High-precision detection method for content of related substances in isoniazide injection

Abstract

The invention provides a high-precision detection method for the content of related substances in an isoniazide injection. The content of four related substances including isonicotinamide, benzoyl hydrazine, 4-cyanopyridine and diisonicotinoyl hydrazine in the isoniazide injection can be accurately measured. The detection method comprises the following steps: S1, preparing a test solution and a mixed reference solution containing isoniazide and four related substances at the same time; s2, performing sample injection detection by using a high performance liquid chromatograph, performing sample injection mixing on the reference substance solution, and determining the retention time and the peak area of each related substance; and injecting the test solution, determining the peak appearance of each related substance according to the retention time, and calculating the content of each related substance through an external standard method. The detection method is high in sensitivity and durability, quantitative analysis can be carried out, the content of isoniazide, isonicotinamide, benzoyl hydrazine, 4-cyanopyridine and diisonicotinoyl hydrazine can be accurately detected, and a reasonable basis is provided for medicine quality control.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a high-precision detection method for the content of related substances in isoniazid injection. Background Art

[0002] Isoniazid, also known as remifen, is a widely used first-line anti-tuberculosis drug. Isoniazid has poor stability. Under conditions of hydrolysis and oxidation, isoniazid can be degraded to produce isonicotinamide, benzohydrazide, 4-cyanopyridine, diisonicotinylhydrazine, and free hydrazine. Free hydrazine, as a genotoxic impurity, needs to be controlled separately. There are many studies on the detection of free hydrazine. At present, isoniazid injection can effectively control free hydrazine through production process and accurate detection. However, there are few related research reports on the detection methods of other related substances in isoniazid injection, such as isonicotinamide, benzohydrazide, 4-cyanopyridine, and diisonicotinylhydrazine.

[0003] Isoniazid injection is included in the United States Pharmacopoeia and the Japanese Pharmacopoeia, but neither of them proposes a method for detecting related substances. The Chinese Pharmacopoeia does not include isoniazid injection, but only includes two preparations, isoniazid for injection and isoniazid tablets. The related substance detection of the two preparations adopts the related substance detection method in isoniazid raw materials included in the Chinese Pharmacopoeia. However, after our company's use and research, it was found that the reproducibility of the related substance detection method in the pharmacopoeia is not ideal, the main component chromatographic peak is bifurcated, and the separation between benzoylhydrazide and 4-cyanopyridine is poor. Summary of the invention

[0004] The purpose of the present invention is to provide a high-precision detection method for the content of related substances in isoniazid injection in view of the drawbacks of the prior art.

[0005] The technical solution adopted by the present invention to solve the technical problem is:

[0006] A high-precision detection method for the content of related substances in isoniazid injection, which can simultaneously realize the accurate detection of the content of isoniazid, and the contents of four related substances, namely, isonicotinamide, benzohydrazide, 4-cyanopyridine, and diisonicotinylhydrazine, in isoniazid injection;

[0007] The detection method comprises the following steps:

[0008] S1. Prepare the test solution and the mixed reference solution containing isoniazid and four related substances;

[0009] S2. Use high performance liquid chromatography for sampling and testing. First, inject the mixed reference solution to determine the retention time and peak area of ​​each related substance; then inject the test solution, determine the peak of each related substance by the retention time, and calculate the content of each related substance by the external standard method.

[0010] In step S1, the preparation process of the test solution is: accurately measure an appropriate amount of isoniazid injection, dilute it with deionized water to prepare the test solution, and the concentration of isoniazid in the test solution is 0.8-1.2 mg / ml, preferably 1.0 mg / ml.

[0011] In step S1, the preparation process of the mixed reference solution is:

[0012] (1) Preparation of stock solutions of relevant substance reference substances

[0013] Accurately weigh appropriate amounts of isonicotinamide reference substance, benzohydrazide reference substance, 4-cyanopyridine reference substance, and diisonicotinylhydrazine reference substance, dissolve in deionized water, and quantitatively dilute to prepare a stock solution of related substance reference substances containing 0.02 mg isonicotinamide, 0.02 mg benzohydrazide, 0.02 mg 4-cyanopyridine, and 0.03 mg diisonicotinylhydrazine per 1 ml;

[0014] (2) Preparation of mixed reference solution

[0015] Take an appropriate amount of isoniazid reference substance, weigh it accurately, add deionized water to dissolve and dilute it to make a solution containing 0.02 mg of isoniazid per 1 ml; accurately measure 5 ml, place it in a 50 ml volumetric flask, add 5 ml of the stock solution of the related substance reference substance, dilute it to the scale with water, shake well, and you will get the mixed reference substance solution.

[0016] It is characterized in that: in the step S2, the chromatographic conditions of the high performance liquid chromatography are:

[0017] Chromatograph: Thermo Vanquish or Agilent 1260 high performance liquid chromatograph;

[0018] Chromatographic column: a chromatographic column with octadecylsilane bonded silica as filler, preferably a Welch Ultimate AQ-C18 chromatographic column with specifications of 4.6 mm×250 mm, 5 μm.

[0019] Mobile phase: phosphate buffer-acetonitrile solution, the volume ratio of phosphate buffer to acetonitrile solution is 94:6-98:2, preferably 97:3;

[0020] Flow rate: 0.9-1.1 ml / min, preferably 1.0 ml / min;

[0021] Detector: UV detector;

[0022] Detection wavelength: 260-270 nm, preferably 265 nm;

[0023] Column temperature: room temperature;

[0024] Injection volume: 10 μl.

[0025] The phosphate buffer is prepared by mixing disodium hydrogen phosphate, triethylamine and deionized water; the concentration of disodium hydrogen phosphate in the phosphate buffer is 0.05-0.1 mol / L, and the volume concentration of triethylamine is 0.05%-3%.

[0026] The pH value of the phosphate buffer is 5.8-6.2, preferably 6.0.

[0027] Through the above technical means, the beneficial effects obtained by the present invention are:

[0028] The present invention provides a high-precision detection method for the content of related substances of isoniazid injection, which can simultaneously realize high-precision detection of the content of isoniazid, and the contents of four related substances, namely, isonicotinamide, benzohydrazide, 4-cyanopyridine, and diisonicotinylhydrazine, in a convenient and fast manner. The detection method of the present invention has high sensitivity and strong durability, can perform quantitative analysis, provides a reasonable basis for drug quality control, and provides guiding significance for the processing technology of drug production.

[0029] This detection method uses a chromatographic column with octadecylsilane bonded silica gel as a filler and a phosphate buffer-acetonitrile solution with appropriate pH and polarity as a mobile phase to effectively separate the chromatographic peaks of the main drug isoniazid and the four related substances, thereby accurately detecting the specific content of the four related substances, which not only improves the quality control of the drug, but also provides guidance for the upstream preparation process control of isoniazid injection through the changes in the content of related substances. Through methodological verification, it is proved that this detection method has good linearity, precision, repeatability, stability, accuracy and durability, and overcomes the difficulties of poor separation of related substances and unsatisfactory reproducibility of existing detection methods.

[0030] The chromatographic conditions of the present invention are highly versatile and durable, do not need to control the column temperature, and detection conditions such as chromatographic instruments, chromatographic columns, and mobile phases are also commonly used options. There is no need to purchase additional expensive equipment, and the detection cost and equipment maintenance cost are low, which is conducive to large-scale promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the chromatogram of the blank solution;

[0032] Figure 2 is the system suitability solution chromatogram;

[0033] Figure 3 It is the chromatogram of mixed reference solution;

[0034] Figure 4 It is the chromatogram of the test solution (batch number: 230301);

[0035] Figure 5 It is the chromatogram of the test solution (batch number: 230302);

[0036] Figure 6 It is the chromatogram of the test solution (batch number: 230303);

[0037] Figure 7 This is the chromatogram of the phosphate buffer solution pH 6.5 mixed reference substance solution of Comparative Example 2;

[0038] Figure 8 This is the chromatogram of the test sample solution reproduced by the 2020 edition of the Chinese Pharmacopoeia method of comparative example 4;

[0039] Fig. 9 This is the chromatogram of the spiked test solution reproduced by the European Pharmacopoeia 11.6 method of Comparative Example 5; DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to understand the content of the present invention, the applicant will further describe the technical solution of the present invention in conjunction with specific embodiments below, but the following content does not limit the present invention in any way.

[0041] In the following examples, the reagents used can be purchased directly from the market. Among them, the manufacturer of isoniazid injection is Shijiazhuang Kaida Bioengineering Co., Ltd.; the isoniazid reference substance comes from the China Food and Drug Inspection Institute, and the impurity reference substances are purchased from SINCO PHARMACHEM. Other reagents used in the method of the present invention are chromatographically pure.

[0042] In the following examples, various detection solutions are prepared, and the preparation methods are as follows:

[0043] 1. Preparation of stock solutions of related substance reference substances

[0044] Accurately weigh appropriate amounts of isonicotinamide reference substance, benzohydrazide reference substance, 4-cyanopyridine reference substance, and diisonicotinylhydrazine reference substance, add deionized water to dissolve and quantitatively dilute to prepare a stock solution of related substance reference substances containing 0.02 mg isonicotinamide, 0.02 mg benzohydrazide, 0.02 mg 4-cyanopyridine, and 0.03 mg diisonicotinylhydrazine per 1 ml.

[0045] 2. Preparation of mixed reference solution

[0046] Take an appropriate amount of isoniazid reference substance, weigh it accurately, add deionized water to dissolve and dilute it to make a solution containing 0.02 mg of isoniazid per 1 ml; accurately measure 5 ml, place it in a 50 ml volumetric flask, add 5 ml of the stock solution of the related substance reference substance, dilute it to the scale with water, shake well, and you will get the mixed reference substance solution.

[0047] 3. Preparation of system suitability solution

[0048] Accurately weigh 10 mg of isoniazid reference substance, place it in a 10 ml volumetric flask, add appropriate amount of deionized water to dissolve it, add 1 ml of related substance reference substance stock solution, dilute to the scale with water, and shake well.

[0049] 4. Preparation of test solution

[0050] Accurately measure an appropriate amount of isoniazid injection and dilute it with deionized water to prepare a test solution. The concentration of isoniazid in the test solution is 0.8-1.2 mg / ml.

[0051] 5. Preparation of spiked test solution

[0052] Accurately measure 1 ml of isoniazid injection, place it in a 50 ml volumetric flask, add 5 ml of the stock solution of the relevant substance reference substance, dilute to the scale with deionized water, and shake well.

[0053] It should be emphasized that the test solution must be freshly prepared before use or stored at 2-8°C for no more than 24 hours to avoid changes in component content due to high temperature.

[0054] The water used in the preparation of the above solutions is all deionized water.

[0055] Example 1

[0056] The method for determining the content of related substances in isoniazid injection comprises the following steps:

[0057] S1. Prepare test solution and mixed reference solution

[0058] S11. Prepare mixed reference solution

[0059] Accurately weigh appropriate amounts of isonicotinamide reference substance, benzohydrazide reference substance, 4-cyanopyridine reference substance, and diisonicotinylhydrazine reference substance, dissolve in deionized water, and quantitatively dilute to prepare a stock solution of related substance reference substances containing 0.02 mg isonicotinamide, 0.02 mg benzohydrazide, 0.02 mg 4-cyanopyridine, and 0.03 mg diisonicotinylhydrazine per 1 ml;

[0060] Take an appropriate amount of isoniazid reference substance, weigh it accurately, add deionized water to dissolve and dilute it to make a solution containing 0.02 mg of isoniazid per 1 ml; accurately measure 5 ml, place it in a 50 ml volumetric flask, add 5 ml of the stock solution of the related substance reference substance, dilute it to the scale with water, shake well, and you will get the mixed reference substance solution.

[0061] S12. Prepare the test solution

[0062] Accurately measure 1 ml of isoniazid injection, place it in a 50 ml volumetric flask, and dilute it with water to make a test solution containing 1.0 mg of isoniazid per 1 ml.

[0063] S2. Sample injection test

[0064] Accurately measure the test solution and the mixed reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0065] The chromatographic detection conditions are:

[0066] Chromatograph: Agilent 1260 high performance liquid chromatograph.

[0067] Chromatographic column: Welch Ultimate AQ-C18 column, 4.6 mm × 250 mm, 5 μm.

[0068] Mobile phase: phosphate buffer-acetonitrile solution, the volume ratio of phosphate buffer to acetonitrile solution is 97%:3%;

[0069] The phosphate buffer solution is prepared by accurately weighing 1.4 g of anhydrous disodium hydrogen phosphate and 1 ml of triethylamine, dissolving them in water and diluting them to 1000 ml, and adjusting the pH value to 6.0 with phosphoric acid;

[0070] Flow rate: 1.0ml / min;

[0071] Detector: UV detector;

[0072] Detection wavelength: 265nm;

[0073] Column temperature: room temperature;

[0074] Injection volume: 10 μl;

[0075] Injector temperature: 5°C.

[0076] Calculated by peak area using the external standard method, the contents of isonicotinamide, 4-cyanopyridine, benzohydrazide and diisonicotinylhydrazine shall not exceed 0.2%. The contents of other individual impurities shall not exceed 0.2% if calculated by the external standard method using the peak area of ​​isoniazid in the mixed reference solution.

[0077] Example 2: Methodological Validation

[0078] 1. Specificity and system suitability experiments

[0079] The specificity of the method was investigated by injecting blank solution, blank auxiliary material solution, system suitability solution, mixed reference solution, and test solution; the system suitability was statistically analyzed by the peak area of ​​the mixed reference solution injected 6 times in a row, and the peak area RSD of each component was required to be ≤5.0%. See the chromatogram for details. Figures 1 to 4The test results of the system suitability solution are shown in Table 1.

[0080] Table 1 System suitability solution results

[0081]

[0082] Conclusion: Blank solvent and blank excipients do not interfere with the determination of main components and impurities. Isoniazid and impurity peaks in the mixed solution and the impurity peaks can be well separated, indicating that the method has good specificity. The mixed reference solution was injected 6 times continuously, and the peak area RSD was less than 5%, and the system applicability met the requirements.

[0083] 2. Sensitivity experiment

[0084] The sensitivity is analyzed by analyzing the chromatographic peak response after stepwise dilution of the reference solution of each component. The signal-to-noise ratio is about 10:1, which is the quantitative limit of the component, and the signal-to-noise ratio is about 3:1, which is the detection limit of the component. Take the quantitative limit solution and inject 6 injections continuously. The RSD of the peak area of ​​each component is required to be ≤10%.

[0085] Table 2 Sensitivity test results

[0086]

[0087] Conclusion: From the above data, it can be seen that this method has good sensitivity.

[0088] 3. Linearity and range

[0089] For isoniazid, isonicotinamide, benzoylhydrazide, 4-cyanopyridine, and diisonicotinylhydrazide, 6 concentration points were studied in the range of LOQ concentration to about 2 times the control limit concentration. Linear relationship was plotted as a function of the measured peak area versus concentration, with concentration as the abscissa (C) and peak area as the ordinate (A), and linear regression was performed. The correlation coefficient r was used to confirm a good linear relationship, requiring r to be no less than 0.998, the response factor RSD ≤ 10%, and the intercept / 100% response value should be ≤ 25%.

[0090] Table 3 Isoniazid linear results

[0091]

[0092] Table 4 Isonicotinamide linear results

[0093]

[0094] Table 5 Benzoyl hydrazide linear results

[0095]

[0096] Table 6 4-cyanopyridine linear results

[0097]

[0098] Table 7 Diisonicotinylhydrazine linear results

[0099]

[0100] Conclusion: The reference solution of each component has a good linear relationship with the peak area in the concentration range from the limit of quantification to 2 times the control limit.

[0101] 4. Precision experiment

[0102] 4.1 Repeatability

[0103] By performing statistical analysis on the detected amount and number of each impurity in 6 parallel prepared spiked test solutions, it is required that the detected amount of each impurity RSD ≤ 10.0% and the detected number be consistent.

[0104] Table 8 Repeatability test results

[0105]

[0106]

[0107] 4.2 Intermediate precision

[0108] Another experimenter independently established the system and re-prepared the reference solution and 6 spiked test solution according to the same method, and conducted the test on different instruments and different dates. It was required that the RSD of each impurity detected in the 12 spiked test solutions under the repeatability item was ≤10.0%, and the number of detected impurities was consistent.

[0109] Table 9 Intermediate precision test results

[0110]

[0111] Conclusion: From the above results, it can be seen that this method has good precision and repeatability.

[0112] 5. Solution stability test

[0113] Inspect the stability of the test solution at room temperature; when the test solution at room temperature shows an unstable trend, inject a sample to inspect the stability under refrigerated conditions. It is required that the content of each impurity should not have an obvious trend of change, and impurities greater than the reporting limit (0.05%) should not appear.

[0114] Table 10 Test solution stability test results

[0115]

[0116] From the above results, it can be seen that when the test solution is placed at room temperature, the unknown impurities have a significant increase trend over time, while when placed under refrigeration for 24 hours, the impurity content does not change significantly, and no new impurities are added, indicating good stability. In order to more accurately control the detection of related substances in the product, it is recommended that the test solution be freshly prepared before use or stored at 2-8°C.

[0117] The stability of the mixed reference solution under refrigerated conditions was investigated, requiring the peak area RSD at different time points to be ≤5.0%.

[0118] Table 11 Results of stability test of reference solution

[0119]

[0120] Conclusion: The mixed reference solution was placed under refrigeration for 57 days, and the RSD of the peak area of ​​each component was less than 5%, indicating good solution stability.

[0121] 6. Accuracy Experiment

[0122] By adding impurity reference solutions of low, medium and high concentrations to the test solution, 3 parallel preparations of each concentration are made, and the ratio between the actual measured result and the added amount is calculated and expressed as recovery rate. The recovery rate of each impurity is required to be between 80% and 120%, and the RSD values ​​of the recovery rates of the 9 samples are all less than 10%.

[0123] Table 12 Isonicotinamide accuracy test results

[0124]

[0125] Table 13 Benzoyl hydrazide accuracy test results

[0126]

[0127] Table 14 4-cyanopyridine accuracy test results

[0128]

[0129]

[0130] Table 15 Diisonicotinylhydrazine accuracy test results

[0131]

[0132] Conclusion: From the above results, it can be seen that the recovery rate of each impurity is between 80% and 120%, and the RSD values ​​of the recovery rates of 9 samples are all less than 10%. This method has good accuracy.

[0133] 7. Durability

[0134] For the determination method of related substances, the flow rate, column temperature, proportion of organic phase components in the mobile phase, and durability of the chromatographic column type were investigated. The test solution and system suitability were determined under various chromatographic conditions.

[0135] Table 16 Durability test results

[0136]

[0137]

[0138] Conclusion: Slight changes in the wavelength, flow rate, mobile phase pH value and mobile phase ratio in the chromatographic conditions did not affect the detection of related substances in this product, indicating that this method has good durability.

[0139] Example 3: Sample testing

[0140] The method of the present invention was used to test three batches of isoniazid injection, 230301, 230302 and 230303. The test results are shown in Table 17 and the chromatograms are shown in Table 18. Figures 4 to 6 .

[0141] Table 17 Test results of related substances in samples

[0142]

[0143] It can be seen from the data in Table 17 that the relevant substances in the three batches of isoniazid injection, 230301, 230302 and 230303, all meet the limit requirements.

[0144] Comparative Example 1

[0145] The method of Example 1 of the present invention was used, an Inertsil ODS-3V 4.6 mm×250 mm, 5 μm chromatographic column was selected, and other conditions were the same as those of Example 1. The results showed that there was baseline fluctuation at the 4-cyanopyridine peak, which interfered with the detection.

[0146] Comparative Example 2

[0147] The method of Example 1 of the present invention was used, and the pH value of the phosphate buffer in the mobile phase was adjusted to 6.5. The other conditions were detected by the method in Example 1. The results showed that the separation between the chromatographic peaks of 4-cyanopyridine and benzoylhydrazide was less than 1.5, which did not meet the requirements. Figure 7 .

[0148] Comparative Example 3

[0149] The method of Example 1 of the present invention was used, the ratio of phosphate buffer to acetonitrile was adjusted to 99:1, and other conditions were detected by the method of Example 1. The results showed that the peak time of diisonicotinylhydrazine was late, and no peak appeared within 60 minutes of program operation.

[0150] Comparative Example 4

[0151] The detection method of related substances under isoniazid in the 2020 edition of the Chinese Pharmacopoeia was reproduced, and the main component chromatographic peak was bifurcated. The results are shown in Figure 8 , and the separation between benzoylhydrazide and 4-cyanopyridine peaks is poor, so the method included in the Chinese Pharmacopoeia cannot accurately control the related substances in isoniazid injection.

[0152] Comparative Example 5

[0153] The method for related substances under isoniazid in European Pharmacopoeia 11.6 was reproduced. The results showed that the baseline separation between benzoyl hydrazide and 4-cyanopyridine peaks was not achieved, and the diisonicotinyl hydrazide peak was not eluted within 60 minutes. The results are shown in Fig. 9 , this method cannot meet the requirements for the detection of related substances in isoniazid injection, and the safety of the drug cannot be well controlled.

[0154] The present application is not limited to the above-mentioned specific implementation modes, which are merely illustrative and not restrictive. Under the guidance of the present application, ordinary technicians in this field can make many forms without departing from the purpose of the present application and the scope of protection of the claims, all of which are within the protection of the present application.

Claims

1. A high-precision detection method for the content of related substances in isoniazid injection, characterized in that: The method can accurately measure the contents of four related substances, namely, isonicotinamide, benzoylhydrazide, 4-cyanopyridine and diisonicotinylhydrazide, in isoniazid injection; The detection method comprises the following steps: S1. Prepare the test solution and the mixed reference solution containing isoniazid and four related substances; S2. Use high performance liquid chromatography for sampling and testing. First, inject the mixed reference solution to determine the retention time and peak area of ​​each related substance; then inject the test solution, determine the peak of each related substance by the retention time, and calculate the content of each related substance by the external standard method.

2. The high-precision detection method for the content of related substances in isoniazid injection according to claim 1, characterized in that: In step S1, the preparation process of the test solution is: accurately measure an appropriate amount of isoniazid injection, dilute it with deionized water to prepare the test solution, and the concentration of isoniazid in the test solution is 0.8-1.2 mg / ml.

3. The high-precision detection method for the content of related substances in isoniazid injection according to claim 2, characterized in that: The concentration of isoniazid in the test solution is 1.0 mg / ml.

4. The high-precision detection method for the content of related substances in isoniazid injection according to claim 1, characterized in that: In step S1, the preparation process of the mixed reference solution is: (1) Preparation of stock solutions of relevant substance reference substances Accurately weigh appropriate amounts of isonicotinamide reference substance, benzohydrazide reference substance, 4-cyanopyridine reference substance, and diisonicotinylhydrazine reference substance, dissolve in deionized water, and quantitatively dilute to prepare a stock solution of related substance reference substances containing 0.02 mg isonicotinamide, 0.02 mg benzohydrazide, 0.02 mg 4-cyanopyridine, and 0.03 mg diisonicotinylhydrazine per 1 ml; (2) Preparation of mixed reference solution Take an appropriate amount of isoniazid reference substance, weigh it accurately, add deionized water to dissolve and dilute it to make a solution containing 0.02 mg of isoniazid per 1 ml; accurately measure 5 ml, place it in a 50 ml volumetric flask, add 5 ml of the stock solution of the related substance reference substance, dilute it to the scale with water, shake well, and you will get the mixed reference substance solution.

5. The high-precision detection method for the content of related substances in isoniazid injection according to claim 1, characterized in that: In step S2, the chromatographic conditions of the high performance liquid chromatography are: Chromatograph: Thermo Vanquish or Agilent 1260 high performance liquid chromatograph; Chromatographic column: chromatographic column with octadecylsilane bonded silica gel as filler; Mobile phase: phosphate buffer-acetonitrile solution, the volume ratio of phosphate buffer to acetonitrile solution is 94:6-98:2; Flow rate: 0.9~1.1ml / min; Detector: UV detector; Detection wavelength: 260~270nm; Column temperature: room temperature; Injection volume: 10 μl.

6. The high-precision detection method for the content of related substances in isoniazid injection according to claim 5, characterized in that: The phosphate buffer is prepared by mixing disodium hydrogen phosphate, triethylamine and deionized water; the concentration of disodium hydrogen phosphate in the phosphate buffer is 0.05-0.1 mol / L, and the volume concentration of triethylamine is 0.05%-3%.

7. The high-precision detection method for the content of related substances in isoniazid injection according to claim 6, characterized in that: The pH value of the phosphate buffer is 5.8-6.

2.

8. The high-precision detection method for the content of related substances in isoniazid injection according to claim 7, characterized in that: The pH value of the phosphate buffer is 6.

0.

9. The high-precision detection method for the content of related substances in isoniazid injection according to claim 5, characterized in that: The volume ratio of the phosphate buffer solution to the acetonitrile solution is 97:

3.

10. The high-precision detection method for the content of related substances in isoniazid injection according to claim 5, characterized in that: The chromatographic column is a Welch Ultimate AQ-C18 chromatographic column with a specification of 4.6 mm×250 mm and 5 μm.

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