CN111812227B

CN111812227B - Analysis method of benserazide impurity A in dopa-serazide compound preparation

Info

Publication number: CN111812227B
Application number: CN202010562591.7A
Authority: CN
Inventors: 张丽君; 卢金萍; 赵周明; 郭晓迪; 沈霞; 章正赞; 娄贵川
Original assignee: Zhejiang Huahai Pharmaceutical Co Ltd
Current assignee: Zhejiang Huahai Pharmaceutical Co Ltd
Priority date: 2020-06-19
Filing date: 2020-06-19
Publication date: 2024-09-17
Anticipated expiration: 2040-06-19
Also published as: CN111812227A

Abstract

The invention provides a method for detecting the content of benserazide impurity A in a compound preparation of dopa serazide, which adopts a hydrophilic chromatographic column, takes aqueous solution containing sodium heptanesulfonate and potassium dihydrogen phosphate with the pH value of 1.7-9.0 as a mobile phase A and methanol as a mobile phase B, and adopts a step gradient elution method to carry out separation and determination on the content of benserazide impurity A in the compound preparation of dopa serazide. The method has the advantages of good separation effect, simple operation, high recovery rate, high method sensitivity, wide linear range and important practical significance in the aspect of quality control in the preparation process.

Description

Analysis method of benserazide impurity A in dopa-serazide compound preparation

Technical Field

The invention belongs to the field of medicine analysis and detection, and particularly relates to an analysis method for detecting benserazide impurity A (2 RS) -2-amino-3-hydroxypropionyl hydrazide in a dopa serazide compound preparation by using HPLC.

Background

The compound tablet of dopa and hydrazine, which is prepared by Roche Holding AG of the original research company, has the trade name Madopar (Meadopa), consists of levodopa and benserazide hydrochloride, and is a medicament for treating parkinsonism or parkinsonism (postencephalitis, arteriosclerosis or poisoning). The composition can increase dopamine content in brain cells, and improve Parkinson's disease. At the same time, the benserazide can prevent the dopa decarboxylase from being converted into dopamine before the levodopa does not enter the brain, thereby increasing the dopamine in the brain, enhancing the curative effect and reducing the adverse reaction of the levodopa. The compound dopa-hydrazine preparation is used as a first-line medicine for treating parkinsonism or parkinsonism, clinical practice shows that the compound dopa-hydrazine preparation can obtain good curative effect, has the same effect as large-dose levodopa, and has much better tolerance. The symptoms of parkinsonism are all obviously improved after long-term administration. The structural formula of the dopa hydrazine is shown as follows:

However, in the analysis and research of the stability and the impurity of the compound preparation of the dopa serazide, the benserazide hydrochloride is unstable to light and humidity, and 3 main degradation impurities, namely benserazide impurity A: (2 RS) -2-amino-3-hydroxypropionyl hydrazine, benserazide impurity B: (2 RS) -2-amino-3-hydroxy-N, N-bis (2, 3, 4-trihydroxybenzyl) propane hydrazide, benserazide impurity C: (2 RS) -2-amino-3-hydroxy-N' - [ (EZ) - (2, 3, 4-trihydroxyphenyl) methylene ] propionyl hydrazine has a growing trend.

However, the existing methods for detecting the benserazide impurity A in the compound preparation of the dopa by using HPLC use common liquid chromatographic columns such as C18, and the methods have the advantages of low concentration sensitivity, small response, large auxiliary material interference, low accuracy and unsatisfactory recovery rate, and the problem of quality is caused by the fact that the detection of the benserazide impurity A content is found to be low in the subsequent sample detection process. Therefore, in order to better control the product quality of the compound preparation of the dopa and to strictly control the content of the benserazide impurity A, a proper method needs to be developed to improve the detection capability.

Disclosure of Invention

The invention provides a method for detecting the content of benserazide impurity A in a compound preparation of dopa serazide, which has the advantages of high specificity, good durability and less interference impurities, and provides a reference for quality control and clinical medication safety of the benserazide impurity A in the compound preparation of dopa serazide.

The method comprises the following steps:

(1) The chromatographic conditions were as follows:

chromatographic column: hydrophilic chromatographic column filled with trifunctional C18 alkyl bonded silica gel or B-type ultra-high purity full porous spherical silica gel;

column temperature: 30 ℃;

Mobile phase a: sodium heptanesulfonate and potassium dihydrogen phosphate are dissolved in water and the pH is adjusted to 2.0 to 7.0 with sodium hydroxide or phosphoric acid;

Mobile phase B: methanol;

Detection wavelength: 210nm;

flow rate: 1.0mL/min;

Elution mode: gradient elution;

(2) Sample solution preparation: dissolving the dopa-hydrazine compound preparation in a diluent to prepare a test solution;

(3) The measuring method comprises the following steps: and (3) injecting the sample solution prepared in the step (2) into a liquid chromatograph, performing high performance liquid chromatography on the sample solution according to the chromatographic conditions in the step (1), and recording a chromatogram.

Further, the ratio of sodium heptanesulfonate, potassium dihydrogen phosphate and water in mobile phase A in step (1) is 1 to 3g:5 to 7g:1L, preferably 2.2g:6.8g:1L.

The elution mode in the step (1) is that the mobile phase A is used for eluting for 10-20 min, and then the mobile phase A and the mobile phase B are used for gradient elution for 15-30 min.

The chromatographic conditions in the further step (1) are as follows:

Chromatographic column: ultimate AQ C18, 18 or WATERS ATLANTIC T;

column temperature: 30 ℃;

mobile phase a:2.2g of sodium heptanesulfonate+6.8 g of potassium dihydrogen phosphate to 1L of water, and adjusting the pH to 2.0 to 7.0 with sodium hydroxide or phosphoric acid;

flow rate: 1.0mL/min;

elution mode: eluting with mobile phase A for 15min, and then eluting with mobile phase A and mobile phase B for 20min.

Further, the detector for detecting the wavelength in the step (1) is selected from ultraviolet detectors (UV detectors).

Further, in the step (2), the diluent is selected from: an organic solvent, or a mixed solvent of water and an organic solvent.

Further, in the step (2), the diluent is selected from: methanol or a mixed solvent of methanol and water.

The compound preparation of the dopa serazide in the step (2) is a tablet or a capsule.

The preparation steps of the sample solution in the step (2) are as follows: the compound preparation of the dopa hydrazine is dissolved by a diluent and the volume is fixed, so as to prepare a solution with 2.5mg of the benserazide in 1ml volume.

And (3) the sample injection amount of the sample solution in the step (3) is 10 mu L.

Compared with the prior art, the method has the advantages that the method can rapidly, effectively and accurately detect the content of the benserazide impurity A in the compound preparation of the dopa serazide, and has good separation effect, simple operation, high recovery rate, high sensitivity and wide linear range. Has important practical significance in the aspects of synthesis and quality control of preparation process.

Drawings

FIG. 1 is a graph showing the HPLC detection result of the sample solution of the dopa hydrazine tablet of comparative example 1.

FIG. 2 is a graph of HPLC detection results for blank solutions.

FIG. 3 is a graph of the results of an HPLC test for the applicability of the system of example 1.

FIG. 4 is a chart showing the localization of impurities in example 1

Fig. 5 is an impurity localization chart of example 1.

FIG. 6 is a graph showing the HPLC detection result of the sample solution of the dopa hydrazine tablet of example 1.

FIG. 7 is a graph of the accuracy of the solution HPLC detection of example 2.

FIG. 8 is a graph of the results of an HPLC test for the applicability of the system of example 2.

FIG. 9 is a graph of the accuracy of the HPLC detection of the solution of example 3.

FIG. 10 is a graph of the results of an HPLC test for the applicability of the system of example 3.

FIG. 11 is a graph of the accuracy of the HPLC detection of the solution of example 4.

FIG. 12 is a graph of the results of the HPLC test for the system of example 4.

Detailed Description

Control information:

Sample information:

Name of the name	Lot number	Source(s)
			Dopa silk hydrazine tablet	R141520001	Hua Hai A
Dopa silk hydrazine tablet blank auxiliary material	2001-330115	Hua Hai A

Comparative example 1 (benserazide hydrochloride API detection method according to EP 9.0)

Chromatographic conditions:

instrument: high performance liquid chromatograph equipped with ultraviolet detector

Chromatographic column: lichrospher ^R 60RP Select B4.6*250mm 5. Mu.m

Mobile phase a:2.2g of sodium heptanesulfonate monohydrate and 6.8g of potassium dihydrogen phosphate- > 900mL of water, 50mL of methanol was added and the pH was adjusted to 3.5.+ -. 0.05 with phosphoric acid.

Mobile phase B:2.2g of sodium heptanesulfonate monohydrate and 6.8g of potassium dihydrogen phosphate → 500mL of water, the pH was adjusted to 3.5.+ -. 0.05 with phosphoric acid, and 500mL of methanol was added.

Dilution liquid: methanol

Detection wavelength: 210nm of

Flow rate: 1.3mL/min

Sample injection volume: 5 mu L

Column temperature: 30 DEG C

Sample cell temperature: 5 DEG C

Run time: 25min

Test solution: weighing 20 pieces of test sample, grinding into powder, taking 1350mg, precisely weighing in a 50mL volumetric flask, adding a diluent to 2/3 of the volumetric flask, shaking for 15min for dissolution, diluting to a scale, mixing uniformly, centrifuging, transferring the supernatant to a liquid phase sample injection vial, and detecting benserazide impurity A by an HPLC method. The result of the chromatogram is shown in FIG. 1.

Preparation of accuracy solution:

Preparing a benserazide impurity A stock solution: the benserazide impurity A reference 12.574mg to 50mL measuring flask is weighed, 70% methanol is added for dissolution and dilution to scale mark, and shaking is carried out uniformly.

TABLE 1-1 preparation of accuracy solutions

The solution 10. Mu.L in Table 1-1 was precisely measured, and the detection and analysis were carried out under the above-mentioned detection conditions, and the results are shown in Table 1-2.

Tables 1-2 accuracy validation results

As can be seen from the results of the above figure 1, the peak time of the levodopa is 4.53min, the peak time of the benserazide impurity A is 4.95min, the response is weak, and the content of the benserazide impurity A in the measured sample is 0.09%; lower than the content measured using the detection method of the present invention. The results in tables 1-2 show that in the accuracy test, the average recovery of the accuracy solution (0.1%) was 48.6%, which did not meet the requirements specified in the pharmacopoeia.

Example 1

Chromatographic conditions:

Chromatographic column: ultimate AQ C18.6X250mm 5 μm (filler: B ultra-high purity full porous spherical silica gel filler)

Mobile phase a:2.2g sodium heptanesulfonate+6.8g potassium dihydrogen phosphate to 1L water, pH=6.0 with 5M sodium hydroxide

Mobile phase B: methanol

Detection wavelength: 210nm of

Flow rate: 1.0mL/min

Sample injection volume: 10 mu L

Column temperature: 30 DEG C

Sample cell temperature: 4 DEG C

Run time: 60min

Time/min	Mobile phase a/%	Mobile phase B/%
			0-15	100	0
15-35	100→30	0→70

Dilution liquid: 70% methanol

Preparing a blank solution:

as with the diluent.

Linear solution 1:

Weighing a proper amount of benserazide impurity A reference substance, and diluting the reference substance by using a diluent until the concentration of the benserazide impurity A is respectively as follows: 1.25, 2.5, 25, 50, 75 micrograms/mL, after shaking to complete dissolution, were detected by HPLC. The regression coefficient (R2) =1.0000 is calculated.

Degree of separation solution (system adaptation solution):

The benserazide impurity A reference substance is weighed into a measuring flask with the volume ranging from 25mg to 100mL, the levodopa impurity B reference substance is weighed into a measuring flask with the volume ranging from about 1.6mg to 50mL, and the benserazide impurity A reference substance and the levodopa impurity B reference substance are respectively dissolved by a diluent to be fixed to be used as stock solution. The method comprises the steps of weighing about 500mg to 50mL of levodopa in a measuring flask, transferring 5mL of benserazide impurity A stock solution, transferring 5mL of levodopa impurity B stock solution to the same 50mL measuring flask, and adding diluent to fix volume to obtain a system applicability solution.

And (3) carrying out detection analysis according to the detection conditions, wherein the sample injection results of the blank solution and the system adaptive solution are shown in fig. 2 and 3. The peak-out sequences of the components in the figure 3 are respectively that the peak-out time of the levodopa, the benserazide impurity A and the levodopa impurity B is 6.212min,7.595min and 8.431min, the separation degree of the components is 3.14,2.09, and the parameters of each color spectrum meet the requirements.

Preparing an accuracy stock solution of benserazide impurity A:

The benserazide impurity A reference 25.86mg to 100mL measuring flask is weighed, 70% methanol is added for dissolution and dilution to scale marks, and the mixture is shaken uniformly to serve as an accuracy intermediate stock solution.

TABLE 2-1 preparation of accuracy solutions

The solution 10. Mu.L in Table 1-1 was precisely measured, and the detection and analysis were carried out under the above-mentioned detection conditions, and the results are shown in Table 2-2.

TABLE 2-2 accuracy validation results

Table 2-2 shows that the recovery of 2.5. Mu.g/mL of accuracy solution 1 was 85.5%; the recovery rate of the 50 mug/mL accurate solution 2 is 95.8%; the recovery rate of the solution 3 with 75 mug/mL accuracy is 96.6%, and the recovery rate meets the specified range of 80-120%. The analysis method provided by the invention can effectively separate the benserazide impurity A, the levodopa impurity B and the levodopa, has the advantages of high separation degree, high response value and strong specificity, can effectively and accurately separate the benserazide impurity A in the compound dopa hydrazine tablet, and is suitable for detecting the benserazide impurity A in the compound dopa hydrazine tablet.

Preparing a test solution:

Weighing 10 pieces of test sample, grinding into powder, taking 1350mg, precisely weighing in a 50mL volumetric flask, adding diluent to 2/3 of the volumetric flask, shaking for 10min for dissolution, diluting to scale, mixing uniformly, centrifuging, transferring supernatant into a liquid phase sample injection vial, and detecting according to the HPLC conditions.

The positioning of each impurity is shown in fig. 4 and 5, wherein the peak time of each impurity is as follows: levodopa impurity a:5.325min, levodopa dimethoxy: 25.182min, benserazide impurity B:31.610min, benserazide impurity C:28.846min.

FIG. 6 is a chromatogram of the test results.

From fig. 6, we can see that the levodopa and the benserazide impurity a can be well separated and are not interfered by auxiliary materials, the sensitivity is high, wherein the sample does not contain the levodopa impurity B, and the content of the benserazide impurity a in the sample is measured to be 0.39%. The method can accurately separate and detect the content of benserazide impurity A in the compound dopa hydrazine tablet.

Example 2

Chromatographic conditions: the pH in mobile phase a was changed, adjusted to ph=7.0 with 5M sodium hydroxide, and the other conditions were unchanged.

Dilution liquid: 70% methanol

Degree of separation solution (system adaptation solution):

The separation degree solution of example 1 was taken and subjected to detection analysis under the above detection conditions, and the system adaptation chromatogram was shown in fig. 8. The peak-out sequence of each component in the figure 8 is respectively 5.988min,4.686min and 8.195min, the separation degree is 2.78,3.88, and the parameters of each color spectrum meet the requirements.

Accuracy (0.1%) solution:

1000mg of levodopa is weighed, 1698mg of blank auxiliary materials are put into a 100ml measuring flask, 1ml of benserazide impurity A accurate stock solution is precisely measured into the same measuring flask, diluent is added to the volume of a 2/3 measuring flask, shaking is carried out for 10min, diluent is added to fix volume, supernatant is centrifugally taken and transferred into a liquid phase sample injection small flask, and the recovery rate is 103.0%. The HPLC chart of the detection result is shown in FIG. 7.

As can be seen from fig. 7, changing the pH value, the recovery rate of the solution with 0.1% accuracy is also satisfactory, which indicates that the analysis method of the present invention can effectively separate the benserazide impurity a, the levodopa impurity B and the levodopa, has high separation degree, high response value and strong specificity, and can effectively and accurately separate the benserazide impurity a in the compound dopa hydrazine tablet.

Example 3

Chromatographic conditions: the pH in mobile phase a was changed, ph=2.0 with phosphoric acid, and the other conditions were unchanged.

Dilution liquid: 70% methanol

Degree of separation solution (system adaptation solution):

the separation degree solution of example 1 was taken and subjected to detection analysis under the above detection conditions, and the system adaptation chromatogram was shown in fig. 10. The peak-out sequence of each component in the figure 10 is 29.710min,30.141min and 30.72min respectively, the separation degree is 2.35,3.38 respectively, and the parameters of each color spectrum meet the requirements.

Accuracy (0.1%) solution:

1000mg of levodopa is weighed, 1698mg of blank auxiliary materials are put into a 100ml measuring flask, 1ml of benserazide impurity A accurate stock solution is precisely measured into the same measuring flask, diluent is added to the volume of a 2/3 measuring flask, shaking is carried out for 10min, diluent is added to fix volume, supernatant is centrifugally taken and transferred into a liquid phase sample injection small flask, and the recovery rate is 82.0%. The HPLC chart of the detection result is shown in FIG. 9.

As can be seen from fig. 9, the PH value is changed, and the solution recovery rate with 0.1% accuracy is also satisfactory, which indicates that the analysis method of the present invention can effectively separate the benserazide impurity a, the levodopa impurity B and the levodopa, has high separation degree, high response value and strong specificity, and can effectively and accurately separate the benserazide impurity a in the compound dopa hydrazine tablet.

Example 4

Chromatographic conditions: other chromatographic conditions were the same as in example 1, except that the chromatographic column was changed to WATERS ATLANTIC t3,250mm 4.6mm;5.0 μm (filler: trifunctional C18 alkyl-bonded silica gel)

Dilution liquid: 70% methanol

Degree of separation solution (system adaptation solution):

The separation degree solution of example 1 was taken and subjected to detection analysis under the above detection conditions, and the system adaptation chromatogram was shown in fig. 12. The peak-out sequence of each component in FIG. 12 is respectively that the peak-out time of the levodopa, the benserazide impurity A and the levodopa impurity B is 6.493min,7.631min and 9.158min, the separation degree of each component is 2.48,3.61, and each chromatographic parameter meets the requirements.

Accuracy (0.1%) solution:

1000mg of levodopa is weighed, 1698mg of blank auxiliary materials are put into a 100ml measuring flask, 1ml of benserazide impurity A accurate stock solution is precisely measured into the same measuring flask, diluent is added to the volume of a 2/3 measuring flask, shaking is carried out for 10min, diluent is added to fix volume, supernatant is centrifugally taken and transferred into a liquid phase sample injection small flask, and the recovery rate is 84.3%. The HPLC chart of the detection result is shown in FIG. 11.

As can be seen from FIG. 11, the chromatographic column is replaced, other chromatographic conditions are not changed, and the recovery rate of the 0.1% accuracy solution meets the requirements, so that the analysis method disclosed by the invention can effectively separate the benserazide impurity A, the levodopa impurity B and the levodopa, has the advantages of high separation degree, high response value and strong specificity, and can effectively and accurately separate the benserazide impurity A in the compound dopa hydrazine tablet, and the method is suitable for detecting the benserazide impurity A in the compound dopa hydrazine tablet.

The above description is merely illustrative of the preferred embodiments of the present invention, and the present invention is not limited to the above embodiments, and any changes and modifications of the present invention fall within the scope of the present invention.

Claims

1. The method for detecting the content of the benserazide impurity A in the compound preparation of the dopa serazide is characterized by using the following chromatographic conditions:

Chromatographic column: a hydrophilic chromatographic column filled with trifunctional C18 alkyl bonded silica gel and B-type ultra-high purity full-porous spherical silica gel;

column temperature: 30 ℃;

Mobile phase a: dissolving sodium heptanesulfonate and potassium dihydrogen phosphate in water, and adjusting the pH to 2.0 to 7.0 with sodium hydroxide or phosphoric acid;

Mobile phase B: methanol;

Detection wavelength: 210 nm;

flow rate: 1.0 mL/min;

Dilution liquid: 70% methanol solvent;

The elution mode is gradient elution, and is specifically as follows:

Time/min Mobile phase a/% Mobile phase B/% 0-15 100 0 15-35 100→30 0→70

Wherein,

The chromatographic column is selected from Ultimate AQ C, WATERS ATLANTIC T3;

The ratio of the sodium heptanesulfonate to the potassium dihydrogen phosphate to the water in the mobile phase A is 1-3 g:5-7 g:1L;

The detection method can effectively separate the benserazide impurity A, the levodopa impurity B and the levodopa, and the benserazide impurity A, the levodopa impurity B and the levodopa have the following structures:

。

2. The method according to claim 1, wherein the ratio of sodium heptanesulfonate, potassium dihydrogen phosphate and water in mobile phase a is 2.2g:6.8g:1l.

3. The detection method according to any one of claims 1-2, characterized in that the detection method specifically comprises the following steps:

(1) Sample solution preparation: dissolving the dopa-hydrazine compound preparation in a diluent to prepare a test solution;

(2) The measuring method comprises the following steps: and (3) injecting the sample solution prepared in the step (1) into a liquid chromatograph, measuring and recording a chromatogram.

4. The method according to claim 3, wherein the compound dobby hydrazine preparation used in the step (1) is a tablet or capsule.

5. A method according to claim 3, wherein the sample solution preparation step in step (1) is as follows: the compound preparation of the dopa hydrazine is dissolved by a diluent and the volume is fixed, so as to prepare a solution with 2.5mg of the benserazide in 1ml volume.

6. The method of claim 3, wherein the sample solution in step (2) is introduced in an amount of 10 μl.