CN111426760B - Method for determining genotoxic impurities in doxofylline raw material medicine - Google Patents

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for determining genotoxic impurities in doxofylline bulk drug. The determination method adopts a gas chromatography-mass spectrometry combined method for determination. The method has high sensitivity, and can simply, quickly and accurately simultaneously determine two genotoxic impurities.

Description

Method for determining genotoxic impurities in doxofylline raw material medicine

Technical Field

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for determining genotoxic impurities in doxofylline bulk drug.

Background

Doxofylline is a derivative of methylxanthine, a bronchodilator, and has effects in relaxing airway smooth muscle and inhibiting asthma by inhibiting phosphodiesterase in smooth muscle cells.

The doxofylline uses vinyl acetate and 2-bromo-1, 1-dimethoxyethane during the synthesis process, and the structure has potential genotoxicity. According to the usage amount of the medicinal preparation, the limit of the genotoxic impurities is 20ppm, and the measurement sensitivity of the genotoxic impurities is difficult to meet the control requirement of medicine inspection by adopting the separation and analysis methods of common liquid chromatography and gas chromatography.

Therefore, from the viewpoints of drug quality control and quality standard research, it is necessary to develop a method for measuring the doxofylline genotoxic impurity, namely vinyl acetate and/or 2-bromo-1, 1-dimethoxyethane, which is simple and convenient to operate and high in sensitivity, so as to control the quality of the raw material drugs and establish the most strict drug quality standard for measurement, and the method is applied to the circulation and use processes of raw materials and preparations.

Disclosure of Invention

Aiming at the defects of the prior art and solving the practical problem that the prior analysis method is lack of determination on two genotoxic impurities, namely vinyl acetate and/or 2-bromo-1, 1-dimethoxyethane, the invention provides a determination method for two genotoxic impurities related to doxofylline bulk drug.

The method for determining genotoxic impurities in doxofylline raw material medicines provided by the invention adopts a gas chromatography-mass spectrometry combined method for determination, and the specific determination method comprises the following steps:

(1) gas chromatography conditions:

a chromatographic column: VF-1301ms gas chromatographic column;

sample inlet temperature: 200 ℃;

interface temperature: 230 ℃;

temperature rising procedure: maintaining at 50 deg.C for 3min, increasing to 110 deg.C at 15 deg.C/min for 2min, and increasing to 200 deg.C at 50 deg.C/min for 5 min;

carrier gas: high purity helium gas;

and (3) sample introduction mode: direct sample introduction, split ratio: 20: 1;

sample introduction amount: 1 mu L of the solution;

solvent retardation: 1min, 4 min-8 min, closing the tester (solvent peak);

(2) mass spectrum conditions:

type of ion source: EI source, 70 eV;

ion source temperature: 230 ℃;

quadrupole temperature: 150 ℃;

an acquisition mode: an ion monitoring mode (SIM) is selected.

Preferably, the chromatographic column is: VF-1301ms (30 m.times.1 μm.times.0.25 mm) gas chromatography column.

Preferably, the genotoxic impurity is vinyl acetate and/or 2-bromo-1, 1-dimethoxyethane.

More preferably, the genotoxic impurities are vinyl acetate and 2-bromo-1, 1-dimethoxyethane, i.e., the assay measures vinyl acetate and 2-bromo-1, 1-dimethoxyethane simultaneously.

Preferably, the genotoxic impurities vinyl acetate and 2-bromo-1, 1-dimethoxyethane are selected under the measurement conditions to have the characteristic ions:

preferably, the assay employs solvent and interfering component excision methods.

Preferably, the measurement sensitivity of the genotoxic impurities of vinyl acetate and 2-bromo-1, 1-dimethoxyethane is 0.1ppm and 0.01ppm respectively.

Preferably, the limits of quantitation of the genotoxic impurities vinyl acetate and 2-bromo-1, 1-dimethoxyethane are 0.7ppm and 0.06ppm, respectively.

Preferably, the assay method comprises the steps of:

1) sample preparation:

preparing a mixed standard series solution: respectively and precisely weighing a proper amount of vinyl acetate and a proper amount of 2-bromo-1, 1-dimethoxyethane reference substance, preparing a single reference substance stock solution by using N, N-dimethylformamide, respectively and precisely weighing a proper amount of the single reference substance stock solution into the same volumetric flask, adding N, N-dimethylformamide for dilution, and fixing the volume to scale to prepare a mixed reference substance stock solution; respectively and precisely absorbing appropriate amount of mixed reference substance stock solution, and diluting to different series concentrations to obtain mixed standard series solution;

preparing a test article: accurately weighing a proper amount of crude doxofylline into a volumetric flask, adding a proper amount of N, N-dimethylformamide, shaking and uniformly mixing, sealing and ultrasonically treating until a sample is completely dissolved, adding N, N-dimethylformamide to a constant volume to scale, shaking uniformly, filtering with a 0.22 mu m filter membrane, and taking filtrate as a test sample;

2) drawing a standard curve:

injecting the mixed standard series solution into a gas chromatography-mass spectrometer, measuring corresponding peak areas, drawing a standard curve by taking the concentration of the standard solution as a horizontal coordinate and the peak area as a vertical coordinate, and listing a mathematical equation and a linear range of the standard curve;

3) and (3) determination:

injecting the sample to be tested into a gas chromatography-mass spectrometer to obtain the chromatographic peak area of the sample;

4) and (3) calculating:

substituting the chromatographic peak area of the sample into the standard curve to calculate the contents of vinyl acetate and 2-bromo-1, 1-dimethoxyethane.

In one embodiment, the assay method comprises the steps of:

1) sample preparation:

preparing a mixed standard series solution: respectively and precisely weighing appropriate amount of vinyl acetate and 2-bromo-1, 1-dimethoxyethane reference substances, preparing single reference substance stock solution with the concentration of the vinyl acetate of 3.846mg/ml and the concentration of the 2-bromo-1, 1-dimethoxyethane of 0.3167mg/ml by using N, N-dimethylformamide, respectively and precisely weighing 1ml of the single reference substance stock solution into the same 100ml volumetric flask, adding the N, N-dimethylformamide for dilution and fixing the volume to scale to prepare mixed reference substance stock solution, respectively and precisely sucking appropriate amount of the mixed reference substance stock solution until the concentrations of the vinyl acetate are respectively 0.769 mu g/ml, 1.23 mu g/ml, 1.54 mu g/ml, 1.92 mu g/ml, 2.31 mu g/ml and 2-bromo-1, 1-dimethoxyethane of 0.0633 mu g/ml, 0.101. mu.g/ml, 0.127. mu.g/ml, 0.158. mu.g/ml, 0.190. mu.g/ml of a mixed standard series solution;

preparing a test article: accurately weighing 0.3g to 10ml of crude doxofylline in a volumetric flask, adding a proper amount of N, N-dimethylformamide, shaking and mixing uniformly, sealing and ultrasonically treating for 1min until the sample is completely dissolved, adding N, N-dimethylformamide to a constant volume to scale, shaking uniformly, filtering with a 0.22 mu m filter membrane, and taking the filtrate as a sample;

2) drawing a standard curve:

injecting the mixed standard series solution into a gas chromatography-mass spectrometer, measuring corresponding peak areas, drawing a standard curve by taking the concentration of the standard solution as a horizontal coordinate and the peak area as a vertical coordinate, and listing a mathematical equation and a linear range of the standard curve;

3) and (3) determination:

precisely injecting the sample into a sample of 1 mu l, and injecting the sample into a gas chromatography-mass spectrometer to obtain the chromatographic peak area of the sample;

4) and (3) calculating:

substituting the chromatographic peak area of the sample into the standard curve to calculate the contents of vinyl acetate and 2-bromo-1, 1-dimethoxyethane.

Compared with the prior art, the invention has the beneficial effects that:

the method for determining the genotoxicity in the doxofylline bulk drug can effectively detect two genotoxicity impurities of vinyl acetate and 2-bromo-1, 1-dimethoxyethane at the same time, greatly improves the determination sensitivity by a gas chromatography-mass spectrometry combined method, can accurately quantify a target compound by a mass spectrometry solvent excision technology, an interference component excision technology and a direct sample injection determination sim ion, and is simple to operate and accurate in result.

The method determines genotoxic impurities in the doxofylline bulk drug by a gas chromatography-mass spectrometry combined method, and effectively reduces the pollution of a sample to a mass spectrometer by adopting a method of solvent excision and interference component excision.

The method determines genotoxic impurities in the doxofylline bulk drug by a gas chromatography-mass spectrometry combined method, adopts a temperature programming method, effectively reduces the loss of stationary liquid of a capillary chromatographic column, and prolongs the service life of the chromatographic column.

The method determines genotoxic impurities in the doxofylline bulk drug by a gas chromatography-mass spectrometry combined method, and greatly purifies the test background by adopting a method of solvent excision and interference component excision. In addition, the program heating and the SIM ion determination can accurately identify the mother ions and the fragment ions of the sample, thereby ensuring the accuracy of the trace determination. The method has the advantages that the measuring sensitivity of the vinyl acetate and the 2-bromine-1, 1-dimethoxyethane can reach 0.1ppm and 0.01ppm respectively, and the method has high sensitivity and specificity.

The method determines genotoxic impurities in the doxofylline raw material medicine through a gas chromatography-mass spectrometry combined method, the quantitative limit of the vinyl acetate and the 2-bromo-1, 1-dimethoxyethane can reach 0.7ppm and 0.06ppm, trace genotoxic impurities can be accurately quantified, and the aims of production control and improvement of determination quality standard are fulfilled.

In a word, the determination method provided by the invention can effectively separate out genotoxic impurities in the doxofylline raw material medicine, has high sensitivity, and can simply, quickly and accurately determine two genotoxic impurities simultaneously.

Related to the invention, Shanxi province food and drug supervision and inspection research institute, the research institute of science and technology in Shaanxi province, 2018, focuses on research and development of quality control and standard improvement research of cantharidin derivative preparations (2018 SF-300). The invention explores and researches the detection of volatile toxic impurities and gene impurities in animal-derived biochemical drugs and semi-synthetic chemical drugs, summarizes experience and model methods for detecting the volatile toxic impurities and the gene toxic impurities by adopting a gas chromatography-mass spectrometry combined method, is invented and designed based on the accumulation of research methods at the early stage of a project, and obtains satisfactory results.

Drawings

FIG. 1 is a GC-MS analysis spectrum of a solvent;

FIG. 2 is a GC-MS analysis spectrum of a labeled simulated sample;

FIG. 3 is a GC-MS analysis of a sample.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto. The examples of the present invention are given for illustrative purposes only and are not intended to limit the present invention. Reagent:

n, N-dimethylformamide: carrying out chromatographic purification; the manufacturer: TEDIA: batch number: 1205415.

comparison products:

vinyl acetate: the purity is 99.9%; the manufacturer: dr. ehrenstorfer GmbH: batch number: g991533;

2-bromo-1, 1-dimethoxyethane: purity 97%: the manufacturer: SIGMA: batch number: MKBD 3673V. Sample preparation: crude products and finished products of the doxofylline raw materials are provided by Shaanxi Bosen biopharmaceutical group, Inc. Sample batch number:

20191016 (finished) 20191016 (crude);

20191022 (finished) 20191022 (crude);

20191027 (finished) 20191027 (crude).

Apparatus and device

Gas chromatography-mass spectrometer: the manufacturer: agilent; the model is as follows: 7890B-5977B;

balance: the manufacturer: a Mettler; the model is as follows: AE-240;

an ultrasonic cleaner: the manufacturer: guangzhou Kejie union; the model is as follows: KM-1030C.

The determination method is a standard curve method specified by pharmacopoeia, and the following experimental processes and experimental methods are not described in detail and all adopt standard operation procedures, such as operation methods of solution preparation and systematic adaptability test.

(1) Gas chromatography conditions

A chromatographic column: VF-1301ms (30 m.times.1 μm.times.0.25 mm);

sample inlet temperature: 200 ℃;

interface temperature: 230 ℃;

temperature rising procedure: maintaining at 50 deg.C for 3min, increasing to 110 deg.C at 15 deg.C/min for 2min, and increasing to 200 deg.C at 50 deg.C/min for 5 min;

carrier gas: high purity helium gas;

and (3) sample introduction mode: direct sample introduction, split ratio: 20: 1;

sample introduction amount: 1 mu L of the solution;

solvent retardation: 1min, 4 min-8 min, closing the tester (solvent peak);

(2) mass spectrum conditions:

type of ion source: EI source, 70 eV;

ion source temperature: 230 ℃;

quadrupole temperature: 150 ℃;

an acquisition mode: selecting an ion monitoring mode (SIM);

selected characteristic ions:

example 1:

1) sample preparation:

preparing a mixed standard series solution: precisely weighing a proper amount of each reference substance, preparing single reference substance stock solution with the concentration of 3.846mg/ml and the concentration of 0.3167mg/ml by using N, N-dimethylformamide, precisely weighing 1ml of the single reference substance stock solution into the same 100ml volumetric flask, adding N, N-dimethylformamide for dilution and fixing the volume to scale to prepare mixed reference substance stock solution, precisely sucking the proper amount of the mixed reference substance stock solution respectively until the concentrations of the vinyl acetate are respectively 0.769 mu g/ml, 1.23 mu g/ml, 1.54 mu g/ml, 1.92 mu g/ml, 2.31 mu g/ml, 0.0633 mu g/ml, 0.101 mu g/ml, 0.127 mu g/ml, N-dimethylformamide and the like, 0.158 mug/ml and 0.190 mug/ml mixed standard series solution;

preparing a test article: accurately weighing 0.3g to 10ml of crude doxofylline (the sample is a simulated sample added with a standard reference substance, and is called a labeled simulated sample for short), adding a proper amount of N, N-dimethylformamide, shaking and mixing uniformly, sealing and ultrasonically treating for 1min until the sample is completely dissolved, adding N, N-dimethylformamide to a constant volume to a scale, shaking uniformly, filtering with a 0.22 mu m filter membrane, and taking the filtrate as a sample;

2) drawing a standard curve:

injecting the mixed standard series solution into a gas chromatography-mass spectrometer, determining corresponding peak areas, drawing a standard curve by taking the concentration of the standard solution as a horizontal coordinate and the peak area as a vertical coordinate, and listing a mathematical equation and a linear range of the standard curve;

3) and (3) determination:

precisely injecting a sample to be tested by 1 mu l, and injecting the sample into a gas chromatography-mass spectrometer to obtain the chromatographic peak area of the sample;

4) and (3) calculating:

substituting the chromatographic peak area of the sample into the standard curve to calculate the contents of vinyl acetate and 2-bromo-1, 1-dimethoxyethane.

The gas chromatograms obtained are shown in FIGS. 1 and 2. FIG. 1 is a GC-MS spectrum of a solvent; FIG. 2 is a GC-MS analysis spectrum of a labeled simulated sample.

By comparing fig. 1 and fig. 2, the impurities were determined: according to the mother ion peak and the daughter ion characteristic fragment of the reference substance in the doxofylline determination map, the impurities 1 and 2 are respectively determined to be vinyl acetate and 2-bromo-1, 1-dimethoxyethane.

The results of the content measurement of the spiked simulated samples are shown in table 1 below.

TABLE 1 content measurement results of spiked simulated samples

As can be seen from Table 1, in the mass spectrometric system, two genotoxic impurities can be detected effectively, and the measurement sensitivity can reach 0.1ppm and 0.01 ppm.

Examples 2 to 7

The same measurement conditions and procedures as in example 1 were used except that the test article was prepared by the following method.

Preparing a test article: accurately weighing 0.3g to 10ml of crude doxofylline and finished product (provided by Shaanxi Bosen biopharmaceutical group Co., Ltd.) in volumetric flasks, adding a proper amount of N, N-dimethylformamide, shaking uniformly, sealing and ultrasonically treating for 1min until the sample is completely dissolved, adding N, N-dimethylformamide to a constant volume to a scale, shaking uniformly, filtering with a 0.22 mu m filter membrane, and taking the filtrate as a test sample.

The gas chromatogram of the measurement is shown in FIG. 3. FIG. 3 is a GC-MS analysis of a sample.

The results of the content measurement of the samples are shown in tables 2 to 7 below.

TABLE 2 results of content measurement of samples

TABLE 3 results of content measurement of samples

TABLE 4 results of content measurement of samples

TABLE 5 results of content measurement of samples

TABLE 6 results of content measurement of samples

TABLE 7 results of content measurement of samples

As can be seen from the results of the measurements in tables 2 to 7, although the synthesis of doxofylline uses vinyl acetate and 2-bromo-1, 1-dimethoxyethane, the genotoxic impurities of vinyl acetate and 2-bromo-1, 1-dimethoxyethane in the crude product and the finished product of doxofylline provided by the Shaanxi Bosen biopharmaceutical group GmbH were not detected. The excellent preparation and purification process of doxofylline of Shaanxi Bosen biopharmaceutical group GmbH is demonstrated.

Example 3 methodological examination

The method for measuring the two genotoxic impurities in the doxofylline bulk drug is examined by methodologies such as specificity, linearity, precision, repeatability, accuracy, stability, detection limit and quantitative limit. The specific investigation method comprises the following steps:

1. linear survey

An appropriate amount of each control was precisely weighed, and N, N-dimethylformamide was used to prepare a mixed standard series solution (hereinafter abbreviated as std1 solution, std2 solution, std3 solution, std4 solution and std5 solution in order of increasing concentration) having a vinyl acetate concentration of 0.769. mu.g/ml, 1.23. mu.g/ml, 1.54. mu.g/ml, 1.92. mu.g/ml and 2.31. mu.g/ml and a 2-bromo-1, 1-dimethoxyethane concentration of 0.0633. mu.g/ml, 0.101. mu.g/ml, 0.127. mu.g/ml, 0.158. mu.g/ml and 0.190. mu.g/ml. And injecting the mixed standard series solution into a gas chromatography-mass spectrometer, measuring the corresponding peak area, drawing a standard curve by taking the concentration of the standard solution as a horizontal coordinate and taking the peak area vertical coordinate, and listing the mathematical equation and the linear range of the standard curve. The results of the linear examination are shown in Table 8.

TABLE 8 Linear survey table

2. Detection limit and quantification limit

The std1 solution in the standard curve was diluted 4-fold to determine the limits of both, and the std1 scale was used as a quantification. The results are shown in Table 9.

TABLE 9 detection limits, quantitation limits for the methods

3. Precision degree

And continuously injecting std3 solution in the standard curve for 6 times, calculating RSD according to the peak area, and obtaining the experimental result shown in the table 10.

TABLE 10 results of precision test

4. Stability test

The standard curve std3 solution is precisely measured and injected at certain time intervals for determination, the chromatogram is recorded, the RSD% of the peak area is calculated, and the calculation result is shown in Table 11.

TABLE 11 results of stability experiments

5. Recovery test

Appropriate amounts of the stock solutions of the mixed control were added to the samples, respectively, to prepare sample labeling solutions having concentrations equivalent to std2 solution, std3 solution, std4 solution, and the samples were prepared according to the sample preparation method, and the recovery test results are shown in table 12.

TABLE 12 recovery rate test results table

6. Repeatability test

6 parts of the sample to be tested are precisely weighed, 1ml of the mixed reference substance stock solution is precisely added, the preparation and the measurement are carried out according to a sample method, and the average content (n is 6) is calculated. The calculation results are shown in Table 13.

TABLE 13 results of repeated experiments

7. Specificity test

Respectively preparing a sample and a sample solution (the concentration is equal to std3 solution), injecting sample according to the method, and determining, wherein the result shows that other components and solvent have no interference to the determined components, and the determination map is shown in figures 1-3.

The results of the above methodology examination illustrate that: the determination method is suitable for determining the contents of genotoxic impurities, namely vinyl acetate and 2-bromo-1, 1-dimethoxyethane in the doxofylline raw material.

Based on the above description of the summary of the invention, a person skilled in the art can apply the invention in its entirety, and all changes that are the same principle or similar are to be considered as included in the scope of the invention.

Claims (8)

1. A method for measuring genotoxic impurities in doxofylline raw material medicine is characterized in that the genotoxic impurities are vinyl acetate and/or 2-bromo-1, 1-dimethoxyethane, and the measuring method adopts a gas chromatography-mass spectrometry combined method for measurement, and the specific measuring conditions are as follows:

(1) gas chromatography conditions:

a chromatographic column: VF-1301ms gas chromatographic column;

sample inlet temperature: 200 ℃;

interface temperature: 230 ℃;

temperature rising procedure: maintaining at 50 deg.C for 3min, increasing to 110 deg.C at 15 deg.C/min for 2min, and increasing to 200 deg.C at 50 deg.C/min for 5 min;

carrier gas: high purity helium gas;

and (3) sample introduction mode: direct sample introduction, split ratio: 20: 1;

sample introduction amount: 1 mu L of the solution;

solvent retardation: closing the measuring device for 1min, 4 min-8 min;

(2) mass spectrum conditions:

type of ion source: EI source, 70 eV;

ion source temperature: 230 ℃;

quadrupole temperature: 150 ℃;

an acquisition mode: an ion monitoring mode is selected.

2. The assay method according to claim 1, wherein the chromatographic column has a specification of: 30 m.times.1 μm.times.0.25 mm.

3. The assay of claim 1, wherein the genotoxic impurities are vinyl acetate and 2-bromo-1, 1-dimethoxyethane, i.e., the assay measures vinyl acetate and 2-bromo-1, 1-dimethoxyethane simultaneously.

4. The assay method according to claim 3, wherein the genotoxic impurities vinyl acetate and 2-bromo-1, 1-dimethoxyethane are, under the assay conditions, the characteristic ions selected as:

5. the assay of claim 1, wherein the assay employs a solvent excision and interfering component excision method.

6. The method according to claim 1, wherein the measurement sensitivity of the genotoxic impurity, vinyl acetate and 2-bromo-1, 1-dimethoxyethane, is 0.1ppm and 0.01ppm, respectively.

7. The assay according to claim 1, wherein the limits of quantitation of the genotoxic impurities vinyl acetate and 2-bromo-1, 1-dimethoxyethane are 0.7ppm and 0.06ppm, respectively.

8. The assay method according to claim 1, comprising the steps of:

1) sample preparation:

preparing a mixed standard series solution: respectively and precisely weighing a proper amount of vinyl acetate and a proper amount of 2-bromo-1, 1-dimethoxyethane reference substance, preparing a single reference substance stock solution by using N, N-dimethylformamide, respectively and precisely weighing a proper amount of the single reference substance stock solution into the same volumetric flask, adding N, N-dimethylformamide for dilution, and fixing the volume to scale to prepare a mixed reference substance stock solution; respectively and precisely absorbing appropriate amount of mixed reference substance stock solution, and diluting to different series concentrations to obtain mixed standard series solution;

preparing a test article: accurately weighing a proper amount of crude doxofylline into a volumetric flask, adding a proper amount of N, N-dimethylformamide, shaking and uniformly mixing, sealing and ultrasonically treating until a sample is completely dissolved, adding N, N-dimethylformamide to a constant volume to scale, shaking uniformly, filtering with a 0.22 mu m filter membrane, and taking filtrate as a test sample;

2) drawing a standard curve:

injecting the mixed standard series solution into a gas chromatography-mass spectrometer, measuring corresponding peak areas, drawing a standard curve by taking the concentration of the standard solution as a horizontal coordinate and the peak area as a vertical coordinate, and listing a mathematical equation and a linear range of the standard curve;

3) and (3) determination:

injecting the sample to be tested into a gas chromatography-mass spectrometer to obtain the chromatographic peak area of the sample;

4) and (3) calculating:

substituting the chromatographic peak area of the sample into the standard curve to calculate the contents of vinyl acetate and 2-bromo-1, 1-dimethoxyethane.

Images