An impurity detection method for neostigmine methylsulfate injection

Abstract

The present invention belongs to the field of impurity detection methods, and particularly relates to an impurity detection method for neostigmine methylsulfate injection. The present invention specifically discloses an impurity detection method for neostigmine methylsulfate injection, which uses an Agilent SB-C18 column, with sodium dihydrogen phosphate buffer solution-acetonitrile (95:5) as mobile phase A and sodium dihydrogen phosphate buffer solution-acetonitrile (30:70) as mobile phase B for gradient elution, the detection wavelength is 215 nm, the injection volume is 20 μl, and the flow rate is 1.0 mL / min; the column temperature is 40 °C; this method has established quality control standards for impurities P0Z8, P0Z12, P0Z13, P0UZ3 and P0UZ4 for the first time, significantly improving the comprehensiveness and effectiveness of impurity detection, and successfully achieving the complete separation of key impurity peaks such as impurity P0Z5 and P0Z8, P0Z9 and P0UZ3, P0Z12 and P0UZ4. This method is not only applicable to laboratory research, but also very suitable for quality control in industrial production, thus ensuring the safety and stability of neostigmine methylsulfate injection and reducing the potential risks for patients.

Description

Technical Field

[0001] The present invention belongs to the field of impurity detection methods, and particularly relates to an impurity detection method for neostigmine methylsulfate injection. Background Art

[0002] As a reversible cholinesterase inhibitor, neostigmine methylsulfate injection is widely used clinically for the treatment of postoperative abdominal distension and myasthenia gravis. The prior art CN118759085A discloses a method for detecting gene impurities in neostigmine methylsulfate injection. The impurities detected by this method are not comprehensive, mostly gene-toxic impurities, and the polymer impurities or other toxic and harmful impurities generated by other processes are not controlled. Moreover, there are defects in aspects such as chromatographic peak symmetry, impurity peak resolution, sensitivity, and chromatogram baseline.

[0003] The prior art CN113651723A discloses that 3-(3-hydroxyphenyl)-1,1-dimethylurea (P0Z8 in this application) is a process impurity generated during the synthesis of neostigmine methylsulfate and may remain during the purification process. This impurity poses a great risk to the quality control of drugs, and the detection methods for impurity P0Z8 in neostigmine methylsulfate injection are not included in the pharmacopoeias of various countries.

[0004] CN110950780A discloses compound I (P0Z12 in this application) and compound II. Because they are similar in structure to the main product, they are likely to remain in neostigmine methylsulfate. Both of these impurities contain aromatic amine characteristic groups, and such compounds are known potential gene-toxic impurities and must be studied and controlled as necessary.

[0005] CN118652197A discloses that the precursor compound of impurity D (P0Z13 in this application) is a definite gene-toxic impurity with mutagenic effects. Therefore, the precursor compound and impurity D have obvious effects on the safety of drugs, and thus this impurity is also one of the key points for supervision.

[0006] In addition, the polymer impurities P0UZ3 and P0UZ4 that may have toxicological risks remaining in the newly confirmed synthesis process of this application are not fully controlled by the existing detection methods, posing potential quality and safety hazards.

[0007] Therefore, the current method for detecting impurities in neostigmine methylsulfate injection by liquid chromatography has certain omissions, which misleads the quality control of neostigmine methylsulfate injection and is not conducive to its quality control. It is necessary to provide a new detection method that can more comprehensively characterize the impurity content and has better guiding significance for its quality control. Summary of the Invention

[0008] The object of the present invention is to provide an impurity detection method for neostigmine methylsulfate injection. According to the process route and the chemical structure of this product, the present application adopts an impurity control strategy that combines source control, in-process control, and end control to conduct a detailed impurity profile analysis of neostigmine methylsulfate injection, so as to solve the defects existing in the existing impurity detection methods for neostigmine methylsulfate injection in the above-mentioned background technology, as well as the problem of difficulty in ensuring the detection of all impurities.

[0009] The impurity detection method for the neostigmine methylsulfate injection includes the following chromatographic conditions:

[0010] Adopt the HPLC method, prepare mobile phase A by mixing sodium dihydrogen phosphate buffer solution and acetonitrile in a volume ratio of 95:5 and adjust the pH; prepare mobile phase B by mixing sodium dihydrogen phosphate buffer solution and acetonitrile in a volume ratio of 30:70 and adjust the pH; use gradient elution for elution, and the chromatographic column is a C18 column;

[0011] Among them, each 1 ml of the test solution contains 0.5 mg of neostigmine methylsulfate injection, and each 1 ml of the reference solution contains 1 μg each of impurities P0Z1, P0Z2, P0Z4, P0Z5, P0Z8, P0Z9, P0Z11, P0Z12, P0Z13, P0Z14, P0UZ3, and P0UZ4.

[0012] Use the aforementioned HPLC to detect the test solution, and the following impurities may be detected:

[0013]

[0014] Preferably, the preparation method of the mobile phase is as follows: add sodium pentanesulfonate to the sodium dihydrogen phosphate buffer solution, adjust the pH of the solution to 3.0, and then prepare mobile phase A by mixing it with acetonitrile in a volume ratio of 95:5; add sodium pentanesulfonate to the sodium dihydrogen phosphate buffer solution, adjust the pH of the solution to 3.0, and then prepare mobile phase B by mixing it with acetonitrile in a volume ratio of 30:70.

[0015] More preferably, the concentration of sodium pentanesulfonate in the mobile phase is 0.8 g / L to 0.9 g / L.

[0016] Preferably, in mobile phase A, the preparation method of the sodium dihydrogen phosphate buffer solution is: weigh 3.12 g of sodium dihydrogen phosphate dihydrate and add it to 1000 ml of water, and adjust the pH value to 3.0 with phosphoric acid; in mobile phase B, the preparation method of the sodium dihydrogen phosphate buffer solution is: weigh 3.12 g of sodium dihydrogen phosphate dihydrate and add it to 1000 ml of water, and adjust the pH value to 3.0 with phosphoric acid.

[0017] Preferably, the chromatographic conditions further include: the elution method is gradient elution, and the elution conditions are shown in the following table:

[0018] 。

[0019] Preferably, the chromatographic column is an Agilent SB-C18 column, 4.6×250 mm, 5 µm.

[0020] More preferably, the chromatographic conditions further include: the flow rate is 1.0 - 1.5 ml per minute; the column temperature is 40 °C; the detection wavelength is 215 nm; the injection volume is 20 μl.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] (1) The method for detecting impurities in neostigmine methylsulfate injection provided by the present invention has established for the first time the quality control standards for impurities P0Z8, P0Z12, P0Z13, P0UZ3 and P0UZ4, significantly improving the comprehensiveness and effectiveness of impurity detection. By precisely controlling the concentration and pH value of the buffer salts in mobile phase A and mobile phase B, the complete separation of key impurity peaks such as P0Z5 and P0Z8, P0Z9 and P0UZ3, P0Z12 and P0UZ4 has been successfully achieved, which is difficult to achieve by ordinary methods;

[0023] (2) The chromatographic performance of this method is excellent, with a stable baseline, high signal-to-noise ratios for each impurity, symmetric peak shapes and no drift. Within the range of 10% - 200% of the impurity concentration limit, the correlation coefficient r is greater than 0.999, and the ratio of the Y-axis intercept to the 100% response value is controlled between 0.05% and 3.94%, showing a good linear relationship. In addition, through the testing of 9 spiked test sample solutions, the recovery rate is stable between 92.74% and 106.79%, verifying the high accuracy of the method;

[0024] (3) The durability and precision of this method have been strictly verified. In the reproducibility investigations carried out in different laboratories, using different equipment and by different experimenters, good precision and durability are shown. These characteristics make this method not only suitable for laboratory research, but also very suitable for the quality control of industrial production, thus ensuring the safety and stability of neostigmine methylsulfate injection and reducing the potential risks for patients to use. Description of the Drawings

[0025] Figure 1 It is the chromatogram of the diluent of the test sample in Example 1 of the present invention.

[0026] Figure 2 It is the chromatogram of the spiked test sample in Example 1 of the present invention.

[0027] Figure 3 The chromatogram of the detection experiment by the method of "Chinese Pharmacopoeia".

[0028] Figure 4 Chromatogram obtained by the method of the European Pharmacopoeia

[0029] Figure 5 Chromatogram obtained by the isocratic elution method of the mobile phase

[0030] Figure 6 Chromatogram obtained by the method of gradient elution program 1 of the mobile phase

[0031] Figure 7 Chromatogram obtained by the method of gradient elution program 2 of the mobile phase

[0032] Figure 8 Chromatogram obtained by the method for the Ultimate AQ-C18 column

[0033] Figure 9 Chromatogram obtained by the method for the Waters XBridge C18 column

[0034] Figure 10 Chromatogram obtained by the method with sodium hexanesulfonate as the ion-pair reagent in the mobile phase

[0035] Figure 11 Chromatogram obtained by the method with the pH value of the mobile phase being 4.0 Detailed implementation manners

[0036] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation manners. Those skilled in the art should understand that the specific implementation manners are only for helping to understand the present invention and should not be regarded as specific limitations to the present invention.

[0037] Neostigmine Methylsulfate Injection is from: Chengdu Yuandong Biopharmaceutical Co., Ltd., batch number 210504;

[0038] Sources of each impurity reference substance:

[0039]

[0040] Example 1

[0041] This example provides a method for detecting impurities in neostigmine methylsulfate injection. The detection method includes the following steps: preparing a test solution from neostigmine methylsulfate injection, using a C18 chromatographic column, and performing gradient elution with a mixed solvent of mobile phase A and mobile phase B as the mobile phase to detect neostigmine methylsulfate and impurities; Instrument: Aglient 1260 high performance liquid chromatograph; Chromatographic column: Agilent SB-C18, 4.6×250mm, 5μm; Detection wavelength: 215nm; Flow rate: 1.0mL / min; Column temperature: 40°C; Concentration of test solution: 0.5mg / mL; Injection volume: 20μL; Mobile phase A: Sodium dihydrogen phosphate buffer solution (weigh 3.12g of sodium dihydrogen phosphate dihydrate into 1000ml of water, adjust the pH value to 3.0 with phosphoric acid, add 0.871g of sodium pentanesulfonate)-acetonitrile (95:5); Mobile phase B: Sodium dihydrogen phosphate buffer solution (weigh 3.12g of sodium dihydrogen phosphate dihydrate into 1000ml of water, adjust the pH value to 3.0 with phosphoric acid, add 0.871g of sodium pentanesulfonate)-acetonitrile (30:70); Perform gradient elution according to Table 1:

[0042] Table 1 Gradient elution conditions

[0043]

[0044] Obtain Figure 1 the chromatogram of the diluent of the sample to be tested, Figure 2 the chromatogram of the spiked test solution, and the specific data in the chromatogram are shown in Table 2 below:

[0045] Table 2

[0046]

[0047] It can be seen from Table 2 that: this method has high sensitivity, the detection limits of each impurity are all below 1ng, and the test solution and the reference solution have good stability under this method. Within 35h of standing at room temperature, the peak areas of each impurity compared with the peak area at 0 point have no obvious change. Within the range of 10% - 200% of the impurity concentration limit, the correlation coefficient r is greater than 0.999, and the ratio of the Y-axis intercept to the 100% response value is controlled between 0.05% - 3.94%, showing a good linear relationship; The results of the accuracy experiment show that: for 9 spiked test solutions at each concentration, the recoveries are all within the range of 92.74% - 106.79%, and the RSD is less than 3.0%, indicating that the proposed method has good accuracy; In summary, it can be seen that the method in Example 1 has excellent chromatographic performance, good stability, high sensitivity and accuracy for each impurity, and the peak shape is symmetrical and there is no drift.

[0048] Comparative Example 1 Detection experiment of the method in Chinese Pharmacopoeia

[0049] The chromatographic conditions of the detection method in the Chinese Pharmacopoeia are shown in Table 3 as follows:

[0050] Table 3 Chromatographic Conditions of the Detection Method in the Chinese Pharmacopoeia

[0051]

[0052] The results of the test samples measured under the above conditions are as Figure 3 shown: It can be seen from Figure 3 that the specificity of the method in the Chinese Pharmacopoeia is poor, and the impurities overlap with the main component.

[0053] Comparative Example 2 Detection Experiment of the Method in the European Pharmacopoeia

[0054] The chromatographic conditions of the detection method in the European Pharmacopoeia are shown in Table 4 as follows:

[0055] Table 4 Chromatographic Conditions of the Detection Method in the European Pharmacopoeia

[0056]

[0057] The results of the test samples measured under the above conditions are as Figure 4 shown: It can be seen from Figure 4 that the specificity of the method in the European Pharmacopoeia is poor, and the impurities overlap with the main component.

[0058] Comparative Example 3: Self-developed Isocratic Elution Conditions

[0059] This comparative example uses an elution method with a different ratio from that in Example 1, and performs isocratic elution with mobile phase A - mobile phase B (20:80), and the other conditions are the same as those in Example 1.

[0060] The results of the test samples measured by the above method are as Figure 5 shown: It can be seen that compared with the gradient method in Example 1, the number of detected impurities is small, and impurity P0Z13 and impurity P0UZ4 are not completely separated (resolution < 1.5).

[0061] Comparative Example 4

[0062] Gradient Elution Condition Screening 1

[0063] This comparative example uses gradient elution conditions different from those in Example 1, and performs gradient elution as shown in Table 5 below, and the other conditions are the same as those in Example 1:

[0064] Table 5

[0065]

[0066] The results show that after the gradient elution conditions are adjusted, the separation of each impurity becomes worse, and impurity P0UZ3 and impurity P0UZ4 overlap with the adjacent chromatographic peaks and cannot be separated and detected. The results are shown in Figure 6 .

[0067] Comparative Example 5

[0068] Gradient elution condition screening 2

[0069] This comparative example uses gradient elution conditions different from those in Example 1, with gradient elution as shown in Table 6 below, and the other conditions are the same as those in Example 1:

[0070] Table 6

[0071]

[0072] The results show that after adjusting the gradient elution conditions, the separation between impurity P0Z8 and the main component becomes worse, and impurity P0UZ4 overlaps with the adjacent chromatographic peak. The results are shown in Figure 7 .

[0073] Comparative Example 6

[0074] Adaptability experiment of different chromatographic columns (the chromatographic column in Example is the best, and the other two chromatographic columns are worse)

[0075] This comparative example uses a chromatographic column different from that in Example 1, and selects Ultimate AQ-C18, 4.6×250mm, 5μm; chromatographic column Waters XBridge C18, 4.6×250mm, 5μm, and the other conditions are the same as those in Example 1. Record the chromatographic parameters such as the retention time, peak shape, and resolution of each impurity under different chromatographic columns.

[0076] The results of the chromatographic column Ultimate AQ-C18, 4.6×250mm, 5μm are shown in Figure 8 , and the results of Waters XBridge C18, 4.6×250mm, 5μm are shown in Figure 9 . It can be seen from the results that on different C18 chromatographic columns, only the chromatographic column (Agilent SB-C18) preferred by the method of the present invention can effectively separate and detect impurities. The resolution of each impurity is greater than 1.5 (meeting the standard for good separation), the retention time is relatively stable, the peak shape is symmetric and sharp, and the baseline of the chromatogram is the best. From Figure 8 , 9 it can be seen that the detection effects of the chromatographic columns Ultimate AQ-C18 and Waters XBridge cannot reach the detection effect of Agilent SB-C18 ( Figure 2 ).

[0077] Comparative Example 7

[0078] This comparative example uses a mobile phase component different from that in Example 1, and uses sodium hexanesulfonate solution as mobile phase A, and the other conditions are the same as those in Example 1.

[0079] The test results show that when sodium hexanesulfonate solution is used as mobile phase A, impurity P0Z8 overlaps with P0Z4 and cannot be detected, and impurity P0UZ3 overlaps with P0Z9. The results are shown in Figure 10 .

[0080] Comparative Example 8

[0081] Comparative Example 8 uses a mobile phase pH value different from that in Example 1, and the other conditions are the same as those in Example 1. Specifically as follows:

[0082] It can be seen from the test results that when the pH value of the mobile phase is adjusted to 4.0, the impurities cannot be completely separated, and impurities such as P0Z13 overlap with the main component. The results are shown in Figure 11 .

Claims

1. An impurity detection method for neostigmine methylsulfate injection, characterized in that, Including the following chromatographic conditions: Using the HPLC method, weigh 3.12 g of sodium dihydrogen phosphate dihydrate into 1000 ml of water, adjust the pH value to 3.0 with phosphoric acid to obtain a sodium dihydrogen phosphate buffer solution, add 0.8 g / L - 0.9 g / L of sodium pentanesulfonate, and then mix with acetonitrile according to a volume ratio of 95:5 to prepare mobile phase A and adjust the pH to 3.0; weigh 3.12 g of sodium dihydrogen phosphate dihydrate into 1000 ml of water, adjust the pH value to 3.0 with phosphoric acid to obtain a sodium dihydrogen phosphate buffer solution, add 0.8 g / L - 0.9 g / L of sodium pentanesulfonate, and then mix with acetonitrile according to a volume ratio of 30:70 to prepare mobile phase B and adjust the pH to 3.0; use a gradient condition of: 0 - 5 min, 100% mobile phase A; 5 - 40 min, 100% - 30% mobile phase A; 40 - 50 min, 30% mobile phase A for gradient elution, and the chromatographic column is a C18 column; Among them, each 1 ml of the test solution contains 0.5 mg of neostigmine methylsulfate injection, and each 1 ml of the reference solution contains 1 μg of impurities P0Z1, P0Z2, P0Z4, P0Z5, P0Z8, P0Z9, P0Z11, P0Z12, P0Z13, P0Z14, P0UZ3, and P0UZ4 respectively; Using the aforementioned HPLC to detect the test solution, the following impurities may be detected: 。 2. The impurity detection method of neostigmine methylsulfate injection according to claim 1, characterized in that, The chromatographic column is an Agilent SB-C18 column, 4.6×250 mm, 5 µm.

3. The impurity detection method of neostigmine methylsulfate injection according to claim 1, wherein, The chromatographic conditions also include: the flow rate is 1.0 - 1.5 mL / min; the column temperature is 40 °C; the detection wavelength is 215 nm; the injection volume is 20 μl.

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