CN117054543B

CN117054543B - Method for detecting genotoxic impurities in antihypertensive drug

Info

Publication number: CN117054543B
Application number: CN202310955111.7A
Authority: CN
Inventors: 孙跃军; 秦晓娜; 李陇刚; 谌宗永
Original assignee: Beijing Sunshine Nuohe Pharmaceutical Research Co ltd
Current assignee: Beijing Sunshine Nuohe Pharmaceutical Research Co ltd
Priority date: 2023-08-01
Filing date: 2023-08-01
Publication date: 2024-04-16
Anticipated expiration: 2043-08-01
Also published as: CN117054543A

Abstract

The invention discloses a detection method of genotoxic impurities in antihypertensive drugs. In particular to the detection of genotoxic impurities (o-chlorobenzaldehyde) in the antihypertensive drug amlodipine besylate. The method comprises the following steps: the content of o-chlorobenzaldehyde as a genotoxic compound B in amlodipine besylate is detected by a gas chromatography method. Research results show that the method for measuring the o-chlorobenzaldehyde in the amlodipine besylate raw material by adopting the gas chromatography has good system applicability and high sensitivity, and is suitable for detecting the o-chlorobenzaldehyde in the amlodipine besylate raw material.

Description

Method for detecting genotoxic impurities in antihypertensive drug

Technical Field

The invention belongs to the technical field of medicine analysis, and particularly relates to detection of genotoxic impurities (o-chlorobenzaldehyde) in amlodipine besylate as a antihypertensive agent.

Background

Amlodipine besylate (amodipine), also known as a complex activating, chemical name 3-ethyl-5-methyl-2- (2-aminoethoxymethyl) -4- (2-chlorophenyl) -1, 4-dihydro-6-methyl-3, 5-pyridinedicarboxylic acid ester benzenesulfonate, a long term calcium channel blocker developed by the american society of feuzter in the 90 s of the 20 th century for the treatment of cardiovascular diseases such as angina, hypertension and congestive cardiac arrest, is a drug consistently recommended by the FDA cardiorenal advisor committee of the united states for the treatment of hypertension.

The genotoxic impurity (genotoxic impurity) is a substance which can directly or indirectly damage DNA, cause gene mutation or have a tendency to cause cancer. It features that it can damage genetic material of human body at low concentration, and has mutagenicity and carcinogenicity, and can seriously threaten human health in the course of medication. According to ICH M7, the o-chlorobenzaldehyde has a warning structure and is controlled according to the 3 rd genotoxic impurities.

ICH M7 states that if mutagenic substances (genotoxic impurities) are impurities in commercially available chemicals, synthesis intermediates, or synthesis byproducts, applicants should use risk-based reasoning to determine which steps should be hazard-rated for such potential impurities, and if it is confirmed that mutagenic-rated steps for these impurities and byproducts are required during synthesis, risk-rated discussions are required.

The impurities are strictly controlled in the compound I, so that the content of the high-toxicity impurities in the crude drug can be controlled from the source, and the pressure of the subsequent process on the impurity removal capacity is reduced.

Through literature research, no relevant report on detection of genotoxic impurities (o-chlorobenzaldehyde) in amlodipine besylate at home and abroad has been found.

Disclosure of Invention

Aiming at the technical blank at present, the invention provides a method for detecting the content of o-chlorobenzaldehyde as a genotoxic impurity compound B in amlodipine besylate (3-ethyl-5-methyl-2- (2-aminoethoxy methyl) -4- (2-chlorophenyl) -1, 4-dihydro-6-methyl-3, 5-pyridine dicarboxylic acid ester benzenesulfonate, compound I).

The method for detecting the content of the o-chlorobenzaldehyde as the genotoxic impurity compound B in amlodipine besylate provided by the invention is to detect the content of the o-chlorobenzaldehyde as the genotoxic impurity compound B in amlodipine besylate by a gas chromatography method;

the method comprises the following steps:

1) Preparing o-chlorobenzaldehyde standard reference substance solution by taking isopropanol as a diluting solvent: precisely weighing an o-chlorobenzaldehyde standard substance, and quantitatively diluting with isopropanol to prepare a solution containing 10 mug (i.e. 10 mug/ml) of o-chlorobenzaldehyde in each 1ml of the solution serving as an o-chlorobenzaldehyde standard substance solution;

2) Preparing a sample solution by taking isopropanol as a diluting solvent: precisely weighing amlodipine besylate raw material, adding isopropanol, shaking, filtering, and taking the subsequent filtrate as a sample solution with the concentration of 100mg/ml;

3) And (3) detection: detecting the genotoxic compound B o-chlorobenzaldehyde in the sample solution by using a gas chromatograph; precisely measuring 1 mu l of each of an o-chlorobenzaldehyde standard reference solution and a test sample solution, directly injecting into a gas chromatograph, recording a chromatogram, and calculating the amount of o-chlorobenzaldehyde in the test sample according to an external standard method by using the peak area;

the gas chromatograph operating conditions were as follows:

chromatographic column: a capillary column taking 6% of cyanopropylphenyl-94% of dimethylpolysiloxane as a fixing solution is taken as a chromatographic column;

heating program: the initial column temperature is 37 ℃ to 43 ℃ and maintained for 5min, and the temperature is increased to 217 ℃ to 223 ℃ at 9 ℃ to 11 ℃ per minute and maintained for 10min;

sample inlet: the temperature is 217 ℃ to 223 ℃; the pre-column pressure is 14kpa to 16kpa; the column flow rate was 3.09ml/min; the split ratio is 2:1; the carrier gas is nitrogen;

a detector: a hydrogen Flame Ionization Detector (FID);

detector temperature: 250 ℃;

sample injection mode: directly sampling; the sample loading was 1.0. Mu.l.

In the step 3), the content of o-chlorobenzaldehyde in the test sample is calculated according to the following formula:

m pairs: weighing the reference substance, and mg; sample a: peak area of the test solution;

d sample: dilution factor of the sample solution; and A pair: peak area of control solution;

m samples: sample weight of the test sample, mg; and D, pairing: dilution of control solution

Research results show that the method for measuring the o-chlorobenzaldehyde in the amlodipine besylate raw material by adopting the gas chromatography has good system applicability and high sensitivity, and is suitable for detecting the o-chlorobenzaldehyde in the amlodipine besylate raw material.

Drawings

FIG. 1 is a graph of a control solution in example 1 of the present invention.

FIG. 2 is a linear spectrum of o-chlorobenzaldehyde measured in example 1 of the present invention.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1

1 instrument and reagent

Table 1: instrument and equipment

Name of the name	Model number	Branding/manufacturer
			Gas chromatograph	Shimadzu GC-2010	Shimadzu (Shimadzu)
Gas chromatograph	Shimadzu GC-2010Plus	Shimadzu (Shimadzu)
			Electronic balance	AB135-S	Meite Teler
Electronic balance	MS105DU	Meite Teler
			Electronic balance	R200D	Sidoris (Sidoris)

Table 2: reagent and reagent

2 method of drafting

2.1 preliminary proposed residual solvent inspection method

The boiling point of the component is higher, the direct sample injection method is adopted for determination, and the proposed detection method is as follows:

(1) Instrument setting parameters

And (5) a direct sample injection method.

Chromatographic column: the capillary column using 6% cyanopropylphenyl-94% dimethylpolysiloxane as the fixing liquid is a chromatographic column (30 m.0.53 mm.3.0 μm, such as OV-624 capillary column).

Heating program: the initial column temperature is 40 ℃, maintained for 5min, and heated to 220 ℃ at 10 ℃/min, and maintained for 10min;

sample inlet: the temperature is 220 ℃; the column front pressure is 15kpa; the column flow rate was 3.09ml/min; the split ratio is 2:1; the carrier gas is nitrogen;

a detector: a hydrogen Flame Ionization Detector (FID);

carrier gas: nitrogen gas; detector temperature: 250 ℃.

Sample injection mode: directly sampling; the amount of sample introduced was 1. Mu.l.

(2) Solution preparation

Standard stock solution: and (3) taking a proper amount of o-chlorobenzaldehyde standard substance, precisely weighing, and quantitatively diluting with isopropanol to prepare a solution with the concentration of about 100 mug o-chlorobenzaldehyde in each 1ml serving as a standard stock solution.

Standard control solution: precisely measuring 2ml of standard stock solution, placing into a 20ml measuring flask, diluting to scale with isopropanol, and shaking to obtain standard reference substance solution.

Test solution: about 200mg of the product is taken, precisely weighed, 2ml of isopropanol is added into the product in precise quantity, the product is shaken and filtered, and the subsequent filtrate is taken as a test sample solution. It is prepared in the immediate use.

(3) Assay

Precisely measuring 1 μl of each of the control solution and the sample solution, directly injecting into a gas chromatograph, and recording the chromatograms. The results were as follows:

the elution sequence of each solvent in the reference substance solution is as follows: isopropanol→o-chlorobenzaldehyde. The amount of o-chlorobenzaldehyde was calculated as peak area by the external standard method, respectively.

Calculation formula

(4) Limit:

o-chlorobenzaldehyde is a warning structure for genotoxic impurities, and has no defined PDE values, so that the limit of the genotoxic impurities is calculated on the basis of TTC values, i.e. the uptake at risk thereof is generally defined as Threshold of Toxicological Concern (TTC). The specific meaning is as follows: a TTC value of "1.5 μg/day", i.e. equivalent to 1.5 μg of genotoxic impurities taken daily, is considered to be an acceptable risk for most drugs (risk of carcinogenesis less than 100000 per 1). According to this threshold, acceptable impurity levels in the active agent can be calculated based on the expected daily intake.

The maximum daily dosage of amlodipine besylate is 14mg, and the limit of o-chlorobenzaldehyde is as follows:

1.5μg*100％/14mg/1000＝0.01％

3 method verification

3.1 applicability of the System, precision of sample introduction

(1) Preparation of the solution

(2) Operating procedure and acceptance criteria

The operation steps are as follows: respectively precisely measuring 1.0 μl of blank solution (isopropanol without o-chlorobenzaldehyde), standard reference solution and standard stock solution, directly injecting into gas chromatograph, and recording chromatogram.

Acceptance criteria: the blank solvent does not interfere with the assay; the separation degree between two peaks in the reference substance solution is not less than 1.5; the control solution is continuously injected for 6 needles, the RSD of the peak area of each solvent is less than 10%, and the RSD value of the retention time of each solvent is less than 2.0%.

(3) Results and conclusions

A. Separation degree investigation result

The separation degree inspection results are shown in the following table, and the chromatograms are shown in the attached figure 1.

Table 1: methodological verification-System suitability investigation results

As can be seen from fig. 1 and table 3: the blank solvent does not interfere with measurement, isopropanol and o-chlorobenzaldehyde in the reference solution sequentially show peaks, the separation degree is more than 1.5, and the theoretical plate number meets the requirements, so that the method has good system applicability.

B. Sample injection precision investigation result

The results of the o-chlorobenzaldehyde sample injection precision investigation are shown in the following table.

Table 2: methodology validation-sample injection precision peak area investigation results

Peak area	1	2	3	4	5	6	RSD(％)
								O-chlorobenzaldehyde	70689	74909	75224	76999	77307	76160	3.20

Table 3: methodology validation-sample introduction precision retention time investigation results

Retention time (min)	1	2	3	4	5	6	RSD(％)
								O-chlorobenzaldehyde	18.481	18.482	18.484	18.488	18.487	18.488	0.02

Conclusion: by adopting a direct sample injection method, the control solution is continuously injected for 6 times, the RSD of the o-chlorobenzaldehyde peak area is less than 10.0%, and the RSD value of the o-chlorobenzaldehyde retention time is less than 2.0%, which indicates that the sample injection precision is good.

In summary, the system applicability and the sample injection precision of the o-chlorobenzaldehyde detection method are good.

3.2 Linear Range

(1) Preparation of the solution

Precisely measuring 4ml of standard stock solution, placing into a 20ml measuring flask, diluting to scale with isopropanol, and shaking to obtain 200% standard curve solution.

3ml of standard stock solution is precisely measured, placed in a 20ml measuring flask, diluted to a scale with isopropanol, and shaken uniformly to obtain 150% standard curve solution.

3ml of standard stock solution is precisely measured, placed in a 25ml measuring flask, diluted to a scale with isopropanol, and shaken uniformly to obtain 120% standard curve solution.

Precisely measuring 2ml of standard stock solution, placing into a 20ml measuring flask, diluting to scale with isopropanol, and shaking to obtain 100% standard curve solution.

Precisely measuring 2ml of standard stock solution, placing into a 25ml measuring flask, diluting to scale with isopropanol, and shaking to obtain 80% standard curve solution.

Precisely measuring 5ml of 120% standard curve solution, placing in a 10ml measuring flask, diluting to scale with isopropanol, and shaking to obtain 60% standard curve solution.

Precisely measuring 5ml of 80% standard curve solution, placing in a 10ml measuring flask, diluting to scale with isopropanol, and shaking to obtain 40% standard curve solution.

Precisely measuring 5ml of 40% standard curve solution, placing in a 10ml measuring flask, diluting to scale with isopropanol, and shaking to obtain 20% standard curve solution.

Precisely measuring 5ml of 20% standard curve solution, placing in a 10ml measuring flask, diluting to scale with isopropanol, and shaking to obtain 10% standard curve solution.

(2) Operating procedure and acceptance criteria

The operation steps are as follows: 1.0 μl of each standard curve solution was precisely measured and directly injected into a gas chromatograph, and the chromatogram was recorded.

Acceptance criteria: the linear regression coefficient is not less than 0.99 in the range of the examined concentration (10% -200% of the limit concentration).

(3) Results and conclusions

The results of linear regression were shown in the following table with concentration and main peak area, and the chromatogram is shown in FIG. 2.

Table 1: methodological validation-linear test results

Conclusion: o-chlorobenzaldehyde has good linear relation between concentration and peak area within the range of 1.015-20.30 mug/ml.

3.3 quantitative limit

(1) Preparation of the solution

O-chlorobenzaldehyde quantitative limiting solution: and (3) taking a 10% standard curve solution under a linear term, and gradually diluting until the signal to noise ratio of a chromatographic peak of the solvent is greater than 10, wherein the standard curve solution is used as a quantitative limiting solution of o-chlorobenzaldehyde.

(2) Operating procedure and acceptance criteria

The operation steps are as follows: 1.0 μl of the quantitative limiting solution was precisely measured and directly injected into a gas chromatograph, and the chromatogram was recorded.

Acceptance criteria: in the quantitative limiting solution, the signal to noise ratio of the o-chlorobenzaldehyde chromatographic peak is more than 10; the concentration is less than the check limit concentration.

(3) Results and conclusions

The quantitative limit examination results are shown in the following table.

Table 1: methodological validation-quantitative limit test results

Solvent(s)	Limit of quantification (μg/ml)	Corresponds to the concentration of the test sample (%)	Limitation (%)	S/N
					O-chlorobenzaldehyde	0.2538	0.00025	0.01％	14.48

Conclusion: the quantitative limit of the detection method of o-chlorobenzaldehyde in amlodipine besylate raw material can meet the detection requirement.

3.4 detection limit

(1) Preparation of the solution

O-chlorobenzaldehyde detection limit solution: and (3) taking a 10% standard curve solution under a linear item, and gradually diluting until the signal to noise ratio of a chromatographic peak of the solvent is greater than 3, and taking the solution as a detection limit solution of o-chlorobenzaldehyde.

(2) Operating procedure and acceptance criteria

The operation steps are as follows: 1.0 mu l of detection limit solution is precisely measured respectively, directly injected into a gas chromatograph, and a chromatogram is recorded.

Acceptance criteria: in the detection limit solution, the signal to noise ratio of the o-chlorobenzaldehyde peak is more than 3; the concentrations were all less than the check limit concentration.

(3) Results and conclusions

The detection limit inspection results are shown in the following table.

Table 1: methodological validation-limit of detection test results

Solvent(s)	Detection limit (mug/ml)	Corresponds to the concentration of the test sample (%)	Limitation (%)	S/N
					O-chlorobenzaldehyde	0.06345	0.000063	0.01％	3.86

Conclusion: the detection limit of the detection method of o-chlorobenzaldehyde in the amlodipine besylate raw material can meet the detection requirement.

3.5 accuracy

(1) Preparation of the solution

3ml of standard stock solution is precisely measured, placed in a 25ml measuring flask, diluted to a scale with isopropanol, and shaken well to obtain 120% of diluent. 2ml of stock solution is precisely measured by the same method, and is respectively put into a 20ml measuring flask and a 25ml measuring flask, diluted to the scale by isopropanol, and uniformly shaken to obtain 100 percent and 80 percent of diluted solution.

80% accuracy solution: precisely weighing about 200mg of C-031812001 batch, precisely adding 2ml of 80% diluent, shaking and filtering to obtain the final product. Preparing 3 parts by the same method; it is prepared in the immediate use.

100% accuracy solution: precisely weighing about 200mg of C-031812001 batch, precisely adding 2ml of 100% diluent, shaking and filtering to obtain the final product. Preparing 3 parts by the same method; it is prepared in the immediate use.

120% accuracy solution: precisely weighing about 200mg of C-031812001 batch, precisely adding 2ml of 120% diluent, shaking and filtering to obtain the final product. Preparing 3 parts by the same method; it is prepared in the immediate use.

Preparing an unlabeled test sample solution: precisely weighing about 200mg of C-031812001 batch, precisely adding 2ml of isopropanol, shaking and filtering to obtain a sample solution. It is prepared in the immediate use.

(2) Operating procedure and acceptance criteria

The operation steps are as follows: 1.0 μl of each solution was measured precisely, directly injected into a gas chromatograph, and the chromatogram was recorded.

Acceptance criteria: in the investigation of the designed concentration (80%, 100% and 120% of the limit concentration), the average recovery rate of the o-chlorobenzaldehyde is between 80% and 120%, and the RSD of 9 parts of recovery rates is less than 10%.

(3) Results and conclusions

The recovery rate of o-chlorobenzaldehyde was calculated as peak area by the external standard method based on recovery = (measured amount-content in sample)/addition amount. The results are shown in the following table.

Table 1: methodological validation-test article measurement results

Lot number	C-031812001(％)
		O-chlorobenzaldehyde	Not detected

Table 2: methodological verification-accuracy test results

Conclusion: the result shows that nine parts of recovery rate of the o-chlorobenzaldehyde is in the range of 80% -120%, the RSD value is less than 10.0%, and the accuracy of the o-chlorobenzaldehyde determination by the method is good.

3.6 repeatability

Because o-chlorobenzaldehyde is not detected by the sample solution, the repeatability of the method is not easy to evaluate, six samples of reference sample solution serving as a standard sample solution are prepared in parallel, RSD of the o-chlorobenzaldehyde content is calculated and measured, and the repeatability of the method is evaluated, wherein the specific test is as follows:

(1) Preparation of the solution

Standard control solution: precisely measuring 2ml of standard stock solution, placing into a 20ml measuring flask, diluting with isopropanol to scale, and shaking to obtain standard reference solution

Adding a labeled test sample solution: precisely weighing about 200mg of C-031812001 batch, precisely adding 2ml of standard reference substance solution, shaking and filtering. 6 parts of the components are prepared by the same method; it is prepared in the immediate use.

(2) Procedure and acceptance criteria

The operation steps are as follows: precisely measuring 1.0 μl of each of the blank solvent, standard reference substance solution and standard test substance solution, directly injecting into gas chromatograph, and recording chromatogram.

Acceptance criteria: the blank solvent does not interfere with the assay; the separation degree of each solvent in the reference substance solution is not less than 1.5; the RSD of the o-chlorobenzaldehyde content of the six test sample solutions is less than 10 percent.

(3) Results and conclusions

The repeatability results are shown in the following table.

Table 1: methodological validation-repeatability investigation results

Solvent(s)	1	2	3	4	5	6	RSD(％)
								O-chlorobenzaldehyde	0.0098	0.0106	0.0098	0.0094	0.0099	0.0097	4.02

Conclusion: by adopting a direct sample injection method, six parts of sample solutions are continuously measured, and RSD of the o-chlorobenzaldehyde is less than 10.0%, which shows that the method has good repeatability.

3.7 solution stability

Because o-chlorobenzaldehyde is not detected by the sample solution, the solution stability of the method is not easy to evaluate, so that the reference sample solution is added into the sample as a standard sample solution, the RSD of the peak area of the o-chlorobenzaldehyde is calculated and measured, and the solution stability of the method is evaluated, wherein the specific test is as follows:

(1) Preparation of the solution

Adding a labeled test sample solution: precisely weighing about 200mg of C-031812001 batch, precisely adding 2ml of standard reference substance solution, shaking and filtering.

(2) Procedure and acceptance criteria

The operation steps are as follows: feeding the same standard reference substance solution or standard sample solution at the time points of 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h and 12h, precisely measuring 1 μl of each standard reference substance solution or standard sample solution, directly injecting into a gas chromatograph, and recording a chromatogram.

Acceptance criteria: and feeding the same standard reference substance solution or standard test substance solution at the time points of 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h and 12h, wherein the RSD of the peak areas of the o-chlorobenzaldehyde detected by the standard reference substance solution and the standard test substance solution is less than 15%.

(3) Results and conclusions

And feeding the same standard reference substance solution or standard test substance solution at the time points of 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h and 12h, wherein the solution stability inspection results are shown in the following table.

Table 1: methodological validation-standard control solution stability investigation results

Table 2: methodological verification-solution stability investigation result of addition of standard test sample solution

Conclusion: feeding the same standard reference substance solution or standard test substance solution at the time points of 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h and 12h, wherein the RSD of the peak area of o-chlorobenzaldehyde in the standard reference substance solution is less than 15%; the RSD value of the peak area of the o-chlorobenzaldehyde in the standard sample solution is more than 15%, and the peak area is gradually reduced. Therefore, the standard reference substance solution has good stability for 12h, and the sample solution needs to be prepared in situ.

3.8 specificity

(1) Preparation of the solution

(2) Procedure and acceptance criteria

The operation steps are as follows: 1.0 mu l of each of the blank solution and the standard reference solution is precisely measured and directly injected into a gas chromatograph, and a chromatogram is recorded.

Acceptance criteria: each solvent was undisturbed in the detection of this method.

(3) Results and conclusions

Conclusion: the blank has no interference, and all solvent peaks can be completely separated, namely, the specificity is good.

3.9 durability

After slightly changing the pre-column pressure (+ -0.2 psi), the temperature rising rate (+ -1 ℃/min), the initial temperature (+ -3 ℃) of the column box and the temperature of the sample inlet (+ -3 ℃) of the column box, the influence of the separation degree result is examined, and the specific test is as follows:

(1) Preparation of the solution

(2) Procedure and acceptance criteria

The operation steps are as follows: after slightly changing the pre-column pressure (+ -1 kpa), the heating rate (+ -1 ℃/min), the initial temperature (+ -3 ℃) of a column box and the temperature (+ -3 ℃) of a sample inlet, precisely measuring 1.0 mu l of each of a blank solvent and a reference solution, directly injecting the solution into a gas chromatograph, and recording a chromatogram.

Acceptance criteria: after slightly changing the pre-column pressure (+ -1 kpa), the heating rate (+ -1 ℃/min), the initial temperature (+ -3 ℃) of a column box and the temperature (+ -3 ℃) of a sample inlet, the blank solvent does not interfere with the measurement; the separation degree between o-chlorobenzaldehyde and the blank solvent in the reference solution should be not less than 1.5.

(3) Results and conclusions

A. Investigation of durability of different column front pressures

The durability test results are shown in the following table after a slight change in the pre-column pressure (+ -1 kpa).

Table 1: methodological verification-durability investigation results of different pre-column pressure separations

Conclusion: after the pre-column pressure (+/-1 kpa) is slightly changed, the retention time of the o-chlorobenzaldehyde peak and the separation degree between the o-chlorobenzaldehyde and the solvent peak slightly change, but the requirement of system applicability is still met, so that the pre-column pressure durability of the o-chlorobenzaldehyde inspection method is better, the pre-column pressure can be selected to be 14kpa-16kpa, and the o-chlorobenzaldehyde product can be inspected.

B. Durability inspection of different column boxes at different heating rates

The durability test results are shown in the following table after a slight change in the column box temperature rise rate (+ -1.0 ℃/min).

Table 1: methodology validation-separation degree durability investigation results of different column boxes at different heating rates

Conclusion: after the temperature rising rate of the column box (+/-1.0 ℃/min) is slightly changed, the retention time of the o-chlorobenzaldehyde peak and the separation degree between the o-chlorobenzaldehyde and the solvent peak slightly change, but still meet the requirement of system applicability, so that the column box temperature rising rate pressure durability of the o-chlorobenzaldehyde inspection method is better, the column box temperature rising rate can be selected to be 9 ℃/min-11 ℃/min, and the o-chlorobenzaldehyde inspection can be performed.

C. Investigation of initial temperature durability of different column boxes

The durability test results are shown in the following table after a slight change in the initial temperature (+ -3 ℃) of the column box.

Table 1: methodological verification-results of durability investigation of initial temperature separation of different bins

Conclusion: after the initial temperature (+/-3 ℃) of the column box is slightly changed, the retention time of the o-chlorobenzaldehyde peak and the separation degree between the o-chlorobenzaldehyde and the solvent peak are slightly changed, but the requirement of system applicability is still met, so that the initial temperature and pressure durability of the column box of the o-chlorobenzaldehyde inspection method are better, the initial temperature of the column box can be selected to be 37 ℃ -43 ℃, and the o-chlorobenzaldehyde can be inspected.

D. Investigation of temperature durability of different sample inlets

The durability test results are shown in the following table after slightly changing the sample inlet temperature (+ -3 ℃).

Table 1: methodological verification-durability investigation results of different headspace equilibrium temperature separations

Conclusion: after the temperature of the sample inlet (+/-3 ℃) is slightly changed, the retention time of the o-chlorobenzaldehyde peak and the separation degree between the o-chlorobenzaldehyde and the solvent peak are slightly changed, but the requirement of system applicability is still met, so that the sample inlet temperature and pressure durability of the o-chlorobenzaldehyde inspection method is better, the temperature of the sample inlet can be selected to be 217 ℃ -223 ℃, and the o-chlorobenzaldehyde can be inspected.

4 test article detection

The results of the analytical method for determining genotoxic impurities by taking amlodipine besylate samples are shown in the following table.

Table 1: test results of test sample

Lot number	C-031812001	C-031812002	C-031808001	C-031610004	Limit of
						O-chlorobenzaldehyde	Not detected	Not detected	Not detected	Not detected	0.01％

Conclusion: the four batches of samples of the product do not detect o-chlorobenzaldehyde and meet the limit regulation, which indicates that the preparation process of the product is stable, the residue of o-chlorobenzaldehyde can be effectively removed, and the measurement results of the batch of raw materials in pilot scale are less than 30% of the limit, so that the raw materials are not limited to the quality standard.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

Claims

1. A method for detecting the content of o-chlorobenzaldehyde as a genotoxic impurity compound in amlodipine besylate is characterized by detecting the content of o-chlorobenzaldehyde as the genotoxic impurity compound in amlodipine besylate by a gas chromatography method;

the method comprises the following steps:

1) Preparing o-chlorobenzaldehyde standard reference substance solution by taking isopropanol as a diluting solvent;

2) Preparing a sample solution by taking isopropanol as a diluting solvent;

3) Precisely measuring an o-chlorobenzaldehyde standard reference solution and a sample solution, directly injecting the o-chlorobenzaldehyde standard reference solution and the sample solution into a gas chromatograph, recording a chromatogram, and calculating the amount of o-chlorobenzaldehyde in the sample according to an external standard method by using a peak area;

the gas chromatograph operating conditions were as follows:

a detector: a hydrogen flame ionization detector;

detector temperature: 250 ℃;

sample injection mode: directly sampling; the sample loading was 1.0. Mu.l.

2. The method according to claim 1, wherein in step 3), the o-chlorobenzaldehyde content of the test sample is calculated according to the following formula:

content of o-chlorobenzaldehyde =

m samples: sample weight of the test sample, mg; and D, pairing: dilution of the control solution.

3. Use of the method according to claim 1 or 2 for quality control of amlodipine besylate.