Impurity detection method of neostigmine methylsulfate injection

Abstract

The invention belongs to the field of impurity detection methods, and particularly relates to an impurity detection method of a neostigmine methylsulfate injection. According to the impurity detection method, Agilent SB-C18 is used as a chromatographic column, sodium dihydrogen phosphate buffer solution-acetonitrile (95: 5) is used as a mobile phase A, sodium dihydrogen phosphate buffer solution-acetonitrile (30: 70) is used as a mobile phase B for gradient elution, the detection wavelength is 215 nm, the sample injection volume is 20 microliters, and the flow velocity is 1.0 mL / min; the column temperature is 40 DEG C; according to the method, quality control standards are established for the impurities P0Z8, P0Z12, P0Z13, P0UZ3 and P0UZ4 for the first time, the comprehensiveness and effectiveness of impurity detection are remarkably improved, and complete separation of key impurity peaks of the impurities P0Z5 and P0Z8, P0Z9 and P0UZ3, P0Z12 and P0UZ4 and the like is successfully achieved. The method is not only suitable for laboratory research, but also very suitable for quality control of industrial production, so that the safety and stability of the neostigmine methylsulfate injection are ensured, and the potential risk of patient use is reduced.

Description

Technical Field

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

[0002] Neostigmine methylsulfate injection is a reversible cholinesterase inhibitor and is widely used in the treatment of postoperative abdominal distension and myasthenia gravis in clinic. The prior art CN118759085A discloses a method for detecting genetic impurities in neostigmine methylsulfate injection. The impurities detected by this method are not comprehensive, mostly genotoxic impurities, and polymer impurities or toxic and harmful impurities produced by other processes are not controlled. In addition, the chromatographic peak symmetry, impurity peak separation, sensitivity and chromatogram baseline are defective.

[0003] Prior art CN113651723A discloses that 3-(3-hydroxyphenyl)-1,1-dimethylurea (P0Z8 in this application) is a process impurity produced during the synthesis of neostigmine methylsulfate, which may remain during the purification process. This impurity poses a great risk to the control of drug quality, and the pharmacopoeias of various countries do not include the detection method of the impurity P0Z8 in neostigmine methylsulfate injection.

[0004] CN110950780A discloses compound I (P0Z12 in the present application) and compound II, which are structurally similar to the main product and are easily left in neostigmine methylsulfate. Both impurities contain aromatic amine characteristic groups. Such compounds are known potential genotoxic impurities and must be studied and controlled as necessary.

[0005] The precursor compound of impurity D (POZ13 in the present application) disclosed in CN118652197A is a clear genotoxic impurity with a mutagenic effect. Therefore, the precursor compound and impurity D have a significant impact on the safety of the drug, and this impurity is also one of the key points of 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 existing detection methods, posing potential quality and safety risks.

[0007] Therefore, the current liquid chromatography method for detecting impurities of neostigmine methylsulfate injection has certain omissions, which is misleading for 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 has a more comprehensive characterization of 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 the product, the present invention adopts an impurity control strategy combining source control, process control and endpoint control, and performs a detailed impurity spectrum analysis on the neostigmine methylsulfate injection to solve the defects of the detection method of the existing impurity detection method for neostigmine methylsulfate injection proposed in the above background technology, as well as the problem that it is difficult to ensure the detection of all impurities.

[0009] The impurity detection method of the neostigmine methylsulfate injection comprises the following chromatographic conditions: The HPLC method was adopted, wherein the mobile phase A was prepared with sodium dihydrogen phosphate buffer solution and acetonitrile in a volume ratio of 95:5 and the pH was adjusted; the mobile phase B was prepared with sodium dihydrogen phosphate buffer solution and acetonitrile in a volume ratio of 30:70 and the pH was adjusted; the elution was performed by gradient elution, and the chromatographic column was 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 each of impurities P0Z1, P0Z2, P0Z4, P0Z5, P0Z8, P0Z9, P0Z11, P0Z12, P0Z13, P0Z14, P0UZ3, and P0UZ4.

[0010] The following impurities may be detected by using the aforementioned HPLC to detect the test solution:

[0011] Preferably, the mobile phase is configured as follows: sodium pentane sulfonate is added to a sodium dihydrogen phosphate buffer solution, the pH of the solution is adjusted to 3.0, and then the mobile phase A is configured with acetonitrile in a volume ratio of 95:5; sodium pentane sulfonate is added to a sodium dihydrogen phosphate buffer solution, the pH of the solution is adjusted to 3.0, and then the mobile phase B is configured with acetonitrile in a volume ratio of 30:70.

[0012] More preferably, the concentration of sodium pentane sulfonate in the mobile phase is 0.8 g / L to 0.9 g / L.

[0013] Preferably, in the mobile phase A, the preparation method of the sodium dihydrogen phosphate buffer solution is: weigh 3.12 g of sodium dihydrogen phosphate dihydrate into 1000 ml of water, and adjust the pH value to 3.0 with phosphoric acid; in the mobile phase B, the preparation method of the sodium dihydrogen phosphate buffer solution is: weigh 3.12 g of sodium dihydrogen phosphate dihydrate into 1000 ml of water, and adjust the pH value to 3.0 with phosphoric acid.

[0014] Preferably, the chromatographic conditions also include: the elution mode is gradient elution, and the elution conditions are shown in the following table: .

[0015] Preferably, the chromatographic column is an Agilent SB-C18 column, 4.6×250 mm, 5 µm.

[0016] More preferably, the chromatographic conditions further include: a flow rate of 1.0-1.5 ml per minute; a column temperature of 40° C.; a detection wavelength of 215 nm; and an injection volume of 20 μl.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The impurity detection method for neostigmine methylsulfate injection provided by the present invention establishes the quality control standards for impurities P0Z8, P0Z12, P0Z13, P0UZ3 and P0UZ4 for the first time, significantly improving the comprehensiveness and effectiveness of impurity detection. By finely controlling the concentration and pH value of the buffer salt in mobile phase A and mobile phase B, the complete separation of key impurity peaks such as impurities P0Z5 and P0Z8, P0Z9 and P0UZ3, and P0Z12 and P0UZ4 was successfully achieved, an effect that is difficult to achieve with ordinary methods; (2) The chromatographic performance of this method is excellent, the baseline is stable, the signal-to-noise ratio of each impurity is high, and the peak shape is symmetrical and drift-free. Within the impurity concentration limit range of 10%~200%, 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 test of 9 spiked test solutions, the recovery rate is stable between 92.74% and 106.79%, verifying the high accuracy of the method; (3) The durability and precision of this method have been rigorously verified. In the reproducibility studies conducted in different laboratories, using different equipment and by different experimenters, good precision and durability were shown. These characteristics make this method not only suitable for laboratory research, but also very suitable for quality control of industrial production, thereby ensuring the safety and stability of neostigmine methylsulfate injection and reducing the potential risks of patient use. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a chromatogram of the diluent of the sample to be tested in Example 1 of the present invention.

[0019] Figure 2 This is a chromatogram of the sample spiked in Example 1 of the present invention.

[0020] Figure 3 Chromatogram of the test experiment based on the Chinese Pharmacopoeia method.

[0021] Figure 4 Chromatogram of the European Pharmacopoeia method test experiment.

[0022] Figure 5Chromatogram of the mobile phase isocratic elution method detection experiment.

[0023] Figure 6 Chromatogram of the mobile phase gradient elution procedure 1 method detection experiment.

[0024] Figure 7 Chromatogram of the mobile phase gradient elution program 2 method detection experiment.

[0025] Figure 8 This is the chromatogram of the Ultimate AQ-C18 column method test experiment.

[0026] Fig. 9 This is the experimental chromatogram for the Waters XBridge C18 column method detection experiment.

[0027] Fig.10 Chromatogram of the mobile phase ion pair reagent detection experiment using sodium hexane sulfonate method.

[0028] Fig.11 The chromatogram of the experiment was detected by the method with the mobile phase pH value of 4.0. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described below with reference to the accompanying drawings and through specific implementations. Those skilled in the art should understand that the specific implementations are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0030] Neostigmine methylsulfate injection was obtained from: Chengdu Yuandong Biopharmaceutical Co., Ltd., batch number 210504; Sources of impurity standards:

[0031] Example 1 The present embodiment provides a method for detecting impurities of neostigmine methylsulfate injection, the method comprising the following steps: preparing the neostigmine methylsulfate injection into a test solution, using a C18 chromatographic column, using a mixed solvent of mobile phase A and mobile phase B as a mobile phase gradient elution, and detecting 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℃; test solution concentration: 0.5mg / mL; injection volume: 20μL; mobile phase A: sodium dihydrogen phosphate buffer solution (weigh 3.12g sodium dihydrogen phosphate dihydrate into 1000ml water, adjust the pH value to 3.0 with phosphoric acid, and add 0.871g sodium pentane sulfonate)-acetonitrile (95:5); mobile phase B: sodium dihydrogen phosphate buffer solution (weigh 3.12g sodium dihydrogen phosphate dihydrate into 1000ml water, adjust the pH value to 3.0 with phosphoric acid, and add 0.871g sodium pentane sulfonate)-acetonitrile (30:70); gradient elution according to Table 1: Table 1 Gradient elution conditions

[0032] get Figure 1 The chromatogram of the sample diluent to be tested, Figure 2 The chromatogram of the spiked test sample is shown in Table 2 below: Table 2

[0033] It can be seen from Table 2 that the method has high sensitivity, and the detection limits of each impurity are all below 1ng. In addition, the test solution and the reference solution have good stability under this method. Within 35 hours at room temperature, the peak area of ​​each impurity has no significant change compared with the peak area at 0. Within the range of 10% to 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; the results of the accuracy experiment show that the recovery rate of 9 spiked test solutions of each concentration is within the range of 92.74% to 106.79%, and the RSD is less than 3.0%, and the proposed method has good accuracy; in summary, it can be seen that the method of Example 1 has superior chromatographic performance, good stability, high sensitivity and accuracy of each impurity, and symmetrical peak shape without drift.

[0034] Comparative Example 1: Testing experiment of the method of "Chinese Pharmacopoeia" The chromatographic conditions of the detection method of the Chinese Pharmacopoeia are shown in Table 3 below: Table 3 Chromatographic conditions of the detection method in the Chinese Pharmacopoeia

[0035] The test results obtained under the above conditions are as follows Figure 3 Shown by: Figure 3 It can be seen that the method of the Chinese Pharmacopoeia has poor specificity and impurities overlap with the main components.

[0036] Comparative Example 2: European Pharmacopoeia Method Test The chromatographic conditions of the European Pharmacopoeia test method are shown in Table 4 below: Table 4 Chromatographic conditions of the European Pharmacopoeia test method

[0037] The test results obtained under the above conditions are as follows Figure 4 Shown by: Figure 4 It can be seen that the European Pharmacopoeia method has poor specificity and impurities overlap with the main components.

[0038] Comparative Example 3: Self-designed isocratic elution conditions This comparative example adopts an elution method with a different ratio from that of Example 1, i.e., isocratic elution with mobile phase A-mobile phase B (20:80), and the other conditions are the same as those of Example 1.

[0039] The test results obtained by the above method are as follows Figure 5 As shown: it can be seen that compared with the gradient method in the embodiment, the number of impurities detected is small, and the impurity P0Z13 and the impurity P0UZ4 are not completely separated (separation degree <1.5).

[0040] Comparative Example 4 Gradient elution condition screening 1 This comparative example adopts different gradient elution conditions from those in Example 1, and adopts the gradient elution shown in Table 5 below, and the other conditions are the same as those in Example 1: Table 5

[0041] The results showed that after the gradient elution conditions were adjusted, the separation of the impurities became worse, and the impurities P0UZ3 and P0UZ4 overlapped with the adjacent chromatographic peaks and could not be separated and detected. Figure 6 .

[0042] Comparative Example 5 Gradient elution condition screening 2 This comparative example adopts different gradient elution conditions from those in Example 1, and adopts the gradient elution shown in Table 6 below, and the other conditions are the same as those in Example 1: Table 6

[0043] The results showed that after the gradient elution conditions were adjusted, the separation between impurity P0Z8 and the main component became worse, and impurity P0UZ4 overlapped with the adjacent chromatographic peak. Figure 7 .

[0044] Comparative Example 6 Adaptability test of different chromatographic columns (the chromatographic column in the example is the best, and the other two chromatographic columns are poor) This comparative example uses a different chromatographic column from that in Example 1, Ultimate AQ-C18, 4.6×250 mm, 5 μm; and Waters XBridge C18, 4.6×250 mm, 5 μm, and the other conditions are the same as in Example 1. The chromatographic parameters such as retention time, peak shape, and separation degree of each impurity under different chromatographic columns are recorded.

[0045] Column Ultimate AQ-C18, 4.6×250mm, 5µm Results see Figure 8 , Waters XBridgeC18, 4.6×250mm, 5µm, see Fig. 9 The results show that on different C18 chromatographic columns, the preferred chromatographic column (AgilentSB-C18) of the method of the present invention can effectively separate and detect impurities, the separation degree of each impurity is greater than 1.5 (meeting the standard of good separation), the retention time is relatively stable, the peak shape is symmetrical and sharp, and the chromatogram baseline is optimal. Figure 8 , 9 It can be seen that the detection effects of the columns Ultimate AQ-C18 and Waters XBridge cannot reach the detection effect of Agilent SB-C18 ( Figure 2 ).

[0046] Comparative Example 7 This comparative example uses a mobile phase component different from that of Example 1, using sodium hexane sulfonate solution as mobile phase A, and other conditions are the same as those of Example 1.

[0047] The test results show that when sodium hexane sulfonate solution is used as mobile phase A, impurities P0Z8 and P0Z4 overlap and cannot be detected, and impurities P0UZ3 and P0Z9 overlap. Fig.10 .

[0048] Comparative Example 8 Comparative Example 8 uses a mobile phase pH value different from that of Example 1, and the other conditions are the same as those of Example 1. The details are as follows: The test results show that when the pH value of the mobile phase is adjusted to 4.0, the impurities cannot be completely separated, and the impurities P0Z13 and others overlap with the main component. Fig.11 .

Claims

1. A method for detecting impurities in neostigmine methylsulfate injection, characterized in that: The chromatographic conditions included the following: The HPLC method was adopted, wherein the mobile phase A was prepared with sodium dihydrogen phosphate buffer solution and acetonitrile in a volume ratio of 95:5 and the pH was adjusted; the mobile phase B was prepared with sodium dihydrogen phosphate buffer solution and acetonitrile in a volume ratio of 30:70 and the pH was adjusted; the elution was performed by gradient elution, and the chromatographic column was 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 each of impurities P0Z1, P0Z2, P0Z4, P0Z5, P0Z8, P0Z9, P0Z11, P0Z12, P0Z13, P0Z14, P0UZ3, and P0UZ4.

2. The impurity detection method of neostigmine methylsulfate injection according to claim 1, characterized in that: The mobile phase is configured as follows: sodium pentane sulfonate is added to a sodium dihydrogen phosphate buffer solution to adjust the solution pH to 3.0, and then configured with acetonitrile in a volume ratio of 95:5 to form mobile phase A; sodium pentane sulfonate is added to a sodium dihydrogen phosphate buffer solution to adjust the solution pH to 3.0, and then configured with acetonitrile in a volume ratio of 30:70 to form mobile phase B.

3. The impurity detection method of neostigmine methylsulfate injection according to claim 2, characterized in that: The concentration of sodium pentane sulfonate in the mobile phase is 0.8 g / L to 0.9 g / L.

4. The impurity detection method of neostigmine methylsulfate injection according to claim 1 or 2, characterized in that: In the mobile phase A, the sodium dihydrogen phosphate buffer solution is prepared by weighing 3.12 g of sodium dihydrogen phosphate dihydrate into 1000 ml of water, and adjusting the pH value to 3.0 with phosphoric acid; in the mobile phase B, the sodium dihydrogen phosphate buffer solution is prepared by weighing 3.12 g of sodium dihydrogen phosphate dihydrate into 1000 ml of water, and adjusting the pH value to 3.0 with phosphoric acid.

5. The impurity detection method for neostigmine methylsulfate injection according to claim 1, characterized in that: The specific conditions of the elution gradient are: 0-5min, 100% mobile phase A; 5-40min, 100%-30% mobile phase A; 40-50min, 30% mobile phase A.

6. 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.

7. The impurity detection method for neostigmine methylsulfate injection according to claim 1, characterized in that: The chromatographic conditions also include: a flow rate of 1.0-1.5 mL / min; a column temperature of 40° C.; a detection wavelength of 215 nm; and an injection volume of 20 μl.

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