A method for determining acetylhydrazine in cefazolin sodium by high performance liquid chromatography-tandem mass spectrometry

Abstract

The present invention provides a method for determining acetylhydrazine in cefazolin sodium by high performance liquid chromatography-tandem mass spectrometry. Chromatographic conditions of the present invention: chromatographic column: Agilent ZORBAX Eclipse XDB-C18 (3.0*150mm, 3.5μm); mobile phase A: aqueous solution of formic acid with a volume concentration of 0.1%; mobile phase B: acetonitrile solution of formic acid with a volume concentration of 0.1%; gradient elution; solvent: aqueous ammonium acetate solution with a pH value of 4.5; derivatization solution: dissolving benzaldehyde in acetonitrile to prepare an acetonitrile solution of benzaldehyde with a volume concentration of 10-15%; test solution: mixing cefazolin sodium, derivatization solution and solvent, fully dissolving, and reacting to obtain. The present invention adopts the principle of furfural derivatization, can accurately determine the content of acetylhydrazine in cefazolin sodium, and has high sensitivity, ensuring that the quality of cefazolin sodium raw materials and preparations thereof can be controlled.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and particularly relates to a method for determining acetyl hydrazine in cefazolin sodium by adopting high performance liquid chromatography-tandem mass spectrometry. Background Art

[0002] Cefazolin sodium is a β-lactam antibiotic used to treat infections caused by sensitive bacteria.

[0003] The presence of impurities in drugs can reduce their efficacy and even cause side effects in some cases. The study of impurities is a dynamic and ongoing process.

[0004] Acetylhydrazine is an intermediate in the synthesis process of 2-mercapto-5-methyl-1,3,4-thiadiazole, the starting material of cefazolin sodium, and there is a risk of it being transferred to the final product, cefazolin sodium.

[0005] The reaction route of 2-mercapto-5-methyl-1,3,4-thiadiazole is as follows: (e.g. Wang Yi, Jiang Jingjie, Chen Jingren. Optimal process conditions for the synthesis of 2-mercapto-5-methyl-1,3,4-thiadiazole [J]. Journal of Natural Science of Harbin Normal University, 1999 (15): 78-82)

[0006]

[0007] The reaction route of cefazolin sodium is as follows: (e.g. Cao Weikai. Study on the synthesis of cefazolin sodium [J]. Chemical Engineering and Pharmaceutical Engineering, 2016 (6): 37-42)

[0008]

[0009] The information of cefazolin sodium, 2-mercapto-5-methyl-1,3,4-thiadiazole, and acetylhydrazine is as follows:

[0010]

[0011] Acetylhydrazine contains a warning structure and therefore has potential genotoxicity (according to the warning structure of mutagenic impurities in the European Commission's Development of structural alerts for the in vivo micronucleus assay in rodents, and the control strategy for potential mutagenic impurities in ICH M7 "Evaluation and Control of DNA Reactive (Mutagenic) Impurities in Drugs to Limit Potential Carcinogenic Risk"), and the control of this compound is particularly important. Due to its high polarity, it is weakly retained under conventional chromatographic detection conditions, resulting in a low method recovery rate. In addition, conventional instruments cannot meet the detection sensitivity requirements, so it is necessary to develop a method that can effectively determine acetylhydrazine in cefazolin sodium. Summary of the invention

[0012] In view of the shortcomings of the prior art, the present invention provides a method for determining acetylhydrazine in cefazolin sodium by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). The present invention adopts the principle of furfural derivatization, can accurately determine the content of acetylhydrazine in cefazolin sodium, and has high sensitivity, ensuring that the quality of cefazolin sodium raw materials and preparations thereof can be controlled.

[0013] To achieve the above object, the technical solution of the present invention is:

[0014] A method for determining acetylhydrazine in cefazolin sodium by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), characterized in that high performance liquid chromatography-tandem mass spectrometry is used;

[0015] Chromatographic conditions: Chromatographic column: Agilent ZORBAX Eclipse XDB-C18 (3.0*150 mm, 3.5 μm); Mobile phase A: 0.1% by volume formic acid in water; Mobile phase B: 0.1% by volume formic acid in acetonitrile; Gradient elution, conditions are as follows:

[0016] At 0 minutes, the volume proportion of mobile phase A is 65-75%, and the volume proportion of mobile phase B is 25-35%; at 1 minute, the volume proportion of mobile phase A is 65-75%, and the volume proportion of mobile phase B is 25-35%; at 5 minutes, the volume proportion of mobile phase A is 15-25%, and the volume proportion of mobile phase B is 75-85%; at 7 minutes, the volume proportion of mobile phase A is 15-25%, and the volume proportion of mobile phase B is 75-85%; at 7.1 minutes, the volume proportion of mobile phase A is 65-75%, and the volume proportion of mobile phase B is 25-35%; at 12 minutes, the volume proportion of mobile phase A is 65-75%, and the volume proportion of mobile phase B is 25-35%;

[0017] Solvent: ammonium acetate aqueous solution with a pH value of 4.5;

[0018] Derivatization solution: dissolve benzaldehyde in acetonitrile to prepare a benzaldehyde acetonitrile solution with a volume concentration of 10-15%;

[0019] Test solution: Cefazolin sodium, derivatization solution and solvent are mixed, fully dissolved, and reacted to obtain a test solution.

[0020] According to the preferred embodiment of the present invention, the chromatographic conditions also include: a mobile phase flow rate of 0.3 ml / min; a column temperature of 40° C.; and an injection volume of 5 μl.

[0021] Preferably, according to the present invention, the gradient elution conditions are: at 0 minutes, the volume of mobile phase A accounts for 70%, and the volume of mobile phase B accounts for 30%; at 1 minute, the volume of mobile phase A accounts for 70%, and the volume of mobile phase B accounts for 30%; at 5 minutes, the volume of mobile phase A accounts for 20%, and the volume of mobile phase B accounts for 80%; at 7 minutes, the volume of mobile phase A accounts for 20%, and the volume of mobile phase B accounts for 80%; at 7.1 minutes, the volume of mobile phase A accounts for 70%, and the volume of mobile phase B accounts for 30%; at 12 minutes, the volume of mobile phase A accounts for 70%, and the volume of mobile phase B accounts for 30%.

[0022] According to a preferred embodiment of the present invention, the preparation method of the solvent is as follows: prepare a 20 mmol / L ammonium acetate aqueous solution, and use glacial acetic acid to adjust the pH value to 4.5.

[0023] According to the preferred embodiment of the present invention, the derivatization solution is an acetonitrile solution of benzaldehyde with a volume concentration of 12%.

[0024] Preferably according to the present invention, in the preparation of the test solution, the mass ratio of cefazolin sodium to the total volume of the derivatization solution and the solvent is 4-6 g / L, preferably 5 g / L.

[0025] Preferably according to the present invention, in the preparation of the test solution, the volume ratio of the mass of cefazolin sodium to the derivatization solution is 480-520 g / L, preferably 500 g / L.

[0026] Preferably according to the present invention, in the preparation of the test solution, the reaction temperature is 55-65° C., and the reaction time is 30-50 minutes; preferably, the reaction temperature is 60° C., and the reaction time is 40 minutes.

[0027] According to the preferred embodiment of the present invention, the reference substance stock solution: the reference substance is dissolved in a solvent to prepare a reference substance stock solution containing 70-80ng of the reference substance per 1ml; the reference substance solution: 1ml of the reference substance stock solution is placed in a 10ml volumetric flask, 0.1ml of the derivatization solution is added, diluted to the scale with the solvent, and shaken well; after sealing, the reference substance solution is obtained by heating in a water bath at 55-65°C for 30-50 minutes. Preferably, the reference substance solution is obtained by heating in a water bath at 60°C for 40 minutes.

[0028] According to the preferred mass spectrometry conditions of the present invention: detection by triple quadrupole tandem mass spectrometer; electrospray ionization source (ESI), acquisition mode is positive ion mode; acetylhydrazine derivative (acetylhydrazine and benzaldehyde reaction to obtain derivatives) monitoring ion pairs and collision voltage (CE) are as follows:

[0029]

[0030] According to a preferred determination method of the present invention, the reference solution is accurately aspirated, injected into a high performance liquid chromatography-tandem mass spectrometer, and the peak area is determined; the test solution is accurately aspirated, injected into a high performance liquid chromatography-tandem mass spectrometer, the peak area is determined, and the acetylhydrazine content in the test sample is calculated by the peak area external standard method.

[0031] The technical features and beneficial effects of the present invention are as follows:

[0032] (1) The present invention adopts the principle of furfural derivatization, first reacts acetylhydrazine in the test sample cefazolin sodium with benzaldehyde to obtain a derivative, and then further detects the content of the derivative to obtain the content of acetylhydrazine in cefazolin sodium. The derivatization temperature of the present invention needs to be appropriate. Too low a temperature will lead to a prolonged reaction time or a low derivatization recovery rate.

[0033] Derivatization principle:

[0034]

[0035] (2) The high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) provided by the present invention can effectively determine the content of acetylhydrazine in cefazolin sodium, has high sensitivity, strong specificity and applicability, high precision and accuracy, and can more accurately ensure the quality controllability of cefazolin sodium raw materials and preparations thereof, and ultimately determine the safety and effectiveness of the products. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 : Test spectrum of the reference substance in Example 1; in the spectrum, the abscissa is time, min, and the ordinate is intensity, cps;

[0037] Figure 2: Cefazolin sodium test spectrum in Example 1; in the spectrum, the abscissa is time, min, and the ordinate is intensity, cps;

[0038] Figure 3 : Standard working curve in Example 2;

[0039] Figure 4 : Spectrum of quantitative limit test in Example 2; in the spectrum, the horizontal axis is time, min, and the vertical axis is intensity, cps;

[0040] Figure 5 : Detection limit test spectrum in Example 2; in the spectrum, the horizontal axis is time, min, and the vertical axis is intensity, cps;

[0041] Figure 6 : Precision test spectrum in Example 2; in the spectrum, the abscissa is time, min, and the ordinate is intensity, cps. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0043] Meanwhile, the experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0044] Example 1

[0045] A method for determining acetylhydrazine in cefazolin sodium by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), using high performance liquid chromatography-tandem mass spectrometry;

[0046] Chromatographic conditions: Agilent ZORBAX Eclipse XDB-C18, (3.0*150mm, 3.5μm); mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile; mobile phase flow rate: 0.3ml / min; column temperature: 40°C; injection volume: 5μl. Gradient elution, conditions are as follows:

[0047]

[0048] Solvent: Ammonium acetate aqueous solution with a pH value of 4.5: prepare a 20 mmol / L ammonium acetate aqueous solution and adjust the pH value to 4.5 with glacial acetic acid.

[0049] Derivatization solution: Accurately measure 6 ml of benzaldehyde, place it in a 50 ml volumetric flask, dissolve it with acetonitrile and dilute to the mark.

[0050] Reference substance stock solution: Take an appropriate amount of reference substance, accurately determine the concentration, add solvent to dissolve and make up to volume, dilute to contain 75 ng of reference substance per 1 ml.

[0051] Reference substance solution: accurately pipette 1 ml of reference substance stock solution into a 10 ml volumetric flask, add 0.1 ml of derivatization solution, dilute to the mark with solvent, and shake well; seal and heat in a 60°C water bath for 40 minutes, remove and cool to room temperature for use.

[0052] Test solution: Take about 50 mg of cefazolin sodium, accurately weigh it, put it in a 10 ml volumetric flask, add 0.1 ml of derivatization solution, dissolve it with solvent and make up the volume, seal it and heat it in a 60 ° C water bath for 40 minutes, take it out and cool it to room temperature for use.

[0053] Mass spectrometry conditions: detection by triple quadrupole tandem mass spectrometer; electrospray ionization source (ESI), acquisition mode is positive ion mode; acetylhydrazine derivative monitoring ion pairs and collision voltage (CE) are shown in the table below.

[0054]

[0055] Determination method: Accurately pipette 5 μl of the above reference solution, inject it into the HPLC-MS / MS instrument, determine the peak area, and measure it 6 times in a row. Accurately pipette 5 μl of the above test solution, inject it into the HPLC-MS / MS instrument, determine the peak area, and calculate the acetylhydrazine content in the test sample by the peak area external standard method.

[0056] The test spectrum of the reference substance is as follows Figure 1 As shown, the cefazolin sodium test spectrum is as follows Figure 2 As shown. It shows that the method of the present invention can effectively detect acetyl hydrazine. 50.13 mg of cefazolin sodium was weighed, and after testing, the content of acetyl hydrazine in cefazolin sodium was 0.14 ppm.

[0057] Example 2

[0058] 1. Standard working curve

[0059] Linear solutions containing acetylhydrazine at concentrations of 0.75, 2.26, 3.77, 7.35, 11.30, and 15.07 ng / ml were prepared in sequence (the solvents used were the same as in Example 1, to which 0.1 mL of the derivatization solution was added, and the derivatization solution was the same as in Example 1), and injected into a high performance liquid chromatography-tandem mass spectrometer in sequence, the chromatographic conditions and mass spectrometry conditions were the same as in Example 1, the ion flow diagram was recorded, a linear regression curve was made with concentration and peak area, and the linear equation and correlation coefficient were calculated. The test results are shown in Table 1 and Figure 3 As shown:

[0060] Table 1 Linear equations and correlation coefficients

[0061]

[0062]

[0063] As shown in Table 1, the concentration of acetylhydrazine is in the range of 0.75 ng / ml to 15.07 ng / ml, and there is a good linear relationship between the concentration and the peak area.

[0064] 2. Limit of Quantitation Test

[0065] A test solution containing acetylhydrazine at a concentration of 0.75 ng / ml was prepared (the solvent used was the same as in Example 1, wherein 0.1 mL of a derivatization solution was added, and the derivatization solution was the same as in Example 1), injected into a high performance liquid chromatography-tandem mass spectrometer, the chromatographic conditions and mass spectrometry conditions were the same as in Example 1, the ion flow diagram was recorded, and the test was performed continuously for 6 times. The test results are shown in Tables 2 and 3. Figure 4 As shown:

[0066] Table 2 Quantitation limit data

[0067] Serial number 1 2 3 4 5 6 average value Signal-to-Noise Ratio 62.6 42.3 52.6 75.9 77.9 109.3 70.1

[0068] As shown in Table 2, the limit of quantification concentration is 0.75 ng / ml.

[0069] 3. Detection limit test

[0070] A test solution containing acetylhydrazine at a concentration of 0.23 ng / ml was prepared (the solvent used was the same as in Example 1, wherein 0.1 mL of a derivatization solution was added, and the derivatization solution was the same as in Example 1), injected into a high performance liquid chromatography-tandem mass spectrometer, the chromatographic conditions and mass spectrometry conditions were the same as in Example 1, the ion flow diagram was recorded, and the test was repeated three times. The test results are shown in Tables 3 and 4. Figure 5 As shown:

[0071] Table 3 Detection limit data

[0072] Serial number 1 2 3 Signal-to-Noise Ratio 19.0 36.3 10.6

[0073] As shown in Table 3, the detection limit concentration is 0.23 ng / ml.

[0074] 4. Precision test

[0075] Precision test solution: Take about 50 mg of cefazolin sodium, accurately weigh, place in a 10 ml volumetric flask, add 1 ml of acetylhydrazine reference substance stock solution (same as Example 1), 0.1 ml of derivatization solution (same as Example 1), dissolve with solvent (same as Example 1) and dilute to scale, shake well, seal and heat in a 60°C water bath for 40 min, take out and cool. Prepare 6 portions in the same way. Inject into a high performance liquid chromatography-tandem mass spectrometer in sequence, the chromatographic conditions and mass spectrometry conditions are the same as in Example 1, record the ion flow diagram, and the test results are shown in Table 4 and Figure 6As shown:

[0076] Table 4 Precision data

[0077]

[0078]

[0079] As can be seen from Table 4, the method of the present invention has good precision.

[0080] 5. Accuracy test

[0081] Accuracy test solution: Take 50 mg of cefazolin sodium, accurately weigh it, put it in a 10 ml volumetric flask, add 0.5 ml, 1.0 ml, and 1.5 ml of acetylhydrazine reference substance stock solution (same as Example 1), and 0.1 ml of derivatization solution (same as Example 1), dissolve it with solvent (same as Example 1) and dilute it to the scale, shake it well, seal it and heat it in a 60°C water bath for 40 minutes, take it out and cool it. Prepare 3 copies of each test solution in the same way. Take the above test solutions and inject them into the high performance liquid chromatography-tandem mass spectrometer in sequence. The chromatographic conditions and mass spectrometry conditions are the same as in Example 1. Record the ion flow diagram. The test results are shown in Table 5 below:

[0082] Table 5 Accuracy data

[0083]

[0084] It can be seen from Table 5 that the method of the present invention has good accuracy.

[0085] Example 3

[0086] The content of acetylhydrazine in the following two representative batches of cefazolin sodium was determined according to the method of Example 1. The test results are shown in Table 6 below:

[0087] Table 6 Test results

[0088]

Claims

1. A method for determining acetylhydrazine in cefazolin sodium by high performance liquid chromatography-tandem mass spectrometry, characterized in that: Using high performance liquid chromatography-tandem mass spectrometry; Chromatographic conditions: Chromatographic column: Agilent ZORBAX Eclipse XDB-C18 (3.0*150 mm, 3.5 μm); Mobile phase A: 0.1% by volume formic acid in water; Mobile phase B: 0.1% by volume formic acid in acetonitrile; Gradient elution, conditions are as follows: At 0 minutes, the volume proportion of mobile phase A is 65-75%, and the volume proportion of mobile phase B is 25-35%; at 1 minute, the volume proportion of mobile phase A is 65-75%, and the volume proportion of mobile phase B is 25-35%; at 5 minutes, the volume proportion of mobile phase A is 15-25%, and the volume proportion of mobile phase B is 75-85%; at 7 minutes, the volume proportion of mobile phase A is 15-25%, and the volume proportion of mobile phase B is 75-85%; at 7.1 minutes, the volume proportion of mobile phase A is 65-75%, and the volume proportion of mobile phase B is 25-35%; at 12 minutes, the volume proportion of mobile phase A is 65-75%, and the volume proportion of mobile phase B is 25-35%; Solvent: ammonium acetate aqueous solution with a pH value of 4.5; Derivatization solution: dissolve benzaldehyde in acetonitrile to prepare a benzaldehyde acetonitrile solution with a volume concentration of 10-15%; Test solution: Cefazolin sodium, derivatization solution and solvent are mixed, fully dissolved, and reacted to obtain a test solution.

2. The method for determining acetylhydrazine in cefazolin sodium by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The chromatographic conditions also include: mobile phase flow rate of 0.3 ml / min; column temperature of 40° C.; injection volume of 5 μl.

3. The method for determining acetylhydrazine in cefazolin sodium by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: Gradient elution conditions: at 0 minutes, the volume proportion of mobile phase A was 70%, and the volume proportion of mobile phase B was 30%; at 1 minute, the volume proportion of mobile phase A was 70%, and the volume proportion of mobile phase B was 30%; at 5 minutes, the volume proportion of mobile phase A was 20%, and the volume proportion of mobile phase B was 80%; at 7 minutes, the volume proportion of mobile phase A was 20%, and the volume proportion of mobile phase B was 80%; at 7.1 minutes, the volume proportion of mobile phase A was 70%, and the volume proportion of mobile phase B was 30%; at 12 minutes, the volume proportion of mobile phase A was 70%, and the volume proportion of mobile phase B was 30%.

4. The method for determining acetylhydrazine in cefazolin sodium by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The solvent is prepared as follows: prepare a 20 mmol / L ammonium acetate aqueous solution and adjust the pH value to 4.5 using glacial acetic acid.

5. The method for determining acetylhydrazine in cefazolin sodium by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The derivatization solution was a 12% by volume solution of benzaldehyde in acetonitrile.

6. The method for determining acetylhydrazine in cefazolin sodium by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In the preparation of the test solution, the mass ratio of cefazolin sodium to the total volume of the derivatization solution and the solvent is 4-6 g / L, preferably 5 g / L; the mass ratio of cefazolin sodium to the volume of the derivatization solution is 480-520 g / L, preferably 500 g / L.

7. The method for determining acetylhydrazine in cefazolin sodium by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In the preparation of the test solution, the reaction temperature is 55-65°C and the reaction time is 30-50 minutes; preferably, the reaction temperature is 60°C and the reaction time is 40 minutes.

8. The method for determining acetylhydrazine in cefazolin sodium by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: Reference substance stock solution: the reference substance is dissolved in a solvent to prepare a reference substance stock solution containing 70-80 ng of the reference substance per 1 ml; reference substance solution: 1 ml of the reference substance stock solution is placed in a 10 ml volumetric flask, 0.1 ml of the derivatization solution is added, diluted to the scale with the solvent, and shaken well; after sealing, heat in a water bath at 55-65° C. for 30-50 minutes to obtain the reference substance solution; preferably, heat in a water bath at 60° C. for 40 minutes to obtain the reference substance solution.

9. The method for determining acetylhydrazine in cefazolin sodium by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: Mass spectrometry conditions: detection by triple quadrupole tandem mass spectrometer; electrospray ionization source (ESI), acquisition mode is positive ion mode; acetylhydrazine derivative (acetylhydrazine and benzaldehyde reaction to obtain derivatives) monitoring ion pairs and collision voltage (CE) are as follows 10. The method for determining acetylhydrazine in cefazolin sodium by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: Determination method: Accurately aspirate the reference solution, inject it into the high performance liquid chromatography-tandem mass spectrometer, and determine the peak area; accurately aspirate the test solution, inject it into the high performance liquid chromatography-tandem mass spectrometer, determine the peak area, and calculate the acetylhydrazine content in the test sample according to the peak area external standard method.

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