A method for detecting sodium azide in a drug or its intermediate

Abstract

The present application belongs to the field of pharmaceutical detection technology, and specifically relates to a method for detecting sodium azide in a drug or its intermediate. The method uses liquid chromatography for detection, and the detection cost is low. The method can achieve low-cost, rapid and accurate quantitative determination of sodium azide.

Description

Technical Field

[0001] The present application belongs to the field of pharmaceutical detection technology, and specifically relates to a method for detecting sodium azide in a drug or its intermediates. Background Art

[0002] Sodium azide is a class of compounds containing three nitrogen-connected structures, generally represented by NaN3. Sodium azide is an inhibitor of the electron transport system and can form coordination compounds with cytochromes to prevent the reduction of the oxidized a3 component of cytochrome oxidase. Sodium azide is highly toxic, so the content of drugs and their intermediates must be strictly controlled.

[0003] Sodium azide is a common key material in the process of drug synthesis. For example, sartan drugs such as irbesartan, valsartan, candesartan; latamoxef sodium, cefoperazone sodium, cefmandol sodium, isosporin drugs; zidovudine and other drugs all use sodium azide as a material in their synthesis. The content of sodium azide in these drugs and their intermediates needs to be strictly controlled.

[0004] At present, the content limit of sodium azide has been written into the standards of some drugs. For example, the raw material quality standard of irbesartan clearly requires that the content limit of sodium azide should not exceed 0.001% by peak area calculated according to the standard method.

[0005] One of the starting materials of latamoxef sodium is 1-methyl-5-mercaptotetrazole, and sodium azide is used as a starting material in the production process of 1-methyl-5-mercaptotetrazole. The source of sodium azide in latamoxef sodium is introduced from the starting material 1-methyl-5-mercaptotetrazole. In order to control the content of sodium azide in the finished product of latamoxef sodium, it can be achieved by controlling the content of sodium azide in 1-methyl-5-mercaptotetrazole.

[0006] The 2020 edition of the Chinese Pharmacopoeia, Part II, Irbesartan Raw Material Standard, discloses a method for detecting sodium azide in Irbesartan raw materials. The specific contents are as follows:

[0007] System suitability solution: Take appropriate amounts of potassium bromide, sodium azide and potassium nitrate, add 90% methanol solution to dissolve and dilute to make a mixed solution containing approximately 0.2 μg of each per 1 ml.

[0008] Chromatographic conditions: an anion exchange column (lonPac AS18 column, or a column with equivalent performance) was used; the detector was a conductivity detector; the detection mode was suppressed conductivity detection; the column temperature was 30°C; potassium hydroxide solution was used as the eluent, and the analytical column concentration gradient elution was performed according to the procedure in the table below; the flow rate was 1.0 ml per minute; the test solution was injected and treated online using the valve switching online matrix elimination method (see Appendix 2); the injection volume was 200 μl.

[0009]

[0010] System suitability requirements In the system suitability solution chromatogram, the separation degree of azide from bromide and nitrate should be greater than 1.5.

[0011] Determination method: Accurately measure the test solution and the reference solution, inject them into the ion chromatograph respectively, and record the chromatogram.

[0012] If a sodium azide peak is evident in the chromatogram of the test solution, the peak area calculated using the external standard method shall not exceed 0.001%.

[0013] In this method, ion chromatography is used for detection, which requires an ion chromatograph with an enrichment device, a pretreatment column, an enrichment column, a switching valve, and a dedicated ion chromatograph column. The ion chromatograph with this function is expensive and has a low utilization rate. Its consumables, such as ion chromatographic columns, conductivity suppressors, and eluents, are all expensive, with a total cost of about RMB 1 million. In addition, the consumables have a short lifespan and the detection cost is high.

[0014] Patent CN108982695A discloses a method for determining azide compounds in drugs or their intermediates by derivatization HPLC. First, at the experimental temperature, a derivatization reaction is carried out on the azide compound using a benzyl chloride-type derivatization reagent for 5 to 60 minutes, and the reaction solution is used as a sample for detection; the derivatization reaction solution generated above is used as a sample, and the derivatization product is determined between 220 and 300 nm by HPLC-DAD method, thereby achieving quantitative detection of azide compounds in drugs or their synthetic intermediates. The present invention can avoid matrix interference caused by the drug or synthetic intermediate itself, establish a highly sensitive, highly specific, simple and universal pre-column derivatization method, and then use HPLC method to determine the residual amount of azide compounds in drugs or their synthetic intermediates, thereby improving the sensitivity and specificity of the azide compound detection method. In this method, the derivatization method is used for detection, and the detection target is the derivatized compound rather than sodium azide itself. The specificity is limited and the operation steps are cumbersome, which is easily affected by the objective environment; each compound, such as cephalosporin compounds, is easy to degrade and the degradation products are complex. The patent provides Figure 4 , Figure 5 , Figure 6 All of them indicate that they contain a large number of other chromatographic peaks, and the derivative products are easily interfered by other chromatographic peaks, which destroys the specificity of the method. Summary of the invention

[0015] Purpose of the invention: The purpose of the present invention is to provide a method for detecting sodium azide in a drug or its intermediate, which adopts liquid chromatography for detection and has low detection cost. The method can realize low-cost, rapid and accurate quantitative determination of sodium azide.

[0016] A method for detecting sodium azide in a drug or its intermediate, wherein the detection method is a high performance liquid chromatography method.

[0017] The drugs are sartans, cephalosporins and zidovudine.

[0018] Preferably, the drug is irbesartan, telmisartan, valsartan, candesartan, latamoxef sodium, cefoperazone sodium, cefuroxime sodium, or zidovudine.

[0019] The intermediate of the latamoxef sodium is 1-methyl-5-mercaptotetrazole.

[0020] The method for detecting sodium azide in the above-mentioned drug or its intermediate comprises the following steps:

[0021] High performance liquid chromatography is used for detection, and a test solution of the drug or its intermediate is directly processed online through a first chromatographic column (column A) and a second chromatographic column (column B) and then directly enters a detector of a liquid chromatograph for detection to obtain the amount of sodium azide in the drug or its intermediate; the first chromatographic column (column A) and the second chromatographic column (column B) are connected by a six-way valve, and the valve is switched within 4-6 minutes using an online valve switching technology.

[0022] Preferably, the first chromatographic column (column A) is an acid-resistant octadecylsilane bonded silica gel column.

[0023] More preferably, the first chromatographic column (column A) is Venusil ASB C18, 4.6×250 mm, 5 μm.

[0024] Preferably, the second chromatographic column (column B) uses sulfonated cross-linked styrene-divinylbenzene copolymer calcium type strong cation exchange resin as filler.

[0025] More preferably, the second chromatographic column (column B) is CHRomegabond CAM Ca2+8% ES INDUSTRIES 300×7.8 mm, 8 μm.

[0026] The detection method of sodium azide in the above-mentioned drug or its intermediate, the chromatographic conditions of the high performance liquid chromatography are: the infusion system comprises a gradient pump A and a constant flow pump B, the gradient pump A uses 0.05% sulfuric acid solution as mobile phase A, methanol as mobile phase B, the flow rate is 1.0 ml / min, and linear gradient elution and valve switching are performed according to the table and figure below, the valve switching time is 4 minutes (pump A→column A→column B→detector, pump B→waste liquid pool) and 6 minutes (pump A→column A→waste liquid pool, pump B→column B→detector); the constant flow pump B uses 0.05% sulfuric acid solution as mobile phase, the flow rate is 0.5 ml per minute; the column temperature is 60°C; the detection wavelength is 210 nm; the injection volume is 100 μl.

[0027]

[0028] The method for detecting sodium azide in the drug or its intermediate of the present invention adopts a high performance liquid chromatograph for detection, and its infusion system adopts a gradient pump A and a constant flow pump B; the connection state of the gradient pump A and the constant flow pump B is shown in Fig. 9 The specific operation is as follows:

[0029] ① 0-4min, the flow path is "pump A→column A→waste liquid pool, pump B→column B→detector". At this time, sodium azide is in column A, and non-sodium azide impurities in the drug flow out of column A and into the waste liquid pool;

[0030] ②4-6min, the flow path is "pump A→column A→column B→detector, pump B→waste pool", at this time, sodium azide flows out of column A and into column B, while the drug or its intermediate remains in column A;

[0031] ③ After 6 minutes, the flow path is "pump A→column A→waste liquid pool, pump B→column B→detector". At this time, the drug or its intermediate is eluted by the gradient pump with the mobile phase with gradually increasing organic phase concentration, and then enters the waste liquid pool; sodium azide is eluted from column B with 0.05% sulfuric acid solution and enters the detector. Specifically, a method for detecting sodium azide in a drug or its intermediate comprises the following steps:

[0032] The chromatographic condition infusion system comprises a gradient pump A and a constant flow pump B, the first chromatographic column (column A) is an acid-resistant silica gel chromatographic column; the second chromatographic column (column B) uses a sulfonated cross-linked styrene-divinylbenzene copolymer calcium type strong cation exchange resin as a filler; the first chromatographic column (column A) and the second chromatographic column (column B) are connected by a six-way valve; the gradient pump A uses 0.05% sulfuric acid solution as mobile phase A, methanol as mobile phase B, and a flow rate of 1.0 ml / min, and performs linear gradient elution and valve switching according to the table and figure below, and the valve switching time is 4 minutes (0 state) and 6 minutes (1 state); the constant flow pump B uses 0.05% sulfuric acid solution as mobile phase, and the flow rate is 0.5 ml per minute; the column temperature is 60°C; the detection wavelength is 210nm; the injection volume is 100μl;

[0033]

[0034] Solvent: 50% methanol in water;

[0035] Linear relationship solution: Take sodium azide reference substance, weigh it accurately, dissolve it in solvent and dilute it quantitatively to make a solution containing about 0.2 mg of sodium azide per 1 ml; accurately measure 1 ml and put it in a 50 ml volumetric flask, dilute it quantitatively to the scale with solvent, and shake it well; accurately measure 1, 2, 3, 4, and 5 ml respectively and put them in a 10 ml volumetric flask, dilute it quantitatively to the scale with solvent, and shake it well to obtain linear relationship test solutions 1, 2, 3, 4, and 5;

[0036] Test solution Weigh 1.0 g of the drug or its intermediate accurately, place in a 10 ml volumetric flask, add solvent to dissolve and quantitatively dilute to the scale, shake well;

[0037] System suitability requires that the linear relationship test 1 solution be used as the sensitivity solution; in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the sodium azide chromatographic peak height should be greater than 10, and a standard curve should be drawn based on the peak area of ​​each component in the linear relationship solution and the corresponding concentration, and the linear regression equation should be calculated. The correlation coefficient (r) should be no less than 0.995;

[0038] Determination method: Accurately measure 100 μl of the test solution and the linear relationship solution and inject them into the liquid chromatograph to record the chromatogram. If there is a chromatographic peak in the chromatogram of the test solution that is consistent with the retention time of sodium azide, calculate according to the linear regression equation to obtain the result.

[0039] Preferably, in the above-mentioned method for detecting sodium azide in a drug or its intermediate, the first chromatographic column (column A) is Venusil ASB C18, 4.6×250 mm, 5 μm.

[0040] Preferably, in the above-mentioned method for detecting sodium azide in a drug or its intermediate, the second chromatographic column (column B) is CHRomegabond CAM Ca 2+ 8% ES INDUSTRIES 300×7.8mm, 8μm.

[0041] One of the preferred technical solutions of the present invention:

[0042] A method for detecting sodium azide in 1-methyl-5-mercaptotetrazole comprises the following steps:

[0043] The chromatographic condition infusion system includes a gradient pump A and a constant flow pump B, a first chromatographic column (column A) (recommended chromatographic column Venusil ASB C18, 4.6×250 mm, 5 μm, or a chromatographic column with equivalent performance), a second chromatographic column (column B) (sulfonated cross-linked styrene-divinylbenzene copolymer calcium type strong cation exchange resin as filler, recommended chromatographic column CHRomegabondCAM Ca 2+8% ES INDUSTRIES 300×7.8mm, 8μm, or a chromatographic column with equivalent performance). The first chromatographic column (column A) and the second chromatographic column (column B) are connected by a six-way valve; the gradient pump A uses 0.05% sulfuric acid solution as mobile phase A and methanol as mobile phase B, with a flow rate of 1.0ml / min, and performs linear gradient elution and valve switching according to the table and figure below, with valve switching time of 4 minutes (0 state) and 6 minutes (1 state); the constant flow pump B uses 0.05% sulfuric acid solution as mobile phase, with a flow rate of 0.5ml per minute; the column temperature is 60°C; the detection wavelength is 210nm; the injection volume is 100μl.

[0044]

[0045] Solvent: 50% methanol in water.

[0046] Linear relationship solution: Take sodium azide reference substance, weigh it accurately, dissolve it in solvent and dilute it quantitatively to make a solution containing about 0.2 mg of sodium azide per 1 ml; accurately measure 1 ml and place it in a 50 ml volumetric flask, dilute it quantitatively to the scale with solvent, and shake well; accurately measure 1, 2, 3, 4, and 5 ml respectively and place them in a 10 ml volumetric flask, dilute it quantitatively to the scale with solvent, and shake well to obtain linear relationship test solutions 1, 2, 3, 4, and 5.

[0047] Test solution: Weigh 1.0 g of 1-methyl-5-mercaptotetrazole, accurately weigh it, place it in a 10 ml volumetric flask, add solvent to dissolve it and quantitatively dilute it to the scale, and shake it well.

[0048] System suitability requires that the linear relationship test 1 solution be used as the sensitivity solution. In the chromatogram of the sensitivity solution, the signal-to-noise ratio of the sodium azide chromatographic peak height should be greater than 10. The standard curve is drawn with the peak area of ​​each component in the linear relationship solution and the corresponding concentration, and the linear regression equation is calculated. The correlation coefficient (r) should be no less than 0.995.

[0049] Determination method: Precisely measure 100 μl of the test solution and the linear relationship solution and inject them into the liquid chromatograph to record the chromatogram. If there is a chromatographic peak in the chromatogram of the test solution with the same retention time as sodium azide, calculate according to the linear regression equation that the sodium azide content shall not exceed 12 ppm.

[0050] Beneficial effects:

[0051] (1) In the method for detecting sodium azide in the drug or its intermediate of the present invention, the method adopts liquid chromatography for detection, and the detection cost is low. Low-cost, rapid and accurate quantitative determination of sodium azide can be achieved. The instrument used in the liquid chromatography is a liquid chromatograph, which is a common instrument in the field of drug detection, and its instrument cost is only 5-20% of that of an ion chromatograph. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 is the liquid chromatogram of blank solvent.

[0053] Figure 2 Liquid chromatogram of sodium azide positioning solution.

[0054] Figure 3 It is the liquid chromatogram of the test solution.

[0055] Figure 4 It is the liquid chromatogram of the test sample mixed solution.

[0056] Figure 5 This is the liquid chromatogram of sodium azide sensitive solution.

[0057] Figure 6 This is the linear relationship diagram of sodium azide.

[0058] Figure 7 The chromatogram obtained for Comparative Example 1 is shown in FIG.

[0059] Figure 8 The chromatogram obtained for Comparative Example 2.

[0060] Fig. 9 It is a schematic diagram of the connection status of the gradient pump A and the constant flow pump B. Among them, Fig. 9 -A is 1 state, Fig. 9 -B is 0 status. DETAILED DESCRIPTION

[0061] The present application will be further described below in conjunction with the accompanying drawings and specific implementation methods so that those skilled in the art can better understand the present application, but the present invention is not limited thereto.

[0062] The experimental instruments, reagents and materials used in the embodiments of the present invention can all be obtained commercially.

[0063] A method for detecting sodium azide in a drug or its intermediate, wherein the detection method is high performance liquid chromatography. The drugs are sartan drugs, cephalosporin drugs and zidovudine. Specifically, they are irbesartan, telmisartan, valsartan, candesartan, latamoxef sodium, cefoperazone sodium, cefmandol sodium and zidovudine. The intermediate of latamoxef sodium is 1-methyl-5-thiol tetrazole.

[0064] A method for detecting sodium azide in a drug or its intermediates comprises the following steps:

[0065] The chromatographic condition infusion system comprises a gradient pump A and a constant flow pump B, the first chromatographic column (column A) is an acid-resistant silica gel chromatographic column; the second chromatographic column (column B) uses a sulfonated cross-linked styrene-divinylbenzene copolymer calcium type strong cation exchange resin as a filler; the first chromatographic column (column A) and the second chromatographic column (column B) are connected by a six-way valve; the gradient pump A uses 0.05% sulfuric acid solution as mobile phase A, methanol as mobile phase B, and a flow rate of 1.0 ml / min, and performs linear gradient elution and valve switching according to the table and figure below, and the valve switching time is 4 minutes and 6 minutes; the constant flow pump B uses 0.05% sulfuric acid solution as mobile phase, and the flow rate is 0.5 ml per minute; the column temperature is 60°C; the detection wavelength is 210nm; the injection volume is 100μl;

[0066]

[0067] Solvent: 50% methanol in water;

[0068] Linear relationship solution: Take sodium azide reference substance, weigh it accurately, dissolve it in solvent and dilute it quantitatively to make a solution containing about 0.2 mg of sodium azide per 1 ml; accurately measure 1 ml and put it in a 50 ml volumetric flask, dilute it quantitatively to the scale with solvent, and shake it well; accurately measure 1, 2, 3, 4, and 5 ml respectively and put them in a 10 ml volumetric flask, dilute it quantitatively to the scale with solvent, and shake it well to obtain linear relationship test solutions 1, 2, 3, 4, and 5;

[0069] Test solution Weigh 1.0 g of the drug or its intermediate accurately, place in a 10 ml volumetric flask, add solvent to dissolve and quantitatively dilute to the scale, shake well;

[0070] System suitability requires that the linear relationship test 1 solution be used as the sensitivity solution; in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the sodium azide chromatographic peak height should be greater than 10, and a standard curve should be drawn based on the peak area of ​​each component in the linear relationship solution and the corresponding concentration, and the linear regression equation should be calculated. The correlation coefficient (r) should be no less than 0.995;

[0071] Determination method: Accurately measure 100 μl of the test solution and the linear relationship solution and inject them into the liquid chromatograph to record the chromatogram. If there is a chromatographic peak in the chromatogram of the test solution that is consistent with the retention time of sodium azide, calculate according to the linear regression equation to obtain the result.

[0072] One of the starting materials of latamoxef sodium is 1-methyl-5-mercaptotetrazole, and sodium azide is used as a starting material in the production process of 1-methyl-5-mercaptotetrazole. The source of sodium azide in latamoxef sodium is introduced from the starting material 1-methyl-5-mercaptotetrazole. In order to control the content of sodium azide in the finished product of latamoxef sodium, it is necessary to control the content of sodium azide in 1-methyl-5-mercaptotetrazole.

[0073] Example 1 Determination of the content of sodium azide in 1-methyl-5-mercaptotetrazolyl and its methodological experiment

[0074] The test methods to be verified are as follows:

[0075] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0076] The chromatographic condition infusion system includes a gradient pump A and a constant flow pump B, a first chromatographic column (column A) (recommended chromatographic column Venusil ASB C18, 4.6×250 mm, 5 μm, or a chromatographic column with equivalent performance), a second chromatographic column (column B) (sulfonated cross-linked styrene-divinylbenzene copolymer calcium type strong cation exchange resin as filler, recommended chromatographic column CHRomegabondCAM Ca 2+ 8% ES INDUSTRIES 300×7.8mm, 8μm, or a chromatographic column with equivalent performance). The first chromatographic column (column A) and the second chromatographic column (column B) are connected via a six-way valve; the specific connection is shown in the figure below.

[0077] Gradient pump A used 0.05% sulfuric acid solution as mobile phase A, methanol as mobile phase B, and the flow rate was 1.0 ml / min. Linear gradient elution and valve switching were performed according to the table and figure below. The valve switching time was 4 minutes (0 state) and 6 minutes (1 state). Constant flow pump B used 0.05% sulfuric acid solution as mobile phase, and the flow rate was 0.5 ml per minute. The column temperature was 60°C. The detection wavelength was 210 nm. The injection volume was 100 μl.

[0078]

[0079] The connection status of gradient pump A and constant flow pump B is shown in Fig. 9 .

[0080] Solvent: 50% methanol in water.

[0081] Linear relationship solution: Take sodium azide reference substance, weigh it accurately, dissolve it in solvent and dilute it quantitatively to make a solution containing about 0.2 mg of sodium azide per 1 ml; accurately measure 1 ml and place it in a 50 ml volumetric flask, dilute it quantitatively to the scale with solvent, and shake well; accurately measure 1, 2, 3, 4, and 5 ml respectively and place them in a 10 ml volumetric flask, dilute it quantitatively to the scale with solvent, and shake well to obtain linear relationship test solutions 1, 2, 3, 4, and 5.

[0082] Test solution: Weigh 1.0 g of 1-methyl-5-mercaptotetrazole, accurately weigh it, place it in a 10 ml volumetric flask, add solvent to dissolve it and quantitatively dilute it to the scale, and shake it well.

[0083] System suitability requires that the linear relationship test 1 solution be used as the sensitivity solution. In the chromatogram of the sensitivity solution, the signal-to-noise ratio of the sodium azide chromatographic peak height should be greater than 10. The standard curve is drawn with the peak area of ​​each component in the linear relationship solution and the corresponding concentration, and the linear regression equation is calculated. The correlation coefficient (r) should be no less than 0.995.

[0084] Determination method: Precisely measure 100 μl of the test solution and the linear relationship solution and inject them into the liquid chromatograph to record the chromatogram. If there is a chromatographic peak in the chromatogram of the test solution with the same retention time as sodium azide, calculate according to the linear regression equation that the sodium azide content shall not exceed 12 ppm.

[0085] This cannot be changed as it involves conversion.

[0086] 1. Methodological test

[0087] Table 1 Summary of method validation information

[0088]

[0089] 1.1 Specificity test

[0090] Solvent: 50% methanol in water

[0091] Sodium azide stock solution: Accurately weigh 19.99 mg of sodium azide, place in a 100 ml volumetric flask, add solvent to dissolve and quantitatively dilute to the scale, and shake well.

[0092] Sodium azide positioning solution: Accurately measure 1 ml of sodium azide stock solution and place it in a 50 ml volumetric flask, quantitatively dilute to the scale with solvent, and shake well.

[0093] Test blank solution: Weigh 1.0019 g of 1-methyl-5-mercaptotetrazole raw material, place in a 10 ml volumetric flask, add solvent to dissolve and quantitatively dilute to the scale, and shake well.

[0094] Test sample mixed solution: Weigh 1.0068 g of 1-methyl-5-mercaptotetrazole raw material, place in a 10 ml volumetric flask, add 3 ml of sodium azide fixing solution to dissolve, add solvent to dissolve and quantitatively dilute to the scale, and shake well.

[0095] Test and results: Precisely measure 100 μl of blank solvent, positioning solution, test solution, and test solution mixture respectively and inject them into the liquid chromatograph to record the chromatogram. The chromatogram is shown in Figure 1 to Figure 4 Conclusion: The retention time of sodium azide is 29.670 minutes; the blank solvent has no chromatographic peak at the chromatographic peak of sodium azide, and has no interference with the detection; the chromatograms of the test solution and the test mixed solution have no chromatographic peaks adjacent to the chromatographic peak of sodium azide, and have no interference with the detection.

[0096] 5.2 Sensitivity test

[0097] Sensitivity solution: Accurately weigh 19.99 mg of sodium azide, place in a 100 ml volumetric flask, add solvent to dissolve and quantitatively dilute to the scale, shake well; Accurately measure 1 ml of the above solution, place in a 50 ml volumetric flask, quantitatively dilute to the scale with solvent, shake well; Accurately measure 1 ml of the above solution, place in a 10 ml volumetric flask, quantitatively dilute to the scale with solvent, shake well.

[0098] Acceptable limit: The signal-to-noise ratio of the sodium azide peak height in the sensitivity solution chromatogram should be greater than 10.

[0099] Test and results: Accurately measure 100 μl of the above solution and inject it into the liquid chromatograph to record the chromatogram. The chromatogram is shown in Figure 5 , as can be seen from the chromatogram, the S / N is 47.57.

[0100] Conclusion: In the sensitivity solution chromatogram, the signal-to-noise ratio of the sodium azide peak height is 47.57, which meets the requirements.

[0101] 5.3 Linear relationship test

[0102] Linear stock solution: Accurately weigh 19.99 mg of sodium azide, place it in a 100 ml volumetric flask, add solvent to dissolve and quantitatively dilute to the scale, shake well; Accurately measure 1 ml of the above solution and place it in a 50 ml volumetric flask, quantitatively dilute to the scale with solvent, and shake well.

[0103] Linear relationship solution: Accurately measure 1, 2, 3, 4, and 5 ml of the above solution respectively and place them in a 10 ml volumetric flask, quantitatively dilute to the scale with solvent, shake well, and obtain linear relationship solutions 1, 2, 3, 4, and 5.

[0104] Acceptable limit: correlation coefficient ≥ 0.995.

[0105] Detection and results: Accurately measure 100 μl of each of the above solutions and inject them into the liquid chromatograph, record the chromatogram, calculate the linear equation parameters, and the results are shown in Table 2.

[0106] Table 2 Sodium azide linear relationship test data

[0107] No Volume(ml) Concentration (μg / 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

[0108] Linear relationship diagram Figure 6 : The linear equation is: A=55379C-3246, R=0.9999.

[0109] Conclusion: Complies with regulations.

[0110] 5.4 Limit of detection and limit of quantification

[0111] Detection limit: Determine the lowest limit at which sodium azide can be detected under the test conditions;

[0112] Limit of quantitation: The lowest amount of sodium azide that can be accurately quantified under the test conditions.

[0113] Signal-to-noise ratio method: Take an appropriate amount of sodium azide reference substance, add solvent to dissolve and dilute to the required concentration, and determine according to the method. The detection limit is calculated based on a signal-to-noise ratio S / N of 2 to 3; the quantification limit is calculated based on a signal-to-noise ratio S / N of 10.

[0114] Intuitive method: The limit of quantitation is determined by the lowest amount of the analyte that can be quantitatively determined and calculated based on the lowest concentration of the accuracy test.

[0115] (1) Signal-to-noise ratio method

[0116] Take an appropriate amount of sodium azide reference substance, add solvent to dissolve and dilute to the required concentration, and determine according to the method. The detection limit is calculated based on a signal-to-noise ratio S / N of 2 to 3; the quantification limit is calculated based on a signal-to-noise ratio S / N of 10.

[0117] Sodium azide quantification limit solution: accurately weigh 19.99 mg of sodium azide, place in a 100 ml volumetric flask, add solvent to dissolve and quantitatively dilute to the scale, shake well; accurately measure 1 ml of the above solution, place in a 50 ml volumetric flask, quantitatively dilute to the scale with solvent, shake well; accurately measure 1 ml of the above solution, place in a 10 ml volumetric flask, quantitatively dilute to the scale with solvent, shake well; accurately measure 2 ml of the above solution, place in a 10 ml volumetric flask, quantitatively dilute to the scale with solvent, shake well.

[0118] Sodium azide detection limit solution: Accurately measure 5 ml of the quantitative limit solution and place it in a 10 ml volumetric flask, quantitatively dilute it to the scale with solvent, and shake well.

[0119] Acceptable criteria: The quantitative limit concentration is less than the concentration of linear relationship test solution 1; the quantitative limit peak area RSD% is less than 10.0%, and the retention time RSD% is less than 2.0%.

[0120] Test and results: 100 μl of each of the above solutions was accurately measured and injected into the liquid chromatograph, and the quantitative limit solution was continuously measured 6 times, and the detection limit solution was measured once. The calculated quantitative limit concentration of sodium azide was 0.07956 μg / ml, the S / N value is shown in Table 3, the quantitative limit was 0.007956 μg, equivalent to 0.007956 ppm of the test sample; the detection limit concentration was 0.03978 μg / ml, the S / N value was 2.89, and the detection limit was 0.003978 μg, equivalent to 0.003978 ppm of the test sample; the calculation results are shown in Table 3.

[0121] Table 3 Sodium azide signal-to-noise ratio method quantitative limit determination data

[0122] frequency 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

[0123] (2) Intuitive method

[0124] Sodium azide quantification limit solution: Accurately weigh 19.99 mg of sodium azide, place in a 100 ml volumetric flask, add solvent to dissolve and quantitatively dilute to the scale, shake well; Accurately measure 1 ml of the above solution, place in a 50 ml volumetric flask, quantitatively dilute to the scale with solvent, shake well; Accurately measure 1 ml of the above solution, place in a 10 ml volumetric flask, quantitatively dilute to the scale with solvent, shake well.

[0125] Acceptable standard: In the chromatogram of the test solution, the retention time of the sodium azide peak RSD% ≤ 2.0%; the peak area RSD% ≤ 5.0%.

[0126] Test and results: Accurately measure 100 μl of the above test solution and inject it into the liquid chromatograph, measure it 6 times continuously, and record the chromatogram. The results are shown in Table 4.

[0127] Table 4 Sodium azide quantification limit solution determination data

[0128] frequency 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

[0129] Conclusion: The quantification limit of the sodium azide peak retention time RSD% is 0.09%; the peak area RSD% is 1.6%.

[0130] 5.5 System precision test

[0131] Determine the result changes corresponding to the instrument completing the same change process repeatedly under the test conditions.

[0132] Acceptable limit: response value RSD% ≤ 6.0%.

[0133] Test and results: 100 μl of the linear relationship solution (3) was accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded and the test was performed 6 times continuously. The results are shown in Table 5.

[0134] Table 5 Sodium azide precision test data

[0135] frequency 1 2 3 4 5 6 Mean RSD% Peak area 62646 63786 63286 63465 62992 63072 63208 0.7

[0136] Conclusion: Complies with regulations.

[0137] 5.6 Accuracy test

[0138] Determine how close the result of the sodium azide assay using the determined method is to the true value.

[0139] Acceptable limit: recovery rate 75% to 120%.

[0140] Accuracy stock solution: Accurately weigh 19.99 mg of sodium azide, place it in a 100 ml volumetric flask, add solvent to dissolve and quantitatively dilute to the scale, shake well; Accurately measure 1 ml of the above solution and place it in a 50 ml volumetric flask, quantitatively dilute to the scale with solvent, and shake well.

[0141] Reference solution: Accurately measure 3 ml of the stock solution and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0142] Accuracy Test solution: Weigh about 1.0 g of 1-methyl-5-mercaptotetrazole raw material into a 10 ml volumetric flask, add each solution according to Table 6, add solvent to dissolve and quantitatively dilute to the scale, and shake well.

[0143] Table 6 Accuracy test solution preparation method

[0144]

[0145] Detection and results: Accurately measure 100 μl of the reference solution and the test solution and inject them into the liquid chromatograph. Record the chromatogram and calculate the measured amount and recovery rate by the external standard method. The results are shown in Table 7.

[0146] Table 7 Sodium azide accuracy test data

[0147]

[0148] Conclusion: The average value of recovery rate 1 is 81.2%, and the RSD% is 2.8%; the average value of recovery rate 2 is 93.0%, and the RSD% is 0.6%; the average value of recovery rate 3 is 96.6%, and the RSD% is 0.5%, all of which meet the requirements.

[0149] 5.7 Repeatability test

[0150] Determine the closeness of the results obtained by the same analyst from multiple samplings of the same test product.

[0151] Repeatable stock solution: Accurately weigh 20.01 mg of sodium azide, place it in a 100 ml volumetric flask, add solvent to dissolve and quantitatively dilute to the scale, shake well; Accurately measure 1 ml of the above solution and place it in a 50 ml volumetric flask, quantitatively dilute to the scale with solvent, and shake well.

[0152] Reference solution: Accurately measure 3 ml of the reproducible stock solution and place it in a 10 ml volumetric flask, quantitatively dilute to the scale with solvent, and shake well.

[0153] Test solution: weigh about 1.0 g of 1-methyl-5-mercaptotetrazole raw material into a 10 ml volumetric flask, add 3.0 ml of reproducible stock solution, dissolve with solvent and quantitatively dilute to scale, shake well. Prepare 6 portions in a row.

[0154] Acceptable limit: RSD%≤6.0%.

[0155] Test and results: Accurately measure 100 μl of the reference solution and the test solution and inject them into the liquid chromatograph, record the chromatogram, calculate the measured amount and recovery rate by external standard method, and calculate the average value and RSD%. The results are shown in Table 8.

[0156] Table 8 Sodium azide repeatability test data

[0157]

[0158] Conclusion: The average recovery rate of sodium azide was 98.0%, and the RSD% was 2.1%, which was in line with the regulations.

[0159] 6. Conclusion

[0160] After detailed verification of the detection method, the specificity, linearity, sensitivity, system precision, accuracy and repeatability of the method all meet the requirements and are suitable for the detection of sodium azide in 1-methyl-5-mercaptotetrazole.

[0161] 7. Test results

[0162] Take 1-methyl-5-mercaptotetrazole raw material and test it according to the law. The results are shown in Table 9.

[0163] Table 9 Sample test results

[0164]

[0165] Comparative Example 1 Method and Spectrum of Using Only Octadecylsilane Bonded Silica Gel Column

[0166] Chromatographic conditions: octadecylsilane bonded silica gel column; mobile phase: 0.1% phosphoric acid solution; flow rate 0.5ml / min, detection wavelength 205nm, column temperature 35℃. Figure 7 .Depend on Figure 7 It can be seen that there is serious interference in the position of the object to be tested in the test sample.

[0167] Comparative Example 2

[0168] Chromatographic conditions: sulfonated cross-linked styrene-divinylbenzene copolymer calcium type strong cation exchange resin as filler; mobile phase: 0.05% sulfuric acid water; flow rate 0.5ml / min, detection wavelength 210nm, column temperature 60℃. Figure 8 .Depend on Figure 8 It can be seen that there is serious interference in the position of the object to be tested in the test sample.

Claims

1. A method for detecting sodium azide in 1-methyl-5-mercaptotetrazole, comprising the following steps: The chromatographic condition infusion system includes a gradient pump A and a constant flow pump B. The first chromatographic column A is Venusil ASB C18, 4.6×250mm, 5μm, and the second chromatographic column B is CHRomegabond CAM Ca 2+ 8% ES INDUSTRIES 300×7.8mm, 8μm; the first chromatographic column A and the second chromatographic column B are connected by a six-way valve; the gradient pump A uses 0.05% sulfuric acid solution as mobile phase A, methanol as mobile phase B, the flow rate is 1.0ml / min, the constant flow pump B uses 0.05% sulfuric acid solution as mobile phase, the flow rate is 0.5ml per minute; the column temperature is 60℃; the detection wavelength is 210nm; the injection volume is 100μl; Pump A elution conditions are as follows: The valve switching time is 4 minutes and 6 minutes, as follows: ① 0-4min, the flow path is pump A→column A→waste pool, pump B→column B→detector; ②4-6min, the flow path is pump A→column A→column B→detector, pump B→waste pool; ③After 6 minutes, the flow path is pump A→column A→waste liquid tank, pump B→column B→detector.

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