An analytical method for determining the content of dextran 20 and sucrose based on HPLC

Abstract

The present application relates to the field of biochemistry, and discloses an analytical method for determining the content of dextran 20 and sucrose based on HPLC, comprising S1, preparing dextran 20 and sucrose gradient standard solutions: dissolving a dextran 20 reference substance and a sucrose reference substance in a CaCl2·2H2O aqueous solution to obtain a standard stock solution, and diluting the standard stock solution to obtain a series of dextran 20 and sucrose gradient standard solutions; S2, preparing a test solution: dissolving a test sample in a CaCl2·2H2O aqueous solution to obtain; S3, setting chromatographic conditions: using an ion exchange chromatographic column and a differential refractive index detector (RID), and performing isocratic elution with a CaCl2·2H2O aqueous solution as a mobile phase; S4, analyzing the test sample: injecting dextran 20 and sucrose gradient standard solutions and the test sample solution, and calculating the dextran 20 and sucrose contents in the test sample solution by an external standard method. The analytical method provided by the invention is simple and easy to operate, and the peak shapes of dextran 20 and sucrose are sharp and symmetrical, with high separation, and is suitable for analyzing the contents of dextran 20 and sucrose.

Description

Technical Field

[0001] The invention relates to the field of biochemistry, and in particular to an analytical method for determining the contents of dextran 20 and sucrose based on HPLC. Background Art

[0002] A mixture of dextran and sucrose is often added to protein drugs as a lyoprotectant to prevent denaturation of protein drugs during freeze drying and storage. Studies have shown that the protective effect of the lyoprotectant depends on its concentration. At low concentrations, the protective effect is positively correlated with the concentration. When the optimal protective concentration is reached, the protective effect no longer increases with the increase in concentration, and even decreases. Excessive concentration of the protective agent will also have an impact on the human body. The optimal protective concentration of dextran and sucrose is 5%. At this concentration, dextran can maximize the storage quality of protein, and sucrose can maximize the connection with protein molecules that have lost water molecules through hydrogen bonds, thereby preventing the direct exposure of the hydrogen bond connection position of the protein molecule and causing protein denaturation. Therefore, it is very important to detect the dextran and sucrose content in protein drugs (lyoprotectants: sucrose, dextran).

[0003] At present, sucrose content is mostly determined by high-performance liquid chromatography-refractive index detector (HPLC-RID), high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD), etc. There are few reports on the determination of dextran content. The inventor retrieved the dextran iron injection included in the British Pharmacopoeia using anthrone-sulfuric acid colorimetry to detect the dextran 20 content. The method has low sensitivity, cumbersome operation and is easily affected by other factors. Wang Li et al. used high-performance gel permeation chromatography (HPGPC) to determine the dextran 70 content in compound dextran 70 eye drops. The dextran 70 obtained by this method has a broad peak shape and many impurities in the spectrum, and the main component peak is easily interfered. The "Establishment and Verification of the Detection Method for Dextran and Sucrose Content in Botulinum Toxin Type A for Injection" reported by Ye Ting et al. used an amino column to separate dextran 20 and sucrose. The method had the following defects: ① The dextran 20 peak had severe tailing; ② The separation degree between the dextran 20 peak and the sucrose peak was difficult to meet the requirement of R ≥ 1.5; ③ The proportion of organic solvent in the mobile phase (5% methanol) was low, and the amino column was easily damaged under the elution of the mobile phase, etc.

[0004] In addition, the 2020 edition of the "Chinese Pharmacopoeia" does not include the method for determining the main component content in the monographs on sucrose and dextran 20. The purity of raw and auxiliary materials is crucial to their subsequent applications. Therefore, it is necessary to propose a technical solution to fill this gap in order to further improve the quality standards of the raw and auxiliary materials sucrose and dextran 20 and ensure their quality. Summary of the invention

[0005] Purpose of the invention: In view of the shortcomings of the prior art, the present invention provides a method for simply and accurately detecting the content of dextran 20 and sucrose, so as to achieve more stringent quality control of the content of dextran 20 and sucrose.

[0006] To achieve the above objectives, the present invention designs the following technical scheme: an analytical method for determining the content of dextran 20 and sucrose based on HPLC, comprising the following steps:

[0007] S1. Preparation of dextran 20 and sucrose gradient standard solutions

[0008] Take appropriate amount of sucrose reference substance and dextran 20 reference substance dried to constant weight, accurately weigh, dissolve in CaCl2·2H2O aqueous solution, transfer to a volumetric flask and dilute to scale, shake well to obtain dextran 20 and sucrose standard stock solutions, then dilute with CaCl2·2H2O aqueous solution to obtain a series of dextran 20 and sucrose gradient standard solutions, filter and set aside;

[0009] S2. Preparation of test solution

[0010] Take botulinum toxin type A for injection Dissolve 4 bottles of freeze-dried preparation in 0.022% CaCl2·2H2O aqueous solution and dilute to a 50ml volumetric flask to obtain the test solution, which is filtered and set aside;

[0011] S3. Set chromatographic conditions

[0012] An ion exchange column and a differential refractive index detector (RID) were used, and isocratic elution was performed with CaCl2·2H2O aqueous solution as the mobile phase;

[0013] S4. Test sample analysis

[0014] Inject dextran 20 and sucrose gradient standard solutions and the test solution, record the chromatograms, and calculate the dextran 20 and sucrose contents in the test solution using the external standard method.

[0015] Preferably, in step S1, the temperature at which the dextran 20 is dried to constant weight is 105° C.; the sucrose reference substance does not need to be dried to constant weight and can be used according to its instructions. For example, the sucrose reference substance of the China Food and Drug Inspection Institute does not need to be dried to constant weight and can be used directly for content determination.

[0016] Preferably, in step S1, the concentration of the dextran 20 and sucrose standard stock solutions is 10 mg / ml, and the concentrations of the dextran 20 and sucrose gradient standard solutions are 1 mg / ml, 1.5 mg / ml, 2 mg / ml, 2.5 mg / ml and 3 mg / ml, respectively.

[0017] Preferably, the filtration in steps S1 and S2 is performed using a syringe filter, and the pore size of the syringe filter is 0.20 to 0.45 μm.

[0018] Preferably, in step S3, the chromatographic column used in the high performance liquid chromatography is an ion exchange chromatographic column. In step S3, the high performance liquid chromatography is isocratic elution, and the mobile phase is a 0.011% to 0.022% CaCl2·2H2O aqueous solution. The 0.011% to 0.022% CaCl2·2H2O aqueous solution can prevent the adsorption of dextran 20 on the chromatographic column. More preferably, the mobile phase is a 0.022% CaCl2·2H2O aqueous solution.

[0019] Preferably, in step S3, the chromatographic column used in the high performance liquid chromatography is a calcium-type strong cation exchange chromatographic column or a sodium-type strong cation exchange chromatographic column.

[0020] Preferably, in step S3, the chromatographic conditions of the high performance liquid chromatography are: column temperature of 50°C to 85°C, flow rate of 0.3ml / min to 0.6ml / min, and detector temperature of 30°C to 55°C.

[0021] The invention provides an analytical method for determining the contents of dextran 20 and sucrose based on HPLC.

[0022] It has the following beneficial effects:

[0023] 1. The present invention adopts ion exchange chromatography column to determine the content of dextran 20 and sucrose. It is found through screening that ion exchange chromatography column is more suitable for the detection of sugars than other chromatography columns.

[0024] 2. The mobile phase of the analytical method provided by the present invention is a CaCl2·2H2O aqueous solution, which is more environmentally friendly than organic solvents; the mobile phase concentration is 0.011% to 0.022%. Compared with high-concentration solutions, low-concentration solutions are safer in use and handling, and reduce the consumption of resources.

[0025] 3. The analytical method provided by the present invention has sharp and symmetrical peaks of dextran 20 and sucrose, high separation, simple and easy analysis process, and accurate and reliable results.

[0026] 4. The analytical method provided by the present invention is used to determine the botulinum toxin type A for injection The methodological validation was carried out using the dextran 20 and sucrose content in the medium as examples, which proved that the method had high sensitivity, strong specificity, high accuracy, good repeatability and intermediate precision, and good durability.

[0027] 5. The analytical method provided by the present invention can be used to analyze botulinum toxin type A for injection In addition to the dextran 20 and sucrose content in water-soluble protein drugs, the method can also be used for: ① Determination of dextran 20 and sucrose content in some water-soluble protein drugs containing dextran 20 and sucrose; ② Determination of dextran 20 and sucrose content in some water-soluble protein drugs containing dextran 20 or sucrose as a single ingredient; ③ Determination of dextran 20 content in raw and auxiliary materials; ④ Determination of sucrose content in raw and auxiliary materials, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The chromatogram of 2 mg / ml dextran 20 and sucrose standard solution in Example 1 of the present invention;

[0029] Figure 2 The chromatogram of the test solution of Example 1 of the present invention;

[0030] Figure 3 It is the specific chromatogram of Example 2 of the present invention; Note: the solid line in the figure is the chromatogram of the test solution; the dotted line is the chromatogram of the blank excipient solution of the prescription amount.

[0031] Figure 4 The chromatogram of the test solution of Comparative Example 1 of the present invention is shown in FIG. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Embodiment 1:

[0034] Please refer to the attached Figure 1 ~Attached Figure 2 The embodiment of the present invention provides an analytical method for determining the content of dextran 20 and sucrose based on HPLC, comprising the following steps:

[0035] S1. Preparation of dextran 20 and sucrose gradient standard solutions

[0036] Take 1.0g of sucrose reference substance and dextran 20 reference substance dried to constant weight at 105℃, accurately weigh, dissolve in 0.022% CaCl2·2H2O aqueous solution, transfer to a 100ml volumetric flask and dilute to the scale, shake well to obtain dextran 20 and sucrose standard stock solutions with a concentration of 10mg / ml. Accurately measure 1ml, 1.5ml, 2ml, 2.5ml and 3ml of dextran 20 and sucrose standard stock solutions into 5 10ml volumetric flasks, dilute with 0.022% CaCl2·2H2O aqueous solution and dilute to the scale to obtain dextran 20 and sucrose gradient standard solutions with concentrations of 1mg / ml, 1.5mg / ml, 2mg / ml, 2.5mg / ml and 3mg / ml, respectively. Filter through a 0.20μm syringe filter and set aside.

[0037] S2. Preparation of test solution

[0038] Take botulinum toxin type A for injection Dissolve 4 bottles of freeze-dried preparation in 0.022% CaCl2·2H2O aqueous solution and make up to the volume in a 50ml volumetric flask to obtain the test solution. Filter with a 0.20μm syringe filter and set aside.

[0039] S3. Set chromatographic conditions

[0040] Instrument: Agilent 1260 liquid chromatograph; chromatographic column: BIO-RAD calcium type strong cation exchange chromatographic column (HPX-87C Column, 300×7.8 mm, 9 μm, filler: sulfonated divinylbenzene / styrene); mobile phase: 0.022% CaCl2·2H2O aqueous solution; flow rate: 0.5 ml / min; column temperature: 60°C; injection volume: 20 μl; detector: differential refractive index detector (RID); detector temperature: 55°C; isocratic elution.

[0041] S4. Test sample analysis

[0042] The injection procedure is as follows:

[0043] 1. System suitability investigation

[0044] name Injection times <![CDATA[0.022% aqueous solution of CaCl2·2H2O]]> 1 2mg / ml Dextran 20 and sucrose standard solution 5

[0045] Inject 2mg / ml dextran 20 and sucrose standard solution 5 times, record the theoretical plate number of dextran 20 peak, the relative standard deviation of the peak area of ​​5 dextran 20 determinations (RSD≤2.0%), the theoretical plate number of sucrose peak, the relative standard deviation of the peak area of ​​5 sucrose determinations (RSD≤2.0%), and the separation R (≥1.5) between the dextran 20 peak and the sucrose peak. After the system suitability study meets the predetermined requirements, the test sample analysis can be carried out.

[0046] 2. Test sample analysis

[0047] name Injection times <![CDATA[0.022% aqueous solution of CaCl2·2H2O]]> 1 1mg / ml Dextran 20 and sucrose standard solution 1 1.5mg / ml Dextran 20 and sucrose standard solution 1 2mg / ml Dextran 20 and sucrose standard solution 1 2.5mg / ml Dextran 20 and Sucrose Standard Solution 1 3mg / ml Dextran 20 and sucrose standard solution 1 <![CDATA[0.022% CaCl2, 2H2O aqueous solution]]> 1 Test solution 1 2mg / ml Dextran 20 and sucrose standard solution 1

[0048] The external standard method was used to calculate the content of dextran 20 and sucrose in the test solution. The purpose of injecting 2 mg / ml dextran 20 and sucrose standard solution at the end of the sequence was to examine whether the system suitability of the HPLC system continued to meet the injection requirements and remained stable during the sequence operation.

[0049] Test results: In this example, the peaks of dextran 20 and sucrose are sharp and symmetrical, with high separation, and botulinum toxin type A for injection The content of dextran 20 is 24.1 mg / bottle, and the content of sucrose is 25.2 mg / bottle. The analysis method in Example 1 was verified by methodology, as shown in Example 2.

[0050] Embodiment 2:

[0051] The embodiment of the present invention provides a specific implementation of an analytical method for determining the content of dextran 20 and sucrose based on HPLC, and the steps are as follows:

[0052] S1-1. Instruments and reagents:

[0053] 1.1 Instruments: Agilent 1260 liquid chromatograph (including: quaternary pump, injector, column oven, differential refractometer), Mettler 1 / 10,000 electronic balance (model: XPE504).

[0054] 1.2 Test drugs: sucrose reference substance (China Food and Drug Inspection Institute, batch number: 111507-202105); dextran 20 reference substance (Denmark Pharmacosmos, batch number: DR4111), calcium chloride, dihydrate (Sinopharm Chemical Co., Ltd., batch number: 20181212), ultrapure water (produced from IQ7010 Milli-Q pure water-ultrapure water all-in-one machine); botulinum toxin type A for injection Lyophilized preparation batches: 20210307, ​​20210308, 20210309.

[0055] S2-2. Analytical methods:

[0056] 2.1 Preparation of dextran 20 and sucrose gradient standard solutions

[0057] Take 1.0g of sucrose reference substance and dextran 20 reference substance dried to constant weight at 105℃, accurately weigh, dissolve in 0.022% CaCl2·2H2O aqueous solution, transfer to a 100ml volumetric flask and dilute to the scale, shake well to obtain dextran 20 and sucrose standard stock solutions with a concentration of 10mg / ml. Accurately measure 1ml, 1.5ml, 2ml, 2.5ml and 3ml of dextran 20 and sucrose standard stock solutions into 5 10ml volumetric flasks, dilute with 0.022% CaCl2·2H2O aqueous solution and dilute to the scale to obtain dextran 20 and sucrose gradient standard solutions with concentrations of 1mg / ml, 1.5mg / ml, 2mg / ml, 2.5mg / ml and 3mg / ml, respectively. Filter through a 0.20μm syringe filter and set aside.

[0058] 2.2 Preparation of test solution

[0059] Take botulinum toxin type A for injection Dissolve 4 bottles of freeze-dried preparation in 0.022% CaCl2·2H2O aqueous solution and make up to the volume in a 50ml volumetric flask to obtain the test solution. Filter with a 0.20μm syringe filter and set aside.

[0060] 2.3 Preparation of prescription amount of blank excipient solution

[0061] Take appropriate amounts of botulinum toxin type A injection stock solution and 10% gelatin solution, dilute with 0.022% CaCl2·2H2O aqueous solution to prepare a solution with a gelatin concentration of about 0.4 mg / ml and a stock solution concentration of about 0.8 ng / ml.

[0062] 2.4 Establishing Chromatographic Parameters

[0063] The chromatographic parameters include: chromatographic column, mobile phase, column temperature, flow rate, injection volume, detector and detector temperature, etc. The chromatographic column is a BIO-RAD calcium-type strong cation exchange chromatographic column (HPX-87C Column, 300×7.8 mm, 9 μm, filler: sulfonated divinylbenzene / styrene), the mobile phase is a 0.022% CaCl2·2H2O aqueous solution, the column temperature is 60°C, the flow rate is 0.5 ml / min, the detector is a differential refractive index detector (RID), the detector temperature is 55°C, and the injection volume is 20 μL.

[0064] 2.5 Test sample analysis

[0065] The injection procedure is as follows:

[0066] 2.5.1 System suitability investigation

[0067] name Injection times <![CDATA[0.022% aqueous solution of CaCl2·2H2O]]> 1 2mg / ml Dextran 20 and sucrose standard solution 5

[0068] Inject 2mg / ml dextran 20 and sucrose standard solution 5 times, record the theoretical plate number of dextran 20 peak, the relative standard deviation of the peak area of ​​5 dextran 20 determinations (RSD≤2.0%), the theoretical plate number of sucrose peak, the relative standard deviation of the peak area of ​​5 sucrose determinations (RSD≤2.0%), and the separation R (≥1.5) between the dextran 20 peak and the sucrose peak. After the system suitability study meets the predetermined requirements, the test sample analysis can be carried out.

[0069] 2.5.2 Test sample analysis

[0070]

[0071]

[0072] The external standard method was used to calculate the content of dextran 20 and sucrose in the test solution. The purpose of injecting 2 mg / ml dextran 20 and sucrose standard solution at the end of the sequence was to examine whether the system suitability of the HPLC system continued to meet the injection requirements and remained stable during the sequence operation.

[0073] S3-3. Methodological Review

[0074] 3.1 System suitability investigation: 2 mg / ml dextran 20 and sucrose standard solution were continuously injected and measured 5 times, and the chromatogram was recorded. The results are shown in Table 1. The theoretical plate number N of dextran 20 peak was all >5000, the theoretical plate number N of sucrose peak was all >4000, the separation R between dextran 20 peak and sucrose peak was all >1.5, the relative standard deviation (RSD) of the peak area of ​​5 dextran 20 measurements was <2.0%, and the relative standard deviation (RSD) of the peak area of ​​5 sucrose measurements was <2.0%.

[0075] Table 1 System suitability investigation results

[0076]

[0077] 3.2 Specificity investigation: Inject the test solution and the blank excipient solution of the prescribed amount once each, and record the chromatogram. For the results, please refer to the attached Figure 3 The test solution had no interference from other chromatographic peaks and the blank excipient solution of the prescription amount did not produce peaks at the retention time of the dextran 20 chromatographic peak and the sucrose chromatographic peak, indicating good specificity.

[0078] 3.3 Linearity test: Take dextran 20 and sucrose gradient standard solution, inject once each under the predetermined chromatographic parameters, and record the chromatogram. Use the gradient standard solution concentration (X) as the horizontal axis and the peak area (Y) as the vertical axis to draw a standard curve and perform linear regression. The results show that dextran 20 and sucrose have a good linear relationship with their peak area in the concentration range of 1mg / ml to 3mg / ml, and the regression equations are Y 右旋糖酐20 =401200.82465X 右旋糖酐20 +3082.89139(r=0.999976),Y 蔗糖 =381631.45490X 蔗糖 +3403.41730 (r=0.999969).

[0079] 3.4 Accuracy and repeatability of linear range: 6 portions of 1 mg / ml dextran 20 and sucrose standard solutions at the low linear concentration and 3 mg / ml dextran 20 and sucrose standard solutions at the high linear concentration were prepared in parallel, and the accuracy and repeatability of the two concentration points were investigated under the predetermined chromatographic parameters. The results are shown in Tables 2 to 5. The recoveries of dextran 20 and sucrose in the two concentrations of dextran 20 and sucrose standard solutions were between 98% and 102%, and the RSDs were all <1%.

[0080] Table 2 Results of the linear low and high point accuracy test - Dextran 20

[0081]

[0082] Table 3 Results of the linear low and high point accuracy test - sucrose

[0083]

[0084]

[0085] Table 4 Linear low and high point repeatability test results - Dextran 20

[0086]

[0087] Table 5 Linear low point and high point repeatability test results - sucrose

[0088]

[0089] 3.5 Accuracy investigation: Take 2 ml of dextran 20 and sucrose standard stock solution, dilute to 10 ml with the prescribed amount of blank auxiliary solution, and obtain the spiked solution. Prepare 6 copies in parallel, inject and measure under the predetermined chromatographic parameters, and record the chromatogram. The results are shown in Table 6 and Table 7. The recovery rates of dextran 20 and sucrose are both between 98% and 101%, indicating that the method has high accuracy.

[0090] Table 6 Dextran 20 accuracy test results

[0091]

[0092] Table 7 Sucrose accuracy test results

[0093]

[0094] 3.6 Repeatability study: 9 test sample solutions were prepared, and 3 batches of test samples were prepared in parallel. The samples were injected and measured under the predetermined chromatographic parameters, and the chromatograms were recorded. The results are shown in Tables 8 and 9. The relative standard deviations (RSDs) of the content (mg / ml) of dextran 20 and sucrose of the same batch of botulinum toxin type A for injection were <2%, indicating that the method had good repeatability.

[0095] Table 8 Dextran 20 repeatability test results

[0096]

[0097] Table 9 Results of sucrose repeatability test

[0098]

[0099] 3.6 Intermediate precision study: 9 test solutions were prepared, 3 batches were prepared in parallel, and the samples were injected and measured under the predetermined chromatographic parameters, and the chromatograms were recorded. Two analysts conducted the above test at different times. The results are shown in Tables 10 and 11. The relative standard deviations (RSDs) of the dextran 20 and sucrose content (mg / ml) of the same batch of botulinum toxin type A for injection measured by the two analysts were both <2%, indicating that the intermediate precision of this method was good.

[0100] Table 10 Results of intermediate precision study of dextran 20

[0101]

[0102]

[0103] Table 11 Results of intermediate precision study of sucrose

[0104]

[0105] According to the verification data of Example 2, the analytical method for determining the content of dextran 20 and sucrose based on HPLC method of the present invention has high sensitivity, strong specificity, high accuracy, good repeatability and intermediate precision, and good durability, and can achieve the determination of botulinum toxin type A for injection. The dextran 20 and sucrose content in the medium are more strictly controlled.

[0106] The analytical method for determining the content of dextran 20 and sucrose based on HPLC described in the present invention can also be used to detect the content of dextran 20 and sucrose in raw and auxiliary materials, as shown in Examples 3 and 4.

[0107] Embodiment 3:

[0108] This embodiment is an analytical method for determining the content of dextran 20 and sucrose based on HPLC for the content detection of dextran 20 in raw and auxiliary materials. The difference from Example 1 is the preparation of the dextran 20 gradient standard solution and the preparation of the test solution, which are as follows:

[0109] Dextran 20 gradient standard solution: Take 1.0g of dextran 20 reference substance dried to constant weight at 105℃, accurately weigh it, dissolve it in 0.022% CaCl2·2H2O aqueous solution, transfer it to a 100ml volumetric flask and make up to the scale, shake well to obtain dextran 20 standard stock solution with a concentration of 10mg / ml. Accurately measure 1ml, 1.5ml, 2ml, 2.5ml and 3ml of dextran 20 standard stock solution into 5 10ml volumetric flasks, dilute with 0.022% CaCl2·2H2O aqueous solution and make up to the scale to obtain dextran 20 gradient standard solutions with concentrations of 1mg / ml, 1.5mg / ml, 2mg / ml, 2.5mg / ml and 3mg / ml, respectively. Filter with a 0.20μm syringe filter and set aside.

[0110] Test solution: Weigh 200 mg of the raw material dextran 20 dried to constant weight at 105°C, accurately weigh, dissolve in 0.022% CaCl2·2H2O aqueous solution and make up to volume in a 100 ml volumetric flask to obtain the test solution, filter with a 0.20 μm syringe filter and set aside.

[0111] The remaining conditions are the same as those in Example 1.

[0112] The calculation formula of the content of dextran 20 in raw and auxiliary materials is:

[0113]

[0114] Where: X 右旋糖酐20含量测定值(mg / ml) It is the measured value of dextran 20 content in each milliliter of test solution.

[0115] X 右旋糖酐20称定值 Weigh the amount of the raw and auxiliary material dextran 20.

[0116] Test results: In this embodiment, the peak shape of dextran 20 is sharp and symmetrical, and there is no interference from other impurity peaks in the chromatogram. The content of dextran 20 in the raw and auxiliary materials is measured to be 100.2%.

[0117] Embodiment 4:

[0118] This embodiment is an analytical method for determining the content of dextran 20 and sucrose based on HPLC for detecting the sucrose content of raw and auxiliary materials. The difference from Example 1 is the preparation of sucrose gradient standard solution and test solution, which are as follows:

[0119] Sucrose gradient standard solution: Take 1.0g of sucrose reference substance, accurately weigh, dissolve in 0.022% CaCl2·2H2O aqueous solution, transfer to a 100ml volumetric flask and dilute to the scale, shake well to obtain a sucrose standard stock solution with a concentration of 10mg / ml. Accurately measure 1ml, 1.5ml, 2ml, 2.5ml and 3ml of the sucrose standard stock solution into 5 10ml volumetric flasks, dilute with 0.022% CaCl2·2H2O aqueous solution and dilute to the scale to obtain sucrose gradient standard solutions with concentrations of 1mg / ml, 1.5mg / ml, 2mg / ml, 2.5mg / ml and 3mg / ml, respectively. Filter through a 0.20μm syringe filter and set aside.

[0120] Test solution: Weigh 200 mg of the raw material sucrose dried to constant weight at 105°C, accurately weigh, dissolve in 0.022% CaCl2·2H2O aqueous solution and make up to volume in a 100 ml volumetric flask to obtain the test solution, filter with a 0.20 μm syringe filter and set aside.

[0121] The remaining conditions are the same as those in Example 1.

[0122] The calculation formula of sucrose content in raw and auxiliary materials is:

[0123]

[0124] Where: X 蔗糖含量测定值(mg / ml) It is the measured value of sucrose content in each milliliter of test solution.

[0125] X 蔗糖称定值 Weigh the amount of sucrose as the raw and auxiliary material.

[0126] Test results: In this example, the sucrose peak is sharp and symmetrical, and there is no interference from other impurity peaks in the chromatogram. The content of sucrose in the raw and auxiliary materials is measured to be 99.9%.

[0127] Comparative Example 1: The analytical method for determining the content of dextran 20 and sucrose based on HPLC in this comparative example comprises the following specific steps:

[0128] The difference from Example 1 is that the chromatographic conditions are set differently, as follows:

[0129] Instrument: Agilent 1260 liquid chromatograph; chromatographic column: NanoChrom calcium type strong cation exchange column (ChromCore Sugar-Ca, 300×7.8 mm, 8 μm, filler: sulfonated divinylbenzene / styrene); mobile phase: 0.022% CaCl2·2H2O aqueous solution; flow rate: 0.3 ml / min; column temperature: 85°C; injection volume: 20 μl; detector: differential refractive index detector (RID); detector temperature: 55°C; isocratic elution.

[0130] The remaining conditions are the same as those in Example 1.

[0131] Test results: In this comparative example, both dextran 20 and sucrose peaks have peak tailing, but the separation between the two meets the test requirements (R ≥ 1.5), and there is no interference from impurities. The content of dextran 20 in the bottle is 24.2 mg / bottle, and the content of sucrose is 25.2 mg / bottle. Figure 4 .

[0132] By comparing the test results of Example 1 with those of Comparative Example 1, it can be seen that the selected BIO-RAD calcium-type strong cation exchange chromatography column (HPX-87C Column, 300×7.8mm, 9μm, filler: sulfonated divinylbenzene / styrene) and NanoChrom calcium-type strong cation exchange chromatography column (ChromCore Sugar-Ca, 300×7.8mm, 8μm, filler: sulfonated divinylbenzene / styrene) can both be used for dextran 20 and sucrose content detection, among which the NanoChrom calcium-type strong cation exchange chromatography column is a domestically produced chromatography column, which meets the demand for localization of materials.

[0133] Although the embodiments and comparative examples of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An analytical method for determining the content of dextran 20 and sucrose based on HPLC, characterized in that: The following steps are involved: S1. Preparation of dextran 20 and sucrose gradient standard solutions Take appropriate amounts of sucrose reference substance and dextran 20 reference substance dried to constant weight, accurately weigh them, dissolve them in CaCl2·2H2O aqueous solution, transfer them to a volumetric flask and dilute to scale, shake well to obtain dextran 20 and sucrose standard stock solutions, and then dilute them with CaCl2·2H2O aqueous solution to obtain dextran 20 and sucrose gradient standard solutions, filter them and set aside; the temperature at which the dextran 20 is dried to constant weight is 105°C; S2. Preparation of test solution Take 4 bottles of lyophilized preparation of botulinum toxin type A for injection, dissolve it in 0.022% CaCl2·2H2O aqueous solution and make up to 50ml volumetric flask to obtain the test solution, filter it and set aside; S3. Set chromatographic conditions The chromatographic column used in the high performance liquid chromatography method is a calcium type strong cation exchange chromatographic column or a sodium type strong cation exchange chromatographic column, and a differential refractive index detector (RID) is used. The CaCl2·2H2O aqueous solution is used as the mobile phase for isocratic elution; the mobile phase of the high performance liquid chromatography method is a 0.011% to 0.022% CaCl2·2H2O aqueous solution; S4. Test sample analysis Inject dextran 20 and sucrose gradient standard solutions and the test solution, record the chromatograms, and calculate the dextran 20 and sucrose contents in the test solution using the external standard method.

2. The analytical method for determining the content of dextran 20 and sucrose based on HPLC according to claim 1, characterized in that: In step S1, the concentration of the dextran 20 and sucrose standard stock solutions is 10 mg / ml, and the concentrations of the dextran 20 and sucrose gradient standard solutions are 1 mg / ml, 1.5 mg / ml, 2 mg / ml, 2.5 mg / ml and 3 mg / ml, respectively.

3. The analytical method for determining the content of dextran 20 and sucrose based on HPLC according to claim 1, characterized in that: In the steps S1 and S2, a syringe filter is used for filtering, and the pore size of the syringe filter is 0.20-0.45 μm.

4. The analytical method for determining the content of dextran 20 and sucrose based on HPLC according to claim 1, characterized in that: In the step S3, the chromatographic conditions of the high performance liquid chromatography are: column temperature of 50°C to 85°C, flow rate of 0.3ml / min to 0.6ml / min, and detector temperature of 30°C to 55°C.

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