CN111595954B

CN111595954B - Method for detecting content of DCC and DCU in indapamide bulk drug

Info

Publication number: CN111595954B
Application number: CN201911130294.9A
Authority: CN
Inventors: 詹志斌; 秦巨波; 谢秀萍; 陈小波; 龚明峰; 张志刚
Original assignee: Grand Pharma China Co ltd
Current assignee: Grand Pharma China Co ltd
Priority date: 2019-11-18
Filing date: 2019-11-18
Publication date: 2022-07-15
Anticipated expiration: 2039-11-18
Also published as: CN111595954A

Abstract

The invention relates to a method for detecting content of DCC and DCU in an indapamide bulk drug, which detects the DCC and DCU in the indapamide bulk drug by using a high performance liquid chromatography, and comprises the following steps: (1) preparing an indapamide raw material drug test solution; (2) respectively preparing DCC reference substance solution, DCU reference substance solution and DCC & DCU reference substance solution; (3) and (3) injecting samples, and respectively detecting the test solution in the step (1) and the reference solution in the step (2) according to the following conditions: chromatographic conditions are as follows: mobile phase: acetonitrile-water; gradient elution: the proportion of acetonitrile is gradually increased from 10-40% to 90-98% in 0-30 min; the column temperature is 33-37 ℃; the flow rate is 1.4-1.6 ml/min; the detection wavelength is 190-210 nm; (4) and calculating the peak areas corresponding to the DCC and DCU of the test sample and the solution of the reference sample to obtain the content of the DCC and DCU. The method for detecting the content of DCC and DCU in the indapamide bulk drug provided by the invention has the advantages of low toxicity of the used reagent, less impurity interference and reliability and stability.

Description

Method for detecting content of DCC and DCU in indapamide bulk drug

Technical Field

The invention relates to a method for detecting the content of impurities in an indapamide raw material medicine, in particular to a method for detecting the content of DCC and DCU in the indapamide raw material medicine.

Background

Indapamide is a sulfanilamide diuretic, has diuretic and calcium antagonistic effects, is a potent and long-acting antihypertensive drug, and is clinically used for treating water and sodium retention in congestive heart failure. DCC (dicyclohexylcarbodiimide) and DCU (dicyclohexylurea) are residues in an indapamide synthesis process, at present, the national standard HG/T5320-2018 only discloses a DCC mass fraction determination method, and the prior art does not have any detection method for DCC and DCU contents, and even does not have a detection method capable of simultaneously detecting DCC and DCU contents.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides the method for detecting the content of DCC and DCU in the indapamide bulk drug, which can detect the content of DCC and DCU at the same time, and has the advantages of low detection limit, time saving and accurate detection result.

The technical scheme for solving the technical problems is as follows: the method for detecting the content of DCC and DCU in the indapamide bulk drug utilizes a high performance liquid chromatography to detect the DCC and DCU in the indapamide bulk drug, and comprises the following steps:

(1) preparing a test solution of the indapamide bulk drug;

(2) respectively preparing DCC reference substance solution, DCU reference substance solution and DCC & DCU reference substance solution;

(3) and (3) injecting samples, and respectively detecting the sample solution in the step (1) and the reference substance solution in the step (2) according to the following conditions:

chromatographic conditions are as follows: mobile phase: acetonitrile-water; gradient elution: the proportion of acetonitrile gradually increases from 10-40% to 90-98% in 0-30 min; the column temperature is 33-37 ℃; the flow rate is 1.4-1.6 ml/min; the detection wavelength is 190-210 nm;

(4) and calculating the peak areas corresponding to the DCC and DCU of the test sample and the solution of the reference sample to obtain the content of the DCC and DCU.

Wherein the chromatographic column is a C18 liquid chromatographic column.

Preferably, the chromatography column is an Agela Venusil C18 Plus, 4.6 x 200mm,5 μm.

Preferably, the column is an Agela Venusil XBP C18, 4.6 x 200mm,5 μm.

Wherein, the chromatographic conditions in the step (3) are as follows: the amount of sample was 10. mu.l.

Preferably, the chromatographic conditions in step (3) are: the detection wavelength was 205 nm.

Preferably, the chromatographic conditions in step (3) are: gradient elution: the proportion of acetonitrile is gradually increased from 35 percent to 95 percent in 0-30 min; the column temperature is 35 ℃; the flow rate is 1.5 ml/min; the detection wavelength was 205 nm.

Wherein, the concentration of the test solution of the indapamide raw material drug in the step (1) is 3-7 mg/ml.

Wherein, the concentration of DCC in the step (1) is 5-10 mug/ml, and the concentration of DCU is 5-10 mug/ml.

1. The invention optimizes the wavelength in the chromatographic condition:

during optimization: a chromatographic column: agela Venusil C18 Plus, 4.6 × 200mm,5 μm; flow rate: 1.5 ml/min; column temperature: 35 ℃; sample introduction amount: 10 mul; mobile phase: acetonitrile-water; gradient elution: the acetonitrile proportion gradually increases from 35% to 95% in 0-30 min.

The DCC and DCU control solution is scanned in the wavelength range of 160-400nm, and the response values of DCC and DCU are higher in the range of 190-210nm according to the chromatogram obtained by scanning, and the optimal wavelength is 205nm in consideration of the terminal absorption interference of the solvent.

2. The invention optimizes the gradient elution condition in the chromatographic condition:

during optimization: a chromatographic column: agela Venusil C18 Plus, 4.6 × 200mm,5 μm; flow rate: 1.5 ml/min; column temperature: 35 ℃; detection wavelength: 205 nm; sample injection amount: 10 mul; the mobile phase is acetonitrile-water.

The initial proportion of acetonitrile was varied to 5%, 10%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, respectively, with separation and retention times being optimal with an initial proportion of acetonitrile of 35%.

The method is used for detecting the content of DCC and DCU in the indapamide bulk drug, the linear range of DCC is 0.9279-14.8464 mu g/ml, the detection limit is 0.4639 mu g/ml, the average recovery rate is more than 99.2%, the linear range of DCU is 0.9336 mu g/ml-14.9372 mu g/ml, the detection limit is 0.4668 mu g/ml, and the average recovery rate is more than 102.4%. The method for detecting the content of DCC and DCU in the indapamide bulk drug has the advantages of low toxicity of used reagents, less impurity interference and reliable and stable detection method, and has important significance for quality control of the indapamide bulk drug.

Drawings

FIG. 1 is a chromatogram of the spiked sample solution of example 1;

FIG. 2 is a chromatogram of the spiked sample solution of example 2;

FIG. 3 is a chromatogram of the spiked sample solution of example 3;

FIG. 4 is a chromatogram of the spiked sample solution of example 4;

FIG. 5 is a chromatogram of the spiked sample solution of example 5;

FIG. 6 is a chromatogram of the test solution numbered 1 in example 6;

FIG. 7 is a chromatogram of the test solution numbered 2 in example 6;

FIG. 8 is a chromatogram of the test solution numbered 3 in example 6;

FIG. 9 is a chromatogram of the test solution numbered 4 in example 6;

FIG. 10 is a chromatogram of the control solution of example 6;

figure 11 is a chromatogram of a DCC control solution of example 6;

FIG. 12 is a chromatogram of a DCU control solution of example 6.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

1. Instrument medicine and reagent

The instrument comprises the following steps: high performance liquid chromatography (model DIONEX Ultimate 3000)

Reagent: acetonitrile (Merck, JA076530), DCC (TCI, AG7CH-QN), DCU (Aladdin, H1809065)

2. Experimental method

2.1 preparation of test solution: accurately weighing 125mg to 25ml of indapamide raw material medicine in a measuring flask, dissolving with acetonitrile, and diluting to a scale.

2.2 preparation of DCC control solution: accurately weighing DCC reference substance 75mg to 100ml measuring flask, dissolving with ethanol and diluting to scale as DCU reference substance stock solution, transferring reference substance stock solution 1ml to 100ml measuring flask, and diluting with acetonitrile to scale.

2.3 preparation of DCU control solution: accurately weighing DCU reference substance 75mg to 100ml measuring flask, dissolving with ethanol and diluting to scale as DCC reference substance stock solution, transferring 1ml to 100ml measuring flask of reference substance stock solution, and diluting to scale with acetonitrile.

2.4 preparation of DCC & DCU control solution: respectively transferring 1ml of DCC reference substance storage solution and DCU reference substance storage solution into the same 100ml measuring flask, and diluting with acetonitrile to scale to obtain reference substance solutions.

Example 1 determination of initial acetonitrile ratio in Mobile phase

Preparing a standard sample solution: DCC and DCU were weighed precisely at 75mg each and placed in the same 200ml measuring flask, diluted to the mark with acetonitrile as a control stock solution. And precisely weighing 125mg of the indapamide raw material medicine, placing the indapamide raw material medicine into a 25ml measuring flask, dissolving the indapamide raw material medicine by using the control stock solution, and diluting the indapamide raw material medicine to a scale to obtain a labeled sample solution.

Chromatographic conditions are as follows: and (3) chromatographic column: agela Venusil C18 Plus 4.6 × 200mm,5 μm; mobile phase: gradient elution with acetonitrile-water, gradually increasing acetonitrile ratio from 5% to 95% in 0-30 min; the column temperature is 35 ℃; the flow rate is 1.5 ml/min; the detection wavelength is 205 nm; the sample size was 10. mu.l, and the results are shown in FIG. 1.

And (4) conclusion: DCC does not peak

Example 2 determination of initial proportions of acetonitrile in the Mobile phase

Preparing a standard sample solution: DCC and DCU were weighed precisely at 75mg each and placed in the same 200ml measuring flask, diluted to the mark with acetonitrile and used as control stock solutions. And precisely weighing 125mg of the indapamide raw material medicine, placing the indapamide raw material medicine into a 25ml measuring flask, dissolving the indapamide raw material medicine by using the control stock solution, and diluting the indapamide raw material medicine to a scale to obtain a labeled sample solution.

Chromatographic conditions are as follows: a chromatographic column: agela Venusil C18 Plus 4.6 × 200mm,5 μm; mobile phase: gradient elution with acetonitrile-water, wherein the acetonitrile ratio is gradually increased from 10% to 95% in 0-30 min; the column temperature was 35 ℃; the flow rate is 1.5 ml/min; the detection wavelength is 205 nm; the amount of sample was 10. mu.l, and the results are shown in FIG. 2.

And (4) conclusion: DCU and DCC both generate peaks, and can be completely separated from other impurity peaks.

Example 3 determination of initial acetonitrile ratio in Mobile phase

Chromatographic conditions are as follows: and (3) chromatographic column: agela Venusil C18 Plus 4.6 × 200mm,5 μm; mobile phase: gradient elution with acetonitrile-water, wherein the acetonitrile ratio is gradually increased from 25% to 95% in 0-30 min; the column temperature is 35 ℃; the flow rate is 1.5 ml/min; the detection wavelength is 205 nm; the sample size was 10. mu.l, and the results are shown in FIG. 3.

And (4) conclusion: DCU and DCC both produce peaks, and can be completely separated from other impurity peaks.

Example 4 determination of initial proportions of acetonitrile in the Mobile phase

Chromatographic conditions are as follows: a chromatographic column: agela Venusil C18 Plus 4.6 × 200mm,5 μm; mobile phase: gradient elution with acetonitrile-water, wherein the acetonitrile ratio is gradually increased from 35% to 95% in 0-30 min; the column temperature was 35 ℃; the flow rate is 1.5 ml/min; the detection wavelength is 205 nm; the amount of sample was 10. mu.l, and the results are shown in FIG. 4.

EXAMPLE 5 determination of initial acetonitrile ratio in Mobile phase

Preparing a standard sample solution: DCC and DCU were weighed precisely at 75mg each and placed in the same 200ml measuring flask, diluted to the mark with acetonitrile as a control stock solution. And (3) precisely weighing 125mg of the indapamide raw material medicine, placing the indapamide raw material medicine into a 25ml measuring flask, dissolving the indapamide raw material medicine with the reference stock solution, and diluting the indapamide raw material medicine to a scale mark to obtain a labeled sample solution.

Chromatographic conditions are as follows: a chromatographic column: agela Venusil C18 Plus 4.6 × 200mm,5 μm; mobile phase: gradient elution with acetonitrile-water, gradually increasing acetonitrile ratio from 45% to 95% in 0-30 min; the column temperature was 35 ℃; the flow rate is 1.5 ml/min; the detection wavelength is 205 nm; the amount of the sample was 10. mu.l, and the results are shown in FIG. 5.

And (4) conclusion: DCU was not completely separated from the adjacent impurity peaks.

Example 6

The analytical method verified the results as follows:

chromatographic conditions are as follows: a chromatographic column: agela Venusil C18 Plus 4.6 × 200mm,5 μm; mobile phase: gradient elution with acetonitrile-water, wherein the acetonitrile ratio is gradually increased from 35% to 95% in 0-30 min; the column temperature is 35 ℃; the flow rate is 1.5 ml/min; the detection wavelength is 205 nm; the amount of sample was 10. mu.l.

1. Applicability of the system:

taking DCC & DCU reference substance solution to continuously repeat sample injection for 6 times, recording chromatogram peak area, and obtaining the following result:

TABLE 1 verification of System Adaptation

2. Specificity

Preparation of linear control stock solution: DCC and DCU were weighed precisely at 75mg each and placed in the same 200ml measuring flask, diluted to the mark with acetonitrile and used as 100% level control solutions.

Preparing a standard sample solution: indapamide bulk drug 125mg is precisely weighed into a 25ml measuring flask, dissolved by the above 100% level reference solution and diluted to the scale.

Respectively injecting a blank solvent (acetonitrile), a DCC reference solution, a DCU reference solution, a DCC & DCU reference solution and a standard sample solution into a high performance liquid chromatograph, and recording a chromatogram, wherein the result is as follows:

TABLE 2 verification results for specialization

3. The linear relationship is:

preparation of linear control stock solution: DCC and DCU were weighed precisely at 75mg each, placed in the same 200ml measuring flask, dissolved in ethanol and diluted to the scale, shaken well. Accurately transferring a proper amount of linear control stock solution, preparing linear solutions with different concentrations according to the following table, diluting the linear solutions to a scale with acetonitrile, and shaking up.

TABLE 3 preparation of solutions for each gradient in the Linear relationship

Level of	Linear stock solution volume (ml)	Constant volume (ml)
			LOQ(12.5％)	0.5	200
25％	1	200
			50％	1	100
100％	2	100
			150％	3	100
200％	2	50

Respectively injecting the linear control solutions with different concentrations into a liquid chromatograph, recording a chromatogram, respectively taking the concentration (mu g/mL) of DCC and the concentration (mu g/mL) of DCU as abscissa, taking the peak area y as ordinate, drawing a standard curve, and calculating a regression equation, wherein the result is as follows:

TABLE 4 verification of the Linear relationship

4. Accuracy:

preparing a background sample solution: accurately weighing 125mg of indapamide raw material medicine, placing the indapamide raw material medicine into a 25ml measuring flask, adding acetonitrile to dissolve and dilute the indapamide raw material medicine to a scale, and shaking up.

Preparing a standard sample solution: 125mg 9 parts of indapamide raw material medicine is precisely weighed, is placed in a 25ml measuring flask, is respectively dissolved and diluted to scales by 50 percent, 100 percent and 200 percent horizontal linear solutions (see a solution preparation table in a linear relation), is shaken up, and is prepared into 3 parts in parallel in each horizontal direction.

And (3) injecting the DCC & DCU reference substance solution, the background sample solution and the standard sample solution into a liquid chromatograph, recording a chromatogram, and calculating the recovery rate of 9 standard samples DCC and DCU according to the peak area by an external standard method, wherein the results are as follows:

TABLE 5 verification results of accuracy

5. Repeatability:

accurately weighing 125mg of indapamide raw material medicine, placing the indapamide raw material medicine into a 25ml volumetric flask, dissolving and diluting to a scale by using a 100% horizontal control solution in a solution preparation table in a linear relation, preparing 6 parts in parallel, using the solution as a standard sample solution, and calculating the recovery rate of DCC and DCU, wherein the result is as follows:

TABLE 6 verification of reproducibility

6. Intermediate precision:

another experimenter was replaced, 125mg of the indapamide drug substance was precisely weighed on a different date and placed in a 25ml measuring flask, and the 100% level control solution in the solution formulation table in the linear relationship was dissolved and diluted to a scale, and 6 parts were prepared in parallel as a spiked sample solution, and the recovery rates of DCC and DCU were calculated as follows:

TABLE 7 results of verification of intermediate precision

7. Detection limit:

gradually diluting DCC & DCU reference substance solution, injecting into a liquid chromatograph, recording chromatogram, and when the signal-to-noise ratio is about 3: the concentration at 1 is the detection limit, and the results are as follows:

table 8 test line experimental results

8. And (4) quantitative limit:

taking DCC & DCU reference substance solution for gradual dilution, injecting the solution into a liquid chromatograph, recording a chromatogram, and when the signal-to-noise ratio is about 10: the concentration at 1 is the limit of quantitation, and the results are as follows:

TABLE 9 Experimental results for quantitation limits

9. Solution stability:

preparing a standard sample solution: 125mg of the indapamide raw material drug is precisely weighed and placed in a 25ml measuring flask, and 100% horizontal control solution in a solution preparation table in a linear relation is dissolved and diluted to a scale to be used as a labeling sample solution.

Placing DCC & DCU reference substance solution and 100% standard sample solution at room temperature, injecting sample at different time points, recording chromatogram peak area, and obtaining the following results:

TABLE 10 verification of solution stability

10. Durability

Preparing a standard sample solution: accurately weighing 125mg of the indapamide raw material medicine, placing the indapamide raw material medicine into a 25ml measuring flask, dissolving the indapamide raw material medicine into 100% horizontal control solution in a solution preparation table in a linear relation, and diluting the solution to a scale to obtain a labeled sample solution.

Taking the standard sample solution, and respectively changing chromatographic conditions: the column temperature is +/-2 ℃; the flow rate is +/-0.2 ml/min; the initial proportion of the mobile phase is +/-2%; the test result of DCC sample adding and recovering is tested by the chromatographic column, and the test result is compared with the standard condition, and the result is as follows:

TABLE 11 verification of durability

The results of the analysis in the table above: the method provided by the invention conforms to ICH methodology verification and related guiding principle requirements of 2015 edition of Chinese pharmacopoeia, can accurately determine DCC and DCU content in the indapamide bulk drug, and provides a reliable detection method for quality control of the bulk drug.

Example 7

Chromatographic conditions are as follows: a chromatographic column: agela Venusil C₁₈Plus 4.6 × 200mm,5 μm; mobile phase: gradient elution with acetonitrile-water, wherein the acetonitrile ratio is gradually increased from 35% to 95% in 0-30 min; the column temperature was 35 ℃; the flow rate is 1.5 ml/min; the detection wavelength is 205 nm; the amount of sample was 10. mu.l.

Taking four different batches of indapamide raw material medicines to prepare test solution, respectively numbering 1-4, and respectively carrying out sample injection detection on the four test solution and reference solution according to the chromatographic conditions, wherein FIG. 6 is a chromatogram of the test solution numbered 1, FIG. 7 is a chromatogram of the test solution numbered 2, FIG. 8 is a chromatogram of the test solution numbered 3, FIG. 9 is a chromatogram of the test solution numbered 4, FIG. 10 is a chromatogram of DCC & DCU reference solution, FIG. 11 is a chromatogram of the DCC reference solution, FIG. 12 is a chromatogram of the DCU reference solution, the contents of DCC and DCU in the test solution are calculated according to an external standard method by comparing with peak areas of DCC and DCU substances in the reference solution, and the calculated contents of DCC and DCU are shown in the following table:

TABLE 12 content of DCC and DCU in Indapamide bulk drugs of different batches

Comparative example gas chromatography detection

Gas chromatography conditions: the stationary phase is a capillary column of 100% polydimethylsiloxane, and the specification is as follows: 30 m.times.0.25 mm.times.0.25 μm;

the carrier gas is nitrogen;

the temperature of a sample inlet is 320 ℃;

the temperature of the detector is 330 ℃;

the sample amount is 1 mul;

split-flow sample injection, split-flow ratio 10: 1;

the column flow rate is 1.5ml/min and is kept for 11.5min, and is increased to 3.5ml/min at 2ml/min and is kept for 15 min; the temperature raising procedure is that the initial temperature is 50 ℃, the temperature is kept for 2min, the temperature is raised to 230 ℃ at the speed of 40 ℃/min, the temperature is kept for 5min, the temperature is raised to 280 ℃ at the speed of 40 ℃/min, and the temperature is kept for 15 min;

background sample solutions and spiked sample solutions were prepared according to the formulation method in example 6 accuracy.

And (3) injecting the reference substance solution, the background sample solution and the standard sample solution into a gas chromatograph, recording a chromatogram, and calculating the recovery rate of 9 standard samples DCC and DCU according to the peak area of an external standard method, wherein the results are as follows:

the DCC and DCU recovery rates obtained by analysis of gas chromatography detection show that the DCC recovery rates are less than 80% and the DCU recovery rates are mostly less than 80%. Furthermore, the average recovery rate of the two is far lower than 90%, which indicates that the accuracy of detection by using a gas chromatography detection method cannot meet the detection requirement on the content of DCC and DCU in the indapamide.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.

Claims

1. The method for detecting the content of DCC and DCU in the indapamide bulk drug is characterized in that the DCC and DCU in the indapamide bulk drug are detected by using a high performance liquid chromatography, and the method comprises the following steps:

(1) preparing an indapamide raw material drug test solution;

(3) and (3) injecting samples, and respectively detecting the test solution in the step (1) and the reference solution in the step (2) according to the following conditions:

chromatographic conditions are as follows: mobile phase: acetonitrile-water; gradient elution: the proportion of acetonitrile gradually increases from 10-35% to 95% in 0-30 min; the column temperature is 33-37 ℃; the flow rate is 1.4-1.6 ml/min; the detection wavelength is 190-210 nm; the chromatographic column is a C18 liquid chromatographic column;

(4) and calculating peak areas corresponding to the DCC and DCU in the sample and the reference solution to obtain the content of the DCC and DCU.

2. The method for detecting the content of DCC and DCU in an indapamide bulk drug according to claim 1, wherein the chromatographic column in step (3) is Agela Venusil C18 Plus, 4.6 x 200mm,5 μm.

3. The method for detecting the content of DCC and DCU in an indapamide bulk drug according to claim 1, wherein the chromatographic column in step (3) is Agela Venusil XBP C18, 4.6 x 200mm,5 μm.

4. The method for detecting the content of DCC and DCU in the indapamide bulk drug according to claim 1, wherein the chromatographic conditions in step (3) are as follows: the amount of sample was 10. mu.l.

5. The method for detecting the content of DCC and DCU in an indapamide raw material drug according to claim 1, wherein the chromatographic conditions in step (3) are as follows: the detection wavelength was 205 nm.

6. The method for detecting the content of DCC and DCU in the indapamide bulk drug according to claim 1, wherein the chromatographic conditions in step (3) are as follows: the column temperature was 35 ℃; the flow rate is 1.5 ml/min; the detection wavelength was 205 nm.

7. The method for detecting the content of DCC and DCU in the indapamide bulk drug according to claim 1, wherein the concentration of the test solution of the indapamide bulk drug in step (1) is 3-7 mg/ml.

8. The method for detecting the content of DCC and DCU in an indapamide crude drug according to claim 1, wherein the concentration of DCC in step (2) is 5-10 μ g/ml, and the concentration of DCU is 5-10 μ g/ml.