Detection method of galanthamine hydrobromide injection enantiomer

Abstract

The invention relates to the technical field of chemical detection analysis, and discloses a detection method of galanthamine hydrobromide injection enantiomer, and a high performance liquid chromatography analysis method provided by the invention adopts a Daexid CHIRALPAK AGP chromatographic column, takes an acetate aqueous solution as a mobile phase A, takes acetonitrile as a mobile phase B, and adopts an isocratic elution mode. The method is high in specificity, wide in application range, good in durability, suitable for large-scale detection of products and capable of saving cost, the enantiomer content in a detection sample can be more accurately calculated through an external standard method, and the method is rapid, simple, convenient, good in specificity, high in sensitivity, wide in application range, low in cost and suitable for application and popularization.

Description

Technical Field

[0001] The invention relates to the technical field of chemical detection and analysis, and in particular to a method for detecting enantiomers of galanthamine hydrobromide injection. Background Art

[0002] Galantamine hydrobromide is a cholinesterase inhibitor that can pass through the blood-brain barrier and antagonize non-depolarizing muscle relaxants. It also has a direct excitatory effect on the N2 cholinergic receptors on the motor end plates, can improve neuromuscular conduction, and has a certain central cholinergic effect. It has intrinsic activity on nicotinic receptors and is mainly used to treat mild to moderately severe dementia in Alzheimer's disease.

[0003] Galantamine hydrobromide Chemical name: (4aS,6R,8aS)-11-methyl-3-methoxy-4a,5,9,10,11,12-hexahydro-6H-benzofurano[3a,3,2-ef][2]benzazepine -6-ol hydrobromide; CAS number: 1953-04-4; molecular formula: C 17 H 21 NO3·HBr; the structural formula is as follows:

[0004]

[0005] Galantamine hydrobromide enantiomers (4R, 6S, 8R isomers) Chemical name: (4aR, 6S, 8aR)-3-methoxy-11-methyl-5,6,9,10,11,12-hexahydro-4aH-[1]benzofurano[3a,3,2-ef]benzazepine -6-ol; CAS number: 60384-53-4; molecular formula: C 17 H 21 NO3; the structural formula is as shown in Formula 2:

[0006]

[0007] Enantiomers may be produced during the synthesis of compounds or during storage. Enantiomers are very important in pharmaceutical chemistry because many drug molecules are optically active, and left-handed and right-handed drugs may have different efficacy. For some drugs, only one optical isomer is effective, while the other may be ineffective or even harmful, so strict control is required in sample testing.

[0008] As degradable impurities of galanthamine hydrobromide, the degradable impurities must be strictly controlled during the storage process of the product. Therefore, the monitoring of galanthamine hydrobromide enantiomers is of great significance for the quality control of galanthamine hydrobromide related preparations.

[0009] In the prior art, there are literature reports on methods for detecting galanthamine enantiomers, most of which use capillary electrophoresis. However, the detection method is not comprehensive, and the calculation method cannot more accurately quantify the content of the enantiomers, and the quantitative limit and detection limit of galanthamine and the enantiomers cannot be verified. Summary of the invention

[0010] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for detecting enantiomers of galanthamine hydrobromide injection. The detection method provided by the present invention can identify the main component and the enantiomers, and can accurately and quantitatively detect the content of the galanthamine enantiomers.

[0011] In order to achieve the above object, the present invention adopts the following technical scheme:

[0012] A method for detecting enantiomers of galantamine hydrobromide injection comprises the following steps:

[0013] S1. Preparation of system suitability solution: Mix the galanthamine hydrobromide racemate reference substance and deionized water to prepare a solution containing 2.0 μg of galanthamine hydrobromide racemate per 1 mL (wherein the content of 4R, 6S, 8R isomers is 1.0 μg / mL) as the system suitability solution;

[0014] S2. Preparation of the test solution: Mix the galanthamine hydrobromide injection to be tested with deionized water to prepare a solution containing 1.0 mg of galanthamine hydrobromide per 1 mL as the test solution;

[0015] S3. Preparation of reference solution: accurately weigh 10 mg of galanthamine hydrobromide 4R, 6S, 8R isomer reference substance, dissolve it in deionized water, place it in a 100 mL volumetric flask, dilute it to the mark with deionized water, shake well, then accurately measure 1 mL of this solution, place it in a 100 mL volumetric flask, dilute it to the mark with deionized water, shake well, and use it as the reference solution;

[0016] S4. Liquid chromatography detection of enantiomers: Take 5 μl of the system suitability solution and inject it into the liquid chromatograph, record the chromatogram, when the mutual separation of the enantiomer chromatographic peaks and the main peak in the chromatogram is not less than 4.5, accurately measure 5 μl of the reference solution and the test solution and inject them into the liquid chromatograph, record the chromatogram, if there is a chromatographic peak in the chromatogram of the test solution that is consistent with the retention time of the enantiomer, calculate the enantiomer content by peak area according to the reference external standard method, the enantiomer content limit range ≤ 0.10%, the specific calculation formula of the enantiomer content is as follows:

[0017]

[0018] A 样: The average peak area of ​​enantiomers in the test solution;

[0019] A 对 : Average peak area of ​​reference solution;

[0020] C 对 : Concentration of reference solution, mg / mL;

[0021] C 样 : Concentration of test solution, mg / mL;

[0022] ω 对 : Enantiomeric purity of reference substance.

[0023] As a preferred technical solution of the present invention, in step S4, the mobile phase A used in the liquid chromatography detection is an acetate solution, and the mobile phase B is acetonitrile.

[0024] As a preferred technical solution of the present invention, the concentration of the acetate solution is 0.1 mol / L, and the pH value is adjusted to 6.3-6.7 with glacial acetic acid.

[0025] As a preferred technical solution of the present invention, the volume ratio of acetate solution to acetonitrile is 95-98:2-5, for example, 95:5, 96:4, 97:3, 98:2 can be selected, but it is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0026] Wherein, the acetate solution is selected from ammonium acetate solution, sodium acetate solution or potassium acetate solution.

[0027] As a preferred technical solution of the present invention, in step S4, the chromatographic conditions of the liquid chromatograph are: using a CHIRALPAK AGP chromatographic column with a specification of 150×4.6 mm and a filler particle size of 5 μm; a column temperature of 15-25°C, isocratic elution, and a flow rate of 0.2-0.6 mL / min; and using an ultraviolet detector as the detector with a detection wavelength of 228 nm.

[0028] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0029] The high performance liquid chromatography analysis method provided by the present invention adopts a CHIRALPAK AGP chromatographic column, an acetate aqueous solution as a mobile phase A, acetonitrile as a mobile phase B, and an isocratic elution mode; the method has strong specificity, a wide range of application, good durability, is suitable for large-scale detection of products, saves costs, and can more accurately calculate the enantiomer content in the detection sample by an external standard method. The method provided by the present invention is fast and simple, has good specificity, high sensitivity, a wide range of application, low cost, and is suitable for popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a chromatogram of a blank solution using chromatographic column 1 in Example 1 of the present invention;

[0031] Figure 2 This is a solution chromatogram of the system suitability of the chromatographic column 1 used in Example 1 of the present invention;

[0032] Figure 3 The chromatogram of the reference substance solution using chromatographic column 1 in Example 1 of the present invention;

[0033] Figure 4 The chromatogram of the test solution using chromatographic column 1 in Example 1 of the present invention;

[0034] Figure 5 is the linear graph of galanthamine in Example 3 of the present invention;

[0035] Figure 6 It is the linear diagram of enantiomers in Example 3 of the present invention. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0037] It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0038] The galanthamine hydrobromide injection test sample (specification 1 mL: 2.5 mg) used in the present invention was provided by Hubei Minkang Pharmaceutical Co., Ltd. and Shanghai Xudong Haipu Pharmaceutical Co., Ltd.; the enantiomer reference substance (i.e., 4R, 6S, 8R isomers) with a purity of 96.35% was provided by Shenzhen Jianyi Biotechnology Co., Ltd.; the racemic reference substance, batch number: R10970, was sourced from USP.

[0039] Example 1

[0040] This example screens the chromatographic columns for the provided method.

[0041] Experimental conditions:

[0042] Equipment: High performance liquid chromatography (HPLC).

[0043] Column 1: CHIRALPAK AGP, 150×4.0 mm, 5 μm; PN: 30714;

[0044] Column 2: CHIRALPAK OJ-RH, 150×4.6 mm, 5 μm; PN: 17724;

[0045] Column 3: CROWNPAK CR(+), 150×4.0 mm, 5 μm; PN: 27714;

[0046] Detection conditions: The detector is an ultraviolet detector, the detection wavelength is 228nm; the column temperature is 25℃; the injection time is 70min; the injection volume is 5μl; the flow rate is 0.2mL / min; and the elution is isocratic.

[0047] Preparation of mobile phase A: Accurately weigh 7.7 g of ammonium acetate in a 2 L beaker, add 1 L of Wahaha purified water to dissolve, and adjust the pH to 6.5 with glacial acetic acid.

[0048] Mobile phase B: acetonitrile.

[0049] The volume ratio of mobile phase A to mobile phase B was 95:5.

[0050] Blank solution: deionized water.

[0051] Preparation of system suitability solution: Mix the galanthamine hydrobromide racemate reference substance and deionized water to prepare a solution containing 2.0 μg of galanthamine hydrobromide racemate per 1 mL, wherein the content of 4R, 6S, 8R isomers is 1.0 μg / mL, which is used as the system suitability solution.

[0052] Preparation of test solution: Mix the galanthamine hydrobromide injection test sample and deionized water to prepare a solution containing 1.0 mg of galanthamine hydrobromide per 1 mL.

[0053] Preparation of reference solution: Accurately weigh 10 mg of enantiomer (4R, 6S, 8R isomer) reference substance, dissolve it in deionized water, place it in a 100 mL volumetric flask, dilute it to the mark with deionized water, shake well, then accurately measure 1 mL of this solution, place it in a 100 mL volumetric flask, dilute it to the mark with deionized water, shake well, and use it as the reference solution.

[0054] Determination method: respectively measure blank solution, system suitability solution, reference solution, and test solution and inject them into the liquid chromatograph using chromatographic column 1, and record the chromatogram. The chromatogram of the blank solution is as follows: Figure 1 The system suitability solution chromatogram is shown in Figure 2 As shown, the chromatogram of the reference solution is Figure 3 As shown, the chromatogram of the test solution is Figure 4 shown.

[0055] Determination method: respectively measure the system suitability solution, reference solution and test solution and inject them into the liquid chromatograph using chromatographic column 2, and record the relevant data.

[0056] Determination method: respectively measure the system suitability solution, reference solution and test solution and inject them into the liquid chromatograph using chromatographic column 3, and record the relevant data.

[0057] The data related to galanthamine and enantiomers in the system suitability solution measured using chromatographic columns 1-3 are shown in Table 1 "Result statistics of chromatographic columns 1-3".

[0058] Table 1 Results of Columns 1-3 (System Suitability Solution)

[0059]

[0060] Conclusion: Under the condition of chromatographic column 2, the peak shape of galanthamine and enantiomers is poor and the separation is low; under the condition of chromatographic column 3, neither galanthamine nor enantiomer peaks appear. It can be seen that not all chromatographic columns are suitable for the detection of galanthamine hydrobromide enantiomers, so the present invention selects chromatographic column 1.

[0061] Example 2

[0062] This example screens the flow rate, column temperature and different mobile phases of the provided method. The chromatographic column selected is CHIRALPAK AGP, 150×4.0 mm, 5 μm, and the other experimental conditions are consistent with those of Example 1.

[0063] Select the chromatographic conditions in Example 1, and accurately measure the system suitability solution and inject it into the liquid chromatograph.

[0064] Determination method: The data related to galanthamine and enantiomers measured using different flow rates are shown in Table 2 "Statistics of results at different flow rates".

[0065] Table 2 Statistics of results at different flow rates

[0066]

[0067] When the flow rates are 0.6mL / min, 0.5mL / min, 0.4mL / min, 0.3mL / min, and 0.2mL / min, respectively, the separation degrees of the enantiomer peak and the galanthamine peak in the system suitability solution are all greater than 4.5; when used at a column pressure of 9Mpa (90bar), it can protect the column and extend its service life. When the flow rate is 0.6mL / min, the column pressure has reached about 90bar. When the flow rate becomes smaller, the enantiomer peak response value is large. After comprehensive consideration, the flow rate is preferably 0.2mL / min.

[0068] Determination method: The data related to galanthamine and enantiomers measured using different column temperatures are shown in Table 3 "Statistics of results at different column temperatures".

[0069] Table 3 Statistics of results at different column temperatures

[0070]

[0071] When the column temperature is between 15°C and 25°C, the separation degrees of the enantiomer peaks and the galanthamine peaks in the system suitability solution are greater than 4.5. At different column temperatures, the peak areas of the enantiomers and galanthamine in the system suitability solution are basically unchanged. The recommended operating temperature of the chromatographic column is 20°C to 25°C. Considering the applicable range of column temperatures of different instruments, the column temperature is preferably 25°C.

[0072] Determination method: The data related to galanthamine and enantiomers measured using different mobile phases are shown in Table 4 "Statistics of results of different mobile phases".

[0073] Table 4: Statistics of results of different mobile phases

[0074]

[0075] When the mobile phase buffer salt is ammonium formate:acetonitrile, the enantiomers in the system suitability solution have peaks, but the peak shape is poor and the separation is poor; when the mobile phase buffer salt is sodium dihydrogen phosphate:acetonitrile, the enantiomers in the system suitability solution do not have peaks, and this method is not applicable; when the mobile phase buffer salt is ammonium acetate:acetonitrile, the enantiomers in the system suitability solution have peaks, the peak shape is good, and the separation is good, so the present invention selects ammonium acetate:acetonitrile as the mobile phase.

[0076] Example 3

[0077] This example verifies the method provided in Example 1 in terms of system applicability, specificity, linearity, limit of quantitation, limit of detection, and stability of the test solution.

[0078] 1) Exclusivity

[0079] Acceptable criteria: The retention time of the main component peak of the system suitability solution and the test solution is consistent with the retention time of the main component peak of the reference solution, and the peak purity of each peak is ≥990. The results are shown in Table 4:

[0080] Table 4 Specificity test results

[0081]

[0082] The blank solvent had no interference in the enantiomer determination, which met the requirements. In the system suitability solution chromatogram, the separation between the galanthamine peak and the enantiomer peak was 10.95, the peak purity of each peak was >990, and the retention time of each peak was consistent with the retention time of each reference solution and the test solution, which met the requirements.

[0083] 2) System applicability

[0084] Acceptable criteria: the separation between the galanthamine peak and the enantiomer peak in the system suitability solution is not less than 4.5, and the RSD of the enantiomer peak area in 6 consecutive injections of the system suitability solution is not greater than 5.0%. The test results are shown in Table 5.

[0085] Table 5 System applicability experimental results

[0086]

[0087] SST-1 to SST-6 represent 6 consecutive injections of system suitability solutions, among which the RSD of the enantiomer peak area meets the acceptance standard of no more than 5.0%; the separation between the enantiomer and galantamine peaks meets the acceptance standard of no less than 4.5; therefore, the system suitability results meet the regulations.

[0088] 3) Linear

[0089] Galantamine linearity test:

[0090] Acceptable criterion: regression coefficient is not less than 0.999.

[0091] Verification results: Galantamine showed good linearity in the range of 0.41 to 1219.50 μg / mL. Figure 5 And as shown in Table 6.

[0092] Table 6 Summary of Galantamine Linearity Experiment Results

[0093]

[0094]

[0095] Enantiomeric linearity experiment:

[0096] Acceptable criterion: regression coefficient is not less than 0.999.

[0097] Verification results: The enantiomers showed good linearity in the range of 0.25-1.51 μg / mL. Figure 6 And as shown in Table 7.

[0098] Table 7 Summary of enantiomer linearity test results

[0099]

[0100] 4) Limit of quantification and limit of detection

[0101] The limit of quantitation is the lowest amount of the analyte in the test sample that can be measured with acceptable precision and accuracy under specified experimental conditions.

[0102] The quantitative limit of this application is demonstrated by analyzing six parallel prepared quantitative limit solutions. The signal-to-noise ratio of the enantiomers and galanthamine in the six quantitative limit solutions should be no less than 10.

[0103] The detection limit refers to the lowest amount of the analyte that can be detected in the test sample under specified test conditions, which does not necessarily require quantitative determination.

[0104] The results of the galanthamine limit of quantitation (LOQ) precision test are shown in Table 8.

[0105] Table 8 Galantamine LOQ precision test results

[0106]

[0107] The results of the precision test of the limit of detection (LOD) of galanthamine are shown in Table 9.

[0108] Table 9 Galantamine detection limit (LOD) precision test results

[0109]

[0110] The quantification limit of galanthamine is 0.41 μg / mL, equivalent to 0.04% of the test solution concentration, the signal-to-noise ratio of the 6-pin quantification limit solution is in the range of 10.1-19.9, the peak area RSD is 3.8%, and the retention time RSD is 0.32%, which meets the requirements. The detection limit of galanthamine is 0.20 μg / mL, equivalent to 0.02% of the test solution concentration, and the signal-to-noise ratio is 3.1, which meets the requirements.

[0111] The results of the enantiomer quantification limit (LOQ) precision test are shown in Table 10.

[0112] Table 10 Enantiomer quantification limit (LOQ) precision test results

[0113]

[0114]

[0115] The results of the enantiomer detection limit (LOD) precision test are shown in Table 11.

[0116] Table 11 Enantiomer detection limit (LOD) precision test results

[0117]

[0118] The quantitative limit concentration of the enantiomer is 0.25μg / mL, equivalent to 0.03% of the test solution concentration, the signal-to-noise ratio of the 6-pin quantitative limit solution is in the range of 12.2-15.8, the peak area RSD is 8.5%, and the retention time RSD is 0.19%, which meets the requirements. The detection limit concentration of 4R, 6S, 8R isomers is 0.13μg / mL, equivalent to 0.01% of the test solution concentration, and the signal-to-noise ratio is 3.0, which meets the requirements.

[0119] 5) Stability of the test solution

[0120] The enantiomer reference solution, system suitability solution, and test solution were placed for a certain period of time, and the galanthamine peak and enantiomer peak area RSD at each time point were detected to be ≤5.0%. The test results are shown in Table 12.

[0121] Table 12 Test solution stability test results

[0122]

[0123] After the enantiomer reference solution was placed for 24 hours, the RSD of the peak area of ​​4R, 6S, and 8R isomers was 2.6%; after the system suitability solution was placed at room temperature for 24 hours, the RSD of the peak area of ​​galanthamine was 3.1%, and the RSD of the peak area of ​​4R, 6S, and 8R isomers was 1.4%, and the separation degrees were all greater than 4.5; after the test solution was placed at room temperature for 24 hours, the RSD of the peak area of ​​galanthamine was 0.70%, indicating that the system suitability solution, reference solution, and test solution had good stability after being placed at room temperature for 24 hours.

[0124] Example 4

[0125] This example tests the accuracy of the calculation results of the external standard method, and the specific test steps are as follows:

[0126] S1. Preparation of system suitability solution: Mix the galanthamine hydrobromide racemate reference substance and deionized water to prepare a solution containing 2.0 μg of galanthamine hydrobromide racemate per 1 mL, wherein the content of 4R, 6S, 8R isomers is 1.0 μg / mL, as the system suitability solution;

[0127] S2. Preparation of reference substance stock solution: accurately weigh 10 mg of galanthamine hydrobromide 4R, 6S, 8R isomer reference substance, dissolve it in deionized water, place it in a 100 mL volumetric flask, dilute it to the mark with deionized water, shake well, then accurately measure 10 mL of this solution, place it in a 100 mL volumetric flask, dilute it to the mark with deionized water, shake well, and use it as the reference substance stock solution;

[0128] S3. Preparation of reference solution: Accurately measure 1 mL of the reference stock solution, place it in a 100 mL volumetric flask, dilute to the mark with deionized water, shake well, and use it as the reference solution;

[0129] S4. Preparation of test sample spike solution: Accurately measure 4 mL of galanthamine hydrobromide injection to be tested and 1 mL of reference stock solution, place them in the same 10 mL volumetric flask, dilute to the mark with deionized water, and prepare a solution containing 1.0 mg of galanthamine hydrobromide and 1.0 μg of 4R, 6S, 8R isomers per 1 mL as the test sample spike solution;

[0130] The experimental conditions in Example 1 were used for detection, wherein the chromatographic column selected was chromatographic column 1: CHIRALPAK AGP, 150×4.0 mm, 5 μm; PN: 30714;

[0131] The test results are shown in Table 13.

[0132] Table 13 External standard method accuracy test results

[0133]

[0134] Finally, it should be noted that the above embodiments do not limit the present invention in any form. For those skilled in the art, some modifications and improvements can be made to the present invention. Therefore, any modification or improvement made without departing from the spirit of the present invention belongs to the scope of protection claimed in the present invention.

Claims

1. A method for detecting enantiomers of galantamine hydrobromide injection, characterized in that: The steps include: S1. Preparation of system suitability solution: Mix the galanthamine hydrobromide racemate reference substance and deionized water to prepare a solution containing 2.0 μg of galanthamine hydrobromide racemate per 1 ml, which is used as the system suitability solution; S2. Preparation of the test solution: Mix the galanthamine hydrobromide injection to be tested with deionized water to prepare a solution containing 1.0 mg of galanthamine hydrobromide per 1 ml as the test solution; S3. Preparation of reference solution: accurately weigh 10 mg of galanthamine hydrobromide 4R, 6S, 8R isomer reference substance, dissolve it in deionized water, place it in a 100 mL volumetric flask, dilute it to the mark with deionized water, shake well, then accurately measure 1 mL of this solution, place it in a 100 mL volumetric flask, dilute it to the mark with deionized water, shake well, and use it as the reference solution; S4. Liquid chromatography detection of enantiomers: Take 5 μl of the system suitability solution and inject it into the liquid chromatograph, record the chromatogram, when the mutual separation of the enantiomer chromatographic peaks and the main peak in the chromatogram is not less than 4.5, accurately measure 5 μl of the reference solution and the test solution and inject them into the liquid chromatograph, record the chromatogram, if there is a chromatographic peak in the chromatogram of the test solution that is consistent with the retention time of the enantiomer, calculate the enantiomer content by peak area according to the reference external standard method, the enantiomer content limit range ≤ 0.10%, the specific calculation formula of the enantiomer content is as follows: A 样 : The average peak area of ​​enantiomers in the test solution; A 对 : Average peak area of ​​reference solution; C 对 : Concentration of reference solution, mg / mL; C 样 : Concentration of test solution, mg / mL; ω 对 : Enantiomeric purity of reference substance.

2. The detection method according to claim 1, characterized in that In step S4, the mobile phase A used in the liquid chromatography detection is an acetate solution, and the mobile phase B is acetonitrile.

3. The detection method according to claim 2, characterized in that: The concentration of the acetate solution was 0.1 mol / L, and the pH was adjusted to 6.3-6.7 with glacial acetic acid.

4. The detection method according to claim 2, characterized in that: The volume ratio of acetate solution to acetonitrile is 95-98:2-5.

5. The detection method according to claim 2, characterized in that: The acetate solution is selected from ammonium acetate solution, sodium acetate solution or potassium acetate solution.

6. The detection method according to claim 1, characterized in that: In step S4, the chromatographic conditions of the liquid chromatograph are: using a CHIRALPAK AGP chromatographic column with a specification of 150×4.6 mm and a filler particle size of 5 μm; a column temperature of 15-25° C., isocratic elution, and a flow rate of 0.2-0.6 mL / min; and using an ultraviolet detector with a detection wavelength of 228 nm.

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