CN109856303B

CN109856303B - High-sensitivity analysis method for genotoxic impurities in pantoprazole sodium

Info

Publication number: CN109856303B
Application number: CN201910037845.0A
Authority: CN
Inventors: 胡方剑; 徐莎; 毛玮; 张昀; 周明
Original assignee: Hangzhou Huadong Medicine Group Biopharmaceutical Co ltd; Hangzhou Zhongmei Huadong Pharmaceutical Co Ltd
Current assignee: Hangzhou Huadong Medicine Group Biopharmaceutical Co ltd; Hangzhou Zhongmei Huadong Pharmaceutical Co Ltd
Priority date: 2019-01-16
Filing date: 2019-01-16
Publication date: 2021-09-03
Anticipated expiration: 2039-01-16
Also published as: CN109856303A

Abstract

The invention discloses a high-sensitivity analysis method of genotoxic impurity 2-chloromethyl-3, 4-dimethoxypyridine hydrochloride (CMDP) in pantoprazole sodium. The method adopts a high performance liquid chromatography-mass spectrometry combined analysis method, uses a mixed solution of ammonium bicarbonate solution and acetonitrile to dilute a sample, uses volatile carbonate solution and acetonitrile as mobile phases, has detection limit and quantification limit of 0.2ppm and 0.6ppm respectively, can separate and detect the CMDP impurity more efficiently, and has the advantages of strong specificity, high sensitivity, simplicity, rapidness and low cost.

Description

High-sensitivity analysis method for genotoxic impurities in pantoprazole sodium

Technical Field

The invention belongs to the technical field of drug analysis and detection, and particularly relates to an analysis method of genotoxic impurities in pantoprazole sodium, and more particularly relates to a method for separating and quantitatively determining 2-chloromethyl-3, 4-dimethoxypyridine hydrochloride (CMDP) by using a high performance liquid chromatography-mass spectrometry combined method.

Background

Pantoprazole sodium is a novel proton pump inhibitor, is used for treating gastric ulcer, duodenal ulcer, esophagitis and the like, and has the characteristics of safety, reliability and small toxic and side effects.

The compound 2-chloromethyl-3, 4-dimethoxypyridine hydrochloride (CMDP) is an important intermediate of pantoprazole sodium, and is a genotoxic impurity, and the residue of the impurity must be strictly controlled. However, the quality standard of the impurities is not recorded in the domestic and foreign pharmacopoeias. According to the rules of the guidelines for the limits of genotoxic impurities issued by the European pharmacopoeia administration (EMEA), the maximum limit of the intake of genotoxic impurities is 1.5 ug/day, according to the Threshold of Toxicological Concerns (TTC) as the threshold for evaluating the majority of genotoxic impurities. The control limit of this impurity was 6.25ppm based on a maximum pantoprazole sodium dose of 240 mg/day (40 mg/tablet, 3 times/day, two tablets at a time). The detection limit is generally about 0.1 of the control limit according to the detection requirement, so the detection limit is below 0.6 ppm. Conventional gas-phase and liquid-phase detection methods cannot meet the detection limit requirement, such as the method for detecting pantoprazole or impurities A-F thereof in a pantoprazole sodium raw material or preparation disclosed in the prior art. In addition, pantoprazole sodium and CMDP are easily degraded, which affects the accuracy of the detection result and increases the difficulty of detection.

The literature Chromatogaphia (2008), 68(5/6), 481-484 discloses a method for detecting CMDP in pantoprazole sodium by GC-MS and RP-LC. Wherein the detection limit of the GC-MS method is 1ppm, the detection limit of the RP-LC method is 3ppm, the detection sensitivity is poor, and the detection requirement cannot be met.

The Journal of pharmaceutical and biological Analysis (2012), 70, 592-. The method can meet the detection requirements, but the method has expensive equipment and complex operation and is not suitable for industrial production.

Therefore, in order to strictly control the content of CMDP, it is an urgent problem to develop a detection and analysis method with high sensitivity, low cost and easy operation.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a high-sensitivity analysis method for genotoxic impurities (CMDP) in pantoprazole sodium. The inventor of the application explores a detection method which meets the requirement of low detection limit and gives consideration to the separation of main components and impurities by repeatedly screening conditions such as a mobile phase, an elution mode, a mass spectrum and the like by using high performance liquid chromatography-mass spectrometry combined equipment, and achieves the purposes of simply, conveniently and effectively separating and analyzing CMDP.

The purpose of the invention can be realized by the following technical scheme:

the invention provides a high-sensitivity analysis method of genotoxic impurity 2-chloromethyl-3, 4-dimethoxypyridine hydrochloride in pantoprazole sodium, which is characterized by comprising the following steps: after the pantoprazole sodium sample is diluted by a diluent, a high performance liquid chromatography-mass spectrometry combined method is adopted, and a carbonate solution and acetonitrile are used as mobile phases for elution.

In a preferred embodiment, the carbonate solution is ammonium bicarbonate solution or ammonium carbonate solution. Because pantoprazole is damaged under the conditions of acid, strong alkali, heating and the like, CMDP is degraded under the acidic condition, and the pH value of a diluent and a mobile phase needs to be strictly controlled in order to ensure the detection accuracy. In the screening process of various alkali or salt solutions, the inventor of the application finds that when the pH of the diluent and the mobile phase is adjusted by adding an alkali buffer solution, the slight difference in the preparation process of the buffer solution causes great difference on the measurement result, so that the pH of the diluent and the mobile phase is adjusted by selecting a single alkali or salt solution. After a large number of screening tests, the pH value can be accurately and conveniently adjusted to be proper alkalescence by adopting volatile carbonate solution, and meanwhile, the stability of a sample in the determination process can be ensured, and the detection requirement can be met.

In a preferred embodiment, the carbonate solution has a concentration of 0.01 to 0.03mol/L, preferably 0.02 mol/L.

In a preferred embodiment, the diluent is a mixed solution of ammonium bicarbonate solution and acetonitrile, and the concentration of the diluted sample is 2 mg/mL.

In a preferred embodiment, the volume ratio of ammonium bicarbonate solution to acetonitrile in the diluent is 5-10: 1, preferably 9: 1.

In a preferred embodiment, the mobile phase is eluted in a gradient elution with a carbonate content of 20-80% by volume in the eluent. By adopting gradient elution and adjusting the proportion of carbonate solution and acetonitrile in the mobile phase, the separation capacity of the system can be improved, the analysis time is shortened, the CMDP is better separated from other components, and the stability of the sample is more effectively ensured.

In a preferred embodiment, the conditions of the gradient elution are:

time (minutes)	Carbonate solution (% V/V)	Acetonitrile (% V/V)
			0	80	20
7	55	45
			10	20	80
10.01	80	20
			15	80	20

In a preferable scheme, the elution rate of the mobile phase is 0.8-1.2 mL/min, and the temperature of the chromatographic column is 38-42 ℃.

In a preferred embodiment, the column used for HPLC is an Agilent Proshell HPH C18 column, 150 x 4.6mm, 2.7 μm.

In a preferred embodiment, the ionization technique of the ion source of the mass spectrum is electrospray ionization, the spray voltage is 3KV, the pressure in the atomizing chamber is 35psi, and the temperature of the drying gas is 350 ℃.

Further, the mass spectrometry conditions are shown in the following table:

compared with the prior art, the method has the following beneficial effects:

1. the analysis method provided by the invention can effectively detect the toxic impurity CMDP of the pantoprazole sodium gene, and has strong specificity;

2. the analysis method provided by the invention can carry out quantitative analysis on CMDP, meets the requirement of 0.6ppm of detection limit, and has high sensitivity;

3. the analysis method provided by the invention is simple to operate, short in time consumption, high in detection accuracy, and capable of reducing the decomposition of the sample;

4. the CMDP detection and analysis method provided by the invention can improve the quality and the medication safety of pantoprazole sodium;

5. the CMDP detection and analysis method provided by the invention provides a good reference for the detection of the genotoxic impurity CMDP in other medicines which may contain CMDP.

Drawings

FIG. 1 is a mass spectrum of a 150301PS batch sample in example 1;

FIG. 2 is a linear relationship diagram of CMDP in the linear examination of example 2.

Detailed Description

The invention is further illustrated with reference to specific examples, which are not intended to limit the invention in any way.

1. The instrument comprises the following steps:

analytical balance, model: sartorius CPA225D (d ═ 0.01 mg); BSA224S (d ═ 0.1 mg);

the high performance liquid chromatograph has model number of Agilent Infinity1260, chromatographic column of Agilent Proshell HPH C18, 4.6 × 150mm, 2.7um, and octadecyl bonded silica gel as filler;

the mass spectrum model is as follows: agilent 6120 single quadrupole mass spectrometer.

2. Reagent:

acetonitrile (chromatographically pure), manufacturer: fisher

Ammonium bicarbonate (chromatographically pure), manufacturer: MREDA

Water (ultrapure water), manufacturer: drech's series

2-chloromethyl-3, 4-dimethoxypyridine hydrochloride (CMDP), batch No.: 09514HDV, Source: sigma, purity: 97 percent

3. Sample preparation:

pantoprazole sodium, batch No.: 150301PS, 150302PS, 150303PS, 150701PS, 150702PS, 150703 PS.

Example 1

Chromatographic conditions are as follows:

mobile phase: 0.02mol/L ammonium bicarbonate solution is used as a mobile phase A, and acetonitrile is used as a mobile phase B;

column temperature: 40 ℃;

flow rate: 1.0 mL/min;

sample introduction amount: 10 uL;

the elution conditions are shown in table 1 below:

TABLE 1

1. Solution preparation and detection

A. Preparation of a test solution:

diluent agent:

3.16g of ammonium bicarbonate solid is taken, water is added to 2L to obtain 0.02mol/L ammonium bicarbonate solution, 900mL of 0.02mol/L ammonium bicarbonate solution is taken, and 100mL of acetonitrile is added to obtain the diluent.

Test solution:

taking a proper amount of a test sample (about 20mg of pantoprazole sodium), precisely weighing, placing into a 10mL measuring flask, adding a diluent to dissolve, fixing the volume, and shaking up.

B. Preparation of control solutions:

taking a CMDP impurity reference substance of about 10mg, precisely weighing, placing in a 10mL measuring flask, adding an appropriate amount of acetonitrile, ultrasonically dissolving, and diluting to a scale with the acetonitrile; precisely measuring 0.5mL, placing in a 50mL measuring flask, adding acetonitrile to dilute to scale, shaking up, precisely measuring 0.6mL, placing in a 50mL measuring flask, adding diluent to scale, shaking up to obtain a solution with a concentration of about 120ng/mL, and using the solution as a CMDP control solution.

C. The detection method comprises the following steps:

and respectively taking 0.2mL, 1.0mL, 1.3mL, 2mL and 2.4mL of CMDP reference solution, and putting the CMDP reference solution into a 20mL measuring flask to be added with the diluent for constant volume. And (3) injecting 10 mu L of each solution into a liquid chromatography-mass spectrometer, and recording the SIM mode spectrum. And performing linear regression by using the peak area of CMDP in the SIM mode to establish a standard curve. And injecting 10 mu L of test solution into a liquid chromatography-mass spectrometer, and recording the SIM mode spectrum. And calculating the CMDP content in the test sample according to the standard curve.

2. Measurement results

According to the method, 6 batches of pantoprazole sodium samples are detected, and the results are shown in table 2:

TABLE 2

Example 2 methodological validation

1. Specialization inspection

A. Solution preparation

CMDP control solution: CMDP control solutions were prepared as in example 1.

Other impurity control solutions: formulated according to the limits specified in USP 40. Taking a proper amount of mixed pantoprazole sodium impurities A, B, C, D and F and an appropriate amount of impurity E reference substances, respectively adding a diluent to quantitatively dilute into solutions containing about 40ug of impurity A, about 30ug of impurity B, about 20ug of impurity C, about 40ug of mixed impurity D and F and about 20ug of impurity E in each 1mL, shaking up, and respectively using as reference substance solutions of each impurity.

Impurity localization solution: and (3) respectively taking 1mL of each impurity reference substance solution, placing the solution into a 10mL measuring flask, adding a diluent to a constant volume to a scale, and respectively taking the solution as each impurity positioning solution.

Mixing the solution: taking a proper amount of pantoprazole sodium sesquihydrate (about 20mg of pantoprazole sodium), precisely weighing, placing in a 10mL measuring flask, respectively adding 1mL of each impurity reference solution, adding a diluent for constant volume, and shaking up to obtain a mixed solution.

Blank solvent: and taking a diluent as a blank solvent.

B. Detection and results

The blank solvent, each impurity-localized solution, and each mixed solution 10uL were injected into the LC-MS, and the spectra were recorded, as shown in Table 3 below.

TABLE 3

Sample (I)	The result of the detection
		Blank solvent	Does not peak
CMDP	6.785min, m/z187.9 ion peak
		Impurity A	Does not peak
Impurity B	Does not peak
		Impurity C	Does not peak
Impurity D&F	Does not peak
		Impurity E	Does not peak
Mixed solution	6.785min, m/z187.9 ion peak

The result shows that the main component of pantoprazole sodium, the impurities A-F and the blank solvent do not interfere with the detection of the impurity CMDP, and the method has good specificity.

2. Detection limit and quantitative limit investigation

Accurately measuring appropriate amount of CMDP reference solution prepared during the specificity test, adding diluent for quantitative dilution, and making into solutions containing CMDP1.2ng and 0.4ng per 1mL respectively.

Precisely measuring 10uL of each solution with different concentrations, respectively injecting into a liquid chromatograph-mass spectrometer, taking the solution with the signal-to-noise ratio of the ion peak in the SIM mode of 10: 1 as a quantitative limiting solution, and taking the solution with the signal-to-noise ratio of 3: 1 as a detection limiting solution. The spectra were recorded and the results are shown in Table 4.

TABLE 4

The result shows that when the concentration of the test sample is 2mg/mL, the CMDP containing 0.2ppm can be detected, and the CMDP containing 0.6ppm can be accurately quantified, which indicates that the method has high detection sensitivity and meets the detection requirement of 6.25ppm of control limit.

3. Linear survey

12ng/mL was used as a 100% limiting concentration solution. CMDP reference solution 0.2mL, 1.0mL, 1.6mL, 2mL, 2.4mL and 3.0mL are precisely measured, respectively placed in 20mL measuring bottles, diluted to the scale with diluent, and made into solutions with limit concentrations of quantitative Limit (LOQ), 50%, 80%, 100%, 120% and 150% as linear solutions.

And precisely measuring 10uL of the linear solution, respectively injecting into a liquid chromatograph-mass spectrometer, and recording the spectrum. The results are given in Table 5 below.

TABLE 5

Series of	Concentration (ng/L)	Area of main peak
			LOQ	1.233	1363.9
50％	6.163	6455
			80％	9.861	10653.8
100％	12.327	13167.7
			120％	14.792	15643
150％	18.490	19216.7

And taking the concentration as an abscissa and the peak area as an ordinate to make a linear regression curve, wherein the linear equation is that Y is 104.2X +161.1, and the correlation coefficient R is 0.999. The CMDP is shown in the concentration range of 1.233-18.490 ng/mL, and the solution concentration and the peak area have a good linear relationship.

4. Accuracy and precision

Accuracy of

The recovery rates of CMDP at 80%, 100% and 120% of the 100% limit concentration were between 90.0% and 105.0%, the evaluated recovery rate was 99.49%, and the RSD was 2.2(n ═ 9), indicating high accuracy of the method.

Precision degree

Sample introduction precision: the CMDP quantitative limit determines the concentration solution, namely 1.233ng/mL concentration solution is continuously injected for 6 times, and the peak area RSD of the SIM ion peak is 6.3 percent, which indicates that the method has high injection precision;

repeatability: 6 parts of a test standard solution with 100% limit concentration prepared from the same batch of test articles (the same batch of test articles about containing pantoprazole 20mg are taken and precisely weighed, the test articles are placed in a 10mL measuring flask, 1.0mL of CMDP mother solution is added with a diluent to be dissolved and diluted to scale, the test is shaken up), the test is repeated for 6 times, the detected amount of CMDP measured for 6 times is 6.365-6.516 ppm, the RSD is 0.9% (n is 6), the test results for 6 times are consistent, and the method has good repeatability.

The RSD of 12 detection results measured at different times by 6 parts of test standard-adding mixed solution with 100% limit concentration prepared by different researchers is 1.2%, and the method has high intermediate precision.

5. Durability

When the chromatographic conditions are slightly changed (the column temperature is 40 +/-2 ℃, the flow rate is 1.0mL/min +/-0.2 mL/min and different chromatographic columns are replaced), the method has no influence on the measurement result basically, the average detection amount of CMDP in the test standard solution with 100% limit concentration measured under different conditions is 6.19ppm, the relative standard deviation RSD of the detection result is 2.0 percent, and the method has good durability.

Example 3

The conditions were the same as in example 1 except that only the mobile phase A was changed to 0.01M ammonium carbonate solution. The same test method was used to test 6 batches of samples from example 1. The results are given in the following table:

sample batch number	CMDP content (%)
		150301PS	Less than 0.2ppm
150302PS	Less than 0.2ppm
		150303PS	Less than 0.2ppm
150701PS	Less than 0.2ppm
		150702PS	Less than 0.2ppm
150703PS	Less than 0.2ppm

The results showed that the results of the detection using 0.01M ammonium carbonate solution as mobile phase A were substantially the same as in example 1.

Example 4

The test results using different diluents and mobile phases were compared, with only changing the diluent and mobile phases and other conditions being the same as in example 1.

The results show that when neutral mobile phases such as water or ammonium acetate solution are used, the CMDP in the sample is unstable, and the accuracy of the detection result is influenced. The mobile phase is alkaline by adding 0.01M sodium hydroxide solution into water, so that the sample has enough stability, but no peak appears in the first 5 minutes during detection, and impurities are possibly not detected, so that the detection result is inaccurate. Furthermore, non-volatile bases such as sodium hydroxide solution are not suitable for mass spectrometric detection and cause great damage to the mass spectrometer. Ammonia is not suitable because it is volatile, which causes the pH of the diluent and mobile phase to change over time. Triethylamine is not suitable for mass spectrometric detection because mass spectrometric residues are serious and can strongly interfere with detection results. When 0.02M and 0.03M ammonium bicarbonate solution are used as mobile phase, the analysis time is short, the solution is stable, the operation is convenient, and a satisfactory detection result can be obtained. However, when 0.04M ammonium bicarbonate solution is used, the detection result is not stable enough, and the reproducibility is not satisfactory, so that ammonium bicarbonate solution with larger concentration is not used. The results of the overall comparison were obtained using 0.01-0.03M ammonium bicarbonate or ammonium carbonate solution as mobile phase A.

It should be noted that the above-mentioned embodiments are further non-limiting detailed descriptions of the technical solutions of the present invention, and are only used for illustrating the technical concepts and features of the present invention. It is intended that the present invention be understood and implemented by those skilled in the art, and not limited thereto. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A high-sensitivity analysis method for genotoxic impurity 2-chloromethyl-3, 4-dimethoxypyridine hydrochloride in pantoprazole sodium is characterized in that: diluting a pantoprazole sodium sample by using a diluent, and eluting by using a carbonate solution and acetonitrile as mobile phases by adopting a high performance liquid chromatography-mass spectrometry combined method, wherein the carbonate solution is an ammonium bicarbonate solution or an ammonium carbonate solution, and the concentration of the carbonate solution is 0.01-0.03 mol/L; wherein the gradient elution conditions are as follows:

2. the assay of claim 1, wherein: the concentration of the carbonate solution is 0.02 mol/L.

3. The assay of claim 1, wherein: the diluent is a mixed solution of ammonium bicarbonate solution and acetonitrile, and the concentration of the diluted sample is 2 mg/mL.

4. The assay of claim 3, wherein: the volume ratio of the ammonium bicarbonate solution to the acetonitrile in the mixed solution is 5-10: 1.

5. The assay of claim 3, wherein: the volume ratio of the ammonium bicarbonate solution to the acetonitrile in the mixed solution is 9: 1.

6. The assay of claim 1, wherein: the elution mode is gradient elution, and the volume content of carbonate in the eluent is 20-80%.

7. The assay of claim 1, wherein: the elution rate of the elution is 0.8-1.2 mL/min, and the temperature of the chromatographic column during elution is 38-42 ℃.

8. The assay of claim 1, wherein: the column used for HPLC was an AgilentProshell HPH C18 column, 150X 4.6mm, 2.7 μm.

9. The assay of claim 1, wherein: the ionization technology of the ion source of the mass spectrum is an electrospray ionization technology, the spray voltage is 3KV, the pressure of an atomization chamber is 35psi, and the temperature of dry gas is 350 ℃.