A method for detecting related substances of carbetocin injection

Abstract

The invention discloses a method for detecting related substances of carbetocin injection, and relates to the technical field of drug detection and analysis. The detection method comprises the following steps: preparing a solution; and detecting related substances of the carbetocin injection by high performance liquid chromatography. The detection method provided by the invention avoids the use of ion pair reagents in the mobile phase by selecting a suitable mobile phase and controlling the conditions of the gradient elution program. On the premise of ensuring effective separation of the main peak and impurities, the detection method solves the problems of poor baseline, low response, and short service life of chromatographic column consumables in the existing high performance liquid chromatography system. The method has the characteristics of strong specificity, good precision, high accuracy, simplicity, high efficiency, low cost, etc., and can be applied to the quality control of carbetocin injection. At the same time, the use cost of chromatographic column consumables is greatly reduced, and the method is more suitable for commercial production.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection and analysis, and in particular to a method for detecting related substances of carbetocin injection. Background Art

[0002] Carbetocin, whose chemical structure is shown in formula (I), is a synthetic long-acting oxytocin octapeptide analog with agonist properties. The drug is formed by dehydration condensation of 8 natural amino acids and 1 mercaptobutyric acid to form a lactam cyclic peptide, of which 7 amino acid residues are chiral and all are L-configuration. Carbetocin has important value in clinical applications in obstetrics and gynecology due to its unique structure and pharmacological effects;

[0003] The formula (I): .

[0004] The control of related substances in drugs is a key link in drug quality control and is essential to ensure the safety and effectiveness of drugs. At present, high performance liquid chromatography (HPLC) is the mainstream choice for the detection of related substances in carbetocin injection. However, the HPLC detection method for related substances in carbetocin injection reported in the prior art has many challenges.

[0005] Traditional high performance liquid chromatography detection methods mostly use phosphoric acid, acetic acid, trifluoroacetic acid and other systems as mobile phases. However, these systems often show some shortcomings in the analysis of peptide drugs, especially carbetocin. Specifically, the separation of carbetocin and its related substances in these systems is poor, the baseline is unstable, the noise is large, and the sensitivity is low. These problems not only affect the accuracy of the analysis, but also limit the wide application of these systems in the analysis of related substances of carbetocin injection.

[0006] In order to improve the analysis effect, some patents try to add ion pair reagents such as PIC B8 low-UV reagent or sodium octane sulfonate to the buffer salt mobile phase. These ion pair reagents can enhance the interaction between the sample components with opposite charges and the stationary phase, so that the detected substances form ion pairs, thereby reducing their polarity and increasing retention time. However, although this method improves the separation effect to a certain extent, it also brings new problems.

[0007] Specifically, the above-mentioned types of ion pair reagents are more harmful to chromatographic columns. They will combine with the stationary phase of the chromatographic column to produce irreversible adsorption, which in turn affects the active sites of the stationary phase. This reaction not only reduces the performance of the chromatographic column, but also greatly shortens the service life of the chromatographic column. At the same time, ion pair reagents are difficult to rinse off the chromatographic column, further exacerbating the damage to the chromatographic column. In addition, ion pair reagents are sensitive to pH values, and high precision is required when preparing the mobile phase, otherwise it will directly affect the repeatability and reproducibility of the experiment.

[0008] Therefore, in view of the many problems existing in the prior art, it is urgent to develop a new method for detecting the related substances of carbetocin injection. The method should be able to overcome the shortcomings of the prior art, improve the accuracy and repeatability of the analysis, and ensure the effective separation of carbetocin and its related substances. At the same time, the method should also protect the chromatographic column from damage, extend its service life, and provide more reliable technical support for the quality control of carbetocin injection. Summary of the invention

[0009] In order to solve the above problems, the present invention provides a method for detecting related substances of carbetocin injection.

[0010] The present invention provides a method for detecting related substances of carbetocin injection, such as Figure 1 As shown, the detection method comprises the following steps:

[0011] Prepare the solution;

[0012] The carbetocin injection-related substances are detected by high performance liquid chromatography;

[0013] Wherein, the solution includes a test solution, a reference solution and a system suitability solution;

[0014] The conditions of the high performance liquid chromatography include:

[0015] Column: Agilent Peptide 4.6 x 150 mm, 2.7 um;

[0016] Mobile phase A: The mobile phase A is composed of a phosphate buffer solution with a volume ratio of (73-78): (22-27) and an acetonitrile aqueous solution with a volume fraction of 80%;

[0017] Mobile phase B: The mobile phase B is composed of a phosphate buffer solution with a volume ratio of (63-68): (32-37) and an acetonitrile aqueous solution with a volume fraction of 80%;

[0018] The pH value of the phosphate buffer is 5.0;

[0019] The gradient elution method was adopted. The gradient elution procedure was as follows in terms of volume percentage:

[0020] .

[0021] Furthermore, the preparation method of the phosphate buffer comprises the following process: dissolving 2.5 g of potassium dihydrogen phosphate in water and diluting to 1000 ml, and then adjusting the pH value of the system to 5.0 with 0.1 mol / L potassium hydroxide solution to obtain the phosphate buffer.

[0022] Furthermore, the mobile phase A is composed of a phosphate buffer solution with a volume ratio of 75:25 and an acetonitrile aqueous solution with a volume fraction of 80%.

[0023] Furthermore, the mobile phase B is composed of a phosphate buffer solution with a volume ratio of 65:35 and an acetonitrile aqueous solution with a volume fraction of 80%.

[0024] Furthermore, the conditions of the high performance liquid chromatography method also include:

[0025] Detection wavelength: 218~222 nm;

[0026] Column temperature: 58~62 ℃;

[0027] Flow rate: 0.8~1.0 ml / min;

[0028] Injection volume: 78~82ul;

[0029] Diluent: 0.5% acetic acid aqueous solution by volume.

[0030] Furthermore, the conditions of the high performance liquid chromatography method also include:

[0031] Detection wavelength: 220 nm;

[0032] Column temperature: 60 °C;

[0033] Flow rate: 0.9 ml / min;

[0034] Injection volume: 80ul;

[0035] Diluent: 0.5% acetic acid aqueous solution by volume.

[0036] Furthermore, the carbetocin injection related substances include impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, and impurity I;

[0037] The impurity A is [Gly9-OH] carbetocin, and its chemical structure is as follows: ;

[0038] The impurity B is [Asp5] carbetocin, and its chemical structure is as follows: ;

[0039] The impurity C is [β-Asp5] carbetocin, and its chemical structure is as follows: ;

[0040] The impurity D is [Glu4] carbetocin, and its chemical structure is as follows: ;

[0041] The impurity E is carbetocin sulfoxide I, and its chemical structure is as follows: ;

[0042] The impurity F is carbetocin sulfoxide II, and its chemical structure is as follows: ;

[0043] The impurity G is [D-Cys6] carbetocin, and its chemical structure is as follows: ;

[0044] The impurity H is [diGly9] carbetocin, and its chemical structure is as follows: ;

[0045] The impurity I is [D-Asn 5 ]Carbetocin, the chemical structure is as follows: .

[0046] Furthermore, the system suitability test results meet the following requirements: the separation between each impurity peak, between the main peak of carbetocin and the adjacent impurity I peak, and between the main peak of carbetocin and the adjacent impurity H peak are not less than 1.5, and the tailing factor of the carbetocin peak is not greater than 2.0;

[0047] The specificity test results satisfy the following requirements: the separation between the impurity peaks and between the main peak of carbetocin and the adjacent peaks is not less than 1.5.

[0048] Furthermore, the preparation of the test solution includes the following process: taking an appropriate amount of carbetocin injection to obtain the test solution;

[0049] The preparation of the reference solution includes the following steps: taking an appropriate amount of carbetocin reference substance, dissolving it in a 0.5% by volume aqueous acetic acid solution, and quantitatively diluting it to make 0.08-0.12 mg of carbetocin per 1 ml, to obtain the reference solution;

[0050] The preparation of the system suitability solution includes the following process: taking appropriate amounts of carbetocin, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, and impurity I reference substances, dissolving them in a 12% to 8% acetonitrile aqueous solution by volume, and quantitatively diluting them to make 0.08 to 0.12 mg of carbetocin and 0.003 to 0.007 mg of each impurity per 1 ml to obtain the system suitability solution.

[0051] The above technical solution provided by the embodiment of the present invention has at least the following advantages compared with the prior art:

[0052] The embodiment of the present invention provides a method for detecting related substances of carbetocin injection. The present invention provides a method for detecting related substances of carbetocin injection, wherein the mobile phase does not contain any traditional reagents such as ion pair reagents, phosphoric acid, acetic acid, trifluoroacetic acid, etc., and while ensuring effective separation of the main peak and impurities, the problems of poor baseline, low response, short service life of chromatographic column consumables, etc. existing in the prior art system are solved, thereby effectively controlling the quality of carbetocin injection.

[0053] Specifically, the beneficial effects of the present invention are embodied in the following aspects:

[0054] 1. Strong specificity: The detection method of the present invention can accurately identify and separate carbetocin and its possible degradation impurities and process impurities, ensuring the specificity and accuracy of the analysis.

[0055] 2. Good precision and high accuracy: By optimizing the mobile phase composition and chromatographic conditions, the present invention achieves effective separation of the main peak and impurities, while improving the precision and accuracy of detection, providing reliable technical support for the quality control of carbetocin injection.

[0056] 3. High sensitivity: The detection method of the present invention has extremely high sensitivity and can detect trace amounts of impurities, ensuring strict control of drug quality.

[0057] 4. Long service life of chromatographic column consumables: Since the mobile phase does not contain any traditional reagents such as ion pair reagents that are harmful to the chromatographic column, the present invention effectively avoids irreversible damage to the chromatographic column, greatly prolongs the service life of the chromatographic column, and reduces the cost of consumables.

[0058] 5. Suitable for commercial production: The detection method of the present invention is not only accurate and reliable, but also easy to operate and low in cost, and is very suitable for quality control needs in commercial production.

[0059] In summary, compared with the prior art, the present invention provides a more superior method for detecting related substances of carbetocin injection. The method not only solves the problems existing in the traditional system, but also improves the specificity, precision, accuracy and sensitivity of the analysis, while reducing the cost of using chromatographic column consumables, and is more suitable for commercial production. Therefore, the present invention has significant beneficial effects and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0061] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0062] Figure 1 A schematic flow chart of a method for detecting related substances of carbetocin injection provided by an embodiment of the present invention;

[0063] Figure 2 chromatogram of the detection method of related substances of carbetocin injection in Example 1 of the present invention;

[0064] Figure 3 is a chromatogram of the detection method of related substances of carbetocin injection in Example 2 of the present invention;

[0065] Figure 4 is a chromatogram of the detection method of related substances of carbetocin injection in Example 3 of the present invention;

[0066] Figure 5 It is a chromatogram of the detection method of related substances of carbetocin injection in Comparative Example 1 of the present invention;

[0067] Figure 6 It is a chromatogram of the detection method of related substances of carbetocin injection in Comparative Example 2 of the present invention;

[0068] Figure 7 It is a chromatogram of the detection method of related substances of carbetocin injection in Comparative Example 3 of the present invention;

[0069] Figure 8 It is a chromatogram of the detection method of related substances of carbetocin injection in Comparative Example 4 of the present invention;

[0070] Fig. 9 It is a chromatogram of the detection method of related substances of carbetocin injection in Comparative Example 5 of the present invention;

[0071] Fig.10 It is a chromatogram of the detection method of related substances of carbetocin injection in Comparative Example 6 of the present invention;

[0072] in, Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Fig. 9 and Fig.10 The meanings of the digital markings in the chromatogram are: 1-Impurity E, 2-Impurity A, 3-Impurity F, 4-Impurity B, 5-Impurity C, 6-Impurity D, 7-Impurity G, 8-Impurity H, 9-Carbetocin, 10-Impurity I, 11-Impurity C + Impurity D, 12-Impurity A + Impurity B, 13-Impurity C + Impurity D, 14-Impurity E + Impurity F, 15-Impurity G + Impurity H. DETAILED DESCRIPTION

[0073] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0074] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0075] It should be noted that the component raw materials involved in the detection method of carbetocin injection related substances provided in the embodiment of the present invention, unless otherwise specified or specifically described, can be directly commercially available products or homemade using existing public detection methods; at the same time, the steps and parameters involved, unless otherwise specified or specifically described, can be carried out according to the existing detection method of carbetocin injection related substances or directly carried out using existing equipment, and the present invention document will not repeat them one by one.

[0076] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

[0077] Example 1

[0078] This example provides a method for detecting related substances of carbetocin injection, and the method for detecting related substances of carbetocin injection comprises the following steps:

[0079] Step (1), preparing solution:

[0080] Step (1.1), prepare the diluent: measure 5 ml of acetic acid and 1000 ml of water, mix well, and obtain a 5% by volume acetic acid aqueous solution;

[0081] Step (1.2), preparing the test solution: taking an appropriate amount of carbetocin injection to obtain the test solution;

[0082] Step (1.3), preparing a reference solution: taking an appropriate amount of carbetocin reference solution and dissolving it in a 0.5% by volume aqueous acetic acid solution and quantitatively diluting it to make 0.08-0.12 mg of carbetocin per 1 ml, to obtain the reference solution;

[0083] Step (1.4), preparing a reference substance stock solution: taking appropriate amounts of carbetocin, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, and impurity I, dissolving them in a 12% to 8% acetonitrile aqueous solution by volume, and quantitatively diluting them to prepare a reference substance stock solution containing 1.0 mg of carbetocin and 0.05 mg of each impurity per 1 ml;

[0084] Step (1.5), preparing a system suitability solution: dissolving 0.1 ml of the reference substance stock solution prepared in step (1.4) in a 0.5% by volume aqueous acetic acid solution and quantitatively diluting the solution to obtain a system suitability solution containing 0.1 mg of carbetocin and 0.005 mg of each impurity per ml;

[0085] Step (1.6), prepare a blank solution: prepare the same as the diluent, measure 5 ml of acetic acid and 1000 ml of water, mix well, and obtain the blank solution;

[0086] Step (1.7), preparing a blank auxiliary material solution: dissolving L-methionine, succinic acid, mannitol and sodium hydroxide in water and diluting to prepare a solution containing about 1 mg of L-methionine, 1.19 mg of succinic acid, 47 mg of mannitol and 0.6 mg of sodium hydroxide per 1 ml, to obtain the blank auxiliary material solution;

[0087] Step (1.8), preparing phosphate buffer: dissolving 2.5 g of potassium dihydrogen phosphate in water and diluting to 1000 ml, and then adjusting the pH value of the system to 5.0 with 0.1 mol / L potassium hydroxide solution to obtain the phosphate buffer;

[0088] Step (2), using high performance liquid chromatography to detect the carbetocin-related substances:

[0089] Step (2.1), HPLC conditions:

[0090] Mobile phase A: The mobile phase A is composed of phosphate buffer with a volume ratio of 75:25 and acetonitrile aqueous solution with a volume fraction of 80%;

[0091] Mobile phase B: The mobile phase B is composed of a phosphate buffer solution with a volume ratio of 65:35 and an acetonitrile aqueous solution with a volume fraction of 80%;

[0092] Column: Agilent Peptide 4.6 x 150 mm, 2.7 um;

[0093] Detection wavelength: 220 nm;

[0094] Column temperature: 60 °C;

[0095] Flow rate: 0.9 ml / min;

[0096] Diluent: 5% acetic acid aqueous solution by volume;

[0097] Injection volume: 80ul;

[0098] The gradient elution method was adopted. The gradient elution procedure was as follows in terms of volume percentage:

[0099] ;

[0100] Step (2.2), detection operation: measure the test solution, reference solution and system suitability solution, etc., inject them into the liquid chromatograph respectively, detect according to the set gradient elution program, and record the chromatogram.

[0101] Typical chromatogram obtained in this example - System suitability solution chromatogram is as follows Figure 2 As shown by Figure 2 It can be seen that: blank solvent and blank excipients do not interfere with the determination of impurities and main peaks; the order of peaks in the chromatogram is: 1-impurity E, 2-impurity A, 3-impurity F, 4-impurity B, 5-impurity C, 6-impurity D, 7-impurity G, 8-impurity H, 9-carbetocin, 10-impurity I; the separation between the impurity peaks, between the main peak of carbetocin and the adjacent impurity I peak, and between the main peak of carbetocin and the adjacent impurity H peak are not less than 1.5, and the tailing factor of the carbetocin peak is not greater than 2.0.

[0102] Therefore, compared with the prior art such as Comparative Example 1, the present invention does not contain ion pair reagents, phosphoric acid, acetic acid, trifluoroacetic acid and other traditional reagents in the mobile phase. By selecting a suitable mobile phase and controlling the conditions of the gradient elution program, it is possible to ensure effective separation of the main peak and impurities, while solving the problems of poor baseline, low response, and short service life of chromatographic column consumables in the prior art system, and effectively controlling the quality of carbetocin injection.

[0103] Example 2

[0104] This example provides a method for detecting related substances of carbetocin injection, which differs from Example 1 only in that the condition parameters of step (2.1) and high performance liquid chromatography are adjusted to:

[0105] Mobile phase A: The mobile phase A is composed of a phosphate buffer solution with a volume ratio of 73:27 and an acetonitrile aqueous solution with a volume fraction of 80%;

[0106] Mobile phase B: The mobile phase B is composed of a phosphate buffer solution with a volume ratio of 68:32 and an acetonitrile aqueous solution with a volume fraction of 80%;

[0107] Column: Agilent Peptide 4.6 x 150 mm, 2.7 um;

[0108] Detection wavelength: 218 nm;

[0109] Column temperature: 58 °C;

[0110] Flow rate: 0.8 ml / min;

[0111] Diluent: 5% acetic acid aqueous solution by volume;

[0112] Injection volume: 78ul;

[0113] The gradient elution method was adopted. The gradient elution procedure was as follows in terms of volume percentage:

[0114] .

[0115] Typical chromatogram obtained in this example - System suitability solution chromatogram is as follows Figure 3 As shown by Figure 3It can be seen that: blank solvent and blank excipients do not interfere with the determination of impurities and main peaks; the order of peaks in the chromatogram is: 1-impurity E, 2-impurity A, 3-impurity F, 4-impurity B, 5-impurity C, 6-impurity D, 7-impurity G, 8-impurity H, 9-carbetocin, 10-impurity I; the separation between the impurity peaks, between the main peak of carbetocin and the adjacent impurity I peak, and between the main peak of carbetocin and the adjacent impurity H peak are not less than 1.5, the tailing factor of the carbetocin peak is not greater than 2.0, and the baseline does not drift downward.

[0116] Example 3

[0117] This example provides a method for detecting related substances of carbetocin injection, which differs from Example 1 only in that the condition parameters of step (2.1) and high performance liquid chromatography are adjusted to:

[0118] Mobile phase A: The mobile phase A is composed of a phosphate buffer solution with a volume ratio of 78:22 and an acetonitrile aqueous solution with a volume fraction of 80%;

[0119] Mobile phase B: The mobile phase B is composed of a phosphate buffer solution with a volume ratio of 63:37 and an acetonitrile aqueous solution with a volume fraction of 80%;

[0120] Column: Agilent Peptide 4.6 x 150 mm, 2.7 um;

[0121] Detection wavelength: 218 nm;

[0122] Column temperature: 58 °C;

[0123] Flow rate: 0.8 ml / min;

[0124] Diluent: 5% acetic acid aqueous solution by volume;

[0125] Injection volume: 82ul;

[0126] The gradient elution method was adopted. The gradient elution procedure was as follows in terms of volume percentage:

[0127] .

[0128] Typical chromatogram obtained in this example - System suitability solution chromatogram is as follows Figure 4 As shown by Figure 4It can be seen that: blank solvent and blank excipients do not interfere with the determination of impurities and main peaks; the order of peaks in the chromatogram is: 1-impurity E, 2-impurity A, 3-impurity F, 4-impurity B, 5-impurity C, 6-impurity D, 7-impurity G, 8-impurity H, 9-carbetocin, 10-impurity I; the separation between the impurity peaks, between the main peak of carbetocin and the adjacent impurity I peak, and between the main peak of carbetocin and the adjacent impurity H peak are not less than 1.5, the tailing factor of the carbetocin peak is not greater than 2.0, and the baseline does not drift downward.

[0129] Comparative Example 1

[0130] This example provides a method for detecting related substances of carbetocin injection disclosed in the prior art (i.e. sodium octane sulfonate is added to the mobile phase as an ion pair reagent), which is different from Example 1 only in that the condition parameters of step (2.1) and high performance liquid chromatography are adjusted to:

[0131] Mobile phase A: The mobile phase A is ammonium acetate buffer, and the preparation of the ammonium acetate buffer includes the following process: weigh 0.37 g of ammonium acetate and 0.533 g of sodium octane sulfonate, dissolve them in water and dilute to 2000 ml to obtain a mixed solution; take 1620 ml of the mixed solution and add acetonitrile to 2000 ml, adjust the pH to 6.0 with 20% phosphoric acid by mass fraction, and obtain the ammonium acetate buffer;

[0132] Mobile phase B: The mobile phase B is composed of equal volumes of mobile phase A and acetonitrile;

[0133] Chromatographic column: Waters XBridgeR C18, 150x3.0mm 3.5um SN: 02353228518829 LC-042#;

[0134] Detection wavelength: 220 nm;

[0135] Column temperature: 60 °C;

[0136] Flow rate: 0.8 ml / min;

[0137] Diluent: water;

[0138] Injection volume: 50ul;

[0139] The gradient elution method was adopted. The gradient elution procedure was as follows in terms of volume percentage:

[0140] .

[0141] Typical chromatogram obtained in this example - System suitability solution chromatogram is as follows Figure 5 ,Depend on Figure 5It can be seen that the peak order is: 2-impurity A, 4-impurity B, 11-impurity C + impurity D, 1-impurity E, 3-impurity F, 7-impurity G, 8-impurity H, 9-carbetocin, 10-impurity I; the separation between the main peak of carbetocin and the adjacent impurity I peak, and between the main peak of carbetocin and the adjacent impurity H peak are not less than 1.5, and the tailing factor of the carbetocin peak is 1.2, but impurities C and impurity D overlap, and the baseline drifts seriously, which will lead to errors in integration, and the mobile phase is added with ion pair reagents, which is not conducive to the long-term use of the chromatographic column.

[0142] Comparative Example 2

[0143] On the basis of Comparative Example 1, the sodium octane sulfonate ion pair reagent in the mobile phase is removed in this example. The only difference from Comparative Example 1 is that in step (2.1), the mobile phase A in the condition parameters of the high performance liquid chromatography is adjusted to: the mobile phase A is an ammonium acetate buffer, and the preparation of the ammonium acetate buffer includes the following process: weighing 0.37 g of ammonium acetate, dissolving it in water and diluting it to 2000 ml to obtain a mixed solution; taking 1620 ml of the mixed solution and adding acetonitrile to 2000 ml, adjusting the pH to 6.0 with 20% phosphoric acid by mass fraction to obtain the ammonium acetate buffer.

[0144] Typical chromatogram obtained in this example - System suitability solution chromatogram is as follows Figure 6 ,Depend on Figure 6 It can be seen that the peak order is: 12-impurity A + impurity B, 13-impurity C + impurity D, 14-impurity E + impurity F, 7-impurity G, 8-impurity H, 9-carbetocin, 10-impurity I; the separation between the main peak of carbetocin and the adjacent impurity I peak, and between the main peak of carbetocin and the adjacent impurity H peak are not less than 1.5, the tailing factor of the carbetocin peak is 1.3, the retention time of impurity A is the same as that of impurity B, the retention time of impurity C is the same as that of impurity D, and the retention time of impurity E is the same as that of impurity F, which results in the inability to accurately detect the above impurities, and the baseline drifts seriously, which will cause errors in the integration.

[0145] Comparative Example 3

[0146] This example provides a method for detecting related substances of carbetocin injection, which is different from Example 1 only in that the mobile phase A is composed of a phosphate buffer solution with a volume ratio of 70:30 and an acetonitrile aqueous solution with a volume fraction of 80%.

[0147] Typical chromatogram obtained in this example - System suitability solution chromatogram is as follows Figure 7 ,Depend on Figure 7It can be seen that the peak order is: 12-impurity A + impurity B, 5-impurity C, 6-impurity D + impurity E, 3-impurity F, 7-impurity G, 8-impurity H, 9-carbetocin, 10-impurity I; the separation between the main peak of carbetocin and the adjacent impurity I peak, and between the main peak of carbetocin and the adjacent impurity H peak are not less than 1.5; the separation of impurity A, impurity B, and impurity C is less than 1.5, and they cannot be effectively separated;; the separation of impurity E and impurity F is less than 1.5, and they cannot be effectively separated, and the baseline drift is serious, resulting in the inability to quantitatively detect the above impurities, and the product quality cannot be accurately controlled.

[0148] Comparative Example 4

[0149] This example provides a method for detecting related substances of carbetocin injection, which is different from Example 1 only in that the mobile phase B is composed of a phosphate buffer solution with a volume ratio of 60:40 and an acetonitrile aqueous solution with a volume fraction of 80%.

[0150] Typical chromatogram obtained in this example - System suitability solution chromatogram is as follows Figure 8 ,Depend on Figure 8 It can be seen that the peak order is: 2-impurity A, 4-impurity B, 5-impurity C, 6-impurity D, 1-impurity E, 3-impurity F, 15-impurity G + impurity H, 9-carbetocin, 10-impurity I; the separation degree of impurity G and impurity H is less than 1.5, and their retention times completely overlap; the separation degree of impurity E and impurity F is 1.3, which is less than 1.5, resulting in the inability to quantitatively detect the above impurities, and the product quality cannot be accurately controlled and detected.

[0151] Comparative Example 5

[0152] This example provides a method for detecting related substances of carbetocin injection, which differs from Example 1 only in that, in terms of volume percentage, the gradient elution procedure is as follows:

[0153] .

[0154] Typical chromatogram obtained in this example - System suitability solution chromatogram is as follows Fig. 9 ,Depend on Fig. 9 It can be seen that the peak order is: 2-impurity A, 4-impurity B, 5-impurity C, 6-impurity D, 1-impurity E, 3-impurity F, 7-impurity G, 8-impurity H, 9-carbetocin, 10-impurity I; the separation between the main peak of carbetocin and the adjacent impurity I peak, and between the main peak of carbetocin and the adjacent impurity H peak are not less than 1.5, and the separation between impurities G and H is less than 1.5, which makes it impossible to accurately detect the above impurities, affecting product quality detection.

[0155] Comparative Example 6

[0156] This example provides a method for detecting related substances of carbetocin injection, which differs from Example 1 only in that, in terms of volume percentage, the gradient elution procedure is as follows:

[0157] .

[0158] Typical chromatogram obtained in this example - System suitability solution chromatogram is as follows Fig.10 ,Depend on Fig.10 It can be seen that the peak order is: 2-impurity A, 4-impurity B, 5-impurity C, 6-impurity D, 1-impurity E, 3-impurity F, 7-impurity G, 8-impurity H, 9-carbetocin, 10-impurity I; the separation between the main peak of carbetocin and the adjacent impurity I peak, and between the main peak of carbetocin and the adjacent impurity H peak are not less than 1.5, the separation between impurities B and C is less than 1.5, and the peak of impurity C is significantly extended, which makes it impossible to accurately detect the above impurities, affecting product quality control.

[0159] Test Case

[0160] In order to verify the scientificity and rationality of the detection method of related substances of carbetocin injection provided by the present invention, this method (taking the detection condition parameters provided in Example 1 as an example) was methodologically validated in accordance with the "Chinese Pharmacopoeia" (2020 Edition, Part 4 9101 Drug Quality Standard Analytical Method Verification Guidelines), and the validation items included system suitability (injection precision), specificity, quantitative limit and detection limit, accuracy, linearity and range, correction factor, precision (repeatability and intermediate precision), solution stability, filter membrane and durability, etc. The test results showed that the above test items all met the requirements.

[0161] Some test results are shown below:

[0162] 1) Impurity localization results: Blank solvent does not interfere with the elution of impurities; Blank excipients do not interfere with the elution of impurities; Test solution does not interfere with the detection of each impurity; The separation between the peak of the substance to be tested and the adjacent peak is not less than 1.5.

[0163] 2) Strong degradation destruction results: Under forced degradation conditions such as high temperature, high light, strong acid, strong alkali, and oxidation, the degradation products in this product can be effectively separated from the main peak, and the purity similarity and purity threshold at the main peak meet the requirements, and no impurities are detected; the mass is balanced before and after destruction (the material balance rate is in the range of 90%~110%), indicating that the chromatographic system of this method is reasonably designed and has strong specificity, which can ensure the accurate and effective detection of related substances of this product.

[0164] 3) Results of the limit of quantification and limit of detection test: The S / N of the limit of quantification solution of each component to be tested was between 10-30, and the peak area RSD was less than 10.0% after 6 consecutive injections. The limit of detection solution was injected 3 times in a row, and the S / N was between 3 and 8, which were in compliance with the regulations. The limit of quantification concentration was below the neglect line of 0.05%, which showed that the detection sensitivity of this method was high and could meet the requirements for the detection of related substances of this product.

[0165] 4) Durability test: After fine-tuning the condition parameters, the blank solvent and blank auxiliary materials did not interfere with the determination of impurities and main peaks; in the system suitability solution chromatogram, the separation between each impurity and the main peak was not less than 1.5; the absolute difference between the total impurity detection amount and the result under standard conditions was less than 0.5%. The above shows that this method has strong anti-interference ability and good durability.

[0166] In summary, the embodiment of the present invention provides a method for detecting related substances of carbetocin injection, which avoids the use of ion pair reagents in the mobile phase by selecting a suitable mobile phase and controlling the conditions of the gradient elution program. On the premise of ensuring effective separation of the main peak and impurities, the problems of poor baseline, low response, and short service life of chromatographic column consumables in the existing high performance liquid chromatography system are solved. The method has the characteristics of strong specificity, good precision, high accuracy, simplicity, high efficiency, and low cost, and can be applied to the quality control of carbetocin injection. At the same time, the cost of using chromatographic column consumables is greatly reduced, and the method is more suitable for commercial production.

[0167] Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which are applicable regardless of the range. In addition, whenever a numerical range is indicated herein, it is intended to include any cited number (fractional or integer) within the indicated range.

[0168] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for detecting related substances of carbetocin injection, characterized in that: The detection method of the carbetocin injection related substances comprises the following steps: Step (1), preparing solution: Step (1.1), prepare the diluent: measure 5 ml of acetic acid and 1000 ml of water, mix well, and obtain a 0.5% by volume acetic acid aqueous solution; Step (1.2), preparing the test solution: taking an appropriate amount of carbetocin injection to obtain the test solution; Step (1.3), preparing a reference solution: taking an appropriate amount of carbetocin reference solution and dissolving it in a 0.5% by volume aqueous acetic acid solution and quantitatively diluting it to make 0.08-0.12 mg of carbetocin per 1 ml, to obtain the reference solution; Step (1.4), preparing a reference substance stock solution: taking appropriate amounts of carbetocin, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, and impurity I, dissolving them in an 8% to 12% acetonitrile aqueous solution by volume, and quantitatively diluting them to prepare a reference substance stock solution containing 1.0 mg of carbetocin and 0.05 mg of each impurity per 1 ml; Step (1.5), preparing a system suitability solution: dissolving 0.1 ml of the reference substance stock solution prepared in step (1.4) in a 0.5% by volume aqueous acetic acid solution and quantitatively diluting the solution to obtain a system suitability solution containing 0.1 mg of carbetocin and 0.005 mg of each impurity per ml; Step (1.6), prepare a blank solution: prepare the same as the diluent, measure 5 ml of acetic acid and 1000 ml of water, mix well, and obtain the blank solution; Step (1.7), preparing a blank auxiliary material solution: dissolving L-methionine, succinic acid, mannitol and sodium hydroxide in water and diluting to prepare a solution containing about 1 mg of L-methionine, 1.19 mg of succinic acid, 47 mg of mannitol and 0.6 mg of sodium hydroxide per 1 ml, to obtain the blank auxiliary material solution; Step (1.8), preparing phosphate buffer: dissolving 2.5 g of potassium dihydrogen phosphate in water and diluting to 1000 ml, and then adjusting the pH value of the system to 5.0 with 0.1 mol / L potassium hydroxide solution to obtain the phosphate buffer; Step (2), using high performance liquid chromatography to detect the carbetocin-related substances: Step (2.1), HPLC conditions: Mobile phase A: The mobile phase A is composed of phosphate buffer with a volume ratio of 75:25 and acetonitrile aqueous solution with a volume fraction of 80%; Mobile phase B: The mobile phase B is composed of a phosphate buffer solution with a volume ratio of 65:35 and an acetonitrile aqueous solution with a volume fraction of 80%; Column: Agilent Peptide 4.6 x 150 mm, 2.7 um; Detection wavelength: 220 nm; Column temperature: 60 °C; Flow rate: 0.9 ml / min; Diluent: 5% acetic acid aqueous solution by volume; Injection volume: 80ul; The gradient elution method was adopted. The gradient elution procedure was as follows in terms of volume percentage: ； Step (2.2), detection operation: measure the test solution, reference solution and system suitability solution, etc., inject them into the liquid chromatograph respectively, detect according to the set gradient elution program, and record the chromatogram; The order of peaks in the chromatogram is: impurity E, impurity A, impurity F, impurity B, impurity C, impurity D, impurity G, impurity H, carbetocin, and impurity I; The impurity A is Carbetocin, the chemical structure is as follows: ; The impurity B is Carbetocin, the chemical structure is as follows: ； The impurity C is Carbetocin, the chemical structure is as follows: ； The impurity D is [Glu4] carbetocin, and its chemical structure is as follows: ； The impurity E is carbetocin sulfoxide I, and its chemical structure is as follows: ； The impurity F is carbetocin sulfoxide II, and its chemical structure is as follows: ； The impurity G is Carbetocin, the chemical structure is as follows: ； The impurity H is Carbetocin, the chemical structure is as follows: ； The impurity I is Carbetocin, the chemical structure is as follows: ； The system suitability test results meet the following requirements: the separation between each impurity peak, between the main peak of carbetocin and the adjacent impurity I peak, and between the main peak of carbetocin and the adjacent impurity H peak are not less than 1.5, and the tailing factor of the carbetocin peak is not greater than 2.0; The specificity test results satisfy the following requirements: the separation between the impurity peaks and between the main peak of carbetocin and the adjacent peaks is not less than 1.5.

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