阅读说明：本技术 一种药物或其中间体中叠氮化钠的检测方法 (Method for detecting sodium azide in medicine or intermediate thereof ) 是由 马桂霞 任霞 邵长凯 李英杰 于 2021-09-26 设计创作，主要内容包括：本申请属于医药检测技术领域,具体涉及一种药物或其中间体中叠氮化钠的检测方法。该方法采用液相色谱法进行检测,采用十八烷基硅烷键合硅胶柱和分子排阻色谱柱联用来实现叠氮化钠的快速分离和检测,检测成本低。该方法可实现叠氮化钠的低成本、快速、准确的定量测定。(The application belongs to the technical field of medicine detection, and particularly relates to a method for detecting sodium azide in a medicine or an intermediate thereof. The method adopts liquid chromatography for detection, realizes rapid separation and detection of sodium azide by combining an octadecylsilane chemically bonded silica column and a molecular exclusion chromatographic column, and has low detection cost. The method can realize low-cost, quick and accurate quantitative determination of the sodium azide.)

1. A method for detecting sodium azide in a drug or an intermediate thereof is characterized in that the detection method is high performance liquid chromatography.

2. The method for detecting sodium azide according to claim 1 or an intermediate thereof, wherein said drug is one selected from the group consisting of sartan drug, cepham drug and zidovudine.

3. The method for detecting sodium azide of a drug or an intermediate thereof according to claim 2, wherein the drug is irbesartan, telmisartan, valsartan, candesartan, latamoxef sodium, cefoperazone sodium, cefamandole nafate, zidovudine.

4. The method for detecting sodium azide in medicine or an intermediate thereof according to claim 3, wherein the intermediate of latamoxef sodium is 1-methyl-5-mercaptotetrazole.

5. The method for detecting sodium azide according to claim 1 or an intermediate thereof, comprising the steps of:

detecting by high performance liquid chromatography, wherein a test solution of the drug or the intermediate thereof is subjected to online treatment by a first chromatographic column and a second chromatographic column and then directly enters a detector of a liquid chromatograph for detection to obtain the amount of sodium azide in the drug or the intermediate thereof; the first chromatographic column is an octadecylsilane chemically bonded silica column, the second chromatographic column is a molecular exclusion chromatographic column, the first chromatographic column and the second chromatographic column are connected through a six-way valve, and valve switching is carried out for 4-6 minutes by adopting an online valve switching technology.

6. The method for detecting sodium azide of claim 5 or an intermediate thereof, wherein said first column is an acid-resistant octadecylsilane chemically bonded silica column.

7. The method for detecting sodium azide according to claim 6 or an intermediate thereof, wherein the first chromatographic column is Venusil ASB C18, 4.6X 250mm, 5 μm.

8. The method for detecting sodium azide according to claim 5 or an intermediate thereof, wherein said second column comprises a sulfonated crosslinked calcium-based strong cation exchange resin of styrene-divinylbenzene copolymer as a filler.

9. The method for detecting sodium azide according to claim 8 wherein the second chromatographic column CHRomegabond CAM Ca2+ 8% ES INDUSTRIES 300X 7.8mm, 8 μm.

10. The method for detecting sodium azide in drug or intermediate thereof according to claim 5, wherein the chromatographic conditions of said HPLC are as follows: the infusion system comprises a gradient pump A and a constant flow pump B, wherein the gradient pump A takes 0.05% sulfuric acid solution as a mobile phase A and methanol as a mobile phase B, the flow rate is 1.0ml/min, linear gradient elution and valve switching are carried out according to the following table and the following figure, and the valve switching time is 4 minutes and 6 minutes; the constant flow pump B takes 0.05 percent sulfuric acid solution as a mobile phase, and the flow rate is 0.5ml per minute; the column temperature is 60 ℃; the detection wavelength is 210 nm; the sample injection volume is 100 mu l;

Technical Field

The application belongs to the technical field of medicine detection, and particularly relates to a method for detecting sodium azide in a medicine or an intermediate thereof.

Background

Sodium azide is a compound containing three nitrogen-linked structures, and is generally prepared by using NaN₃And (4) showing. Sodium azide is an inhibitor of the electron transport system and forms a complex with cytochrome, preventing the reduction of the oxidized form of the a3 component of cytochrome oxidase. Sodium azide is highly toxic and therefore, strict control of the drug is necessaryAnd the content of its intermediate sodium azide.

Sodium azide is a common key material in the process of drug synthesis. Sartan drugs such as irbesartan, valsartan, candesartan, and the like; latamoxef sodium, cefoperazone sodium, cefamandole nafate, isosporins; the zidovudine and other medicaments are synthesized by using sodium azide as a material. The content of sodium azide in the medicaments and the intermediates thereof needs to be strictly controlled.

At present, the content limit of sodium azide is written in the standards of partial medicines, such as irbesartan, and the content limit of sodium azide is definitely required in the quality standard of raw materials so as to be less than 0.001 percent calculated by peak area according to the standard method.

One of starting materials of latamoxef sodium is 1-methyl-5-mercapto tetrazole, and sodium azide is used as the starting material in the production process of the 1-methyl-5-mercapto tetrazole. The source of sodium azide in latamoxef sodium is introduced into 1-methyl-5-mercapto tetrazole which is a starting material. In order to control the content of sodium azide in the latamoxef sodium finished product, the content of sodium azide in 1-methyl-5-mercaptotetrazole can be controlled.

A method for detecting sodium azide in irbesartan raw materials is disclosed in the second irbesartan raw material standard of China pharmacopoeia 2020 edition, and the specific contents are as follows:

system applicability solution appropriate amounts of potassium bromide, sodium azide and potassium nitrate were taken, dissolved in 90% methanol solution and diluted to make mixed solutions each containing about 0.2. mu.g per 1 ml.

Chromatographic conditions were performed using an anion exchange chromatography column (lonPac AS18 column, or equivalent performance column); the detector is a conductivity detector; the detection mode is inhibition conductance detection; the column temperature is 30 ℃; taking potassium hydroxide solution as eluent, and performing gradient elution of the concentration of the analytical column according to the following table procedure; the flow rate was 1.0ml per minute; sample introduction is carried out on the sample solution by a valve switching online matrix elimination method (see attached 2) and then online treatment is carried out; the injection volume was 200. mu.l.

System applicability requires that the separation degree of azide, bromide ion and nitrate in a system applicability solution chromatogram should be greater than 1.5.

The measurement method comprises precisely measuring the test solution and the reference solution, respectively injecting into an ion chromatograph, and recording chromatogram.

If a sodium azide peak appears in the chromatogram of the limit test solution, the peak area is calculated according to an external standard method, and the peak area is not more than 0.001 percent.

In the method, ion chromatography is adopted for detection, and an ion chromatograph with an enrichment device, a pretreatment column, an enrichment column, a switching valve and a special ion chromatographic column are required. The ion chromatograph with the function has the advantages of high price, low utilization rate, high price of consumables such as an ion chromatographic column, a conductance suppressor, leacheate and the like, comprehensive cost of about one million RMB, long service life of the consumables and high detection cost.

The patent CN108982695A discloses a method for determining azide in a drug or an intermediate thereof by a derivatization HPLC method, firstly, performing derivatization reaction on the azide by using a biphenyl acyl chloride derivatization reagent for 5-60 min at an experimental temperature, and detecting a reaction solution as a sample; and (3) taking the generated derivatization reaction liquid as a sample, and determining a derivatization product between 220 and 300nm by using an HPLC-DAD method, so as to realize quantitative detection of the azide compound in the drug or the synthetic intermediate thereof. The method can avoid matrix interference caused by the drug or the synthetic intermediate, establish a simple and universal pre-column derivatization method with high sensitivity and specificity, measure the residual quantity of the azide in the drug or the synthetic intermediate thereof by an HPLC method, and improve the sensitivity and specificity of the azide detection method. In the method, a derivatization method is adopted for detection, the detection target is a derivatized compound instead of sodium azide, the specificity is limited, the operation steps are complicated, and the method is easily influenced by objective environment; each compound such as a cephalosporin compound is easy to degrade and the degradation product is complex, and figures 4, 5 and 6 provided by the patent all show that the derivative product contains a large number of other chromatographic peaks and is easy to interfere with the other chromatographic peaks, so that the specificity of the method is damaged.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a method for detecting sodium azide in a drug or an intermediate thereof, which adopts liquid chromatography for detection, adopts low-cost acid-resistant octadecyl silane bonded silica gel column and molecular exclusion chromatographic column to realize the rapid separation and detection of the sodium azide and has low detection cost. The method can realize low-cost, quick and accurate quantitative determination of the sodium azide.

A method for detecting sodium azide in a drug or an intermediate thereof is high performance liquid chromatography.

The medicines are sartan medicines, cephalosporin medicines and zidovudine.

Preferably, the medicament is irbesartan, telmisartan, valsartan, candesartan, latamoxef sodium, cefoperazone sodium, cefamandole nafate or zidovudine.

The latamoxef sodium intermediate is 1-methyl-5-mercapto tetrazole.

The method for detecting the sodium azide in the medicine or the intermediate thereof comprises the following steps:

detecting by high performance liquid chromatography, wherein a test solution of the drug or the intermediate thereof is subjected to online treatment by a first chromatographic column and a second chromatographic column and then directly enters a detector of a liquid chromatograph for detection to obtain the amount of sodium azide in the drug or the intermediate thereof; the first chromatographic column is an octadecylsilane chemically bonded silica column, the second chromatographic column is a molecular exclusion chromatographic column, the first chromatographic column and the second chromatographic column are connected through a six-way valve, and valve switching is carried out for 4-6 minutes by adopting an online valve switching technology.

Preferably, the first chromatographic column is an acid-resistant octadecylsilane bonded silica column.

More preferably, the first chromatographic column is Venusil ASB C18, 4.6X 250mm, 5 μm.

Preferably, the second column is packed with a sulfonated crosslinked calcium styrene-divinylbenzene copolymer strong cation exchange resin, and the second column is CHRomegabond CAM Ca2+ 8% ES INDUSTRIES 300X 7.8mm, 8 μm.

In the method for detecting sodium azide in the medicine or the intermediate thereof, the chromatographic conditions of the high performance liquid chromatography are as follows: the infusion system comprises a gradient pump A and a constant flow pump B, wherein the gradient pump A takes 0.05% sulfuric acid solution as a mobile phase A and methanol as a mobile phase B, the flow rate is 1.0ml/min, linear gradient elution and valve switching are carried out according to the following table and the following figure, and the valve switching time is 4 minutes (pump A → column A → waste liquid, pump B → column B → detector) and 6 minutes (pump A → column B → detector, pump B → waste liquid); the constant flow pump B takes 0.05 percent sulfuric acid solution as a mobile phase, and the flow rate is 0.5ml per minute; the column temperature is 60 ℃; the detection wavelength is 210 nm; the injection volume was 100. mu.l.

The method for detecting the sodium azide in the medicine or the intermediate thereof adopts a high performance liquid chromatograph to detect, and a gradient pump A and a constant flow pump B are adopted in an infusion system; the chromatographic column system adopts an octadecylsilane chemically bonded silica column and a molecular exclusion chromatographic column which are connected in series, the middle part of the chromatographic column system is connected in series through a six-way valve, and the sodium azide is separated by adopting an online valve switching technology. The specific mechanism of separation is as follows: firstly, utilizing the different retention capacities of sodium azide and a drug or an intermediate thereof on an octadecylsilane chemically bonded silica column, selecting a mobile phase with low organic phase concentration, wherein the sodium azide flows out of the chromatographic column firstly, and the drug or the intermediate flows out of the chromatographic column later; step two, allowing sodium azide to flow out of an octadecylsilane chemically bonded silica column, and allowing the octadecylsilane chemically bonded silica to enter a molecular exclusion chromatographic column connected with the chromatographic column in series; thirdly, cutting off the connection of the two chromatographic columns through a conversion valve so as to prevent the medicine or the intermediate from entering the size exclusion chromatographic column; fourthly, executing a pump gradient program to elute the octadecylsilane chemically bonded silica column by using a mobile phase of a high-concentration organic phase, and selecting a mobile phase equilibrium chromatographic column with low organic phase concentration by using the pump gradient program after the medicine and the intermediate flow out to carry out the next detection; and fifthly, flowing the sodium azide out of the size exclusion chromatographic column while performing the fourth step, and entering a detector to finish detection.

The connection state of the gradient pump A and the constant flow pump B is shown in figure 9.

Specifically, the method for detecting sodium azide in the drug or the intermediate thereof comprises the following steps:

the chromatographic condition transfusion system comprises a gradient pump A and a constant flow pump B, and the first chromatographic column is an acid-resistant silica gel chromatographic column; the second chromatographic column takes sulfonated cross-linked styrene-divinylbenzene copolymer calcium type strong cation exchange resin as a filling agent; the first chromatographic column and the second chromatographic column are connected through a six-way valve; the gradient pump A uses 0.05% sulphuric acid solution as mobile phase A, methanol as mobile phase B, the flow rate is 1.0ml/min, the following table and the following chart are used for carrying out linear gradient elution and valve switching, and the valve switching time is 4 minutes (1 state) and 6 minutes (0 state); the constant flow pump B takes 0.05 percent sulfuric acid solution as a mobile phase, and the flow rate is 0.5ml per minute; the column temperature is 60 ℃; the detection wavelength is 210 nm; the sample injection volume is 100 mu l;

solvent: 50% methanol water;

taking a sodium azide reference substance as a linear relation solution, precisely weighing, adding a solvent for dissolution, and quantitatively diluting to prepare a solution containing about 0.2mg of sodium azide in every 1 ml; precisely measuring 1ml, putting into a 50ml measuring flask, quantitatively diluting to scale with a solvent, and shaking up; precisely measuring 1ml, 2ml, 3ml, 4 ml and 5ml respectively, putting into a 10ml measuring flask, quantitatively diluting to scale with a solvent, and shaking up to obtain linear relation test solutions 1, 2, 3, 4 and 5;

weighing 1.0g of the medicine or the intermediate thereof in the sample solution, precisely weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and quantitatively dilute to a scale, and shaking up;

the applicability of the system requires that a solution of a linear relation test 1 is taken as a sensitivity solution; in a sensitivity solution chromatogram, the signal-to-noise ratio of the high peak of a sodium azide chromatogram should be greater than 10, a standard curve is drawn according to the peak area and the corresponding concentration of each component in a linear relation solution, a linear regression equation is calculated, and the correlation coefficient (r) should be not less than 0.995;

the measurement method precisely measures 100. mu.l of each of the test solution and the linear relationship solution, and injects them into a liquid chromatograph, and records the chromatogram. If a chromatographic peak consistent with the retention time of the sodium azide exists in the chromatogram of the test solution, calculating according to a linear regression equation to obtain the sodium azide chromatographic sample.

Preferably, in the method for detecting sodium azide in the above-mentioned drug or intermediate thereof, the first chromatographic column is Venusil ASB C18, 4.6 × 250mm, 5 μm.

Preferably, in the method for detecting sodium azide in drug or intermediate thereof, the second chromatographic column is CHRomegabond CAM Ca²⁺8％ES INDUSTRIES 300×7.8mm，8μm。

One of the preferred technical schemes of the invention is as follows:

a method for detecting sodium azide in 1-methyl-5-mercapto tetrazole includes the following steps:

the chromatographic condition infusion system comprises a gradient pump A and a constant flow pump B, a first chromatographic column (recommended chromatographic column Venusil ASB C18, 4.6X 250mm, 5 μm, or equivalent chromatographic column), a second chromatographic column (recommended chromatographic column CHRomegabond CAM Ca) using sulfonated crosslinked styrene-divinylbenzene copolymer calcium type strong cation exchange resin as filler²⁺8% ES INDUSTRIES 300X 7.8mm, 8 μm, or equivalent performance columns). The first chromatographic column and the second chromatographic column are connected through a six-way valve; the gradient pump A uses 0.05% sulphuric acid solution as mobile phase A, methanol as mobile phase B, the flow rate is 1.0ml/min, the following table and the following chart are used for carrying out linear gradient elution and valve switching, and the valve switching time is 4 minutes (1 state) and 6 minutes (0 state); the constant flow pump B takes 0.05 percent sulfuric acid solution as a mobile phase, and the flow rate is 0.5ml per minute; the column temperature is 60 ℃; the detection wavelength is 210 nm; the injection volume was 100. mu.l.

Solvent: 50% methanol water.

Taking a sodium azide reference substance as a linear relation solution, precisely weighing, adding a solvent for dissolution, and quantitatively diluting to prepare a solution containing about 0.2mg of sodium azide in every 1 ml; precisely measuring 1ml, putting into a 50ml measuring flask, quantitatively diluting to scale with a solvent, and shaking up; precisely measuring 1ml, 2ml, 3ml, 4 ml and 5ml respectively, putting into a 10ml measuring flask, quantitatively diluting with solvent to scale, and shaking up to obtain test solutions 1, 2, 3, 4 and 5 with linear relationship.

Weighing 1.0g of 1-methyl-5-mercapto tetrazole in the sample solution, precisely weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and quantitatively dilute to a scale, and shaking up.

System applicability requires taking the linear relationship test 1 solution as the sensitivity solution. In a sensitivity solution chromatogram, the signal-to-noise ratio of the high peak of the sodium azide chromatogram should be greater than 10, a standard curve is drawn according to the peak area and the corresponding concentration of each component in a linear relation solution, a linear regression equation is calculated, and the correlation coefficient (r) should be not less than 0.995.

The measurement method precisely measures 100. mu.l of each of the test solution and the linear relationship solution, and injects them into a liquid chromatograph, and records the chromatogram. If a chromatographic peak consistent with the retention time of the sodium azide exists in the chromatogram of the test solution, the content of the sodium azide is not more than 12ppm according to the calculation of a linear regression equation.

Has the advantages that:

(1) in the method for detecting the sodium azide in the medicine or the intermediate thereof, the liquid chromatography is adopted for detection, and the acid-resistant octadecyl silane bonded silica gel column and the molecular exclusion chromatographic column with lower cost are combined to realize the rapid separation and detection of the sodium azide, so that the detection cost is low. Can realize the low-cost, quick and accurate quantitative determination of the sodium azide. The instrument used in the liquid chromatography is a liquid chromatograph, which is a common instrument in the field of medicine detection, and the instrument cost is only 5-20% of that of an ion chromatograph. Acid-resistant octadecylsilane bonded silica columns and size exclusion chromatography columns are commonly used chromatography columns, and the cost is much lower than that of ion chromatography columns.

(2) In the method for detecting the sodium azide in the medicine or the intermediate thereof, the rapid separation and detection of the sodium azide are realized on line through two chromatographic columns. The first chromatographic column is a silica gel column, and because sodium azide is not retained on the silica gel column, but the drug or an intermediate thereof can be generally retained on the silica gel column for a long time and cannot be eluted by a 0.05% sulfuric acid solution, the first C18 column is used for separating partial substances with similar polarities except the sodium azide and the sodium azide, and other substances are adsorbed in the C18 column; the second chromatographic column is sulfonated cross-linked calcium type strong cation of styrene-divinylbenzene copolymer, is a molecular exclusion chromatographic column, is separated according to the size of molecular weight, and is equivalent to separating sodium azide and partial substances with similar polarity of the sodium azide according to the size of the molecular weight. The six-way valve is added between the two chromatographic columns, so that the rapid separation of sodium azide in the 1-methyl-5-mercapto tetrazole is realized.

Drawings

FIG. 1 is a blank solvent liquid chromatogram,

FIG. 2 is a liquid chromatogram of a sodium azide positioning solution.

FIG. 3 is a liquid chromatogram of the test solution.

FIG. 4 is a liquid chromatogram of the sample mixture solution.

FIG. 5 is a liquid chromatogram of a sodium azide sensitive solution.

FIG. 6 is a linear relationship diagram of sodium azide.

FIG. 7 is a chromatogram obtained in comparative example 1.

FIG. 8 is a chromatogram obtained in comparative example 2.

Fig. 9 is a schematic view of the connection state of the gradient pump a and the constant flow pump B. In which FIG. 9-A shows a 1 state and FIG. 9-B shows a 0 state.

Detailed Description

The present application will now be described more fully hereinafter with reference to the accompanying drawings, in which some embodiments of the invention are shown and described, but which are not intended to limit the invention thereto.

Laboratory instruments, reagents and materials used in the examples of the present invention are commercially available.

A method for detecting sodium azide in a drug or an intermediate thereof is high performance liquid chromatography. The medicines are sartan medicines, cephalosporin medicines and zidovudine. In particular irbesartan, telmisartan, valsartan, candesartan, latamoxef sodium, cefoperazone sodium, cefamandole nafate and zidovudine. The latamoxef sodium intermediate is 1-methyl-5-mercapto tetrazole.

A method for detecting sodium azide in a drug or an intermediate thereof comprises the following steps:

the chromatographic condition transfusion system comprises a gradient pump A and a constant flow pump B, and the first chromatographic column is an acid-resistant silica gel chromatographic column; the second chromatographic column takes sulfonated cross-linked styrene-divinylbenzene copolymer calcium type strong cation exchange resin as a filling agent; the first chromatographic column and the second chromatographic column are connected through a six-way valve; the gradient pump A takes 0.05% sulfuric acid solution as a mobile phase A and methanol as a mobile phase B, the flow rate is 1.0ml/min, linear gradient elution and valve switching are carried out according to the following table and the following chart, and the valve switching time is 4 minutes and 6 minutes; the constant flow pump B takes 0.05 percent sulfuric acid solution as a mobile phase, and the flow rate is 0.5ml per minute; the column temperature is 60 ℃; the detection wavelength is 210 nm; the sample injection volume is 100 mu l;

solvent: 50% methanol water;

taking a sodium azide reference substance as a linear relation solution, precisely weighing, adding a solvent for dissolution, and quantitatively diluting to prepare a solution containing about 0.2mg of sodium azide in every 1 ml; precisely measuring 1ml, putting into a 50ml measuring flask, quantitatively diluting to scale with a solvent, and shaking up; precisely measuring 1ml, 2ml, 3ml, 4 ml and 5ml respectively, putting into a 10ml measuring flask, quantitatively diluting to scale with a solvent, and shaking up to obtain linear relation test solutions 1, 2, 3, 4 and 5;

weighing 1.0g of the medicine or the intermediate thereof in the sample solution, precisely weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and quantitatively dilute to a scale, and shaking up;

the applicability of the system requires that a solution of a linear relation test 1 is taken as a sensitivity solution; in a sensitivity solution chromatogram, the signal-to-noise ratio of the high peak of a sodium azide chromatogram should be greater than 10, a standard curve is drawn according to the peak area and the corresponding concentration of each component in a linear relation solution, a linear regression equation is calculated, and the correlation coefficient (r) should be not less than 0.995;

the measurement method precisely measures 100. mu.l of each of the test solution and the linear relationship solution, and injects them into a liquid chromatograph, and records the chromatogram. If a chromatographic peak consistent with the retention time of the sodium azide exists in the chromatogram of the test solution, calculating according to a linear regression equation to obtain the sodium azide chromatographic sample.

One of starting materials of latamoxef sodium is 1-methyl-5-mercapto tetrazole, and sodium azide is used as the starting material in the production process of the 1-methyl-5-mercapto tetrazole. The source of sodium azide in latamoxef sodium is introduced into 1-methyl-5-mercapto tetrazole which is a starting material. In order to control the content of sodium azide in the latamoxef sodium finished product, the content of sodium azide in 1-methyl-5-mercaptotetrazole needs to be controlled.

Example 11 determination of sodium azide content in methyl-5-Mercaptotetrazole and methodological experiment thereof

The detection method to be verified is as follows:

measuring by high performance liquid chromatography (China pharmacopoeia 2020 edition four-part general regulation 0512).

The chromatographic condition infusion system comprises a gradient pump A and a constant flow pump B, a first chromatographic column (recommended chromatographic column Venusil ASB C18, 4.6X 250mm, 5 μm, or equivalent chromatographic column), a second chromatographic column (recommended chromatographic column CHRomegabond CAM Ca) using sulfonated crosslinked styrene-divinylbenzene copolymer calcium type strong cation exchange resin as filler²⁺8% ES INDUSTRIES 300X 7.8mm, 8 μm, or equivalent performance columns). The first chromatographic column and the second chromatographic column are connected through a six-way valve; specific connections are shown in the following figures.

The gradient pump A uses 0.05% sulphuric acid solution as mobile phase A, methanol as mobile phase B, the flow rate is 1.0ml/min, the following table and the following chart are used for carrying out linear gradient elution and valve switching, and the valve switching time is 4 minutes (1 state) and 6 minutes (0 state); the constant flow pump B takes 0.05 percent sulfuric acid solution as a mobile phase, and the flow rate is 0.5ml per minute; the column temperature is 60 ℃; the detection wavelength is 210 nm; the injection volume was 100. mu.l.

The connection state of the gradient pump A and the constant flow pump B is shown in figure 9.

Solvent: 50% methanol water.

Taking a sodium azide reference substance as a linear relation solution, precisely weighing, adding a solvent for dissolution, and quantitatively diluting to prepare a solution containing about 0.2mg of sodium azide in every 1 ml; precisely measuring 1ml, putting into a 50ml measuring flask, quantitatively diluting to scale with a solvent, and shaking up; precisely measuring 1ml, 2ml, 3ml, 4 ml and 5ml respectively, putting into a 10ml measuring flask, quantitatively diluting with solvent to scale, and shaking up to obtain test solutions 1, 2, 3, 4 and 5 with linear relationship.

Weighing 1.0g of 1-methyl-5-mercapto tetrazole in the sample solution, precisely weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and quantitatively dilute to a scale, and shaking up.

System applicability requires taking the linear relationship test 1 solution as the sensitivity solution. In a sensitivity solution chromatogram, the signal-to-noise ratio of the high peak of the sodium azide chromatogram should be greater than 10, a standard curve is drawn according to the peak area and the corresponding concentration of each component in a linear relation solution, a linear regression equation is calculated, and the correlation coefficient (r) should be not less than 0.995.

The measurement method precisely measures 100. mu.l of each of the test solution and the linear relationship solution, and injects them into a liquid chromatograph, and records the chromatogram. If a chromatographic peak consistent with the retention time of the sodium azide exists in the chromatogram of the test solution, the content of the sodium azide is not more than 12ppm according to the calculation of a linear regression equation.

Here, no changes are made to the conversion.

1. Methodology test

TABLE 1 summary of methodological validation information

1.1 specificity test

Solvent: 50% methanol water

Sodium azide stock solution: precisely weighing 19.99mg of sodium azide, placing the sodium azide in a 100ml measuring flask, adding a solvent to dissolve the sodium azide, quantitatively diluting the sodium azide to a scale mark, and shaking up.

Sodium azide positioning solution: precisely measuring 1ml of sodium azide stock solution, placing the sodium azide stock solution into a 50ml measuring flask, quantitatively diluting the sodium azide stock solution to a scale mark by using a solvent, and shaking up.

Blank solution of test sample: 1.0019g of 1-methyl-5-mercapto tetrazole raw material is weighed, placed in a 10ml measuring flask, dissolved by adding a solvent, quantitatively diluted to a scale and shaken up.

Mixed solution of test sample: 1.0068g of 1-methyl-5-mercapto tetrazole raw material is weighed, placed in a 10ml measuring flask, dissolved by adding 3ml of sodium azide positioning solution, dissolved by adding a solvent, quantitatively diluted to a scale and uniformly shaken.

Detection and result: precisely measuring blank solvent, positioning solution, sample solution, and sample mixed solution, respectively, 100 μ l each, injecting into liquid chromatograph, and recording chromatogram, which is shown in fig. 1-4. And (4) conclusion: the sodium azide retention time was 29.670 minutes; the blank solvent has no chromatographic peak at the chromatographic peak of the sodium azide, and has no interference to detection, and the chromatogram of the test solution and the mixed solution of the test solution has no chromatographic peak at the adjacent position of the chromatographic peak of the sodium azide, and has no interference to detection.

5.2 sensitivity test

Sensitivity solution: precisely weighing 19.99mg of sodium azide, putting the sodium azide into a 100ml measuring flask, adding a solvent to dissolve the sodium azide, quantitatively diluting the sodium azide to a scale, and shaking up; precisely measuring 1ml of the solution, putting the solution into a 50ml measuring flask, quantitatively diluting the solution to a scale with a solvent, and shaking up; precisely measuring 1ml of the solution, putting the solution into a 10ml measuring flask, quantitatively diluting the solution to a scale with a solvent, and shaking up.

Acceptable limits: in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the peak height of the sodium azide is more than 10.

Detection and result: precisely measuring 100 μ l of the solution, injecting into a liquid chromatograph, and recording chromatogram, wherein the chromatogram is shown in FIG. 5, and the S/N is 47.57.

And (4) conclusion: in the chromatogram of the sensitive solution, the signal-to-noise ratio of the peak height of the sodium azide is 47.57, which meets the specification.

5.3 Linear relationship test

Linear stock solution: precisely weighing 19.99mg of sodium azide, putting the sodium azide into a 100ml measuring flask, adding a solvent to dissolve the sodium azide, quantitatively diluting the sodium azide to a scale, and shaking up; precisely measuring 1ml of the solution, putting the solution into a 50ml measuring flask, quantitatively diluting the solution to a scale with a solvent, and shaking up.

Linear relationship solution: precisely measuring the solutions 1, 2, 3, 4 and 5ml respectively, placing into a 10ml measuring flask, quantitatively diluting with solvent to scale, and shaking to obtain solutions 1, 2, 3, 4 and 5 with linear relationship.

Acceptable limits: the correlation coefficient is more than or equal to 0.995.

Detection and result: precisely measuring 100 μ l of each solution, injecting into a liquid chromatograph, recording chromatogram, and calculating linear equation parameters, wherein the results are shown in Table 2.

TABLE 2 sodium azide Linear relationship test data

No	Volume (ml)	Concentration (μ g/ml)	Peak area
				Linear relationship test solution 1	1	0.3978	19259
Linear relationship test solution 2	2	0.7956	40207
				Linear relationship test solution 3	3	1.193	62793
Linear relationship test solution 4	4	1.591	84842
				Linear relationship test solution 5	5	1.989	107085

The linear relationship is shown in FIG. 6: obtaining a linear equation: a is 55379C-3246 and R is 0.9999.

And (4) conclusion: compliance with the regulations.

5.4 detection and quantitation limits

Detection limit: determining the minimum amount of sodium azide that can be detected under the detection conditions;

and (4) quantitative limit: the minimum amount of sodium azide that can be accurately quantified under the assay conditions is determined.

Signal-to-noise ratio method: taking a proper amount of sodium azide reference substances, adding a solvent to dissolve and dilute the sodium azide reference substances to a required concentration, and measuring according to a method, wherein the detection limit is calculated by a signal-to-noise ratio S/N of 2-3; the limit of quantitation is based on a signal-to-noise ratio S/N of 10.

An intuitive method comprises the following steps: the limit of quantitation is determined by the minimum amount of the test substance that can be quantitatively determined, calculated as the minimum concentration of the accuracy test.

(1) Signal to noise ratio method

Taking a proper amount of sodium azide reference substances, adding a solvent to dissolve and dilute the sodium azide reference substances to a required concentration, and measuring according to a method, wherein the detection limit is calculated by a signal-to-noise ratio S/N of 2-3; the limit of quantitation is based on a signal-to-noise ratio S/N of 10.

Sodium azide quantitative limiting solution: precisely weighing 19.99mg of sodium azide, putting the sodium azide into a 100ml measuring flask, adding a solvent to dissolve the sodium azide, quantitatively diluting the sodium azide to a scale, and shaking up; precisely measuring 1ml of the solution, putting the solution into a 50ml measuring flask, quantitatively diluting the solution to a scale with a solvent, and shaking up; precisely measuring 1ml of the solution, putting the solution into a 10ml measuring flask, quantitatively diluting the solution to a scale with a solvent, and shaking up; precisely measuring 2ml of the solution, putting the solution into a 10ml measuring flask, quantitatively diluting the solution to a scale with a solvent, and shaking up.

Sodium azide detection limiting solution: precisely measuring 5ml of the limiting solution, putting the limiting solution into a 10ml measuring flask, quantitatively diluting the limiting solution to a scale with a solvent, and shaking up.

Acceptance criteria: the quantitative limit concentration is less than the concentration of the linear relation test solution 1; the quantitative peak limit area RSD percent is less than 10.0 percent, and the retention time RSD percent is less than 2.0 percent.

Detection and result: precisely measuring 100 μ l of each of the above solutions, injecting into a liquid chromatograph, continuously measuring for 6 times with quantitative limiting solution, and measuring for 1 time with detection limiting solution. Calculating to obtain the quantitative limit concentration of the sodium azide of 0.07956 mug/ml, wherein the S/N value is shown in Table 3, the quantitative limit is 0.007956 mug and is equivalent to 0.007956ppm of a test sample; the detection limit concentration is 0.03978 mug/ml, the S/N value is 2.89, the detection limit is 0.003978 mug, which is equivalent to 0.003978ppm of the test article; the calculation results are shown in Table 3.

TABLE 3 sodium azide SNR quantitation limit determination data

Number of times	1	2	3	4	5	6	RSD％
								Retention time	29.689	29.593	29.584	29.647	29.624	29.717	0.2
Peak area	2895	2659	2430	2502	2822	2584	6.9
								S/N	10.42	7.98	11.02	11.10	9.74	8.37	NA

(2) Direct viewing method

Sodium azide quantitative limiting solution: precisely weighing 19.99mg of sodium azide, putting the sodium azide into a 100ml measuring flask, adding a solvent to dissolve the sodium azide, quantitatively diluting the sodium azide to a scale, and shaking up; precisely measuring 1ml of the solution, putting the solution into a 50ml measuring flask, quantitatively diluting the solution to a scale with a solvent, and shaking up; precisely measuring 1ml of the solution, putting the solution into a 10ml measuring flask, quantitatively diluting the solution to a scale with a solvent, and shaking up.

Acceptance criteria: in the chromatogram of the quantitative limit test solution, the retention time RSD of the sodium azide peak is less than or equal to 2.0 percent; the peak area RSD percent is less than or equal to 5.0 percent.

Detection and result: precisely measuring 100 μ l of the sample solution, injecting into a liquid chromatograph, continuously measuring for 6 times, and recording chromatogram. The results are shown in Table 4.

TABLE 4 sodium azide quantitative limiting solution determination data

Number of times	1	2	3	4	5	6	RSD％
								Retention time	29.664	29.618	29.652	29.677	29.617	29.646	0.09
Peak area	19305	18977	18516	19074	18670	19030	1.6

And (4) conclusion: the visual method quantitative limit sodium azide peak retention time RSD% is 0.09%; the peak area RSD% was 1.6%.

5.5 System precision test

Determining that the instrument repeatedly completes the result change corresponding to the same change process under the detection condition.

Acceptable limits: the response value RSD percent is less than or equal to 6.0 percent.

Detection and result: precisely measuring 100 μ l of the linear relation solution (3), injecting into a liquid chromatograph, recording chromatogram, and continuously measuring for 6 times. The results are shown in Table 5.

TABLE 5 sodium azide precision test data

Number of times	1	2	3	4	5	6	Mean value	RSD％
									Peak area	62646	63786	63286	63465	62992	63072	63208	0.7

And (4) conclusion: compliance with the regulations.

5.6 accuracy test

And determining the degree of the detection result of the sodium azide by the determined method to be close to the true value.

Acceptable limits: the recovery rate is 75-120%.

Accuracy stock solutions: precisely weighing 19.99mg of sodium azide, putting the sodium azide into a 100ml measuring flask, adding a solvent to dissolve the sodium azide, quantitatively diluting the sodium azide to a scale, and shaking up; precisely measuring 1ml of the solution, putting the solution into a 50ml measuring flask, quantitatively diluting the solution to a scale with a solvent, and shaking up.

Control solution: precisely measuring 3ml of the accurate stock solution, putting the accurate stock solution into a 10ml measuring flask, quantitatively diluting the accurate stock solution to a scale with a solvent, and shaking up.

Accuracy test solution: weighing about 1.0g of 1-methyl-5-mercapto tetrazole raw material, placing the raw material into a 10ml measuring flask, adding each solution according to the table 6, adding a solvent for dissolution, quantitatively diluting to a scale, and shaking up.

TABLE 6 method for preparing accuracy test solution

Detection and result: precisely measuring 100 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, recording chromatogram, and calculating the measured amount and recovery rate by external standard method, the results are shown in Table 7.

TABLE 7 sodium azide accuracy test data

And (4) conclusion: the average value of the recovery rate 1 is 81.2 percent, and the RSD percent is 2.8 percent; the average recovery rate 2 is 93.0%, and the RSD% is 0.6%; the average value of the recovery rate 3 is 96.6%, and the RSD% is 0.5%, which both meet the specification.

5.7 repeatability test

The same sample is determined, and the results are tested by the same analyst through multiple sampling.

Repeated stock solutions: precisely weighing 20.01mg of sodium azide, putting the sodium azide into a 100ml measuring flask, adding a solvent to dissolve the sodium azide, quantitatively diluting the sodium azide to a scale, and shaking up; precisely measuring 1ml of the solution, putting the solution into a 50ml measuring flask, quantitatively diluting the solution to a scale with a solvent, and shaking up.

Control solution: precisely measuring 3ml of repetitive stock solution, placing the repetitive stock solution into a 10ml measuring flask, quantitatively diluting the repetitive stock solution to a scale with a solvent, and shaking up.

Test solution: weighing about 1.0g of 1-methyl-5-mercapto tetrazole raw material, placing the raw material into a 10ml measuring flask, adding 3.0ml of repetitive stock solution, adding a solvent for dissolving, quantitatively diluting to a scale, and shaking up. 6 parts are prepared continuously.

Acceptable limits: RSD percent is less than or equal to 6.0 percent.

Detection and result: precisely measuring 100 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, recording chromatogram, calculating the measured amount and recovery rate by external standard method, and calculating average value and RSD. The results are shown in Table 8.

TABLE 8 sodium azide repeatability test data

And (4) conclusion: the average recovery rate of sodium azide is 98.0 percent, and the RSD percent is 2.1 percent, which meets the specification.

6. Conclusion

Through detailed verification of the detection method, the specificity, linear relation, sensitivity, system precision, accuracy and repeatability of the method all meet the requirements, and the method is suitable for detecting sodium azide in 1-methyl-5-mercaptotetrazole.

7. The result of the detection

1-methyl-5-mercapto tetrazole as a raw material was taken, and the results of the detection by the method are shown in Table 9.

TABLE 9 results of sample examination

Comparative example 1 method and pattern for bonding silica gel column using octadecylsilane only

Chromatographic conditions are as follows: octadecylsilane chemically bonded silica gel column; mobile phase: 0.1% phosphoric acid solution; the flow rate is 0.5ml/min, the detection wavelength is 205nm, and the column temperature is 35 ℃. The map is shown in FIG. 7. It can be seen from fig. 7 that the level to be measured in the test sample has a severe disturbance.

Comparative example 2 method and profile using size exclusion chromatography column only

Chromatographic conditions are as follows: sulfonated crosslinked styrene-divinylbenzene copolymer calcium type strong cation exchange resin is used as a filler; mobile phase: 0.05% sulfuric acid water; the flow rate is 0.5ml/min, the detection wavelength is 210nm, and the column temperature is 60 ℃. The map is shown in FIG. 8. It can be seen from fig. 8 that the level of the object to be measured in the test sample has a severe disturbance.