CN112611819B - Method for measuring related substances in benfotiamine raw material and preparation thereof - Google Patents
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- 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
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- G—PHYSICS
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- 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
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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
- G01N30/04—Preparation or injection of sample to be analysed
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- 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
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Abstract
The invention discloses a method for measuring related substances in benfotiamine raw materials and preparations thereof, which comprises the following steps: (1) preparing a test solution, a control solution and a system applicability solution; (2) setting high performance liquid detection conditions: the chromatographic column uses octadecylsilane chemically bonded silica as a filler, the specification is 4.6mm multiplied by 150mm, the particle size of the filler is 5 mu m, sodium heptanesulfonate solution is used as a mobile phase A, methanol is used as a mobile phase B, and the flow rate is 0.8ml/min for gradient elution; (3) respectively sucking the test solution and the control solution, injecting into a high performance liquid chromatograph, and performing HPLC analysis. The invention has the advantages of high sensitivity, high precision, accurate content measurement result, good specificity and linear relation and the like, and can be used for quality control of benfotiamine raw materials and preparation samples.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a benfotiamine raw material and a method for measuring related substances in a preparation thereof.
Background
The chemical name of benfotiamine is S- { (Z) -2- [ (4-amino-2-methylpyrimidin-5-yl) methyl-formylamino]-5-phosphonooxy-pent-2-en-3-yl } benzoic acid thioester, a lipid soluble derivative of vitamin B1 having the CAS number: 22457-89-2 with molecular formula C 19 H 23 N 4 O 6 PS, molecular weight: 466.45, chemical structure:
benfotiamine is gradually widely applied to the aspects of medicines and dietary supplements after being synthesized in the 60 th of the 20 th century. As a derivative of fat-soluble vitamin B1, benfotiamine has better bioavailability than water-soluble vitamin B1, is easier to absorb in a human body than water-soluble vitamin B1, has more durable action, and has a plurality of special medicinal functions. In clinical medicine, benfotiamine is mainly used for preventing and treating vitamin B1 deficiency and metabolic disorder related diseases, and neuropathic pain generated by neuropathy caused by alcoholism and the like. Studies show that benfotiamine has great potential in the field of treating diabetic complications and senile dementia.
Benfotiamine is currently widely marketed in the United states, Japan, Europe and other places around the world, and dosage forms on the market include tablets, granules and the like. In order to ensure the safety and effectiveness of benfotiamine, research and control on related substances of bulk drugs are required. By analyzing the synthesis process route of the product and combing the impurity spectrum, the impurities related to the product, such as process by-products, degradation products and the like, mainly comprise the following 10 impurities.
The impurities generated in the process of synthesis of the product are as follows:
impurity A: 4-methyl-3- [ (2-methyl-4-amino-5-pyrimidinyl) methyl ] -5- (2-benzoyloxyethyl) thiazolium
Impurity B: thio-S- (6-formyl-2, 7-dimethyl-6, 9,10, 11-tetrahydro-5H-pyrimidine [4,5-b ] [1,5] diazepin-8-yl) benzoate
Impurity C: thiosylate S- { (Z) -2- [ (4-benzoylamino-2-methylpyrimidin-5-yl) methyl) -formylamino ] -5-phosphonooxy-pent-2-en-3-yl } benzoate
Impurity D: thio-S, S '- ((3Z, 3' Z) - ((hydroxyphosphoryl) bis (oxy) bis (4- (N- ((4-amino-2-methylpyrimidin-5-yl) methyl) carboxamide) pent-3-en-1, 3-diyl) dibenzoate
Impurity E: 5- (aminomethyl) -4-amino-2-methylpyrimidine
Impurity F: 5- (carboxamidomethyl) -4-amino-2-methylpyrimidine
Impurity G: benzoic acid
Impurity H: 2-carbonyl-3-benzoylthioester-5-phosphoric acid pentyl ester
Thiamine hydrochloride:
thiamine phosphate:
disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a method for determining related substances of benfotiamine, which can effectively separate a plurality of impurities and has the advantages of high detection sensitivity, wide linear range and good accuracy of each impurity.
The technical scheme is as follows: in order to solve the technical problems, the invention adopts the following technical scheme:
a method for measuring related substances in a benfotiamine raw material and a preparation thereof comprises the following steps:
(1) preparing a test solution, a control solution and a system applicability solution;
(2) setting high performance liquid detection conditions: the chromatographic column uses octadecylsilane chemically bonded silica as a filler, the specification is 4.6mm multiplied by 150mm, the particle size of the filler is 5 mu m, sodium heptanesulfonate water solution is used as a mobile phase A, methanol is used as a mobile phase B, and the flow rate is 0.8ml/min for gradient elution;
(3) respectively sucking the test solution and the control solution, injecting into a high performance liquid chromatograph, and performing HPLC analysis.
Wherein, the related substances comprise: 4-methyl-3- [ (2-methyl-4-amino-5-pyrimidinyl) methyl ] -5- (2-benzoyloxyethyl) thiazolium, thio-S- (6-formyl-2, 7-dimethyl-6, 9,10, 11-tetrahydro-5H-pyrimidine [4,5-b ] [1,5] diazanan-8-yl) benzoate, thio-S- { (Z) -2- [ (4-benzoylamino-2-methylpyrimidin-5-yl) methyl) -formylamino ] -5-phosphonooxy-pent-2-en-3-yl } benzoate, thio-S, S' - ((3Z, 3' Z) - ((hydroxyphosphoryl) bis (oxy) bis (4- (N- ((4-amino-2-methylpyrimidin-5-yl) methyl) carboxamide) pent-3-ene-1, 3-diyl) dibenzoate thioester, 5- (aminomethyl) -4-amino-2-methylpyrimidine, 5- (carboxamidomethyl) -4-amino-2-methylpyrimidine, benzoic acid, 2-carbonyl-3-benzoylthiocarbonyl-5-phosphate pentyl ester, thiamine hydrochloride, thiamine phosphate.
The preparation method of the test solution comprises the following steps: accurately weighing benfotiamine, adding a diluent to dissolve and dilute the benfotiamine into a solution containing 1mg/mL of benfotiamine;
the preparation method of the control solution comprises the following steps: precisely measuring 1mL of a test solution, and diluting the test solution by using a diluent to prepare a solution with the benfotiamine content of 1 mu g/L;
the preparation method of the system adaptive solution comprises the following steps:
(S1) weighing 4-methyl-3- [ (2-methyl-4-amino-5-pyrimidinyl) methyl ] -5- (2-benzoyloxyethyl) thiazolium, S- (6-formyl-2, 7-dimethyl-6, 9,10, 11-tetrahydro-5H-pyrimidine [4,5-b ] [1,5] diazanan-8-yl) benzoic acid thioester, S- { (Z) -2- [ (4-benzoylamino-2-methylpyrimidin-5-yl) methyl) -formylamino ] -5-phosphonooxy-pent-2-en-3-yl } benzoic acid thioester, S, respectively, s '- ((3Z, 3' Z) - ((hydroxyphosphoryl) bis (oxy) bis (4- (N- ((4-amino-2-methylpyrimidin-5-yl) methyl) carboxamide) pent-3-ene-1, 3-diyl) dibenzoate thioester, 5- (aminomethyl) -4-amino-2-methylpyrimidine, 5- (formylaminomethyl) -4-amino-2-methylpyrimidine, benzoic acid, 2-carbonyl-3-benzoylthiocarbonyl-5-phosphate amyl ester, thiamine hydrochloride, thiamine phosphate, made up with a diluent of a control stock solution of impurity containing 500. mu.g of each control per 1 mL;
(S2) precisely weighing benfotiamine, adding a diluent to dissolve, taking the impurity reference substance storage solution, and preparing a mixed solution containing 1 mu g of each reference substance and 0.2mg of benfotiamine per 1ml to obtain the system adaptability solution.
Wherein the diluent is a solution with the volume ratio of the mobile phase A to the mobile phase B being 80: 20.
In the step (2), the chromatographic column is Agilent Zorbax SB-C18.
In the step (2), the concentration of sodium heptanesulfonate in the sodium heptanesulfonate aqueous solution is 2.0g/L, and the pH value of the sodium heptanesulfonate solution is 3.2.
In step (2), the gradient elution procedure is as follows: reducing the volume percentage of the mobile phase A to 60 percent and increasing the volume percentage of the mobile phase B to 40 percent in 0-10 min; for 10-18min, the volume percentage of the mobile phase A is reduced to 35 percent, and the volume percentage of the mobile phase B is increased to 65 percent; 18-25min, the volume percentage of the mobile phase A is 35%, and the volume percentage of the mobile phase B is 65%; 25-25.1min, the volume percentage of the mobile phase A is increased to 80 percent, and the volume percentage of the mobile phase B is decreased to 20 percent from 65 percent; 25.1-35min, the volume percentage of the mobile phase A is 80%, and the volume percentage of the mobile phase B is 20%.
| Elution time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0.0 | 80.0 | 20.0 |
| 10.0 | 60.0 | 40.0 |
| 18.0 | 35.0 | 65.0 |
| 25.0 | 35.0 | 65.0 |
| 25.1 | 80.0 | 20.0 |
| 35 | 80.0 | 20.0 |
Wherein the detection wavelength of the ultraviolet detector of the liquid chromatograph is 244 nm.
Has the advantages that:
the invention discloses a method for detecting benfotiamine and related substances thereof, which has the advantages of high sensitivity, high precision, accurate content measurement result and good specificity and linear relation and can be used for quality control of benfotiamine raw materials and preparation samples. The invention adopts the ion pair reagent to greatly improve the defects of poor retention capacity and weak response of partial impurities in a reversed phase chromatographic system, realizes the high-sensitivity and high-accuracy qualitative and quantitative research on the new impurities of the benfotiamine, and lays a foundation for the quality evaluation of the benfotiamine.
Drawings
FIG. 1 is a chromatogram of a blank solution in the resolution examination in the examples;
FIG. 2 is a chromatogram of a system suitability solution in a resolution review in an example; (the retention time of each impurity is 6.563min for thiamine phosphate, 8.140min for F, 8.727min for H, 10.677min for benfotiamine, 13.827min for G, 16.930min for hydrochloride, 17.140min for E, 18.557min for C, 18.987min for B, 20.103min for D and 20.440min for A)
FIG. 3 is a chromatogram of a sensitive solution in the sensitivity examination in the examples.
Detailed Description
The drawings are for illustrative purposes only and are not to be construed as limiting the patent.
The method for detecting a substance relating to benfotiamine according to the present invention will be described in further detail below by way of examples.
Example 1: determination of related substances of benfotiamine
(1) High performance liquid chromatography conditions
The chromatographic column is an octadecylsilane chemically bonded silica column, the model is Agilent Zorbax SB-C18(4.6mm multiplied by 150mm, 5 μm), 2.0g/L sodium heptanesulfonate solution (pH value is adjusted to 3.2 by phosphoric acid) is used as a mobile phase A, methanol is used as a mobile phase B, gradient elution is carried out according to the following table 1, and the flow rate is 0.8ml per minute; the detection wavelength is 244 nm; the column temperature was 35 ℃.
TABLE 1 gradient elution
(2) Solution preparation
Test solution: taking a proper amount of benfotiamine, precisely weighing, adding a diluent (mobile phase A-mobile phase B (80:20), V/V) for ultrasonic dissolution and dilution to prepare a solution containing about 1mg of benfotiamine per 1 ml.
Control solution: 1.0ml of the test sample solution was measured precisely and diluted with a diluent to give a solution containing about 1. mu.g of benfotiamine per 1 ml.
Impurity reference stock solution: weighing appropriate amount of thiamine hydrochloride reference, thiamine phosphate reference, impurity A reference, impurity B reference, impurity C reference, impurity D reference, impurity E reference, impurity F reference, impurity G reference and impurity H reference, dissolving [ mobile phase A-mobile phase B (80:20), V/V ] with diluent, and diluting to obtain solution containing each impurity 500 μ G per 1 ml.
System applicability solution: taking a proper amount of benfotiamine reference substance, precisely weighing, adding a diluent to perform ultrasonic dissolution, taking another proper amount of the impurity reference substance stock solution, and preparing a mixed solution containing about 1 mu g of each impurity and 0.2mg of benfotiamine per 1 ml.
Blank solution: diluent [ mobile phase A-mobile phase B (80:20), V/V ];
and precisely taking 10 mu l of each of the test solution, the reference solution and the system applicability solution, injecting into a liquid chromatograph, and recording a chromatogram. If there is impurity peak in the chromatogram of the sample solution, the content of impurity is calculated according to the main component self-contrast method added with correction factor. Peaks in the chromatogram of the test solution that are 0.3 times (0.03%) smaller than the area of the main peak of the control solution are ignored.
(3) Degree of separation investigation
The test was carried out by preparing a blank solution, a system test solution, a test solution and a control solution according to the methods in the examples. The map is shown in figure 1 and figure 2. The result shows that the chromatographic condition of the invention has no interference in the blank solution, and the main components and the impurities can be effectively separated, thereby showing that the method has good specificity.
(4) Sensitivity investigation
Preparing impurity reference substance storage solutions according to the method in the reference example, precisely measuring a proper amount of each impurity reference substance storage solution, gradually diluting and measuring by using a diluent, and observing the detection sensitivity of each impurity by taking the signal-to-noise ratio of about 10:1 as a quantitative limit. The results are shown in Table 2 and the pattern is shown in FIG. 3. The result shows that the method has good sensitivity.
TABLE 2 sensitivity examination results
| Name of impurity | Concentration (μ g/ml) | Peak area | Signal to noise ratio | Corresponding to the concentration percentage of the test sample |
| Benfotiamine | 0.0493 | 0.0319 | 20.7 | 0.005％ |
| Thiamine phosphate | 0.0356 | 0.0175 | 13.7 | 0.004％ |
| Thiamine hydrochloride | 0.0527 | 0.0289 | 20.7 | 0.005％ |
| Impurity A | 0.0437 | 0.0191 | 18.2 | 0.004％ |
| Impurity B | 0.0546 | 0.0388 | 13.7 | 0.005％ |
| Impurity C | 0.0528 | 0.0310 | 26.0 | 0.005％ |
| Impurity D | 0.0436 | 0.0137 | 13.8 | 0.004％ |
| Impurity E | 0.0500 | 0.0340 | 32.1 | 0.005％ |
| Impurity F | 0.0542 | 0.0488 | 35.4 | 0.005％ |
| Impurity G | 0.0542 | 0.0246 | 17.4 | 0.005％ |
| Impurity H | 0.0486 | 0.0187 | 11.7 | 0.005％ |
(5) Line survey
Preparing impurity reference substance stock solutions according to the method in the embodiment, precisely measuring a proper amount of each impurity reference substance stock solution, and diluting with a diluent to prepare a series of concentrations as linear solutions of each component. The results of the measurements are shown in Table 3 below. The result shows that the linear relation of each component to be detected is good by adopting the chromatographic condition of the invention.
TABLE 3 results of Linear examination
(6) Correction factor
Calibration factors were determined using a standard curve method. The results of taking the correction factors measured from the above linear results are shown in Table 4.
TABLE 4 correction factors
(7) Investigation of solution stability
And respectively taking 10 mu l of the system test solution and 10 mu l of the control solution at different time, injecting into a liquid chromatograph, and recording the chromatogram. The results are shown in tables 5 and 6. The result shows that the system test solution is placed for 24 hours at room temperature, the peak area of each impurity is compared with 0 hour, and the ratio is between 90.0% and 110.0%; the reference solution is placed at room temperature for 24 hours, the main peak area RSD is less than 2.0 percent, and the reference solution meets the requirements, which shows that the solution stability in the method is good, and each test solution is stable within at least 24 hours when placed at room temperature.
TABLE 5 stability test of solutions of related substances-systematic test solution
TABLE 6 stability test of solutions of related substances-control solution
(8) Accuracy survey
Precisely weighing 9 parts of benfotiamine sample, respectively adding impurity reference substance solutions with impurity limits of 50%, 100% and 150%, adding a diluent to dissolve and dilute to a scale, and taking the solution as a test sample solution. Precisely measuring each sample solution to be tested, diluting 1000 times, and using as a reference solution. The sample solution and the control solution were measured precisely and 10. mu.l of each solution was injected into a liquid chromatograph, a chromatogram was recorded, and the recovery rate was calculated from each impurity [ (measured amount-local amount)/added amount ], and the results are shown in Table 7. The result shows that the recovery rate of each impurity is between 90% and 110%, and the RSD is less than 10.0%, which shows that the method has good accuracy and is suitable for detecting related substances of the product.
TABLE 7 accuracy calculation Table
| Name of impurity | Range of recovery | Average recovery rate | RSD |
| Impurity A | 93.00％～99.51％ | 97.05％ | 3.65％ |
| Impurity B | 90.01～102.59％ | 96.52％ | 6.56％ |
| Impurity C | 97.63％～102.74％ | 99.71％ | 2.69％ |
| Impurity D | 92.93％～101.69％ | 97.99％ | 4.63％ |
| Impurity E | 91.69％～104.04％ | 98.67％ | 6.41％ |
| Impurity F | 98.79％～108.34％ | 103.90％ | 4.63％ |
| Impurity G | 97.10％～105.49％ | 101.28％ | 4.14％ |
| Impurity H | 92.17％～94.96％ | 93.34％ | 1.55％ |
| Thiamine phosphate | 92.65％～103.18％ | 98.70％ | 5.51％ |
| Thiamine hydrochloride | 94.21％～109.82％ | 103.93％ | 8.16％ |
In conclusion, the method for determining related substances in benfotiamine has the advantages of strong specificity, high sensitivity, good linear relation and high accuracy.
It should be understood that the above-mentioned examples of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (5)
1. A method for measuring related substances in a benfotiamine raw material and a preparation thereof is characterized by comprising the following steps:
(1) preparing a test solution, a control solution and a system applicability solution;
(2) setting high performance liquid detection conditions: the chromatographic column uses octadecylsilane chemically bonded silica as a filler, the specification is 4.6mm multiplied by 150mm, the particle size of the filler is 5 mu m, sodium heptanesulfonate solution is used as a mobile phase A, methanol is used as a mobile phase B, and the flow rate is 0.8ml/min for gradient elution; the concentration of sodium heptanesulfonate in the sodium heptanesulfonate solution is 2.0g/L, and the pH value of the sodium heptanesulfonate solution is 3.2;
(3) respectively sucking the test solution and the control solution, injecting into a high performance liquid chromatograph, and performing HPLC analysis;
the related substances comprise: 4-methyl-3- [ (2-methyl-4-amino-5-pyrimidinyl) methyl ] -5- (2-benzoyloxyethyl) thiazolium, S- (6-formyl-2, 7-dimethyl-6, 9,10, 11-tetrahydro-5H-pyrimidine [4,5-b ] [1,5] diazepin-8-yl) benzoic acid thioester, S- { (Z) -2- [ (4-benzoylamino-2-methylpyrimidin-5-yl) methyl) -formylamino ] -5-phosphonooxy-pent-2-en-3-yl } benzoic acid thioester, S' - ((3Z, 3' Z) - ((hydroxyphosphoryl) bis (oxy) bis (4- (N- ((4-amino-2-methylpyrimidin-5-yl) methyl) carboxamide) pent-3-ene-1, 3-diyl) dibenzoate thioester, 5- (aminomethyl) -4-amino-2-methylpyrimidine, 5- (carboxamidomethyl) -4-amino-2-methylpyrimidine, benzoic acid, 2-carbonyl-3-benzoylthiocarbonyl-5-phosphate amyl ester, thiamine hydrochloride, thiamine phosphate;
the gradient elution procedure was as follows:
reducing the volume percentage of the mobile phase A to 60 percent and increasing the volume percentage of the mobile phase B to 40 percent in 0-10 min;
for 10-18min, the volume percentage of the mobile phase A is reduced to 35 percent, and the volume percentage of the mobile phase B is increased to 65 percent;
18-25min, the volume percentage of the mobile phase A is 35%, and the volume percentage of the mobile phase B is 65%;
25-25.1min, the volume percentage of the mobile phase A is increased to 80 percent, and the volume percentage of the mobile phase B is decreased to 20 percent from 65 percent;
25.1-35min, the volume percentage of the mobile phase A is 80%, and the volume percentage of the mobile phase B is 20%.
2. The method for measuring the related substances in the benfotiamine raw material and the preparation thereof according to claim 1, wherein the preparation method of the test solution comprises the following steps: accurately weighing benfotiamine, adding a diluent to dissolve and dilute the benfotiamine into a solution containing 1mg/mL of benfotiamine;
the preparation method of the control solution comprises the following steps: precisely measuring 1mL of a test solution, and diluting the test solution by using a diluent to prepare a solution with the benfotiamine content of 1 mu g/L;
the preparation method of the system applicability solution comprises the following steps:
(S1) weighing 4-methyl-3- [ (2-methyl-4-amino-5-pyrimidinyl) methyl ] -5- (2-benzoyloxyethyl) thiazolium, S- (6-formyl-2, 7-dimethyl-6, 9,10, 11-tetrahydro-5H-pyrimidine [4,5-b ] [1,5] diazanan-8-yl) benzoic acid thioester, S- { (Z) -2- [ (4-benzoylamino-2-methylpyrimidin-5-yl) methyl) -formylamino ] -5-phosphonooxy-pent-2-en-3-yl } benzoic acid thioester, S, respectively, s '- ((3Z, 3' Z) - ((hydroxyphosphoryl) bis (oxy) bis (4- (N- ((4-amino-2-methylpyrimidin-5-yl) methyl) carboxamide) pent-3-ene-1, 3-diyl) dibenzoate thioester, 5- (aminomethyl) -4-amino-2-methylpyrimidine, 5- (formylaminomethyl) -4-amino-2-methylpyrimidine, benzoic acid, 2-carbonyl-3-benzoylthioester-5-phosphate amyl ester, thiamine hydrochloride, thiamine phosphate, dissolved and diluted with a diluent to make a control stock solution of impurity containing 500. mu.g of each control per 1 mL;
(S2) precisely weighing benfotiamine, adding a diluent to dissolve, taking the impurity reference substance storage solution, and preparing a mixed solution containing 1 mu g of each reference substance and 0.2mg of benfotiamine per 1ml to obtain a system applicability solution.
3. The method for determining the related substances in the benfotiamine raw material and the preparation thereof as claimed in claim 2, wherein the diluent is a mixture of a mobile phase A and a mobile phase B, the volume ratio of which is 80:20, respectively.
4. The method for measuring the related substances in the benfotiamine raw material and the preparation thereof according to claim 1, wherein in the step (2), the chromatographic column is Agilent Zorbax SB-C18.
5. The method for measuring a material and a related substance in a formulation thereof as recited in claim 1, wherein the detection wavelength of the ultraviolet detector of the liquid chromatograph is 244 nm.
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