A method for detecting methylhydrazine in ceftriaxone sodium for injection

Abstract

This invention provides a method for detecting methylhydrazine in ceftriaxone sodium for injection. The analyte, ceftriaxone sodium, is dissolved in a solvent to obtain a test solution, which is then detected using high-performance liquid chromatography (HPLC). The chromatographic conditions are as follows: column: octadecylsilane-bonded silica gel; mobile phase: 0.5% phosphoric acid solution-acetonitrile, gradient elution; detector: ultraviolet detector, detection wavelength 264 nm; injection volume: 30 μL; flow rate: 0.8 mL / min–1.0 mL / min; column temperature: 38℃–42℃. This method can effectively detect the content of methylhydrazine in ceftriaxone sodium for injection, thereby effectively controlling product quality and improving drug safety. Furthermore, the detection method provided by this invention has high sensitivity, with a limit of quantitation (LOQ) of 3.61 ng / ml and a limit of detection (LOD) of 1.81 ng / ml, making it highly suitable for industrial production and impurity detection.

Description

Technical Field

[0001] This invention relates to methods for testing the quality of pharmaceuticals, and more particularly to a method for detecting methylhydrazine in ceftriaxone sodium for injection. Background Technology

[0002] Ceftriaxone sodium for injection is a third-generation semi-synthetic cephalosporin. This long-acting, broad-spectrum cephalosporin has strong bactericidal activity against both Gram-positive and Gram-negative bacteria. It was first launched in Switzerland by Roche in 1982, and subsequently in China and the UK. Furthermore, ceftriaxone sodium for injection is listed in the WHO Essential Medicines List and the National Essential Medicines List (2018 Edition), representing one of the most effective drugs required by the healthcare system.

[0003] Methylhydrazine is a starting material in the synthesis of ceftriaxone sodium for injection. According to reports, its TD50 in mice is 7.55 mg / kg / day, and it is classified as a Group 1 mutagenic and carcinogenic impurity according to ICH-M7 classification. Currently, some journals have reported the use of packed adsorption microextraction-high performance liquid chromatography (HPLC) to detect methylhydrazine in process water. However, this method requires imported equipment—a packed adsorption microextraction device from CE GmbH in Australia—and involves repeated extraction of the sample up to 15 times, resulting in a complex pretreatment process and low detection sensitivity. Furthermore, this method detects methylhydrazine in process water, and no methods for detecting methylhydrazine in ceftriaxone sodium for injection have been reported to date.

[0004] To ensure drug quality and the safety and efficacy of clinical use, it is essential to analyze and study this impurity in ceftriaxone sodium for injection. Therefore, developing a simple and accurate method for detecting methylhydrazine in ceftriaxone sodium for injection is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting methylhydrazine in ceftriaxone sodium for injection. This method can accurately detect the content of methylhydrazine in ceftriaxone sodium for injection, providing a basis for controlling the content of methylhydrazine in ceftriaxone sodium for injection.

[0006] To achieve the above object, the technical solution of the present invention is:

[0007] A method for detecting methylhydrazine in ceftriaxone sodium for injection, the method being high performance liquid chromatography, with the following chromatographic conditions:

[0008] Column: Octadecylsilane-bonded silica gel as the packing material;

[0009] Detection wavelength: 264 nm; injection volume: 30 μL; flow rate: 0.8 mL / min - 1.0 mL / min; column temperature: 38℃ - 42℃;

[0010] Detector: Ultraviolet detector;

[0011] Mobile phase A: 0.5% phosphoric acid aqueous solution; Mobile phase B: acetonitrile;

[0012] Perform gradient elution according to the following gradient procedure:

[0013] From 0 to 25 min, the volume ratio of mobile phase A to mobile phase B is 40:60; from 25 to 30 min, the volume ratio of mobile phase A to mobile phase B is 36:64; from 30 to 33 min, the volume ratio of mobile phase A to mobile phase B is 10:90; from 33 to 40 min, the volume ratio of mobile phase A to mobile phase B is 10:90; from 40 to 40.1 min, the volume ratio of mobile phase A to mobile phase B is 40:60; and from 40.1 min to 50 min, the volume ratio of mobile phase A to mobile phase B is 40:60.

[0014] The test solution was prepared as follows: 10 mg of ceftriaxone sodium was accurately weighed and placed in a vial. 610 μL of solvent was accurately added to dissolve the ceftriaxone. 200 μL of 8.0 mg / mL fluorenemethyloxycarbonyl chloride solution and 100 μL of 0.5 mol / L sodium bicarbonate solution were added sequentially. The mixture was vortexed for 30 seconds and allowed to stand for 30 minutes. 90 μL of 2 mol / L citric acid solution was accurately added, and the reaction was terminated by vortexing.

[0015] The reference standard stock solution is prepared as follows: accurately weigh an appropriate amount of methylhydrazine sulfate reference standard, dissolve it in solvent and quantitatively dilute it to prepare a solution containing 180 ng of methylhydrazine per 1 mL.

[0016] The reference solution was prepared as follows: 100 μL of the reference stock solution was accurately measured and placed in a vial. 510 μL of solvent, 200 μL of 8.0 mg / mL fluorene methoxycarbonyl chloride solution, and 100 μL of 0.5 mol / L sodium bicarbonate solution were added sequentially. The mixture was vortexed for 30 seconds, allowed to stand for 30 minutes, and then 90 μL of 2 mol / L citric acid solution was accurately added. The reaction was terminated by vortexing.

[0017] The system suitability solution was prepared as follows: 10 mg of ceftriaxone sodium was accurately weighed and placed in a vial. 510 μL of solvent was added to dissolve the ceftriaxone. Then, 100 μL of the stock solution of the reference standard, 200 μL of 8.0 mg / mL fluorenyl methoxycarbonyl chloride solution, and 100 μL of 0.5 mol / L sodium bicarbonate solution were added sequentially. The mixture was vortexed for 30 seconds, allowed to stand for 30 minutes, and then 90 μL of 2 mol / L citric acid solution was added to terminate the reaction by vortexing.

[0018] The solvent is a mixture of acetonitrile and water in a 1:1 volume ratio.

[0019] Preferably, the method for calculating the content of methylhydrazine is the external standard method.

[0020] This invention provides a method for detecting methylhydrazine in ceftriaxone sodium for injection. This method can effectively detect the content of methylhydrazine in ceftriaxone sodium for injection, thereby effectively controlling product quality and improving drug safety. In addition, the detection method provided by this invention has high sensitivity, with a limit of quantitation of 3.61 ng / mL and a limit of detection of 1.81 ng / mL. Attached Figure Description

[0021] Figure 1 The high-performance liquid chromatogram is for a blank solvent.

[0022] Figure 2 This is a high-performance liquid chromatogram of the sensitivity solution.

[0023] Figure 3 This is a high-performance liquid chromatogram of the system suitability solution.

[0024] Figure 4 This is the high-performance liquid chromatogram of the test solution.

[0025] Figure 5 The graph shows the linear relationship of methylhydrazine. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments.

[0027] In the following embodiments, the various processes and methods not described in detail are conventional methods known in the art, and the reagents used, unless otherwise specified, are commercially available analytical grade or chromatographic grade.

[0028] Information on the instruments and equipment used is shown in Table 1.

[0029] Table 1

[0030]

[0031] Information on the reagents used is shown in Table 2.

[0032] Table 2

[0033]

[0034] Example 1

[0035] A method for detecting methylhydrazine in ceftriaxone sodium for injection includes the following steps:

[0036] (1) Preparation of test solution, reference solution and system suitability solution

[0037] Test solution: Accurately weigh 10 mg of ceftriaxone sodium and place it in a vial. Accurately add 610 μL of solvent to dissolve it. Then, accurately add 200 μL of 8.0 mg / mL fluorene methoxycarbonyl chloride solution and 100 μL of 0.5 mol / L sodium bicarbonate solution in sequence. Vortex for 30 seconds, let stand for 30 minutes, and then accurately add 90 μL of 2 mol / L citric acid solution and vortex to terminate the reaction.

[0038] Reference stock solution: Accurately weigh an appropriate amount of methylhydrazine sulfate reference standard, dissolve it in solvent and dilute quantitatively to prepare a solution containing 180 ng of methylhydrazine per 1 mL.

[0039] Reference solution: Accurately measure 100 μL of the reference stock solution and place it in a vial. Then, accurately add 510 μL of solvent, 200 μL of 8.0 mg / mL fluorene methoxycarbonyl chloride solution, and 100 μL of 0.5 mol / L sodium bicarbonate solution in sequence. Vortex for 30 seconds, let stand for 30 minutes, and then accurately add 90 μL of 2 mol / L citric acid solution. Vortex to terminate the reaction.

[0040] System adaptability solution: Accurately weigh 10 mg of ceftriaxone sodium and place it in a vial. Add 510 μL of solvent to dissolve it. Then, add 100 μL of the reference stock solution, 200 μL of 8.0 mg / mL fluorenyl methoxycarbonyl chloride solution, and 100 μL of 0.5 mol / L sodium bicarbonate solution in sequence. Vortex for 30 seconds, let stand for 30 minutes, and then add 90 μL of 2 mol / L citric acid solution to terminate the reaction by vortexing.

[0041] All solvents used above are a mixture of acetonitrile and water in a 1:1 volume ratio.

[0042] (2) Chromatographic conditions

[0043] Column: Octadecylsilane-bonded silica gel as the packing material;

[0044] Detection wavelength: 264 nm; injection volume: 30 μL; flow rate: 0.9 mL / min; column temperature: 40 °C;

[0045] Mobile phase A: 0.5% phosphoric acid-water solution;

[0046] Mobile phase B: Acetonitrile;

[0047] Perform gradient elution according to the table below:

[0048] Table 3

[0049]

[0050] (3) Measurement

[0051] Accurately measure 10 μL of the reference solution and the test solution, inject them into the high performance liquid chromatograph, record the chromatograms, and calculate the amount of methylhydrazine using the external standard method.

[0052]

[0053] —A S For the peak area of ​​the test sample, A R The peak area of ​​the reference standard;

[0054] —C R The concentration of the reference standard (ng / mL), —C S The concentration is the sample concentration (mg / mL).

[0055] (4) Results

[0056] The test samples were three batches of large-scale production samples manufactured by the applicant, and none of them tested positive for methylhydrazine (see Table 4).

[0057] Table 4

[0058]

[0059] Example 2: Determination of detection limit and quantitation limit

[0060] The detection limit of methylhydrazine was 1.81 ng / mL (equivalent to 10% of the limit concentration), and the quantitation limit of methylhydrazine was 3.61 ng / mL (equivalent to 20% of the limit concentration). The signal-to-noise ratio and peak area RSD both met the requirements.

[0061] The results for the limit of detection solution and limit of quantitation solution are shown in Tables 5 and 6.

[0062] Table 5 Results of the detection limit test

[0063]

[0064] Table 6 Results of Limit of Quantitation Test

[0065]

[0066] Example 3: Linear Correlation Test

[0067] Reference solutions at different concentrations (20%, 50%, 100%, 120%, and 150%) were taken, and two injections were performed for each concentration. The peak areas were recorded. The results of the linearity and range tests are shown in Table 7.

[0068] Table 7. Results of Linearity and Range Tests

[0069]

[0070] Conclusion: The target impurity showed good linearity in the range of 3.6136 ng / mL to 27.1020 ng / mL, with R0... 2 It is 0.9998.

[0071] Example 4: Accuracy Test

[0072] The accuracy solution was tested, and the spiked recoveries at three concentrations (3*3 samples) were all between 94.7% and 100.0%, with a 9-needle recovery RSD of 1.9%, which met the requirements and indicated good method accuracy.

[0073] The results of the accuracy test are shown in Table 8.

[0074] Table 8 Accuracy Test Results

[0075]

[0076] Example 5: Repeatability Test

[0077] Weigh out the reproducible solutions of the following concentrations, inject each solution once, record the peak area, and calculate the RSD value of the methylhydrazine content.

[0078] Table 9 Results of Repeatability Tests

[0079]

[0080] Conclusion: In the 6 repeatable spiked samples, the RSD of methylhydrazine content was 1.2%, which met the requirements, and the method had good repeatability.

[0081] Example 6: Solution Stability Test

[0082] Inject the reference solution and the test solution, which were placed at room temperature, and examine the changes in the content of target impurities in the reference solution and the test solution within 25 hours. The stability results are shown in Table 10.

[0083] Table 10 Stability Test Results

[0084]

[0085] Conclusion: After being placed at room temperature for 25 hours, the content of target impurities in both the reference solution and the test solution was within the range of 80.0% to 120.0% compared with 0 hours, indicating that the solutions were stable.

[0086] Example 7:

[0087] The mobile phase flow rate and column temperature were changed in the chromatographic conditions, while all other conditions remained the same as in Example 1, and the detection was performed.

[0088] Flow rate: 0.8 mL / min; Column temperature: 42℃.

[0089] Methylhydrazine was not detected in the ceftriaxone sodium for injection sample.

[0090] Example 8:

[0091] The mobile phase flow rate and column temperature were changed in the chromatographic conditions, while all other conditions remained the same as in Example 1, and the detection was performed.

[0092] Flow rate: 1.0 mL / min; Column temperature: 38℃.

[0093] Methylhydrazine was not detected in the ceftriaxone sodium for injection sample.

Claims

1. A method for detecting methylhydrazine in ceftriaxone sodium for injection, characterized in that, Includes the following steps: Prepare the test solution and the reference solution. The test solution is prepared as follows: Take 10 mg of ceftriaxone sodium for injection, place it in a vial, add 610 μL of solvent to dissolve it, then add 200 μL of 8.0 mg / mL fluorene methoxycarbonyl chloride solution and 100 μL of 0.5 mol / L sodium bicarbonate solution sequentially, vortex for 30 seconds, let stand for 30 minutes, add 90 μL of 2 mol / L citric acid solution, and vortex to terminate the reaction; the solvent is a mixture of acetonitrile and water in a 1:1 volume ratio. The test sample was a large-scale production sample produced by Hebei Huamin Pharmaceutical Co., Ltd. of North China Pharmaceutical Group, with batch numbers BDY290801, BDY290802 or BDY290803. The detection was performed using high-performance liquid chromatography (HPLC). The HPLC conditions were as follows: Column: Octadecylsilane-bonded silica gel as the packing material; Mobile phase A: 0.5% phosphoric acid-water solution; Mobile phase B: acetonitrile; Gradient elution; The elution conditions are: The detector is an ultraviolet detector; the detection wavelength is 264 nm. The content of methylhydrazine was calculated based on the spectrum.

2. The method for detecting methylhydrazine in ceftriaxone sodium for injection according to claim 1, characterized in that, The reference standard stock solution was prepared as follows: Weigh the methylhydrazine sulfate reference standard, dissolve it in solvent and dilute it quantitatively to prepare a solution containing 180 ng of methylhydrazine per 1 mL; The reference solution was prepared as follows: 100 μL of the reference stock solution was placed in a vial, and 510 μL of solvent, 200 μL of 8.0 mg / mL fluorene methoxycarbonyl chloride solution and 100 μL of 0.5 mol / L sodium bicarbonate solution were added sequentially. The mixture was vortexed for 30 seconds, allowed to stand for 30 minutes, and then 90 μL of 2 mol / L citric acid solution was added to terminate the reaction by vortexing. The solvent is a mixture of acetonitrile and water in a 1:1 volume ratio.

3. The method for detecting methylhydrazine in ceftriaxone sodium for injection according to claim 1, characterized in that, The chromatographic column has the following specifications: 4.6 mm × 250 mm, 5 μm, and a column temperature of 38℃-42℃.

4. The method for detecting methylhydrazine in ceftriaxone sodium for injection according to claim 1, characterized in that, The flow rate of the mobile phase is 0.8 mL / min - 1.0 mL / min.

5. The method for detecting methylhydrazine in ceftriaxone sodium for injection according to claim 1, characterized in that, The injection volume was 30 μL.

6. The method for detecting methylhydrazine in ceftriaxone sodium for injection according to claim 1, characterized in that, The method for calculating the content of methylhydrazine is the external standard method.

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