CN116136517A - A kind of detection method of methylhydrazine in ceftriaxone sodium for injection - Google Patents

Abstract

The invention provides a method for detecting methylhydrazine in ceftriaxone sodium for injection. The test substance ceftriaxone sodium is dissolved in a solvent to obtain the test solution, which is detected by high performance liquid chromatography. The chromatographic conditions are: Chromatographic column: filler is octadecylsilane bonded silica gel; mobile phase: 0.5% phosphoric acid solution - acetonitrile, gradient elution; detector: ultraviolet detector, detection wavelength 264nm; injection volume: 30μL, flow rate: 0.8mL/ min-1.0mL/min, column temperature: 38°C-42°C. The method can effectively detect the content of methylhydrazine in ceftriaxone sodium for injection, thereby effectively controlling the product quality and improving the drug safety of the drug. In addition, the detection method provided by the present invention has higher sensitivity and a quantitative limit concentration It is 3.61ng/ml, and the detection limit concentration is 1.81ng/ml, which is very suitable for industrial production and impurity detection.

Description

A kind of detection method of methylhydrazine in ceftriaxone sodium for injection

technical field

The invention relates to a method for detecting the quality of medicines, in particular to a method for detecting methylhydrazine in ceftriaxone sodium for injection.

Background technique

Ceftriaxone sodium for injection is the third generation semi-synthetic cephalosporin. It was first listed in Switzerland in 1982 by Roche, followed by listings in China and the UK. In addition, Ceftriaxone Sodium for Injection is on the WHO Model List of Essential Medicines and the "National List of Essential Medicines (2018 Edition)", and is the most effective medicine needed by the health system.

Methylhydrazine is the 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 class 1 mutagenic carcinogen impurity according to ICH-M7. At present, there are journals reporting the use of packed adsorption microextraction-high performance liquid chromatography to detect methylhydrazine in process water, but this method requires the use of imported devices - Australia CE company filled adsorption microextraction device, repeated extraction of samples 15 times, The pretreatment process is complicated, and the detection limit sensitivity is low. Moreover, what it detects is methylhydrazine in process water, but there is no relevant report on the detection method of methylhydrazine in ceftriaxone sodium for injection.

In order to ensure the quality of medicines and the safety and effectiveness of clinical medication, it is necessary to analyze and study the impurity in ceftriaxone sodium for injection. Therefore, it is of great significance to develop a simple and accurate detection method for methylhydrazine in ceftriaxone sodium for injection.

Contents of the invention

The object of the present invention is to provide a kind of detection method of methylhydrazine in ceftriaxone sodium for injection, this detection method can accurately detect the content of methylhydrazine in ceftriaxone sodium for injection, for controlling the content of methylhydrazine in ceftriaxone sodium for injection The content of methylhydrazine provides the basis.

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

A detection method for methylhydrazine in ceftriaxone sodium for injection, the method is high performance liquid chromatography, and the chromatographic conditions are:

Chromatographic column: Octadecylsilane bonded silica gel as filler;

Detection wavelength: 264nm; Injection volume: 30μL; Flow rate: 0.8mL/min-1.0mL/min; Column temperature: 38°C-42°C;

Detector: UV detector;

Mobile phase A: 0.5% phosphoric acid-water solution, mobile phase B: acetonitrile;

Gradient elution was performed according to the following gradient program:

0-25min, the volume ratio of mobile phase A to mobile phase B is 40:60; 25-30min, the volume ratio of mobile phase A to mobile phase B is 36:64; 30-33min, mobile phase A The volume ratio to mobile phase B is 10:90; 33-40min, the volume ratio of mobile phase A to mobile phase B is 10:90, 40-40.1 min The volume ratio of mobile phase A to mobile phase B 40:60, 40.1min-50min The volume ratio of mobile phase A to mobile phase B is 40:60.

The test solution was prepared according to the following method: take 10 mg of ceftriaxone sodium, accurately weighed, put it in a sample injection vial, accurately add 610 μL of solvent to dissolve it, and then accurately add 200 μL of 8.0 mg/mL fluorenylmethoxycarbonyl chloride solution, 0.5 mol /L sodium bicarbonate solution 100 μL, vortex for 30 seconds, let stand for 30 minutes, precisely add 2mol/L citric acid solution 90 μL and vortex to terminate the reaction.

The reference substance stock solution was prepared according to the following method: Accurately weigh an appropriate amount of methylhydrazine sulfate reference substance, add solvent to dissolve and quantitatively dilute to make a solution containing 180ng of methylhydrazine per 1mL.

The reference substance solution was prepared according to the following method: accurately measure 100 μL of the reference substance stock solution, put it into the sample injection vial, and sequentially add 510 μL of solvent, 200 μL of 8.0 mg/mL fluorenyl methaneoxycarbonyl chloride solution, and 100 μL of 0.5 mol/L sodium bicarbonate solution , vortex for 30 seconds, let stand for 30 minutes, and precisely add 90 μL of 2mol/L citric acid solution and vortex to terminate the reaction.

The system suitability solution was prepared according to the following method: take 10 mg of ceftriaxone sodium, accurately weigh it, place it in a sample injection vial, add 510 μL of solvent to dissolve it, add 100 μL of the reference substance stock solution, and 8.0 mg/mL fluorenylmethoxycarbonyl chloride solution in sequence 200 μL, 100 μL of 0.5 mol/L sodium bicarbonate solution, 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 volume ratio of 1:1.

Preferably, the method for calculating the content of the methylhydrazine is an external standard method.

The invention provides a method for detecting methylhydrazine in ceftriaxone sodium for injection. The method can effectively detect the content of methylhydrazine in ceftriaxone sodium for injection, thereby effectively controlling product quality and improving the quality of medicines. Medication is safe. In addition, the detection method provided by the present invention has high sensitivity, the quantification limit concentration is 3.61 ng/mL, and the detection limit concentration is 1.81 ng/mL.

Description of drawings

Fig. 1 is the high performance liquid chromatogram of blank solvent.

Figure 2 is a high performance liquid chromatogram of the sensitivity solution.

Figure 3 is a high performance liquid chromatogram of the system suitability solution.

Fig. 4 is the high performance liquid phase chromatogram of need testing solution.

Fig. 5 is a linear relationship diagram of methylhydrazine.

Detailed ways

The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments.

In the following examples, various processes and methods that are not described in detail are conventional methods well known in the art, and the reagents used are commercially available analytically pure or chromatographically pure without indicating their sources and specifications.

The information about the equipment used is shown in Table 1.

Table 1

The information of the reagents used is shown in Table 2.

Table 2

Example 1

A method for detecting methylhydrazine in ceftriaxone sodium for injection, comprising the steps of:

(1) Preparation of test solution, reference solution and system adaptability solution

Need test solution: Take 10 mg of ceftriaxone sodium, accurately weighed, put in the sample injection vial, accurately add 610 μL of solvent to dissolve, and accurately add 200 μL of 8.0 mg/mL fluorenylmethoxycarbonyl chloride solution, 0.5mol/L bicarbonate successively Sodium solution 100 μL, vortex for 30 seconds, let stand for 30 minutes, add 90 μL 2mol/L citric acid solution precisely and vortex to terminate the reaction.

Reference substance stock solution: Accurately weigh an appropriate amount of methylhydrazine sulfate reference substance, dissolve it with a solvent, and quantitatively dilute to make a solution containing 180ng of methylhydrazine per 1mL.

Reference substance solution: Accurately measure 100 μL of the reference substance stock solution, put it into the sample injection vial, add 510 μL of solvent, 200 μL of 8.0 mg/mL fluorenylmethoxycarbonyl chloride solution, 100 μL of 0.5 mol/L sodium bicarbonate solution, and vortex for 30 Seconds, let stand for 30 minutes, precisely add 90 μL of 2mol/L citric acid solution and vortex to terminate the reaction.

System suitability solution: Take 10 mg of ceftriaxone sodium, weigh it accurately, place it in a sample injection vial, add 510 μL of solvent to dissolve, add 100 μL of reference substance stock solution, 200 μL of 8.0 mg/mL fluorenylmethoxycarbonyl chloride solution, 0.5mol 100 μL/L sodium bicarbonate solution, vortex for 30 seconds, let stand for 30 minutes, add 90 μL 2mol/L citric acid solution and vortex to terminate the reaction.

The solvent used above is a mixture of acetonitrile and water in a volume ratio of 1:1.

(2) Chromatographic conditions

Chromatographic column: Octadecylsilane bonded silica gel as filler;

Detection wavelength: 264nm; Injection volume: 30μL; Flow rate: 0.9mL/min; Column temperature: 40℃;

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

Mobile phase B phase: acetonitrile;

Carry out gradient elution according to the following table:

table 3

(3) Determination

Precisely measure 10 μL of the reference substance solution and the test solution, inject it into a high-performance liquid chromatograph, record the chromatogram, and calculate the amount of methylhydrazine by the external standard method.

_—AS is the peak area of the test product, _AR is the peak area of the reference substance;

—C _R is the concentration of the reference substance (ng/mL), —C _S is the concentration of the sample (mg/mL).

(4) Results

The test samples were 3 batches of large-scale production samples produced by the applicant, and methylhydrazine was not detected, see Table 4.

Table 4

Embodiment 2: Determination of detection limit and quantitative limit

The detection limit concentration of methylhydrazine is 1.81ng/mL (equivalent to 10% of the limit concentration), the quantitative limit concentration of methylhydrazine is 3.61ng/mL (equivalent to 20% of the limit concentration), and the signal-to-noise ratio and peak area RSD are both meet the requirements.

The results of the detection limit solution and quantification limit solution are shown in Table 5~Table 6.

Table 5 Detection limit test results

Table 6 Quantitative limit test result

Example 3: Linear Dependency Test

Take reference substance solutions with different concentrations of 20%, 50%, 100%, 120%, and 150% of the limit, inject 2 injections for each concentration, and record the peak area. The linearity and range test results are shown in Table 7.

Table 7 Linearity and range test results

Conclusion: The target impurity has a good linear relationship in the range of 3.6136ng/mL~27.1020ng/mL, and the ^R2 is 0.9998.

Embodiment 4: Accuracy test

Taking the accuracy solution, the recovery rate values of the standard addition at three concentrations (3*3 samples) were all between 94.7% and 100.0%, and the RSD of the 9-pin recovery rate was 1.9%, which met the requirements and the accuracy of the method was good.

The accuracy test results are shown in Table 8.

Table 8 Accuracy test results

Embodiment 5: repeatability test

Weigh the repeating solutions with the following concentrations, inject 1 needle each, record the peak area, and calculate the RSD value of the methylhydrazine content.

Table 9 Repeatability test results

Conclusion: The RSD of the content of methylhydrazine in the 6 repeated spiked samples was 1.2%, which met the requirements, and the method had good repeatability.

Embodiment 6: solution stability test

Take the reference substance solution placed at room temperature and the sample injection of the test substance stability solution, investigate the change of the target impurity content in the reference substance solution and the test substance solution within 25h, and the stability results are shown in Table 10.

Table 10 Stability test results

Conclusion: After being placed at room temperature for 25 hours, the target impurity contents in the reference solution and the test solution were both in the range of 80.0% to 120.0% compared with 0 hours, and the solutions were stable.

Embodiment 7:

Change the mobile phase flow velocity and the column temperature in the chromatographic condition, and others are all the same as embodiment 1, detect,

Flow rate: 0.8mL/min; column temperature: 42°C.

No methylhydrazine was detected in the sample of ceftriaxone sodium for injection.

Embodiment 8:

Change the mobile phase flow velocity and the column temperature in the chromatographic condition, and others are all the same as embodiment 1, detect,

Flow rate: 1.0 mL/min; Column temperature: 38°C.

No methylhydrazine was detected in the sample of ceftriaxone sodium for injection.

Claims (9)

1. a detection method for methylhydrazine in ceftriaxone sodium for injection, is characterized in that, comprises the steps: prepare need testing solution and reference substance solution, adopt high performance liquid chromatography to detect, the high performance liquid chromatography The conditions are: Chromatographic column: Octadecylsilane bonded silica gel as filler; Mobile phase A: 0.5% phosphoric acid-water solution, mobile phase B: acetonitrile; gradient elution; Calculate the content of methylhydrazine according to the spectrum. 2. the detection method of methylhydrazine in ceftriaxone sodium for injection according to claim 1 is characterized in that, elution condition is: 0-25min, the volume ratio of mobile phase A phase and mobile phase B phase is 40 :60; 25-30min, the volume ratio of mobile phase A to mobile phase B is 36:64; 30-33min, the volume ratio of mobile phase A to mobile phase B is 10:90; 33-40min, mobile phase The volume ratio of phase A to mobile phase B is 10:90, 40-40.1 min The volume ratio of mobile phase A to mobile phase B is 40:60, 40.1 min-50 min Mobile phase A to mobile phase B The volume ratio is 40:60. 3. the detection method of methylhydrazine in ceftriaxone sodium for injection according to claim 1, is characterized in that, need testing solution is prepared according to the following method: get ceftriaxone sodium 10mg, put in the sampling vial, add Dissolve in 610 μL of solvent, add 200 μL of 8.0 mg/mL fluorenylmethoxycarbonyl chloride solution, 100 μL of 0.5 mol/L sodium bicarbonate solution in sequence, vortex for 30 seconds, let stand for 30 minutes, add 90 μL of 2 mol/L citric acid solution, and vortex to stop Reaction; the solvent is a mixture of acetonitrile and water in a volume ratio of 1:1. 4. the detection method of methylhydrazine in ceftriaxone sodium for injection according to claim 1 is characterized in that, reference substance stock solution is prepared according to the following method: take by weighing methylhydrazine sulfate reference substance, solubilizer dissolves and quantitatively dilutes , to make a solution containing 180ng of methylhydrazine per 1mL; The reference substance solution was prepared according to the following method: Measure 100 μL of the reference substance stock solution, put it into a sample injection vial, add 510 μL of solvent, 200 μL of 8.0 mg/mL fluorenylmethoxycarbonyl chloride solution, and 100 μL of 0.5 mol/L sodium bicarbonate solution, and vortex Spin for 30 seconds, let stand for 30 minutes, add 90 μL of 2mol/L citric acid solution and vortex to terminate the reaction; The solvent is a mixture of acetonitrile and water in a volume ratio of 1:1. 5. the detection method of methylhydrazine in ceftriaxone sodium for injection according to claim 1 is characterized in that, the specification of described chromatographic column is: 4.6mm * 250mm, 5 μ m, column temperature: 38 ℃-42 ℃. 6. the detection method of methylhydrazine in ceftriaxone sodium for injection according to claim 1 is characterized in that, detector is: ultraviolet detector; Detection wavelength is: 264nm. 7. the detection method of methylhydrazine in ceftriaxone sodium for injection according to claim 1 is characterized in that, the flow velocity of mobile phase is: 0.8mL/min-1.0mL/min. 8. the detection method of methylhydrazine in ceftriaxone sodium for injection according to claim 1, is characterized in that, sample size is: 30 μ L. 9. the detection method of methylhydrazine in the ceftriaxone sodium for injection according to claim 1 is characterized in that, the method for calculating the content of methylhydrazine is an external standard method.

Images