CN116297922A - Method for determining isomer in berberine hydrochloride - Google Patents

Method for determining isomer in berberine hydrochloride Download PDF

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CN116297922A
CN116297922A CN202310139694.6A CN202310139694A CN116297922A CN 116297922 A CN116297922 A CN 116297922A CN 202310139694 A CN202310139694 A CN 202310139694A CN 116297922 A CN116297922 A CN 116297922A
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solution
mobile phase
berberine hydrochloride
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chromatographic column
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刘玲艳
王勇军
陶忠保
周慧琴
王彬
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Changzhou Yabang Pharmaceutical Co Ltd
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Changzhou Yabang Pharmaceutical Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
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Abstract

The invention relates to the technical field of chemical analysis, and discloses a method for detecting isomers in berberine hydrochloride. The method utilizes high performance liquid chromatography, adopts a chromatographic column with octadecylsilane chemically bonded silica as a filler, wherein the mobile phase A is formic acid aqueous solution with the volume fraction of 0.1 percent, the mobile phase B is methanol solution, the flow rate of the mobile phase is 0.4 mL/min-0.5 mL/min, gradient elution is adopted, a detector is an ultraviolet absorption detector, the detection wavelength is 226 nm-230 nm, the sample injection amount is 10-50 mu L, and the column temperature of the chromatographic column is 38-42 ℃. The method has high sensitivity, good repeatability, accurate content measurement result and good specificity and linearity, and can be used for quality control of isomers in berberine hydrochloride.

Description

Method for determining isomer in berberine hydrochloride
Technical Field
The invention belongs to the field of analytical chemistry, and relates to a method for detecting isomers in berberine hydrochloride, which provides a basis for formulating quality standards of berberine hydrochloride bulk drugs, thereby improving the safety quality of the drugs.
Background
Berberine hydrochloride is western medicine, and the real medicine name is berberine. It is an antibacterial alkaloid extracted from Coptidis rhizoma and cortex Phellodendri, and has effects of clearing heat and detoxicating. As explained by traditional Chinese medicine, the medicine is cool and can remove pathogenic heat or deficiency heat and eliminate harmful poison. It can be used for resisting pathogenic microorganism, relieving inflammation and fever, and has inhibiting effect on Bacillus dysenteriae, bacillus tuberculosis, pneumococcus, staphylococcus aureus, streptococcus, typhoid bacillus, diphtheria bacillus, and amoeba, etc., with the highest inhibiting effect on Bacillus dysenteriae.
The alkali of berberine hydrochloride is named as 5, 6-dihydro-9, 10-dimethoxy benzo [ g ]]-1, 3-benzodioxol [5, 6-alpha ]]Quinolizine hydrochloride dihydrate of formula C 20 H 18 ClNO 4 ·2H 2 O has a molecular weight of 407.85 and a structural formula:
Figure SMS_1
by analyzing the synthetic process route of the crude drug, the isomer generated in the process of the product is 6, 7-dihydro-10, 11-dimethoxybenzo [ g ] -1, 3-benzodioxol [4, 5-alpha ] quinolizine hydrochloride, and the structural formula is as follows:
Figure SMS_2
in order to ensure the safety and effectiveness of the berberine hydrochloride medicine and also directly influence the quality of the final product, the isomer generated in the synthesis process of the raw material medicine is required to be researched and controlled, and the separation and detection method of the isomer is not reported in the prior art, so that the isomer in the finished berberine hydrochloride is not convenient to control.
Disclosure of Invention
The invention provides a method for determining isomers in berberine hydrochloride, which utilizes a high performance liquid chromatography, adopts a chromatographic column with octadecylsilane chemically bonded silica as a filler, wherein a mobile phase A is formic acid aqueous solution with volume fraction of 0.1%, a mobile phase B is methanol solution, the flow rate of the mobile phase is 0.4-0.5 mL/min, gradient elution is adopted, a detector is an ultraviolet absorption detector, the detection wavelength is 226-230 nm, the sample injection amount is 10-50 mu L, and the column temperature of the chromatographic column is 38-42 ℃. Wherein the isomers are: 6, 7-dihydro-10, 11-dimethoxybenzo [ g ] -1, 3-benzodioxol [4, 5-alpha ] quinolizine hydrochloride (impurity C) having the structure:
Figure SMS_3
further, the mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is methanol solution.
Further, the detection wavelength is 228nm, the column temperature of the chromatographic column is 40 ℃, and the sample injection amount is 20 mu L.
Further, the mobile phase flow rate was 0.4mL/min.
Further, the column was a Waters X-Select CSH C18 column, 150X 4.6mm in size and 2.5 μm in packing size.
Further, the gradient elution procedure employed is as follows:
Figure SMS_4
the method for measuring the isomer in the berberine hydrochloride provided by the invention comprises the following steps:
(1) Preparing a sample solution and a system applicability solution;
(2) Setting high performance liquid phase detection conditions: the chromatographic column is Waters X-Select CSH C18X 4.6mm, the particle size of the filler is 2.5 mu m, 0.1% formic acid aqueous solution is taken as mobile phase A, methanol solution is taken as mobile phase B, gradient elution is carried out at the flow rate of 0.4ml/min, the detection wavelength is 228nm, the sample injection amount is 20 mu L, and the column temperature is 40 ℃;
(3) And respectively sucking the sample solution and the system applicability solution, and injecting the sample solution and the system applicability solution into a high performance liquid chromatograph for HPLC analysis.
The preparation method of the sample solution comprises the following steps: precisely weighing a proper amount of berberine hydrochloride test sample, adding a diluent (mobile phase A: mobile phase B=50:50, V/V) to dissolve and dilute into a solution containing 1mg/ml berberine hydrochloride;
the preparation method of the system applicability solution comprises the following steps: accurately weighing about 10mg of impurity C reference substance, placing into a 100ml measuring flask, adding diluent for dissolution, and fixing volume to scale to obtain stock solution. Accurately weighing berberine hydrochloride reference substance about 10mg, accurately weighing 1ml stock solution, placing into 10ml measuring flask, adding diluent for dissolution, and fixing volume to scale. (berberine hydrochloride about 1mg/ml, impurity C about 10 mug/ml);
in step (2), the gradient procedure is as follows:
Figure SMS_5
according to the invention, the charged Waters X-Select CSH C18 column is selected to effectively separate the product from the isomer, so that high-sensitivity and high-accuracy qualitative and quantitative research on the isomer in the berberine hydrochloride is realized, and a foundation is laid for quality control of the isomer in the berberine hydrochloride.
Drawings
FIG. 1 is a liquid chromatogram of a blank solution.
Fig. 2 is a liquid chromatogram of a sample solution of berberine hydrochloride.
FIG. 3 is a system applicability solution liquid chromatogram of berberine hydrochloride and isomers.
Detailed Description
In connection with the following examples, the analytical methods according to the present invention are described in further detail as applied to the detection of isomers in berberine hydrochloride.
Example 1: method for detecting isomer in berberine hydrochloride
1. Chromatographic conditions:
instrument: shimadzu LC-20A high performance liquid chromatograph
Chromatographic column: waters X-Select CSH C18X 4.6mm with a filler particle size of 2.5 μm
Mobile phase: mobile phase A is formic acid water solution with volume fraction of 0.1%, mobile phase B is methanol solution
Mobile phase flow rate: gradient elution is adopted at the concentration of 0.4mL/min,
the detector is an ultraviolet absorption detector, the detection wavelength is 228nm, the sample injection amount is 20 mu L, and the column temperature of the chromatographic column is 40 DEG C
The gradient elution procedure was as follows:
Figure SMS_6
2. sample preparation:
test solution: precisely weighing a proper amount of berberine hydrochloride test sample, adding a diluent (mobile phase A: mobile phase B=50:50, V/V) to dissolve and dilute into a solution containing 1mg/ml berberine hydrochloride;
system applicability solution: accurately weighing about 10mg of impurity C reference substance, placing into a 100ml measuring flask, adding diluent for dissolution, and fixing volume to scale to obtain stock solution. Accurately weighing berberine hydrochloride reference substance about 10mg, accurately weighing 1ml stock solution, placing into 10ml measuring flask, adding diluent for dissolution, and fixing volume to scale. (berberine hydrochloride about 1mg/ml, impurity C about 10 mug/ml).
3. Detection method
Precisely measuring the solution of the sample and the solution suitable for the system, injecting into a high performance liquid chromatograph, performing HPLC analysis, and recording a chromatogram.
Example 2: method for detecting isomer in berberine hydrochloride
The detection method according to example 1 was performed, and the difference from example 1 was that: the flow rate was changed to 0.35ml/min.
Example 3: method for detecting isomer in berberine hydrochloride
The detection method according to example 1 was performed, and the difference from example 1 was that: the flow rate was changed to 0.45ml/min.
Example 4: method for detecting isomer in berberine hydrochloride
The detection method according to example 1 was performed, and the difference from example 1 was that: the column temperature was changed to 38 ℃.
Example 5: method for detecting isomer in berberine hydrochloride
The detection method according to example 1 was performed, and the difference from example 1 was that: the column temperature was changed to 42 ℃.
Example 6: method for detecting isomer in berberine hydrochloride
The detection method according to example 1 was performed, and the difference from example 1 was that: and replacing another chromatographic column with the same type.
Example 7: method for detecting isomer in berberine hydrochloride
The detection method according to example 1 was performed, and the difference from example 1 was that: changing the initial mobile phase ratio: mobile phase a: b is 73:27.
Example 8: method for detecting isomer in berberine hydrochloride
The detection method according to example 1 was performed, and the difference from example 1 was that: changing the initial mobile phase ratio: mobile phase a: b is 67:33.
The results of examples 1 to 8 above are summarized as follows:
Figure SMS_7
analysis results: comparing the detection results of the embodiment 1, wherein the system applicability of the embodiments 2-8 meets the requirements; the absolute value of the content difference of the corrected impurity C is less than or equal to 0.05 percent, the absolute value of the content difference of all single impurities is less than or equal to 0.05 percent, and the absolute value of the total impurity content difference is less than or equal to 0.10 percent according to the requirements.
The detection method of the present invention was validated according to example 1 and includes items such as specificity, limit of detection, limit of quantification, linearity, correction factor, precision, accuracy, durability, solution stability, etc. The results are summarized as follows:
the exclusive results show that: the blank diluent has no detectable peak at the peak of known impurities, and has no interference to the measurement of the solution of the sample; the separation degree of the impurity C and the adjacent peaks of the system applicability solution is more than 1.5, and the system applicability solution meets the standard regulation. In the sensitivity solution, the signal to noise ratio of the main component is more than 10, and meets the requirements.
The detection limit and the quantitative limit result show that: the signal-to-noise ratio (S/N) of the detection limit concentration is more than 3, the RSD of 6-needle peak areas of continuous sample injection with 10% linear concentration as the quantitative limit is less than or equal to 10.0%, and the S/N is more than 10, so that the method meets the requirements.
The linearity and correction factor results show that: the berberine hydrochloride is in the range of 0.47-9.41 mug/ml and the impurity C is in the range of 0.51-10.17 mug/ml, in the linear regression equation, the absolute value of the intercept is smaller than 10% of the peak area of 100%, and the correlation coefficient r is larger than 0.99, so that the method meets the requirements. The factor of impurity C relative to berberine hydrochloride is 1.35.
And (3) the system sample injection precision results show that: the peak area of each component in the reference solution and the RSD of the retention time of each component are less than or equal to 2 percent, which accords with the regulations.
The repeatability results show that: RSD% of the content of the related substances measured in 6 samples is less than or equal to 20%, and meets the standard regulation. The absolute value of the content difference of the corrected impurity C is less than or equal to 0.05 percent, the absolute value of the maximum unknown single impurity content difference is less than or equal to 0.05 percent, and the absolute value of the total impurity content difference is less than or equal to 0.10 percent according to the regulation.
The intermediate precision results show that: RSD% of the content of the related substances measured in 6 parts of the test sample of the second person is less than or equal to 20%, and RSD% of the content of the related substances measured in 12 parts of the test sample of the second person is less than or equal to 20%, which meet the regulations.
The accuracy results show that: the accuracy solution recovery rate of the quantitative limit level is 80% -120%; the recovery rate of other level accuracy solutions is between 90% and 108%, and the recovery rate result RSD is less than or equal to 10%, which meets the regulations.
Stability results show that: the RSD% of each peak area in the control solution at each time point within 66 hours is less than or equal to 10%, and the control solution is stable within 66 hours. And compared with 0 hour, the absolute value of the content difference value of the corrected impurity C is less than or equal to 0.05%, the absolute value of the content difference value of all single impurities is less than or equal to 0.05%, the absolute value of the total impurity content difference value is less than or equal to 0.10% at each time point in the sample solution for 72 hours, and the sample solution is stable within 72 hours.
According to the result, the method for detecting the isomer in the berberine hydrochloride has satisfactory specificity, linearity, detection limit, quantitative limit, precision, accuracy, durability and solution stability, and is suitable for detecting the isomer in the berberine hydrochloride.

Claims (6)

1. A method for determining isomer in berberine hydrochloride, which uses high performance liquid chromatography, adopts a chromatographic column with octadecylsilane chemically bonded silica as filler, wherein mobile phase A is formic acid water solution with volume fraction of 0.1%, mobile phase B is methanol solution, the flow rate of mobile phase is 0.4 mL/min-0.5 mL/min,gradient elution is adopted, the detector is an ultraviolet absorption detector, the detection wavelength is 226 nm-230 nm, the sample injection amount is 10-50 mu L, and the chromatographic column temperature is 38-42 ℃. Wherein the isomers are: 6, 7-dihydro-10, 11-dimethoxybenzo [ g ]]-1, 3-benzodioxol [4, 5-alpha ]]Quinolizine hydrochloride (impurity C), having the structure:
Figure QLYQS_1
2. in some embodiments, mobile phase a is a 0.1% aqueous formic acid solution and mobile phase B is a methanol solution.
3. In some embodiments, the detection wavelength is 228nm, the column temperature is 40 ℃, and the sample volume is 20 μl.
4. In some embodiments, the mobile phase flow rate is 0.5mL/min.
5. In some embodiments, the chromatographic column is a Waters X-Select CSH C18 column, with a specification of 150X 4.6mm, and a packing particle size of 2.5 μm.
6. In some embodiments, the gradient elution procedure employed is as follows: the method for measuring the isomer in the berberine hydrochloride provided by the invention comprises the following steps: (1) preparing a test solution and a system applicability solution; (2) setting high performance liquid phase detection conditions: the chromatographic column is Waters X-Select CSH C18X 4.6mm, the particle size of the filler is 2.5 mu m, 0.1% formic acid aqueous solution is taken as mobile phase A, methanol solution is taken as mobile phase B, gradient elution is carried out at the flow rate of 0.4ml/min, the detection wavelength is 228nm, the sample injection amount is 20 mu L, and the column temperature is 40 ℃; (3) And respectively sucking the sample solution and the system applicability solution, and injecting the sample solution and the system applicability solution into a high performance liquid chromatograph for HPLC analysis. The preparation method of the sample solution comprises the following steps: precisely weighing a proper amount of berberine hydrochloride test sample, adding a diluent (mobile phase A: mobile phase B=50:50, V/V) to dissolve and dilute into a solution containing 1mg/ml berberine hydrochloride; the preparation method of the system applicability solution comprises the following steps: accurately weighing about 10mg of impurity C reference substance, placing into a 100ml measuring flask, adding diluent for dissolution, and fixing volume to scale to obtain stock solution. Accurately weighing berberine hydrochloride reference substance about 10mg, accurately weighing 1ml stock solution, placing into 10ml measuring flask, adding diluent for dissolution, and fixing volume to scale. (berberine hydrochloride about 1mg/ml, impurity C about 10 mug/ml); in step (2), the gradient procedure is as follows:
Figure QLYQS_2
CN202310139694.6A 2023-02-21 2023-02-21 Method for determining isomer in berberine hydrochloride Pending CN116297922A (en)

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