Method for detecting content of N-chlorosuccinimide

Abstract

The invention relates to a method for detecting the content of N-chlorosuccinimide (NCS), and mainly relates to a method for detecting the content of N-chlorosuccinimide (NCS) in N-bromosuccinimide (NBS). According to the method, an NBS test solution and an NCS reference solution are prepared by adopting an ion chromatography method and selecting a 0.005-0.1 mol / L sodium hydrogen sulfite solution as a diluent. The method provided by the invention has high sensitivity, high reproducibility and good specificity, can effectively solve the defects of the existing detection method, can accurately and sensitively detect the content of NCS in NBS with good reproducibility, effectively controls the drug quality and ensures the medication safety.

Description

Technical Field

[0001] The present disclosure relates to the field of pharmaceutical analysis, and particularly to a method for detecting the content of trace or ultra-trace N-chlorosuccinimide (NCS) contained in N-bromosuccinimide (NBS). Background Art

[0002] N-bromosuccinimide (NBS) is an important organic synthesis reagent, which is widely used in the fields of medicine, pesticides, and materials science. NBS is mainly used as a brominating reagent in organic synthesis to introduce bromine atoms. However, NBS often contains the impurity N-chlorosuccinimide (NCS), which will have an adverse effect on the reaction results in some applications. For example, during the synthesis of sugammadex, NCS will participate in the reaction and generate chlorinated sugammadex, thus affecting the quality and safety of the drug. Therefore, accurately detecting the content of NCS in NBS is of great significance for controlling drug quality and ensuring drug safety. However, the currently commonly used content detection methods all have some deficiencies in the face of such detection issues.

[0003] First of all, traditional titration methods cannot accurately distinguish the bromine element and chlorine element in NBS and NCS, resulting in inaccurate detection results. Although the commonly used liquid chromatography and gas chromatography in the laboratory can be used to detect NCS, due to the similar properties of NBS and NCS, the separation difficulty is large, and both components are easily degraded, resulting in very poor reproducibility of the detection results.

[0004] Therefore, in order to monitor the content of NCS in NBS, improve drug quality and drug safety, developing a method for detecting the content of trace or ultra-trace N-chlorosuccinimide (NCS) contained in N-bromosuccinimide (NBS) that is accurate, sensitive and has good reproducibility is an urgent problem to be solved at present.

[0005] In recent years, ion chromatography has been widely used in the field of analysis. Ion chromatography has high sensitivity, high selectivity and good reproducibility, and can effectively separate and detect various ions. However, there is currently no research report on the application of ion chromatography to the above topics, and the bromine in N-bromosuccinimide (NBS) and the chlorine in N-chlorosuccinimide (NCS) are not the detection objects that ion chromatography is good at. Developing corresponding detection methods still faces many challenges.

[0006] The present invention uses ion chromatography to detect the content of NCS in NBS, realizes a simple and reliable ionization scheme by selecting a special diluent, and realizes the separation of NCS in NBS and the accurate detection of NCS by selecting chromatographic conditions. Summary of the Invention

[0007] The object of the present invention is to provide a method capable of accurately detecting the content of NCS in NBS. This method has high sensitivity, high reproducibility and good specificity, and can effectively solve the deficiencies of existing detection methods.

[0008] The method of the present invention uses ion chromatography. The NBS test solution and the NCS reference solution are prepared with a sodium bisulfite diluent with a chloride ion content not exceeding 0.002% (including 0.002%). The NBS test solution and the NCS reference solution are prepared by selecting a sodium bisulfite solution with a concentration of 0.005 - 0.1 mol / L as the diluent. The sodium bisulfite solution can reduce bromine in NBS to bromide ions and chlorine in NCS to chloride ions, thereby achieving accurate detection of the NCS content.

[0009] The detector of the ion chromatograph is a conductivity detector (CD), and the chromatographic column is an anion exchange chromatographic column with a pore size in the range of 10 - 50 nanometers. The specific model is Dionex IonPac AS11 - HC (4 * 250 mm). The anion suppressor of the ion chromatograph is a suppressor with a maximum working current in the range of 300 - 500 mA.

[0010] The mobile phase consists of water and 10 - 30 mmol / L sodium hydroxide. In one embodiment, the gradient elution program is as follows:

[0011] 0 - 10 minutes: 50% - 70% water, 30% - 50% sodium hydroxide;

[0012] 10 - 25 minutes: 5% - 15% water, 85% - 95% sodium hydroxide;

[0013] 25 - 35 minutes: 5% - 15% water, 85% - 95% sodium hydroxide;

[0014] 35 - 45 minutes: 50% - 70% water, 30% - 50% sodium hydroxide.

[0015] The method of the present invention has good system suitability. When the reference solution is injected continuously for at least 5 injections, the RSD of the main peak peak area is not greater than 10.0%. The concentration range of the reference solution is 1 - 10 μg / mL. The preparation method includes the following steps:

[0016] First step: Weigh accurately about 10 mg of NCS reference substance, place it in a 15 mL centrifuge tube, add 10 mL of diluent to dissolve it, and shake well;

[0017] Second step: Accurately measure 0.1 mL from it, place it in a 15 mL centrifuge tube, add 4.9 mL of diluent, and shake well to obtain the reference mother solution;

[0018] Step 3: Precisely measure 0.5 mL of the reference stock solution, place it in a 15 mL centrifuge tube, add 4.5 mL of the diluent, shake well, and you will obtain a solution (the NCS concentration is approximately 2 μg / mL).

[0019] The detection limit of the test solution in the said detection method is 0.05 - 0.10 μg / mL, which is equivalent to 0.005% - 0.010% of the test sample. The quantification limit is 0.15 - 0.25 μg / mL, which is equivalent to 0.015% - 0.025% of the test sample.

[0020] Compared with the existing methods, the present invention first realizes a method for determining the content of trace or ultratrace N-chlorosuccinimide (NCS) contained in N-bromosuccinimide (NBS), and has the following technical advantages:

[0021] High sensitivity: By selecting sodium bisulfite solution as the diluent, the bromine in NBS can be reduced to bromide ions, and the chlorine in NCS can be reduced to chloride ions, thus realizing the accurate detection of the NCS content.

[0022] High reproducibility: Through the system suitability test and specificity determination, the reproducibility and specificity of the detection method are ensured.

[0023] Simple operation: The method of the present invention is simple to operate and is applicable to quality control in actual production.

[0024] Wide application range: The method of the present invention is not only applicable to the detection of the NCS content in NBS, but can also be extended to the detection of other similar compounds. Description of the Drawings

[0025] Figure 1 It is a chromatogram showing the detection of NCS in NBS by liquid chromatography;

[0026] Figure 2 It is the RSD when detecting NCS in NBS by gas chromatography;

[0027] Figure 3 It is the chromatogram of the test solution of NBS;

[0028] Figure 4 It is the chromatogram showing the detection of the blank in Example 1 of the present invention;

[0029] Figure 5 It is the chromatogram showing the detection of the reference in Example 1 of the present invention;

[0030] Figure 6 It is the chromatogram showing the detection of the test sample in Example 1 of the present invention;

[0031] Figure 7It represents the chromatogram of the 100% spiked test sample in Example 1 of the present invention;

[0032] Figure 8 It represents the chromatogram for detecting the detection limit in Example 3b of the present invention;

[0033] Figure 9 It represents the chromatogram for detecting the quantitation limit in Example 3b of the present invention. Detailed implementation manners

[0034] The following specifically describes each element of the present disclosure.

[0035] For example, in the synthesis process of the common drug sugammadex, NBS is used as a key reagent to participate in the reaction, and the impurity NCS therein will also participate in the reaction and will be transferred and remain in the active pharmaceutical ingredient as chlorinated sugammadex. Through research, there is no removal effect on chlorinated sugammadex in the synthesis process. In order to make the content of the impurity chlorinated sugammadex in sugammadex qualified, it is necessary to strictly control the content of NCS in NBS, that is, to control the content of the derivative chlorinated sugammadex of NCS in sugammadex. However, there is currently no detection method for detecting the content of NCS in NBS. The purpose of the present invention is to provide a detection method that can accurately detect the content of NCS in NBS, so as to effectively control the content of the impurity chlorinated sugammadex in sugammadex, reduce the occurrence of side effects of patients' medications, and ensure the safety of medication.

[0036] According to the content disclosed in the following examples, when implementing the method of the present invention, in the specific examples, a sodium bisulfite solution with a concentration of about 0.01 mol / L is selected as a diluent to prepare the NBS test solution and the NCS reference solution, creating a unique ion chromatography method, avoiding the problem that the bromine element and chlorine element in NBS and NCS have similar properties and interfere with the detection when using the titration method, and the problem that NCS is extremely easy to degrade and the reproducibility of the detection results is poor when using liquid or gas chromatography. The bromine in NBS is completely reduced to bromide ions by the sodium bisulfite solution, and the chlorine in NCS is completely reduced to chloride ions, and the content of NCS in NBS is determined by detecting the chloride ions. As a basic principle, NBS is a good brominating reagent, and the NCS reagent is mainly used as a chemical raw material to regulate low-energy chlorination reactions and is a good chlorinating reagent. When detecting NCS in NBS, due to their similar properties and both being unstable and prone to losing bromine or chlorine themselves. In the detection process, a sodium bisulfite solution is selected as the diluent, and the bromine in NBS and the chlorine in NCS are reduced to bromide ions and chloride ions by the reducibility of sulfite ions, and then the ion chromatography theory is used to use a chromatographic column for specifically detecting anions to develop a method for separating bromide ions and chloride ions, and accurately detect the content of chloride ions therein.

[0037] In the method development stage, liquid chromatography and gas chromatography were attempted. Due to the similar properties of NBS and NCS, their peaks were difficult to separate, and both NBS and NCS were extremely prone to degradation, resulting in inaccurate detection. In liquid chromatography, both NBS and NCS would degrade rapidly, and NBS and NCS could not achieve baseline separation (see Figure 1 , the arrow indicates the peak positions of NBS and NCS). When using gas chromatography, in the chromatogram with HP-5, the peaks of NBS and NCS could be baseline-separated. However, due to the rapid degradation of NBS, the low sensitivity of NCS itself, and the susceptibility to interference during the detection process, when using the area normalization method to detect the spiked sample at a concentration of 0.1%, the peak of NCS was submerged by the baseline and the peak could not be seen. At a concentration of 0.2%, the results changed greatly and the reproducibility was poor. See Figure 2 .

[0038] The inventors conducted in-depth research and innovative improvements, and attempted to change to the detection method using an ion chromatograph. The method of the present invention was developed, which improved the sensitivity and completely eliminated the situation where NBS and NCS degraded rapidly and had similar properties, interfering with the detection. From the results of the examples, under typical detection environments and recommended conditions, the detection limit of chloride ions in this detection method reached 0.07 μg / mL, which was equivalent to 0.007% of the test sample.

[0039] A sodium bisulfite solution with a suitable concentration is the key to the present invention. A sodium bisulfite solution with a concentration of 0.005 - 0.1 mol / L as a diluent can rapidly and completely ionize the halogens in NBS and NCS, and the by-products generated by the reaction, through the selection of appropriate chromatographic conditions, will not affect the resolution of the chromatographic peaks of the target substances.

[0040] In some embodiments, the sodium bisulfite solution is 0.005 - 0.02 mol / L.

[0041] As the recommended detection conditions, typically the following conditions are used. However, as long as the detection conditions meet the spirit of the present invention, they are included in the protection scope of the present invention:

[0042] When preparing the solutions required as the test targets, including diluents, test sample solutions, and reference substance solutions. The diluent consists of a sodium bisulfite solution with a concentration of 0.005 - 0.1 mol / L. The test sample solution is obtained, for example, by accurately weighing about 10 mg of NBS, adding 10 mL of the diluent, dissolving, and shaking well. The preparation of the reference substance solution includes three steps: First, accurately weigh about 10 mg of the NCS reference substance, add 10 mL of the diluent, dissolve, and shake well; then accurately measure 0.1 mL from it, add 4.9 mL of the diluent, and shake well to obtain the mother solution of the reference substance; finally, accurately measure 0.5 mL of the mother solution of the reference substance, add 4.5 mL of the diluent, and shake well to obtain a reference substance solution with a concentration of about 2 μg / mL.

[0043] The determination of the detection limit and the quantitation limit is accomplished by preparing a sensitivity stock solution, a quantitation limit solution, and a detection limit solution. The sensitivity stock solution is prepared by dissolving 10 mg of the NCS reference substance in 10 mL of the diluent, and then accurately measuring 0.4 mL and adding it to 19.6 mL of the diluent. The quantitation limit solution is obtained by adding 0.5 mL of the sensitivity stock solution to 49.5 mL of the diluent, and the detection limit solution is obtained by adding 1 mL of the quantitation limit solution to 2 mL of the diluent. By measuring the peak areas and signal-to-noise ratios of these solutions, the detection limit and the quantitation limit are determined to be 0.05 - 0.10 μg / mL and 0.15 - 0.25 μg / mL, respectively.

[0044] During the actual detection process, the test solution and the reference solution are injected separately, the peak area of chloride ions is recorded, and the content of NCS in the test solution is calculated based on the peak area and concentration of the reference solution. By this method, the content of NCS in NBS can be detected accurately, sensitively, and with good reproducibility, effectively controlling the drug quality and ensuring the safety of drug use.

[0045] In the subsequent system suitability test, the reference solution is injected continuously for 5 injections, the peak area of the main peak is recorded, and the RSD value is calculated to ensure that the RSD is not more than 10.0%. In the specificity determination, the diluent, the reference solution, the chloride ion location solution, and the test solution are analyzed by ion chromatography to ensure that there is no interference from impurity peaks at the elution position of NCS.

[0046] The present invention uses ion exchange chromatography for detection. General chromatographic columns are based on ion exchange mechanism and ion exclusion mechanism. An important detection condition in the present invention is to select a suitable anion exchange chromatographic column, preferably a high-capacity anion exchange chromatographic column with a polymer matrix. For example, the chromatographic column is an anion exchange chromatographic column with a pore size in the range of 10 - 50 nanometers. Specific models can be exemplified as Dionex IonPac AS11-HC (4*250mm). Dionex IonPac AS11-HC is a high-performance anion exchange chromatographic column designed for ion chromatography analysis, capable of effectively separating and detecting various anions. This chromatographic column adopts advanced polymer matrix technology, has a high capacity factor and good peak shape characteristics, and can effectively separate and detect multiple anions, including chloride ions, bromide ions, iodide ions, etc. The pore size range of Dionex IonPac AS11-HC chromatographic column is 10 - 50 nanometers, which can provide high separation efficiency, ensuring the accuracy and reliability of the detection results. In addition, this chromatographic column also has a good peak shape, which can reduce peak tailing and peak fronting phenomena, improving the sensitivity and reproducibility of the detection. In the present invention, Dionex IonPac AS11-HC chromatographic column is used to detect the content of N-chlorosuccinimide (NCS) in N-bromosuccinimide (NBS). By selecting a sodium bisulfite solution with a concentration of 0.005 - 0.1 mol / L as the diluent, the bromine in NBS is reduced to bromide ions, and the chlorine in NCS is reduced to chloride ions. Utilizing the high separation efficiency and good peak shape characteristics of Dionex IonPac AS11-HC chromatographic column, the accurate detection of the NCS content is achieved. However, the present invention is not necessarily limited to using this type of chromatographic column, and chromatographic columns of the same type can be used in the present invention. The present invention has ensured very good compatibility with most anions through the setting of the type and concentration of the diluent. This type of chromatographic column can ensure high sensitivity, high reproducibility and good specificity of the detection method, effectively solve the deficiencies of the existing detection methods, accurately, sensitively and with good reproducibility detect the content of NCS in NBS, and effectively control the drug quality and ensure drug safety.

[0047] The detector of the ion chromatograph is a conductivity detector (CD), and the anion suppressor of the ion chromatograph is a suppressor with a maximum working current in the range of 300 - 500 mA.

[0048] The method uses gradient elution, where mobile phase A is water and mobile phase B is an aqueous sodium hydroxide solution with a concentration of 10 - 30 mmol / L.

[0049] The mobile phase consists of water and sodium hydroxide with a concentration of 10 - 30 mmol / L. In one embodiment, the gradient elution program is as follows:

[0050] 0 - 10 minutes: 50% - 70% water, 30% - 50% sodium hydroxide;

[0051] 10 - 25 minutes: 5% - 15% water, 85% - 95% sodium hydroxide;

[0052] 25 - 35 minutes: 5% - 15% water, 85% - 95% sodium hydroxide;

[0053] 35 - 45 minutes: 50% - 70% water, 30% - 50% sodium hydroxide.

[0054] In order to further optimize the chromatographic time, the gradient elution program of the mobile phase can also be slightly adjusted to:

[0055] 0 - 8 minutes: 50% - 70% water, 30% - 50% sodium hydroxide;

[0056] 8 - 20 minutes: 5% - 15% water, 85% - 95% sodium hydroxide;

[0057] 20 - 30 minutes: 5% - 15% water, 85% - 95% sodium hydroxide;

[0058] 30 - 40 minutes: 50% - 70% water, 30% - 50% sodium hydroxide.

[0059] The method uses gradient elution, where mobile phase A is water and mobile phase B is an aqueous solution of 20 mmol / L sodium hydroxide. The gradient elution program of the mobile phase is:

[0060] T (min) Mobile Phase A (%) Mobile Phase B (%) 0-10 60 40 10-25 60 40 25-35 10 90 35-45 60 40

[0061] The elution gradient can be adjusted according to the specific situation of the selected chromatographic column. The above are recommended gradients and are not used to limit the protection scope of the present invention.

[0062] To further illustrate the elements of the present disclosure, the following is described in conjunction with embodiments. However, the protection scope of the present disclosure is not limited to the following embodiments.

[0063] Embodiment

[0064] Experimental reagents:

[0065]

[0066] 2. Instrumentation and equipment:

[0067] Ion chromatograph (Thermo ICS-6000SP), electronic balance, plastic centrifuge tubes, ultrapure water instrument (Millipore), chromatographic column (Dionex IonPac AS11-HC(4*250mm)), pre-column (Dionex IonPac AG11-HC(4*50mm)), anion suppressor (Dionex ASRS300)

[0068] Establishment of content detection method and data statistics

[0069] Solution Preparation and Detection

[0070] a. Diluent

[0071] Weigh 1.04 g of sodium bisulfite and dissolve it in 1000 mL of water to obtain a 10 mmol / L sodium bisulfite solution as the diluent.

[0072] b. Test solution (NBS containing a small amount of NCS impurity)

[0073] Precisely weigh about 10 mg of NBS and place it in a 15 mL centrifuge tube. Add 10 mL of diluent to dissolve it and shake well.

[0074] c. Reference solution

[0075] Precisely weigh about 10 mg of NCS reference substance and place it in a 15 mL centrifuge tube. Add 10 mL of diluent to dissolve it and shake well. Precisely measure 0.1 mL from it and place it in a 15 mL centrifuge tube, then add 4.9 mL of diluent and shake well to obtain the mother reference solution. Precisely measure 0.5 mL of the mother reference solution and place it in a 15 mL centrifuge tube, then add 4.5 mL of diluent and shake well, that is, obtain (the NCS concentration is about 2 μg / mL). Prepare two portions in parallel to obtain reference solution I and reference solution II.

[0076] d. System suitability criteria

[0077] Inject reference solution I continuously for 5 injections, and the RSD of the main peak area should not be greater than 10.0%; inject reference solution II for 1 injection, and compared with reference solution I, the RD should not be greater than 10.0%. The calculation formula is as follows:

[0078]

[0079] Where:

[0080] A is the ratio of the average peak area of the impurity in reference solution I to its concentration;

[0081] B is the ratio of the peak area of the impurity in reference solution II to its concentration.

[0082] e. Inspection process

[0083] Inject the diluent once.

[0084] Inject the reference solution Ⅰ five times.

[0085] Inject the reference solution Ⅱ once.

[0086] Inject the test solution once each.

[0087] Inject the reference solution Ⅰ once.

[0088] f. Calculation formula:

[0089]

[0090] In the formula:

[0091] As is the peak area of NCS in the test solution;

[0092] Ar is the average peak area of NCS in the reference solution Ⅰ;

[0093] Mr is the weighed amount of NCS in the reference solution I, mg;

[0094] P is the content of the NCS reference substance, %;

[0095] Ms is the weighed amount of the test sample, mg;

[0096] Dr is the dilution factor of the reference solution;

[0097] Ds is the dilution factor of the test solution;

[0098] Instrument Parameters for Chromatography

[0099] Ion Chromatography System: Thermo ICS-6000SP Chromatographic Column: Dionex IonPac AS11-HC (4 * 250 mm) Pre-column: Dionex IonPac AG11-HC (4 * 50 mm) Anion Suppressor: Dionex ASRS300 Detector: CD Column Flow Rate: 1.0 ml / min Injection Volume: 10 μl Mobile Phase: Mobile Phase A: Water; Mobile Phase B: 20 mmol / L Sodium Hydroxide

[0100] Eluent gradient:

[0101] T (min) Mobile Phase A (%) Mobile Phase B (%) 0 60 40 10 60 40 25 10 90 35 10 90 35.1` 60 40 45 60 40

[0102] Results of the system suitability test in Example 1

[0103] Taking the system suitability results of a sequence as an example, inject the reference solution Ⅰ continuously five times and the reference solution Ⅱ once. The peak areas of each impurity obtained and the RD results obtained by comparing the reference solution Ⅱ with Ⅰ are shown in Table 1.

[0104] Table 1 System suitability results

[0105]

[0106]

[0107] The results showed that: for the reference solution I, when injected continuously for 5 times, the RSD of the peak area of NCS was 1.64%, less than 10.0%; for the reference solution II, when injected once, compared with the reference solution I, the RD value was 1.66%, less than 10.0%, and the RSD of the NCS back-calculated standard was 1.53, not greater than 10.0%, meeting the requirements of system suitability.

[0108] Specificity determination in Example 2

[0109] Specificity was determined by the interference of diluent, reference solution, chloride ion location solution, and test solution in ion chromatography analysis. The results are shown in Table 2.

[0110] Table 2 Specificity results

[0111]

[0112] The results showed that: there was no chromatographic peak interference at the elution peak of NCS (chloride ion) in the blank solution, and there was no impurity peak interference at the elution peak of NCS in the test solution, meeting the requirements.

[0113] Example 3 Detection limit and quantitation limit

[0114] a. Preparation of sensitivity stock solution

[0115] Weigh 10 mg of NCS reference substance and place it in a 15 ml centrifuge tube. Add 10 ml of diluent to dissolve it. Accurately measure 0.4 ml and transfer it to a centrifuge tube, then add 19.6 ml of diluent to obtain the sensitivity stock solution.

[0116] b. Preparation of quantitation limit and detection limit solutions

[0117] Accurately measure 0.5 mL of the sensitivity stock solution and transfer it to a 50 mL centrifuge tube. Add 49.5 ml of diluent and mix well to obtain the quantitation limit solution. Accurately measure 1 mL of the quantitation limit solution and transfer it to a 15 ml centrifuge tube, then add 2 ml of diluent and mix well to obtain the detection limit solution.

[0118] Table 3 Determination results of detection limit and quantitation limit

[0119]

[0120] The results showed that: the quantitation limit concentration of NCS was 0.20 μg / mL, approximately equivalent to 0.02% of the test sample, the signal-to-noise ratio was greater than 10, when injected continuously for 6 times, the RSD of the peak area was 9.88%, not greater than 15%; the detection limit concentration was 0.07 μg / mL, approximately equivalent to 0.007% of the test sample, the signal-to-noise ratio was greater than 3, meeting the requirements.

[0121] Example 4 Linearity and range

[0122] Linear stock solution: Take the sensitivity stock solution under Example 3a as the linear stock solution.

[0123] Preparation of linear solutions: Respectively take the above linear stock solution and prepare linear solutions with corresponding concentrations, linearly covering the range from the quantitation limit to 200% of the limit concentration.

[0124] Table 4-1 Preparation of linear solutions

[0125]

[0126] Table 4-2 NCS linear test results

[0127]

[0128]

[0129] The results show that for NCS in the concentration range of 0.2 μg / mL to 4 μg / mL, the concentration and peak area have a good linear relationship, the correlation coefficient r = 0.9996 > 0.990, and the absolute value of the Y-axis intercept is less than 25% of the peak area of the impurity limit solution.

[0130] Example 5 Accuracy

[0131] Accuracy is investigated by recovery rate, and samples at 50%, 100%, and 150% of the limit concentration are used for confirmation. Three replicates are prepared for each concentration.

[0132] Solution preparation

[0133] Reference solution: Weigh accurately about 10 mg of NCS reference substance and place it in a 15-mL centrifuge tube. Add 10 mL of diluent to dissolve, shake well. Then accurately measure 0.1 mL from it and place it in a 15-mL centrifuge tube, add 4.9 mL of diluent, and shake well to obtain the reference stock solution. Accurately measure 0.5 mL of the reference stock solution and place it in a 15-mL centrifuge tube, add 4.5 mL of diluent, and shake well to obtain (NCS concentration is about 2 μg / mL). Prepare two replicates in parallel to obtain reference solution Ⅰ and reference solution Ⅱ.

[0134] Accuracy stock solution: Take the sensitivity stock solution under Example 3a as the accuracy stock solution.

[0135] Test solution: Weigh accurately about 10 mg of NBS and place it in a 15-mL centrifuge tube. Add 10 mL of diluent to dissolve, shake well.

[0136] Spiked test solution:

[0137] Preparation of R1-50% solution:

[0138] Take 10 mg of this product, accurately weigh it, place it in a 15-ml centrifuge tube, add 0.5 ml of the accuracy stock solution, add 9.5 ml of the diluent, shake well, and you will get it.

[0139] Preparation of the R2-100% solution:

[0140] Take 10 mg of this product, accurately weigh it, place it in a 15-ml centrifuge tube, add 1 ml of the accuracy stock solution, add 9 ml of the diluent, shake well, and you will get it.

[0141] Preparation of the R3-150% solution:

[0142] Take 10 mg of this product, accurately weigh it, place it in a 15-ml centrifuge tube, add 1.5 ml of the accuracy stock solution, add 8.5 ml of the diluent, shake well, and you will get it.

[0143] The results are shown in Table 5.

[0144] Table 5 Results of accuracy test

[0145]

[0146] The results show that: the recovery ranges of NCS for the 9 samples measured are all between 80.0% and 120.0%; the RSD% of the recovery is 3.78%, less than 10.0%, meeting the requirements.

[0147] Example 6 Precision

[0148] Two experimenters independently prepared 2 portions of 100% reference substance solutions, 1 portion of test substance solution, and 6 portions of R2-100% solutions for NCS determination on different dates according to the requirements of Example 5. The results are shown in Table 6.

[0149] Table 6 Results of precision test - NCS

[0150]

[0151] The results show that: the NCS recovery contents of the 6 portions of repeatability test substance spiked solutions are 104.49% - 107.20%; the RSD is 1.00%, less than 15.0%, meeting the requirements; the NCS recovery contents of the 6 portions of intermediate precision test substance spiked solutions are 113.39% - 116.97%; the RSD is 1.27%, less than 15.0%, meeting the requirements; the RSD of the NCS recovery contents of the 12 portions of test substance spiked solutions measured is 4.84%, less than 20.0%, meeting the requirements, indicating that the precision of this method is good.

[0152] Example 7 Solution stability

[0153] Prepare 100% reference solution (reference solution) and R2 - 100% (spiked test sample) solution according to Example 5, and place them at room temperature respectively. The reference solution is detected at different time points, and the absolute value of the change is obtained based on the results measured at different times and the initial result to investigate the stability of the reference solution and the spiked test sample solution. The results are shown in the following table.

[0154] Table 7 Determination Results of Reference Solution (NCS)

[0155]

[0156] Table 8 Determination Results of Spiked Test Sample Solution (NCS)

[0157]

[0158] The results show that: at room temperature, when the reference solution is placed for 90 h, the maximum change value of the peak area is 8.11%, which is less than 10.0%, indicating that the reference solution is stable within 90 h; when the 100% spiked test sample solution is placed for 84 h, the maximum change value of the peak area is 2.26%, which is less than 20.0%, indicating that the 100% spiked test sample solution is stable within 84 h.

[0159] Example 8 Durability

[0160] Prepare two portions of reference solution, one portion of test sample solution and two portions of spiked test sample solution respectively according to item 3.5, inject samples under normal chromatographic conditions and adjusted parameter schemes respectively, and calculate the change value % of the NCS recovery rate in the spiked test sample solution under each condition. The specific durability parameter schemes and results are shown in the following table.

[0161] Table 9 Durability Parameter Adjustment (Chromatographic Condition Investigation)

[0162] Adjustment Items Specified Value Variable 1 Variable 2 Initial Proportion of Phase B % 40 38 42 Column Temperature (°C) 40 38 42 Flow Rate (mL / min) 1.0 0.9 1.1

[0163] Table 10 Summary of Recovery Results of Durability Test

[0164]

[0165]

[0166] The results showed that: under the 6 adjusted durability conditions, the reference solution I was injected continuously for 5 times, and the maximum RSD of the peak area was 1.55%, less than 10.0%; the maximum calculated RD of the two reference solutions was 1.42%, less than 10.0%, and the system suitability met the requirements under each condition. The NCS recovery rates measured under 5 conditions were in the range of 80.0% - 120.0%; the maximum change value of the recovery rates of the known impurities measured under normal conditions and the 6 conditions was 3.03%, less than 20.0%. Therefore, the method had good durability under slight changes in chromatographic conditions.

[0167] Test Results of Multiple Batches in Example 9

[0168] a. Diluent Solution

[0169] Weigh 1.04 g of sodium bisulfite and dissolve it in 1000 mL of water to obtain a 10 mmol / L sodium bisulfite solution as the diluent.

[0170] b. Test Solution

[0171] NBS test solution (batch number DS230407): Accurately weigh 10.23 mg of NBS and place it in a 15 mL centrifuge tube. Add 10 mL of diluent to dissolve it and shake well.

[0172] NBS test solution (batch number DS230410): Accurately weigh 10.26 mg of NBS and place it in a 15 mL centrifuge tube. Add 10 mL of diluent to dissolve it and shake well.

[0173] NBS test solution (batch number DS230411): Accurately weigh 10.02 mg of NBS and place it in a 15 mL centrifuge tube. Add 10 mL of diluent to dissolve it and shake well.

[0174] NBS test solution (batch number DS230412): Accurately weigh 10.79 mg of NBS and place it in a 15 mL centrifuge tube. Add 10 mL of diluent to dissolve it and shake well.

[0175] c. Reference Solution

[0176] Accurately weigh 10.12 mg of NCS reference substance and place it in a 15 mL centrifuge tube. Add 10 mL of diluent to dissolve it and shake well. Accurately measure 0.1 mL from it and place it in a 15 mL centrifuge tube. Add 4.9 mL of diluent and shake well to obtain the reference stock solution. Accurately measure 0.5 mL of the reference stock solution and place it in a 15 mL centrifuge tube. Add 4.5 mL of diluent and shake well to obtain reference solution I (NCS concentration is about 2 μg / mL). Accurately weigh 10.26 mg of NCS reference substance and prepare reference solution II in parallel.

[0177] Detect according to the following experimental parameters:

[0178]

[0179]

[0180] Gradient:

[0181] T (min) Mobile Phase A (%) Mobile Phase B (%) 0 60 40 10 60 40 25 10 90 35 10 90 35.1` 60 40 45 60 40

[0182] The experimental results are as follows

[0183] Name Peak Area of NCS Control 1-1 0.111 Control 1-2 0.111 Control 1-3 0.112 Control 1-4 0.115 Control 1-5 0.114 RSD (%) 1.64 Control 2-1 0.118 RD (%) 1.66 Standard Substance 1 Recovery-1 0.114 RSD (%) 1.53

[0184]

[0185] Investigation on the mass of sodium sulfite in Example 10

[0186] Prepare a blank solution: Weigh 1.04 g of sodium bisulfite and dissolve it in 1000 mL of water to obtain a sodium bisulfite solution, which is used as a diluent.

[0187] Sodium bisulfite containing different chloride ion contents was used, all configured to a concentration of 0.1 mol / L, and injected for detection. Chloride ion peak areas of different sizes were obtained. The results are shown in Table 11. When the chloride ion content in sodium bisulfite is less than 0.002%, its peak area is the smallest. When the concentration of sodium bisulfite is designed to be 0.01 mol / L, it not only ensures that all chloride ions in NCS are reduced, but also ensures that the interference peak area of chloride ions in the blank solvent (0.01 mol / L sodium bisulfite aqueous solution) is below the detection limit. Therefore, it is determined that the chloride ion content in sodium bisulfite shall not exceed 0.002%.

[0188] Table 11.

[0189] Reagent Chloride Ion Content % Peak Area of 0.01 mol / L Sodium Bisulfite 0.002 0.0113 Sodium Bisulfite 0.006 0.0307 Sodium Bisulfite 0.159 0.8724

[0190] The present disclosure illustrates the detailed method of the present disclosure through the above embodiments, but the present disclosure is not limited to the above detailed method, that is, it does not mean that the present disclosure must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement to the present disclosure, the equivalent substitution of each raw material of the present disclosure product, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present disclosure.

Claims

1. A method for detecting the content of N-chlorosuccinimide NCS, characterized in that: The invention adopts ion chromatography to detect the content of N-chlorosuccinimide (NCS) in N-bromosuccinimide (NBS), and adopts sodium bisulfite with a chloride ion content of no more than 0.002% as a diluent to prepare an NBS test solution and an NCS reference solution.

2. The method according to claim 1, characterized in that The NBS test solution and the NCS reference solution are prepared using a 0.005-0.1 mol / L sodium bisulfite solution as a diluent, preferably a 0.005-0.02 mol / L sodium bisulfite solution as a diluent.

3. The method according to claim 1, characterized in that The detector of the ion chromatograph in the ion chromatography method is a conductivity detector CD.

4. The method according to claim 1, characterized in that: The chromatographic column of the ion chromatograph in the ion chromatography method is an anion exchange chromatographic column with a pore diameter in the range of 10-50 nanometers.

5. The method according to claim 4, characterized in that The chromatographic column used is Dionex IonPac AS11-HC.

6. The method according to claim 1, characterized in that The anion suppressor of the ion chromatograph in the ion chromatography method is a suppressor with a maximum operating current in the range of 300-500 mA.

7. The method according to claim 1, characterized in that The method uses gradient elution, wherein mobile phase A is water and mobile phase B is a 10-30 mmol / L sodium hydroxide aqueous solution.

8. The method according to claim 7, characterized in that The gradient elution program of the mobile phase is: 0-10 minutes: 50%-70% water, 30%-50% sodium hydroxide; 10-25 minutes: 5%-15% water, 85%-95% sodium hydroxide; 25-35 minutes: 5%-15% water, 85%-95% sodium hydroxide; 35-45 minutes: 50%-70% water, 30%-50% sodium hydroxide.

9. The method according to claim 7, characterized in that: The gradient elution program of the mobile phase is: 0-8 minutes: 50%-70% water, 30%-50% sodium hydroxide; 8-20 minutes: 5%-15% water, 85%-95% sodium hydroxide; 20-30 minutes: 5%-15% water, 85%-95% sodium hydroxide; 30-40 minutes: 50%-70% water, 30%-50% sodium hydroxide.

10. The method according to claim 1, characterized in that The detection limit of the test solution of the detection method is 0.05-0.10 μg / mL, which is equivalent to 0.005%-0.010% of the test product.

11. The method according to claim 1, characterized in that: The limit of quantification of the detection method is 0.15-0.25 μg / mL, which is equivalent to 0.015%-0.025% of the test sample.

12. The method according to claim 1, characterized in that In the method, when the reference solution is continuously sampled for at least 5 injections, the RSD of the main peak area is not greater than 10.0%.

13. The method according to claim 1, characterized in that The concentration range of the reference solution is 1-10 μg / mL.

14. The method according to claim 13, characterized in that The preparation method of the reference substance solution comprises the following steps: Step 1: Accurately weigh about 10 mg of NCS reference substance, place it in a 15 mL centrifuge tube, add 10 mL of diluent to dissolve, and shake well; Step 2: Accurately measure 0.1 mL from the solution, place it in a 15 mL centrifuge tube, add 4.9 mL of diluent, shake well, and obtain the mother solution of the reference substance; Step 3: Accurately measure 0.5 mL of the reference substance stock solution, place it in a 15 mL centrifuge tube, add 4.5 mL of the diluent, and shake well to obtain a reference substance solution with an NCS concentration of 2 μg / mL.

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