A method for detecting a mivacurium double bond z configuration isomer by liquid chromatography

Abstract

The application belongs to the technical field of compound detection, and discloses a liquid chromatography detection method for a double-bond Z configuration isomer of mivacurium chloride. In the test process, a chromatographic column is a tricosylsilane bonded silica gel chromatographic column, and a gradient elution is adopted in the elution mode. In the elution process, a mobile phase A and a mobile phase B are both mixed solutions of organic solvents and aqueous solutions. The aqueous solution is an aqueous solution with or without added salt substances. The salt substances are selected from at least one of potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, ammonium acetate, ammonium formate, sodium bicarbonate and ammonium bicarbonate. The aqueous solution is added with a pH regulator. The organic solvents are selected from at least one of methanol, isopropyl alcohol and acetonitrile.

Description

Technical Field

[0001] This application relates to the field of compound detection technology, and in particular to a liquid chromatography method for detecting the Z-configuration isomer of micous chloride double bond. Background Technology

[0002] Micuronium chloride is a non-depolarizing neuromuscular blocking agent used as an adjunct to general anesthesia to relax skeletal muscles, facilitating endotracheal intubation and mechanical ventilation. Neuromuscular blocking agents are classified into depolarizing and non-depolarizing agents based on their mechanism of action, and into short-acting, intermediate-acting, and long-acting agents based on their duration of action. Depolarizing drugs have serious side effects, such as cardiac arrest, malignant hyperthermia, and arrhythmias. Short-acting drugs are generally safer than long-acting drugs, while long-acting drugs can cause severe brain damage.

[0003] Micuronium chloride is currently the only short-acting, non-depolarizing muscle relaxant in clinical practice. It has a rapid onset of action, short duration of action, no accumulation, and rapid recovery. Micuronium chloride is not dependent on organ metabolism and is rapidly degraded by cholinesterase in plasma. It has no potential neurotoxicity and can be widely used in neurosurgical practice. Micuronium chloride is the only muscle relaxant that does not require routine antagonism, has a much lower incidence of residual muscle relaxation than other non-depolarizing muscle relaxants, has no significant histamine release, and has a low incidence of allergic reactions.

[0004] Micucurbita chloride is a compound with multiple chiral centers. Commercially available drugs are mixtures of three isomers: trans-trans, trans-cis, and cis-cis, typically in proportions of 52-62%, 34-40%, and 4-8%, respectively. The Z-configuration isomer of micucurbita chloride (ZZO) is an isomer caused by the different relative positions of the substituents at both ends of the double bond in the molecule, as described by a sequence rule. It is difficult to separate and quantify using conventional chromatographic columns (such as C18 columns), and this impurity is easily overlooked because it co-elutes with the main peak of micucurbita chloride. According to the guidelines of ICH Q3A and Chinese Pharmacopoeia 2025 Edition 9102, it is necessary to study known impurities.

[0005] Currently, there are very few analytical methods for micudium chloride, mainly focusing on degradation impurities and optical isomers. For example, US4761418 reports a chromatographic separation method for detecting three isomers and four impurities of micudium chloride, CN107782821A discloses a method for detecting three isomers and related substances of micudium chloride, and CN117434167A discloses a method for detecting (E)-oct-4-ene-1,8-diacid in micudium chloride. In addition to the above patented methods, the applicant has also reviewed a large number of Chinese and foreign patent and non-patent literatures and found that there are currently no literature reports on the detection of the double bond Z-isomer (ZZO) of micudium chloride using high performance liquid chromatography. Chinese patent application 201910686614.2 discloses an HPLC detection method for intermediate isomers of micu chloride. However, upon further observation of the scheme, it can be seen that it mainly detects isomers of intermediates in the preparation process of micu chloride. Whether it can distinguish between micu chloride and the Z-type isomer of micu chloride double bond remains unknown.

[0006] The problem this solution aims to solve is: how to provide a detection method that can detect the content of the Z-isomer of the double bond in micuronium chloride. Summary of the Invention

[0007] The purpose of this application is to provide a method for detecting the content of the Z-isomer of the double bond in micuronium chloride. This method improves the accuracy of the detection of the Z-isomer content by optimizing the chromatographic column.

[0008] To achieve the above objectives, this application discloses a liquid chromatography detection method for the Z configuration isomer of micous chloride double bond. During the test, the chromatographic column is a triacontylsilane-bonded silica gel column, and the elution method is gradient elution. During the elution process, both mobile phase A and mobile phase B are mixtures of organic solvent and aqueous solution; The aqueous solution may be an aqueous solution with or without added salts; The salt is selected from at least one of potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, ammonium acetate, ammonium formate, sodium bicarbonate, and ammonium bicarbonate. The aqueous solution contains a pH adjuster, and the pH value of the aqueous solution is 2.0 to 7.0; The organic solvent is selected from at least one of methanol, isopropanol, and acetonitrile.

[0009] Preferably, the chromatographic column is a triacontylsilane-bonded silica column with dimensions of 30–250 mm × 2.1–4.6 mm and a diameter of 1.6–5.0 μm.

[0010] Preferably, the volume ratio of the aqueous solution to the organic solvent in the mobile phase A is 5-9:1-5; The volume ratio of organic solvent to aqueous solution in the mobile phase B is 1-10:1-10.

[0011] Preferably, during the test, the column temperature of the chromatographic column is 15-50℃, the detector is an ultraviolet detector with a detection wavelength of 190-280nm, the flow rate is 0.4-2.0mL / min, and the injection volume is 5-50μL.

[0012] Preferably, the gradient elution conditions are as follows: Minute 0: Mobile phase A is 98–91.5%, mobile phase B is 2–8.5%; At 23 minutes: mobile phase A was 98–91.5%, and mobile phase B was 2–8.5%. At 26 minutes: mobile phase A was 87-75%, and mobile phase B was 13-25%. At 32 minutes: Mobile phase A was 70-50%, and mobile phase B was 30-50%. At 35 minutes: mobile phase A was 45-40%, and mobile phase B was 55-60%. At 35.5 min: mobile phase A was 98–91.5%, and mobile phase B was 2–8.5%; At 40 min: mobile phase A was 98–91.5%, and mobile phase B was 2–8.5%; Or the conditions for a gradient elution procedure are: Minute 0: Mobile phase A is 92-88%, mobile phase B is 8-12%; At 20 minutes: Mobile phase A was 92-88%, and mobile phase B was 8-12%. At 23 minutes: mobile phase A was 87-83%, and mobile phase B was 13-17%. At 27 minutes: mobile phase A was 55-45%, and mobile phase B was 45-55%. At 31 min: mobile phase A was 55-45%, and mobile phase B was 45-55%. At 31.5 min: mobile phase A was 92-88%, and mobile phase B was 8-12%; At 35 minutes: mobile phase A was 92-88%, and mobile phase B was 8-12%.

[0013] Preferably, the pH adjuster is selected from at least one of phosphoric acid, formic acid, and acetic acid.

[0014] Preferably, when the aqueous solution is an aqueous solution with added salts, the concentration of salts in the aqueous solution is 0.01 to 0.15 mol / L.

[0015] Preferably, it includes the following steps: Step 1: Take micuronium chloride and dilute it with mobile phase A to obtain a test solution with a concentration of micuronium chloride of 0.1-2.0 mg / ml; Step 2: Take the Z-configuration isomer of micuronium double bond and dilute it with mobile phase A to obtain a reference solution with a concentration of 0.1-2.0 μg / ml of micuronium double bond Z-configuration isomer; Step 3: Take the reference solution and the test solution, inject them into the high performance liquid chromatograph, and record the chromatogram; Step 4: Calculate the content of the Z configuration isomer of the double bond of micous chloride using the external standard method based on the peak area.

[0016] The beneficial effects of this application are: This application provides a liquid chromatography method for the detection of the Z-configuration isomer of micuronium chloride double bonds. The method uses a triacontylsilane-bonded silica column as the detection column and employs gradient elution to separate peak 1 (the trans-trans isomer) from the cis-trans isomers of micuronium chloride. Utilizing the relatively constant ratio of the three isomers, quantification is achieved by detecting the peak area of ​​the Z-configuration trans-trans isomers of micuronium chloride using an external standard method. This method avoids the use of short-life, expensive chiral columns and highly toxic normal-phase reagents such as n-hexane, which are unfriendly to personnel, instruments, and the environment. This effectively reduces enterprise costs and embodies the concept of green chemistry. Furthermore, the method is simple, easy to operate, efficient, and rapid, achieving quantitative detection of the Z-configuration isomer of micuronium chloride double bonds within 40 minutes. It exhibits good sample stability, ensuring product quality and is more conducive to enterprise production and daily testing, improving efficiency and significantly reducing detection costs. This method fills the technological gap in the detection of the Z-configuration isomer of micuronium chloride double bonds and has practical application value. Attached Figure Description

[0017] Figure 1 The high-performance liquid chromatogram obtained by detecting the spiked solution of the test sample in Example 1 of this application; Figure 2 The high-performance liquid chromatogram (190 nm) obtained by detecting the spiked solution of the test sample in Example 2 of this application is shown. Figure 3 The high performance liquid chromatogram (280 nm) obtained by detecting the spiked solution of the test sample in Example 2 of this application is shown. Figure 4 Example 3 of this application shows the high-performance liquid chromatogram obtained by detecting the spiked solution of the test sample; Figure 5 Example 4 of this application shows the high-performance liquid chromatogram obtained by detecting the spiked solution of the test sample; Figure 6 Example 5 of this application shows the high-performance liquid chromatogram obtained by detecting the spiked solution of the test sample; Figure 7 Example 6 of this application shows the high-performance liquid chromatogram obtained by detecting the spiked solution of the test sample; Figure 8 The high-performance liquid chromatogram obtained from the detection of the test solution in Comparative Example 1 of this application is shown below. Figure 9 The high-performance liquid chromatogram obtained from the detection of the test solution in Comparative Example 2 of this application is shown below. Figure 10The high-performance liquid chromatogram obtained from the detection of the test solution in Comparative Example 3 of this application; Figure 11 This is the high-performance liquid chromatogram obtained from the detection of the reference solution in Comparative Example 4 of this application; Figure 12 The high-performance liquid chromatogram obtained from the detection of the test solution in Comparative Example 4 of this application; Figure 13 The high-performance liquid chromatogram obtained from the detection of the spiked solution of the test sample in Comparative Example 5 of this application; Figure 14 The high-performance liquid chromatogram obtained from the blank solution in Part II, Method Validation - Specificity Detection; Figure 15 The high-performance liquid chromatogram obtained for the reference solution in Part II, Method Validation - Specificity Detection; Figure 16 The high-performance liquid chromatogram obtained for Part II, Method Validation - Specificity Detection of the Test Solution; Figure 17 The high-performance liquid chromatogram obtained from the spiked solution of the test sample in Part II, Method Validation - Specificity Detection; Figure 18 High-performance liquid chromatogram of the solution used for the detection limit of the method validation in Part II; Figure 19 High-performance liquid chromatogram obtained from the limit of quantitation solution for method validation in Part II; Figure 20 This is a schematic diagram of the linear regression equation for calculating the peak area of ​​the ZZO anti-anti isomers for the methodology validation in Part II. Detailed Implementation

[0018] The present application will now be clearly and completely described in conjunction with its embodiments. It should be noted that, unless otherwise specified in the embodiments, conditions are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0019] Part 1: Validation of the Liquid Chromatography Detection Method

[0020] Example 1

[0021] Main experimental instruments and chromatographic conditions Instrument: Agilent 1260 II; Chromatographic column: triacontylsilane-bonded silica gel as the packing material (ArtChrom C30 4.6mm×250mm, 5μm); Mobile phase A: 100 mmol / L potassium dihydrogen phosphate solution (dissolve 13.6 g of potassium dihydrogen phosphate in 1000 mL of water and adjust the pH to 3.0 with phosphoric acid) - acetonitrile (volume ratio 75:25); Mobile phase B: Acetonitrile: Phosphoric acid aqueous solution (adjust the pH of pure water to 3.0 with phosphoric acid) = 8:2 (volume ratio); Wavelength: 210nm; Flow rate: 0.9 mL / min; Column temperature: 30℃; Injection volume: 10 μL; The gradient elution program is shown in Table 1; Table 1 Gradient elution program for Example 1 Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 20 90 10 23 85 15 27 50 50 31 50 50 31.5 90 10 35 90 10

[0022] Blank solution: Mobile phase A.

[0023] Reference solution: Accurately weigh an appropriate amount of ZZO reference standard, dissolve and dilute it with mobile phase A to prepare a solution containing approximately 1 μg per 1 mL.

[0024] Spiked solution for test sample: Accurately weigh an appropriate amount of micuronium chloride sample and ZZO reference standard, dissolve and dilute with mobile phase A to prepare a mixed solution containing approximately 1 mg micuronium chloride and approximately 1 μg ZZO per 1 mL.

[0025] It should be noted that the reference solution is mainly used to observe the peak position of ZZO and as an external standard during the test; in addition, since the ZZO content in some micuronium chloride is low, the addition of ZZO to the spiked solution of the test sample is mainly used to actively introduce impurities to test the detection capability of the detection system.

[0026] Assay: Accurately measure 10 μL each of blank solution, reference solution, and spiked test solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the results using the peak area of ​​the ZZO trans-trans isomer according to the external standard method. See results below. Figure 1 Compared with Table 2: Table 2 Test results of Example 1 project Retention time (min) Resolution Micudium chloride trans-trans isomer 20.722 - ZZO anti-trans isomer 23.059 2.62 Micudium chloride transcis isomer 25.085 2.40

[0027] The above results indicate that under these chromatographic conditions, the micuronium trans-trans isomer, the ZZO trans-trans isomer, and the micuronium trans-cis isomer are well separated.

[0028] Example 2 Main experimental instruments and chromatographic conditions Instrument: Agilent 1260 II; Chromatographic column: triacontylsilane-bonded silica gel as the packing material (ArtChrom C30 4.6mm×250mm, 5μm); Mobile phase A: 75 mmol / L potassium dihydrogen phosphate solution (dissolve 10.2 g of potassium dihydrogen phosphate in 1000 mL of water and adjust the pH to 3.0 with phosphoric acid) - acetonitrile (volume ratio 75:25); Mobile phase B: Acetonitrile: Phosphoric acid aqueous solution (adjust the pH of pure water to 3.0 with phosphoric acid) = 8:2 (volume ratio); Wavelengths: 190nm, 280nm; Flow rate: 1.0 mL / min; Column temperature: 50℃; Injection volume: 10 μL; The gradient elution program is shown in Table 3; Table 3 Gradient elution program table for Example 2 Time (min) Mobile phase A (%) Mobile phase B (%) 0 91.5 8.5 23 91.5 8.5 26 80 20 32 50 50 35 45 55 35.5 91.5 8.5 40 91.5 8.5

[0029] Blank solution: Mobile phase A.

[0030] Reference solution: Accurately weigh an appropriate amount of ZZO reference standard, dissolve and dilute it with mobile phase A to prepare a solution containing approximately 1 μg per 1 mL.

[0031] Spiked solution for test sample: Accurately weigh an appropriate amount of micuronium chloride sample and ZZO reference standard, dissolve and dilute with mobile phase A to prepare a mixed solution containing approximately 1 mg micuronium chloride and approximately 1 μg ZZO per 1 mL.

[0032] Assay: Accurately measure 10 μL each of blank solution, reference solution, and spiked test solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the results using the peak area of ​​the ZZO trans-trans isomer according to the external standard method. See results below. Figures 2-3 Compared with Tables 4 and 5: Table 4 Test results of Example 2 (190nm) project Retention time (min) Resolution Micudium chloride trans-trans isomer 23.151 - ZZO anti-trans isomer 25.866 2.06 Micudium chloride transcis isomer 28.214 2.03

[0033] Table 5 Test results of Example 2 (280nm) project Retention time (min) Resolution Micudium chloride trans-trans isomer 23.149 - ZZO anti-trans isomer 25.978 3.16 Micudium chloride transcis isomer 28.213 3.04

[0034] The above results indicate that under these chromatographic conditions, the micuronium trans-trans isomer, the ZZO trans-trans isomer, and the micuronium trans-cis isomer are well separated.

[0035] Example 3 Main experimental instruments and chromatographic conditions Instrument: Agilent 1260 II; Chromatographic column: triacontylsilane-bonded silica gel as the packing material (ArtChrom C30 4.6mm×250mm, 5μm); Mobile phase A: 75 mmol / L potassium dihydrogen phosphate solution (dissolve 10.2 g of potassium dihydrogen phosphate in 1000 mL of water and adjust the pH to 3.0 with phosphoric acid) - acetonitrile (volume ratio 75:25); Mobile phase B: Acetonitrile: Phosphoric acid aqueous solution (adjust the pH of pure water to 3.0 with phosphoric acid) = 8:2 (volume ratio); Wavelength: 210nm; Flow rate: 1.0 mL / min; Column temperature: 15℃; Injection volume: 5 μL; The gradient elution program is shown in Table 6; Table 6 Gradient elution program table for Example 3 Time (min) Mobile phase A (%) Mobile phase B (%) 0 91.5 8.5 23 91.5 8.5 26 80 20 32 50 50 35 45 55 35.5 91.5 8.5 40 91.5 8.5

[0036] Blank solution: Mobile phase A.

[0037] Reference solution: Accurately weigh an appropriate amount of ZZO reference standard, dissolve and dilute it with mobile phase A to prepare a solution containing approximately 1 μg per 1 mL.

[0038] Spiked solution for test sample: Accurately weigh an appropriate amount of micuronium chloride sample and ZZO reference standard, dissolve and dilute with mobile phase A to prepare a mixed solution containing approximately 1 mg micuronium chloride and approximately 1 μg ZZO per 1 mL.

[0039] Assay: Accurately measure 5 μL each of blank solution, reference solution, and spiked test solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the results using the peak area of ​​the ZZO trans-trans isomer according to the external standard method. See results below. Figure 4 Compared with Table 7: Table 7 Test results of Example 3 project Retention time (min) Resolution Micudium chloride trans-trans isomer 24.076 - ZZO anti-trans isomer 27.600 2.98 Micudium chloride transcis isomer 29.133 1.95

[0040] The above results indicate that under these chromatographic conditions, the micuronium trans-trans isomer, the ZZO trans-trans isomer, and the micuronium trans-cis isomer are well separated.

[0041] Example 4 Main experimental instruments and chromatographic conditions Instrument: Agilent 1260 II; Chromatographic column: Triacontylsilane-bonded silica gel column (ArtChrom C30 4.6mm×250mm, 5μm); The mobile phase consisted of 75 mmol / L potassium dihydrogen phosphate solution (10.2 g of potassium dihydrogen phosphate was dissolved in 1000 mL of water, and the pH was adjusted to 3.0 with phosphoric acid) and acetonitrile (volume ratio 75:25). Mobile phase B: Acetonitrile: Phosphoric acid aqueous solution (adjust the pH of pure water to 3.0 with phosphoric acid) = 8:2 (volume ratio); Wavelength: 210nm; Flow rate: 2.0 mL / min; Column temperature: 30℃; Injection volume: 10 μL; The gradient elution program is shown in Table 8; Table 8 Gradient elution program table for Example 4 Time (min) Mobile phase A (%) Mobile phase B (%) 0 91.5 8.5 23 91.5 8.5 26 80 20 32 50 50 35 45 55 35.5 91.5 8.5 40 91.5 8.5

[0042] Blank solution: Mobile phase A.

[0043] Reference solution: Accurately weigh an appropriate amount of the Z configuration isomer of micous chloride (ZZO) reference standard, dissolve and dilute it in mobile phase A to prepare a solution containing approximately 1 μg per 1 mL.

[0044] Spiked solution for test sample: Accurately weigh an appropriate amount of micuronium chloride sample and ZZO reference standard, dissolve and dilute with mobile phase A to prepare a mixed solution containing approximately 1 mg micuronium chloride and approximately 1 μg ZZO per 1 mL.

[0045] Assay: Accurately measure 10 μL each of blank solution, reference solution, and spiked solution of test sample, inject them into the liquid chromatograph, record the chromatogram, and calculate the results using the peak area of ​​the ZZO trans-trans isomer according to the external standard method. See results below. Figure 5 Compared with Table 9: Table 9 Test results of Example 4 project Retention time (min) Resolution Micudium chloride trans-trans isomer 12.913 - ZZO anti-trans isomer 14.472 1.72 Micudium chloride transcis isomer 16.037 1.64

[0046] Conclusion: The above results indicate that under these chromatographic conditions, the micuronium trans-trans isomer, the ZZO trans-trans isomer, and the micuronium trans-cis isomer are well separated.

[0047] Example 5 Main experimental instruments and chromatographic conditions Instrument: Agilent 1260 II; Chromatographic column: Triacontylsilane-bonded silica gel column (ArtChrom C30 4.6mm×250mm, 5μm); The mobile phase consisted of 75 mmol / L potassium dihydrogen phosphate solution (10.2 g of potassium dihydrogen phosphate was dissolved in 1000 mL of water, and the pH was adjusted to 3.0 with phosphoric acid) and acetonitrile (volume ratio 75:25). Mobile phase B: Acetonitrile: Phosphoric acid aqueous solution (adjust the pH of pure water to 3.0 with phosphoric acid) = 8:2 (volume ratio); Wavelength: 210nm; Flow rate: 0.4 mL / min; Column temperature: 30℃; Injection volume: 50 μL; The gradient elution program is shown in Table 10; Table 10 Gradient elution program table for Example 5 Time (min) Mobile phase A (%) Mobile phase B (%) 0 91.5 8.5 57.5 91.5 8.5 65 80 20 80 50 50 87.5 45 55 88.75 91.5 8.5 100 91.5 8.5

[0048] Blank solution: Mobile phase A.

[0049] Reference solution: Accurately weigh an appropriate amount of the Z configuration isomer of micous chloride (ZZO) reference standard, dissolve and dilute it in mobile phase A to prepare a solution containing approximately 1 μg per 1 mL.

[0050] Spiked solution for test sample: Accurately weigh an appropriate amount of micuronium chloride sample and ZZO reference standard, dissolve and dilute with mobile phase A to prepare a mixed solution containing approximately 1 mg micuronium chloride and approximately 1 μg ZZO per 1 mL.

[0051] Assay: Accurately measure 50 μL each of blank solution, reference solution, and spiked solution of test sample, inject them into the liquid chromatograph, record the chromatogram, and calculate the results using the peak area of ​​the ZZO trans-trans isomer according to the external standard method. See results below. Figure 6 Compared with Table 11: Table 11 Test results of Example 5 project Retention time (min) Resolution Micudium chloride trans-trans isomer 54.588 - ZZO anti-trans isomer 61.676 2.69 Micudium chloride transcis isomer 67.315 2.10

[0052] Conclusion: The above results indicate that under these chromatographic conditions, the micuronium trans-trans isomer, the ZZO trans-trans isomer, and the micuronium trans-cis isomer are well separated.

[0053] Example 6 Main experimental instruments and chromatographic conditions Instrument: Agilent 1260 II; Chromatographic column: Triacontylsilane-bonded silica gel column (ArtChrom C30 4.6mm×250mm, 5μm); The mobile phase consisted of 75 mmol / L potassium dihydrogen phosphate solution (10.2 g of potassium dihydrogen phosphate was dissolved in 1000 mL of water, and the pH was adjusted to 3.0 with phosphoric acid) and acetonitrile (volume ratio 75:25). Mobile phase B: Acetonitrile: Phosphoric acid aqueous solution (adjust the pH of pure water to 3.0 with phosphoric acid) = 8:2 (volume ratio); Wavelength: 210nm; Flow rate: 1.0 mL / min; Column temperature: 30℃; Injection volume: 10 μL; The gradient elution program is shown in Table 12; Table 12 Gradient elution program table for Example 6 Time (min) Mobile phase A (%) Mobile phase B (%) 0 91.5 8.5 23 91.5 8.5 26 80 20 32 50 50 35 45 55 35.5 91.5 8.5 40 91.5 8.5

[0054] Blank solution: Mobile phase A.

[0055] Reference solution: Accurately weigh an appropriate amount of the Z configuration isomer of micous chloride (ZZO) reference standard, dissolve and dilute it in mobile phase A to prepare a solution containing approximately 1 μg per 1 mL.

[0056] Spiked solution for test sample: Accurately weigh an appropriate amount of micuronium chloride sample and ZZO reference standard, dissolve and dilute with mobile phase A to prepare a mixed solution containing approximately 1 mg micuronium chloride and approximately 1 μg ZZO per 1 mL.

[0057] Assay: Accurately measure 10 μL each of blank solution, reference solution, and spiked solution of test sample, inject them into the liquid chromatograph, record the chromatogram, and calculate the results using the peak area of ​​the ZZO trans-trans isomer according to the external standard method. See results below. Figure 7 Compared with Table 13: Table 13 Test results of Example 6 project Retention time (min) Resolution Micudium chloride trans-trans isomer 21.207 - ZZO anti-trans isomer 23.744 2.61 Micudium chloride transcis isomer 26.033 2.30

[0058] Conclusion: The above results indicate that under these chromatographic conditions, the micuronium trans-trans isomer, the ZZO trans-trans isomer, and the micuronium trans-cis isomer are well separated.

[0059] Comparative Example 1 Main experimental instruments and chromatographic conditions Instrument: Agilent 1260 II Prime; Column: Octadecylsilane-bonded silica gel as packing material (Agilent Poroshell 120EC-C18, 150 mm × 3.0 mm, 2.7 μm); Column temperature: 30℃; Detection wavelength: 280nm; Mobile phase A: 75 mmol / L potassium dihydrogen phosphate solution: methanol: acetonitrile = 75:20:5 (v / v); Mobile phase B: acetonitrile:water solution volume ratio = 8:2, wherein the water solution is phosphoric acid adjusted to pure water to pH 3.0; Flow rate: 0.8 mL / min; Injection volume: 5 μL; The gradient elution program is shown in Table 14; Table 14 Gradient elution program for Comparative Example 1 Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 3.6 95 5 4.5 90 10 7.5 85 15 9.1 77.5 22.5 16.3 75 25 19.6 57.5 42.5 27.0 57.5 42.5 27.5 100 0 30.0 100 0

[0060] Blank solution: Mobile phase A.

[0061] Reference solution: Accurately weigh an appropriate amount of ZZO reference standard, dissolve and dilute it with mobile phase A to prepare a solution containing approximately 1 μg per 1 mL.

[0062] Spiked solution for test sample: Accurately weigh an appropriate amount of micuronium chloride sample and ZZO reference standard, dissolve and dilute with mobile phase A to prepare a mixed solution containing approximately 1 mg micuronium chloride and approximately 1 μg ZZO per 1 mL.

[0063] Assay: Accurately measure 5 μL each of blank solution, reference solution, and spiked test solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the results using the peak area of ​​the ZZO trans-trans isomer according to the external standard method. See results below. Figure 8 .

[0064] The results showed that the trans-trans isomers of micous chloride and impurity O trans-trans isomers co-eluted without separation.

[0065] Comparative Example 2 Main experimental instruments and chromatographic conditions Instrument: Agilent 1260 II; Chromatographic column: silica gel coated with amylose-tris(3,5-dimethylphenylcarbamate) (CHIRALCELAD-H, 4.6 mm × 250 mm, 5 μm); Mobile phase: 0.1% hexane acetic acid solution - anhydrous ethanol (1:9); Wavelength: 280 nm; Flow rate: 1.0 mL / min; Column temperature: 30℃; Injection volume: 20 μL; Washing method: Isocratic washing for 20 minutes Blank solution: anhydrous ethanol.

[0066] Reference solution: Accurately weigh an appropriate amount of ZZO reference standard, dissolve and dilute it with anhydrous ethanol to prepare a solution containing approximately 15 μg per 1 mL.

[0067] Test solution: Accurately weigh an appropriate amount of mico chloride sample, dissolve and dilute with anhydrous ethanol to prepare a solution containing approximately 1 mg per 1 mL.

[0068] Assay: Accurately measure 20 μL each of blank solution, reference solution, and test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the results using the peak area of ​​the ZZO trans-trans isomers according to the external standard method. See results below. Figure 9 .

[0069] Results: No obvious chromatographic peaks were detected in either the reference solution or the test solution.

[0070] Comparative Example 3 Main experimental instruments and chromatographic conditions Instrument: Agilent 1260 II; Chromatographic column: Silica gel coated with cellulose-tris(4-methylbenzoate) (CHIRALCEL OJ-H, 4.6 mm × 250 mm, 5 μm); Mobile phase: 0.1% hexane acetic acid solution - anhydrous ethanol (1:9); Wavelength: 280 nm; Flow rate: 1.0 mL / min; Column temperature: 30℃; Injection volume: 20 μL; Washing method: Isocratic washing for 20 minutes Blank solution: anhydrous ethanol.

[0071] Reference solution: Accurately weigh an appropriate amount of ZZO reference standard, dissolve and dilute it with anhydrous ethanol to prepare a solution containing approximately 15 μg per 1 mL.

[0072] Test solution: Accurately weigh an appropriate amount of mico chloride sample, dissolve and dilute with anhydrous ethanol to prepare a solution containing approximately 1 mg per 1 mL.

[0073] Assay: Accurately measure 20 μL each of blank solution, reference solution, and test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the results using the peak area of ​​the ZZO trans-trans isomers according to the external standard method. See results below. Figure 10 .

[0074] Results: No obvious chromatographic peaks were detected in either the reference solution or the test solution.

[0075] Conclusion: The above results indicate that micuronium chloride is strongly retained in normal-phase chiral systems, and even anhydrous ethanol, a strong elution solvent, cannot elute it at 90%. Therefore, normal-phase chiral chromatographic columns cannot effectively separate micuronium chloride and its double-bond Z-isomer.

[0076] Comparative Example 4 Main experimental instruments and chromatographic conditions Instrument: Agilent 1260 II; Chromatographic column: Cellulose-tris-(3,5-dimethylphenylcarbamate) coated reversed-phase chiral column (CHIRALCEL OD-RH 4.6mm×150mm, 5μm); Mobile phase A: 75 mmol / L potassium dihydrogen phosphate solution (dissolve 10.2 g of potassium dihydrogen phosphate in 1000 mL of water and adjust the pH to 3.0 with phosphoric acid) - methanol - acetonitrile (volume ratio 75:20:5); Mobile phase B: Acetonitrile: Phosphoric acid aqueous solution (adjust the pH of pure water to 3.0 with phosphoric acid) = 8:2 (volume ratio); Wavelength: 280 nm; Flow rate: 1.0 mL / min; Column temperature: 25℃; Injection volume: 10 μL; The gradient elution program is shown in Table 15; Table 15 Gradient elution program for Comparative Example 4 Time (min) Mobile phase A (%) Mobile phase B (%) 0 85 15 15 85 15 20 70 30 22 40 60 26 40 60 26.5 85 15 30 85 15

[0077] Blank solution: acetonitrile-water (25:75).

[0078] Reference solution: Accurately weigh an appropriate amount of ZZO reference standard, dissolve and dilute it with acetonitrile-water (25:75) to prepare a solution containing approximately 10 μg per 1 mL.

[0079] Test solution: Accurately weigh an appropriate amount of Micourium chloride sample, dissolve and dilute it with acetonitrile-water (25:75) to prepare a solution containing approximately 1 mg per 1 mL.

[0080] Assay: Accurately measure 10 μL each of blank solution, reference solution, and test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the results using the peak area of ​​the ZZO trans-trans isomer according to the external standard method. See results below. Figures 11-12 and Table 16:

[0081] Table 16 Test results of Comparative Example 4 project Retention time (min) Theoretical number of plates Micudium chloride trans-trans isomer 9.929 636 ZZO anti-trans isomer 12.819 1029 Micudium chloride transcis isomer 14.968 573

[0082] The above results indicate that using a reversed-phase chiral column can significantly increase the retention time differences of micuronium chloride trans-trans isomer, ZZO trans-trans isomer, and micuronium chloride trans-cis isomer. However, the three target peaks have low theoretical plate numbers, short and wide peak shapes, and severe tailing, resulting in a method sensitivity far lower than that of Example 6.

[0083] Comparative Example 5 It is basically the same as Example 6, except that: Column: Octadecylsilane-bonded silica gel as packing material (Agilent Poroshell 120EC-C18, 150 mm × 3.0 mm, 2.7 μm); Wavelength: 280nm; Measurement results: Reference Figure 13At a wavelength of 280 nm, the peak width of the correlation peak will decrease and the separation will be better, but the trans-trans isomer of micous chloride and the trans-trans isomer of impurity O will still co-elute and not be separated.

[0084] Methodological validation of the liquid chromatography detection method in Example 6, Part Two: Example 6 Methodological Validation: 1. Operating Steps a. Main experimental instruments and chromatographic conditions: Instrument: Agilent 1260 II; Chromatographic column: triacontylsilane-bonded silica gel as the packing material (ArtChrom C30 4.6mm×250mm, 5μm); Mobile phase A: 75 mmol / L potassium dihydrogen phosphate solution (dissolve 10.2 g of potassium dihydrogen phosphate in 1000 mL of water and adjust the pH to 3.0 with phosphoric acid) - acetonitrile (volume ratio 75:25); Mobile phase B: Acetonitrile: Phosphoric acid aqueous solution (adjust the pH of pure water to 3.0 with phosphoric acid) = 8:2 (volume ratio); Wavelength: 210 nm; Flow rate: 1.0 mL / min; Column temperature: 30℃; Injection volume: 10 μL; The gradient elution program is shown in Table 17; Table 17 Gradient elution program table for Example 6 Time (min) Mobile phase A (%) Mobile phase B (%) 0 91.5 8.5 23 91.5 8.5 26 80 20 32 50 50 35 45 55 35.5 91.5 8.5 40 91.5 8.5

[0085] b. Solution preparation: Blank solution: Mobile phase A.

[0086] ZZO reference stock solution: Weigh 20 mg of ZZO reference standard accurately, place it in a 20 ml volumetric flask, add diluent to dissolve and quantitatively dilute to prepare a solution containing approximately 1 mg per ml, as reference stock solution 1. Accurately measure 1 ml of reference stock solution 1 and place it in a 50 ml volumetric flask, add diluent to quantitatively dilute to prepare a solution containing approximately 20 μg per ml, as reference stock solution 2.

[0087] Limit of Quantification Solution: Accurately measure 0.1 ml of the reference stock solution 2 into a 20 ml volumetric flask, and add diluent to quantitatively dilute to prepare a solution containing approximately 0.1 μg per ml.

[0088] Reference solution: Accurately measure 1 ml of reference stock solution 2 into a 20 ml volumetric flask, add diluent to quantitatively dilute to prepare a solution with a concentration of approximately 1 μg / ml.

[0089] Detection limit solution: Accurately measure 3 ml of the quantitation limit solution and place it in a 10 ml volumetric flask. Add diluent to dilute quantitatively to the mark.

[0090] Series of reference solutions: Accurately measure 0.5 ml, 0.8 ml, 1 ml, 1.2 ml, and 1.5 ml of reference stock solution 2 into different 20 ml volumetric flasks, and quantitatively dilute with diluent to prepare linear solutions with concentrations of approximately 0.5, 0.8, 1, 1.2, and 1.5 μg / ml.

[0091] Test solution: Weigh 20 mg of micuronium chloride accurately, place it in a 20 ml volumetric flask, add diluent to dissolve and quantitatively dilute to prepare a solution containing approximately 1 mg per ml.

[0092] Spiked solutions for test samples: Weigh 20 mg of micuronium chloride accurately and place it in a 20 ml volumetric flask. Add 0.1 ml, 1 ml, and 1.2 ml of reference stock solution 2, respectively. Dilute quantitatively to the mark with diluent to prepare low, medium, and high concentration spiked solutions for the test sample.

[0093] 2. Method Validation: a. Exclusivity Determination: Inject blank solution, reference solution, and medium-concentration spiked solution of test sample separately, and record the chromatograms.

[0094] Test results: The test results are shown in Table 18 and... Figures 14-17 Table 18 Specificity Test Results

[0095] Conclusion: The blank solution did not interfere with the detection of the ZZO trans-trans isomers. The minimum resolution between the ZZO trans-trans isomers and adjacent peaks was 2.30, indicating that the method has good specificity.

[0096] b. Limit of Detection and Limit of Quantification Measurement: Inject the detection limit solution three times consecutively and the quantitation limit solution six times consecutively. The signal-to-noise ratio of the ZZO trans-trans isomer peak in the detection limit solution should be no less than 3, and the signal-to-noise ratio of the ZZO trans-trans isomer peak in the quantitation limit solution should be no less than 10, and the RSD of the peak area should not exceed 20%.

[0097] Test results: The test results are shown in Table 19 and... Figures 18-19 .

[0098] Table 19 Results of Limit of Detection and Limit of Quantitation Tests

[0099] Conclusion: At the detection limit concentration of 0.03038 μg / ml (approximately 3% of the quality control limit), the ZZO reference solution has an average signal-to-noise ratio of 12.9, with all signal-to-noise ratios greater than 3; at the quantitation limit concentration of 0.1013 μg / ml (approximately 10% of the quality control limit), the average signal-to-noise ratio is 41.5, with all signal-to-noise ratios greater than 10, and the peak area RSD is 3.0%, meeting the requirements. The above data indicate that this method has high sensitivity and can meet the requirements for quantitative detection of ZZO.

[0100] c. Linearity and Range Determination: Inject the limit of quantitation solution and the series of reference solutions separately, two injections for each, record the chromatogram, and calculate the linear regression equation based on the concentration and the peak area of ​​the ZZO trans-trans isomer. The correlation coefficient R should not be less than 0.99.

[0101] Test results: The test results are shown in Table 20 and Figure 20 .

[0102] Table 20 Linearity and Range Test Results linear Concentration (µg / ml) Average peak area Limit of Quantification 0.1013 3.325 1 0.5064 16.590 2 0.8102 27.080 3 1.013 33.553 4 1.215 39.890 5 1.519 50.341

[0103] Conclusion: For ZZO in the concentration range of 0.1013 μg / ml to 1.519 μg / ml, the linear regression equation is y=33.092x-0.025, and the correlation coefficient R is 1.000, which meets the requirements and shows good linearity.

[0104] d. Accuracy Determination: Inject the reference solution, test solution, and low, medium, and high concentration spiked test solutions separately. Prepare one part of the reference solution for two injections, one part of the test solution for one injection, and prepare three parallel parts of each low, medium, and high concentration spiked test solution for one injection. Record the chromatograms. Evaluate the recovery results using the nine spiked solutions. The recovery rate should be between 80% and 120%, and the RSD (n=9) of the recovery rate should not exceed 20%.

[0105] Measurement results: The test results are shown in Table 21.

[0106] Table 21 Accuracy Test Results

[0107] Conclusion: The recovery rate of ZZO was between 82.6% and 90.1%, and the RSD (n=9) of the recovery rate was 2.9%, which met the requirements. The accuracy of this method was good.

[0108] e. Repetitive Determination: Inject the reference solution and the spiked solution of the test sample separately. Prepare one part of the reference solution and inject it twice. Prepare six parallel parts of the spiked solution of the test sample and inject each part once. Record the chromatogram. Evaluate the results of the ZZO content determination using the six parts of the spiked solution of the test sample of the test sample. The RSD (n=6) of the content should not exceed 20%.

[0109] Measurement results: The test results are shown in Table 22.

[0110] Table 22 Repeatability Test Results project 1 2 3 4 5 6 RSD ZZO content (%) 0.09560 0.08594 0.08892 0.08795 0.09113 0.08716 3.9％

[0111] Conclusion: The RSD of ZZO content in the six spiked solutions of the test samples was 3.9%, which meets the requirements, indicating that the repeatability of this method is good.

[0112] f. Intermediate precision Determination: According to the operation under the "Repeatability" section, different experimenters use different instruments to perform the operation, record the chromatogram, and evaluate the ZZO content determination results using the medium concentration test sample spiked solution. The intermediate precision RSD (n=6) of the ZZO content of 6 medium concentration test sample spiked solutions should not exceed 20%. The content RSD (n=12) of the ZZO content of the medium concentration test sample spiked solutions is calculated by combining the results with the repeatability results.

[0113] Measurement results: The test results are shown in Table 23.

[0114] Table 23 Precision Test Results

[0115] Conclusion: The RSD of ZZO content in the intermediate precision of the 6 spiked test samples was 2.4%, and the RSD of ZZO content in the repeatability of the 6 spiked test samples (n=12) was 3.7%, which meets the requirements and indicates that the precision of this method is good.

[0116] g. Durability Determination: Examine the flow rate variation of ±0.1 ml / min and column temperature variation of ±5℃. Prepare one part of reference solution and one part of medium-concentration test sample spiked solution, and inject them into each one. After changing the chromatographic conditions, evaluate the resolution between the ZZO trans-trans isomers and adjacent peaks in the medium-concentration test sample spiked solution, as well as the rate of change of the ZZO content determination results compared with the original conditions. The resolution should not be less than 1.5, and the rate of change should not exceed 20%.

[0117] Test results: The test results are shown in Table 24.

[0118] Table 24 Durability Test Results

[0119] Conclusion: Under different flow rates and column temperatures, the separation degree between the ZZO trans-trans isomer and the adjacent peak is greater than 1.5. The ZZO content determination results show a change rate of less than 20% compared with the original conditions, which meets the requirements and indicates that the method has good robustness.

[0120] h. Solution stability Determination: Prepare one reference solution and one medium-concentration test sample spiking solution. After standing at room temperature for 0h, 8h, and 24h, inject one sample at each time point. Evaluate the results by the change rate of the response factor of the reference solution at 0h and the change rate of the ZZO content determination result of the medium-concentration test sample spiking solution at each time point. The change rate of the response factor should not exceed 10%, and the change rate of the ZZO content determination result should not exceed 20%.

[0121] Test results: The test results are shown in Table 25.

[0122] Table 25 Solution stability test results

[0123] Conclusion: After 24 hours at room temperature, the ZZO response factor change rate of the reference solution was 1.0%, and the content change rate of the medium-concentration test sample spiked solution was 1.7%, which met the requirements, proving that the reference solution and the medium-concentration test sample spiked solution had good stability within 24 hours.

[0124] i. Application of detection methods Determination: Inject blank solution, reference solution, and test solution. Prepare one part of the reference solution and inject it twice. Prepare two parts of the test solution for each of the three batches of test samples and inject them once. Record the chromatogram. If there is a chromatographic peak in the test solution with the same retention time as the reference solution, calculate the peak area according to the external standard method.

[0125] Test results: The test results are shown in Table 26.

[0126] Table 26 Test results of ZZO content in three batches of micuronium chloride raw material batch number content G230703 0.003% G230704 Not detected G230705 Not detected

[0127] In summary, the detection method of the present invention has strong specificity, high sensitivity, good linearity, wide range, high accuracy, good precision, good robustness and stability, and is suitable for the detection of the content of double bond Z / E isomers in micudium chloride.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A liquid chromatography method for detecting the Z-configuration isomer of micuronium chloride double bond, characterized in that, During the test, the chromatographic column was a triadecylsilane-bonded silica column, and the elution method was gradient elution; During the elution process, both mobile phase A and mobile phase B are mixtures of organic solvent and aqueous solution; The aqueous solution may be an aqueous solution with or without added salts; The salt is selected from at least one of potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, ammonium acetate, ammonium formate, sodium bicarbonate, and ammonium bicarbonate. The aqueous solution contains a pH adjuster, and the pH value of the aqueous solution is 2.0 to 7.0; The organic solvent is selected from at least one of methanol, isopropanol, and acetonitrile.

2. The liquid chromatography detection method for the Z-configuration isomer of micuronium chloride double bond according to claim 1, characterized in that, The chromatographic column is a triacontylsilane-bonded silica column with dimensions of 30–250 mm × 2.1–4.6 mm and a diameter of 1.6–5.0 μm.

3. The liquid chromatography detection method for the Z-configuration isomer of micuronium chloride double bond according to claim 1, characterized in that, The volume ratio of the aqueous solution to the organic solvent in the mobile phase A is 5-9:1-5; The volume ratio of organic solvent to aqueous solution in the mobile phase B is 1-10:1-10.

4. The liquid chromatography detection method for the Z-configuration isomer of micuronium chloride double bond according to claim 1, characterized in that, During the test, the column temperature was 15–50℃, the detector was an ultraviolet detector with a detection wavelength of 190–280 nm, the flow rate was 0.4–2.0 mL / min, and the injection volume was 5–50 μL.

5. The liquid chromatography detection method for the Z-configuration isomer of micuronium chloride double bond according to claim 1, characterized in that, The gradient elution conditions are as follows: Minute 0: Mobile phase A is 98–91.5%, mobile phase B is 2–8.5%; At 23 minutes: mobile phase A was 98–91.5%, and mobile phase B was 2–8.5%. At 26 minutes: mobile phase A was 87-75%, and mobile phase B was 13-25%. At 32 minutes: Mobile phase A was 70-50%, and mobile phase B was 30-50%. At 35 minutes: mobile phase A was 45-40%, and mobile phase B was 55-60%. At 35.5 min: mobile phase A was 98–91.5%, and mobile phase B was 2–8.5%; At 40 min: mobile phase A was 98–91.5%, and mobile phase B was 2–8.5%; Or the conditions for a gradient elution procedure are: Minute 0: Mobile phase A is 92-88%, mobile phase B is 8-12%; At 20 minutes: Mobile phase A was 92-88%, and mobile phase B was 8-12%. At 23 minutes: mobile phase A was 87-83%, and mobile phase B was 13-17%. At 27 minutes: mobile phase A was 55-45%, and mobile phase B was 45-55%. At 31 min: mobile phase A was 55-45%, and mobile phase B was 45-55%. At 31.5 min: mobile phase A was 92-88%, and mobile phase B was 8-12%; At 35 minutes: mobile phase A was 92-88%, and mobile phase B was 8-12%.

6. The liquid chromatography detection method for the Z-configuration isomer of micuronium chloride double bond according to claim 1, characterized in that, The pH adjuster is selected from at least one of phosphoric acid, formic acid, and acetic acid.

7. The liquid chromatography detection method for the Z-configuration isomer of micuronium chloride double bond according to claim 1, characterized in that, When the aqueous solution is an aqueous solution with added salts, the concentration of salts in the aqueous solution is 0.01–0.15 mol / L.

8. The liquid chromatography detection method for the Z-configuration isomer of micuronium chloride double bond according to claim 1, characterized in that, Includes the following steps: Step 1: Take micuronium chloride and dilute it with mobile phase A to obtain a test solution with a concentration of micuronium chloride of 0.1-2.0 mg / ml; Step 2: Take the Z-configuration isomer of micuronium double bond and dilute it with mobile phase A to obtain a reference solution with a concentration of 0.1-2.0 μg / ml of micuronium double bond Z-configuration isomer; Step 3: Take the reference solution and the test solution, inject them into the high performance liquid chromatograph, and record the chromatogram; Step 4: Calculate the content of the Z configuration isomer of the double bond of micous chloride using the external standard method based on the peak area.

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