prepare 13 stock solutions with a concentration of 1-3 mg / mL. b. Measure 0.5-1.5 mL of itcatide hydrochloride and each impurity stock solution, and add mobile phase A to prepare 13 positioning solutions with a concentration of 40-60 µg / mL. c. Measure 0.5-1.5 mL of each of the stock solutions of etakatide hydrochloride and each impurity, add mobile phase A to prepare a solution containing 40-60 µg of etakatide hydrochloride and each impurity per 1 mL, which is the mixed solution; d. Take 10 µL each of the etakatide hydrochloride localization solution, each impurity localization solution, and the mixed solution, and detect them by high performance liquid chromatography. Record the retention times of etakatide hydrochloride and each impurity, and observe the separation of etakatide hydrochloride and each impurity in the mixed solution, as well as the separation between each impurity.

10. The method for separating etakatide hydrochloride and impurities as described in any one of claims 1 to 9 is used in the analysis, detection, or content determination of etakatide hydrochloride and impurities.

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