Detection method and application of related substances in glycopyrronium bromide neostigmine injection

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a detection method and application of related substances of glycopyrronium bromide neostigmine injection. The related substances of the glycopyrronium bromide neostigmine injection are detected by adopting a high performance liquid chromatography, taking an aqueous solution of potassium dihydrogen phosphate and sodium heptanesulfonate as a mobile phase A and taking acetonitrile as a mobile phase B for gradient elution. In addition, the content of related substances can be calculated according to a main component reference substance external standard method with correction factors. The detection method provided by the invention has relatively high specificity and sensitivity, and excellent precision, accuracy and durability.

Description

[0001] This application claims priority to the prior application filed by the applicant with the State Intellectual Property Office on January 3, 2025, with patent application number 202510011115.9, entitled "A method and application for detecting related substances in glycopyrronium bromide neostigmine injection", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method and application for detecting related substances in glycopyrronium bromide neostigmine injection. Background Technology

[0003] The chemical name of glycopyrronium bromide is: (RS)-[3-(SR)-hydroxy-1,1-dimethylpyrrolidinyl]-α-cyclopentylmandelate, and its chemical structural formula is as follows:

[0004]

[0005] Glycerone bromide is an atropine-like quaternary ammonium anticholinergic drug, a quaternary ammonium muscarinic receptor antagonist with strong inhibitory effects on gastric acid secretion and mild gastrointestinal antispasmodic effects. This drug can regulate gastrointestinal motility, reduce gastric acid secretion and free acid concentration, and inhibit excessive secretion from the trachea and bronchi. Early formulations were used to treat gastric and duodenal ulcers, chronic gastritis, and excessive gastric acid secretion; marketed dosage forms include tablets, injections, and oral solutions. In addition, this drug has a stronger anti-salivation effect than atropine, and its duration of action is longer. Later studies found that glycopyrronium bromide is a selective M receptor antagonist, with high selectivity for cholinergic M3 receptors, and 3-5 times more selective for M3 and M1 receptors than for M2 receptors. Novartis developed glycopyrronium bromide into an inhaled powder formulation, which was launched in 2012 for long-term relief of symptoms in adults with chronic obstructive pulmonary disease (COPD). Furthermore, because the quaternary ammonium group of this drug restricts its passage across lipid cell membranes such as the blood-brain barrier, adverse reactions related to the central nervous system occur very rarely. This drug is 5 to 6 times more potent than an equivalent amount of atropine; its anti-salivation effect is better than atropine, and its sedative effect is milder than scopolamine; its adverse reactions such as increased heart rate, blurred vision, and fever are milder than atropine; delayed mydriasis is more pronounced when atropine is administered systemically, but this drug causes only a slight change; when used in combination with neostigmine to correct competitive muscle relaxant overdose, this drug results in less tachycardia and better anti-salivation compared to atropine combined with neostigmine.

[0006] The chemical name of neostigmine methylsulfate is N,N,N-trimethyl-3-[(N,N-dimethylamino)formyloxy]anilinium monomethyl sulfate, and the chemical structural formula is as follows:

[0007]

[0008] Neostigmine methylsulfate is a commonly used reversible anticholinesterase drug, which is a parasympathetic stimulant. The drug can inhibit the activity of cholinesterase, interfere with the degradation of acetylcholine, and indirectly stimulate nicotinic and muscarinic receptors to control muscle contraction without crossing the blood-brain barrier. In addition, the drug can directly stimulate the nicotinic receptors (N2 receptors) on the motor endplate of skeletal muscle, and its action characteristics are weak on the glands, eyes, cardiovascular and bronchial smooth muscles, can promote gastric contraction and increase gastric acid secretion, and promote the peristalsis of small and large intestines, especially the colon, thereby preventing intestinal relaxation and promoting the downward movement of intestinal contents. The product has strong skeletal muscle excitatory effect, but weak central effect. Therefore, neostigmine methylsulfate is commonly used for severe and emergency myasthenia gravis, respiratory prolongation caused by arrow poison agent (tubocurarine), postoperative and postpartum intestinal paralysis inhibition, gastrointestinal dysfunction, postoperative and postpartum urinary difficulty. It can be used for the treatment of excessive poisoning of competitive muscle relaxants, paroxysmal supraventricular tachycardia. It is also used for the treatment of movement disorders after brain trauma, paralysis in the recovery period of tuberculous meningitis, poliomyelitis and encephalitis, optic atrophy and neuritis, and other diseases. It is also used for various relaxation paralysis, muscle and nerve disorders in internal medicine, gynecology and otolaryngology.

[0009] At present, there is no detection method for the related substances of glycopyrronium bromide and neostigmine injection in the pharmacopoeias of various countries. The detection method of related substances in the quality standards of neostigmine methylsulfate and glycopyrronium bromide in the pharmacopoeias of various countries is only applicable to single preparation, and cannot simultaneously consider the separation and detection of two active ingredients and related impurities. At present, there are few literatures reporting the detection method of related substances of the compound preparation.

[0010] In summary, there is still no effective detection method for the related substances of glycopyrronium bromide and neostigmine injection at present, which cannot effectively monitor the impurity situation of glycopyrronium bromide and neostigmine injection. Therefore, it is of great significance to develop an impurity detection method suitable for the product for the quality control of glycopyrronium bromide and neostigmine injection. SUMMARY

[0011] The present application provides a detection method for the related substances of glycopyrronium bromide and neostigmine injection, which has good specificity and high sensitivity, can effectively detect the related substances of glycopyrronium bromide and neostigmine injection, and has superior precision, accuracy and durability, thereby providing a solution for the detection of related substances of glycopyrronium bromide and neostigmine injection.

[0012] To this end, the present application provides, in one aspect, a method for detecting related substances of glycopyrronium bromide neostigmine injection, comprising:

[0013] The method comprises the following steps: adopting high performance liquid chromatography, using potassium dihydrogen phosphate and sodium heptanesulfonate aqueous solution as mobile phase A, and acetonitrile as mobile phase B for gradient elution, and detecting related substances of glycopyrronium bromide neostigmine injection.

[0014] In a preferred embodiment of the present application, the gradient elution procedure is shown in the following table:

[0015] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 10 90 10 20 65 35 25 65 35 40 35 65 41 90 10 50 90 10

[0016] In a preferred embodiment of the present application, the concentration of potassium dihydrogen phosphate solution in the mobile phase A is 0.01 mol / L to 0.1 mol / L, and more preferably, 0.05 mol / L; the concentration of sodium heptanesulfonate solution is 0.001 mol / L to 0.006 mol / L, and more preferably, 0.003 mol / L; and the pH value is 2.8 to 3.2, and preferably, 3.0.

[0017] In a preferred embodiment of the present application, the flow rate of the mobile phase is 0.8 to 1.2 ml / min, and more preferably, 1.0 ml / min.

[0018] In a preferred embodiment of the present application, the chromatographic column uses octadecylsilane bonded silica gel as the filler, and more preferably, the model of the chromatographic column is Aglient poroshell 120EC-C18, 4.6*150mm, 4μm.

[0019] In a preferred embodiment of the present application, the column temperature of the chromatographic column is 35℃ to 45℃, and more preferably, 40℃.

[0020] In a preferred embodiment of the present application, the detector is an ultraviolet detector, and more preferably, the detection wavelength of the detector is 213 to 217 nm, and further preferably, 215 nm.

[0021] In a preferred embodiment of the present application, the injection amount is 5 to 20μl, and more preferably, 10μl.

[0022] In the preferred embodiments of the present application, the detection method further comprises:

[0023] Preparation of system suitability solution: take glycopyrronium bromide reference substance, neostigmine methylsulfate reference substance, GL-impurity A reference substance, GL-impurity B reference substance, GL-impurity C reference substance, XS-impurity A reference substance, XS-impurity B reference substance, and XS-impurity C reference substance, respectively, add appropriate amount of acetonitrile to dissolve, and then dilute with diluent to prepare a solution containing 0.5 mg of glycopyrronium bromide, 1 μg of GL-impurity A, 0.5 μg of GL-impurity B, 5 μg of GL-impurity C, 2.5 mg of neostigmine methylsulfate, 25 μg of XS-impurity A, 0.25 μg of XS-impurity B, and 2.5 μg of XS-impurity C per 1 ml;

[0024] Preparation of test solution: take the product to prepare a solution containing 0.5 mg of glycopyrronium bromide and 2.5 mg of neostigmine methylsulfate per 1 ml;

[0025] Preparation of reference solution: take glycopyrronium bromide reference substance and neostigmine methylsulfate reference substance, respectively, and dilute with diluent to prepare a solution containing 0.1 mg of glycopyrronium bromide and 0.5 mg of neostigmine methylsulfate per 1 ml;

[0026] Sensitivity solution: take appropriate amount of reference solution, and add diluent to prepare a solution containing 0.25 μg of neostigmine methylsulfate and 0.05 μg of glycopyrronium bromide per 1 ml;

[0027] The diluent is mobile phase A-mobile phase B (90:10).

[0028] In the preferred embodiments of the present application, the detection method further comprises:

[0029] The content of neostigmine methylsulfate and glycopyrronium bromide in the glycopyrronium bromide and neostigmine methylsulfate injection is calculated by the principal component reference external standard method with correction factor,

[0030] The calculation formula of the content of glycopyrronium bromide impurities is:

[0031]

[0032] Wherein, C1 is the concentration of glycopyrronium bromide in the reference solution, mg / ml; A1 is the peak area of glycopyrronium bromide in the reference solution; Ai is the peak area of GL-impurity A / GL-impurity B / GL-impurity C in the test solution; B1 is the labeled amount of glycopyrronium bromide in the test, 0.5 mg; F is the correction factor of impurities, which is calculated by linearity, F = slope of glycopyrronium bromide / slope of impurities;

[0033] The calculation formula of the content of neostigmine methylsulfate impurities is

[0034]

[0035] Wherein, C2 is the concentration of neostigmine methylsulfate in the control solution, mg / ml; A2 is the peak area of neostigmine methylsulfate in the control solution; Ai is the peak area of XS-impurity A / XS-impurity B / XS-impurity C in the test solution; B2 is the labeled amount of neostigmine methylsulfate in the test solution, 2.5 mg; F is the correction factor of the impurity, which is calculated by linear calculation, F = slope of neostigmine methylsulfate / slope of the impurity;

[0036] Wherein, when the content of the related substances of the glycopyrronium bromide is determined, the correction factor F of GL-impurity A is 0.1, the correction factor F of GL-impurity B is 0.7, and the correction factor F of GL-impurity C is 0.5;

[0037] When the content of the related substances of the neostigmine methylsulfate is determined, the correction factor F of XS-impurity A is 0.8, the correction factor F of XS-impurity B is 0.7, and the correction factor F of XS-impurity C is 0.6.

[0038] Another aspect of the present application provides the use of the detection method of the present application in detecting the related substances of glycopyrronium bromide neostigmine injection.

[0039] Still another aspect of the present application provides a quality control standard for the related substances of glycopyrronium bromide neostigmine injection, wherein the detection method of the present application is used to detect the related substances.

[0040] Wherein, in the chromatogram of the test solution, if there is an impurity peak, the content of GL-impurity A, GL-impurity B and GL-impurity C in the test solution is calculated by the external standard method of the main component, and the content of each of them should not exceed 0.2%, 0.1% and 1.2% of the labeled amount of glycopyrronium bromide, respectively; the content of XS-impurity A, XS-impurity B and XS-impurity C in the test solution is calculated by the external standard method of the main component, and the content of each of them should not exceed 1.0%, 0.01% and 0.1% of the labeled amount of neostigmine methylsulfate, respectively; the content of other single unknown impurity should not exceed 0.2% by the area normalization method; and the total content of other impurities should not exceed 1.0%.

[0041] The detection method of the present application is verified by system suitability test, single impurity positioning test, quantitative limit detection limit test, linearity test and accuracy test, and it is proved that the detection method of the related substances of glycopyrronium bromide neostigmine injection of the present application has the following beneficial effects:

[0042] 1. The detection method of the present application has good system suitability;

[0043] 2. In the detection method of the present application, the related substances can be well separated from the main peak and each impurity peak.

[0044] 3、The detection method of the present application has high sensitivity, and the concentration of the related substances that can be effectively detected is lower than the reporting limit;

[0045] 4、The detection method of the present application has high accuracy;

[0046] 5、In the detection method of the present application, the related substances show good linear relationship within a certain concentration range. BRIEF DESCRIPTION OF DRAWINGS

[0047] The accompanying drawings are used to provide a further understanding of the technical solutions of the present application, and constitute a part of the specification, and are used together with the embodiments of the present application to explain the technical solutions of the present application, and do not constitute a limitation on the technical solutions of the present application.

[0048] Figure 1 The chromatogram of the blank solution in the system suitability test of Example 1;

[0049] Figure 2 The chromatogram of the system suitability solution in the system suitability test of Example 1;

[0050] Figure 3 The chromatogram of the sensitivity solution in the system suitability test of Example 1;

[0051] Figure 4 The chromatogram of the blank auxiliary material solution in the specificity test of Example 2;

[0052] Figure 5 The chromatogram of the limit of quantification-mixed solution in the limit of detection and limit of quantification test of Example 3;

[0053] Figure 6 The chromatogram of the limit of quantification-neostigmine impurity C in the limit of detection and limit of quantification test of Example 3;

[0054] Figure 7 The chromatogram of the limit of quantification-glycopyrronium bromide + glycopyrronium bromide impurity B in the limit of detection and limit of quantification test of Example 3;

[0055] Figure 8 The chromatogram of the 100% linearity of the main components glycopyrronium bromide and neostigmine methylsulfate in the linearity test of Example 4;

[0056] Figure 9 The chromatogram of the 100% linearity of the impurity mixed solution of glycopyrronium bromide and neostigmine methylsulfate in the linearity test of Example 4;

[0057] Figure 10 The chromatogram of the 100% linearity of neostigmine methylsulfate impurity C in the linearity test of Example 4;

[0058] Figure 11Chromatogram of the 100% accuracy solution of the gromyronium bromide and neostigmine methylsulfate impurity mixture solution in the accuracy test of Example 5;

[0059] Figure 12 Chromatogram of the 100% accuracy solution of neostigmine methylsulfate impurity C in the accuracy test of Example 5;

[0060] Figure 13 Chromatogram of the 0 h control solution in the solution stability test of Example 6;

[0061] Figure 14 Chromatogram of the 0 h test solution in the solution stability test of Example 6;

[0062] Figure 15 Chromatogram of the GLXS2 107 0601 batch test solution of Example 7. DETAILED DESCRIPTION

[0063] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively used to explain and illustrate the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of protection intended by the present application. It should be noted that the examples in the present application and the features in the examples can be combined with each other in any manner without conflict.

[0064] Unless otherwise specified, the potassium dihydrogen phosphate, sodium heptanesulfonate, phosphoric acid, gromyronium bromide neostigmine injection, gromyronium bromide reference substance, neostigmine methylsulfate reference substance, GL-impurity A reference substance, GL-impurity B reference substance, GL-impurity C reference substance, XS-impurity A reference substance, XS-impurity B reference substance, XS-impurity C reference substance, acetonitrile, etc. used in the following examples can be obtained by purchase.

[0065] Example 1: System suitability test of gromyronium bromide neostigmine injection

[0066] 1. Instruments (Agilent HPLC 1260) and chromatographic conditions

[0067] (1) Chromatographic column: Agilent poroshell 120 EC-C18, 4.6*150 mm, 4 μm;

[0068] (2) Mobile phase A: 0.05 mol / L potassium dihydrogen phosphate solution (containing 0.003 mol / L sodium heptanesulfonate, and the pH value is adjusted to 3.0 by phosphoric acid);

[0069] (3) Mobile phase B: acetonitrile;

[0070] (4) Elution gradient: as shown in the following table.

[0071] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 10 90 10 20 65 35 25 65 35 40 35 65 41 90 10 50 90 10

[0072] (5) Diluent: Mobile phase A - Mobile phase B (90:10). In the following examples, the diluent used is the same as in Example 1 unless otherwise specified.

[0073] (6) Detection wavelength: 215 nm.

[0074] (7) Injection volume: 10 μl.

[0075] (8) Flow rate: 1.0 ml / min.

[0076] (9) Column temperature: 40.0 °C.

[0077] 2. Experimental procedure (1) Preparation of solutions

[0078] A. Blank solution (Diluent)

[0079] B. System suitability solution

[0080] Weigh about 10 mg of GL-impurity A, GL-impurity B, GL-impurity C reference substance, accurately, respectively, in different 100 ml volumetric flasks, dissolve and dilute to the mark with diluent, and shake well to obtain each impurity stock solution.

[0081] Weigh about 12.5 mg of XS-impurity A, XS-impurity B, XS-impurity C reference substance, accurately, respectively, in different 50 ml volumetric flasks, dissolve and dilute to the mark with diluent, and shake well to obtain each impurity stock solution.

[0082] Weigh 5 mg of glycopyrronium bromide reference substance and 25 mg of neostigmine methylsulfate reference substance, accurately, in a 10 ml volumetric flask, add 0.1 ml, 0.05 ml, 0.5 ml of GL-impurity A, B, C stock solution, respectively, and 1 ml, 0.01 ml, 0.1 ml of XS-impurity A, B, C stock solution, dilute to the mark with diluent, and shake well to obtain the solution.

[0083] C. Test solution

[0084] Take the product and inject directly.

[0085] D. Reference solution

[0086] Take glycopyrronium bromide reference substance and neostigmine methylsulfate reference substance, respectively, accurately, dilute with diluent to obtain a solution containing about 0.1 mg of glycopyrronium bromide and 0.5 mg of neostigmine methylsulfate per 1 ml.

[0087] E. Sensitivity solution

[0088] Take the control solution appropriate amount, add diluent to make each 1 ml containing neostigmine methylsulfate 0.25 μg, glycopyrronium bromide 0.05 μg solution.

[0089] (2) Respectively take the above solution injection liquid chromatograph, record chromatogram.

[0090] 3, experimental results and analysis

[0091] System applicability results are shown in the following table.

[0092]

[0093]

[0094] The results showed that the blank solution had no interference on the detection of related substances; the signal-to-noise ratio (S / N) of neostigmine peak in the sensitivity solution was 36, the separation degree of glycopyrronium bromide peak and glycopyrronium bromide impurity B peak in the system suitability solution was 3.55; the RSD of glycopyrronium bromide peak area in the control solution was 0.1%, the RSD of retention time was 0.1%, the RSD of neostigmine peak area was 0.1%, the RSD of retention time was 0.1%, and the system suitability was good.

[0095] In this example, the chromatograms of the blank solution, system suitability solution and sensitivity solution are shown in Figures 1-3, respectively. Figures 1-3

[0096] Example 2: Specificity test of glycopyrronium bromide neostigmine injection (known impurity positioning test)

[0097] 1, instrument (Agilent HPLC 1260) and chromatographic conditions

[0098] The same as example 1.

[0099] 2, experimental steps (1) solution preparation

[0100] A, blank solution (diluent)

[0101] B, blank excipient solution

[0102] Take citric acid 0.80 g, disodium hydrogen phosphate dodecahydrate 1.164 g, dilute with water to 20 ml volumetric flask, and you get it.

[0103] C, each impurity positioning solution

[0104] Take GL-impurity A, GL-impurity B, GL-impurity C reference substance about 10 mg each, accurately weigh, respectively in different 100 ml volumetric flask, dissolve and dilute to the mark with diluent, get each impurity stock solution;

[0105] ​Take about 12.5 mg of each of the XS-impurity A, XS-impurity B and XS-impurity C reference substances, accurately weigh, and respectively place in different 50 ml volumetric flasks. Dissolve and dilute to the mark with the diluent, shake well to obtain the impurity stock solutions.

[0106] Take 1 ml, 0.5 ml and 5 ml of the GL-impurity A, B and C stock solutions respectively, and 10 ml, 0.1 ml and 1 ml of the XS-impurity A, B and C stock solutions respectively, and respectively place in different 100 ml volumetric flasks. Add the diluent to dilute to the mark, shake well to obtain the solutions.

[0107] D. System suitability solution

[0108] Take about 10 mg of each of the GL-impurity A, GL-impurity B and GL-impurity C reference substances, accurately weigh, and respectively place in different 100 ml volumetric flasks. Dissolve and dilute to the mark with the diluent, shake well to obtain the impurity stock solutions.

[0109] Take about 12.5 mg of each of the XS-impurity A, XS-impurity B and XS-impurity C reference substances, accurately weigh, and respectively place in different 50 ml volumetric flasks. Dissolve and dilute to the mark with the diluent, shake well to obtain the impurity stock solutions.

[0110] Take 5 mg of the glycopyrronium bromide reference substance and 25 mg of the neostigmine methylsulfate reference substance, accurately weigh, and place in a 10 ml volumetric flask. Add 0.1 ml, 0.05 ml and 0.5 ml of the GL-impurity A, B and C stock solutions respectively, and 1 ml, 0.01 ml and 0.1 ml of the XS-impurity A, B and C stock solutions respectively. Add the diluent to dilute to the mark, shake well to obtain the solution.

[0111] E. Test solution

[0112] Take the product, and directly inject the sample.

[0113] (2) Respectively inject the above solutions into the liquid chromatograph, and record the chromatogram.

[0114] 3. Experimental results and analysis

[0115] The retention times of the impurities are shown in the following table.

[0116] Name Retention time (min) Glycopyrronium bromide 32.065 Neostigmine 14.256 GL- Impurity A 7.837 GL- Impurity B 33.872 GL- Impurity C 40.756 XS- Impurity A 4.492 XS- Impurity B 6.154 XS- Impurity C 25.749

[0117] The results show that the blank solution and the blank excipient do not interfere with the detection of the related substances; in the chromatogram of the system suitability solution, the separation degree of the glycopyrronium bromide peak and the glycopyrronium bromide impurity B peak is not less than 1.5; the separation degree of the main peak and the adjacent impurities, and the separation degree of each impurity peak are all greater than 1.5 (the minimum separation degree between impurities is 1.67).

[0118] In this example, the chromatogram of the blank excipient solution is shown in the following figure. Figure 4 ​

[0119] Example 3: Limit of detection and limit of quantitation test for glycopyrronium bromide neostigmine injection

[0120] 1. Instruments (Agilent HPLC 1260) and chromatographic conditions

[0121] The same as in Example 1.

[0122] 2. Experimental procedure (1) Preparation of solutions

[0123] A. Stock solution of reference substance

[0124] About 10 mg of glycopyrronium bromide reference substance was accurately weighed into a 100-ml volumetric flask, dissolved and diluted to the mark with diluent, and shaken to give a glycopyrronium bromide stock solution.

[0125] About 10 mg of neostigmine methylsulfate reference substance was accurately weighed into a 20-ml volumetric flask, dissolved and diluted to the mark with diluent, and shaken to give a neostigmine methylsulfate stock solution.

[0126] B. Stock solution of impurities

[0127] About 10 mg of GL-impurity A, GL-impurity B, and GL-impurity C reference substances were accurately weighed into different 100-ml volumetric flasks, dissolved and diluted to the mark with diluent, and shaken to give stock solutions of the respective impurities.

[0128] About 12.5 mg of XS-impurity A, XS-impurity B, and XS-impurity C reference substances were accurately weighed into different 50-ml volumetric flasks, dissolved and diluted to the mark with diluent, and shaken to give stock solutions of the respective impurities.

[0129] C. LOQ (limit of quantitation) solution

[0130] The reference substance stock solution and the stock solutions of the respective impurities were quantitatively diluted to solutions in which the signal-to-noise ratio of the peak height of each component was greater than 10, to serve as the limit of quantitation solution.

[0131] D. LOD (limit of detection) solution

[0132] The reference substance stock solution and the stock solutions of the respective impurities were quantitatively diluted to solutions in which the signal-to-noise ratio of the peak height of each component was about 3, to serve as the limit of detection solution.

[0133] (2) The limit of quantitation solution and the limit of detection solution were injected into the liquid chromatograph, respectively, and the chromatogram was recorded.

[0134] 3. Experimental results and analysis

[0135] The test results are shown in the following table.

[0136]

[0137]

[0138] The results show that the quantitative limit concentration of each impurity is less than the report limit, and the content of the related substances can be accurately controlled.

[0139] In this example, the chromatograms of the quantitative limit-mixed solution, the quantitative limit-neostigmine impurity C, and the quantitative limit-glycopyrronium bromide + glycopyrronium bromide impurity B are shown in FIGS. 1-3, respectively. Figures 5-7

[0140] Example 4: Linear test of glycopyrronium bromide neostigmine injection

[0141] 1. Instrument (Agilent HPLC 1260) and chromatographic conditions

[0142] The same as in Example 1.

[0143] 2. Experimental steps (1) Solution preparation

[0144] A. Reference substance stock solution

[0145] About 10 mg of glycopyrronium bromide reference substance was accurately weighed, placed in a 100 ml volumetric flask, dissolved and diluted with diluent to the mark, and shaken to obtain a glycopyrronium bromide stock solution.

[0146] About 10 mg of neostigmine methylsulfate reference substance was accurately weighed, placed in a 20 ml volumetric flask, dissolved and diluted with diluent to the mark, and shaken to obtain a neostigmine methylsulfate stock solution.

[0147] B. Impurity stock solution

[0148] About 10 mg of GL-impurity A, GL-impurity B, and GL-impurity C reference substances were accurately weighed, respectively placed in 100 ml, 100 ml, and 20 ml volumetric flasks, dissolved and diluted with diluent to the mark, and shaken to obtain the respective impurity stock solutions.

[0149] About 12.5 mg of XS-impurity A, XS-impurity B, and XS-impurity C reference substances were accurately weighed, respectively placed in different 50 ml volumetric flasks, dissolved and diluted with diluent to the mark, and shaken to obtain the respective impurity stock solutions.

[0150] C. Main component linearity was prepared according to the following table

[0151]

[0152] D. GL-impurity A, GL-impurity B, GL-impurity C, XS-impurity A, and XS-impurity B were prepared according to the following table

[0153] ​

[0154] E, XS- Impurity C linearly prepared according to the following table

[0155]

[0156]

[0157] (2) The above solution was injected respectively and the chromatogram was recorded, with the concentration as the abscissa and the peak area as the ordinate, to make a linear regression equation.

[0158] 3. Experimental results and analysis

[0159] The linear results are shown in the following table respectively.

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170] The results show that glycopyrronium bromide has a good linear relationship in the concentration range of LOQ~200% (0.000275 mg / ml~0.0110 mg / ml).

[0171] GL-Impurity A has a good linear relationship in the concentration range of LOQ~150% (0.0000303 mg / ml~0.00151 mg / ml).

[0172] GL-Impurity B has a good linear relationship in the concentration range of LOQ~150% (0.000233 mg / ml~0.000700 mg / ml).

[0173] GL-Impurity C has a good linear relationship in the concentration range of LOQ~150% (0.0000741 mg / ml~0.00741 mg / ml).

[0174] Glycopyrronium bromide and neostigmine methylsulfate showed good linearity in the LOQ~200% concentration range (0.000129 mg / ml~0.0515 mg / ml, 0.0000861 mg / ml~0.0344 mg / ml in terms of neostigmine).

[0175] XS-impurity A showed good linearity in the LOQ~150% (0.0000464 mg / ml~0.0232 mg / ml) concentration range.

[0176] XS-impurity B showed good linearity in the LOQ~150% (0.0000754 mg / ml~0.000377 mg / ml) concentration range.

[0177] XS-impurity C showed good linearity in the LOQ~150% (0.0000796 mg / ml~0.00398 mg / ml) concentration range.

[0178] In this example, the chromatograms of the 100% linearity of the main components glycopyrronium bromide and neostigmine methylsulfate, the 100% linearity of the mixed solution of glycopyrronium bromide and neostigmine methylsulfate impurities, and the 100% linearity of neostigmine methylsulfate impurity C are shown in FIGS. 1-3, respectively. Figures 8-10

[0179] Example 5: Accuracy test of glycopyrronium bromide and neostigmine injection

[0180] 1. Instruments (Agilent HPLC 1260) and chromatographic conditions

[0181] The same as Example 1.

[0182] 2. Experimental steps (1) Solution preparation

[0183] A. Blank excipient stock solution

[0184] Take citric acid 8.0 g, disodium hydrogen phosphate dodecahydrate 11.64 g, and place them in a 50 ml volumetric flask. Add water to constant volume to the mark, and you get it.

[0185] B. Glycopyrronium bromide stock solution (5 mg / ml)

[0186] Take glycopyrronium bromide 0.1 g, accurately weigh, and place it in a 20 ml volumetric flask. Dissolve and dilute to the mark with water, and you get it.

[0187] C. Neostigmine methylsulfate stock solution (25 mg / ml)

[0188] Take neostigmine methylsulfate 0.5 g, accurately weigh, and place it in a 20 ml volumetric flask. Dilute to the mark with water, and you get it.

[0189] ​D, Impurity stock solution

[0190] Take about 10 mg of GL-Impurity A, GL-Impurity B, GL-Impurity C reference substance respectively, accurately weigh, respectively in 100 ml, 100 ml, 20 ml volumetric flask, dissolved and diluted with diluent to the mark, shake, get each impurity stock solution;

[0191] Take about 12.5 mg of XS-Impurity A, XS-Impurity C reference substance respectively, accurately weigh, respectively in different 50 ml volumetric flask, dissolved and diluted with diluent to the mark, shake, get each impurity stock solution;

[0192] Take about 12.5 mg of XS-Impurity B reference substance, accurately weigh, in 50 ml volumetric flask, take 5.0 ml in 50 ml volumetric flask, dissolved and diluted with diluent to the mark, shake, get XS-Impurity B stock solution;

[0193] E, Base solution

[0194] Take 1.0 ml of glycopyrronium bromide stock solution and 1.0 ml of neostigmine methylsulfate stock solution, add 1.0 ml of auxiliary blank solution, in 10 ml volumetric flask, dilute with water to the mark, get it.

[0195] F, Mixed impurity LOQ limit solution

[0196] Precisely take 2 ml of GL-Impurity A, 1 ml of GL-Impurity C stock solution, 2 ml of neostigmine impurity A stock solution, in 20 ml volumetric flask, dilute with diluent to the mark, shake;

[0197] Take 3 ml of the above mixed solution, in 100 ml volumetric flask, add 2.5 ml of GL-Impurity B stock solution, 3 ml of XS-Impurity B stock solution, dilute with diluent to the mark, shake;

[0198] Take 0.5 ml of the above mixed solution, add 0.5 ml of auxiliary blank stock solution, 0.5 ml of glycopyrronium bromide stock solution, 0.5 ml of neostigmine methylsulfate stock solution, in 5 ml volumetric flask, dilute with water to the mark, get it.

[0199] Prepare 3 copies in parallel.

[0200] G, Mixed impurity 100% limit solution

[0201] Precisely take 0.5 ml of GL-Impurity A, 0.25 ml of GL-Impurity B, 0.5 ml of GL-Impurity C stock solution, 0.5 ml of XS-Impurity B stock solution, in 5 ml volumetric flask, dilute with diluent to the mark, shake;

[0202] Take the above mixed solution 0.5ml, add auxiliary blank stock solution 0.5ml, XS-impurity A stock solution 0.5ml, glycopyrronium bromide stock solution 0.5ml, methionine neostigmine stock solution 0.5ml, 5ml volumetric flask, dilute to the mark with water, get.

[0203] Prepare 3 copies in parallel.

[0204] H, mixed impurities 120% limit solution

[0205] Respectively, take GL-impurity A, B, C stock solution 1.2ml, 0.6ml, 1.2ml, neostigmine impurity B stock solution 1.2ml, 10ml volumetric flask, dilute to the mark with diluent, shake well;

[0206] Take the above mixed solution 0.5ml, add auxiliary blank stock solution 0.5ml, XS-impurity A stock solution 0.6ml, glycopyrronium bromide stock solution 0.5ml, methionine neostigmine stock solution 0.5ml, 5ml volumetric flask, dilute to the mark with water, get.

[0207] Prepare 3 copies in parallel.

[0208] I, mixed impurity control solution

[0209] Respectively, take GL-A, B, C stock solution 1ml, 0.5ml, 1ml, XS-impurity B stock solution 1ml, 10ml volumetric flask, dilute to the mark with diluent, shake well;

[0210] Take the above mixed solution 1ml, add XS-impurity A stock solution 1ml, 10ml volumetric flask, dilute to the mark with diluent, get.

[0211] J, XS-impurity C-LOQ limit solution

[0212] Take XS-impurity C stock solution 1ml, 10ml volumetric flask, dilute to the mark with water, shake well;

[0213] Take 3ml, 100ml volumetric flask, dilute to the mark with water, shake well;

[0214] Take the above solution 0.5ml, add auxiliary blank stock solution 0.5ml, glycopyrronium bromide stock solution 0.5ml, methionine neostigmine stock solution 0.5ml, 5ml volumetric flask, dilute to the mark with water, get.

[0215] Prepare 3 copies in parallel.

[0216] K, XS-impurity C-100% limit solution

[0217] Accurately take 1.2 ml of the XS-impurity C stock solution into a 10-ml volumetric flask, dilute to the mark with diluent, and mix.

[0218] Take 0.5 ml of the above mixture solution, add 0.5 ml of the blank stock solution of adjuvant, 0.5 ml of the stock solution of glycopyrronium bromide, and 0.5 ml of the stock solution of neostigmine methylsulfate into a 5-ml volumetric flask, dilute to the mark with water, and you get the solution.

[0219] Prepare 3 samples in parallel.

[0220] L, XS-impurity C-120% limit solution

[0221] Accurately take 1.2 ml of the XS-impurity C stock solution into a 10-ml volumetric flask, dilute to the mark with diluent, and mix.

[0222] Take 0.5 ml of the above mixture solution, add 0.5 ml of the blank stock solution of adjuvant, 0.5 ml of the stock solution of glycopyrronium bromide, and 0.5 ml of the stock solution of neostigmine methylsulfate into a 5-ml volumetric flask, dilute to the mark with water, and you get the solution.

[0223] Prepare 3 samples in parallel.

[0224] M, XS-impurity C control solution

[0225] Accurately take 1 ml of the XS-impurity C stock solution into a 100-ml volumetric flask, dilute to the mark with diluent, and mix.

[0226] (2) Sample separately and record the chromatogram, and calculate the recovery rate.

[0227] 3. Experimental results and analysis

[0228] The linear results are shown in the following tables, respectively.

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237] The results show that the recovery of each impurity at the limit of quantification, 100% and 120% test levels is between 85% and 115%, and the accuracy of the present application is good.

[0238] In the present embodiment, the chromatogram of the 100% accuracy solution of the mixed solution of impurities of glycopyrronium bromide and neostigmine methylsulfate and the chromatogram of the 100% accuracy solution of impurity C of neostigmine methylsulfate are shown in FIGS. 1 and 2, respectively. Figures 11-12

[0239] Example 6: Solution stability test of glycopyrronium bromide and neostigmine injection

[0240] 1. Instruments (Agilent HPLC 1260) and chromatographic conditions

[0241] The same as Example 1.

[0242] 2. Experimental procedure (1) Solution preparation

[0243] A. Test solution

[0244] Take the product and directly inject the sample.

[0245] B. Reference solution

[0246] Take about 10 mg of glycopyrronium bromide reference substance, accurately weigh, and place in a 10 ml volumetric flask. Dissolve and dilute to the mark with diluent, and mix well.

[0247] Take about 10 mg of neostigmine methylsulfate reference substance, accurately weigh, and place in a 20 ml volumetric flask. Accurately add 2 ml of the above glycopyrronium bromide solution, dissolve and dilute to the mark with diluent, and shake well.

[0248] (2) Take the test solution and the reference solution at room temperature, and inject the sample at different time points, respectively, and record the chromatogram.

[0249] 3. Experimental results and analysis

[0250] The results show that the reference solution is stable within 67 h, and the test solution is stable within 60 h.

[0251] In the present embodiment, the chromatogram of the reference solution at 0 h and the chromatogram of the test solution at 0 h are shown in FIGS. 3 and 4, respectively. Figures 13-14

[0252] Example 7: Determination of related substances of glycopyrronium bromide and neostigmine injection

[0253] 1. Instruments (Agilent HPLC 1260) and chromatographic conditions

[0254] The same as Example 1.

[0255] 2. Experimental procedure (1) Solution preparation​​

[0256] A, blank solution (diluent)

[0257] B, system suitability solution

[0258] Take about 10 mg of GL-impurity B and GL-impurity C reference substances respectively, accurately weigh, and respectively place in different 100 ml volumetric flasks, dissolve and dilute to the mark with diluent, shake well, and obtain each impurity stock solution;

[0259] Take about 12.5 mg of XS-impurity A and XS-impurity B reference substances respectively, accurately weigh, and respectively place in different 50 ml volumetric flasks, dissolve and dilute to the mark with diluent, shake well, and obtain each impurity stock solution;

[0260] Take 5 mg of glycopyrronium bromide reference substance and 25 mg of neostigmine methylsulfate reference substance, accurately weigh, and place in a 10 ml volumetric flask. Add 0.05 ml and 0.5 ml of GL-impurity B and C stock solutions respectively, and 1 ml and 0.01 ml of XS-impurity A and B stock solutions respectively, and dilute to the mark with diluent, shake well, and obtain.

[0261] C, test solution

[0262] Take the product directly for sampling.

[0263] D, reference solution

[0264] Take glycopyrronium bromide reference substance and neostigmine methylsulfate reference substance respectively, accurately weigh, and dilute with diluent to prepare a solution containing about 0.1 mg of glycopyrronium bromide and 0.5 mg of neostigmine methylsulfate per 1 ml.

[0265] E, sensitivity solution

[0266] Take the reference solution, add diluent to prepare a solution containing 0.25 μg of neostigmine methylsulfate and 0.05 μg of glycopyrronium bromide per 1 ml.

[0267] (2) Inject the above solutions into the liquid chromatograph respectively, and record the chromatogram.

[0268] (3) Use the above detection method to check the related substances of three batches of glycopyrronium bromide neostigmine injection for 12 months, and the results are as follows:

[0269]

[0270] The results show that GL-impurity B (%) is not detected, which is lower than 0.1% of the quality standard; GL-impurity C (%) is 0.85% and 0.84% respectively, which is lower than 1.2% of the quality standard; XS-impurity A is 0.12% respectively, which is lower than 1.0% of the quality standard; XS-impurity B (%) is not detected, which is lower than 0.01% of the quality standard; the maximum unknown single impurity is 0.07%, 0.06%, 0.06%, which is lower than 0.2% of the quality standard; the total impurities are 0.28%, 0.27%, 0.27% respectively, which is lower than 1.0% of the quality standard. Among them, GL-impurity A and XS-impurity C are confirmed as process impurities, which are not determined in the quality standard, so they are not detected.

[0271] In the present embodiment, the chromatogram of GLXS21070601 batch test sample solution is shown in Figure 1. Figure 15

[0272] Finally, it should be pointed out that the above embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.​

Claims

1. A method for detecting related substances of glycopyrronium bromide neostigmine injection, comprising: using high performance liquid chromatography, using an aqueous solution of potassium dihydrogen phosphate and sodium heptanesulfonate as mobile phase A and acetonitrile as mobile phase B for gradient elution to detect related substances of glycopyrronium bromide neostigmine injection, wherein the related substances of glycopyrronium bromide are 5-nitrobenzene-1, 3-dicarboxylic acid (GL-impurity A), 1-methylpyrrolidin-3-yl-2-cyclopentyl-2-hydroxy-2-phenylacetic acid (GL-impurity B), 2-cyclopentyl-2-hydroxy-2-phenylacetic acid (GL-impurity C), and the related substances of neostigmine methylsulfate are 3-hydroxy-N, N, N-trimethyl aniline methylsulfate (XS-impurity A), 3-dimethylamino phenol (XS-impurity B), 3- (dimethylamino) -phenyl-dimethylamino carbamate (XS-impurity C).

2. The detection method according to claim 1, wherein, The concentration of potassium dihydrogen phosphate solution in the mobile phase A is 0.01mol / L-0.1mol / L, preferably 0.05mol / L; the concentration of sodium heptanesulfonate solution is 0.001mol / L-0.006mol / L, preferably 0.003mol / L; the pH value is 2.8-3.2, preferably 3.

0.

3. The detection method according to claim 1 or 2, wherein, The flow rate of the mobile phase is 0.8-1.2ml / min, preferably 1.0ml / min.

4. The assay of any one of claims 1-3, wherein, The chromatographic column uses octadecylsilane-bonded silica gel as the filler, and preferably, the type of the chromatographic column is Aglientporoshell 120EC-C18, 4.6*150mm, 4μm.

5. The detection method according to any one of claims 1 to 4, wherein, The column temperature of the chromatographic column is 35℃-45℃, preferably 40℃.

6. The assay of any one of claims 1-5, wherein, The detector is an ultraviolet detector, and preferably, the detection wavelength of the detector is 213-217nm, more preferably 215nm.

7. The assay method according to any one of claims 1 to 6, wherein, The injection volume is 5-20μl, preferably 10μl.

8. The assay of any one of claims 1-7, wherein, Further comprising: Preparation of system suitability solution: take appropriate amounts of glycopyrronium bromide reference substance, neostigmine methylsulfate reference substance, GL-impurity A reference substance, GL-impurity B reference substance, GL-impurity C reference substance, XS-impurity A reference substance, XS-impurity B reference substance, and XS-impurity C reference substance, dissolve them in appropriate amount of acetonitrile, and then dilute with diluent to prepare a solution containing 0.5mg of glycopyrronium bromide, 1μg of GL-impurity A, 0.5μg of GL-impurity B, 5μg of GL-impurity C, 2.5mg of neostigmine methylsulfate, 25μg of XS-impurity A, 0.25μg of XS-impurity B, and 2.5μg of XS-impurity C per 1ml; Preparation of test sample solution: take the product to prepare a solution containing 0.5mg of glycopyrronium bromide and 2.5mg of neostigmine methylsulfate per 1ml; Preparation of reference substance solution: take appropriate amounts of glycopyrronium bromide reference substance and neostigmine methylsulfate reference substance, and dilute with diluent to prepare a solution containing 0.1mg of glycopyrronium bromide and 0.5mg of neostigmine methylsulfate per 1ml; Sensitivity solution: take appropriate amount of reference substance solution, and dilute with diluent to prepare a solution containing 0.25μg of neostigmine methylsulfate and 0.05μg of glycopyrronium bromide per 1ml; The diluent is mobile phase A-mobile phase B (90:10).

9. The assay of any one of claims 1-8, wherein, Also included are: The content of the related substances in the glycopyrronium bromide neostigmine injection is calculated by the principal component calibration sample external standard method with a correction factor, The calculation formula of the content of the glycopyrronium bromide impurities is: Wherein, C1 is the concentration of glycopyrronium bromide in the calibration sample solution, mg / ml; A1 is the peak area of glycopyrronium bromide in the calibration sample solution; Ai is the peak area of GL-impurity A / GL-impurity B / GL-impurity C in the test sample solution; B1 is the labeled amount of glycopyrronium bromide in the test sample, 0.5 mg; F is the correction factor of the impurities, which is calculated by linear, F = slope of glycopyrronium bromide / slope of impurities; The calculation formula of the content of the neostigmine methylsulfate impurities is Wherein, C2 is the concentration of neostigmine methylsulfate in the calibration sample solution, mg / ml; A2 is the peak area of neostigmine methylsulfate in the calibration sample solution; Ai is the peak area of XS-impurity A / XS-impurity B / XS-impurity C in the test sample solution; B2 is the labeled amount of neostigmine methylsulfate in the test sample, 2.5 mg; F is the correction factor of the impurities, which is calculated by linear, F = slope of neostigmine methylsulfate / slope of impurities; Wherein, when the content of the related substances of glycopyrronium bromide is determined, the correction factor F of GL-impurity A is 0.1, the correction factor F of GL-impurity B is 0.7, and the correction factor F of GL-impurity C is 0.5; When the content of the related substances of neostigmine methylsulfate is determined, the correction factor F of XS-impurity A is 0.8, the correction factor F of XS-impurity B is 0.7, and the correction factor F of XS-impurity C is 0.

6.

10. The detection method of any one of claims 1-9 is used for detecting the related substances of glycopyrronium bromide neostigmine injection.

11. A quality control standard for related substances in glycopyrronium bromide neostigmine injection, wherein, The detection method of any one of claims 1-9 is used for detecting the related substances.

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