阅读说明：本技术 雷尼替丁类药物中亚硝胺类物质的高效液相色谱分析方法 (High performance liquid chromatography analysis method for nitrosamines in ranitidine drugs ) 是由 史加桂 孙征宇 卫耿虎 于 2020-10-14 设计创作，主要内容包括：本发明公开了一种雷尼替丁类药物中亚硝胺类物质的高效液相色谱分析方法,包括如下步骤：(1)样品处理；(2)样品溶液配制；(3)对照溶液配制；(4)色谱分析。本发明通过对雷尼替丁类药物进行干燥、研磨处理及密封条件下低温配置、测试前过滤等工艺条件的设置,一方面确保了N-二甲基亚硝胺测试结果的稳定、准确；另一方面极大地降低了样品对色谱系统的污染,减少了后期的使用维护,节约维护成本；在上述样品处理的基础上,使用常规的高效液相色谱仪就可以进行试验分析,且准确度高,结果的重现性好,不需要大额资金的投入,节约成本,适合工业化生产应用,市场前景广阔。(The invention discloses a high performance liquid chromatography analysis method of nitrosamine substances in ranitidine drugs, which comprises the following steps: (1) processing a sample; (2) preparing a sample solution; (3) preparing a control solution; (4) and (4) performing chromatographic analysis. According to the invention, the Ranitidine drug is dried and ground, and the process conditions of low-temperature configuration under a sealed condition, filtration before testing and the like are set, so that on one hand, the stability and accuracy of the test result of the N-dimethylnitrosamine are ensured; on the other hand, the pollution of the sample to a chromatographic system is greatly reduced, the later use and maintenance are reduced, and the maintenance cost is saved; on the basis of the sample treatment, the conventional high performance liquid chromatograph can be used for experimental analysis, the accuracy is high, the reproducibility of results is good, the investment of large amount of funds is not needed, the cost is saved, and the method is suitable for industrial production and application and has wide market prospect.)

1. A high performance liquid chromatography analysis method for nitrosamines in ranitidine drugs is characterized by comprising the following steps:

(1) sample treatment: drying ranitidine medicine, and grinding into powder;

(2) preparing a sample solution: precisely weighing the powder ground in the step (1), adding a precisely measured solvent, sealing, mixing into a sample solution at low temperature, and carrying out constant volume sealing and placement;

(3) preparing a control solution: accurately weighing nitrosamine substance reference substances, and preparing a reference solution by using water;

(4) and (3) chromatographic analysis: the sample solution and the control solution were separately chromatographed using a chromatographic column.

2. The method of claim 1, further comprising a filtration step of filtering the hermetically sealed sample solution with a needle filter before the chromatography step.

3. The HPLC analysis method for nitrosamines in ranitidine drugs according to claim 2, wherein the filtration membrane of the needle filter is a hydrophilic polytetrafluoroethylene membrane.

4. The method for analyzing a nitrosamine compound in a ranitidine compound according to claim 1, wherein in the step (2), the solvent is dichloromethane; the concentration of the sample solution was 0.5 g/ml.

5. The HPLC analysis method for nitrosamines in ranitidine drugs according to claim 1, wherein in said step (2), said mixing conditions are as follows: temperature, ultrasonic treatment or oscillation for 5-10 min.

6. The method for analyzing a nitrosamine compound in a ranitidine compound according to claim 1, wherein in the step (3), the solvent is dichloromethane; the concentration of the control solution was 1 ppm.

7. The HPLC analysis method for nitrosamines in ranitidine drugs according to claim 1, wherein in step (4), the specification of the chromatographic column is: agilent C18, 4.6 mm. times.150 mm, 3.5 μm.

8. The method for high performance liquid chromatography of a nitrosamine in a ranitidine compound according to claim 1, wherein in said step (4), said chromatographic conditions are as follows:

detection wavelength: 230 nm;

flow rate of mobile phase: 0.8 ml/min;

and (3) an elution mode: gradient elution;

column temperature: 35 ℃;

solution sample introduction amount: 20 mu L of the solution;

ambient temperature: less than 15 ℃.

9. The method for analyzing nitrosamines in ranitidine, according to claim 8, wherein said mobile phase comprises mobile phase a and mobile phase B; wherein in the mobile phase A, the mixing volume ratio of phosphate buffer solution to acetonitrile is 98: 2; and in the mobile phase B, the mixing volume ratio of the phosphate buffer solution to the acetonitrile is 78: 22.

10. The HPLC analysis method for nitrosamines in ranitidine drugs according to claim 8, wherein the gradient elution process conditions are as follows:

in 0-15 min, the volume of the mobile phase A is decreased from 100% to 0%, and the volume of the mobile phase B is increased from 0% to 100%;

in 15-23 min, the volume of the mobile phase A is 0%, and the volume of the mobile phase B is 100%;

at 23-24 min, the volume of the mobile phase A is increased from 0% to 100%, and the volume of the mobile phase B is decreased from 100% to 0%;

and at 24-30 min, the volume of the mobile phase A is 100%, and the volume of the mobile phase B is 0%.

Technical Field

The invention relates to the technical field of medicine detection, in particular to a high performance liquid chromatography analysis method for nitrosamines in ranitidine medicines.

Background

After the ranitidine drugs spontaneously find that certain amounts of nitrosamine substances, such as N-dimethylnitrosamine (NDMA), are contained, the FDA and the national food and drug inspection research institute of the United states formulate various liquid mass methods for detecting the substances, all the detection methods except the mass spectrometer with the triple quadrupole are required, the sensitivity requirement on instruments is very high, common laboratories do not have detection equipment with corresponding conditions, the instruments have high use requirement and high maintenance difficulty, and the ranitidine drugs are not suitable for the needs of most enterprises or scientific research units for daily quantitative analysis of the substances. Nitrosamine substances (NDMA) basically have the phenomenon of conjugate elution in the existing detection method, the conditions in the experiment are harsh, and the data reproduction and the accurate determination of the content are very difficult to achieve.

Disclosure of Invention

The invention mainly solves the technical problem of providing a high performance liquid chromatography analysis method for nitrosamines in ranitidine drugs, which can solve the problems in the prior art.

In order to solve the technical problems, the invention adopts a technical scheme that: the high performance liquid chromatography analysis method of N-dimethyl nitrosamine is provided, which comprises the following steps:

(1) sample treatment: drying ranitidine hydrochloride and grinding into powder;

(2) preparing a sample solution: precisely weighing the ranitidine hydrochloride powder ground in the step (1), adding a precisely measured solvent, sealing, mixing at a low temperature to obtain a sample solution, and fixing the volume, sealing and placing;

(3) preparing a control solution: precisely weighing a reference substance, and preparing a reference solution by using water;

(4) and (3) chromatographic analysis: the sample solution and the control solution were separately chromatographed using a chromatographic column.

In a preferred embodiment of the present invention, a filtration process is further included before the chromatography process, wherein the sample solution hermetically placed is filtered by using a needle filter.

In a preferred embodiment of the invention, the filtering membrane of the needle type filter is a hydrophilic polytetrafluoroethylene membrane.

In a preferred embodiment of the present invention, in the step (2), the solvent is dichloromethane; the concentration of the sample solution was 0.5 g/ml.

In a preferred embodiment of the present invention, in the step (2), the mixing conditions are: temperature (lower than 15 ℃), ultrasonic treatment or oscillation, 5-10 min.

In a preferred embodiment of the present invention, in the step (3), the solvent is dichloromethane; the concentration of the control solution was 1 ppm.

In a preferred embodiment of the present invention, in the step (4), the specification of the chromatographic column is: agilent C18, 4.6 mm. times.150 mm, 3.5 μm.

In a preferred embodiment of the present invention, in the step (4), the chromatographic conditions are:

detection wavelength: 230nm (wavelength range modified to enable detection of NDMA);

flow rate of mobile phase: 0.8 ml/min;

and (3) an elution mode: gradient elution;

column temperature: 35 ℃;

solution sample introduction amount: 20 mu L of the solution;

ambient temperature: less than 15 ℃.

In a preferred embodiment of the present invention, the mobile phase comprises mobile phase a and mobile phase B; wherein in the mobile phase A, the mixing volume ratio of phosphate buffer solution to acetonitrile is 98: 2; and in the mobile phase B, the mixing volume ratio of the phosphate buffer solution to the acetonitrile is 78: 22.

In a preferred embodiment of the present invention, the process conditions of the gradient elution are as follows:

in 0-15 min, the volume of the mobile phase A is decreased from 100% to 0%, and the volume of the mobile phase B is increased from 0% to 100%;

in 15-23 min, the volume of the mobile phase A is 0%, and the volume of the mobile phase B is 100%;

at 23-24 min, the volume of the mobile phase A is increased from 0% to 100%, and the volume of the mobile phase B is decreased from 100% to 0%;

and at 24-30 min, the volume of the mobile phase A is 100%, and the volume of the mobile phase B is 0%.

The invention has the beneficial effects that: according to the high performance liquid chromatography analysis method for nitrosamines in ranitidine drugs, the ranitidine drugs are dried and ground, and the process conditions of low-temperature configuration under a sealed condition, filtration before testing and the like are set, so that on one hand, the stability and accuracy of an N-dimethyl nitrosamine test result are ensured; on the other hand, the pollution of the sample to a chromatographic system is greatly reduced, the later use and maintenance are reduced, and the maintenance cost is saved; on the basis of the sample treatment, the conventional high performance liquid chromatograph can be used for experimental analysis, the accuracy is high, the reproducibility of results is good, the investment of large amount of funds is not needed, the cost is saved, and the method is suitable for industrial production and application and has wide market prospect.

Drawings

FIG. 1 is a chromatogram obtained by a high performance liquid chromatography analysis method of nitrosamines in ranitidine drugs.

Detailed Description

The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention easier to understand by those skilled in the art, and thus will clearly and clearly define the scope of the invention.

Referring to fig. 1, an embodiment of the present invention includes:

example 1

The invention discloses a high performance liquid chromatography analysis method of nitrosamine substances in ranitidine drugs, taking ranitidine hydrochloride as a sample, testing the content of N-dimethylnitrosamine (NDMA) in the ranitidine drugs as an example,

the method comprises the following steps:

(1) sample treatment: taking about 1g of ranitidine hydrochloride raw material medicine, drying, then grinding the ranitidine hydrochloride raw material medicine into powder in a dry mortar, and keeping the drying in the grinding process to prevent moisture absorption. Because ranitidine hydrochloride has the characteristic of extremely easy moisture absorption, the N-dimethyl nitrosamine contained in ranitidine hydrochloride can be better dissolved into diluent after drying treatment and grinding of components under the drying condition;

(2) preparing a sample solution: immediately and precisely weighing 0.5g of ground ranitidine hydrochloride powder after grinding, adding the powder into a 10ml container with a plug to ensure that the powder does not remain on the wall of the container, then precisely adding 5ml of dichloromethane, sealing, carrying out ultrasonic dispersion or oscillation treatment for 5-10min at low temperature to prepare a sample solution with the concentration of 0.5g/ml, and carrying out constant volume sealing and placing; the precision weighing and the sealing preservation ensure the accuracy of the constant volume, and are beneficial to improving the measurement precision; ultrasonic dispersion or oscillation treatment is carried out under the low temperature condition, so that the N-dimethyl nitrosamine in the sample is not influenced by the external environment temperature to change the generated amount, and the measurement precision is improved;

(3) preparing a control solution: accurately weighing an N-dimethyl nitrosamine reference substance, and diluting the N-dimethyl nitrosamine reference substance into a solution with the concentration of 1ppm by using deionized water as a reference solution;

(4) and (3) chromatographic analysis: filtering the sample solution which is sealed and placed by using a needle filter of which the filter membrane is a hydrophilic polytetrafluoroethylene membrane, storing the filtrate in a sample bottle, and respectively carrying out chromatographic analysis on the sample solution and a control solution by using a chromatographic column; wherein the filtration is used for removing a large amount of ranitidine hydrochloride so that the ranitidine hydrochloride does not remain in the chromatographic column;

the chromatographic column adopts octadecylsilane chemically bonded silica as a filling agent, and the specification is as follows: agilent C18, 4.6mm × 150mm, 3.5 μm; the chromatographic column of the type can avoid the conjugate elution phenomenon of a ranitidine hydrochloride sample;

the chromatographic conditions are as follows:

detection wavelength: 230nm, at which the absorption peak of N-dimethylnitrosamine is maximum;

flow rate of mobile phase: 0.8ml/min, and the high separation degree of the N-dimethyl nitrosamine and the adjacent impurity peaks can be ensured at the flow rate;

column temperature: the ranitidine hydrochloride can be eluted in a chromatographic column at the temperature of 35 ℃ to a high degree;

ambient temperature: less than 15 ℃, so that the N-dimethyl nitrosamine in the sample is not influenced by external temperature to cause quantity change;

solution sample introduction amount: 20 mu L, wherein the sample amount can ensure that the chromatographic peak of the N-dimethyl nitrosamine is normal and can sufficiently reach a certain detection limit;

mobile phase: comprises a mobile phase A and a mobile phase B; wherein in the mobile phase A, the mixing volume ratio of phosphate buffer solution to acetonitrile is 98: 2; in the mobile phase B, the mixing volume ratio of phosphate buffer solution to acetonitrile is 78: 22; the preparation method of the phosphate buffer solution comprises the following steps: putting 6.8ml of phosphoric acid into 900ml of water, adding 8.6ml of sodium hydroxide solution with the mass concentration of 50%, adding water to 2000ml, and adjusting the pH value to 7.10 +/-0.05 by using the phosphoric acid or the sodium hydroxide solution with the mass concentration of 50%;

the elution mode of the mobile phase is gradient elution, which comprises the following specific steps:

in 0-15 min, the volume of the mobile phase A is decreased from 100% to 0%, and the volume of the mobile phase B is increased from 0% to 100%;

in 15-23 min, the volume of the mobile phase A is 0%, and the volume of the mobile phase B is 100%;

at 23-24 min, the volume of the mobile phase A is increased from 0% to 100%, and the volume of the mobile phase B is decreased from 100% to 0%;

and at 24-30 min, the volume of the mobile phase A is 100%, and the volume of the mobile phase B is 0%.

The analytical results were as follows:

(1) specificity

The solvent blank is free of interference; other impurity peaks in the sample should be non-interfering with the examination of NDMA.

(2) Sensitivity of the probe

The signal to noise ratio was 28.9.

(3) Repeatability of

The RSD of the peak area of the NDMA control solution after continuous sample injection for 6 times is 3.46 percent, and the repeatability is good.

(4) Recovery rate

The recovery rate of the method is 94.1%.

The obtained spectrogram is shown in figure 1, wherein the peak at 4.298 min is NDMA peak, the peak at 18.710 min is ranitidine hydrochloride drug tiutane peak, and the other peaks are other impurity peaks.

The high performance liquid chromatography analysis method of the nitrosamine substances in the ranitidine medicines has the following advantages:

1. the conventional high performance liquid chromatograph can be used for experimental analysis, the accuracy is high, the reproducibility of results is good, large capital investment is not needed, and the cost is saved;

2. through the setting of process conditions such as sample drying, grinding treatment, low-temperature configuration under a sealing condition, filtering before testing and the like, on one hand, the stability and accuracy of the test result of the N-dimethyl nitrosamine are ensured; on the other hand, the pollution of the sample to a chromatographic system is greatly reduced, the later use and maintenance are reduced, and the maintenance cost is saved;

3. by selective use of the chromatographic column, the phenomenon of conjugate elution is effectively avoided;

4. the method has the advantages of simple experimental conditions, short process and convenient implementation and operation, is very suitable for production control and use, and is beneficial to industrial application.

In the description of the present invention, it should be noted that the terms "upper", "lower", "left", "right", "inner", "outer", and the like indicate orientations or positional relationships based on orientations or positional relationships shown in the drawings or orientations or positional relationships that are conventionally arranged when the products of the present invention are used, and are used for convenience of description and simplicity of description only, and do not indicate or imply that the devices or elements indicated must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.