Method for determining content of impurity rhodamine hydrobromide in anisodamine hydrobromide bulk drug

Abstract

The invention provides a method for determining the content of an impurity, namely erythrogallogenine hydrobromide, in an anisodamine hydrobromide raw material medicine. The detection method provided by the invention can be used for effectively controlling the erythrogoumine hydrobromide in the anisodamine hydrobromide bulk drug, so that the erythrogoumine hydrobromide is accurately controlled, and the quality of the anisodamine hydrobromide bulk drug is more effectively ensured.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceuticals, and particularly relates to a method for determining the content of cuscohygrine hydrobromide, an impurity in anisodamine hydrobromide bulk drug. Background Art

[0002] Anisodamine hydrobromide is a compound obtained by extraction, separation, purification and salification from the medicinal material of Scopolia tangutica Maxim., and is an anticholinergic drug that blocks M cholinergic receptors. The effect of anisodamine hydrobromide is similar to or slightly weaker than that of atropine. It has the effects of relaxing smooth muscle, relieving vasospasm, improving microcirculation, and has an analgesic effect. However, its effects of mydriasis and inhibiting glandular secretion are weaker, and it rarely causes central excitatory symptoms. Clinically, it is used for the following diseases: 1. Septic shock; 2. Vascular disorders; 3. Various neuralgias; 4. Smooth muscle spasm; 5. Vertigo; 6. Ocular fundus diseases; 7. Sudden deafness, etc.; 8. It can be used for the treatment of organophosphorus pesticide poisoning.

[0003] Since the medicinal material of Scopolia tangutica Maxim. contains a certain amount of cuscohygrine, cuscohygrine hydrobromide may remain during the preparation of anisodamine hydrobromide. The presence of cuscohygrine hydrobromide will lead to a decrease in the purity of the bulk drug and an increase in impurities, with the risk of introducing adverse reactions. Therefore, it is necessary to quantify cuscohygrine hydrobromide to ensure the quality of the product and improve the safety of drug use.

[0004] Most of the existing methods for detecting cuscohygrine in the medicinal material of Scopolia tangutica Maxim. are thin-layer chromatography methods, which have low sensitivity and poor specificity. Therefore, it has become an urgent problem to provide a detection method that can accurately quantify cuscohygrine hydrobromide in anisodamine hydrobromide bulk drug, thereby effectively controlling the quality of the product and improving the safety of drug use. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for determining the content of cuscohygrine hydrobromide, an impurity in anisodamine hydrobromide bulk drug. The method provided by the present invention is simple and can accurately quantify cuscohygrine hydrobromide, thereby effectively controlling the quality of the product and improving the safety of drug use.

[0006] The present invention provides a method for determining the content of cuscohygrine hydrobromide, an impurity in anisodamine hydrobromide bulk drug, comprising the following steps:

[0007] A) Prepare a test sample solution, a cuscohygrine hydrobromide reference solution and a system suitability solution respectively;

[0008] B) Perform high performance liquid chromatography detection on the test sample solution, the cuscohygrine hydrobromide reference solution and the system suitability solution;

[0009] The conditions for the high performance liquid chromatography (HPLC) detection include: the chromatographic column is selected from octadecylsilyl-bonded silica gel chromatographic columns;

[0010] The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is a 0.03% - 0.1% trifluoroacetic acid solution, and mobile phase B is acetonitrile;

[0011] Gradient elution is adopted, and the elution program is as follows:

[0012] From 0 to 10 min, 95% mobile phase A and 5% mobile phase B; from 10 to 15 min, mobile phase A decreases from 95% to 0, and mobile phase B increases from 5% to 100%; from 15 to 50 min, mobile phase A remains at 0 and mobile phase B remains at 100%;

[0013] C) Calculate the content of cuscohygrine hydrobromide in the test sample according to the peak areas of the test sample solution, cuscohygrine hydrobromide reference substance solution and system suitability solution in the HPLC chromatogram.

[0014] Preferably, the preparation of the test sample solution includes:

[0015] Take the raw material drug of anisodamine hydrobromide to be tested, dissolve and dilute it with a diluent to obtain the test sample solution;

[0016] The diluent is a mixed solution of acetonitrile and water with a volume ratio of (75:25) - (85:15);

[0017] In the test sample solution, the concentration of the raw material drug of anisodamine hydrobromide is 10 - 30 mg / ml.

[0018] Preferably, the preparation of the cuscohygrine hydrobromide reference substance solution includes:

[0019] Dissolve and dilute the cuscohygrine hydrobromide reference substance with a diluent to obtain the cuscohygrine hydrobromide reference substance solution;

[0020] The diluent is a mixed solution of acetonitrile and water with a volume ratio of (75:25) - (85:15);

[0021] In the cuscohygrine hydrobromide reference substance solution, the concentration of the cuscohygrine hydrobromide reference substance is 10 - 30 μg / ml.

[0022] Preferably, the preparation of the system suitability solution includes:

[0023] Dissolve and dilute the raw material drug of anisodamine hydrobromide and the cuscohygrine hydrobromide reference substance with a diluent to obtain the system suitability solution;

[0024] The diluent is a mixed solution of acetonitrile and water with a volume ratio of (75:25) - (85:15);

[0025] In the system suitability solution, the concentration of anisodamine hydrobromide raw material medicine is 10-30 mg / ml, and the concentration of cuscohygrine hydrobromide reference substance is 10-30 μg / ml.

[0026] Preferably, the column temperature for the high performance liquid chromatography detection is 25-35°C, and the drift tube temperature is 35-55°C.

[0027] Preferably, the flow rate of the mobile phase is 0.5-1.5 ml / min.

[0028] Preferably, the detector for the high performance liquid chromatography detection is an evaporative light scattering detector.

[0029] Preferably, the injection volume for the high performance liquid chromatography detection is 10-100 μL.

[0030] Preferably, the chromatographic column is an Agilent ZORBAX SB-Aq chromatographic column with a specification of 250*4.6 mm, 5 μm, or a Shimadzu SymmetryC 18 4.6×250 mm 5 μm.

[0031] Preferably, in step C), according to the peak areas of the test sample solution, cuscohygrine hydrobromide reference substance solution and system suitability solution in the high performance liquid chromatography chart, the content of cuscohygrine hydrobromide in the anisodamine hydrobromide is calculated by the standard curve method of the logarithm of the peak area of cuscohygrine hydrobromide and the concentration.

[0032] Compared with the prior art, the present invention provides a method for determining the content of cuscohygrine hydrobromide, an impurity in anisodamine hydrobromide raw material medicine, comprising the following steps: A) preparing a test sample solution, a cuscohygrine hydrobromide reference substance solution and a system suitability solution respectively; B) performing high performance liquid chromatography (HPLC) detection on the test sample solution, the cuscohygrine hydrobromide reference substance solution and the system suitability solution; the conditions for the HPLC detection include: the chromatographic column is selected from an octadecylsilane bonded silica gel chromatographic column; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is a 0.03% - 0.1% trifluoroacetic acid solution, and mobile phase B is acetonitrile; gradient elution is adopted, and the elution program is: 0 - 10 min, 95% mobile phase A, 5% mobile phase B; 10 - 15 min, mobile phase A decreases from 95% to 0, and mobile phase B increases from 5% to 100%; 15 - 50 min, mobile phase A remains at 0, and mobile phase B remains at 100%; C) calculating the content of cuscohygrine hydrobromide in the test sample according to the peak areas of the test sample solution, the cuscohygrine hydrobromide reference substance solution and the system suitability solution in the HPLC chromatogram. The detection method provided by the present invention can effectively control cuscohygrine hydrobromide in anisodamine hydrobromide raw material medicine, achieve accurate control of cuscohygrine hydrobromide, and more effectively ensure the quality of anisodamine hydrobromide raw material medicine. Description of the Drawings

[0033] Figure 1 The HPLC chromatogram of the blank solution;

[0034] Figure 2 The HPLC chromatogram of the cuscohygrine hydrobromide reference substance solution;

[0035] Figure 3 The HPLC chromatogram of the system suitability solution;

[0036] Figure 4 The HPLC chromatogram of the test solution;

[0037] Figure 5 The HPLC chromatogram of the detection limit of cuscohygrine hydrobromide;

[0038] Figure 6 The linear relationship diagram of cuscohygrine hydrobromide;

[0039] Figure 7 The ultraviolet absorption spectrum of cuscohygrine hydrobromide;

[0040] Figure 8 The HPLC chromatogram of the reference substance solution of Comparative Example 2;

[0041] Figure 9 The HPLC chromatogram of the spiked test solution of Comparative Example 2;

[0042] Figure 10 It is the HPLC chromatogram of the reference solution for Comparative Example 4. Detailed implementation mode

[0043] The present invention provides a method for determining the content of cuscohygrine in anisodamine hydrobromide raw material medicine, which comprises the following steps:

[0044] A) Prepare a test sample solution, a reference solution of cuscohygrine hydrobromide and a system suitability solution respectively;

[0045] B) Perform high performance liquid chromatography detection on the test sample solution, the reference solution of cuscohygrine hydrobromide and the system suitability solution;

[0046] The conditions for the high performance liquid chromatography detection include: the chromatographic column is selected from an octadecylsilane chemically bonded silica gel chromatographic column;

[0047] The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is a 0.03% - 0.1% trifluoroacetic acid solution, and mobile phase B is acetonitrile;

[0048] Gradient elution is adopted, and the elution program is:

[0049] 0 - 10 min, 95% mobile phase A, 5% mobile phase B; 10 - 15 min, mobile phase A decreases from 95% to 0, and mobile phase B increases from 5% to 100%; 15 - 50 min, mobile phase A remains at 0, and mobile phase B remains at 100%;

[0050] C) Calculate the content of cuscohygrine in the test sample according to the peak areas of the test sample solution, the reference solution of cuscohygrine hydrobromide and the system suitability solution in the high performance liquid chromatogram.

[0051] The present invention first prepares a test sample solution, a reference solution of cuscohygrine hydrobromide and a system suitability solution.

[0052] Specifically, the preparation of the test sample solution includes:

[0053] Take the anisodamine hydrobromide raw material medicine to be tested, dissolve and dilute it with a diluent to obtain a test sample solution;

[0054] The diluent is a mixed solution of acetonitrile and water with a volume ratio of (75:25) - (85:15), which can be 75:25, 80:20, 85:15, or any ratio between (75:25) - (85:15);

[0055] In the test sample solution, the concentration of the anisodamine hydrobromide raw material medicine is 10 - 30 mg / ml, which can be 10, 15, 20, 25, 30, or any value between 10 - 30 mg / ml.

[0056] The preparation of the cuscohygrine hydrobromide reference solution includes:

[0057] Dissolve and dilute the cuscohygrine hydrobromide reference substance with a diluent to obtain the cuscohygrine hydrobromide reference solution;

[0058] The diluent is a mixed solution of acetonitrile and water with a volume ratio of (75:25) to (85:15), which can be 75:25, 80:20, 85:15, or any ratio between (75:25) and (85:15);

[0059] In the cuscohygrine hydrobromide reference solution, the concentration of the cuscohygrine hydrobromide reference substance is 10 - 30 μg / ml, which can be 10, 15, 20, 25, 30 μg / ml, or any value between 10 - 30 μg / ml.

[0060] The preparation of the system suitability solution includes:

[0061] Dissolve and dilute the anisodamine hydrobromide raw material drug and the cuscohygrine hydrobromide reference substance with a diluent to obtain the system suitability solution;

[0062] The diluent is a mixed solution of acetonitrile and water with a volume ratio of (75:25) to (85:15), which can be 75:25, 80:20, 85:15, or any ratio between (75:25) and (85:15);

[0063] In the system suitability solution, the concentration of the anisodamine hydrobromide raw material drug is 10 - 30 mg / ml, which can be 10, 15, 20, 25, 30 mg / ml, or any value between 10 - 30 mg / ml, and the concentration of the cuscohygrine hydrobromide reference substance can be 10, 15, 20, 25, 30 μg / ml, or any value between 10 - 30 μg / ml.

[0064] Then, perform high - performance liquid chromatography detection on the test sample solution, the cuscohygrine hydrobromide reference solution, and the system suitability solution;

[0065] The conditions for the high - performance liquid chromatography detection include:

[0066] The chromatographic column is selected from octadecylsilane - bonded silica gel chromatographic columns. In some specific embodiments of the present invention, the chromatographic column 1 is an Agilent ZORBAX SB - Aq chromatographic column, with a specification of 250 * 4.6 mm, 5 μm. The chromatographic column 2 is a Shimadzu SymmetryC 18 4.6×250 mm 5 μm. Preferably, it is the chromatographic column 1.

[0067] The detector for the high - performance liquid chromatography detection is an evaporative light - scattering detector.

[0068] The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is a 0.03% - 0.1% trifluoroacetic acid solution, and mobile phase B is acetonitrile. Among them, the mass concentration of the trifluoroacetic acid solution can be 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, or any value between 0.03% and 0.1%, preferably 0.05%.

[0069] Gradient elution is adopted, and the elution program is as follows:

[0070] From 0 to 10 min, 95% mobile phase A and 5% mobile phase B; from 10 to 15 min, mobile phase A decreases from 95% to 0, and mobile phase B increases from 5% to 100%; from 15 to 50 min, mobile phase A remains at 0 and mobile phase B remains at 100%.

[0071] The column temperature for the high - performance liquid chromatography detection is 25 - 35°C, which can be 25, 30, 35, or any value between 25 - 35°C. The drift tube temperature is 35 - 55°C, which can be 35, 40, 45, 50, 55, or any value between 35 - 55°C.

[0072] The injection volume for the high - performance liquid chromatography detection is 10 - 100 μL, which can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or any value between 10 - 100 μL.

[0073] The flow rate of the mobile phase is 0.5 - 1.5 ml / min, which can be 0.5, 1, 1.5, or any value between 0.5 - 1.5 ml / min.

[0074] According to the peak areas of the test sample solution, cuscohygrine hydrobromide reference solution, and system suitability solution in the high - performance liquid chromatography chart, calculate the content of cuscohygrine in the test sample.

[0075] Specifically, according to the peak areas of the test sample solution, cuscohygrine hydrobromide reference solution, and system suitability solution in the high - performance liquid chromatography chart, calculate the content of cuscohygrine hydrobromide in anisodamine hydrobromide by the standard curve method of the logarithm of the peak area of cuscohygrine hydrobromide and its concentration.

[0076] Among them, the specific method of the standard curve method of the logarithm of the peak area of cuscohygrine hydrobromide and its concentration is as follows: Take the logarithm of the peak area and concentration of the linear cuscohygrine hydrobromide reference solution to make a regression curve. Calculate the regression equation, and the equation r should be greater than 0.98.

[0077] The detection method provided by the present invention can effectively control cuscohygrine in the raw material drug of anisodamine hydrobromide, achieve accurate control of cuscohygrine, and more effectively ensure the quality of the raw material drug of anisodamine hydrobromide.

[0078] To further understand the present invention, the method for determining the content of cuscohygrine in the raw material drug of anisodamine hydrobromide provided by the present invention will be described below in conjunction with embodiments. The protection scope of the present invention is not limited by the following embodiments.

[0079] Example 1

[0080] 1. Instruments and HPLC chromatographic conditions

[0081] Agilent 1260Ⅱ type high performance liquid chromatograph (quaternary pump, degassing unit, column oven, auto sampler); Agilent 1260ⅡInfinity evaporative light scattering detector; Mettler Toledo MS105DU type one in one hundred thousandth analytical balance; Mettler Toledo FE28 type pH meter.

[0082] Chromatographic conditions: The chromatographic column is packed with octadecylsilyl bonded silica gel (Agilent ZORBAX SB-Aq 250*4.6mm, 5μm); The mobile phase includes mobile phase A and mobile phase B. The mobile phase A is 0.05% trifluoroacetic acid solution, and the mobile phase B is acetonitrile. The specific gradient elution program is shown in Table 1; The flow rate is 1.0 ml per minute; The column temperature is 30°C; The drift tube temperature is 40°C.

[0083] The gradient elution program is shown in Table 1

[0084] Table 1 Elution program

[0085] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 95 5 10 95 5 15 0 100 50 0 100

[0086] 2. Materials

[0087] Trifluoroacetic acid (source: Macklin, batch number: C11666012, analytical pure), acetonitrile (source: OCEANPAK, batch number: 22100329G403, chromatographic pure), raw material drug of anisodamine hydrobromide (source: Chengdu No.1 Raw Material Drug Co., Ltd., batch numbers: 20221201, 20230107, 20230108); Cuscohygrine reference substance (source: Chengdu No.1 Raw Material Drug Co., Ltd., batch number: 20200206); Purified water (source: Watson Group Co., Ltd., batch number: 202211119).

[0088] 3. Determination of cuscohygrine in the raw material drug of anisodamine hydrobromide

[0089] S1. The diluent is a mixed solution of acetonitrile and water with a volume ratio of 80:20, i.e., the blank solution.

[0090] S2. Take 10 mg of the control substance of cuscohygrine hydrobromide, place it in a 20-ml volumetric flask, dissolve and dilute it to the mark with the diluent, and shake well to obtain the stock solution of the control substance of cuscohygrine hydrobromide.

[0091] Control solution: Accurately measure 2 ml of the stock solution of the control substance of cuscohygrine hydrobromide, dissolve and dilute it to 50 ml with the diluent to obtain the control solution of the control substance of cuscohygrine hydrobromide (content 20 μg / ml).

[0092] S3. Take 0.2 g of the raw material drug of anisodamine hydrobromide (batch number 20221201), place it in a 10-ml volumetric flask, dissolve and dilute it to the mark with the diluent, and shake well to obtain the test solution.

[0093] S4. Take 0.2 g of the raw material drug of anisodamine hydrobromide (batch number 20221201), place it in a 10-ml volumetric flask, accurately measure 400 μl of the stock solution of the control substance of cuscohygrine hydrobromide, dissolve and dilute it to the mark with the diluent, and shake well to obtain the system suitability solution.

[0094] S5. Using high performance liquid chromatography, inject 20 μl of the blank solution described in step S1, 20 μl of the control solution described in step S2, 20 μl of the test solution described in step S3, and 20 μl of the system suitability solution in step S4 into the liquid chromatograph under the same detection conditions to obtain chromatograms as Figures 1 - 4 shown. Figure 1 The HPLC chromatogram of the blank solution; Figure 2 The HPLC chromatogram of the control solution of the control substance of cuscohygrine hydrobromide; Figure 3 The HPLC chromatogram of the system suitability solution; Figure 4 The HPLC chromatogram of the test solution.

[0095] S6. Determination method: Accurately measure an appropriate amount of the stock solution of the control substance of cuscohygrine hydrobromide, prepare control solutions with concentrations of 10, 15, 20, 25, and 30 μg / ml with 80% acetonitrile. Take the raw material drugs of anisodamine hydrobromide in batches of 20221201, 20230107, and 20230108, and prepare test solutions with a concentration of 0.02 g / ml with 80% acetonitrile. Accurately measure 20 μl of each of the above solutions, inject them into the liquid chromatograph, and record the chromatograms. Draw a regression curve with the logarithm of the concentration of the control substance of cuscohygrine hydrobromide and the peak area, and calculate the regression equation. Substitute the peak area of the test solution into the regression equation to calculate the content of the control substance of cuscohygrine hydrobromide in the raw material drug of anisodamine hydrobromide.

[0096] The test results showed that cuscohygrine was not detected in the three batches of samples of anisodamine hydrobromide with batch numbers 20221201, 20230107, and 20230108.

[0097] 4. Method validation:

[0098] 1). Specificity

[0099] Accurately measure 20 μl of the blank solution described in step S1, 20 μl of the reference solution described in step S2, 20 μl of the test solution described in step S3, and 20 μl of the system suitability solution in step S4 and inject them into the liquid chromatograph respectively. Record the chromatogram. The typical chromatogram is shown in Figures 1 - 4 As shown, it can be seen from the figure that the blank solvent does not interfere with the sample determination, and other chromatographic peaks in the test solution do not interfere with the detection of cuscohygrine.

[0100] 2). Limit of quantitation

[0101] The limit of quantitation (LOQ) is determined according to the signal-to-noise ratio method. Specifically, take the reference stock solution of cuscohygrine at 0.5 mg / ml and dilute it step by step. Detect the signal-to-noise ratio after each dilution until the signal-to-noise ratio approaches 10. This concentration is the limit of quantitation concentration. Six tests were carried out, namely limit of quantitation -1 to limit of quantitation -6. The results are shown in Table 2, and the typical chromatogram is shown in Figure 5 , and the RSD of the peak area of the limit of quantitation concentration solution is 7.76%, which is less than 10%, indicating that the sensitivity of this method is relatively high.

[0102] Table 2 Results of the limit of quantitation of cuscohygrine

[0103] Name Peak Area S / N Quantitation Limit - 1 3.587 14.01 Quantitation Limit - 2 3.444 14.00 Quantitation Limit - 3 3.866 14.84 Quantitation Limit - 4 3.074 14.33 Quantitation Limit - 5 2.968 13.03 Quantitation Limit - 6 3.351 14.69 RSD (%) 7.76 -

[0104] 3). Linearity

[0105] Prepare a reference solution of cuscohygrine with gradient concentrations in the range of 10 - 120 μg / ml. Accurately measure 20 μl of each and inject it into the liquid chromatograph. Record the chromatogram. Make a logarithm of the peak area and concentration and calculate the regression equation. The linearity graph is shown in Figure 6 , for cuscohygrine in the concentration range of 10 - 120 μg / ml, the linear equation is y = 1.6588x - 1.2281, and the correlation coefficient r = 0.998 is greater than 0.995, indicating a good linear correlation between the logarithm of the concentration and the logarithm of the peak area.

[0106] Table 3 Results of the linearity of cuscohygrine hydrobromide

[0107]

[0108] 4). Precision

[0109] 4.1 Intermediate precision

[0110] Inject the reference solution under "specificity" continuously for 6 times, record the retention time and peak area, and calculate the RSD. The results are shown in Table 4. It can be seen that the RSD of the retention time for 6 consecutive determinations is ≤1.0%, and the RSD of the peak area is ≤2.0%, both of which meet the requirements, indicating that the injection precision of this method is good.

[0111] Table 4 Intermediate precision results

[0112] Name Retention Time Peak Area Reference Solution 1 6.277 275.841 Reference Solution 2 6.275 272.627 Reference Solution 3 6.279 283.544 Reference Solution 4 6.273 284.460 Reference Solution 5 6.273 281.508 Reference Solution 6 6.274 282.597 RSD 0.04％ 1.70％

[0113] 4.2 Repeatability

[0114] Preparation of spiked test solution: Take 0.2 g of the test sample (specific batch number 20221201), weigh accurately, add 1 ml of the reference stock solution of cuscohygrine hydrobromide at 0.2 mg / ml, dissolve with the diluent and make up the volume to 10 ml to prepare a solution containing 20 mg of anisodamine hydrobromide raw material per ml as the spiked test solution. (Prepare 6 portions in parallel). Accurately measure 20 μl of each of the above solutions and inject them into the liquid chromatograph, and record the chromatogram. The results are shown in the following table. From the results in the table, it can be seen that the RSD of the determination results of cuscohygrine hydrobromide in 6 samples is ≤10.0%, indicating that the repeatability of this method is good.

[0115] Table 5 Methodology of cuscohygrine hydrobromide - Repeatability

[0116] Name Content (%) Repeatability Solution 1 0.10 Repeatability Solution 2 0.10 Repeatability Solution 3 0.10 Repeatability Solution 4 0.10 Repeatability Solution 5 0.11 Repeatability Solution 6 0.10 RSD 3.6％

[0117] 5). Accuracy

[0118] Select 3 different concentrations of 80%, 100%, and 120%, and prepare 3 test solution portions for each concentration for determination, and evaluate with the determination results of 9 samples.

[0119] S1. Preparation of reference stock solution: Take the reference substance of cuscohygrine hydrobromide, weigh accurately, dissolve and dilute with the diluent to prepare a solution containing about 0.2 mg of cuscohygrine hydrobromide per 1 ml as the reference stock solution.

[0120] S2. 80% accuracy test solution: Take 0.2 g of anisodamine hydrobromide raw material with batch number 20221201, weigh accurately, place it in a 10 ml volumetric flask, add 0.8 ml of the reference stock solution, and then dissolve and dilute with the diluent to the scale to prepare the 80% accuracy test solution (prepare 3 portions in parallel).

[0121] S3. 100% accuracy test solution: Take 0.2 g of anisodamine hydrobromide raw material medicine with batch number 20221201, accurately weigh it, place it in a 10-ml volumetric flask, add 1.0 ml of reference stock solution, then add diluent to dissolve and dilute to the scale, as the 100% accuracy test solution (prepare 3 portions in parallel).

[0122] S4. 120% accuracy test solution: Take 0.2 g of anisodamine hydrobromide raw material medicine with batch number 20221201, accurately weigh it, place it in a 10-ml volumetric flask, add 1.2 ml of reference stock solution, then add diluent to dissolve and dilute to the scale, as the 120% accuracy test solution (prepare 3 portions in parallel).

[0123] Precisely measure 20 μl of each of the above solutions, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Table 6 below. It can be seen from the results in Table 6 that the recovery rate of cuscohygrine is between 80.0% and 120.0%, the average recovery rate is 98.45%, and the RSD is 1.47%, indicating that the accuracy of this method is good.

[0124] Table 6 Accuracy result table

[0125]

[0126] Example 2

[0127] Under the conditions of the determination method provided in Example 1, adjust the chromatographic conditions and examine the durability of the method. The durability chromatographic conditions are examined as shown in Table 7.

[0128] Table 7

[0129]

[0130] Chromatographic column 2 is: Shimadzu SymmetryC 18 4.6×250 mm 5 μm.

[0131] Solution preparation:

[0132] Blank solution: Diluent.

[0133] Reference stock solution: Take an appropriate amount of cuscohygrine hydrobromide, accurately weigh it, dissolve it with diluent and prepare a solution containing about 0.2 mg of cuscohygrine per 1 ml.

[0134] Reference solution: Precisely measure an appropriate amount of the reference stock solution, add diluent to prepare a solution containing about 20 μg of cuscohygrine per 1 ml.

[0135] Test solution: Take 0.2 g of anisodamine hydrobromide raw material medicine (batch number 20221201), accurately weigh it, dissolve it with diluent and prepare a solution containing about 20 mg of anisodamine hydrobromide per 1 ml.

[0136] Spiked test solution: Take 0.2 g of anisodamine hydrobromide raw material, accurately weigh it, place it in a 10-ml volumetric flask, accurately measure 1 ml of the reference stock solution and add it, dissolve it with the diluent and make up the volume to the mark.

[0137] Under each chromatographic condition, take the blank solution, reference solution, test solution, and spiked test solution, accurately pipette 20 μl respectively, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in the following table. From the results, after adjusting the chromatographic conditions, the blank solution does not interfere with the determination, and the recovery rate of the spiked test solution is between 92% and 108%, indicating that the method has good durability.

[0138] Table 8

[0139]

[0140] Comparative Example 1

[0141] Take 10 mg of cuscohygrine reference substance, dissolve it with methanol to prepare a solution containing 1 mg per 1 ml as the stock solution for standby. Measure an appropriate amount of the stock solution and dilute it with methanol to prepare a series of cuscohygrine reference solutions with different concentrations (0.05 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.5 mg / ml). Take the above reference solutions, use methanol as the blank, and perform absorbance scanning with a UV spectrophotometer at 190 nm to 800 nm. See Figure 7 , and the results show that cuscohygrine has no obvious UV absorption at 190 nm to 800 nm.

[0142] Comparative Example 2

[0143] On the basis of Example 1, change the chromatographic conditions:

[0144] Chromatographic condition 1: Use 10 mmol / L sodium acetate - acetonitrile (volume ratio 90:10) as the mobile phase, column temperature: 30 °C, mobile phase flow rate 1.0 ml / min, the detector is an evaporative light scattering detector, and the drift tube temperature is 40 °C.

[0145] Diluent: Mobile phase.

[0146] Blank solution: Diluent.

[0147] Reference stock solution: Take an appropriate amount of cuscohygrine hydrobromide, accurately weigh it, dissolve it with the diluent and prepare a solution containing about 0.2 mg / ml of cuscohygrine per 1 ml.

[0148] Reference solution: Accurately measure an appropriate amount of the reference stock solution, add the diluent to prepare a solution containing about 20 μg of cuscohygrine per 1 ml.

[0149] Test solution: Weigh accurately 0.2 g of anisodamine hydrobromide raw material drug (batch number 20221201), dissolve it with a diluent, and prepare a solution containing about 20 mg of anisodamine hydrobromide per 1 ml.

[0150] Spiked test solution: Weigh accurately 0.2 g of anisodamine hydrobromide, place it in a 10-ml volumetric flask, accurately pipette 1 ml of the reference stock solution, add it, dissolve it with a diluent, and make up the volume to the mark.

[0151] The results are shown in Figure 8 : The retention time of cuscohygrine is about 2 min, the peak elutes too fast and the chromatographic peak bifurcates.

[0152] Comparative Example 3

[0153] On the basis of Example 1, change the chromatographic conditions:

[0154] Chromatographic condition 2: Using octadecylsilyl as the stationary phase, 10 mmol / L sodium acetate as mobile phase A, and acetonitrile as mobile phase B, perform gradient elution according to the following table. The flow rate of the mobile phase is 1.0 ml / min, the column temperature is 30 °C, the detector is an evaporative light scattering detector, and the drift tube temperature is 40 °C.

[0155] Table 9

[0156] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 95 5 10 95 5 11 0 100 40 0 100 41 95 5 50 95 5

[0157] Diluent: Mobile phase A.

[0158] Blank solution: Diluent.

[0159] Reference stock solution: Weigh accurately an appropriate amount of cuscohygrine hydrobromide, dissolve it with a diluent, and prepare a solution containing about 0.2 mg / ml of cuscohygrine per 1 ml.

[0160] Reference solution: Accurately pipette an appropriate amount of the reference stock solution, add a diluent to prepare a solution containing about 20 μg of cuscohygrine per 1 ml.

[0161] Test solution: Weigh accurately 0.2 g of anisodamine hydrobromide raw material drug (batch number 20221201), dissolve it with a diluent, and prepare a solution containing about 20 mg of anisodamine hydrobromide per 1 ml.

[0162] Spiked test solution: Weigh accurately 0.2 g of anisodamine hydrobromide, place it in a 10-ml volumetric flask, accurately pipette 1 ml of the reference stock solution, add it, dissolve it with a diluent, and make up the volume to the mark.

[0163] The results are shown in Figure 9:The retention time of cuscohygrine is about 5 min. The main component (anisodamine hydrobromide) can be completely eluted within 50 min of collection, but the chromatographic peak of cuscohygrine is split, and the solvent peak interferes with the chromatographic peak of cuscohygrine.

[0164] Comparative Example 4

[0165] On the basis of Example 1, the mobile phase was changed. Chromatographic condition 1: The mobile phase was water - acetonitrile (volume ratio 90∶10), the column temperature was 30 °C, the flow rate of the mobile phase was 1.0 ml / min, the detector was an evaporative light scattering detector, and the drift tube temperature was 40 °C.

[0166] Diluent: Mobile phase.

[0167] Blank solution: Diluent.

[0168] Stock solution of reference substance: Take an appropriate amount of cuscohygrine hydrobromide, weigh accurately, dissolve it with the diluent and prepare a solution containing about 0.2 mg / ml of cuscohygrine in each 1 ml.

[0169] Reference substance solution: Accurately measure an appropriate amount of the stock solution of reference substance, add the diluent to prepare a solution containing about 20 μg of cuscohygrine in each 1 ml.

[0170] Results: See Figure 10, Figure 10 which is the chromatogram of the reference substance solution of Comparative Example 4. It can be seen from Figure 10 that the elution of cuscohygrine hydrobromide is too early.

[0171] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for determining the content of cuscohygrine hydrobromide, an impurity in anisodamine hydrobromide bulk drug, which is characterized in that, It includes the following steps: A) Prepare a test sample solution, a scopolamine hydrobromide reference solution, and a system suitability solution respectively; B) Perform high performance liquid chromatography (HPLC) on the test sample solution, the scopolamine hydrobromide reference solution, and the system suitability solution; The conditions for the HPLC include: the chromatographic column is selected from octadecylsilane bonded silica gel chromatographic columns; The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is a 0.03% - 0.1% trifluoroacetic acid solution, and mobile phase B is acetonitrile; Gradient elution is adopted, and the elution program is: 0 - 10 min, 95% mobile phase A, 5% mobile phase B; 10 - 15 min, mobile phase A decreases from 95% to 0, and mobile phase B increases from 5% to 100%; 15 - 50 min, mobile phase A remains at 0, and mobile phase B remains at 100%; C) Calculate the content of scopolamine hydrobromide in the test sample according to the peak areas of the test sample solution, the scopolamine hydrobromide reference solution, and the system suitability solution in the HPLC chromatogram.

2. The method according to claim 1, wherein The preparation of the test sample solution includes: Take the scopolamine hydrobromide raw material to be tested, dissolve and dilute it with a diluent to obtain the test sample solution; The diluent is a mixed solution of acetonitrile and water with a volume ratio of (75:25) - (85:15); In the test sample solution, the concentration of the scopolamine hydrobromide raw material is 10 - 30 mg / ml.

3. The method according to claim 1, wherein The preparation of the scopolamine hydrobromide reference solution includes: Dissolve and dilute the scopolamine hydrobromide reference with a diluent to obtain the scopolamine hydrobromide reference solution; The diluent is a mixed solution of acetonitrile and water with a volume ratio of (75:25) - (85:15); In the scopolamine hydrobromide reference solution, the concentration of the scopolamine hydrobromide reference is 10 - 30 μg / ml.

4. The method according to claim 1, characterized in that The preparation of the system suitability solution includes: Dissolve and dilute the scopolamine hydrobromide raw material and the scopolamine hydrobromide reference with a diluent to obtain the system suitability solution; The diluent is a mixed solution of acetonitrile and water with a volume ratio of (75:25) - (85:15); In the system suitability solution, the concentration of the scopolamine hydrobromide raw material is 10 - 30 mg / ml, and the concentration of the scopolamine hydrobromide reference is 10 - 30 μg / ml.

5. The method according to claim 1, characterized in that, The column temperature for the HPLC is 25 - 35 °C, and the drift tube temperature is 35 - 55 °C.

6. The method according to claim 1, wherein The flow rate of the mobile phase is 0.5 - 1.5 ml / min.

7. The method according to claim 1, wherein The detector for the HPLC is an evaporative light scattering detector.

8. The method according to claim 1, wherein The injection volume for the HPLC is 10 - 100 μL.

9. The method according to claim 1, characterized in that, The chromatographic column is an Agilent ZORBAX SB-Aq chromatographic column with a specification of 250*4.6mm, 5μm, or a Shimadzu SymmetryC 18 4.6×250mm 5μm.

10. The method according to claim 1, characterized in that, In step C), according to the peak areas of the test sample solution, the scopolamine hydrobromide reference solution, and the system suitability solution in the HPLC chromatogram, calculate the content of scopolamine hydrobromide in the scopolamine hydrobromide by the standard curve method of the logarithm of the peak area and concentration of scopolamine hydrobromide.

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